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NATURE AND IMPORTANCE OF OXYGEN-CONSUMING
MICROBES IN TERMITE GUT 8

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JOHN TIMOTHY WERTZ

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NATURE AND IMPORTANCE OF OXYGEN-CONSUMING MICROBES IN
TERMITE HINDGUTS

By

John Timothy Wertz

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Microbiology and Molecular Genetics

2006

ABSTRACT

NATURE AND IMPORTANCE OF OXYGEN-CONSUMING MICROBES IN
TERMITE HINDGUTS

By
John Timothy Wertz

In termite hindguts, the fermentative production of acetate — a major carbon
and energy source for the termite - depends upon the efficient removal of inwardly
diffusing Oz by microbes residing on and near the hindgut wall. However, little is
known about the identity of these microorganisms or the substrates used to support
their respiratory activity. A cultivation-based approach was used to isolate potentially
important 02 consuming organisms within the hindgut of Reticulitermes flavipes.
Highest recoveries of colonies were obtained on plates incubated under hypoxia (2%
02); and the increased recoveries were attributed to novel, rod-shaped, obligately
microaerophilic Neisseriaceae that shared 99.7% 168 rDNA identity, but were <95%
similar to any other known bacteria. Nearly identical organisms were isolated or their
16S rRNA genes amplified from geographically separated and genetically distinct
populations of Reticulitermes. PCR-based procedures implied that the isolates were
autochthonous to the hindgut, were associated with the hindgut wall, and comprised
ca. 3% of the hindgut bacterial community.

Further characterization of Neisseriaceae representative strain TAM-0N1
revealed that the isolate used a limited range of energy sources that included
acetate. On solid medium, the optimal 02 concentration for growth was 1%, with no
growth above 4% or in the absence of 02, though TAM-0N1 could adapt to higher
(16%) oxygen concentrations in liquid medium and expressed both catalase and

superoxide dismutase enzymes. These data suggest TAM-0N1 and related strains

warrant recognition as a new genus and species, for which the name 'Stenoxybacter
acetivorans’ gen. nov., sp. nov. is proposed.

Enzymes expressed by TAM-0N1 indicative of growth on acetate included
acetate kinase (ACK; EC 2.7.2.1) and phosphotransacetylase (PTA; EC 2.3.1.8), but
not acetyl-CoA synthetase (EC 6.2.1.1). TAM-0N1 did not appear to possess typical
glyoxylate cycle enzymes, suggesting that it has an alternative pathway to replenish
TCA cycle intermediates or can obtain these compounds in situ. All “Stenoxybacter’
isolates possessed ccoN, which encodes the oxygen-reducing subunit of the high-
affinity cbbg-type cytochrome oxidase. “Stenoxybactef-specific transcripts of ccoN,
ack and pie were detected in hindguts of R. flavipes by RT-PCR, suggesting the
population is oxidizing acetate and consuming oxygen in situ. The maximum
contribution of the “Stenoxybacter‘ strains to total hindgut 0; consumption was
approximately 0.1 - 2.0%, similar to other hindgut isolates. Experiments designed to
estimate the relative contribution of major microbial groups to total hindgut 0;
consumption suggested that protozoa may be more important to 02 reduction than
previously thought.

Concurrent with the above work, a novel method for the detection and
isolation of specific microorganisms, termed “Plate Wash PCR,’ was developed. A
result of this effort was the isolation of novel acidobacteria and verrucomicrobia from
termite guts and from soil. Such microbes have often been detected in these
environments but few have been previously isolated. Further investigation revealed
that only the verrucomicrobia were autochthonous to the termite gut, and that both
the verrucomicrobia and acidobacteria isolates are minor members of the gut

community and probably have little to no impact on overall hindgut 02 consumption.

Copyright by
JOHN TIMOTHY WERTZ

2006

ACKNOWLEDGEMENTS

My earnest and most heartfelt gratitude is extended to the many people
that supported me throughout my dissertation research. First and foremost, I am
ever grateful for the support (both financial and emotional) given to me by my
family; especially my wife Kristine, my parents Tim and Janien, and my sister
Catherine. For as much love and effort that I put into this dissertation, they put
one hundred times more into me. This dissertation is as much theirs as mine. My
highest regards and sincerest appreciation to my advisor, Dr. John Breznak, who
took a wide-eyed college graduate and made a scientist out of him. His
contagious enthusiasm for discovery and unflinching requirement for nothing less
than excellence has set a standard I will spend a lifetime emulating. My success
as a scientist, present and future, is because of this excellent tutelage. I would
also like to thank my colleagues over the past five years, especially Bradley
Stevenson, Joseph Graber, Jorge Rodrigues, James McKinlay, Stephanie
Eichorst, Kristin Huizinga, and Uri Levine. My appreciation also to Kwi Kim, who,
in addition to providing superior technical advice, looked out for me in the
laboratory from day one. I would also like to extend my appreciation to the
members of my committee, who have been nothing but encouraging and
consistently helpful: Dr. Robert Britton, Dr. Edward Walker, Dr. Thomas Whittam,
and especially Dr. Thomas Schmidt, who has been like a second advisor, going
above and beyond to ensure my present and future success, scientific and

othenrvise.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................ viii
LIST OF FIGURES ................................................................................ ix
CHAPTER 1: INTRODUCTION ................................................................ 1
Global Impact of Termites ............................................................... 1
Nature of Termite Gut Prokaryotes ................................................... 2
Function of Termite Hindgut Microbial Symbionts ................................. 8
The Importance of 02 Consumption in Termite Guts ........................... 13
Linking Community Structure and Function ....................................... 16
Dissertation Research .................................................................. 17
References ................................................................................ 19

CHAPTER 2: “STENOXYBACTER ACETIVORANS” GEN. NOV., SP. NOV.,
AN ACETATE OXIDIZING OBLIGATE MICROAEROPHILE AMONG DIVERSE

02 CONSUMING BACTERIA FROM TERMITE GUTS ................................. 26
Introduction ................................................................................ 26
Materials & Methods .................................................................... 28
Results & Discussion ................................................................... 43

Isolation and enumeration of putative 02 consuming organisms. ........43
Autochthony of the TAM-strain community .................................... 49
Geographical distribution ........................................................... 53
Relationship to oxygen .............................................................. 54
Substrate utilization of strain TAM-DN1 ........................................ 58
Rationale for the proposal of TAM-0N1 and related strains as a new
genus and species ................................................................... 62
Description of “Stenoxybactef' gen. nov ....................................... 63
Description of “Stenoxybacter acetivorans” sp. nov ......................... 63
Acknowledgements ..................................................................... 64
References ................................................................................ 65

CHAPTER 3: PHYSIOLOGICAL ECOLOGY OF “STENOXYBACTER

ACETIVORANS” IN TERMITE GUTS ...................................................... 71
Introduction ................................................................................ 71
Materials & Methods .................................................................... 76
Results ..................................................................................... 90

In situ location of “S. acetivorans” ............................................... 9O
Enzymes and genes relevant to acetate oxidation and 02 consumption
in vifm ................................................................................... 91

vi

Genes expressed by “S. acetivorans” in situ ................................ 101
Oxygen consumption by “S. acetivorans” and other components of the

R. flavipes gut ....................................................................... 104
Discussion ............................................................................... 109
Conclusions .............................................................................. 123
Acknowledgements .................................................................... 125
References .............................................................................. 126

CHAPTER 4: SUMMARY ..................................................................... 132
APPENDIX: Cover Page ..................................................................... 139
NEW STRATEGIES FOR THE CULTIVATION AND DETECTION OF
PREVIOUSLY UNCULTURED MICROBES ............................................. 140
Abstract ................................................................................... 141
Introduction .............................................................................. 142
Materials & Methods .................................................................. 144
Results 8. Discussion .................................................................. 155

Specificity and sensitivity of PCR and PWPCR ............................ 155

Treatment effects and isolation of Acidobacten'a and

Venucomicmbia .................................................................... 1 56

Properties of Acidobacten‘a and Verrucomicrobia isolates .............. 160

Overview of the PWPCR-based isolation procedure ..................... 164
Acknowledgements .................................................................... 166
References .............................................................................. 167

vii

LIST OF TABLES

Table 2.1. Total cultivable bacteria from the guts of geographically separated
Reticulifermes workers incubated under Gog-enriched oxic and hypoxic

atmospheres ....................................................................................... 44
Table 3.1. Gene-targeted broad range and “Stenoxybacter acetivorans"-specific
PCR primers used in this study ................................................................ 82
Table 3.2. Enzymes and genes relevant to acetate oxidation and 02
consumption in acetate-grown cells of “S. acetivorans” ................................. 93
Table 3.3. Substrate-specific oxygen consumption rates of TAM-1, Citrobacter
sp. RFC-10, and whole R. flavipes guts ................................................... 106
Table 3.4. Effect of diet on gut microbial communities and 02 consumption rates
of whole guts of R. flavipes ................................................................... 107
Table 3.5. Estimated contribution of “Stenoxybactef’ sp., Enterobacten‘aceae,
and lactic acid bacteria to the total hindgut 02 consumption rate .................. 118
Table A1. PCR primers used for Plate Wash PCR .................................... 151

viii

LIST OF FIGURES

Figure 1.1. A worker larva of the eastern subterranean termite, Reticulitermes
flavipes, is pictured above an extracted midgut and hindgut from a separate
worker. The hindgut paunch is labeled PA. Scale bar = 1 mm. Adapted from
Breznak and Leadbetter, 2002 .................................................................. 3

Figure 1.2. Phase-contrast light (top) and scanning electron (bottom)
micrographs of microorganisms within the hindgut paunch fluid (A) and
associated with the epithelial wall (B). The hindgut fluid harbors protozoa (P), a
remarkable morphological diversity of spirochetes (white arrows) including those
attached to some protozoans, as well as other rod- or vibrioid- shaped
prokaryotes (black arrows). Undigested wood particles can also be seen (W).
Associated with the epithelia are a diversity of mostly non-protozoal and non-
spirochetal microbes, some of which form microcolonies (arrows). Scale bars
represent 0.01 mm (A) and 10 um (B). Adapted from Breznak and Leadbetter,
2002 (A) and Breznak and Pankratz (B) ...................................................... 6

Figure 1.3. Diagrammatic representation of carbon flow in the termite
Reticulitermes flavipes. Numbers indicate the flux rates (in nmoI C-h°‘-termite")
estimated from microinjection of radiolabeled substrates (T holen et al., 2000) or
direct measurements of C02 and CH4 emission (Odelson and Breznak, 1983).
Lactate and pynlvate have not been directly measured in hindgut fluid and are
therefore represented in brackets. Question marks indicate hypothetical
pathways for which little or no experimental support exists. The thickness of
arrows indicates the relative contribution of the pathway .............................. 10

Figure 1.4. Radial gradients of oxygen (0) and hydrogen (0) in an agarose-
embedded hindgut of Reticulitennes flavipes as measured by microelectrodes.
The insert represents the hindgut paunch and indicates the zones of hypoxia and
anoxia. Adapted from ane, 1998 ........................................................... 15

Figure 2.1 . Composite photograph showing colony morphotypes on two separate
isolation plates. Arrows point to the unique colony morphotype present on the
plate incubated in a hypoxic atmosphere (A) that were not on the plate incubated
in COz-enriched air (B) .......................................................................... 45

Figure 2.2. Transmission electron (A), scanning electron (B) and phase-contrast
light (C) micrographs of termite gut Neisseriaceae strain TAM-0N1. Arrows point
to intracellular granules that resemble poly-B-hydroxybutyrate. Scale bars are 1
pm (A, B) and 10 um (C) ........................................................................ 46

Figure 2.3. Maximum likelihood-based 16S rRNA gene phylogeny of isolates

obtained from Reticulitermes flavipes collected from Dansville, Ml. Isolates
obtained in this study are shown in boldface. Many closely related isolates are

ix

condensed as gray trapezia with the number of sequences in parentheses.
Aquifex pyrophilus was used as an outgroup (not shown) ............................. 50

Figure 2.4. Dilution-to-extinction PCR of DNA from termite hindguts and
degutted termite bodies. A. R. flavipes DNA amplified with Verrucomicrobia-
specific 16S rDNA primers. (1) 1 kb DNA ladder; (2-9) one gut-equivalent DNA
serially diluted 1:2 8x; (10-13) one body-equivalent DNA serially diluted 1:2 4x;
(14) positive control Venucomicrobia st. TAV-1 DNA. B. R. flavipes DNA
amplified with Acidobacten'a-specific 16S rDNA primers. (1) 1 kb DNA ladder, (2-
8) two gut-equivalents’ DNA serially diluted 1:2 7x; (9-15), two body-equivalents’
DNA serially diluted 1:2 7x; (16) positive control Acidobacten'um capsuletum
DNA. C. Purified DNA from Venucomicrobia strain TAV-1 amplified with
Venucomicrobia—specific 16$ rDNA primers as a control. (1) 1 kb DNA ladder, (2-
9) 100 ng DNA serially diluted 1:16 8x; (10) 100 ng E. coli DNA; (11) negative
control without DNA .............................................................................. 51

Figure 2.5. Maximum likelihood-based phylogeny of PCR-generated 16S rRNA
gene clones from R. flevipes termite guts (51 clones; black rectangle), termite
nest soil (29 clones; gray rectangles) and adjacent non-tennite inhabited forest
soil (25 clones; white rectangles) obtained with a termite gut Neisseriaceae-
specific primer set. Closely related clones were condensed into a single
rectange, with the number of clones given in parentheses. Rectangles are
aligned for ease of comparison; such alignment is not intended to imply equal
evolutionary time. Scale bar represents 0.1 change per nucleotide..................52

Figure 2.6. Neighbor-joining analysis of 5 microsatellite loci (44 alleles) of 43

individual worker termites collected at different geographical locations. R. flavipes
workers were collected from Dansville, MI (DN); Raleigh, NC (NC); Spring Arbor,
MI (SA); Janesville, WI (JN); and Woods Hole, MA (WH). R. santonensis workers
were collected from Foret de la Coubre, France (FC) ................................... 55

Figure 2.7. Maximum likelihood-based 16S rRNA gene phylogeny of termite gut
Neisseriaceae isolates and clones. Isolates were obtained from R. flavipes
collected at: Dansville, Ml (DN); Raleigh, NC (NC); Spring Arbor, (SA); and
Janesville, WI (JN). 16S rRNA gene clones were obtained from R. flavipes
collected at Woods Hole, MA (WH). Isolates were also obtained from R.
santonensis from Forét de la Coubre, France (FC). Lactococcus lactis was used
as an outgroup (not shown). Numbers at nodes represent percentage of topology
conservation after 100 bootstrap samplings. Bar represents 0.1 change per
nucleotide ........................................................................................... 56

Figure 2.8. 02 tolerance of Neisseriaceae strain TAM-0N1. (A) Growth on solid
medium in Wolfe bottles containing a headspace of: 0% (1), 2% (2), 4% (3), 6%
(4) v/v oxygen. (B) Growth in liquid medium under an atmosphere of: 0% (A), 2%
(2), 4% (3), 8% (4), and 16% (5) v/v oxygen ............................................... 59

Figure 2.9. Final cell yield (In) and lag time (o) of TAM-DNl cells grown in liquid
medium under a headspace of 0% to 8% oxygen ........................................ 60

Figure 2.10. Co-utilization of acetate (0) and succinate (A) by TAM-DN1 during
growth (9) ........................................................................................... 61

Figure 3.1. Common pathways for acetate activation and metabolization.
Enzymes involved in acetate activation are: acetate kinase (ACK),
phosphotransacetylase (PTA), or acetyl-CoA synthetase (ACS). Once activated,
acetyl-CoA proceeds through the TCA cycle. Typically, if acetate is the sole
carbon source, replenishment of TCA cycle intermediates drawn off for
biosynthesis such as 2-oxoglutarate or oxaloacetate is accomplished by the
combination of isocitrate lyase (ICL) and malate synthase A (MSA). For
biosynthesis of glucose from acetate, the anapleurotic enzyme
phosphoenolpyruvate carboxykinase (PEPCk) is used ................................. 74

Figure 3.2. In situ association of “S. acetivorans" with the gut epithelial wall.
Quantification of product intensity after PCR of termite gut fluid (gray bars) or
sliced and washed gut epithelium (black bars) with “Stenoxybactef-specific (A)
or spirochete-specific (B) 16S rDNA primers. Error bars represent standard
deviation (n=3) .................................................................................... 92

Figure 3.3. Maximum likelihood-based phylogenetic analysis of the deduced
amino acid sequence (178 positions) of acetate kinase from selected “8.
acetivorans” isolates (boldface) and R. flavipes guts (boldface+italics). Numbers
within R. flavipes clusters represent the number of sequences within that cluster.
The acetate kinase from Neurospora crassa was used as an outgroup. Scale bar
represents 0.1 change per amino acid ...................................................... 96

Figure 3.4. Maximum likelihood-based phylogenetic analysis of the deduced
amino acid sequence (201 positions) of phosphotransacetylase from selected “8.
acetivorans” isolates (boldface). Escherichia coli PTA is used as an outgroup.
Scale bar represents 0.1 change per amino acid ......................................... 97

Figure 3.5. Maximum likelihood-based phylogenetic analysis of the evolutionarily
related malate synthase A, malate synthase G, and malyI-coA lyase proteins.
The deduced amino acid sequences (170 positions) from selected “8.
acetivorans” isolates (boldface) group within the malate-synthase G cluster. The
malyI-coA lyase cluster is used as an outgroup. Scale bar represents 0.1 change
per amino acid .................................................................................... 98

Flgure 3.6. Maximum likelihood-based phylogenetic analysis of the deduced
amino acid sequence (136 positions) of the Oz-reducing (ccoN) subunit of the
cbb3-type cytochrome oxidase from selected “8. acetivorans” isolates (boldface)
and R. flavipes guts (boldface+italics). The ccoN subunit from Magnetospirillum

xi

magnetotacticum is used as an outgroup (not shown). Scale bar represents 0.1
change per amino acid .......................................................................... 99

Figure 3.7. Gelstar-stained agarose gel electrophoresis of RT-PCR products for
(A) acetate kinase (ack); (B) phosphotransacetylase (pie); and (C) the Orbinding
subunit of the cbb3 terminal oxidase (ccoN) from R. flavipes gut homogenate
RNA with “S. acetivorans”-specific, gene-targeted primers. Lane 1, 1 kb DNA
ladder. Lane 2, RT-PCR product using R. flavipes gut RNA as template. Lane 3,
RT-PCR with R. flavipes gut RNA without the addition of reverse transcriptase
enzyme. Lane 4, RT-PCR with TAM-0N1 DNA as template. Lane 5, RT-PCR
without the addition of template ............................................................. 102

Figure 3.8. Maximum likelihood-based phylogenetic analysis of the deduced
amino acid sequences of RT-PCR products depicted in Fig. 3.7. All RT-PCR
products clustered specifically with the respective genes from “S. acetivorans”
isolates. (A) acetate kinase (170 positions); (B) phosphotransacetylase (182
positions); (C) ccoN subunit of cbb3 oxidase (124 positions). Neisseria
meningitidis was used as an outgroup for all phylogenetic analyses (not shown).
Scale bars represent 0.1, 0.05 or 0.025 changes per amino acid .................. 100

Figure 3.9. The citramalate cycle - an alternative to the glyoxylate cycle for
acetate assimilation in organisms lacking isocitrate lyase activity. Key enzymes
are: citramalate synthase (CMS), B-methylmalyl-CoA lyase (MMC), malyl-CoA
lyase (MLC) and malate synthase A or G (MS). It is not yet known if the product
of the CMS reaction is citramalate or citramalyI-CoA. Figure adapted from
(Meister et al. 2005) ............................................................................ 1 14

Figure 3.10. Correlation between R. flavipes whole-gut oxygen consumption rate
and the number of gut protozoa (A) or cultivable prokaryotes (B). r2 values for the
best-fit regression lines are 0.95 (A) and 0.56 (B). Termites were maintained for
11 days on diets of cellulose (V), cellulose with antibiotics (o), starch (A), and
starch with antibiotics (0). Wood control (I) .............................................. 121

Figure A.1. Plate Wash PCR method to detect growth, and monitor isolation, of
targeted bacteria. Of the three medium and incubation conditions shown in this
diagram (A, B, C), growth of targeted bacteria is represented only in "C'........150

Flgure A.2. Detection of Venucomicrobium spinosum within a collection of
diverse bacteria isolated from soil. (A) A single V. spinosum colony is shown
among 94 other colonies growing on an agar plate. (B) Plate Wash PCR with
Venucomicrobia-specific primers using template in which V. spinosum colony
material represented: 1 part in: 95 (Le. plate in panel A; lane1); 1 part in: 189
(lane 2); 1 part in: 471 (lane 3); 1 part in: 941 (lane 4); and 1 part in: 9,401 (lane
5). Plate Wash PCR of a control plate lacking V. spinosum (lane 6); negative
control (no DNA, lane 7); and V. spinosum DNA (50 ng; lane 8) are also shown in
Panel B. Sizes (kb) of markers in lane M are given to the left ...................... 158

xii

Figure A.3. Influence of medium additives and incubation conditions on CFU
recovered from soil (panel A) and on the occurrence of Acidobacten’a among the
isolates (panel B). The label “air” refers to the concentration of 002 (0.03% v/v) in
normal air. Data in panel (A) represent the mean CF U recovered from soil among
n samples. Error bars represent sample standard deviation. Data in panel (B)
represent the frequency with which Acidobacten’a were detected among the
plates in panel A using Plate Wash PCR. These data were subjected to chi-
square analyses with a Bonferroni error rate adjustment. Statistical significance
(a = 0.10, df = 1) is indicated by an * ...................................................... 159

Figure A.4. Maximum likelihood tree (left) of subdivisions 1-4 of the phylum
Acidobacteria based on 16S rRNA gene sequences from organisms in culture, as
well as PCR-generated clones from soil. Isolates obtained in this study are
shown in boldface. Bootstrap values for branchpoints of the major subdivisions
are given. Branchpoints conserved in all analyses with bootstrap values >75%
are represented as closed circles; bootstrap values of 50 - 74% are represented
as open circles. Subdivisions 2-4 are labeled. bracketed, and condensed as grey
trapezia with the number of sequences represented in parentheses. 16S rRNA
gene sequences of members of subdivisions 6-8 were used as outgroups (not
shown). The scale bar represents 0.10 changes per nucleotide. The scanning
electron micrograph (right) shows K8889 cells trapped in an extracellular

matrix .............................................................................................. 162

Figure A.5. Maximum likelihood tree of subdivisions 1-4 and 6 of the phylum
Vermcomicrobia based on 16S rRNA gene sequences from organisms in culture
as well as PCR-generated clones from environmental samples (Panel A).
Isolates obtained in this study are shown in boldface. Bootstrap values for
branchpoints of the major subdivisions are given. Branchpoints conserved in all
analyses with bootstrap values >75% are represented as closed circles;
bootstrap values of 50 - 74% are represented as open circles. Subdivisions 1-3
and 6 are labeled, bracketed, and condensed as grey trapezia with the number of
represented sequences in parentheses to simplify presentation of the tree. 16S
rRNA gene sequences of members of the phylum Planctomycetes were used as
an outgroup (not shown). The scale bar represents 0.10 changes per nucleotide.
The scanning electron micrograph of TAV2 (B) shows the doublet cell
morphology shared by all TAV isolates; and that of TAV1 (C) shows the
encapsulation of the cells in an extracellular matrix, a morphological feature not
shared by TAV2,3, or 4 ........................................................................ 163

xiii

Chapter 1

Introduction

" The termite hindgut is filled wall-to-wall with these fascinating
morphologies of organisms. I was really gripped with it in the same
way you might be interested in going through a rainforest - you don‘t

have to be a scientist to appreciate that this is a beautiful place."
- J. Leadbetter (as quoted in 47)

Global Impact of Termites

Termites are one of the most abundant and ecologically relevant soil-
dwelling insects on earth. In some areas, such as tropical forests, termite
biomass (39-110 glmz) surpasses that of grazing herbivores (0.013 - 17.5
g/m2)(60). Together with their gut-associated microbial symbionts, termites have
evolved to thrive on the most abundant form of biomass on the planet,
“lignocellulose” (Iignin, cellulose, and hemicellulose), the primary components of
wood and other terrestrial plant material, and residues derived from it (eg.
humus). Mineralization of a significant portion of the ingested plant material,
chiefly cellulose and hemicellulose, has significant ecological consequences. It is
estimated that globally, termites consume about 2.2 — 5.1% of the 136 x 10“" g of
dry plant material produced from photosynthesis (11), contributing 2% and 4% to
the biogeochemical cycling of the greenhouse gasses 002 and CH4, respectively

(60). Less obvious, but locally dramatic, are termite-mediated changes in the

physical, chemical and biological properties of soils that may contribute
significantly to biogeochemical cycling of carbon, nitrogen, and nutrients (25).
Termites can be categorized into two groups, the so—called “lower"
termites, which are phylogenetically basal, feed on sound or degraded wood, and
harbor protozoan symbionts within their hindguts, or the “higher” termites, which
are phylogenetically derived, include wood, soil, litter, and fungus-feeders, have
highly compartmentalized guts, and typically lack gut protozoans. Seventy-five
percent of the estimated 2,600 species of termites belong to a single family of
higher termite, the Termitidae (31). The remaining 25% of termite species are
distributed among six recognized families of lower termites (Tennopsidae, Hodo-,
Kan-, Masto-, Rhino- and Serritennitidae). The gut microbiota of the lower
termite Reticuliterrnes flavipes (Kollar, Rhinoterrnitidae) is perhaps the most well

studied (7, 8, 17), and so it was used as a model in this dissertation (Figure 1.1).

Nature of Termite Gut Prokaryotes

The ability of termites to thrive on a relatively refractory and nitrogen-poor
(approx 0.05% N w/w) food resource is due to the dense populations of
symbionts harbored in their guts. Only recently, through cultivation-independent
16S rDNA-based techniques, has the full scope of prokaryotic diversity in the
termite gut begun to be revealed. In two of the most comprehensive studies of
termite gut microbial diversity thus far, Hongoh et al. (26, 27) analyzed a total of
2304 bacterial 16S rRNA gene clones from 32 termite colonies representing four

species of Reticulitennes and four species of the higher termite,

 

Hindgut

Figure 1.1. A worker larva of the eastern subterranean termite, Reticulitennes
flavipes, is pictured above an extracted midgut and hindgut from a separate
worker. The hindgut paunch is labeled PA. Scale bar = 1 mm. Adapted from
Breznak, 1984.

Microcerotennes. In total, these studies have identified 367 bacterial phylotypes
(using the criterion of 97% sequence identity) from Reticuliterrnes sp. and 228
phylotypes from Microcerotermes sp. The 16S rRNA gene sequences from both
termites together encompass 17 bacterial divisions, including the candidate
Termite Group 2 and Termite Group 3 divisions. Estimates of overall diversity
based on these studies indicate the termite gut may harbor over 700 different
phylotypes (27).

At the division level, the dominant phylotypes in guts of lower termites
appear to be similar. In the case of Reticulitermes speratus (26, 27), R.
santonensis (69), Coptotennes fonnosanus (56), and Cryptotennes domesticus
(44), the 16S rRNA gene libraries are dominated by members of the divisions
Spirochaetes, Bacteroidetes, and Firmicutes. These divisions appear to be the
most phylotype-rich; in R. speratus, the Clostn'diales (Firmicutes) were
represented by 100 unique phylotypes, the Spirochaetes by 61 phylotypes, and
the Bacteroidetes by 31 phylotypes. Also consistently detected were members of
the bacterial divisions TM7, OP11, Acidobacten'a, Planctomycetes,
Verrucomicrobia and Endomicmbia that are represented by few, or no, cultivars
(28, 59).

The guts of four species of the wood-feeding higher termite,
Microcerotermes, were also dominated by members of the divisions
Spirochaefes, Bacteroidetes, and Finnicutes (26). However, statistical
comparison by I-LIBSHUFF revealed these clones were quite different at the

nucleotide sequence level from the clones from Reticulitermes gut homogenate

(P = 0.0001). Analysis of 16S rDNA clones from guts of the soil-feeding higher
termite, Cubitennes orthognathus, revealed that Clostn'diales (approximately
70% of the total number of clones), instead of Spirochaetes (10%) or
Bacteroidetes (10%) appeared to dominate (53). Clustering analysis between
conspecific and congeneric termites has revealed that the gut bacterial
composition appeared to be determined by (in order of importance): (i) the
feeding habit; (ii) the genus of the termites; and (iii) the location of the termite
colony (26).

Microscopic examination of the microbiota near the hindgut epithelia and
within the hindgut fluid of lower termites like Reticulitennes flavipes and
Coptotennes fonnosanus clearly reveal radial differences in community structure
(12). In R. flavipes, a distinctive, epithelium-associated community consists of
mostly rod- or filament-shaped prokaryotes, whereas the fluid chiefly consists of
spirochetes and protozoa (Figure 1.2) (8, 12). Using fluorescent in situ
hybridization (FISH) with group-specific probes, Berchtold et al. also
demonstrated differences in the axial distribution of microbiota in the gut of
Mastotennes darwiniensis (4). Whereas the posterior region was colonized
preferentially by Gram-positive cocci and rod-shaped and filamentous bacteria of
the Cytophaga—Flexibacter—Bacteroides (CFB) division, morphologically different
microorganisms were located in the anterior paunch and were mostly associated
with the flagellates.

Yang et al. (69) and Nakajima et al (38) dissected and separated hindguts

of R. santonensis and R. speratus, respectively, into two fractions: a “wall

 

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Figure 1.2. Phase-contrast light (top) and scanning electron (bottom)
micrographs of microorganisms within the hindgut paunch fluid (A) and
associated with the epithelial wall (B). The hindgut fluid harbors protozoa
(P), a remarkable morphological diversity of spirochetes (white arrows)
including those attached to some protozoans, as well as other rod- or
vibrioid- shaped prokaryotes (black arrows). Undigested wood particles
can also be seen (W). Associated with the epithelia are a diversity of
mostly non-protozoal and non-spirochetal microbes, some of which
form microcolonies (arrows). Scale bars represent 0.01 mm (A) and

10 um (B). Adapted from Breznak, 2000 (A) and Breznak and Pankratz,
1977 (B).

fraction” that represented the hindgut epithelium and associated organisms; and
a “fluid fraction” primarily consisting of lumen-associated microbes. From each
fraction, 16S rRNA gene clone libraries were constructed. For R. speratus, the
wall fraction was dominated by clones grouping with the Actinobacteria,
Firmicutes and Bacteroidetes. Spirochaetes and members of the candidate
phylum Endomicrobia were more numerous in the hindgut luminal fraction (38).
Statistical analysis of the libraries by LIBSHUFF revealed that the two libraries
were quite distinct (P = 0.0001). Similar libraries from R. santonensis had less
distinction between wall and fluid communities, however (69). Clones related to
Spirochaetes and Mycoplasmatales were clearly localized to the fluid fraction, but
there appeared to be no uniquely wall-associated phylotypes. A LIBSHUF F
comparison of the libraries (not reported) revealed a statistically significant
difference between the fluid library and the wall (P = 0.02), but not vice-versa (P
= 0.3), indicating the wall-associated community is a subset of the luminal
microbiota. This may be due to insufficient separation of the gut fractions, a
continual sloughing-off of wall-associated microbes into the lumen, or as ability of
facultative anaerobes or aerotolerant anaerobes in the lumen to also colonize the
gut epithelium.

Unfortunately, the number of pure culture isolates from termite guts is a
poor reflection of the diversity revealed by microscopic and 16S rRNA gene
analyses (8, 14). The more frequently encountered isolates, obtained from a
number of different termites, include Enterobacten’aceae (mostly Citrobacter or

Enterobacter sp.), members of Firmicutes (including species of Lactococcus,

Enterococcus, Staphylococcus and Lactobacillus), and Bacteroides (1, 2, 21, 55,
62). More recently, sulfate—reducing bacteria and methanogenic Archaea have
been isolated (33-35). However, it is painfully apparent that about 90% of all
prokaryotes seen by direct microscopic counts, or 99% of all bacterial phylotypes
found by 16S rRNA methods (26), still elude cultivation. This underscores the
fact that new strategies for isolation of phylotypes not-yet-represented in culture
(e.g. provision of nutrients and incubation in atmospheres that better mimic the in
situ environment) and new ways to rapidly differentiate between successful and
unsuccessful enrichment or isolation attempts continue to be necessary. Only
after painstaking, long-ten'n development of habitat-simulating enrichment media
were the first termite gut spirochetes, Treponema primitia and 7'. azotonutn'cium
coaxed into pure culture (23, 36). The effort was worthwhile: subsequent studies
revealed in the isolates metabolic pathways previously unknown in spirochetes —
COz-reductive acetogenesis and nitrogen fixation - that would have continued to

go unnoticed without the availability of pure cultures for study (36, 37).

Function of Termite Hindgut Microbial Symbionts

Though pure culture isolates of termite gut microbes are limited in number,
more than a century of elegant and detailed research has provided significant
insights into the basis for the symbiotic interaction between the termite and its
hindgut microbial symbionts: to facilitate decomposition of plant material into
utilizable sources of carbon and energy; and to provide fixed or recycled nitrogen

necessary for termite growth and survival.

Immediately upon ingestion of woody material, endogenous
cellulases secreted primarily by the midgut epithelium begin to hydrolyze a
portion of the cellulose into soluble oligosaccharides (30, 39, 64, 65) (Figure 1.3).
Some of the released glycosides cross the midgut epithelium and can proceed
through glycolysis within the termite tissue, but limited activity of pyruvate
dehydrogenase prevents further oxidation of pyruvate (41, 58). Instead, pyruvate
and glycosides may be stored, used as biosynthetic precursors, or transported
into the hindgut and metabolized by the gut microbiota (58). The main energy
requirements of the termite are met by oxidation of other metabolites produced
fermentatively by hindgut symbionts (see below).

A majority of ingested cellulose passes through the midgut into the
hindgut, and in lower termites it is then phagocytosed by anaerobic protozoa
(11). Using their own cellulase enzymes, the protozoa hydrolyze cellulose to
soluble oligosaccharides, which are largely fermented by the protozoa to acetate
according to equation 1. Some oligosaccharides, possibly from hemicellulose
degradation as well, may also be secreted by the protozoa to support a

population of microbes upon which they feed (42, 66-68).

1) [Celeoe] 4' 2H20 —’ ZCH3COOH+ 2COz + 4H2

Thus the hindgut protozoa fulfill two essential roles in lower termites: (i) their

phagocytic activity increases cellulose retention in the hindgut, allowing a

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majority of cellulose to be metabolized; and (ii) produce acetate, a utilizable
source of carbon and energy for the termite.

Fermentation of released glycosides by protozoa and anaerobic bacteria
is a major source of acetate in the hindgut (48, 51, 54, 55). Additional acetate is
produced by COz-reductive acetogenesis by bacteria such as spirochetes (6, 13,

36) using H2 and CO; released from glucose fermentation according to equation

2:

2) 4H2 + 2COz —> CH3COOH + 2HzO

Recently, Tholen et al. provided evidence for acetate production from lactate. By
microinjecting radiolabeled substrates into intact hindguts they were able to
demonstrate that up to one-third of the carbon flux in living termites proceeds via
a previously unrecognized pool of lactate (61). An absence of measurable lactate
within the hindgut implies a very rapid turnover of this small pool to acetate,

possibly by Iactate-fennenting homoacetogens (54, 55) according to equation 3:
3) 203H303 —’ 3CH3COOH
As the above pathways indicate, the hindgut system is almost completely
homoacetogenic. Acetate dominates the hindgut fatty acid pool, comprising up to

98 mol% of all volatile fatty acids with concentrations as high as 80 mM (43).

Acetate is absorbed through the hindgut epithelium, oxidized in termite tissues,

11

and serves as the major energy source for the insect (24, 43). By measuring the
rate of acetate production in the hindgut, Odelson and Breznak clearly
demonstrated that microbially produced acetate could account for 71 -1 00% of
the C02 produced by termites, thereby supporting up to 100% of their daily
respiratory requirements (43). Thus the overall sum of reactions in the hindgut

and termite tissue can be summarized in equation 4:

4) [CGH1206] + 602 _’ 6C02 4' 6H20

Utilization of lignocellulose as the primary source of carbon and energy
also requires supplementation of this nitrogen-poor diet with fixed or recycled
nitrogen. Breznak et al. (10) and Benemann et al. (3) were the first to
demonstrate nitrogen fixation in termites and attribute it to hindgut prokaryotes.
Subsequently, N2 fixation has been confirmed in more than 20 species of
termites (8). Several strains of Nz-fixing bacteria, including enterobacteria as well
as spirochetes, have been isolated from a number of different termites (22, 33,
37, 50), and molecular biological studies suggest that additional, not-yet-cultured
nitrogen-fixing organisms remain to be identified. For example, cultivation-
independent experiments have demonstrated that a diversity of nifl-I genes
(encoding dinitrogenase reductase) present in termite hindguts resemble
homologous genes in clostridia, y- and b—Proteobacten'a and Archaea (40, 45,
46). However, the relative importance to termite nitrogen requirements of

microbes bearing these genes remains to be established. An assessment of in

12

situ expression of nifH genes in the hindgut of Neotennes koshunensis by Noda
et al. demonstrated that while several phylogenetic classes of nifl-l genes were
represented among the hindgut microbiota, only a few were actually expressed in
situ (40). It now appears that termite gut treponemes may be the origin of such
expressed genes (37).

Other members of the hindgut community can also contribute to termite
nitrogen economy by recycling the nitrogen in the excretory product uric acid
which is secreted into the midgut-hindgut junction through Malpighian tubules
(48, 49). Potrikus and Breznak isolated Streptococcus, Bacteroides, and
Citrobacter strains from hindguts of R. flavipes that could anaerobically degrade
uric acid to acetate, CO2 and NH3 (48, 49, 51, 52). Additionally, they showed that
microbial degradation of ‘sN-uric acid in situ resulted in liberation of 15N that was

subsequently assimilated into termite macromolecules (49).

The Importance of 02 Consumption in Termite Guts

The demonstration that approximately 60% of the termite hindgut is
persistently hypoxic (<4% 02 vlv) has been described as “the most significant
recent advance in our understanding of metabolism in the hindgut” (57).
Historically, the termite gut was considered to be a completely anoxic system (9,
11), an assumption dating to early observations of the 02-sensitivity of the
protozoa (19). Subsequent observations of termite dependency upon the 02-
sensitive microbial processes of carbohydrate dissimilation (29), reductive

acetogenesis (13), and in some cases nitrogen fixation (10), solidified this notion.

l3

Other data, however, seemed to suggest at least some portion of the
hindgut was oxic. For example: (i) aerotolerant (21, 55), facultative, or 02-
requiring (62) microorganisms were frequently represented among hindgut
isolates; (ii) termites exposed to hyperbaric levels of 02 resulted in the death of
hindgut spirochetes and protozoa (both groups known to be anaerobes), but
increased the viable cell counts of bacteria 6- to 10-fold on culture plates
incubated in air (63); and (iii) the large surface area-to-volume ratio of the hindgut
indicates equilibrium with atmospheric oxygen should occur throughout the gut
unless mechanisms existed to block or remove it (5, 15).

The anoxic gut paradigm was not challenged, however, until Veivers et al.,
employing redox indicator dyes, noted that termites fed antibiotic drugs were no
longer able to maintain the low redox potential (Eh = -150 to -250 mV) typical of
normal guts (63). These results suggested that the gut bacteria may be oxygen
consumers that retain anoxic conditions in the hindgut lumen by removing
inwardly diffusing 02. However, experiments meant to directly detect oxygen in
the gut were not done until the observation that in situ degradation of lignin
model compounds was 02-dependent (18, 32) prompted Brune and coworkers
(16, 20) to use microelectrodes to provide a micrometer scale resolution of 02
gradients within agarose-embedded, intact hindguts of Reticulitennes flavipes
and Nasutitennes lujae (Figure 1.4). The results clearly revealed the presence of
a peripheral, hypoxic zone in the hindgut with 02 partial pressures of 30 mbar
(30% air saturation) at the epithelial surface that rapidly decrease to anoxia 150-

200 um inward. The measurements indicated that a majority of the hindgut

14

Partial pressure of gases (mbar)

 

 

 

 

0 20 40 60 80 100
o 1 1 2 1 1 J n l l 1 I
300 .
: . Agarose
600 -
E I
3 .
5 900 - ______ o . --- - . .
3 - - /( ‘ _Epithelium
o . (ca. 10 um)

  

  

1200 ‘ .
' Anoxrc

, Gut

1500 .
. Hypoxic

Figure 1.4. Radial gradients of oxygen (0) and hydrogen (0) in an agarose-
embedded hindgut of Reticulitennes flavipes as measured by microelectrodes.
The insert represents the hindgut paunch and indicates the zones of hypoxia
and anoxia. Adapted from Brune, 1998.

15

(approx. 60% of the total hindgut volume) was hypoxic, whereas the luminal
portion of the hindgut paunch remained anoxic (15). The presence of a steep 02
gradient at the epithelial wall supported the previous conclusion by Veivers et al.
that some members of the microbiota on or near the hindgut wall were an
important “oxygen sink“ (15, 16).

The realization that a majority of the hindgut may be exposed to some
level of oxygen caused a paradigm shift in which the essential role of O2
consuming microorganisms to termite vitality became clear (16, 57). The termite
is dependent upon the 02 consuming members of the peripheral gut microbiota
for the creation and maintenance of anoxic conditions within the hindgut lumen
so that the incomplete oxidation of dietary polysaccharides to acetate can occur.
If oxygen were allowed to diffuse in too far, as with incubation of termites under a
hyperbaric (30% 02) atmosphere or removal of the O2 consuming bacteria with
antibiotic drugs (63), acetate production would cease and the termite would
eventually die. Though the importance of the 02 consuming microbiota to the
continued survival of the termite was clear, upon initiation of this Dissertation,
little was known about the identity of the microbes responsible for 02
consumption in the hindgut or the substrate(s) used to support their in situ

activity.

Linking Community Structure and Function

Elegant physiological studies have appraised the symbiotic function and

importance of the termite gut microbiota in toto. Molecular techniques such as

16

16S rRNA-based identification have considerably advanced our knowledge of the
nature of the hindgut symbiotic community. However, our ability to relate a
particular function to a specific microbial population has been hampered by a
lack of a sufficient number of pure culture isolates. Though molecular methods
exist that allow metabolic, phylogenetic, and localization information to be
combined (e.g. catalyzed reporter deposition FISH (CARD-FISH), large-insert
libraries, functional gene arrays), these methods are encumbered by technical
difficulty and high cost. The most cost-effective and direct route to linking
structure and function is to use a combination of in vivo molecular techniques
with in vitro physiological studies; such methods first require, however, the

isolation of target microorganisms in culture.

Dissertation Research

The research presented in this dissertation focuses on the nature and
importance of 02 consumption within termite hindguts. Chapter 2 discusses the
isolation, enumeration, and identification of aerobic organisms from R. flavipes
guts, including a group of abundant, but hitherto unrecognized, obligately
microaerophilic B—Proteobacten’a. The characterization and classification of these
novel microorganisms as a new genus and species within the Neisseriaceae is
discussed. Chapter 3 presents evidence for in situ acetate oxidation and 02
consumption by these microaerophilic B—Proteobacten'a within the guts of R.
flavipes, and analyzes, through feeding experiments, the importance of the

bacterial and protozoal hindgut populations to overall hindgut O2 consumption.

17

Chapter 4 summarizes the main conclusions of these studies. Finally, an
appendix chapter, based upon a recent publication, reports the development of a
novel method (“Plate Wash PCR”) for the detection and isolation of previously
uncultivated microorganisms, including potentially important 02 consumers from

termite guts.

18

10.

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Microbiol. Lett. 164:389-395.

Ohkuma, M., S. Mode, and T. Kudo. 1999. Phylogenetic diversity of
nitrogen fixation genes in the symbiotic microbial community in the gut of
diverse termites. Appl. Environ. Microbiol. 65:4926-4934.

Ohkuma, M., S. Noda, R. Usami, K. Horikoshl, and T. Kudo. 1996.
Diversity of nitrogen fixation genes in the symbiotic intestinal microflora of
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2752.

Platoni, K. 2005. Bug Juice: Could termite guts hold the key to the world's
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Potrikus, C. J., and J. A. Breznak. 1981. Gut bacteria recycle uric acid
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Potrikus, C. J., and J. A. Breznak. 1977. Nitrogen-fixing Enterobacter
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25

Chapter 2

“Stenoxybacter acetivorans” gen. nov., sp. nov., an Acetate
Oxidizing Obligate Microaerophile Among Diverse 02

Consuming Bacteria from Termite Guts

Introduction

Wood-feeding termites depend upon a dense and phylogenetically diverse
community of hindgut-associated microbes to contribute to the insects nitrogen
economy, as well as to assist in the degradation of wood polysaccharides
(cellulose and hemicelluloses) into short-chain fatty acids used by the host for
energy (10, 12). In most termites, microbially-produced acetate dominates the
hindgut fatty acid pool, comprising up to 98 mol% of all volatile fatty acids and
existing at concentrations as high as 80 mM (34). Acetate is absorbed through
the hindgut epithelium and serves as the major oxidizable energy source for the
insect, capable of supporting up to 100% of the termites’ daily respiratory
requirements (34). However, production of acetate depends on the maintenance
of anoxia in the hindgut lumen (9).

Early measurements of the redox potential of hindguts, taken together with
the presence of strictly anaerobic microbes, led to the hypothesis that termite
guts were devoid of oxygen (7, 50). By contrast, other data suggested that some
portion of the hindgut contained 02. For example, aerotolerant (41, 51),

facultative, or 02-requiring (14, 49) hindgut microorganisms were frequently

26

represented among isolates, and degradation of lignin model compounds in vivo
was found to be O2-dependent (15, 28). However, it was not until recently that
direct measurements of 02 concentrations in hindguts were made with
microelectrodes, thereby providing a fine-scale resolution of O2 gradients within
intact hindguts embedded in agarose (14, 18). Results clearly demonstrated the
presence of a peripheral hypoxic zone, with 02 at partial pressures of about 30
mbar (30% air saturation) at the epithelial surface decreasing steeply to anoxia a
distance of 150-200 um inward. This indicated that a majority (approx. 60%) of
the hindgut contains significant levels of O2, undoubtedly a result of its small size
and large surface area to volume ratio (13), and it implied that some members of
the microbiota on and/or near the hindgut wall constituted an important “oxygen
sink” (13, 14). Consistent with this interpretation were earlier experiments
showing that termite hindguts rapidly become completely oxic (implied by the
color of redox dyes) when termites were fed antibacterial drugs (51), and termites
exposed to hyperbaric levels of 02 results in the death of hindgut spirochetes and
protozoa (both groups known to be anaerobes), but leads to a 6- to 10-fold
increase in overall hindgut bacterial colony counts on culture plates incubated in
air (51). However, both treatments decrease the ability of termites to survive on a
diet of wood or cellulose.

It seems clear that the O2-consuming members of the peripheral hindgut
microbiota are critical in creating anoxic conditions for the incomplete oxidation of
wood polysaccharides to acetate (the main energy source for the insect) by

carbohydrate ferrnenters, and for the anaerobic metabolism of CO2-reducing

27

acetogens. However, our understanding of this 02-consuming microbiota is
meager, as is the nature of the substrate(s) that fuels their respiratory activity,
although acetate itself seems a likely candidate given its high rate of oxidation to
CO2 in termite hindguts (31) and the inferred abundance of (not-yet-
characterized) acetate oxidizers from most-probable-number enumerations (32).
Accordingly, we sought to explore the nature and activity of such microbes by
using a cultivation approach that included acetate as an oxidizable substrate in
isolation media, as well as incubation atmospheres that included hypoxia (2%
02) based on the hypothesis that specialized members of the 02-consuming
microbiota are so highly adapted to life in hypoxia that they cannot grow in air
(21% 02). Results of that effort have led to the isolation of several previously
uncultivated bacteria from termite guts, including members of the
Venucomicrobia and Acidobacten'a (47), and a dominant, novel group of
microaerophilic, acetate-oxidizing fl-Proteobacteria, “Stenoxybacter acetivorans”
gen. nov. sp. nov., whose description constitutes the subject of the present

chapter.

Materials and Methods
Termites
Reticulitennes flavipes (Kollar) (Rhinotermitidae) were collected near
Dansville, Ml., Spring Arbor, MI., Raleigh, NC., Woods Hole, MA., and Janesville,

WI. Reticulitennes santonensis (Feytaud) was collected near Foret de la Coubre,

28

France. Zootennopsis angusticollis were from laboratory cultures (31) and
Coptotennes fonnosanus were collected near Ft. Lauderdale, FL.

R. flavipes and C. fonnosanus termites were either degutted within hours
of collection or maintained in laboratory nests as described previously (11, 34).
R. santonensis were maintained in the laboratory as described in (53) for one

year previous to use.

Isolation and cultivation of bacteria.

Guts from approx. 50 worker termites were extracted with sterile forceps
under a hypoxic, CO2 enriched atmosphere (2% 02, 5% CO2, 93% N2) within a
flexible vinyl chamber equipped with an oxygen sensor and controller (Coy
Laboratory Products, Grass Lake, MI). Extracted guts were transferred to and
homogenized in a sterile glass tissue homogenizer containing 2 ml of a buffered
salts solution (BSS) composed of (per liter): KH2PO4, 0.2 g; NH4CI, 0.25 g; KCI,
0.5 g; CaCl2 - 2H20, 0.15 9; NaCl, 1.0 g; MgCI2 . 6 H20, 0.62 g; Na2SO4, 2.84 g;
and 3-(N-morpholino)propanesulfonic acid (MOPS), 10 mM. The 338 was
adjusted to pH 7.0 and was always pre-incubated under the hypoxic atmosphere
for at least 12 hours before use. Serial 10-fold dilutions of gut homogenate were
prepared in B88, and 0.1 ml aliquots of each dilution were spread onto six plates
of ACY medium, which consisted of 888 amended with 10 mM sodium acetate,
0.05% wlv Bacto casamino acids, 0.05% wlv Bacto yeast extract, 0.01% vlv each
of a trace element solution and a vitamin solution (47), and 1.5% vlv Bacto agar.

The pH of ACY medium was adjusted to 7.0. After plating, three of the plates

29

from each dilution were retained within the hypoxic chamber; the remaining three
plates of each dilution were removed from the chamber and placed in a glass
dessicator jar that was subject to six cycles of evacuation and filling with CO2-
enriched air (5% vlv CO2, balance air). Every two days the CO2-enriched air
atmosphere in the dessicator jars was replaced. All incubations were held at
room temperature (22-23°C)

After approximately 20 days, when new colony formation was subsiding,
plates that contained well-separated colonies were selected for colony
enumeration, isolation, and identification. Isolates were obtained by using two
methods. First, plates were examined under a dissecting microscope and
individual colonies were picked with a sterile inoculating loop and streaked for
isolation on homologous medium. The number of colonies of similar morphology
on each plate was noted, and an effort was made to have at least one
representative of each colony type streaked for isolation. For each colony, two
duplicate plates were streaked: one was incubated under hypoxia, whereas the
companion plate was incubated in CO2-enriched air. Subcultures were
continually checked for purity and the ability to form colonies under hypoxia
and/or CO2-enriched air. Second, specific groups of organisms were targeted for
cultivation by using the Plate Wash PCR method described previously (47), with
the following forward primers: Acd31f (5’ - GAT CCT GGC TCA GAA TC - 3’, for
members of the Acidobacten‘a domain (4)); End197f ( 5' - GCA GCA ATG CGT
TTI’ GAG - 3’, for members of the Endomicrobia domain (this study)); Pla40f (5’
- CGG RTG GAT TAG GCA TG - 3’, for members of the Planctomycete domain

30

(Kristin Huizinga, personal communication»; and Ver53f (5’ — TGG CGG CGT
GGW TAA GA— 3’, for members of the Ven'ucomicrobia domain (47)). The
reverse primer in all cases was the general bacterial reverse primer 1492r
(below). Colonies were re-streaked for isolation until they were determined to be
pure based on uniform colony and cell morphology, the latter determined by
examination of wet mounts by phase contrast microscopy. Preliminary screening
of pure cultures for acetate utilization was done by streaking colonies onto plates
of ACY medium with and without acetate. Robust growth on acetate-containing
medium, but little or no growth on medium lacking acetate, was a presumptive
indicator of acetate utilization. Isolated strains belonging to the Verrucomicrobia
division begin with the letters TAV, and strains of the novel acetate-oxidizing,
microaerophilic ,B-Proteobacten'a described herein (i.e. “Stenoxybacter

acetivorans“ gen. nov. sp. nov.) begin with the letters TAM.

Bacterial DNA extraction and PCR-related procedures

For rapid phylogenetic identification of isolated strains, a Ioopfull of colony
material was removed with a sterile inoculating loop and placed in 500 pl of
sterile 888 in a 1.5 ml polypropylene centrifuge tube and centrifuged for 10 min
at 4000 x g. The supernatant liquid was removed, and total DNA in the cell pellet
was extracted by using the Bactozol DNA extraction kit (Molecular Research
Center, Cincinnati, Ohio). Briefly, the cell pellet was suspended in 100 pl of 1x
Bactozyme lysis solution and allowed to incubate at room temperature or 50°C

until the suspension cleared. Four hundred microliters of DNAzoI was added, and

31

the lysate was incubated at room temperature for 10 minutes. The DNA was
precipitated by adding 300 pl absolute ethanol and incubating at room
temperature for an additional 10 minutes. The precipitated DNA was harvested
by centrifugation at 14,000 x g for 15 minutes, washed in one milliliter of 75%
ethanol, re-centrifuged, and allowed to air-dry. The DNA pellet was then
redissolved in 250-500 pl of sterile water, pH 7.5. Occasionally, DNA
redissolution was aided by incubation at 60°C for 1-12 hours.

For each isolate, the 16S rRNA gene was amplified via the polymerase
chain reaction (PCR) using the general primers 8f (5’-AGA GTT TGA TCC TGG
CTC AG-3') and 1492r (5’-GGT TAC C'l'l' GTT ACG ACT T-3’), which target
regions of the gene common to most Bacteria (52). The 25 pl reaction mixture
contained: 25 - 100 ng DNA template, 1x reaction buffer (lnvitrogen, Carlsbad,
Calif.), 1.5 mM MgCI2, 0.25 mM of each deoxynucleoside triphosphate, 0.2 pM of
each primer, and 0.625 U Taq polymerase (lnvitrogen). PCR mixtures were
incubated in a model PT-100 thermal cycler (MJ Research, Inc., Watertown,
Mass.) as follows: (i) 3 min at 95°C; (ii) 30 cycles of 45 sec at 95°C, 45 s at 56°C,
45 s at 72°C; and (iii) 5 min at 72°C. Five-microliter samples of each PCR
reaction mixture was analyzed by electrophoresis on 1.0% agarose gels
prepared in 0.5x Tris-borate-EDTA gel with 10 pg/ml ethidium bromide (39).
Fluorescent bands of the PCR products were visualized by UV transillumination,
and images were captured by using a Kodak electrophoresis documentation and

analysis system 290 (Eastman Kodak).

32

To estimate the total number of “S. acetivorens”, Acidobacten’a or
Verrucomicrobia cells in R. flevipes termites, as well as their primary anatomical
location, a dilution-to-extinction PCR approach was used. DNA was extracted
from 50-100 freshly collected intact termites, termite guts, and degutted termite
bodies by using a MoBio Ultraclean Soil DNA extraction kit (MoBio Laboratories,
Carlsbad, CA.) after homogenization in a Mini-BeadBeater-8 (BioSpec Products,
Inc., Bartlesville, OK.) operating at full speed for 45 s according to the DNA
isolation protocol. Purified DNA was normalized on a per-terrnite equivalent basis
and serially diluted in dilution buffer (10 mMTris-HCI (pH 8.0) containing 50 ng/pl
calf thymus DNA as a carrier). As a control, 100 ng purified DNA from TAM-DN1
or Ven'ucomicrobia strain TAV-1 (47) was also serially diluted in dilution buffer.
Each dilution was used as template in a PCR reaction using either the forward
primer TAM203f (5’ - GCT TCG CAA GGA CCT CAC - 3’; specific for 16S rRNA
gene of “S. acetivorans” strains based on phylogenetic analysis, below), Ver53f
or Acd31f combined with the general reverse primer 1492r (above). The PCR
mixture was identical to that described above, and a total of 30 PCR cycles was
done. Five-microliter samples of each PCR reaction mixture was analyzed by
electrophoresis on 1.0% agarose-0.5x Tris-borate-EDTA gel stained with 1x
Gelstar nucleic acid stain (Cambrex, East Rutherford, NJ). Fluorescent bands of
the PCR products were visualized, captured and analyzed as above.

An estimate of the in situ abundance of the organisms was based on
comparison of the extinction point (lowest amount of DNA that resulted visually

obvious amplification) of the known quantity of purified TAM-DN1 or TAV-1 DNA

33

to the extinction point of corresponding target DNA obtained from termite
samples, taken together with the determined genome size and 16S rRNA gene
copy number for “S. acetivorans" (3.2 Mb and 4 copies, respectively (below)) and
Venucomicrobia strain TAV-1 (4.0 Mb and 1 copy, respectively (below)).

To evaluate the autochthony of “S. acetivorans” for R. flavipes, termites
were freshly collected from a forested natural preserve in Dansville, Ml, along
with termite “nest soil” (is. soil at the interface of a fallen log on which termites
were feeding and through which they were actively tunneling), and separate,
“non-nest soil“ that showed no evidence of termites or termite activity. Soil
samples were collected with sterile spatulas and placed in Whirl-Pak bags
(Nasco, Fort Atkinson, WI). DNA was extracted from termite guts (100 guts) and
soil samples (19) within hours of collection by using the MoBio Ultraclean Soil
DNA extraction kit as described above. The purified DNA was used in PCR
reactions as above, with the “S. acetivorans”-specific 16S rRNA gene fonlvard
primer TAM203f and general reverse primer 1492r. PCR amplified DNA was
cloned into TOP10 E. coli using the plasmid vector pCR2.1 (TA Clone Kit,
lnvitrogen). The partial sequence of randomly selected clones was determined
using the TAM203f primer. Only sequences longer than 500 bp were used for
subsequent analyses. The sequences were imported into the ARB software (32),
aligned, and phylogenetic trees constructed (as described below) using 503
shared nucleotide positions. The statistical program LIBSHUFF was used to

determine statistical relatedness of the clones (40).

34

Sequencing and Phylogenetic analysis.

Prior to sequencing, unreacted dNTP’s and primers in the PCR reactions
were digested and dephosphorylated using ExoSap-IT (USB, Cleveland, Ohio).
The reaction mixture contained 1.3 pl PCR amplified DNA, 0.5 pl ExoSAP-IT
enzyme mix, and 3.2 pl sterile water. Incubation was according to the ExoSAP-IT
protocol.

Partial 16S rRNA gene sequences for each isolate was determined with
Applied Biosystems cycle sequencing technology (Applied Biosystems, Foster
City, Calif.), with the general bacterial primer 8f. Sequence chromatograms were
checked for quality, and the initial identification of each isolate was determined
by using the BLAST search tool in the Genbank nucleotide database (2) or the
Ribosomal Database Project (http:l/cme.rdp.msu.edu) (17). From the 29 total
isolates representing “S. acetivorans”, a subset of 15 that represented the
apparent phylogenetic breadth of the group were chosen for nearly-full length
sequencing of the 16S rRNA gene. Fourfold coverage of each nucleotide position
was obtained by using the same method described previously (47), but omitting
primers F2, R4“, Acd31f, and Ver53f, and adding primer 8f. Individual 16S rRNA
gene sequence reads for each isolate were manually edited and assembled by
using the Contig Assembly Program tool contained within the program BioEdit
(http:lIwww.mbio.ncsu.edulBioEdit/bioedit.html).

For phylogenetic analyses, the partial or nearly full 16S rRNA gene
sequence of each isolate or clone was aligned against a 16S rRNA gene

sequence database in the ARB software package (http:l/www.arb-home.del)

35

(32). Ambiguities in the sequence alignments were corrected manually, where
possible. For each phylogenetic tree, only unambiguous alignment positions
present in every sequence were used. Maximum likelihood phylogenetic trees

were constructed in ARB using the FastDNAML routine (32).

Termite microsatellite DNA analysis.

Eight worker termites were selected from each of the six collection sites
(Dansville, Ml; Spring Arbor, MI; Janesville, WI; Raleigh, NC; Woods Hole MA;
and Forét de la Coubre, Fra.) and individual degutted bodies were placed in 550
pl Bead solution (MoBio Ultraclean Soil DNA kit, MoBio Labs) in separate Bead
tubes and homogenized in a Mini-BeadBeater-8 operating at full speed for 1 min.
Purified termite body DNA was obtained according to the MoBio Ultraclean Soil
DNA kit protocol. The resulting DNA was used in individual, non-multiplexed
polymerase chain reactions using 5’-Hex-Iabeled PCR primers 5-10, 6-1, 11-1,
11-2, and 21-1 as described by Vargo (50). Each 50 pl reaction contained: 10 ng
of termite DNA per reaction, 1x reaction buffer (Invitrogen, Carlsbad, Calif.), 1.5
mM MgCI2, 0.25 mM each deoxynucleoside triphosphate, 0.2 pM each primer,
and 0.625 U Taq polymerase (lnvitrogen). PCR mixtures were incubated in a
model PT-100 thermal cycler (MJ Research, Inc., Watertown, Mass.) as follows:
(i) 3 min at 94°C; (ii) 35 cycles of 30 sec at 94°C, 50 s at 60°C, 1.0 min at 72°C,
and (iii) 5 min at 720°C. The PCR products were precipitated by using 1I10
volume 3 M sodium acetate (pH 5.0) with 2 volumes 100% ethanol. The DNA

pellet was washed in 75% EtOH, air-dried, and suspended in 50 pl sterile water,

36

pH 7.0. Two to three hundred nanograms of the purified PCR product were
separated by using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems,
Foster City, Calif.). Peak sizes were quantified with the Genotyper software
(Applied Biosystems), and alleles at each locus were binned according to
fragment size :t 1.5 bp to accommodate variability during electrophoretic
separation of DNA fragments.

The total number of alleles at each locus for all termites was categorized
using the Microsoft Excel spreadsheet. For all possible alleles, a “1” was denoted
where an allele was present in a given termite, and a “0" was denoted where an
allele was absent. Based on this table, the Jaccard coefficient (Jc) was
calculated for all pairwise comparisons using Estimates (16). A distance matrix
was created using Microsoft Excel where the Jaccard distance (Jd) was
calculated as 1-(Jc). This matrix was uploaded into MEGA (29) for cluster

analysis.

Cultivation, Nutrition and Physiological studies.

Routine cultivation of TAM isolates was carried out in liquid medium that
contained: BSS, 0.05% Bacto yeast extract, 10 mM sodium acetate (BYA
medium). Isolates were typically inoculated into 15 ml medium in a 50 ml sterile
Erlenmeyer flask capped with a sterile, plastic 30 ml beaker (Nalgene) and
incubated at 22-23°C with shaking (250 rpm). Though CO2 was not required for

growth, the TAM strains were incubated in a CO2-enriched hypoxic atmosphere

37

(2% O2, 5% CO2, balance N2) within a flexible vinyl chamber. Final pH of media
in the CO2-enriched atmosphere was 6.5.

Substrate utilization of TAM-DN1 was performed by using butyl rubber-
stoppered, 18 mm anaerobe tubes (Bellco, \fineland, NJ; no. 2048-00150)
containing 5 ml of BSS supplemented with 0.05% Bacto yeast extract and 10 mM
of the test substrate. The headspace of tubes (ca. 22 ml) consisted of 2% O2, 5%
CO2 and 93% N2. Test cultures were inoculated with 1% WV exponential phase
culture growing in BYA medium and incubated at 22-23°C with the tubes held
horizontally and shaken at 150 rpm. The cell yield was determined by measuring
the optical density of the cultures at 600 nm with a Milton Roy Spectronic 20
calorimeter. A 50% increase of cell yield above the “No Substrate“ after passage
through two successive transfers was considered evidence of the ability to utilize
the substrate.

The ability of TAM-DN1 to utilize acetate and succinate simultaneously
was tested in 500 ml screw-cap glass media storage bottles (Bellco no.5636-
00533) to which an 18 mm anaerobe tube (described above) was permanently
affixed. Bottles containing 100 ml of BYA medium supplemented with 10 mM
succinate were inoculated with a 1% vlv exponential phase culture growing in
BYA medium and were incubated within a flexible vinyl chamber containing a 2%
O2, 5% C02, 93% N2 atmosphere (described above) with shaking at 250 rpm.
Growth was monitored by measuring the optical density of cultures at 600 nm as
described above. Simultaneous with growth measurements, 1 ml of culture fluid

was removed, centrifuged at 12,000 x g for 10 minutes, filtered through a 0.2 pm

38

pore size filter, and stored at -20 until used for organic acid analysis. Organic
acids were quantified by high performance liquid chromatography (HPLC)
Meters, Milford, MA) on a 300- by 7.8-mm Aminex HPX-87H column (Bio-Rad,
Hercules, CA) at 23°C with 4 mM H2804 as the eluent at a flow rate of 0.6
mein. Organic acids were detected with a Waters 2487 UV detector at 210 nm
and calibrated with homologous standards.

The oxygen tolerance of TAM-DN1 was determined by two approaches.
First, the ability of cells to grow in liquid medium under defined headspace
concentrations of 02 was evaluated. To do this, butyl rubber-stoppered 18 mm
anaerobe tubes containing 5 ml of anoxic BYA medium were prepared in air
(21% 02) or under a headspace of 100% N2. To the tubes that contained a 100%
N2 headspace, air was injected to attain a final headspace percentage of 0.5, 1,
2, 4 or 8% 02 (after the overpressure was released). To attain a headspace
percentage of 12 or 16% 02, pure oxygen was injected. The tubes were then
sterilized by autoclaving. Sterilized tubes were inoculated with a 5% inoculum of
exponential phase TAM-DN1 cells growing in BYA medium, and incubated
horizontally at 22-23°C with shaking at 150 rpm. Growth was monitored
spectrophotometrically as described above. Time in lag phase was estimated as
described in Lenski et al. (30) and total protein was quantified by the BCA
method (45) with bovine serum albumin as a standard. Final cell yield was based
on the assumption that protein constitutes 55% of cell dry weight (33). In the
second approach, the ability of cells to grow on the surface of agar medium

under various concentrations of 02 was evaluated. To do this, 1.5% agar was

39

incorporated into liquid BYA medium, which was then heated to dissolve the
agar, and 4 ml was dispensed into each of a number of Wolfe Anaerobic Agar
Bottles fitted with a screw cap (Bellco no. 2535-50020). The bottle plates were
then autoclaved, placed on their sides, and the medium allowed to solidify.
Bottles were then brought into an anoxic chamber (10% H2, 5% CO2, 85%
N2)(Plas Labs, Lansing, MI), uncapped, and streaked with an exponential-phase
culture of TAM-DN1. The bottles were then stoppered with a butyl-rubber stopper
held in place by a screw cap possessing a small hole as an injection port. Pure,
sterile 02 was then injected into the bottles (60 ml average headspace volume) to
attain final concentrations of 0.5, 1, 2, 4, 6, 8, 12, 16 and 21% 02 in the
headspace (after release of overpressure), after which they were incubated in an
upright position at 22-23°C.

The ability of strain TAM-DN1 to grow under anoxia was tested by using 5
ml BYA medium that had been deoxygenated under vacuum and added to butyl-
rubber stoppered 18 mm anaerobe tubes under 100% N2. After autoclaving, the
BYA medium was supplemented with 10 mM (final conc.) of one of the following
from a sterile stock solution: potassium nitrate, potassium nitrite, sodium sulfate,
sodium fumarate or D-glucose. For tubes containing sodium fumarate, 10 ml of
100% hydrogen was also added to the headspace of some tubes. The tubes
were inoculated with a 2% vlv exponential phase culture growing on BYA
medium under hypoxia. Tubes were then incubated at 22-23°C horizontally,

shaking at 150 rpm.

40

Genomic properties.

The genome size of TAM-DN1 and TAV-1 was estimated by pulsed-field
gel electrophoresis of restriction endonuclease digestions of total DNA according
to previously published protocols (8, 22). Quantification of 16S rRNA gene copy
number for TAM-DN1 and TAV-1 was determined according to the methods

described in (26, 27).

Enzyme Assays

“S. acetivorans” strain TAM-DN1 cells were grown in 100 mL BYA medium
in 500 ml sidearm bottles (above) under a hypoxic atmosphere shaking at 250
rpm. At mid-log phase (at which growth was known to be acetate—dependent,
representing approximately 5 x 108 cells/ml) the entire culture volume was
centrifuged at 10,000 x g for 10 min at 4°C, washed with 20 ml sonication buffer
(10 mM EDTA, 50 mM Tris-HCI, pH 7.0), re-centrifuged and resuspended in 10
ml of the same buffer. Cells were disrupted in an ice water bath by sonication (3
x 30 3 each) with a Branson Model 450 sonifier (power setting of 5, 50% duty
cycle) equipped with a 1/z" threaded-body step horn with flat tip. To remove
undisrupted cells and debris, the sonicate was centrifuged at 12,000 x g for 60
min at 4°C. The resulting supernatant liquid was considered to be the crude cell
extract and was distributed into a SIide-A-Lyzer dialysis cassette (3 ml to 12 ml
capacity, 3500 molecular weight cutoff; Pierce, Rockford, IL) and dialyzed for 12

hours in two liters dialysis buffer (50 mM Tris-HCI, pH 7.0). The dialyzed crude

41

extract was removed from the cassette and used immediately for assays of
enzyme activities.

Catalase was assayed by measuring the rate of decrease in A240 of H202
according to Beers et al. (6). Superoxide dismutase activity was measured by the
xanthine/xanthine oxidase-cytochrome c reduction method (20). NAD(P)H
oxidase and peroxidase activities were assayed as described previously (46).

A qualitative, colorimetric test for cytochrome C oxidase was done by
spreading TAM-DN1 colony material onto a piece of generic filter paper wetted
with a 1% solution of tetramethyl-p-phenylenediamine dihydrochloride (Sigma) as

described previously (44).

Microscopy

Phase-contrast micrographs were prepared from wet mounts on
agar-coated slides (36). Images were captured on a Zeiss Axioskop microscope
(Carl Zeiss, Inc., Thomwood, NY) equipped with 3 SPOT charge-coupled-device
digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Cells were
prepared for electron microscopy by the staff at the Center for Advanced
Microscopy at Michigan State University. Electron micrographs were obtained
with a JEOL 6400V scanning electron microscope with a LaB6 emitter or JEOL
2200FS 200 kV field emission transmission electron microscope (JEOL-USA,

lnc., Peabody, MA).

42

Results and Discussion
Isolation and enumeration of putative O2-consuming organisms.

For termites collected from Dansville, Ml, the total CFU appearing on
plates of ACY isolation medium was 9.2 (:I: 1.0) x 105 per gut equivalent for plates
incubated in 5% CO2-enriched air and 12 (t 2.4) x 105 per gut equivalent for
plates incubated in hypoxia (5% C02, 2% O2, balance N2). The consistent and
noticeable, though not statistically significant, increase in total CF U seen with
plates incubated under hypoxia was also observed with termites collected from
Spring Arbor, MI, (3% increase); Janesville, WI, (29% increase); Raleigh, NC,
(8% increase); and Forét de la Coubre, Fra., (12% increase) (Table 2.1). In every
case, this increase was due to a single colony type whose morphology was
distinct enough to be easily differentiated from the rest (Figure 2.1). Such
colonies accounted for as many as 10% of all CF U appearing on plates
incubated under hypoxia and were especially abundant in guts of R. flavipes
collected from the Michigan sites. Phase contrast, scanning and transmission
electron microscopy of cells comprising such colonies revealed that they were
thin, nonmotile rods (0.5 x 5 pm) with a Gram-negative type cell wall and outer
membrane (Figure 2.2a-c). They also accumulate intracellular granuals that
appear morphologically similar to poly-B-hydroxybutrate (PHB). Similar granules
accumulate in organisms like Azospin’llum under conditions of low 02 and a high
CIN ratio (25, 48). The isolates were cytochrome C oxidase positive and could
only be subcultured on plates incubated in hypoxia. Moreover, the isolates also

displayed robust colony growth only if acetate was included in ACY medium.

43

Table 2.1. Total cultivable bacteria from the guts of geographically separated
Reticulitennes workers incubated under C02enriched oxic and hypoxic
atmospheres.

 

 

Termites Total‘ Total'
(Collection location) cog-Enriched Air Hypoxia
R. flavipes

Dansville, MI 9.2 1 1.0 12 :I: 2.4
Spring Arbor, MI 6.2 :t 2.7 6.4 :l: 1.0
Janesville, WI 10 d: 0.8 14 :t 1.7
Raleigh, NC 12 :l: 1.5 13 :t 2.6

R. santonensis
Forét de la Coubre, Fra. 15 t 5.4 17 :l: 3.2

 

‘ Total based on direct colony counts of isolation media and are given as colony
forming units (CFU) per gut equivalent x105. Mean i s.d. n=3.

” 5% 002, 95% Air
° 2% 02, 5% co2, 93% N2

 

Figure 2.1. Composite photograph showing colony morphotypes on
two separate isolation plates. Arrows point to the unique colony
morphotype present on the plate incubated in a hypoxic atmosphere (A)
that were not on the plate incubated in COz-enriched air (B).

45

 

 

 

 

 

 

Figure 2.2. Transmission electron (A), scanning electron (B) and phase-
contrast light (C) micrographs of termite gut Neisseriaceae strain TAM-DN1.
Arrows point to intracellular granules that resemble poly-B-hydroxybutyrate.
Scale bars are 1 pm (A, B) and 10 pm (C).

46

Preliminary phylogenetic identification of 18 randomly selected strains
(designated with the prefix “TAM”) purified from such colonies revealed that their
16S rRNA genes were 99.7% similar to each other and grouped within the family
Neisseriaceae in the phylum p—Proteobacten‘a (Figure 2.3). However, they were
only 94.1% similar to their closest known relative, Eikenella corrodens. The in
situ abundance of the TAM strains in guts of Dansville-collected R. flavipes,
based on plate counts, was 1.0 x 105 CF U-gut". This is in close agreement with
the 2.2 x 105 cells-gut'1 estimated by quantitative dilution-to-extinction PCR
(based upon the estimated 3.2 Mb genome size and 4 rrs copies present in TAM-
DN1 (data not shown». Therefore, the TAM strains are estimated to comprise
between 1.6% and 3.5% of the total prokaryotic population in R. fiavipes guts
(49). Owing to their abundance, their phylogenetic novelty, and their apparent
ability to oxidize acetate only under hypoxic conditions, suggesting that they
might be true microaerophiles specialized to live within the hypoxic peripheral
region of R. fiavipes hindguts, strains of these bacteria were chosen for further
examination, with representative strain TAM-DN1 subject to the most detailed
characterization.

The bulk of the other colonies present on isolation plates proved to be
members of the families Streptococcaceae, Enterococcaceae, and
Enterobacten’aceae (Figure 2.3), however these occurred with equal frequency
on plates incubated under CO2-enriched air or the hypoxic gas mixture. Not
surprisingly, only colonies of the latter bacteria displayed more robust growth on

acetate-containing medium. Members of the Streptococcaceae and

47

Enterococcaceae are capable of growth in the presence of oxygen and can use
02 as an electron acceptor (49), but are not known to oxidize acetate as an
energy source; their appearance on isolation plates was almost certainly
supported by energy-yielding nutrients (e.g. sugars, amino acids) present in the
yeast extract and casamino acids components of ACY isolation medium. Based
on 528 aligned 16S rDNA nucleotides, the Streptococcaceae isolates were most
closely related to the genus Lactococcus, and were 97.1% identical to each
other. The Enterococcaceae had a 98.0% 16S rRNA gene sequence identity to
each other and grouped with the genus Enterococcus (564 positions). The
Enterobacteriaceae isolates grouped most closely to the genera Citrobacter and
Enterobacter, all known to be capable of acetate oxidation, and were 96.8%
similar based on 457 nucleotide positions.

With the exception of the Neisseriaceae, the isolates above were typical of
organisms isolated from termite guts previously or whose 16S rRNA genes were
represented in clone libraries prepared from gut homogenates of various termite
species (1, 5, 19, 23, 24, 37, 38, 41, 42, 49, 53). A single 16S rRNA gene clone
closely related to the Neisseriaceae isolates obtained here was obtained from a
gut wall fraction prepared from R. santonensis (53). Othenlvise, these organisms
have not previously been encountered or isolated from termite guts, perhaps
because most previous cultivation efforts have employed incubations in air or
under anoxia.

Recent molecular phylogenetic methods have revealed the presence of

not-yet-cultivated organisms related to members of the Verrucomicrobia,

48

Acidobacteria, Planctomyces and candidate phylum Endomicrobia in the hindgut
of termites (24, 35). As these organisms might also be important to O2
consumption in situ, we used “Plate Wash PCR” (47) in early experiments to
screen for the presence of such bacteria on plates inoculated with diluted gut
homogenates from Dansville, MI specimens of R. fiavipes. By using this
technique, members of the phyla Vermcomicrobia and Acidobacteria were
isolated and were described previously (47) (F igure 2.3). However, dilution-to-
extinction PCR implied that the in situ abundance of the Verrucomicrobia was
relatively low (ca. 2x103 cells-gut“), albeit localized to the gut (Figure 2.4). With
Acidobacteria-specific primers, PCR products were obtained from both termite
gut DNA as well as DNA from degutted bodies, suggesting that the Acidobacteria
isolates cannot be localized strictly to the gut region. Furthermore, PCR
amplification of Acidobacteria from gut DNA was only successful with DNA
concentrations representing 2 % of a gut equivalent (Figure 2.4). These results
suggest the gut-associated Verrucomicrobia and Acidobacteria in R. flavipes,

while intrinsically interesting, are but minor members of the microbial community.

Autochthony of the TAM-strain community

In order to determine whether the TAM strains were autochthonous
members of the R. flavipes gut community as opposed to allochthonous,
transient inhabitants, 16S rRNA gene clone libraries were prepared from termite
guts, termite nest soil, and non-termite associated forest soil by using PCR with

TAM specific primer sets, and the libraries were analyzed by the LIBSHUFF

49

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strain
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Figure 2.4. Dilution-to-extinction PCR of DNA from termite hindguts and degutted
termite bodies. A. R. flavipes DNA amplified with Verrucomicrobia-specific 16$
rDNA primers. ( 1) 1 kb DNA ladder; (2-9) one gut-equivalent DNA serially diluted
1:2 8x; (10-13) one body-equivalent DNA serially diluted 1:2 4x; (14) positive
control Venucomicrobia st. TAV-1 DNA. B. R. flavipes DNA amplified with
Acidobacteria-specific 16$ rDNA primers. (1) 1 kb DNA ladder; (2-8) two gut-
equivalents’ DNA serially diluted 1:2 7x; (9-15), two body-equivalents' DNA

serially diluted 1:2 7x; (16) positive control Acidobacterium capsulatum DNA.

C. Purified DNA from Verrucomicrobia strain TAV-1 amplified with Verrucomicrobia-
specific 16S rDNA primers as a control. (1) 1 kb DNA ladder; (2-9) 100 ng DNA
serially diluted 1:16 8x; (10) 100 ng E. coli DNA; (11) negative control without DNA.

51

   

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Simonsiella steedae
Alysiella filiformls

 

 

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Hydrogenophilus thermoluteolus

0.10

Figure 2.5. Maximum likelihood-based phylogeny of PCR-generated 16$ rRNA
gene clones obtained with a termite gut Neisseriaceae-specific primer set from
R. flavipes termite guts (51 clones; black rectangle), termite nest soil (29 clones;
gray rectangles), and adjacent but non-termite inhabited forest soil (25 clones;
white rectangles). Closely related clones were condensed into a single rectangle,
with the number of clones given in parentheses. Rectangles are aligned for ease
of comparison only; such alignment is not intended to imply equal evolutionary
time. Scale bar represents 0.1 change per nucleotide.

52

program (40). Results clearly indicate the presence of a closely related (average
98.4% 16S rDNA identity) population in the termite gut that is phylogenetically
distinct from 16S rRNA genes amplified from the termite nest soil or surrounding
non-termite associated forest soil (P = 0.001) (Figure 2.5). Interestingly, however,
there appears to be a cluster of clones from termite nest soil that are genetically
related to the termite gut clones. No clones from the forest soil clustered with this

group, suggesting they are termite-nest specific.

Geographical Distribution

In order to determine whether TAM-like strains were present in guts of
geographically and genetically distinct populations of R. fiavipes, TAM isolates or
TAM-like 16S rRNA gene clones were obtained from hindguts of Reticulitennes
specimens collected from different sites, which were themselves examined by
microsatellite DNA analysis. Five separate microsatellite loci for eight individual
worker termites collected from each of the six different sites were amplified by
PCR as described previously (50). Combining all termites, forty-four distinct
alleles were amplified from the 5 different loci. Clustering analysis of the alleles
revealed that most of the termites clustered according to their geographical origin
(Figure 2.6). Not surprisingly, termites collected from Raleigh, NC and Woods
Hole, MA clustered more closely together, as did those collected from Dansville
and Spring Arbor, MI. Interestingly, R. fiavipes collected in Janesville, WI,
clustered with R. santonensis collected in France. However, recent studies have

suggested the synteny of R. flavipes and R. santonensis (3), which our data

53

would seem to support. Between collection sites the results clearly indicate the
genetic distinctiveness of the termites, presumably the result of divergent
evolution over long periods of time. Within a collection site, there is also a high
degree of variation. This may be because worker termites were collected while
foraging, and termites from several genetically different colonies may have been
collected together from the same food source. No isolates related to the TAM
organisms were obtained from the two non-Reticulitennes genera, Zootennopsis
angusticollis (Hagen) (Tennopsidae) and Coptotennes fonnosanus (Shiraki)
(Rhinotermitidae) tested in this study.

The TAM isolates and 16S rRNA gene sequences obtained from the
genetically distinct and geographically separated Reticulitermes are closely
related (99.8% 16S rRNA gene identity) (Figure 2.7). This further supports the
autochthony of the TAM isolates and suggests these symbionts occupy an

important ecological niche within hindguts of Reticulitennes termites.

Relationship to oxygen

One of the most striking and readily apparent properties of the TAM-
strains was their robust growth under hypoxia, but inability to grow anaerobically
(by fermentation or with alternate electron acceptors, below) or in air (or CO2-
enriched air). Accordingly, experiments were done with strain TAM-DN1 to
examine the O2-sensitivity of cells by monitoring their growth on or in acetate-
containing solid or liquid medium under various concentrations of oxygen. On

solid medium, where cells are in direct contact with the headspace gas phase,

54

8A8

 

 

 

 

8A3 ‘
SA6 S A 1
SA4
SA2
DN8
DN7
0N4;
DN6
DN5
DN3 DN2
DN1
JN8
JN6
JN7
JN4
0.1 JN2
_

 

 

FC5

SA7

N07
8 A5 N06 N C4

NC5

NC8

N03

#VW7

 

—WHZ

FC6 FCZ

Figure 2.6. Neighbor-joining analysis of 5 microsatellite loci (44 alleles) of

43 individual worker termites collected at different geographical locations.

R. flavipes workers were collected from Dansville, Ml (DN); Raleigh, NC
(NC); Spring Arbor, MI (SA); Janesville, WI (JN); and Woods Hole, MA (VVH).
R. santonensis workers were collected from Forét de la Coubre, France (FC).

55

TAM-JN1
TAM-JN3
TAM-JN2
TAM-FC2
TAM-FC3
TAM-FC5
- TAM-DN1
TAM-DN2b
TAM-SA2
TAM-8A3
67 TAM-DN3b
TAM-DN7b
100 TAM-DN9b
TAM-NC1
TAM-N05
98 TAM-WH2
, 100 Ek JAM-W24
100 _r— i ene a cone ens
'I Neisseria gonorrhoeae
Chromobacten'um violaceum

 

 

 

 

0.10

 

Figure 2.7. Maximum likelihood-based 16S rRNA gene phylogeny of termite gut
Neisseriaceae isolates and clones. Isolates were obtained from R. fiavipes
collected at: Dansville, Ml (DN); Raleigh, NC (NC); Spring Arbor, MI (SA); and
Janesville, WI (JN). 16S rRNA gene clones were obtained from R. fiavipes
collected at Woods Hole, MA (WI-I). Isolates were also obtained from R.
santonensis from Forét de la Coubre, Fra (FC). Lactococcus lactis was used as
an outgroup (not shown). Numbers at nodes represent percentage of topology
conservation after 100 bootstrap samplings. Bar represents 0.1 change per
nucleotide.

56

TAM-DN1 was unable to form colonies at 02 concentrations higher than 4%
(Figuie 2.8a). Based on the number of colonies on these plates, the optimum 02
concentration for growth on solid media is between 1 and 2%. By contrast, in
broth cultures, where oxygen diffusion to cells is more limited by a relatively large
volume of liquid, a 1% vlv inoculum of 2% O2-grown TAM-DN1 was able to
initiate growth at 02 concentrations at least as high as 16% (Figure 2.8b). Under
these conditions, however, the duration of the lag phase increased linearly with
the initial 02 concentration (Figure 2.9). Nevertheless, the final cell yield also
increased proportionally with increasing 02 concentration (Figure 2.9). This
observation suggests that, within limits, cells require a period of adaptation to
deal with elevated concentrations of 02 (or reactive oxygen species generated by
metabolism in the presence of elevated concentrations of O2), and the duration of
that adaptation is proportional to the 02 concentration to which they are initially
exposed, but once adapted they can consume all of the 02 for energy
generation, which is translated into a corresponding increase in cell biomass.

This microaerophilic nature of TAM-DN1 prompted assays for
oxyprotective enzymes. Under in vitro cultivation conditions TAM-DN1 does
express catalase (99 U-mg. mot") and superoxide dismutase (32 U-mg. prot.")
enzymes. No NAD(P)H oxidase or peroxidase activity was detected.

These observations indicate that TAM-DN1 and related strains are
obligate microaerophiles and probably reside in the hypoxic region of hindguts,
on or near the gut wall. Expression of oxyprotective enzymes like catalase and

superoxide dismutase, as well as their ability to adapt in liquid medium to

57

headspace 02 concentrations up to 16% may be important for transfer to, and
colonization of, guts of newly hatched larvae and recently-molted colony mates,
whose microbiota is essentially absent or drastically reduced, respectively, and
whose 02 content is almost certainly substantially higher than when fully

colonized.

Substrate Utilization by TAM-DN1

Substrates utilized by strain TAM-DN1 for growth included acetate, acetyl-
acetate, succinate, butyrate, glutamate, glutamine, fumarate and casamino acids.
Of these, acetate (60-80 mM), butyrate (2 mM) (34), and a number of free amino
acids including glutamate (1.7 mM) and glutamine (1.0 mM) (21, 43) have been
detected in termite hindgut fluid. HPLC analysis of substrate utilization by TAM-
DN1 revealed that the isolate was able to co-utilize acetate and succinate when
both were available in the culture medium (Figure 2.10). TAM-DN1 was able to
achieve approximately twice the final cell yield (2.3 x 109 cells/ml) when grown on
both substrates than on acetate alone (1.2 x 109 cellsImI)(data not shown). The
rate of acetate utilization by TAM-DN1 was 0.09 pmol-min'I-mg protein" when
succinate was present in the medium, and 0.12 pmol~min"-mg protein" without
added succinate. Substrates not utilized for growth included cellobiose, maltose,
glucose, xylose, arabinose, lactate, pyruvate, citrate, forrnate, propionate, 2-
oxoglutarate, malate, maleic acid, benzoate, threonine, glycine. TAM-DN1 was
not able to grow anaerobically on acetate using fumarate (with or without H2 in

the headspace), nitrate, nitrite, or sulfate as electron acceptors.

58

 

Figure 2.8. 02 tolerance of Neisseriaceae st. TAM-DN1. (A) Growth
on solid medium in Wolfe bottles containing a headspace of: 0% (1),
2% (2), 4% (3), 6% (4) vlv oxygen. (B) Growth in liquid medium
under an atmosphere of: 0% (1), 2% (2), 4% (3), 8% (4) and 16% (5)
v/v oxygen.

59

Lag Time (hours 0)

 

20 I ' I

15-

10-
i

 

-1100
:1000
L900
:800
.700
loco
1500
.400
1300
lzoo

 

 

I

2 4

—1

Headspace 02 (Percent)

I

6

Figure 2.9. Final cell yield (I) and lag time (o) of TAM-DN1 cells
grown in liquid medium under a headspace of 0% to 8% oxygen.

60

Final Cell Yield (pg dry weight I)

 

lllll

1

Growth (00500)

1

0.1

 

g 100

:10

 

0.1

 

' I ' I ' I ' l

16 20 24 28 32

Time (hours)

44

Figure 2.10. Co-utilization of acetate (0) and succinate (A) by TAM-DN1

during growth (9).

61

Substrate Concentration (mM)

TAM-DN1 was not able to grow fermentatively on glucose. In BYA medium,
TAM-DN1 grown at 22°C, 30°C, 37°C and 42°C had the shortest generation time

(3.4 hours) at 30°C. No growth occurred above 37°C.

Rationale for the proposal of TAM-DN1 and related strains as a new genus
and species

The molecular and physiological characterization of isolate TAM-DN1 and
related strains suggests the cultivars are sufficiently distinct from any other
known bacteria to warrant their classification as a novel genus and species within
the domain Bacteria. The distant (94.1%) 16S rRNA gene identity to their closest
cultivated relative, coupled with their microaerophilic phenotype, substrate
utilization spectra, autochthony to the termite gut and apparent specialization to
the hypoxic periphery has distinguished these isolates from any other known
genus and species of Bacteria. The proposed name “Stenoxybacter acetivorans”
appears in quotations throughout this dissertation to signify that the name has
not been validly published, and to reflect a desire that the first description of
“Stenoxybacter acetivorans”, for comparative work or bibliographies not be cited
as this chapter but rather the paper derived from it that will be published within

the primary literature.

62

Description of “Stenoxybacter” gen. nov.

Stenoxybacter (Sten.o.xy.bac' ter. Gr. adj. stenos narrow, Gr. adj. oxys
acid/sour, in combined words indicating oxygen (N.L. oxygenium), NL. masc. n.
bacter rod/bacterium, N.L. masc. n. Stenoxybacter, rod with a narrow oxygen
range). The genus description is, at present, the same as the type species

“Stenoxybacter acetivorans“.

Description of “Steoxybacter acetivorans” sp. nov.

Stenoxybacter acetivorans (a.ce.ti.vo' rans. L. neut. n. acetum
vinegar/acetic acid. L. pres. part. vorans devouring, N.L. pres. part. acetivorans,
acetate consuming). The type strain is TAM-DN1.

Cells are thin (0.5 pm x 5 pm) rods with an undulating, Gram-negative
type outer membrane and cell wall. Cells contain electron translucent inclusions
that resemble poly-B-hydroxybutyrate. Cytochrome C oxidase, catalase, and
superoxide dismutase positive. On solid BYA medium, colonies have a crenate,
low convex morphology. Optimum oxygen concentration for growth on solid
medium is between 1 and 2% O2. Optimum temperature for growth in liquid
medium is 30°C. The cells have a 3.2 Mb genome with 4 16S rRNA gene copies

per cell.

63

Acknowledgements

I would like to thank Ed Vargo for sending R. flavipes termites from North
Carolina, Andreas Brune for providing R. santonensis from France, and Nan-Yao
Su for providing a collection of C. fonnosanus. I would also like to thank Bradley
Stevenson for assistance with Plate Wash PCR, hypoxic cultivation, and
molecular techniques, James McKinlay for help with HPLC analyses, Kwi Kim for
determining the genome size and rrs copy number of TAM-DN1, Joseph Graber
for assistance with anoxic cultivation techniques, and Thomas Schmidt for help
with primer design and microsatellite analyses. My gratitude as well to Hans

Trtiper for providing help with nomenclature and etiology.

10.

11.

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70

Chapter 3

Physiological Ecology of “Stenoxybacter acetivorans” in

Termite Guts

Introduction

In termite hindguts, fermentative production of acetate - a major carbon
and energy source for the insect - depends upon the efficient removal of
inwardly diffusing 02 by microbes residing on and near the hindgut wall (11).
Acetate, present in abundance (60-80 mM) within the hindgut fluid, is the most
likely electron donor supporting the respiratory activity of such microorganisms
(40). Chapter 2 discussed the isolation and identification of microorganisms from
Reticulitennes flavipes guts that may be important 02 consumers in situ.
Abundant among these isolates were strains of a novel, obligately
microaerophilic B-Proteobacten’um, “Stenoxybacter acetivorans” gen. nov. sp.
nov., that appeared to be autochthonous in hindguts of R. fiavipes and present in
genetically distinct populations of Reticulitennes collected from different
geographical locations. Owing to their abundance, obligately microaerophilic
nature, and ability to utilize a relatively narrow range of substrates, including
acetate, as energy sources, it seemed reasonable to hypothesize that “S.
acetivorans“ resides in the hypoxic, peripheral region of R. fiavipes hindguts and
uses a high-affinity terminal oxidase for acetate-driven respiratory activity in situ.

Among the information needed to test this hypothesis, however, were the nature

71

of “S. acetivorans” enzymes (and their encoding genes) specific to acetate
utilization and oxygen consumption.

Key enzymes specific to acetate utilization by microbes include those that
activate acetate to acetyI-CoA prior to its entry into dissimilatory and assimilatory
pathways. Among these are acetate kinase (ACK; EC 2.7.2.1) and
phosphotransacetylase (PTA; EC 2.3.1.8), which catalyze the conversion of
acetate first to acetyl-phosphate, then to acetyl-CoA, respectively (Figure 3.1).
ACK and PTA are the primary enzymes of acetate activation in organisms such
as Corynebacterium glutamicum and Methanosarcina therrnophila (1, 20). By
contrast, in many other organisms such as the Enterobacten’aceae, the ACK/PTA
enzyme pair functions in the reverse direction, primarily during anaerobic growth
on carbohydrates wherein acetate is a major fermentation product and is
excreted. This allows additional ATP production by substrate-level
phosphorylation (14, 27, 28, 41). When grown on acetate as a carbon and
energy source, the Enterobacten'aceae and Bacillus subtilis express an acetate-
inducible acetyl-CoA synthetase (ACS; EC 6.2.1.1) which catalyzes the
conversion of acetate, ATP and CoA, to acetyl-CoA, AMP, and inorganic
pyrophosphate (PPi) (27, 28) (Figure 3.1). Kinetic data have demonstrated that
acetyl-CoA synthetase has a high affinity for acetate (Km = 0.2 mM), whereas
acetate kinase is a low affinity enzyme (Km = 7.0 - 22 mM) (1, 28). However,
inasmuch as PPi is often hydrolyzed by endogenous pyrophosphatase, which
serves to pull the ACS-mediated reaction, acetate activation to acetyI-CoA by

ACS consumes two high energy phosphate bonds compared to one consumed

72

by the ACK/PTA enzyme pair. Other, less widely distributed acetate-activating
enzymes include a pyrophosphate-dependent acetylkinase (EC 2.7.2.12) found
in Entamoeba histolytica (61) and an ADP-forming acetyl-CoA synthetase (EC
6.2.1.13) found in Archaea and some Eukarya, and which is structurally and
evolutionarily unrelated to ACS (25, 37).

When acetate is the only carbon and energy source for a bacterium such
as Escherichia coli, further catabolism of acetyI-CoA occurs via the TCA cycle.
However, the TCA cycle alone does not allow any net assimilation of acetate
carbon. Under these conditions, the anapleurotic glyoxylate cycle is derepressed
and serves to replenish TCA cycle intermediates derived from acetate
catabolism, but drawn off for biosynthesis of cell material (14, 17) (Figure 3.1).
The two characteristic and key enzymes of the glyoxylate cycle are isocitrate
lyase (ICL; EC 4.1.3.1) and malate synthase A (MSA; EC 2.3.3.9). Each turn of
the cycle results in the net formation of one molecule of malate from two
molecules of acetyl-CoA. Malate can then be oxidized to oxaloacetate, an
important precursor for amino acid synthesis and gluconeogenesis.

Among high-affinity oxidases used to mediate terminal electron transfer to
oxygen, especially during aerobic respiration at low ambient oxygen
concentrations, are the cbb3-type cytochrome oxidases. These have been
referred to as “specialized enzymes at the heart of microbial metabolism” (43).
Many so-called microaerophilic microorganisms or organisms that spend at least
part of their life in low 02 environments, such as the N2-fixing root nodule

symbiont Bradyrhizobium japonicum, or the gastric mucosa-colonizing pathogen

73

GIucose—6-phosphate Acetate
| ATP

Gluconeogenesis \r‘I‘

 
   
  
    
 

ATP
ACK

 

  

ADP 096
I 7 Ace l-Phos hate
Phosphoenolpyruvate 00A ty p A2“:
‘ Pi PTA ACS PPi
PEPCk AcetyI-CoA
Aspartate <— Oxaloacetate Citrate
| NADH
\ NADy \
I Malate MSA ACGWI'COA lsocitrate
Lysine I \J IC NAD+
Glyoxylate
Fumarate
FADH
NADH
002
FAD Succinate 2-Oxoglutarate
Glutamate
GTP NAD+

GDP

002 NADH
Figure 3.1. Common pathways for acetate activation and metabolization.
Enzymes involved in acetate activation are: acetate kinase (ACK), phospho-
transacetylase (PTA), or acetyI-coA synthetase (ACS). Once activated, acetyl-
coA proceeds through the TCA cycle. If acetate is the sole carbon source,
replenishment of TCA cycle intermediates drawn 011‘ for biosynthesis such as
2-oxoglutarate or oxaloacetate is accomplished through the action of the
glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase A (MSA).
For biosynthesis of glucose from acetate, the anapleurotic enzyme phospho-
enolpyruvate carboxykinase (PEPCk) is used.

74

Helicobacter pylori, utilize a cbb3-type terminal cytochrome oxidase (34, 46). In
contrast to the well-studied cytochrome aa3 oxidase, which has an estimated Km
for oxygen of 0.1 to 1 pM, the cbba oxidases have estimated Km’s in the
nanomolar range (eg. 7 nM for B. japonicum) (46). The ability of organisms
utilizing these high-affinity enzymes to “pull” oxygen down to very low
concentrations presumably minimizes the production of reactive oxygen species
(to which microaerophiles may be particularly sensitive) and also allows O2-labile
processes such as N2-fixation to occur, as with B. japonicum bacteroids in root
nodules (46). The typically greater energy yield of aerobic vs. anaerobic
metabolism may give a competitive edge to organisms with a cbb3 -type terminal
oxidase in low-O2 environments. However, physiological studies with cells, as
well as experiments with purified cbb3 oxidase reconstituted into phospholipid
vesicles, suggest that the cbb3-type oxidase is significantly less efficient at
transducing energy than cytochrome aa3 (51).

In this chapter, a combination of molecular and physiological approaches
were used to: (i) determine the primary location of “S. acetivorans" in situ; (ii)
identify key genes and gene products involved in acetate utilization and oxygen
consumption by “S. acetivorans” in vitro; and (iii) determine whether these same
genes (and by inference, the gene products) are being expressed by cells of “S.
acetivorans” in situ. Experiments were also done to estimate the potential
contribution of “S. acetivorans” to overall 02 consumption in guts of R. flavipes,
as well as the relative contribution of major microbial groups (i.e. bacteria and

protozoa) to O2 consumption.

75

Materials and Methods

Termites.
Reticulitennes fiavipes (Kolla r) (Rhinotermitidae) were collected near
Dansville, and Spring Arbor, MI. The termites were either degutted within hours

of collection or maintained in laboratory nests as described previously (8, 40).

Microorganisms and Growth Conditions

“Stenoxybacter acetivorans” strain TAM-DN1 and Citrobacter strain RFC-
10 were isolated from the guts of Reticulitennes fiavipes collected in Dansville,
Ml, as described in Chapter 2. Routine cultivation was carried out in liquid BYA
medium under a hypoxic (2% O2, 5% CO2, 93% N2) atmosphere (Chapter 2).

Optical density readings of cells were made in an 18 mm anaerobe tube
using a Spec-20 spectrophotometer at a wavelength of 600 nm. A standard curve
relating optical density to cell number was done by using a Petroff-Hauser
counting chamber. Cells were counted at 400x magnification. Optical density
(and hence cell number) was related to protein concentration by centrifuging 1 ml
of cells at a given optical density in a 1.5 ml centrifuge tube for 5 min at 4000 x g.
The supernatant was removed, and the cells were suspended in 100 pl lysis
buffer (1% vlv sodium dodecyl sulfate (SDS) in BSS, (above)). Complete lysis of
the cells was observed microscopically. Total protein was measured by the BCA

method (52) using bovine serum albumin (dissolved in lysis buffer) as a standard.

76

In Situ Location of the “Stenoxybacter” population

To evaluate the approximate location of the TAM community within the
termite hindgut, four R. fiavipes termites were degutted and the guts pooled in
100 pl of B88 on one-half of a 100 x 15 mm sterile Petri dish under a dissecting
microscope. With a razor blade, the midgut was separated from the hindgut as
close to the midgut-hindgut junction as possible. The guts were then transferred
into a fresh 100 pl drop of BSS, sliced longitudinally, and washed by gentle
agitation using sterile forceps. The 100 pl of B88 (now visibly dense with gut
microbes) was transferred into a sterile, 10 ml conical centrifuge tube. The
remaining sliced gut fragments were transferred to a separate 1.5 ml centrifuge
tube containing 0.5 ml of BSS and vigorously agitated for 60 s with the aid of a
Vortex mixing device. Gut fragments were allowed to settle, the supernatant was
removed and added to the previous 10 ml conical tube. This portion of the
microbiota was referred to as the “fluid fraction”. The gut fragments were
transferred, using sterile forceps, to a 1 ml Bead Tube (Mo-Bio Ultraclean Fecal
DNA isolation kit, Mo Bio Laboratories, Carlsbad, CA) containing 500 pl of Bead
Solution for DNA extraction. These gut fragments constituted the “wall fraction.”
This process was repeated for a total of 50 guts. The fluid fraction was
centrifuged at 4000 x g for 10 minutes to pellet cells, the supernatant was
removed, the pellet suspended in 500 pl of Bead Solution, and transferred to a

dry 1 ml Bead Tube for DNA extraction.

77

DNA was obtained from the fluid and wall fractions using a Mo-Bio
Ultraclean Fecal DNA Isolation Kit afier bead homogenization as described
above. “Stenoxybacter” or spirochete-specific 16S rRNA genes were PCR
amplified from each fraction using primers TAM203f or 63f (5'-CAT GTC GAC
GTY TTA AGC ATG CAA GT-3'; domain Spirochaetes (32)) as described above.
Each reaction contained one gut-equivalent termite DNA made up to 50 ng with
calf thymus DNA. After PCR incubation, five-microliter samples of each reaction
mixture was analyzed by electrophoresis on 1.0% agarose-0.5x Tris-borate-
EDTA gel stained with 1x Gelstar nucleic acid stain. The fluorescent bands of the
PCR products were digitally captured as described above. The amount of PCR
product in each band was quantified using the Kodak 1D electrophoresis analysis
software by dividing the total, background-subtracted intensity of each band by

the total band area (in pixels).

Enzyme assays.

“S. acetivorans” strain TAM-DN1 cells were grown in 100 ml BYA medium
under a hypoxic atmosphere (2% O2, 5% CO2, 93% N2) shaking at 250 rpm in a
500 ml media bottle with an 18 mm anaerobe tube attached as described in
Chapter 2. When the culture had reached mid-log phase (during which growth
was known to be acetate-dependent and which represented approximately 5 x
10" cells/ml) the cells were centrifuged at 10,000 x g for 10 min at 4°c, washed
with 20 ml sonication buffer (10 mM EDTA, 50 mM Tris-HCI, pH 7.0), re-

centrifuged and suspended in 10 ml of the same buffer. Cells were disrupted in

78

an ice water bath by sonication 3 x 30 s each with a Branson Model 450 sonifier
(power setting of 5, 50% duty cycle) equipped with a Va” threaded-body step horn
with flat tip. To remove undisrupted cells and debris, the sonicate was
centrifuged at 12,000 x g for 60 min at 4°C. The resulting supernatant liquid was
considered to be the crude cell extract and was distributed into a Slide-A-Lyzer
dialysis cassette (3 ml to 12 ml capacity, 3500 molecular weight cutoff ; Pierce,
Rockford, IL) and dialyzed for 12 hours in two liters dialysis buffer (50 mM Tris-
HCI, pH 7.0). The dialyzed crude extract was removed from the cassette and
used immediately for assays of enzyme activities.

Acetyl-CoA synthetase (AMP-fonning, EC ) was assayed by two different
methods: (i) measurement of acetate- and CoA-dependent formation of AMP
from ATP according to Oberlies et al. (38) and (ii) measurement of acetate- and
CoA-dependent formation of inorganic pyrophosphate from ATP, according to the
protocol supplied with the kit for enzymatic detection of pyrophosphate (Sigma-
Aldrich, St. Louis, MO). Pyrophosphate-acetate phosphotransferase was
assayed according to the same method, except that the rate of acetate-
dependent disappearance of PPi was followed. Acetyl-CoA synthetase (ADP-
forming) was measured by determining the rate of acetate and CoA-dependent
ADP formation from ATP to the oxidation of NADH in the presence of
phosphoenolpyruvate, pyruvate kinase and lactate dehydrogenase according to
(48). Acetate kinase activity was determined by using the hydroxylamine method
of Jones and Lipmann (24) as modified by Brown (10). Phosphotransacetylase

was determined according to the method of Whiteley (59). Malate synthase

79

activity was assayed according to the method of Carpenter and Merkler (13).
lsocitrate lyase was assayed according to the same protocol as malate synthase,
substituting isocitrate for glyoxylate. Protein content of cell extracts was
measured by the BCA assay (52) with bovine serum albumin as a standard. All
absorbance measurements were made using a Perkin-Elmer Lambda 14 UVNIS

spectrophotometer and Perkin-Elmer UVWlnLab software.

PCR primer design

(I) Broad specificity primers.

Deduced amino acid sequences for genes encoding known acetate kinase
(ack), phosphotransacetylase (pta), acetyl-CoA synthetase (acs), isocitrate lyase
(aceA), malate synthase A (aceB), malate synthase G (gch), and the O2-
reducing subunit of the cbb3-type cytochrome oxidase (ccoN) were obtained from
completed, annotated microbial genomes through the Comprehensive Microbial
Resource at The Institute for Genomic Research (TIGR, http:/lcmr.tigr.orgltigr-
scripts/CMRICmrHomePage.cgi) based on a gene name search. Additional
sequences were obtained though Genbank (3). The amino acid sequences for
each enzyme of interest were aligned using ClustalW, and the alignments were
uploaded into the Blocks Multiple Alignment Processor of the CODEHOP online
primer design tool (47). The blocks were then imported into the CODEHOP
program. For all primers, the default search criteria were used, except that
Ralstonia eutropha replaced Homo sapiens as a model for codon bias.

Suggested primers from the CODEHOP output were selected with an effort to

80

obtain a maximum distance between the forward and reverse primers in order to
allow amplification of as much genetic information as possible (Table 3.1).
(ii) “S. acetivorans”-specific primers.

The ClustaIW protein alignment tool inside the ARB software package
(http:lIwww.arb—homedel) (33) was used to align the deduced amino acid
sequences of the functional genes from known organisms with those amplified by
PCR from the “S. acetivorans” strains using the broad specificity primers. Visual
inspection of the “S. acetivorans” strain sequences typically revealed areas of
conservation specific to the “S. acetivorans" clade. Additional primer pairs were
then designed to target these specific regions (Table 3.1). The sequence
specificity of candidate primers was checked by searching the ARB database

and performing a BLAST search.

PCR

The optimal annealing temperature for each primer pair was determined
by temperature gradient PCR with DNA from “S. acetivorans" TAM-DN1 or E. coli
DNA. The 25 pl reaction mixture contained: 25 - 100 ng DNA template, 1x
reaction buffer (lnvitrogen, Carlsbad, CA), 1.5 mM MgCI2, 0.25 mM each
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(lnvitrogen). PCR mixtures were incubated in a model PTO-200 DNA Engine
gradient thermal cycler (MJ Research, Watertown, MA) as follows: (i) 3 min at
95°C; (ii) 30 cycles of 45 s at 95°C, 45 s at 50-62°C, 45 s at 72°C, and (iii) 5 min

at 72°C.The PCR products were then visualized on a 0.5x TBE, 1% agarose gel

81

 

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83

with ethidium bromide stain. Once the optimal annealing temperature was
determined, the amplicon was cloned by using the TA Cloning Kit (lnvitrogen)

and transformed into TOP10 E. coli. Several clones were selected for

sequencing.

Sequencing and Phylogenetic analysis.

Prior to sequencing, unreacted dNTP’s and primers from the PCR
reactions were removed using ExoSap-IT (USB). The reaction mixture contained
1.3 pl PCR-amplified DNA, 0.5 pl ExoSAP-IT enzyme mixture, and 3.2 pl sterile
water. The reaction was incubated according to the ExoSAP-IT protocol.

The partial gene sequence was determined with Applied Biosystems cycle
sequencing technology (Applied Biosystems), using the broad specificity or “S.
acetivorans”-specific fonivard primer corresponding to the gene of interest (T able
3.1). Sequences were quality checked by hand, and the initial identification of
each sequence was determined by using the BLASTx search tool in the Genbank
protein database. Each nucleotide sequence was converted to the deduced
amino acid sequence by using Transeq (www.ebi.ac.uklembossltranseq).

For phylogenetic analyses, the sequences were aligned against a
database populated with protein sequences from TIGR as well as Genbank by
using the ClustalW protein alignment algorithm within the ARB software package
(33). Ambiguities in the sequence alignments were corrected manually. Only

alignment positions present in every sequence were used in subsequent

84

phylogenetic analyses. Phylogenetic trees were constructed in ARB using the

maximum likelihood routine for protein sequences.

RNA extraction and purification

All RNA work was done with reagents, pipette tips, and tools that were
RNase-free. Approx. 100 R. fiavipes termites were degutted and the guts
immediately placed in a 1.5 ml centrifuge tube on dry ice. One ml of RNA protect
reagent was then added, and the contents were immediately transferred into a
sterile glass tissue homogenizer followed by thorough homogenization for 3 min.
RNA was purified from the homogenate according to the protocol for bacteria in
the RNeasy RNA purification kit (Qiagen, Valencia, CA). The RNA was quantified
by optical density at 260 nm measured using a Perkin-Elmer Lambda 14 UVNIS
spectrophotometer. After quantification, the RNA was treated with DNase I as
follows: to a 0.5 ml RNase-free microcentrifuge tube, 1 pg RNA, 1 U DNasel
(amplification grade; lnvitrogen). and RNase-free water was added for a final
volume of 10 pl. The reaction was incubated at room temperature for 15 minutes,
after which the DNase was inactivated by the addition of 1 pl of 25 mM EDTA
and incubation for 10 min at 65°C. The DNA-free RNA was stored at -80°C until

used.

Reverse Transcriptase PCR
First-strand cDNA synthesis was done following the protocol described for

Superscript lll reverse transcriptase (lnvitrogen). To a nuclease-free 0.5 ml

85

 

centrifuge tube, 2 pmol of gene-specific reverse primer, 0.5-1 pg R. flavipes gut
RNA, 1 pl 10 mM dNTP mix (Roche, Indianapolis, IN), and sterile, RNAse-free
water was added for a total volume of 13 pl. The mixture was heated to 65°C for
5 minutes, then placed on ice for >1 min. After brief centrifugation, 4 pl buffer, 1
pl 0.1 M DTT, and 200 U Superscript Ill reverse transcriptase (lnvitrogen) were
added. The reaction was then incubated at 55°C for 1-2 hours, heated to 70°C
for 15 minutes, then stored at -20°C until PCR amplification.

PCR amplification of the first-strand cDNA was done according to the PCR
methods described above, using 2-4 pl of the first-strand cDNA synthesis
reaction mixture, the annealing temperature of the specific primer set (Table 3.1),

and extending the total number of PCR cycles to 35.

Oxygen Uptake Measurements

“Stenoxybacter acetivorans” strain TAM-DN1 and Citrobacter sp. RFC-10
were grown on BYA media, or BYA media modified to contain 0.75 mM NH4 (a
concentration known to limit growth to approximately half the cell yield othenrvise
attained with excess NH.) and 20 mM sodium acetate. These modifications were
made so that in vitro growth of termite gut isolates would more closely mimic the
low-nitrogen, acetate-rich environment of the termite gut. The cells were
harvested upon entry into stationary phase by centrifugation (10,000 x g, 10 min,
4°C), washed 2x in insect Ringer’s solution (per liter; 7.5 9 NaCl, 0.35 g KCI, 0.21
9 CaCl, pH 7.0) and resuspended at 10x initial concentration in the same buffer.

Oxygen uptake rates were measured under rapid stirring in a 2-ml glass HPLC

86

vial fitted with a screw-cap having a central hole through which a narrow Clark-
type oxygen electrode (Diamond General, Ann Arbor, MI) was placed and sealed
with dental wax. Prior to use, the oxygen electrode was calibrated by immersion
in insect Ringer’s solution that had been vigorously bubbled with air for >15 min
(100% air saturation), or that had been degassed under vacuum and bubbled
with 100% N2 (0% air saturation). The cell suspensions were vigorously aerated
by shaking, added to the 02 uptake chamber and oxygen consumption was
measured before and after the addition of substrate. Sodium acetate, sodium
succinate, sodium lactate or D-glucose were the test substrates and were added
individually to a final concentration of 10 mM.

For measurements of oxygen uptake by termite guts, guts were removed
from R. flavipes worker termites that had been maintained in the laboratory for
approximately 8 months. Such termites were either untreated (controls) or were
fed (for eleven days prior to gut removal) on diets intended to eliminate major
components of the gut microbiota (bacteria or cellulolytic protozoa; below).
Extracted guts were immediately placed in 2 ml insect Ringer's solution within the
02 uptake chamber, as described above. For each experiment, 10-20 guts were
used. The guts were first allowed to settle, then the overlying buffer was
aspirated by using an 18-guage needle attached to a 5 ml syringe. The aspirated
buffer was then immediately replaced with fresh, fully aerated buffer, and 02
uptake was measured as described above, with and without the addition of 10

mM (final concentration) sodium acetate.

87

Elimination of Gut Microbes

To determine the relative contribution of major components of the gut
microbiota (cellulolytic protozoa and bacteria) to oxygen consumption by termite
guts, either or both groups of microbes were largely eliminated from guts by pre-
feeding the termites on agarose food cubes containing starch (known to eliminate
cellulolytic protozoa (58)), a mixture of antibacterial drugs, or a combination of
both. Control termites for these experiments were fed for the same period of time
on agarose food cubes containing microgranular CC41 cellulose powder
(Whatman, Brentford, UK). To prepare such food cubes, a solution of 1% wlv
agarose in insect Ringer’s solution (above) was heated to boiling, and 10% wlv
cellulose or 5% wlv cornstarch was added under rapid stirring. When desired, the
antibiotics ampicillin, cefoperazone and vancomycin (800 pglml each, final conc.)
were incorporated after the agarose had cooled but before it solidified. The
agarose suspensions were allowed to solidify in trays, and the final thickness of
the suspension was approximately one-half inch. Once solidified, the agarose
was cut into two-inch squares, and each square was placed within a 100 x 15
mm sterile Petri dish to which 20-40 termites were added. The Petri dishes were
placed within a humid chamber for 11 days, with the agarose food squares
replaced every three days.

To determine the efficacy of such treatments in removing gut microbes,
viable cell counts were of bacteria were made every second day of treatment, as
were direct microscopic counts of protozoa. For viable cell counts, twelve

termites from each treatment were degutted and the guts were placed into 2 ml

88

of 1x basal salts solution (53) buffered with 10 mM MOPS (pH 7.0). The guts
were thoroughly homogenized with a sterile glass tissue homogenizer, and the
homogenate was serially diluted in 10-fold increments in the same buffer.
Samples from each dilution were plated onto a complex medium containing (per
liter): KH2PO4, 0.2 g; NH4CI, 0.25 g; KCI, 0.5 g; CaCl2 - 2H2O, 0.15 9; NaCl, 1.0
g; MgCI2 - 6 H20, 0.62 g; Na2SO4, 2.84 9; brain heart infusion medium (BD
Franklin Lakes, NJ) 3.7 g; casamino acids 1.0 g; and cellobiose, D-glucose, D-
xylose, maltose, sodium pyruvate, sodium lactate, and sodium acetate, 1 mM
each. The medium was buffered by inclusion of morpholinopropanesulfonic acid
(MOPS; 10 mM final conc.) and adjusted to pH 7.0 prior to being autoclaved.
Plates were incubated at 23°C under hypoxia (93% N2, 5% C02, 2% 02) for 15
days before colonies were counted.

For quantification of protozoa, four termites were degutted, and the guts
placed within 100 pl insect Ringer's solution on a sterile Petri dish under a
dissecting microscope. Each gut was sliced longitudinally with a razor blade, and
the gut contents were washed out of the gut and into the Ringer’s solution briefly
by agitation while being held with sterile forceps. The 100 pl suspension
containing gut fluid and microorganisms, but not sliced guts, was added to a
sterile, 1.5 ml centrifuge tube and briefly centrifuged to pellet the cells. The pellet
was resuspended in 10% neutral buffered formalin (per liter: 37% formalin, 100
ml; Na2HPO4, 6.5 g; NaH2PO4, 4.0 9; pH 7.0) and incubated at 4°C overnight.
The fixed cells were again collected by centrifugation and then resuspended in a

mixture of equal volumes of insect Ringer’s and absolute ethanol and placed at -

89

20°C until counted. For counting, 10 pl of the cell suspension was transferred
onto a well of an 8-well Teflon coated slide (each well having an area of 28.26
mm), allowed to dry, and washed briefly in water. The cells were stained with
freshly-prepared (5-[4,6-dichlorotriazin-2-yl] aminofluorescein (DTAF, 0.2 mglml;
prepared in a buffer containing 0.05 M Na2HPO4 and 0.15 M NaCl; pH 9.0) for 30
minutes followed by 3 washes in the same buffer for 30 minutes each. Slides
were air-dried, coverslips were mounted with Entellan (Merck) preservative, and ‘
cells were visualized at 100x magnification by UV epifluorescence with a Zeiss
Axioskop equipped with a DTAF-specific filter set. Protozoa in at least 20 fields of

view were counted.

Results

In situ location of “S. acetivorans”

Results of the PCR-based method for assessing the in situ location of the
TAM organisms suggested that they are more closely associated with the
epithelial wall as opposed to the gut fluid (Figure 3.2a). DNA extracted from the
epithelium and adherent microorganisms (wall fraction) amplified with TAM-
specific 16S rDNA primers required 24 amplification cycles before a quantifiable
product could be detected. Twenty-five cycles were required before a product
could be detected using DNA from non- or loosely-adherent wall-associated
microbes (“fluid fraction”) with the same primer set, suggesting less TAM-specific

16S rRNA genes were present in the “fluid fraction” of the gut. This is supported

90

by the fact that a significantly greater amount of PCR product was obtained from
the wall fraction at least through 27 PCR cycles. Clone libraries prepared from
PCR products resulting from amplification with wall fraction DNA confirmed the
reaction was TAM-specific (data not shown).

To test the validity of this method, the TAM-specific primers were replaced
by primers specific for the 16S rRNA gene of spirochetes, bacteria known to be
present primarily in the gut fluid rather than attached to the epithelium (8). As
expected, a quantifiable PCR product was obtained after 13 cycles from fluid
fraction DNA, but not wall fraction DNA (Figure 3.2b). A greater amount of PCR
product was obtained from the fluid fraction than the wall fraction at least through
17 PCR cycles.

Enzymes and Genes Relevant to Acetate Oxidation In Vitro

In an effort to identify enzymes specific to acetate oxidation by “S.
acetivorans”, crude extracts of acetate-grown cells were examined for enzyme
activities associated with the activation of acetate to acetyI-CoA. The results
revealed the presence of acetate kinase and phosphotransacetylase, but not
acetyI-CoA synthetase (AMP-forming) or the less widely distributed ADP-forming
synthetase, nor PPi-acetate phosphotransferase (Table 3.2). However, AMP-
forming acetyl-CoA synthetase activity could be readily detected in reaction
mixtures to which authentic acetyl-CoA synthetase (purified from Saccharomyces
cerevisiae, Sigma) was added. Acetate kinase activity was dependent on the

presence of both acetate and ATP in the reaction mixture; and

91

J

Net Intensity I Band Area
5 2.3 .345 €33 .3

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Net Intensity I Band Area

 

   

13 14 15 16 17
Number of PCR Cycles

Figure 3.2. In situ association of “S. acetivorans” with the gut epithelial wall.
Quantification of product intensity after PCR of termite gut fluid (gray bars) or
sliced and washed gut epithelium (black bars) with “Stenoxybactef- specific (A)
or spirochete—specific (B) 168 rDNA primers. Error bars represent standard
deviation (n=3).

Table 3.2. Enzymes and genes relevant to acetate oxidation and 02-
consumption in acetate-grown cells of “S. acetivorans”.

 

 

Enzyme Specific Activity‘ Gene Amplified
Acetate Activation
Acetate Kinase 3.7 :l: 0.7 ack
Phosphotransacetylase 1.1 :l: 0.5 pta
AcetyI-coA Synthetase 0.0 none
(AM P-forming)
Acetyl-coA Synthetase 0.0 n.d.
(ADP-forming)
PPi-Acetate 0.0 n.d.
Phosphotransferase
Glyoxylate Cycle
lsocitrate Lyase 0.0 none
Malate Synthase 0.5 :l: 0.3 9ch
Oxygen Reduction
cbb3-type Cytochrome n.d. ccoN
Oxidase

 

" Specific activities are expressed as micromoles product formed per minute per
milligram protein (mean :t sd)(n=3). n.d., not determined.

93

phosphotransacetylase activity was dependent on the presence of both acetyl-
phosphate and coenzyme A. Interestingly, robust acetate kinase activity was also
observed in crude extracts of succinate-grown cells (9.4 t 1.9 U-mg prot.“),
suggesting that this enzyme may be constitutively synthesized in “S.
acetivorans”.

Not surprisingly, PCR amplification of “S. acetivorans” genomic DNA with
broad specificity primers readily revealed the presence of genes encoding
acetate kinase (ack) and phosphotransacetylase (pta), but not acetyl-CoA
synthetase (Table 3.2). However, the acs gene was readily amplified by using the
same primer set and Escherichia coli genomic DNA as a control. Upon
translation, the deduced amino acid sequence of the ack PCR product was 69%
identical to its closest relative, an acetate kinase from Neissefia meningitidis
22491 (Genbank accession number CAB84946) (Figure 3.3). Moreover, the ACK
sequences from 11 different strains of “S. acetivorans” were 99.8% identical to
each other, (based on 178 amino acids).

The same broadly specific acetate kinase primers were also used to PCR
amplify R. flavipes gut homogenate DNA, and a clone library was constructed.
Phylogenetic analysis of the deduced amino acid sequence from 44 clones
revealed that a majority formed a distinct cluster (R. fiavipes ACK cluster 1, 26
clones) whose closest relative was an acetate kinase from Clostridium
thermoceIIum (Figure 3.3). Other clones grouped together with an acetate kinase

from Treponema denticola (R. flavipes ACK clusters 2.1 and 2.2; 7 and 3 clones,

94

respectively). Less abundant were clones related to Treponema pallidum,
Bacteroides thetaiotaomicron, and Mycobacterium vanbaalenii.

The ~800 nucleotide amplicon from “S. acetivorans” TAM-DN1 DNA
obtained by using the broad specificity phosphotransacetylase primers bore 75%
deduced amino acid identity to its closest known relative, a
phosphotransacetylase from Neissen'a meningitidis M058 (AAF41056) (Figure
3.4). The deduced amino acid sequences of phosphotransacetylases from
different TAM strains were 93% identical (based on 201 amino acids).

Bacteria that oxidize acetate aerobically usually employ the TCA cycle to
do so, and such bacteria typically possess a glyoxylate bypass for replenishment
of TCA cycle intermediates drawn off for biosynthesis. Accordingly, and as
potential targets for inferring in situ activity, two key enzyme activities of the
glyoxylate bypass were sought in crude extracts of TAM-DN1, i.e. isocitrate lyase
and malate synthase A (Fig. 3.1). Although glyoxylate- and acetyI-CoA-
dependent putative malate synthase A activity was detected, isocitrate lyase
activity was not (Table 3.2). Moreover, despite multiple attempts to PCR amplify
the genes encoding these two enzymes, including a re—design of primers and
application of less stringent amplification conditions, no amplification products
resulted from PCR with primers targeted to isocitrate lyase or malate synthase A,
although amplification of these genes always occurred with E. coli control DNA.
However, TAM-DN1 DNA amplified with malate synthase (3 primers resulted in a
~1100 nucleotide product bearing 64% deduced amino acid sequence identity to

malate synthase G from Pseudomonas syn'ngeae pv. tomato st. D03000

95

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Figure 3.4. Maximum likelihood-based phylogenetic analysis of the
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from selected “8. acetivorans” isolates (boldface). Escherichia coli PTA is
used as an outgroup (not shown). Scale bar represents 0.1 change per
amino acid.

97

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Figure 3.6. Maximum likelihood-based phylogenetic analysis of the deduced
amino acid sequence (136 positions) of the Oz-reducing (ccoN) subunit of the
cbb3-type cytochrome oxidase from selected “S. acetivorans” isolates (boldface)
and R. flavipes guts (bold+italics). The ccoN subunit from Magnetospin'llum
magnetotacticum is used as an outgroup (not shown). Scale bar represents

0.1 change per amino acid.

99

(AAOS4024) (Figure 3.5). Phylogenetic analysis revealed that the deduced
amino acid sequences of malate synthase G PCR products from 8 different “S.
acetivorans” strains were 99.5% identical and grouped specifically with malate
synthase G enzymes, not the phylogenetically related malate synthase A or
malyl-CoA lyase enzymes (Figure 3.5) (35). In light of the apparent absence of
isocitrate lyase, the role of malate synthase G in “S. acetivorans” remains to be
determined.

Inasmuch as “S. acetivorans” strains were shown to be microaerophilic
(Chapter 2), it was hypothesized that the cells possessed a high-affinity cbb3-
type oxidase as their terminal respiratory enzyme. To explore this possibility,
broad specificity primers were designed that targeted ccoN, the gene encoding
the Oz-reducing subunit of the cbb3 cytochrome oxidase (Table 3.1). PCR
amplification of “S. acetivorans” TAM-DN1 genomic DNA yielded a 2 kb product
whose deduced amino acid sequence was 96% identical (based on 125 amino
acids) to a cbb3—type cytochrome oxidase from Chmmobacterium violaceum
ATCC 12472 (NP_900844). Sequencing and phylogenetic analysis revealed the
ccoN subunit to be highly conserved among the TAM strains (99.8% sequence
identity).

The same broad specificity ccoN primers were also used to construct a
clone library from R. flavipes gut homogenate DNA. Phylogenetic analysis of 82
clones from this library revealed that a majority of the clones fell within distinct
groups (R. flavipes clusters 1.1 and 1.2; 63 and 4 clones, respectively), whose

closest relative was the ccoN from Dechloromonas aromatica (Figure 3.6). Other

100

clones grouped with ccoN from Verrucomicrobia isolate TAV-1, also cultivated
from termite guts (R. flavipes cluster 2, 5 clones) (See Appendix and (53)). Six
clones (R. flavipes cluster 3) formed a cluster related to Campylobacterjejuni,

and four grouped together with the ccoN genes from the TAM isolates.

Genes Expressed by “S. acetivorans” In Situ

Owing to the high degree of within-species similarity of “S. acetivorans”
genes presumably important to acetate-supported aerobic respiration (i.e. ack,
pta and ccoN, above), coupled with their substantial sequence divergence from
homologues present in other known organisms or PCR amplified from termite gut
homogenates, an effort was made to design “S. acetivorans”-specific primers to
examine (by reverse transcriptase PCR; RT-PCR) whether these same genes
were expressed in vitro and in situ. To do this, “8. acetivorans”-specific primer
pairs for these genes were designed (Table 3.1 ) and used in conventional PCR
amplification with purified R. flavipes gut homogenate DNA as template. Upon
cloning and sequencing of the PCR products, all were identical, or nearly so, to
ack, pta, and ccoN genes from the TAM isolates (data not shown).

When these same primers were used in individual RT-PCR reactions with
R. flavipes gut homogenate RNA as a template, each reaction usually yielded a
single PCR product of the same size as the analogous product obtained by
using, as a template, DNA from acetate-grown TAM-DN1 (Figure 3.7).
Importantly, no products were obtained if reverse transcriptase was omitted from

the RT-PCR reactions, indicating that any products formed in the complete

101

A12345

 

B12345

 

C 12345

 

Figure 3.7. Gelstar-stained agarose gel electrophoresis of RT-PCR products
for (A) acetate kinase (ack); (B) phosphotransacetyalse (pta); and (C) the 02-
binding subunit of the cbb3 terminal oxidase (ccoN) from R. flavipes gut
homogenate RNA with “S. acetivorans”-specific, gene-targeted primers.

Lane 1, 1 kb DNA ladder. Lane 2, RT-PCR product using R. flavipes gut RNA
as template. Lane 3, RT—PCR with R. flavipes gut RNA without the addition of
reverse transcriptase enzyme. Lane 4, RT—PCR with TAM-DN1 DNA as
template. Lane 5, RT—PCR without the addition of template.

102

A R. flavi es ack RT-PCR product 1
TAM-D 4b
TAM-DN6b
TAM-DN8b
TAM-DN10b
TAM-DN1 1b
TAM-DN13b
TAM-DN12b
TAM-DN7b
R. flavipes ack RT-PCR product 2
TAM-DN1
TAM-DNZD

0.05

 

 

TAM-DN2b
R. flavipes pta RT-PCR Product 2
R. flavipes pta RT-PCR Product 1
R. flavr’rJes pta RT-PCR Product 3
TAM-DN
TAM-DN3b
TAM-DN13b
TAM-DN5b
TAM-DN4b
R. flavipes pta RT-PCR Product 4
TAM-DN8b

TAM-DN14b
l l TAM-DNQb
TAM-DN10b

0.10

TAM-DN1
_‘:" TAM-DN4b
TAM-DN3b
TAM-DN2b
TAM-DN9b
R. flavipes ccoN RT-PCR Product 1
R. flavrpes ccoN RT-PCR Product 3
, R. flavrpes ccoN RT-PCR Product 4
I —— R. flavipes ccoN RT-PCR Product 2
Chromobacten’um

violaceum

  
   
 
   
  
  

 

 

 

 

 

0.025

 

Figure 3.8. Maximum likelihood-based phylogenetic analysis of the deduced
amino acid sequences of RT-PCR products depicted in Fig. 3.7. All RT—PCR
products clustered specifically with the respective genes from “S. acetivorans"
isolates. (A) acetate kinase (170 positions); (8) phosphotransacetylase (182
positions); (C) ccoN subunit of cbb3 oxidase (124 positions). Neissena
meningitidis was used as an outgroup for all phylogenetic analyses (not shown).
Scale bars represent 0.1, 0.05 or 0.025 changes per amino acid.

103

reaction mixtures were derived from RNA (presumably mRNA) and not from DNA
contamination of the template. Upon cloning and sequencing of the correctly-
sized RT-PCR products, phylogenetic analysis revealed that they grouped
specifically with the ack, pta and ccoN gene products from “S. acetivorans”
isolates (Figure 3.8). These results indicated that expression of ack, pta and
ccoN is not only relevant to acetate-dependent growth of “S. acetivorans” in vitro,
but to growth and/or survival in situ as well.

It was noticed that, in RT-PCR reactions with the ack-targeting primer pair,
an additional product was produced that was slightly smaller (450 bp) than the
anticipated ack fragment (600 bp)(Figure 3.7). However, cloning and sequencing
of material from this band revealed that it was related to a gene encoding a
hypothetical protein in Drosophila melanogaster and probably arose from non-

specific amplification of RNA from termite gut tissue.

Oxygen Consumption by “S. acetivorans” and Other Components of the R.
flavlpes Gut

In an effort to estimate the potential contribution of “S. acetivorans" to 02
consumption by guts of R. flavipes, per-cell rates of 02 consumption were
determined for “S. acetivorans" grown in vitro without, and with, NH4 limitation.
The latter condition was imagined to be a possible mimic of what these cells
might be experiencing in situ, as the food source of R. flavipes (wood) is
relatively low in N (ca. 0.05% wlv for sound wood). When grown on NH4 limiting

medium, the 02 consumption rate of TAM-DN1 was 1.5 (:l: 0.4) x 10'5 pmol-min'

104

‘-cell" (mean 4. s.d.) in buffer supplemented with acetate and 1.0 (:l: 0.2) x 10'5
pmol-min'1-cell'1 in succinate-supplemented buffer (Table 3.3). The 0;
consumption rates in medium with no NH4 limitation were similar 9.9 (:l: 0.6)

x 10'5 and 9.5 (t 0.4) x 10'5 pmol-min‘1-cell'1 in acetate- or succinate-
supplemented buffer, respectively. Little or no oxygen consumption was
observed when glucose or lactate was added to the buffer, or in buffer without
added substrate. Per-cell rates of 02 consumption by Citrobacter str. RFC-10,
another abundant organism isolated with “S. acetivorans”, were about half that of
“S. acetivorans" on these same substrates (Table 3.3). However, Citrobacter
RFC-10 also displayed lactate- and glucose-supported 02 consumption.

02 consumption rates for intact extracted guts of R. flavipes in acetate-
supplemented buffer were 1013 :t 141 pmol-min‘1-gut". However, the oxygen
consumption rates for guts incubated in buffer without acetate (969 t 256
pmol-min'1-gut") were not significantly different than for guts incubated with
acetate (p=0.80). These data suggested that respiration by intact guts was
probably largely, if not entirely, supported by endogenous acetate, which is
present in relatively high concentrations in R. flavipes hindgut fluid (ca. 80 mM).

To estimate the relative contribution of bacteria and protozoa to 02 uptake
by whole guts, termites were fed on artificial diets intended to eliminate all or
most of these particular microbial communities from the gut, after which
measurements were made of whole gut 02 consumption (Table 3.4). Compared

to the normal diet of wood, even a seemingly innocuous shift to a short-tenn diet

105

Table 3.3. Substrate-specific oxygen consumption rates of “Stenoxbactef' st.
TAM-DN1, Citrobacter st. RF C-1 0, and whole R. flavipes guts.

 

“Stenoxybacter’ Citrobacter st. R. flavipes Whole

 

Substrate' st. TAMO-DN1 RFC-.10 Guts
(pmfllggngcell (pmzzlgglcell ( pm ollml nl gut)
None 0.0 0.0 969 :t 256
Acetate 1.5 t 0.4 0.6 i 0.2 1013 :l: 141
Succinate 1.0 t 0.2 0.5 :t 0.2 n.d.
Lactate 0.0 1.0 t 0.3 n.d.
Glucose 0.0 0.8 :l: 0.2 n.d.

 

" Final substrate concentration was 10 mM in fully aerated insect Ringers
solution. n.d. not determined. All values represent mean t standard deviation
(n=3).

b Cultivated in nitrogen-limited medium that also contained an excess of acetate.

106

 

Table 3.4. Effect of diet on gut microbial communities and 0; consumption rates
of whole guts of R. flavipes.

 

 

Diet‘ bac$:rlit;7aar:ll?a:la (Celt/2322103? gngzggtigrid
(CFU/git x 10 ) (pmol/minlgut)
Wood (control) 4.5 1: 0.5 9.6 :t 1.5 1013 1 141
Cellulose 3.2 :I: 0.7 6.6 :l: 0.3 740 :l: 86
Cellulose + Ab 0.004 :l: 0.002 3.3 :l: 1.5 348 i 65
Starch 4.0 :t 0.5 2.3 :l: 0.3 436 1 108
Starch + Ab 0.003 :1: 0.001 0.7 :l: 0.1 286 :l: 92

 

‘ Termites were maintained on diet for 11 days. Ab, antibiotic cocktail [ampicillin,
cephoperazone, vancomycin (800 ug/mL each)]. Values are the mean :l: standard
deviation of the mean.

b For each treatment, twelve guts were pooled, homogenized and plated onto
carbon-supplemented 1/10 brain-heart infusion media and incubated in hypoxia
(2% Oz, 98% N2). Colonies were counted 14 days after plating (n=4).

° Determined by direct counts of DTAF-stained gut fluid (n=3).

d Buffer supplemented with 10 mM acetate, pH 7.0 (n=3).

107

of pure cellulose in agarose resulted in a moderate, but statistically significant
drop in both bacterial viable cells (p = 0.04) counts and protozoan numbers (p =
0.008), as well as a drop in whole gut 02 consumption (p = 0.04). However,
incorporation of a triple-antibiotic cocktail (800 pglml each ampicillin,
cefoperazone, vancomycin) into such a diet drastically reduced bacterial CF U by
a factor of 103, but it also resulted in a 50% drop in total protozoan numbers. The
0; consumption rate of whole guts extracted from such termites was a little less
than half that of cohorts fed cellulose alone.

Termites fed on a diet of cornstarch retained relatively high numbers of
bacterial CFU per gut, but lost about two-thirds of their protozoa compared to
cellulose-fed controls. This was expected: starch is known to almost totally
eliminate cellulolytic protozoa from termite guts (15, 58), probably because it is
so readily hydrolyzed, even by salivary amylases present in the termite itself, that
protozoa no longer have a selective advantage for retention in the gut. Moreover,
it is a substrate on which wood-feeding termites can survive indefinitely.
Nevertheless, the 02 consumption rate of whole guts from starch-fed termites
(436 1 108 pmol-min'1-gut") was only about 60% that of the cellulose-fed controls
(p = 0.003). Incorporation of antibiotics into the starch diet not surprisingly
decreased the bacterial CFU drastically, and it also further reduced protozoan
numbers and whole gut 02 consumption rates to the lowest value seen in the
entire experiment (286 1 92 pmol-min'1-gut'1; Table 3.4).

Although facultative and strictly aerobic bacteria, including obligate

microaerophiles like “8. acetivorans”, would be expected to augment the

108

respiratory activity of the gut tissue itself and contribute to the 02 uptake rates
seen with extracted whole guts, the data presented in Table 3.4 suggest that
protozoa, long thought to be strict anaerobes, also make a substantial
contribution to 0; consumption. This is discussed further below, as are
cautionary considerations in interpreting results from feeding experiments in

which major components of the gut microbiota are disrupted or eliminated.

Discussion
In situ location of “S. acetivorans”

The obligate microaerophilic phenotype of the “S. acetivorans” strains
suggested that their in situ location was within the “hypoxic zone,” which extends
from the gut epithelial wall to approximately 150 - 200 um inward (11). However,
direct visualization of the cells within the termite hindgut by fluorescent in situ
hybridization (FISH) (5, 55) with a fluorescently-labeled, “S. acetivorans”-specific
16S rRNA probe was not possible owing to the overpowering autofluorescence of
the termite tissue. Attempts to (i) decrease the autofluorescenoe with various
fixatives, blocking reagents, HCl and H202 washes (6, 55); and (ii) to amplify the
TAM strain-specific signal by tyramide signal amplification (42) or hybridization
with dual fluorescently labeled probes (6) was ultimately unsuccessful. Therefore,
an alternative, PCR-based method was used to identify the general (wall- or fluid-
associated) location of the “Stenoxybacter’ cells within the hindgut. By using this
method, which involved physical separation of the hindgut epithelium (and its

associated microbiota) from the luminal community, the location of the spirochete

109

population within the gut fluid could be correctly identified (Figure 3.2b). After
substitution of the spirochete-specific 16S rDNA primers with “Stenoxybactef—
specific primers, the results suggested that the “Stenoxybacter’ population was
primarily wall-associated (Figure 3.2a). Thus, the microaerophilic phenotype of
the “Stenoxybacter’ population is consistent with their in situ location on, or
associated with, the hindgut wall, an area known to contain oxygen-consuming

microorganisms (1 1).

In vitro and In situ Acetate Oxidation by TAM-DN1

Consistent with the acetate-rich environment of the termite gut, “S.
acetivorans” strain TAM-DN1 expresses low affinity acetate kinase enzyme
(Kmam. = 7 - 22 mM) rather than the high-affinity acetyl-CoA synthetase
(Kmm. = 200 uM)(60). Given that the phosphotransacetylase reaction (1.1
pmol-min‘1-mg protein") (most likely the rate-limiting step) can account for the
entire experimentally-derived rate of acetate consumption by TAM-DN1 (0.12
umol-min'1-mg protein") (Chapter 2), the ACK/PTA pathway is most likely the
sole method for activating acetate in “S. acetivorans” cells. The lack of activity for
other acetate-activating enzymes such as AMP- or ADP-forming acetyl-CoA
synthetase and PPi-acetate phosphotransferase support this conclusion.
Furthermore, the expression of acetate kinase by TAM-DN1 cells during
succinate—dependent growth suggests the ACK/PTA pathway is constitutive;
regulation of this pathway may be unnecessary within the acetate-rich termite

hindgut environment.

110

As mentioned above, the experimentally derived acetate consumption rate
for TAM-DN1 was 0.12 pmol-min"-mg protein". By relating cell number to protein
content, this rate can be expressed as 1.1 x 10"5 nmol-hour"-cell". Assuming the
in situ “Stenoxybacter’ population to be approximately 1 x 105 cells-gut'1 (Chapter
2), and to the extent that the in vitro measurements might reflect the in situ
conditions, the estimated acetate oxidation rate for the TAM population in the
termite gut is 0.1 nmol-gut"-hour". This is approximately 4.6% of the 2.4
nmol-gut"-hour" acetate oxidation rate as measured by microinjection of
radiolabeled acetate into R. flavipes hindguts (56), and 0.5% of the estimated
20.2 nmol-terrnite"-hour" acetate production rate measured in laboratory
maintained termites (40). Therefore, though both the “Stenoxybactef' population
and the termite are sharing the same substrate, the bacteria probably have
minimal impact on the acetate resources available to meet the daily energy
requirements of the termite host.

PCR amplification of R. flavipes gut DNA with the broad-range acetate
kinase primers revealed a diversity of ack genes (Figure 3.3). Considering that
the termite gut is primarily an acetogenic system, and the ACK/PTA pathway is
bidirectional (Figure 3.1), these results reflect the fact that many gut organisms
may operate this pathway in the direction of acetate and ATP production when
fermenting mono- and polysaccharides (60). A majority of ack genes amplified
from the termite gut clustered with the ack genes of known acetate-producing
anaerobes, such as Clostn'dium and Treponema species (Figure 3.3). However,

the distant phylogenetic relationship of the “Stenoxybactef' ack and pta genes to

111

any other ack or pta genes in R. flavipes hindgut organisms allowed the facile
design of “Stenoxybactef-specific gene-targeted primers. By using these
“Stenoxybactef-specific primers in reverse-transcriptase PCR reactions with R.
flavipes hindgut RNA, the in situ expression of ack and pta by the
“Stenoxybacter’ population was confirmed (Figure 3.7 and 3.8). This, coupled
with the ability of TAM-DN1 to co-utilize acetate and succinate (Chapter 2), and
the inability to grow on other substrates found within the termite gut fluid such as
cellobiose, xylose and lactate, supports the conclusion that the “Stenoxybacter”
strains are almost certainly using acetate in situ.

When acetate is the only carbon and energy source for a bacterium the
TCA cycle alone does not allow any net assimilation of acetate carbon. Under
these conditions, the anapleurotic glyoxylate cycle serves to replenish TCA cycle
intermediates derived from acetate catabolism, but drawn off for biosynthesis of
cell material (14, 17) (Figure 3.1). As further possible evidence of in situ acetate
oxidation, the ability of TAM-DN1 to operate a glyoxylate cycle was examined.
Activity for the first enzyme in the glyoxylate cycle, isocitrate lyase, was not
detected in cell-free extract of TAM-DN1 (Table 3.2), even though cells were
harvested during acetate-dependent growth. Attempts to amplify the isocitrate
lyase gene from TAM-DN1 using broad-range primers were also unsuccessful.
However, apparent malate synthase activity was detected. E. coli is known to
contain two distinct malate synthase isoforms, malate synthase A (MSA) and
malate synthase G (MSG) (36). Malate synthase A is involved in metabolization

of glyoxylate formed from the dissimilation of acetate, whereas malate synthase

112

G metabolizes glyoxylate formed primarily from growth on glycolate (36). Both
isoenzymes condense glyoxylate with acetyl-CoA to form malate, and both
display similar enzyme kinetics. PCR amplification and sequencing results clearly
reveal that TAM-DN1 possesses a malate synthase G, whereas no malate
synthase A, or the related malyl-CoA lyase, was identified (Table 3.2, Figure 3.5)
(35).

This seeming paradox of growth on CZ compounds without a complete
glyoxylate cycle has been reported in other organisms (2, 26, 35), and has led to
the proposal of an alternative anapleurotic pathway, the citramalate cycle (22)
(Figure 3.9). In this pathway, the requirement for isocitrate lyase is surmounted
by the action of B-methylmalyl-CoA lyase. This enzyme cleaves B-methylmalyl-
CoA to form propionyI-CoA and glyoxylate. Glyoxylate then condenses with
acetyl-CoA to form malate in the typical malate synthase-type reaction, which
can involve malate synthase A, G, or malyl-CoA lyase (35).

Enzymatic assays for the key enzymes B-methylmalyl-CoA lyase and
malyl-CoA lyase in the citramalate cycle are available in the literature (35).
However, the malyl-CoA and B-methylmalyl-CoA substrates are not commercially
available and must be synthesized in the laboratory. Therefore, for purposes of
timeliness and practicality, these assays with TAM-DN1 extract were not pursued
further.

The possibility exists that TAM-DN1 uses a citramalate or similar cycle as
an anapleurotic pathway if acetate is the sole carbon and energy source.

However, it is likely that the “Stenoxybactef' population does not rely solely on

113

Acetyl-CoA
C02 Pyruvate

CMS
Oxaloacetate Citramalate
/ (Citramalyl-CoA)
Malate B-methylmalyl-CoA

\a Succinyl-CoA

A MMC
C02

Propionyl-CoA

  

MCL
or
MS

Acetyl-coA
Glyoxylate

Figure 3.9. The citramalate cycle - an alternative to the glyoxylate cycle

for acetate assimilation in organisms lacking isocitrate lyase activity. Key
enzymes are citramalate synthase (CMS), B-methylmalyl-CoA lyase (MMC)
malyl-coA lyase (MLC) and malate synthase A or G (MS). It is not yet known
if the product of the CMS reaction is citramalate or citramalyl-CoA. Figure
adapted from Meister et al. 2005.

114

acetate for carbon and energy in situ. Compounds such as amino acids and
volatile fatty acids that can be used for biosynthesis or regeneration of TCA cycle
intermediates have been measured in termite gut fluid (19, 40, 50). Furthermore,
lysis of hindgut organisms though normal cell death or predation by protozoa (39)
certainly releases some amount of lipids, nucleotides, etc. into the termite fluid
upon which the “Stenoxybactef' population may rely. This notion is supported by
the inefficacy of attempts to cultivate TAM-DN1 on a completely defined medium

- the presence of a small amount of yeast extract is thus far required for growth.

In vitro and In situ 02 consumption by TAM-DN1

TAM-DN1 reduces oxygen through the use of at least one terminal
cytochrome oxidase, as verified by the PCR amplification of ccoN, the 02-
reducing subunit of a cbb3-type haem-copper oxidase (Figure 3.5). The presence
of a high-affinity (Kmom... = 7 nM) oxidase is not surprising given the
microaerophilic nature of the “Stenoxybacter” isolates and their phylogenetic
relationship to Eikenella comodens and Neisseria spp., which also contain Cbba-
type terminal oxidases (23). Given that approximately 60% of the hindgut volume
is exposed to a persistently low level of 02, a diversity of ccoN genes within the
hindgut is expected. PCR amplified with broad-range primers, a majority of ccoN
sequences from R. fiavipes gut DNA clustered with DechIoromonas aromatica,
an organism known to degrade aromatic compounds (16). Whereas organisms

related to DechIoromonas aromatica have not been isolated or described from

115

termite guts, Oz-dependent aromatic ring cleavage of lignin model compounds is
known to occur (12).

Though the hindgut contains a diversity of organisms with ccoN genes,
RT-PCR with R. flavipes gut RNA and ccoN-targeted “Stenoxybactef-specific
primers revealed that the In situ population of “S. acetivorans” appears to be
expressing the 02 reducing enzyme (Figures 3.7 and 3.8). Though the cbba-type
cytochrome oxidase has also been implicated in the reduction of nitrate (44),
TAM-DN1 is unable to use nitrate for anaerobic respiration in vitm. The high-
affinity for oxygen (Kmom... = 7 nM) suggests that locally, the “Stenoxybacter”
population could be very effective at consuming oxygen to extremely low levels,
possibly supporting the Oz-labile processes of Nz-fixation, 002-reduction and
methanogenesis by other gut organisms.

Given the abundance of “Stenoxybacter’ cells within the termite gut as
measured by colony counts and semi-quantitative PCR (Chapter 2) and insofar
as the in vitro 02 consumption rates (Table 3.4) represent maxima not
necessarily reflective of the in situ situation, a maximum rate of oxygen
consumption within the termite gut by the “Stenoxybacter” population of 1.4 - 3.6
pmol-min"-gut‘1 is estimated. Similarly, quantification of the in situ abundance of
Enterobacten'aceae coupled with the ()2 consumption rate of Citmbacter sp.
RFC-10 yields a maximum potential 02 consumption rate of 0.2 pmol-min"-gut"
for the Enterobacten’aceae within the termite hindgut. Estimates of 02
consumption rates for the lactic acid bacteria, based on Enterococcus st. Rfl.6,

were 3.0 pmol-min"-gut" (57).

116

In this study, an 02 consumption rate for whole termite guts of 1013
pmol-min"-gut" was measured. This is approximately 6x higher than the
estimated 178 pmol-min"-gut" measured by Brune et a! with microelectrodes
(11). Given the fact that two completely different methodologies were used
(microelectrode-agarose chamber vs. Clark-type 02 uptake chamber), this
difference should perhaps be expected. Furthermore, 02 consumption estimates
by the microelectrode technique admittedly measured 0; consumption only
within the hindgut paunch region (~1/5 of the total hindgut tissue), and probably
underestimates total hindgut respiration. However, the rates measured in this
study are, in all likelihood, an over-estimation, as the whole extracted gut (midgut
+ hindgut) was used. Furthermore, the whole—gut 02 consumption rates
measured in this study were higher than the respiration rate of the entire termite
(860 pmol-min"-termite") measured by Odelson et al. (40). Because the
measurements by Odelson et al. did not require dissection of the termite, they
are certainly more accurate. Therefore, the true hindgut 02 consumption rate
most likely lies somewhere between 178 and 860 pmol-min"-termite".

Table 3.5 gives the respective contributions of the “Stenoxybacter”,
Enterobacten‘aceae, and lactic acid bacteria to the total 02 consumption within
the termite gut, based on both microelectrode and 02 uptake chamber methods.
The fact that each population contributes roughly 1% to the total suggests that
rather than a single group of microorganisms being responsible for a majority of
the oxygen consumption, the task is spread out among many. Therefore, even if

a fraction of the estimated 700 phylotypes (21) within the termite gut are

117

Table 3.5. Estimated contribution to the total hindgut 02 consumption rate by
three hindgut bacterial communities.

 

In situ In situ Oz % %
abundance consumption contribution contribution
(cells-gut" (pmol-min" to total ' to total °
)

 

x 10 gut")
“Stenoxybacter” , d
strains 1.0 — 2.2 1.5 - 3.6 0.8 — 2.0 0.1 — 0.4
Enterobacteriaceae 0.4 b 0.2 ° 0,1 mm
Lactic acid 6
bacteria 19 3.0 1.7 0.3

 

‘ Based upon quantification of colony forming units and semi-quantitative
dilution-to-extinction PCR.

b Based solely on colony forming units.

° Estimated from most-probable number studies by Tholen et al. 1997, using
Enterococcus sp. RfLG as a model organism.

" Using TAM-DN1 as the model organism for the “Stenoxybacter” population.

‘ Using Citrobacter sp. RFC-10 as a model organism for the Enterobacten‘aceae
community.

'Assuming total gut 0; consumption is 178 pmol-gut"~min" based on
microelectrode measurements (Brune et al. 1995).

9 Assuming total gut 02 consumption is 1013 pmol-gut"-min" based on
measurements in this study.

118

responsible for 1% of the total 0; consumption, the task of removing 02 from the
hindgut system for the continued fermentative production of acetate is
accomplished. It is interesting to note that an ability to consume 02, through
respiratory or other enzymes, has been discovered in many so-called “strict” or
“aerotolerant” anaerobes such as members of the Bacteroidetes,
Enterococcaceae, Desulfovibn'o, and Methanobrevibacter (4, 31,49, 57). Many
of these, or closely related species, are termite gut symbionts, some, such as the
methanoarcheae are known to inhabit the hindgut epithelial wall (29, 30).
Therefore, these atypical 02 consumers may, together with other wall-associated
microbes such as “S. acetivorans”, contribute significantly to the overall 02

respiration within the termite gut.

Relative Contribution of Major Microbial Groups to Hindgut 02
Consumption

Total removal of inwardly diffusing oxygen in the termite gut is most likely
accomplished by some combination of 02 reduction by hindgut bacteria, archaea,
protozoa, and epithelial cell mitochondria. To increase our understanding of the
microbial contribution to the total 02 consumption in the gut, termites were
maintained on diets that included antibiotic drugs and/or starch that resulted in
the removal of a majority of termite gut bacteria and/or protozoa (9, 45).
Antibiotics that target the bacterial cell wall were chosen specifically in order to
minimize possible damage to the epithelial mitochondria, thereby reducing any

confounding variables.

119

The results reveal that the size of the per-gut protozoan population is
highly correlated with the whole-gut 02 consumption rate (r2 = 0.95, Figure 3.10),
whereas the correlation between the whole-gut 0; consumption rate and the
number of cultivable bacteria and archaea is much weaker (r2 = 0.56). These
analyses reveal that a disruption to the protozoan population has a greater effect
on the whole-gut 0; consumption rate than does a disruption in the bacterial
population.

Given the complexity of the termite gut and the unknown pleiotropic
effects of even the smallest disruption, the reason for a high correlation between
02 consumption rate and number of protozoa is unclear. It may be that protozoa
are directly involved in 02 consumption in the hindgut. This may be analogous to
rumen ciliates that are able to use 02 as a terminal electron acceptor if present in
very low concentrations (18). Direct visualization of the attachment of the
protozoan Pyrsonympha vertens to the epithelium (placing it, at least in part,
within the hypoxic zone) was frequently seen in transmission electron micrograph
section of R. flavipes hindguts (8). Given the microscopic volume of the hindgut,
the large number of protozoa, and their constant motion, it would be difficult to
imagine that a majority of protozoa would not encounter some level of oxygen, if
only periodically. If the protozoa are directly involved in oxygen consumption in
the hindgut, a similar analysis using higher termites, which by and large do not
contain gut protozoa, would be interesting.

Second, the protozoa may indirectly and selectively affect the 02

consuming prokaryotes through predation, production of vitamins, cofactors,

120

>

Whole-Gut 02 Consumption Rate

Whole-Gut 02 Consumption Rate

(pmol/min/gut)

(pmol/minlgut)

12C!)-
1100-

 

 

mj I r I I I I I I I l I I T
0 2000 4000 6000 8000 10000 12000
Protozoa (cells/gut)

 

 

 

100 1000 10000 100000
Cultivable Prokaryotes (CF U/gut)

Figure 3.10. Correlation between R. flavipes whole-gut oxygen consumption
rate and the number of gut protozoa (A) or cultivable prokaryotes (B).

r2 values for the best-fit regression lines are 0.95 (A) and 0.56 (B). Termites
were maintained for 11 days of diets of cellulose (V), cellulose with
antibiotics (o), starch (A), and starch with antibiotics (0). Wood control (I).

121

substrates, or some other unknown mechanism. In this case the loss of the
protozoa may directly or indirectly affect the 02 consuming community, resulting
in a decrease in whole-gut 02 consumption.

A final explanation for the seemingly high correlation between 02
consumption and gut protozoa includes the possibility that some of the protozoan
symbionts are 02 consumers. These symbionts could have a protective role,
removing 02 from the immediate proximity of the protozoans to allow for
anaerobic fermentation. Furthermore, aerobic oxidation of protozoan—derived H2
by ecto- or endosymbionts would improve the relative energy yield to the
eukaryote by consumption of a fermentation endproduct (7). Only a low incidence
of aerobic oxidation of H2 by most-probable number studies was found in R.
flavipes (57), however the homogenization procedure certainly resulted in the
lysis of a majority of the protozoa. A lack of pure culture isolates of prokaryotic
symbionts of protozoa, including members of the abundant Candidatus
Endomicrobia (formerly Termite Group 1)(54) division make current testing of this
question difficult.

A possible confounding reason for the weak correlation between whole-
gut 0; consumption and the number of bacteria and archaea may be due to a
shift in the composition of this community (and hence metabolic rates and
capabilities) without a shift in the total number of organisms. 16S rRNA-based
identification of the bacteria and archaea within the gut after completion of the

feeding experiments would resolve this question.

122

When introducing “directed" changes into the termite hindgut community,
one is always confounded by the complex, immeasurable, and innumerate
confounding interrelationships between the microbiota. A disruption to one
population, community, or even cell may have a “rippling” effect on the rest of the
community that renders any results far from absolute. Until there is a mechanism
by which one microbe can be selectively manipulated with the guarantee of
having no effect on any other microbe, any conclusions based on disruptions to
the gut community will be replete with caveats. However, as that time may never
exist, such measurements remain useful as a first approximation of in situ

behavior that can often offer insights that lead to new, testable hypotheses.

Conclusions

In this chapter the physiological ecology of “Stenoxybecter acetivorans”
within the R. flavipes hindgut is described. The “Stenoxybacter’ population was
primarily associated with a hypoxic area of the hindgut known to contain 02
consuming microorganisms. Consistent with their existence in a high (60 — 80
mM) acetate-containing environment, these organisms express a low affinity
pathway for acetate—activation and a high affinity respiratory oxidase. Detection
of “Stenoxybactef-specific expression of these genes in situ indicates the
population respires 02 and metabolizes acetate in the termite hindgut. Estimates
suggest the maximal in situ respiratory capability of the “Stenoxybactef'
population is 0.1 to 2% of the total 02 reduction in the hindgut, similar to

estimates of other abundant Orconsuming isolates from the R. flavipes hindgut.

123

Disruption of the gut microbiota by maintaining termites on selective diets
revealed that disruption of the protozoal community had a greater effect on the
whole-gut 02 consumption rate than did disruption of the bacterial community.
This suggests a need for future studies that further explore the relationship

between 02 consumption and the hindgut protozoa.

124

Acknowledgements
I would like to thank Dr. Thomas Schmidt for help with primer design and
phylogenetic analyses, Kwi Kim for assistance with RNA work, and Dr. Bradley

Stevenson for helpful discussion and analysis of cbba terminal oxidases.

125

 

 

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131

Chapter 4

Summary

The historical view of the termite hindgut as an anaerobic fermentation
chamber has led to a greater understanding of Oz-sensitive hindgut processes
such as cellulose digestion, acetogenesis, methanogenesis, and H2 production
(2, 4). However, the realization that a majority of the hindgut is exposed to some
level of oxygen caused a paradigm shift in which the important role of 02
consuming microorganisms to termite vitality became clear (3, 11). This
dissertation has investigated the nature and importance of the 02 consuming
microbiota in the hindguts of the termite Reticulitennes flavipes.

In Chapter 2, a cultivation-based approach, using acetate containing
media and incubation conditions that included hypoxia (2% 02, 5% C02, 93% N2)
was used to test the hypotheses that acetate was a likely electron donor for
aerobic respiration in the gut, and that some members of the Oz-consuming
microbiota were microaerophiles, highly adapted to the “hypoxic zone” on and
near the hindgut epithelial wall. Among the numerically dominant isolates were
members of the Enterobacteriaceae, Enterococcaceae and Streptococcaceae,
closely related to strains previously isolated from termite guts (1, 5, 8-10, 13).
However, highest recoveries were obtained on acetate-containing plates
incubated under hypoxia, an increase due almost exclusively to a single, easily
distinguishable colony type. Phase contrast and electron microscopy of cells

comprising such colonies revealed they were thin, nonmotile rods that appeared

132

to accumulate intracellular poly-B-hydroxybutryate. They were able to be
subcultured only on plates incubated under hypoxia and displayed robust colony
growth only if acetate was included in the medium, suggesting they were acetate-
oxidizing microaerophiles. Phylogenetic identification of these, and similar strains
(designated with the prefix “TAM”) isolated from Reticulitennes workers collected
from widely separated locations within the US and one location in France
revealed they shared >99% 16S rRNA gene identity, but only were <95% similar
to any other known bacteria, including the closest cultivated relative, Eikenella
corrodens.

Though other intrinsically interesting microorganisms were isolated,
including the first Venucomicrobia and Acidobacteria isolated from termites (12)
(See Appendix), the estimated in situ abundance, apparent microaerophilic
phenotype, and distant phylogenetic relationship to any other known bacteria
suggested the TAM organisms were unique and potentially important 02
consuming microbes in the guts of Reticulitennes. Therefore, the characterization
and physiological ecology of the TAM isolates became the focus of this
dissertation.

PCR-based procedures implied the TAM isolates were autochthonous to
the hindgut and comprised ca. 5% of the hindgut bacterial community. In vitro
characterization of representative strain TAM-DN1 revealed that besides acetate,
acetyl-acetate, succinate, butyrate, fumarate, glutamate, glutamine and
casamino acids, few other substrates, including common organic acids and

carbohydrates, were growth-supporting. This is perhaps a reflection of the

133

relatively stable, acetate-rich hindgut environment. When cultivated on solid
medium in direct contact with the atmosphere, the oxygen tolerance of TAM-DN1
was equally narrow. Their ability to adapt to higher headspace 02 concentrations
in liquid medium, as well as express of oxyprotective enzymes like catalase and
superoxide dismutase, may be important for colonization of the guts of newly
hatched larvae or recently-malted colony mates under conditions of substantially
higher 02 content. The proposed classification of the TAM strains as
“Stenoxybacter acetivorans” gen. nov., sp. nov. is a reflection of their distinct
physiology and phylogeny.

The observations described in Chapter 2 suggested that “Stenoxybacter’
st. TAM-DN1 and related strains were likely to be involved in oxygen
consumption and acetate oxidation within the termite gut. Chapter 3 described
the combination of in vitro and in situ molecular and physiological approaches
used to test these hypotheses.

As inferred from its obligate microaerophilic phenotype, and confirmed
with PCR, the primary location of the “Stenoxybacter’ cells in situ appears to be
in close association with the hindgut wall, a hypoxic region characterized by a
persistent inward flux of 02(3). Through enzyme assays and PCR experiments
with gene-specific primers, key genes and gene products involved in acetate
utilization and oxygen consumption by “S. acetivorans" were detected. The use
of an acetate-activating pathway with a low Km for acetate, and a terminal
oxidase possessing a high affinity for oxygen, suggested that “S. acetivorans" is

well-adapted to life within the hypoxic zone of termite hindguts. Detection of key

134

acetate utilization and 02 consuming gene transcripts from R. flavipes hindguts
revealed that the genes (and by inference, the gene products) are being
expressed by cells of “S. acetivorans” in situ. Estimates of the potential
contribution of “S. acetivorans” to overall 0; consumption in guts of R. flavipes
indicate the organisms may be responsible for approximately 0.1 to 2% of the
total hindgut 02 consumption. Finally, feeding treatments that largely eliminated
major microbial groups (i.e. bacteria and protozoa) from the R. flavipes hindgut
revealed the hindgut protozoa may have a more significant role, directly or
indirectly, to 02 consumption within the hindguts of lower termites than was
previously recognized. .

This study represents the first dedicated effort to isolate and characterize
the O; consuming microbiota in termite hindguts. As a result, new, fundamental
information about the nature, function, and importance of “Stenoxybacter
acetivorans” within the hindgut community has been realized. Furthermore, the
putative importance of the hindgut Eukarya, an abundant but often overlooked
potential oxygen sink (owing to their “strict” anaerobic metabolism) was
suggested. The discovery of “Stenoxybacter acetivorans”, an abundant, but
heretofore undetected population of 02 consuming microbes within the termite
gut reveals the need for further refinements to cultivation techniques in order to
better mimic the in situ environment. Further study of these microorganisms will
likely reveal new principles of growth and survival under reduced 02

concentrations, a poorly understood phenomenon often encountered in nature.

135

As the quotation by Dr. Jared Leadbetter that opened this dissertation
suggested, the diversity of termite gut microbes astounds today as much as it did
100 years ago. At once invigorating to behold and challenging to study,
fundamental answers to a host of biological, biochemical and ecological
questions lie within a microliter of hindgut fluid. After all, Dr. Leadbetter notes “if
only one or two of those two hundred species were doing something useful for
the termite, evolution would have booted the freeloaders millions of years ago”
(7). VIfith such a profusion of complexity, an increase in understanding of only a
fraction of the symbionts will serve as a foundation to piece together the whole.
Dr. Robert E. Hungate, whom, after 45 years studying the termite gut and bovine

rumen advised (6):

“The total activity (metabolism) of the ecosystem should be
measured, and the precision and validity of the ecological analysis
should be tested by the algebraic addition of the individual activities
and comparison of the sum with the measured activity of the total

system. Adherence to this goal will do much to prevent over-
inflation of the ecologist’s ego concerning his ecological

accomplishments!”

Study of the termite gut and associated symbionts will, without doubt, lead to new

discoveries and surprises, as well as prevent many egos from over-inflation for

the next one hundred years.

136

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138

Appendix

Previously published as:
Stevenson, Bradley 8. Stephanie A. Eichorst, John T. Wertz, Thomas M.
Schmidt, John A. Breznak. New Strategies for Cultivation and Detection of

Previously Uncultured Microbes. Appl. Environ. Microbiol. 70:4748-4755.

Preface

Of particular abundance in soils, members of the Acidobacteria,
Planctomycetes and Verrucomicrobia divisions often also comprise a small
subset of 168 rRNA gene clones in libraries from termite gut homogenates.
Members of the candidate division Endomicrobia have not been identified in soil,
but are also abundant within the guts of lower termites. To facilitate as thorough
an examination of microbial 02 consumption in termite hindguts as possible, it
was desirable to screen for representatives of these divisions during the isolation
of Oz-consuming bacteria described in Chapter 2. Thus, in conjunction with
ongoing work in the laboratory to isolate Acidobacteria, Planctomycetes and
Verrucomicrobia from soil, a team effort was made to develop a facile, high-
throughput method to facilitate the detection and isolation of these microbes from
soil (3.8. and SE.) and soil invertebrates (J.W.). The fruit of this effort, the
isolation of novel Verrucomicrobia and Acidobacteria from soil and R. flavipes
guts, is presented in this Appendix, and is contemporaneous with experiments

described in Chapter 2.

139

New Strategies for the Cultivation and Detection of Previously Uncultured
Microbes

BRADLEY S. STEVENSON", STEPHANIE A. EICHORST, JOHN T. WERTZ,

THOMAS M. SCHMIDT, AND JOHN A. BREZNAK

Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI

140

ABSTRACT

An integrative approach was used to obtain pure cultures of previously
uncultivated Acidobacteria and Verrucomicrobia from agricultural soil and from
the gut of wood-feeding termites. Some elements of the cultivation procedure
included: the use of agar media with little or no added nutrients; relatively long
periods of incubation (over 30 days); protection of cells from exogenous
peroxides; and inclusion of humics or a humic analogue (anthraquinone
disulfonate) and “quorum signaling” compounds (acyl homoserine lactones) in
growth media. Air, hypoxic (1—2% 02, vlv) and anoxic incubation atmospheres
were also used, some with elevated concentrations of CO; (5%, vlv), the latter of
which treatments resulted in a significantly greater occurrence of Acidobacteria
on isolation plates. A simple, high-throughput, PCR-based surveillance method
(“Plate Wash PCR") was developed that greatly facilitated the detection and
ultimate isolation of target bacteria from among as many as 1,000 colonies of
non-target microbes growing on the same agar plates. Results illustrate the
power of integrating culture methods with molecular techniques to isolate

bacteria from phylogenetic groups underrepresented in culture.

141

INTRODUCTION

Cultivation-independent molecular techniques have illuminated the
enormous microbial diversity that exists on our planet and have served to define
nearly 40 phylum-level divisions existing within the Bacteria domain alone (23).
Most of these divisions, however, are poorly represented by cultured organisms,
and at least 13 remain “candidate divisions” represented only by environmental
gene sequences (23). The Acidobacteria and Verrucomicrobia are among those
Bacteria divisions represented by a large diversity of 16S rRNA genes, which
occur in particular abundance in soils, but contain few cultured members (3, 11,
12, 17, 20, 21, 23, 36,48). Hence, our appreciation of the physiological diversity
of Acidobacteria and Verrucomicrobia is limited, as is our knowledge of their role
in global biogeochemical cycles. Clearly, a better understanding of these
divisions would be attained by having a greater diversity of their members
available in pure culture for detailed study.

The intrinsic selectivity of any given medium and incubation condition
imposes limits on the nature, number, and diversity of microbes recovered from
natural samples. It follows, then, that the application of isolation procedures that
better mimic conditions existing in the habitat from which the samples are
obtained could increase the likelihood of retrieving previously uncultured
organisms. Recent efforts to accomplish this have met with some success by
using: (i) relatively low concentrations of nutrients (1, 13-15, 19, 43, 45, 50); (ii)
non-traditional sources of nutrients, signaling molecules, or inhibitors (of

undesired organisms) (9, 10, 13, 31); and (iii) relatively lengthy periods of

142

incubation (19, 22, 24-26, 33, 39, 40), sometimes directly in the natural
environment from which the inoculum was obtained (26).

For soil microbes, some of which may have become adapted to
elevated concentrations of CO2 and lower-than-atrnospheric concentrations of O2
(38), the composition of the incubation atmosphere may be an important
consideration. Elevated CO2 is rarely used in incubation atmospheres for
isolation of soil microbes, yet CO2 could be important for metabolic processes
other than pure autotrophy. Likewise, transition of soil microbes to fully aerobic
conditions on plating in air may be a stressful event. This would be especially
true if cells were not immediately equipped to cope with reactive oxygen species
(ROS) like hydrogen peroxide (H202), superoxide (02') or hydroxyl radical (OH-)
produced by their own metabolism or present in media as a result of autoclaving
(reviewed in 29). Even with facultative anaerobes like Escherichia coli, an abrupt
transition of anaerobically-grown cells to aeration can severely retard growth of
certain mutants (27). It is also noteworthy that the cultivability (in air) of E. coli
and Vibrio vulnificus following starvation is greatly improved if plating media are
supplemented with catalase or pyruvate, two compounds known to eliminate
H202 (5, 34). Such observations suggest that incubation atmospheres enriched
with CO2 and/or limited in 02, as well as the incorporation of agents to detoxify
ROS in the plating media, should be included among treatments seeking to
recover previously uncultured microbes.

Whatever cultivation approach is tried, however, one is ultimately

confronted with the need to evaluate its success. This is a potentially arduous

143

task if, as in this study, many different media and incubation conditions are being
tested and little or nothing is known about the microbes sought other than their
16S rRNA gene sequences. Accordingly, some high throughput screening
method is desirable. To deal with this, we developed a simple, high-throughput,
PCR-based procedure, “Plate Wash PCR”, that facilitated the surveillance of
isolation plates for the presence of target organisms and the ultimate recognition
of colonies comprised of them. The results of this endeavor constitute the

substance of the present paper.

MATERIALS AND METHODS
Sample collection and manipulation.

Soil samples were collected between August 2001 and October 2002 from
the Long Term Ecological Research (LTER) site located at the Michigan State
University WK. Kellogg Biological Station (KBS) in Hickory Comers, Michigan.
The KBS-LTER site includes a large-scale replicated field experiment with
treatments representing different cropping systems and types of management,
several successional sites, and unmanaged forested sites
(http:lIwww.lter.kbs.msu.edu). Soil core (2 cm diameter x 10 cm depth) samples
were taken from each of five permanent sampling stations distributed across one
of four replicate fields (replicate 1) of the Never Cultivated Successional (NC8)
treatment, which is representative of “native” soil.

Collected soil cores were stored at 4°C (usually for less than 48 h) until

they were homogenized under a hypoxic, CO2-enriched atmosphere (2% O2, 5%

144

CO2, balance N2) contained within a flexible vinyl hypoxic chamber fitted with an
oxygen sensor/controller (Coy Laboratory Products, Grass Lake, MI).
Approximately 30 g of soil was added to 100 ml of phosphate-buffered saline
(PBS; pH 7.0) containing 224 mM sodium pyrophosphate as a dispersal agent,
and 1 mM dithiothreitol (DTT) as a reducing agent (47). The suspension was
stirred vigorously for 30 min and allowed to settle for 30 min. An aliquot of the
supernatant was serially diluted in the same buffer and spread onto various
media with at least three replicate plates per dilution.

Termites, Reticulitennes flavipes (Kollar) (Rhinotermitidae), were collected
near Dansville, MI and either used immediately or maintained in the laboratory as
described previously (8, 35). Guts from 25-50 worker larvae were extracted
under a hypoxic atmosphere (described above) with sterile forceps and pooled in
a glass tissue homogenizer containing 2 ml of a sterile basal salts solution based
on “freshwater medium” described by Wlddel and Bak (49), and contained (per
liter): KH2PO4, 0.2 g; NH4CI, 0.25 9; KCl 0.5 g; CaCl2-2H2O, 0.15 9; NaCl, 1.0 g;
MgCI2-6H2O, 0.62 g; Na2804, 2.84 g; and MOPS (pH 7.0), 10 mM. After
homogenization, the homogenate was diluted serially in the basal salts solution
and spread onto various media.

The total numbers of microbes per gram (dry wt) soil or per termite gut
were determined by direct microscopic count after staining with 5-(4,6-
dichlorotriazine-2-yl) aminofluorescein (DTAF) following a protocol described by

J. Bloem (4). Soil moisture content was determined by baking three replicate

145

samples of soil at 80°C to constant mass. Soil moisture content was then used

with total direct counts to estimate the number of cells/g (dry wt) of soil.

Cultivation Conditions and Screening.

The basal medium used for cultivation of soil bacteria was a modification
of the basal salts solution described above and contained (per liter): KH2PO4, 0.2
g; NH4CI, 0.25 g; KCI, 0.5 g; CaCI2-2H2O, 0.15 9; NaCl, 1.0 g; MgCI2-6H2O, 0.62
g; Na2SO4, 2.84 g; HEPES (pH 6.8), 10 mM; trace element solution (below), 1 ml;
vitamin B12 solution (50 mg/l), 1 ml; and mixed vitamin solution (below), 1ml;
Bacto Agar (Becton, Dickinson, and Company, Franklin Lakes, NJ) , 15 9; final
pH adjusted to 6.8 - 7.0. The trace element stock solution contained (per liter):
FeCl2-4H2O, 1.5 g; CoCl2-6H2O, 190 mg; MnCl2-4H2O, 100 mg; ZnCl2, 70 mg;
H3803, 6 mg; Na2MoO4-2H2O, 36 mg; NiCl2-6H20, 24 mg; CaCI2-2H2O, 2 mg; HCI
(25% vlv), 10 ml (49). The mixed vitamin stock solution contained (per liter): 4-
aminobenzoic acid, 40 mg; D-(+)-biotin, 10 mg; nicotinic acid, 100 mg; Ca-0(+)-
pantothenate, 50 mg; pyridoxamine dihydrochloride, 100 mg; and thiamine
dihydrochloride, 100 mg (49). All solutions were heat sterilized; except for the
trace element and mixed vitamin solutions, which were passed through a 0.22
pm filter. Variations in the medium composition above included the incorporation
of some or all of the following (per liter): 3 mixture of organic carbon substrates
(yeast extract, Bacto protease peptone #3, casamino acids, and dextrose
(Becton, Dickinson», 0.05 g each; catalase (bovine liver, Sigma-Aldrich, Inc.),

2,000 U (spread onto individual plates containing 30 ml of solidified medium just

146

 

 

prior to inoculation) or 130,000 U (added to 1 liter of cooled, molten agar just
prior to pouring plates); soil extract (44), 100 ml; disodium anthraquinone-Z, 6-
disulfonate (AQDS), 2 g; and an N-acyl homoserine lactone “cocktail” (acyl-
HSLs) prepared in ethyl acetate acidified with 0.1% (vlv) acetic acid and
containing N-(butyryl, heptanoyl, hexanoyl, B-ketocaproyl, octanoyl, and
tetradecanoyI)-DL-homoserine lactones (Sigma-Aldrich Inc.), used at a final
concentration of 1 pM each in the media. The pH range of the prepared media
was 5.9 - 6.4, depending upon medium composition. Cultivation of termite gut
microbes was done with the same basal medium (above) containing also (per
liter): sodium acetate, 2.46 9: and/or a combination of yeast extract and peptone,
0.1 g each.

Incubation atmospheres used were: air (unamended); CO2-enriched (5%
vlv) air; 2% O2 and 5% CO2, balance N2 (termed hypoxic); or 5% CO2 and 10%
H2, balance N2 (termed anoxic). lncubations under atmospheres other than air
were carried out in glass dessicator jars, the flexible vinyl hypoxic chamber
(above), or a Plexiglas anoxic chamber (PIas-Labs Inc., Lansing, MI). All
incubations were maintained under low light conditions at room temperature (21-
23°C).

Primary screening for growth of target organisms was done after 30 days
or more of incubation by sacrificing at least one replicate agar plate from selected
treatments containing between 30 and 300 colonies and subjecting it to Plate
Wash PCR (PWPCR) with group-specific primers (below). Remaining plates from

successful treatments were then used as a source of colonies that were picked

147

individually, or removed in groups by swabbing sectors of the plate, for patching
or streaking on homologous medium. For picking isolated colonies, many of
which were invisible to the naked eye, plates were held under a dissecting
microscope and illuminated with cool white light from a fiber-optic illuminator
positioned at about a 45° angle from the horizontal plate surface. When
subcultures were grown, individual colonies or defined pools of them were again
subjected to screening by the PCR with specific primers. This process was
continued until individual colonies of target organisms were ultimately identified

and obtained as pure cultures (Fig. A.1).

PCR and Plate Wash PCR.

The polymerase chain reaction (PCR) was carried out with primers
targeting regions of 16S rRNA-encoding genes common to nearly all bacteria, or
specific to the phyla Acidobacteria and Vermcomicrobia (Table A1). Unless
otherwise stated, each 25 pl reaction mixture contained approximately 50 ng of
template DNA, 1x reaction buffer (lnvitrogen, Carlsbad, CA), 1.5 mM MgCI2, 0.25
mM of each dNTP, 0.2 pM of each fonrvard (F) and reverse (R) primer, and 0.625
units of Taq DNA polymerase (lnvitrogen). Reactions were incubated in a model
PT-100 thermal cycler (MJ Research Inc., Watertown, MA) for the following
amplification schedule: 950°C, 3 min; 30 cycles of [95.0°C, 30 sec; (see Table
A1 for annealing temp), 30 sec; 720°C, 45 sec]; and 720°C for 10 min.

Preliminary experiments, to determine optimum PCR conditions with the

Acidobacteria-targeting (Acd31 F: 1 492 R) and Verrucomicrobia-targeting

148

(Ver53Fz1492R) primer pairs, were done by using template DNA from
Acidobacterium capsulatum (ATCC 51196) and Verrucomicrobium spinosum
(ATCC 43997), respectively. Optimum reaction conditions were determined
across a gradient of annealing temperatures (50 - 65°C) and MgCI2
concentrations (1 - 2.5 mM) by using a PTO-200 DNA Engine gradient
thennocycler (MJ Research, South San Francisco, CA). Sensitivity of target gene
detection was determined by performing the PCR reactions with Acidobacteria-
targeting primers and decreasing amounts of A. capsulatum DNA mixed with
non-target DNA (Eco/i K12) to yield 50 ng total DNA per reaction. Sensitivity was
also determined by using the Verrucomicrobia-targeting primer set with
decreasing amounts of genomic DNA from a termite-associated Verrucomicrobia
division isolate TAV1 (described below) in a 1:2 mass ratio with E. coli K12 DNA.
Direct, group-specific PCR amplification of 16S rDNA genes in environmental
samples was carried out with group-specific primers (see above) and 50 ng of
DNA from soil or from 50 termite guts. Genomic DNA was extracted using the
Ultraclean Soil or Fecal DNA Kits as per manufacturer’s protocols (MoBio
Laboratories, Carlsbad, CA).

Plate Wash PCR (PWPCR) was simply the PCR in which template DNA
was obtained from the aggregate of colonies present on an isolation plate (Fig.
A.1). To do this, the surface of the agar medium was flooded with 2 ml of Bead
Solution from the Ultraclean Fecal DNA Kit (MoBio Laboratories), and then a
sterile spreader was used to suspend as much colony material as possible. The

bead solution with suspended cells was transferred to a dry bead tube from the

149

 

 

    

   
 

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screen with targeted organisms
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Figure A.1. Plate Wash PCR method to detect growth, and monitor isolation of,
targeted bacteria. Of the three media and incubation conditions shown in this
diagram (A, B, C), growth of targeted bacteria is represented only in “C”.

150

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151

DNA Kit, 50 ul of lysozyme solution (50 mglml, Sigma-Aldrich, St. Louis, MO)
was added, and the tube was incubated for 45 minutes in a 56°C water bath.
Alter incubation, DNA extraction was carried out according to the manufacturer’s
protocol, except that a Mini-BeadBeater-8 (BioSpec Products Inc., Bartlesville,
OK) operating at full speed for 45 sec was used for physical disruption of the
cells. The concentration and purity of each DNA sample was estimated by
absorbance at wavelengths from 220 to 320 nm (41).

The ability of PWPCR to detect a colony of a target organism from among
a large excess of non-target organisms was examined by performing PWPCR on
simulated isolation plates. A laboratory collection of 26 different bacterial isolates
obtained from KBS LTER soil [various a-, B-, and v-Proteobacteria; Bacillus spp.
(phylum Firmicutes); Arthrobacter spp. (phylum Actinobacteria), and Cytophaga-
and FIavobacterium-Iike strains (phylum Bacteroidetes)], were each inoculated
onto 3 or 4 sites (94 total) on plates of R2A agar (Difco, Detroit, MI), a medium
commonly used for isolating environmental heterotrophs. Some plates were also
inoculated at one site with V. spinosum (ATCC 43997), and then all plates were
incubated in air, at room temperature, and in the dark for 6 days. At the end of
the incubation period, plates with (positive) and without (negative) V. spinosum
were used for PWPCR individually, and after diluting template DNA extracted
from positive plates with DNA extracted from negative plates.

PCR products were analyzed by electrophoresis of 5 ul samples of
reaction mixtures on 1% agarose gels at 100 volts in 0.5x TBE. PCR products

were visualized by UV illumination after staining with 1x Gelstar nucleic acid stain

152

(Cambrex, East Rutherford, NJ), and images were captured by using a Kodak
Electrophoresis Documentation and Analysis System (EDAS) 290 (Eastman
Kodak).

Sequence determination and phylogenetic analyses.

PCR amplified 168 rRNA genes from environmental samples, PWPCR, or
bacterial isolates were cloned directly into E. coli using the plasmid vector
pCR2.1 or pCR4.0 (T OPO TA cloning kit, lnvitrogen). Restriction fragment
length polymorphism (RFLP) analyses were used to identify common and unique
clones. The partial sequence of each clone was determined with the Applied
Biosystems cycle sequencing technology (Applied Biosystems, Foster City, CA),
the 168 rRNA gene primer 531R (5’-TAC CGC GGC TGC TGG GAC-3’), and/or
vector primers. Preliminary phylogenetic affiliation of each clone was determined
by sequence comparison to the Genbank nucleotide database using BLAST (2),
or to the Ribosomal Database Project II database using the sequence match tool
(18). Nearly full-length sequence (at least 4-fold coverage) of the 168 rRNA
gene from isolates and selected clones was obtained by using primers
complementary to the multiple cloning site of pCR2.1 or pCR4.0 (F2 and R4“,
see Table A1); Acd31F, Ver53F and 1492R (Table AI); and 338F (5’-CTC CTA
CGG GAG GCA GCA GT-3’), 531 R (above), 776F (5’-AGC AAA CAG GAT TAG
ATA CCC TGG-3’), 810R (5’-GGC GTG GAC TI'C CAG GGT ATC T-3’), and
1087F (5’-GGT TAA GTC CCG CAA GGA-3’) with the Applied Biosystems cycle

153

sequencing technology and either an ABI Prism® 3100 Genetic Analyzer or ABI
Prism 3700 DNA Analyzer (Applied Biosystems).

Contiguous sequences for each isolate were assembled with the Vector
NTI software package (lnformax). These were inserted into, and aligned against,
a 16S rRNA gene sequence database in the ARB software package
(http:lewarb-homedel) (32), along with any other available phylum-specific
sequences (>500 nt) from Genbank (http:lIwww.ncbi.nIm.nih.gov/), the
Ribosomal Database Project II (http:l/rdp.cme.msu.edul) (18), or our own
environmental clones. Aligned Acidobacteria and Verrucomicrobia sequences
greater than 1250 nt in length were used to generate phylogenetic trees using
maximum likelihood based on 1097 shared nucleotides for the Acidobacteria and
1050 nucleotides for the Verrucomicrobia. The minimum evolutionary distance

method in PAU P* was used for bootstrap analyses of the same data (46).

Treatment effects on cultivability.

In order to determine which, if any, treatments had a significant impact on
overall cultivability or the cultivability of Acidobacteria from soil, CFU/g soil (dry
wt) and PWPCR results were compared for each treatment and used in a chi
square test for goodness of fit with a Bonferroni error rate adjustment (37, 42).
Colonies used to determine CFU/g soil (dry wt) had a minimum diameter of 0.2
mm and were visible using a colony counter fitted with a 1.5 x magnifying lens.
For overall cultivability, the average CFU/g soil (dry wt) was used as the

expected value and that for a particular treatment was used as the observed

154

value. For Acidobacteria cultivability, the expected value was the probability of
detection using PWPCR among all treatments multiplied by the number of agar
plates used for a given treatment, where as the observed value was the number
of times Acidobacteria were detected for a particular treatment. A total of 63

treatments were screened for this analysis.

Nucleotide sequence accession numbers.

Partial 16S rRNA gene sequences (ca. 1400 bases) from isolates K3889,
TAA43, TAA48, TAA166, TAV1, TAV2, TAV3, and TAV4 have been deposited in
the EMBL, GenBank, and DDBJ nucleotide sequence databases under

accession numbers AY587227 through AY587234.

RESULTS 8. DISCUSSION

Specificity and Sensitivity of PCR and PWPCR

Only targeted 16S rRNA genes were amplified during the PCR with group-
specific primers in control reactions run in the presence of E. coli DNA, or
following PWPCR of a diverse collection of soil bacteria (Fig. A.2). Amplification
with the PCR and as little as 16 pg of A. capsulatum DNA and 93.75 fg of V.
spinosum DNA yielded a visible amplimer. The specificity of each primer set was
also confirmed by sequence analysis of clones obtained after amplification with
the PCR using soil or termite gut community DNA and after PWPCR of simulated
or experimental isolation plates. Of more than 100 such clones examined, all

corresponded to the 16S rRNA gene targeted by the primer pair.

155

By using PWPCR, the equivalent of a single V. spinosum colony could be
detected on plates among a background of at least 940 non-target colonies
composed of 26 different soil bacteria from six major phylogenetic groups (Fig.
A.2). Considering the small amount of the V. spinosum colony material relative to
that of the other bacteria, it should be quite possible to detect colonies of
targeted microbes among a much larger number of non-target colonies of similar
size. RFLP analysis revealed that only Verrucomicrobia-specific rDNA was
amplified despite the diversity of non-specific DNA in each sample (data not

shown).

Treatment Effects and Isolation of Acidobacteria and Verrucomicrobia.
Based on direct microscopic counts, 1.411 0.16 x 109 (n = 3) DTAF-
stainable microbes were present in each gram (dry wt) of soil. In cultivation
experiments, recoveries ranged from 4.0 x 107 to 9.7 x 107 CFU/gram of soil (dry
wt), or roughly 4.0 to 7.0% of the total microbial community based on direct
counts. These recoveries were higher than the “1% or less” recoveries commonly
cited, but similar to those from other studies that have used low nutrient
concentrations and long incubation times (19, 24). No single treatment
significantly increased the overall recovery of soil bacteria relative to any other
(Fig. A.3a), which suggests that the longer incubation times used for all
experiments may be responsible for our higher recoveries of bacteria. When
PWPCR results were compiled from the same experiments, however, one

treatment, used individually or in combination with other treatments, had a

156

significant positive effect on the occurrence of soil Acidobacteria on plates: this
was the presence of 5% CO2 in incubation atmospheres (Fig. A.3b). Incubation
of media in atmospheres with 5% CO2 resulted in a slight acidification (about half
of a pH unit) and, therefore, could also be responsible for the increase in
cultivation of Acidobacteria. Incubation of plates under hypoxia or
supplementation of media with catalase or acyl-HSLs also tended to elicit a
greater occurrence of Acidobacteria, whereas supplementation of media with an
organic nutrient mixture appeared to have the opposite effect. The addition of
humics in the form of soil extract or the humic analogue AQDS had no apparent
effect on the occurrence of Acidobacteria (data not shown). While these latter
treatments were not statistically significant in this study, they may ultimately
prove to be so if examined individually and systematically in a large-scale
experiment.

PWPCR-based identification of primary isolation plates containing
Acidobacteria enabled us to make an informed selection of companion treatment
plates from which to prepare subcultures for additional PCR-based screening
(see Methods). Ultimately, soil Acidobacteria strain K8889 was isolated from soil
that was plated on basal medium supplemented with catalase, acyl-HSLs, and a
mixture of organic carbon substrates (described above), and incubated under an
atmosphere of CO2-enriched air.

A PWPCR-based strategy, similar to that used for the isolation of
Acidobacteria from soil, was used for the isolation of previously uncultivated

microbes from termite guts. The overall recovery of viable prokaryotes from

157

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Figure A.2. Detection of Verrucomicrobium spinosum within a collection of
diverse bacteria isolated from soil. (A) A single V. spinosum colony is shown
among 94 other colonies growing on an agar plate. (B) Plate Wash PCR with
Verrucomicrobia-specific primers using template in which V. spinosum colony
material represented: 1 part in 95 (Le. plate in panel A; lane 1); 1 part in 189
(lane 2); 1 part in 471 (lane 3); 1 part in 941 (lane 4); and 1 part in 9401 (lane
5). Plate Wash PCR of a control plate lacking V. spinosum (lane 6); negative
control (no DNA, lane 7); and V. spinosum DNA (50 ng; lane 8) are also shown
in Panel B. Sizes (kb) of markers in lane M are given to the left.

158

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Figure A.3. Influence of medium additives and incubation conditions on CF U
recovered from soil (panel A) and on the occurrence of Acidobacteria among
the isolates (panel B). The label “air” refers to the concentration of C02

(0.03% vlv) in normal air. Data in panel (A) represent the mean CFU recovered
from soil among n samples. Error bars represent sample standard deviation.
Data in panel (B) represent the frequency with which Acidobacteria were
detected among the plates in panel A using Plate Wash PCR. These data
were subjected to chi-square analyses with a Bonferroni error rate adjustment.
Statistical significance (a = 0.10, df = 1) is indicated by an *.

159

termite guts (9.7% of the direct microscopic count) was marginally higher than
that from soil, with an estimated 4.5 x 105 CFU per gut equivalent. As with
primary isolation plates from soil inocula, PWPCR revealed the presence of
Acidobacteria on some of the plates, and by using analogous procedures
Acidobacteria strains TAA43, TAA48, and TAA166 were subsequently isolated
on plates containing basal medium supplemented with yeast extract and peptone
(0.1% wlv each) and incubated under an atmosphere of CO2-enriched air. By
using Vemrcomicrobia-specific primers, Verrucomicobia were also detected by
PWPCR on primary isolation plates of all compositions inoculated with termite
gut homogenate and incubated under air and hypoxic atmospheres enriched with
5% CO2. Media used for cultivation of termite associated microorganisms
contained yeast extract and peptone, with or without acetate and/or catalase.
From such plates, four termite gut Verrucomicrobia (strains TAV1 through TAV4)
were isolated, assisted by PWPCR surveillance of subcultures. TAV1 and TAV2
were isolated from plates containing basal medium with yeast extract, peptone,
and acetate; TAV3 and TAV4 were isolated from the same medium without
acetate. All TAV isolates were obtained from plates incubated under CO2-

enriched air.

Properties of Acidobacteria and Verrucomicrobia Isolates.

All of the Acidobacteria isolates belong to subdivision 1 of the
Acidobacteria (Fig. AA) (23). Based on 16S rRNA gene sequence similarity, the
nearest cultivated relatives to strains K8889 and TAA166, are Ellin351 (97%)

160

and Ellin337 (98%), respectively (40). TAA166 is the nearest cultivated relative to
TAA43 and TAA48 (96.1%), the latter of which are identical to each other. All
Acidobacteria isolates are short rods (0.5 pm x 1 pm) that divide via binary
fission and form slightly opaque colonies after 4-5 days, which reach a maximum
of 1mm diam in 14-16 days. Soil Acidobacteria isolate K8889 and, to a lesser
extent, the termite gut Acidobacteria isolates produce copious amounts of an
extracellular (apparently capsular) material (Fig. A.4), which made colonies hard
to disrupt and was presumably responsible for their flocculent growth in liquid
cultures.

The termite-associated Verrucomicrobia isolates (T AV1 -4) belong to
subdivision 4 of the phylum Verrucomicrobia (Fig. A5) (23). Based on 16S rRNA
gene sequence similarity, the nearest cultivated relative to TAV1 is Opitutus
terrae strain VeSm 13 (94.2%). Strains TAV2, TAV3, and TAV4 have 16S rRNA
gene sequences virtually identical to each other, and the nearest cultivated
relative to these isolates is Opitutus tenae strain PB90-1 (93%). TAV1 shares
only 92.7% sequence similarity to the other TAV isolates. All of the terrnite-
associated Ven'ucomicrobia isolates are facultative anaerobes, obtaining
significantly higher population densities in liquid culture under CO2-enriched air
and hypoxic atmospheres, than under CO2-enriched anoxic atmospheres. “TAV"
cells are 0.25-0.50 pm in diameter and occur almost exclusively in pairs (Fig.
A.5b). Additionally, TAV1 produces an abundance of extracellular (apparently
capsular) material (Fig. A.5c). The TAV isolates were detected on primary

isolation plates after 30 days and subculture plates after 14 days. On the original

161

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163

isolation media, they formed very small (< 0.5 mm), white, round, mucoid
colonies that were only visible with a dissecting microscope. After isolation and
several passages in the laboratory, however, all TAV isolates formed larger
colonies (2-4 mm) in 2-5 days on RZA in air. Preliminary results from studying the
distribution and abundance of these targeted phylogenetic groups suggest that
Verrucomicrobia are autochthonous to the guts of R. flavipes and not
allochthonous contaminants derived from soil, whereas the opposite is true for
the Acidobacteria (J.T. Wertz, B.S. Stevenson, and J.A. Breznak, Abstr. 103rd

Gen. Mtg, Am. Soc. Microbial, abstr. N-223, 2003).

Overview of the PWPCR-Based Isolation Procedure.

Given the variety of individual cultivation treatments and treatment
combinations used in this study, as well as the various sources of inocula, the
detection and isolation of Acidobacteria and Verrucomicrobia would have been
extremely difficult without the PWPCR method. One of the most time-consuming
aspects of any isolation procedure is the screening, picking and subculture of
colonies from primary isolation plates, and if low nutrient conditions are used to
prevent overgrowth by non-desired organisms, most colonies on such plates will
be fairly small. Indeed, colonies of the Acidobacteria and Verrucomicrobia strains
isolated in this study would have been easily overlooked without the aid of a
dissecting microscope. However, the PWPCR procedure economizes on time by
directing one to treatment plates known to contain the target organism(s). Hence,

owing to its simplicity, utility and relatively low cost, we anticipate that PWPCR

164

will become widely used as an adjunct to creative approaches for isolation of
novel, sought-after organisms. The only requirement is at least one specific and
reliable primer in the pair used for the PCR.

As with any method, PWPCR also has some limitations. For PWPCR,
the sought-after organisms must be capable of growth on plates solidified with
agar (or an agar substitute) and also capable of being harvested from such
plates. This would eliminate organisms that either cannot grow on solid media or
that, like certain spirochetes (7) and spirilla (16), form largely subsurface colonies
difficult to harvest by simple plate washing. However, the key element of PWPCR
is the PCR with a specific primer pair, so as long as sufficient cell material can be
obtained to make a DNA template, either by harvesting cells from liquid cultures
or removing subsurface colony material by coring, surveillance of cultures is
possible. Thus, our results underscore the power of integrating various cultivation
conditions with molecular biology to retrieve some of the “not-yet-cultured

majority” of microbes on our planet (6, 39).

165

ACKNOWLEDGEMENTS
This work was supported by grants from the United States Department of
Agriculture (2001-35107-09939) and the National Science Foundation (MCB-
0135880; and lBN-0114505).
The authors thank Les Dethlefsen for assistance with the statistical

analyses.

166

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