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ISOLATION, CHARACTERIZATION AND FUNCTIONAL
SIGNIFICANCE or BACTERIOPHAGBS IN THE RUNBN
NICROBIAL ECOSYSTEN

BY
TNEODROS TADBSB

A DISSERTATION

submitted to
Michigan State University
in partial fulfilment of the requirement
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Science

1993

ABSTRACT

There are limited reports in the literature concerning
the occurrence of bacteriophages in the rumen. No particular
attempts have been made to study the interactions of ruminal
bacteriophages and predominant cellulolytic rumen bacteria.
Furthermore, there is no information available regarding their
host range or physiochemical characteristics. In this study,
wild type cellulolytic bacteria and a ruminal spirochete,
Treponema saccharophilum, were isolated by anaerobic roll tube
technique. The morphology of these bacteria were determined
by Gram staining followed by microscopic examination and their
fermentation by-products determined by gas chromatography (GC)
and high performance liquid chromatography (HPLC)
respectively. Isolation of ruminal phages were carried out by
the double agar overlay method. To monitor cell lysis and
regrowth of infected cells, optical density readings (600 nm)
were determined with a spectrophotometer. Morphologic
features of the free phages and those attached or within
bacteria were identified by transmission electron microscopy
after negative staining or thin sectioning. Five
morphologically distinct bacteriophages were identified in
ruminal fluid from a cow fed alfalfa hay. A phage possessing
an icosahedral head symmetry (100 x100 nm) and a short rigid
tail (150 x 20 nm); a phage with a cuboidal head (110 nm) and
a short flexible tail (290 nm); a cluster of phage particles
with distinctly hexagonal heads (50 x 50 nm) and no tail; a

phage with an icosahedral head (40 nm) and sheathed tail (120

nm); and filamentous like-phage particles, variable in length
(100 to 400 nm) were observed. A bacteriophage associated
with an identified ruminal spirochete, Treponena
saccharophilum, was also identified. Of particular'importance
was the isolation of a bacteriophage, resembling the lambda
phage for E. coli in its morphologic and physiologic
characteristics. This phage was temperate for a ruminal
cellulolytic anaerobe, Ruminococcus albus, but obligately
lytic to another ruminal anaerobe, Butyrivibrio.tibrisolvens,
strains Dl or 49. The phage appeared to have a wide host
range specificity, and infected other ruminal strains
including: Fibrobacter succinogenes, $85; Bacteroides
amylophilus, H18; Selenomonas ruminantium, H04; Ruminococcus
flavefaciens, FDl; and Ruminococcus albus, 7 and 8. The total
genome of the phage as determined by pulsed field gel
electrophoresis (PFGE) technique was found to be 48.5 Kb. The
temperate nature of the phage was studied by hybridization
techniques. Restriction fragments (smaI) of the phage were
shown to hybridize with restriction fragments of R. albus DNA,
indicating that the phage was indeed harbored in the

cellulolytic anaerobe.

ACKNOWLEDGMENTS

I would first of all like to extend my deepest
gratitude to Dr. Melvin T. Yokoyama for being such a great
advisor, committee chair, and guide, for offering me an
opportunity to do research of my interest. Your advice has always
been pertinent and seasoned with experience. My graduate
committee members, Dr. Steven Rust, Dr. Margaret Benson, Dr.
Werner Bergen, Dr. Robert Cook and Dr. Roger Maes, I thank each
of you for the valuable input and guidance in the program.

My thanks goes to Dr. Susan Hengemuehle, for her continual
help in providing laboratory supplies whenever needed.

I wish to extend sincere thanks to Dr. John Garlach, Dept of
Medical Technology, for allowing me to use their pulsed field gel
electrophoresis technique equipment.

I thank Dr. Paul Coussens, Dept. of Animal Science, for
allowing me to use some of his laboratory equipment and his time
for discussion.

My thanks would not be complete without acknowledging Dr.
Karen Klomparens, Center for Electron Optics, for her assistance
in electron microscopy.

The author is indebted to the Rackham Foundation and the

Department of Animal science for supporting this project.

iv

TABLE OF CONTENTS

List of Tables.......................................viii
List of Figures.................. ..... ...............ix
List of Terminology..................................x
Introduction.........................................1
Literature Review....................................6
1. Ruminal bacteriophage-Host interaction..........11
2. Morphologic Diversity of Rumen Bacteriophages...14
3. Properties of Rumen Bacteriophages..............16
Materials and Methods...............................20
1.0 Bacterial Cultures...........................20
1.1. Isolation of Cellulolytic Bacteria............21
1.1.2. Isolation of Ruminal Spirochete.............22
1.2. Bacterial Cell Count..........................23
1.2.1. Spectrophotometer Analysis..................23
1.3. Fermentation Products Analysis. ............. ..23
1.3.1. Statistical Analysis........................25
2.0 Bacteriophage Isolation......................26
2.1. Bacteriophage Plaque Assays...................26
2.1.2. Double Agar Layer Method....................27

2.2. Bacteriophage Extraction and Propagation......27

TABLE 0? CONTENTS
3.0 Induction of Lysogenic Bacteria..........7§§gg
3.1. Mitomycin C Induction....................29
3.1.2. UV- light Induction....................30
4.0 Thin Sectioning...........................30
4.1. Negative Staining.......................31
4.2. Scanning Electron Microscopy............31
5.0 Genomic DNA Preparation...................32
5.1. Pulsed Field Gel Electrophoresis.........33
5.1.2. Hybridization Techniques...............33
Results....... ...... .............. ...... ..36
Isolation of Ruminal Bacteriophages.......36
Bacteriophage-Spirochete Interaction......43
Phage-Butyrivibrio Species Interaction....46
Phage-Ruminococcus albus Interaction......47
Host Range Specificity of R. albus phage..61
Temperature Sensitivity of R. albus phage.63
PH Sensitivity of R. albus phage ........ ..65
Induction 0f R. albus Culture.............65

Genomic and Hybridization Studies
Of R. albus Phage....OOOOOOOOOOOOOOOOOO9.075

Effect of Phage on Sugar Fermentation.....80

vi

TABLE OF CONTENTS
_£éQ§
Discussion...............................84
Conclusion...............................97
Implication of R. albus Phage............99

ReferenceSOOOO0.0.0.0...OOOOOOOOOOOOOOOOOlos

vii

List of Tables

Table m
1. Host Range specificity of R. albus phage......62
2. Temperature sensitivity of R. albus phage.....64
3. PH sensitivity of R. albus phage..............64

4. Effect of R. albus phage on the fermentation
of xylan by mixed rumen bacterial species.....82

5. Effect of R. albus phage on the fermentation
of GC-RF by mixed rumen bacterial species.....83

viii

List of Figures

£19m: .2191:

1a.

1b.

1c.

1d.

1e.

23.

2b.

3a.

3b.

4a.

4b.

5a.

Transmission electron micrograph of phage
particle with icosahedral head...............39

Transmission electron micrograph of phage
particle with cuboidal head..................39

Transmission electron micrograph of phage
particle with hexagonal head.................41

Transmission electron micrograph of phage
particle with icosahedral head and sheathed

tailOOOOOOO...O0.0COO...OOOOOOOOOOOOOOOOOOOOO41

Transmission electron micrograph of
filamentous like phage particles.............42

Transmission electron micrograph of phage
particle attaching to T. saccharophilum......45

Transmission electron micrograph of phage
particle penetrating outer sheath of
T. saCCharophilumOOOOOOOOOO0.00.00.00.000000045

Transmission electron micrograph of phage
particle within B. fibrisolvens, strain

D10.00.0.000....OOOOOOOOOOOOOOOOOOOOOOOOOO0.049

Transmission electron micrograph of phage
particle within B. fibrisolvens, strain

49.I0.00.00...0.00000000000.0.0.0.0000000000049

Plate showing plaque formation by a ruminal
phage onR. albus.0.0.0000...OOOOOOOOOOOOOOOOSI

Transmission electron micrograph of phage
partial-es Of R. albus00000000.0.0.0000000000051

Growth curve of R. albus after infection
WithR. albus phageOOOOOOOO...OOOOOOOOOOOOOOOSS

ix

List of Figures

11921;: Jim
5b. Growth curve of B. fibrisolvens, strain D1

after infection with R. albus phage..........57
5c. Growth curve of B. fibrisolvens, strain 49

after infection with R. albus phage..........59
5d. Transmission electron micrograph of

filamentous like phage particle found

WithBO fibrisOIVQHSOOOOOOOOOOOOOOOOOO0.0.0.060
6a. Transmission electron micrograph of phage

particle induced from R. a1bus...............67
6b. Transmission electron micrograph of phage

mutants induced from R. albus................67
7a. Effect of UV irradiaton on growth of

R. albus culture......... ...... ..............70
7b. Effect of mitomycin C or UV irradiation on

Growth of B. fibrisolvens,strain Di culture..72
7c. Effect of mitomycin C or Uv irradiation on

Growth of B. fibrisolvens,strain 49 culture..74
8a. Pulsed field gel electrophoresis of

R. albus phage DNA...........................77
8b. Pulsed field gel electrophoresis of DNA's

from R. albus, B. fibrisolvens, strain D1

and 49....O0.0...OOOOOOOOOOOOOOOOOOOOOOOOO0.079
8c. Autoradiograph showing hybridization of

R. albus phage DNA to R. albus DNA fragments.79

Indicator

Induction

Integration

Lysate

Lysogen

Lysogenization

Lysogeny

Pseudolysogeny

Plaque

Temperate

Transduction

Terminology

A bacterial strain sensitive to a
specified kind of phage, used to identify
phage.

Experimental elicitation of phage
development in lysogen.

The insertion of one genetic element into
another to form a single chromosome.

Disrupted bacteria. Phage strains are
usually preserved and used in the form of
lysates of infected cultures.

A bacterium that possesses and transmits
to its progeny the power to produce phage
particles and is therefore prone to lysis.

The process following infection of
bacteria with phage and leading to
establishment of lysogeny.

A stable coexistence of a phage
within DNA of the host bacterium.

Short term coexistence of host and phage,
as opposed to true lysogeny which allows
the viral genome to remain for an

indefinite time inside the infected cell
without killing it or producing virions.

A circular clearing caused by local growth
of phage and lysis of bacteria on the
surface of a nutrient agar plate..

Term applied to a phage able to lysogenize
its host.

Transfer of bacterial markers from one
cell to another by phage particles.

xi

Transfection

Virulency

Superinfection

Prophage

Host Restriction

Multiplicity of
Infection

Generalized
Transducing
Phage

Specialized
Transducing
Phage

Virion

Introduction of cloned viral DNA into
cells treated by a calcium/phosphate
procedure which favors the uptake of viral
DNA by the cell.

A virulent or intemperate phage species is
one unable to lysogenize its host.

Reinfection by phage of a cell that
carries a prophage, or is otherwise
already infected.

Integrated phage DNA within a bacterium
chromosome.

A phenomenon in which a bacterium type x
is able to distinguish a phage that has
been grown in a type x bacterium from one
grown in a different type such as Y and is
able to prevent the phage grown in Y from
carrying out a successful infection.

The ratio in the number of phage to that
of the bacteria in one particular
infection assay.

A phage which can produce particles
containing only bacterial DNA.

A phage which occasionally produce
particles containing both phage and

bacterial DNA sequences.

A complete virus, that is the virus
DNA is completely enclosed within
protein coat.

xii

INTRODUCTION

Ruminal microorganisms play an essential role in the
digestive processes of the ruminant. These
microorganisms consisting primarily of bacteria, protozoa
and fungi occuring in ecological balance, will
efficiently degrade and ferment food material ingested by
the animal. The fermentation end products along with the
microbial protein synthesized in the rumen are used by
ruminants to meet their nutritional requirements for
growth and other energy requiring processes. Ruminal
microorganisms have been intensively studied and the
information from such studies have provided a better
understanding of these microorganisms in the efficient
degradation and fermentation of feed stuffs. Although a
great deal is known about the predominant bacteria and
protozoa, there is a major gap in our knowledge of the
ruminal bacteriophages within the complex ruminal
ecosystem. Bacteriophages are viruses which are capable
of infecting only bacteria. A typical bacteriophage
contains only a few different types of molecules, usually
several hundred protein molecules of one to ten types
(depending on the complexity of the phage) and one
nucleic acid molecule (Freifelder, 1983). There are
large numbers and many different types of bacteriophages

in the rumen (Paynter, 1969; Ritchie, 1970; Klieve,

2

1988). Their occurrence in the rumen indicates that they
may be a constant feature of the microbial population.
Since their discovery, bacteriophages have been regarded
as being important in the ecology of their host bacteria.
For many years, their influence on bacteria was thought
to be solely on the elimination of sensitive bacteria by
the process we now call virulency. However, recent work
has identified other significant functions of
bacteriophages, such as control of toxigenicity in
bacterial species and their capacity to transfer genetic
information from one host to another. A bacteriophage
may influence a bacterial population in the following
ways:

1. By the elimination of sensitive bacteria, a
virulent phage may cause the replacement of such a
bacteria, by a phage resistant mutant differing in
ecological function from the original bacteria.

2. A temperate phage may lyse a certain proportion
of an original bacterial population and lysogenize the
remainder. Apart from conferring protection against the
phage concerned, lysogenization may cause specific
structural changes in the host’s surface membrane as a
result of prophage formation.

3. Transduction (Terminology, xi ) of genetic
properties may occur with or without lysogenization and

may be effected by either virulent or temperate phages.

3

All three of the above properties of a given
bacteriophage may offer some bacterial hosts ecological
advantages in which to operate. This may be particularly
true in the complex ruminal ecosystem, in which
bacteriophage activity may be important in determining
bacterial relationships, and in influencing the numbers
and kinds of bacteria in the rumen.

Although the occurrence of bacteriophages in the
rumen is documented, virtually nothing is known about
their origin, host range and specificity, virulence,
induction and effects on fermentation efficiency. The
lysogenic state of microorganisms is widely distributed
and is the normal condition of microorganisms in a given
stage of their evolution. The majority of bacteriophages
found in the rumen may exist in harmony with their hosts
in a state of lysogeny or pseudolysogeny (Klieve, 1989),
and are probably being induced periodically. The
periodic loss of important fiber degrading bacterial
species from the rumen and the high turnover of the
bacterial population in the rumen of forage fed ruminants
(Hungate, 1966) might also be explained by the activities
of the bacteriophages. Major ’blooms’ of bacteriophages
have been observed in the rumen (Klieve, personal
communications) . Any factor which affects the balance of
the ruminal microbial population and their rate of

metabolism of ingested feed may have an important effect

4
on the nutrition of the host animal. At present the
range of types of bacteriophages isolated from the rumen
is limited and appears unrepresentative to the numerous

bacterial species occurring in the rumen.

The objectives of this thesis research were:

1. To demonstrate the occurrence of
bacteriophages in the rumen ecosystem and
to identify phages which infect specific
ruminal bacterial species.

2. To determine the host specificity and
range, induction, and effects of the
isolated ruminal bacteriophages on their
bacterial hosts.

3. To determine the effects of the
bacteriophage on the interactions between
ruminal bacterial species in mixed and
cocultures.

The knowledge acquired from these studies will
provide a better understanding of host-parasite
relationships in the rumen ecosystem. The knowledge
which would be gained from this research could also be
used to develop new approaches for genetically modifying
rumen bacterial species by using isolated bacteriophages
for genomic libraries and as cloning vectors. 'The use of

bacteriophages in genetic engineering is likely to

5
increase, as they exist everywhere in the bacterial world
and transformation by plasmid DNA is not practical in all
bacteria. In general, the potential of ruminal phages
have not been fully exploited. Phages in the rumen have
never been assayed for transducing ability, which can

possibly be used to modify particular bacterial species.

6
LITERATURE REVIEW

Bacteriophages seem to occur everywhere in the
bacterial world. In order to perpetuate their progeny,
bacteriophages multiply in either of two ways. In
productive growth, a bacteriophage undergoes four main
steps: adsorption, infection, multiplication proper and
release. Once a bacteriophage adsorbs to the host
receptor, it injects its DNA.molecule into the bacterium
and directs the synthesis of numerous gene products,
which promote autonomous replication of the phage DNA,
its packaging into phage particles, and lysis of the cell
to release about one hundred progeny particles, all
within 50 min. This sequence of events is called either
the " lytic", " virulent", " vegetative", or "
productive" cycle. A phage which initiates lytic cycles
only is called " virulent" (xii). A bacteriophage can
also replicate as part of the host bacterial chromosome.
The injected DNA first directs the synthesis of a gene
product called integrase, which promotes insertion of the
phage chromosome into the DNA of the bacterial host, and
then express other genes that act promptly to repress
autonomous DNA replication and most phage function. When
these processes take place, the phage DNA is inserted
into the bacterial chromosome at a characteristic
insertion sites The inserted phage DNA is called

prophage (xii). Its replication contributes to viral

7
growth indirectly, because every cell carrying a prophage
is a potential source for production of phage particles.
The phage that undergoes a stable symbiosis with the
bacterium is known as temperate. The bacterium that
harbors the prophage is called lysogenic, because it has
acquired the potential to produce phages and thus to
lyse. Moreover, it has become immune against
superinfection by homologous phages and sometimes has new
metabolic or antigenic properties that persist as long as
the prophage is present. It may even reproduce more
rapidly than its non-lysogenic parent. This phage-host
equilibrium may break down spontaneously or upon
induction by some stimuli, usually ultraviolet light or
mitomycin C. The lysogenic state is beneficial to the
phage and its host: it ensures perpetuation of the phage
and confers at least one new property to the bacterium,
anti-phage immunity, which has an evident survival value.
Lysogeny occurs in many, if not all branches of
Eubacteria. The frequency with which an infecting
bacteriophage initiates lysogenization rather than
productive phage growth depends on the state of the
bacteria, the temperature, and the genotypes of the phage
and the host. Many bacteriophages carry mutant phages
which cannot replicate, but may be visualized in lysate
of induced cells. They are variable in morphology,

including complete phages with full or empty heads,

8

headless tails or tailless heads, and polyheads and
polysheaths. Some phages are also known to undergo
pseudolysogeny or carrier state. This refers to the
simultaneous presence of phages and bacterial cells in
the same culture. Phage DNA is not integrated and only
some of the cells of the bacterial culture are infected.
Any' phage, virulent. or 'temperate, is theoretically,
capable of generalized transduction (xii). This means
that in the course of phage multiplication a 'headful’ of
bacterial DNA is randomly packaged into the capsid of a
phage. The transducing particle, which has none of the
normal phage genes, is nonviable. By contrast, the
transducing phage in specialized transduction (xii), is
always temperate, usually requires induction, and
contains both phage and host DNA.

The bacterial viruses exhibit a greater diversity in
form than any other group. There are six basic
morphological types of bacteriophages. The most complex
of these, Group A, has a head with a hexagonal outline
which. may' or' may' not be elongated. A ‘tail ‘with a
contractile sheath is attached to the head. The second
group in order of structural complexity, Group B, also
has a six sided head and a tail. However, the tail is
relatively flexible; it may or may not have terminal
appendages. The tail is longer than the head diameter

and has no contractile apparatus. The third type, Group

9
C, also has a tail and a six sided head, but the tail is
shorter than the head.diameter or maximal dimension, and
may also have appendages attached to it, but, again it is
non-contractile. The fourth type, Group D, has no tail,
but is still six sided in outline though of symmetrical
appearance. Each apex of the hexagon.has a knob or large
capsomere on it. In the fifth group, Group E, the large
capsomeres are absent and the virion shows a simple
regular hexagonal outline. The sixth group, Group F, is
quite unlike the others. The virion has the form of a
long flexible filament with no additional structures of
any kind attached to it. Bacteriophages have been
isolated from a large proportion of bacterial genera.
The present overall picture of the distribution of the
six basic morphological forms may be a distorted one.
One or two fundamental facts are, however, apparent.
First, a basic morphological type of phage is not
restricted.toiany one bacterial genus or species; that is
to say, contractile phages have been isolated for both G
(-) and G (+) bacteria of widely separated genera.
Furthermore, a single phage is highly specific, its
activity being usually restricted to closely related

species of a single genus (Bradley, 1967).

10

Q 90
W t

L ll

A ’ /
1

Basic morphological types of bac teriOphages.

 

 

 

 

 

 

 

 

11

1. Ruminal Bacteriophage- Host Interaction

High numbers of morphologically different types of
bacteriophages have been detected in the rumen of sheep
(Hoogenraad, 1967) and cattle (Paynter et al., 1969;
Ritchie et al., 1970). Paynter et al. (1969) examined
the ruminal contents for a period of one year from a cow
fed alfalfa hay and detected bacteriophages, both
attached and unattached to bacteria, at concentrations of
about 5x107 per ml in all samples. Similarly, Ritchie et
al. (1970) characterized bacteriophages in the rumen of
cattle and sheep, and found more than 125 morphologically
distinct types, with 65 different bacterial species
showing intracellular phage infections. In a recent
study by Klieve and Bauchop (1988), 26 distinct types of
bacteriophages were identified and large numbers of
phages, 2 x 107 to 1 x108 were present in ruminal fluid
from sheep and cattle. Although, it is obvious from the
literature that indigenous rumen bacterial species are
infected with bacteriophages, the detection of
bacteriophages associated with specifically identified
rumen bacterial species has been very limited.
Hoogenraad et al.(1967) showed that ruminal spirochetes
were heavily infected with bacteriophages. Orpin and
Munn (1974) reported that a bacteriophage was associated
with Eadie’s Oval and also with an unidentified,

obligately anaerobic Gm (-) rod which effected the

12
biohydrogenation of unsaturated fatty acids.
Bacteriophages have also been shown to infect transient
serratia species (Adams et al., 1966), and Fusobacterium
necrophorum (Tamada et al., 1985). Bacteriophages have
also been detected for temporal species such as
Bifidobacterium ruminale (Matteuzzi and Sozzi, 1971),
which is found in the rumen of young preruminant calves
on a milk diet. The only rumen bacteria which has been
studied in detail with regard to bacteriophage is
Streptococcus bovis, which is found in high numbers when
a high starch diet is fed (Iverson and Millis, 1976a;
Tarakanov, 1974). Lysogeny of the ruminal Streptococcus
species has been well established (Iverson and Millis,
1976a, Tarakanov, 1974). A majority of streptococcal
strains harbor temperate phages that can be readily
induced by ultraviolet light and mitomycin C. In some
cases, induction can also occur spontaneously. For most
of these studies, lysis of the culture was observed
following induction, and temperate phages were identified
by lysis of the appropriate indicator strain or, more
often, by detection of phage particles using electron
microscopy. In a series of studies, Iverson and Millis
(1976a, 1976b, 1977) isolated. and. «characterized
bacteriophages which infect S. Davis, and reported that
only one strain of S. bovis was lysogenic. Of the 23

strains of S. bovis tested, only 4 percent were inducible

13
by mitomycin C. In contrast, Tarakanov (1974, 1976)
reported that lysogeny was common in S. bovis strains,
which were isolated from the rumen of sheep and cattle.
Fifty five percent (10/18) of S.bovis strains from sheep
were lysogenic; 39 percent were induced by UV - light
irradiation, while 27 percent (5/18) were spontaneously
induced. Of the 48 S. bovis cultures obtained from cows,
50 percent were lysogenic, and about 84 percent were
spontaneously induced and, only 13 percent were induced
using chloroform. The differences in the lysogenic
characteristics of isolated S.bovis strains in both
studies have been attributed to variation in
geographical area and variability of individual animals
(Iverson, 1976a). However, as lysogeny is normally a
stable characteristic in microorganisms, the difference
in lysogenic cultures due to geographic location seems
remote. In spite of the differences, lysogeny is
believed to be the source of bacteriophages in the rumen
of cattle and sheep (Tarakanov, 1974). The attention
focused on the lysogenic nature of S. bovis is based on
the possibility that lytic phages occurring in ruminal
fluid originate from phages harbored by the lysogen.
Recently, similar observations have been made by
Lockington et al. (1988), who reported on the isolation
and characterization of a ruminal temperate bacteriophage

from Selenomonas ruminantium, an important propionic acid

14

producing ruminal bacteria. Klieve et al. (1989)
reported the induction of a temperate bacteriophage in a
wide range of ruminal bacteria by mitomycin C. Of 38
ruminal bacterial species investigated, 9 (23.7%),
namely, Streptococcus intermedius AR36, Streptococcus
bovis AR3., Eubacteria ruminantium ARZ, AR35.,
Bacteroides ruminicola subsp.brevis, AR29, AR30, AR32.,
Butyrivibrio fibrisolvens AR14. , and Ruminococcus
flavefaciens, produced phage- like particles. All these
accumulated evidence clearly demonstrate the ubiquity of
temperate phages which are carried as lysogens by ruminal
microorganisms.

Apart from these studies, no research has yet been
conducted to determine whether bacteriophages infect
other predominant rumen bacterial species, especially

those species responsible for plant polymer degradation.

2. Morphologic Diversity of Rumen Bacteriophages

Rumen bacteriophages interacting with ruminal
bacterial species have been subjected to extensive
morphologic characterizations. Structural identification
and measurement of phage heads, tails, collars, and base
plates by electron ‘microscopy has demonstrated the
presence of a diverse number of morphologic types in the
rumen. Paynter et al. (1969) examined bacteriophages

isolated from cattle ruminal fluid. The dimensions of

15
the phages found in the cow were different from any of
those found in sheep (Hoogenraad et al., 1967) or from
bovine phages active against S.durans (Brailsford, 1968) .
Nevertheless, each phage contained a tail and a
polyhedral shaped head. Base plate-like structures were
evident only on two of the six bovin phages seen. The
dimensions in all the phages varied and ranged from 103
by 103 to 137 by 128 nm for the head and from 666 by 34
to 134 by 29 nm for the tail. Similarly, Ritchie et
al.(1970) examined the morphologically different phage-
like entities in sheep and cattle which were fed normal
diets. The most prevalent types of phages had
contractile tails and cuboidal heads. Their overall
length ranged from less than 70 nm to greater than 80 nm,
with head diameters varying from less than 30 nm to
greater than 15 nm. The next numerous types were those
with noncontractile tails with no apparent tail sheath.
Matteuzzi and Sozzi (1971) identified bacteriophages
specific for.Bifidobacterium thermophilum, from rumen of
a cow fed high carbohydrate diet. These phages had
octahedral heads and long curved tails with base plates.
The morphology of S. bovis phages isolated from bovine
rumen, lysogenic cultures of S. Davis, and abbatoir
ruminal wastes was examined and compared by Iverson and
Millis (1976b). Phages isolated from the rumen had

hexagonal heads with a diameter of 65 nm, and

16
noncontractile tails of 175 nm in length, terminating
into a knob with short spikes. However , phages from
lysogenic cultures of S. bovis, and 50 percent of the
phages isolated from abbatoir wastes had hexagonal heads
measuring 50 nm. in. diameter, and. tails with short
noncontractile spikes which were 32 nm long. Klieve et
al. (1988) identified 26 morphologically distinct types
of bacteriophages, from ruminal fluids of cattle and
sheep, collected over one year. On the basis of the
scheme of Bradley (1967), they were classified as
follows: Group A has long contractile tails; Group B has
long, noncontractile tails; and Group C has short,
noncontractile tails. The remaining phages were newly
classified (Klieve et al., 1989) as: Group D, which are
tailless, icosahedral and have a large capsomere; and

Group F, which are filamentous phages.

3. Properties of Rumen Bacteriophages

Brailsford et al. (1968) elucidated the properties
of a transient ruminal species, S. durans phage isolate,
from bovine rumen, fecal samples and lysogenic strains of
S. durans. All phages appeared to be similar in their
lytic activity, and produced plaques or a zone of lysis
whose sizes ranged between 1 and 5 mm in diameter. All
phages were sensitive to temperatures between 10 C and 70

C. They were completely inactivated at 70 C for 1 hr.

17
The viability of the phage was not affected by being in
medium between a pH of 5 and 9 for 24 h. In a detailled
study by Tarakanov (1976) to characterize S. bovis phages
isolated from sheep rumen and lysogenic cultures of S
bovis isolated from cows, all phages produced round,
transparent, smooth plaques with a fairly distinct edge
and diameters of 0.5 to 2 mm. Sixty eight percent of the
phages were inactivated after three hours of incubation
in ruminal contents. Their degree of inactivation
increased with the increase:in contact.time, that is 85.5
to 97 percent inactivation took place in 30 hour
incubation period. However, the phages reacted
differently to treatment with different concentrations of
acetic acid. Increasing the concentration of acetic acid
to 0.1065 mmole per ml, which is the upper possible limit
of this acid in the ruminal fluid, led to greater but
still incomplete inactivation of these phages. Because
of this phenomenon, acetic acid was assumed to be one of
the links in the chemical regulation of the number of S.
bovis phages in the rumen. The effect of urea on these
phages was also studied, since urea has a denaturing
effect on the protein structure of phage particles and
urea is consumed by ruminants. Ninety percent of the
phages were inactivated within 15 min of exposure to urea
(8M) in culture. The effect of trisodium citrate (0.01%)

on the phages of S. Davis also resulted in a high degree

18
of phage growth inhibition. Inhibition in the presence
of citrate was believed to be a factor limiting the
normal development of the infectious processes. The
isolated phages for S. bovis were also shown to have
different sensitivity to phenol and chloroform.

The functional role of phages in the rumen ecosystem
is yet to be determined, but because phages are present
in all ruminal fluid samples examined and there are
several morphologically different types in rumen fluid at
one time suggests that phage activity is important in the
dynamics of the microbial population, and could be
responsible for preventing wide shifts in the types of
bacteria in the rumen, by keeping particular species in
check when their numbers exceed the ecological balance.
The only in vivo studies by Orpin and Munn (1974) to
determine the role of ruminal bacteriophages showed that
Eadie’s oval, strain E02, normally present at 106-107per
ml in the rumen of sheep, decreased to zero in 10 days
when the rumen was inoculated with an isolated
bacteriophage which infected the E02 strain. Control
inoculation of the uninfected strain in other sheep
resulted in no decrease in the numbers of this ruminal
bacterium under similar conditions. In contrast, Iverson
and Millis (1977) reported that the rapid succession of
S.bovis strains with differing sensitivities to

bacteriophages prevented their attempts to eliminate

19
S.bovis from the rumen with their isolated virulent
bacteriophages. Clearly, there is a need to better
understand the relationship of bacterial strain
sensitivity to bacteriophage infection and the temperate
and obligatory lytic nature of the bacteriophage in the

r umen .

20
MATERIALS AND METHODS
1. Bacterial Cultures

All bacterial species used in this study have been
shown to be involved.in.either the ruminal degradation of
plant polymers, in nutritional cross-feeding,
interspecies hydrogen transfer, growth factor
requirements or biohydrogenation of polyunsaturated fatty
acids. .Although, most of the bacteria used in this study
were laboratory strains which had been kept in culture
collections for many years, it was also important to use
new isolates of the major cellulose degrading bacterial
species to avoid any possible mutational and physiologic
changes after years of cultivation. Once selectively
isolated and presumptively identified, these wild
bacterial strains were used to screen for virulent and
temperate bacteriophages in pure cultures. The ruminal
bacterial species used in the research were: R. albus,
strain 7, strain 8, wild type; R. flavefaciens, strain
FD1; B. fibrisolvens, strain D1, strain 49, strain A38;
Fibrobacter succinogenes, strain S85; B. amylophilus,
strain H18; T. saccharophilum, wild type; T. bryantii,
strain B25. Butyrivibrio fibrisolvens strains D1 and A38
were from the culture collection of M.P. Bryant, Univ. of
Illinois, Urbana, 11 and strain 49 was obtained from R.B.
Hespell, USDA, Peoria, II. All working cultures were

maintained on 30% rumen fluid containing glucose-

21
cellobiose- starch (GCS-RF) medium (Bryant, 1962) agar
slants held at 4C. In cultivation of bacterial strains,
a loopful of the fluid of syneresis from agar slant
cultures is inoculated into GCF-RF liquid medium, under
anaerobic condition, and incubated at 39C for 24 h..About
0.1 ml of the bacterial culture:is then subcultured every
24 h. into fresh liquid medium (4.0 ml). The composition
of the medium consists of (9/1): glucose, 0.6;
cellobiose, 0.6; starch, 0.6; yeast extract, 2.0;
trypticase, 5.0; resazurin, 1.0; Mineral 1, 75 ml/l;
mineral.2, 75 ml/l; cysteine-HCl, 20 ml/l; NaCQ,(8%), 50
ml/l. Mineral I consisted of (g/l): CaC12.2H20, 1.6;
10590,, 6.0; (mm2 $0,, 6.0; Mgso,.7H,0, 2.5; NaCl, 12.0.

Mineral II consisted of (g/l): KZHP04, 6.0.

1.1. Isolation 0f Cellulolytic Bacteria

Ruminal cellulolytic bacterial species used in the
study were isolated from the rumen using the Hungate
anaerobic technique (1950, 1970) as modified by Bryant
(1972). Ruminal fluid was obtained from a rumen
fistulated cow maintained on an alfalfa hay diet.
Ruminal fluid was strained through four layers of
cheesecloth, and then centrifuged at 1075 x g at 4 C
twice to remove large debris. The ruminal fluid
supernatant was serially diluted (10'l - 1040]) into an

anaerobic dilution medium containing (m1 / 300 ml) mineral

22
I, 22.5; mineral II, 22.5; resazurin, 0.3, Nafiflh, 15.0;
and cysteine-HCl, 6.0. The highest four dilutions (106-
10‘0) were inoculated into anaerobic roll tubes containing
ground cellulose (2mg/ml; Whatman filter paper, Balston
Ltd.) and agar medium. After 24 h. of incubation at 39
C, well isolated bacterial colonies were picked from the
highest dilutions and transferred into liquid medium with
cellulose (0.7 mg/ml) as substrate and then subcultured
back into liquid medium containing cellobiose (0.7mg/ml)
Bacterial species were characterized between transfers on
the basis of their morphology by Gram stain. Once pure
isolates were obtained, cells were identified on the
basis of cellular morphology, culture characteristics,
and fermentation products. Colonies of Ruminococcus albus
were white. The cells were spherical, with adjoining
sides of diplococci flattened. Cells occurred
predominantly as pairs, with a few numbers occurring
singly and only occasionally chains of up to four cells.
The cells were Gm (-) . Volatile fatty acids were
determined by the method of Playne (1985). All bacterial
isolates were maintained in 30% GCS- RF agar (2 %) medium

and maintained at 4 C.

1.1.2. Isolation 0f Ruminal Spirochete
The ruminal spirochete, Treponema saccharophilum,

was isolated as described for cellulolytic bacterial

23
isolates, except the medium used contained pectin
(0.7mg/ml) and rifampin (1 mg/ml) as selective media
(Paster, 1985). On the bases of size, morphological
features, and substrate degrading ability, the ruminal
spirochete was identified as T. saccharophilum as
previously isolated and characterized by Faster and

Canale-Parola (1985).

1.2. Bacterial Cell Count

The method of choice for measuring the cell number
of the microorganisms was both an optical one in a
spectrophotometer (Bausch and Lomb, spectronic 70
colorimeter) and bacterial colony count in a roll tube

containing agar media.

1.2.1. Spectrophotometer Analysis
Optical density (CD) of all bacterial cultures and
bacterial cultures infected with bacteriophages were
determined by spectrophotometry readings at 600 nm. In
all experiments of bacterial cultures, an optical density
of culture was measured. An 0D value of 0.3 to 0.5

represented approximately 107-1Ifi cells per ml.

1.3. Fermentation Product Analysis
Bacterial fermentation by-products were determined

by gas-liquid chromatography using a Hewlett-Packard 5890

24
A gas chromatograph (Mt View. CA.) equipped with a 3 m
(SS) column packed with GP 15% SP-1220. Nitrogen was the
carrier gas. Temperature conditions were: oven, 125 C.,
injector, 180 C., FID, 180 C. Ruminococcus albus was
identified by its fermentation products profile as
determined by gas-liquid chromatography (Salanitro,
1975). A pure culture of R. albus was inoculated into
only cellulose (0.7 mg/ml) or cellobiose (0.7 mg/ml)
containing liquid media under anaerobic condition.
Following incubation at 39 C, aliquot samples were taken
at 24, 36 and 48 h. period and were centrifuged at 1075
x g twice for 20 min. The supernatant was filtered
through a Millipore membrane filter ( 0.2 um pore size).
One ml of the filtrate was placed in a vial with the
addition of 75 ul of 1M sulfuric acid. All samples were
kept at 4 C until analyzed by gas liquid chromatography.
Fermentation by-products obtained from Ruminococcus albus
treated cultures were analyzed by high performance liquid
chromatography (HPLC). Each ruminal bacterial species
was initially grown in 30% GCS-RF media for 24 h at 39 C.
After centrifugation at 4 C for 20 min, pellets were
pooled into fresh GCS-RF media and were incubated for 24
h at 39 C. The mixed culture (consisting of B.
fibrisolvens, strain D1, and strain 49; R. albus, strain
7 and strain 8 and wild type; R. flavefaciens, strain

FD1; Fibrobacter succinogenes, strain S85) was

25
centrifuged at 4 C for 20 min. The pellet was suspended
in dilution media and transfer immediately into media
containing xylan (0.5%) or glucose—cellobiose (G0) (0.5%)
as substrate. Since some ruminal bacteria do not grow
well without rumen fluid, branched chain volatile fatty
acids (isobutyric,1 mM; N-valeric, 0.9 mM; isovaleric,
0.9mM; 2- methylbutyric, 0.9 mM; and phenylacetic, 0.9
mM) were added. The cultures were allowed to grow for 3
h at 39 C and were inoculated with the R. albus phage at
a multiplicity of infection (M.0.I.) of 1:100. The
infected cultures were incubated at 39 C and aliquots in
3 ml quantities were taken at 16 h and 36 h. Samples
were centrifuged at 4 C for 20 min and the supernatant
were filtered through a Millipore filter membrane (20 um-
pore-size). The filtrates were transferred into small
vials with the addition of 75 ul of 1 M sulfuric acid.

The samples were kept at 4 C until analyzed.

1.3.1 Statistical analysis

The molar concentration of succinate, lactate,
formats, acetate, propionate and butyrate was determined
from 10 ul of each sample. Analysis of mean was
performed using the Bonferonni t test procedure of SAS

(1987).

26

2. Bacteriophage Isolation

Ruminal fluid (IL) was obtained from a rumen
fistulated cow maintained on an alfalfa hay diet,
strained through four layers of cheesecloth and
centrifuged twice at 1075 x g for 20 min. The supernatant
was passed through a sterile Millipore membrane filter
(0.45 um pore size), and concentrated as described by
Tamada et a1. (1985). Ten ml of filtrate was added to
1.2 ml. of 5M NaCl solution, containing 0.6 gm of
polyethylene glycol (PEG 6000). The mixture was held at
4 C overnight, then centrifuged at 1075 x g for 20 min.
The pellet containing the concentrated bacteriophage was
resuspended in GCS — RF medium and used for infection of

ruminal bacteria.

2.1. Bacteriophage Plaque Assays

Virulence of the bacteriophage was determined by
plaque diameter size and rate of plaque formation
(Cluzel, 1987), using the double agar overlay technique
of Gratia (1936) as described by Adams (1959). Motile
bacteria, which do not produce confluent lawns, were
examined in liquid medium inoculated with known
concentrations of bacteriophages, with optical density
readings (600 nm) of inoculated cultures measured against

control cultures.

27

2.1.2. Double Agar Layer Method

All bacterial species except for T. saccharophilum
were tested for bacteriophage infection by the technique
of Gratia (1936) as described by Adams (1959). About 0.1
ml of the concentrated bacteriophage suspension was
inoculated into exponentially growing cultures (0.4 ml)
of ruminal bacterial species and incubated at 39 C for 20
min to allow for phage adsorption. The inoculated
culture was added into a tube containing soft agar (0.7%)
media kept at 47 C, gently vortexed and poured directly
onto a sterile petri dish containing a bottom agar layer
(2%) media. Plates were inverted and placed in a
desiccator and the desiccator was flushed with carbon
dioxide (100 %) for 20 to 30 min. The desiccator was then
sealed and incubated at 39 C for 24 h. Plates were
examined for plaque formation after 24 and 48 h. When
plaques were observed, their diameter was measured. The

plates were also photographed immediately.

2.2. Bacteriophage Extraction and Propagation

Plates that showed plaque formation were carefully
scraped off the top agar and placed into tubes containing
GCS - RF medium. The tubes were held for 24 h to allow
diffusion of phage from the agar. After centrifugation
at 4302 x:g for 20 min, the supernatant was recovered and

stored at 4 C for phage identification by electron

28
microscopy. Phage was usually grown in conditions that
was optimal for its bacterial host. Propagation of the
phage on solid medium was the most frequently used
technique because it produced higher titers than
propagation in liquid cultures. In liquid medium, the
medium is inoculated with a known bacteria to phage
ratio, and the mixture incubated until lysis occurred,
which is not always visible. The ratio of bacteria to
phage, or multiplicity of infection (M.0.I.),was 1:100.
The bacteria inoculum was adjusted by optical density.
Propagation on solid medium is as described in the

section on the double agar method.

3. Induction of Lysogenic Bacteria

In screening for temperate bacteriophage, early
logarithmic culture of the ruminal bacterial species was
induced using' mitomycin C (Cluzel, 1987) or by' UV
irradiation (Eisensark, 1967). Induction by UV-light or
mitomycin C apparently involves inactivation of
repressor. Ultraviolet light induction has been
explained by assuming that some product ('inducing
substance') either a DNA precursor or degradation product
formed as a result of irradiation, complexes with and
inactivates the phage repressor (Ben-Gurion, 1967;
Hertman, 1967), as a result of which the prophage is

excised from the host chromosome, followed by autonomous

29
DNA replication, phage production, and lysis. The
inactivation may be indirect since it depends on a
bacterial gene (Rec A) as well as on the structure of

the repressor.

3.1. Mitomycin C Induction

Mid-log phase cultures of bacteria (1 ml) were
inoculated into a 30 % GCRF medium containing, mitomycin
C to give a final concentration of 5 and 10 ug per ml.
Mitomycin C is an anti-tumor antibiotic complex(C”HmNJ%)
produced by a soil microorganism, Streptomyces
caespitosus (Siuta, 1974). The mitomycin C treated
cultures and the control cultures were incubated at 39 C
for 15 to 30 min. The cultures were pelleted by
centrifugation at 1075 x g for 20 min. The pellet was
added into fresh GCSRF medium without mitomycin C, and
further incubated in a shaking water bath (39C) for 6 h.
All cultures were covered with aluminum foil as
protection from exposure to light. The cultures were
centrifuged at 1075 x g for 20 min and the supernatant
was examined for presence of phage against an indicator
strain by the double agar method, and by electron
microscopy. Also, the CD of mitomycin C induced cultures
were followed for their lytic activity at intervals of 20

min for about 24 h.

30

3.1.2. UV LIGHT INDUCTION

Mid- log phase growing cultures of ruminal bacterial
species (1 ml) poured onto sterile petri dish, or
confluent lawn of bacteria, were exposed to UV light for
15, 45, 60, 120 sec from a distance of 25 cm from a
bactericidal UV lamp in a hood. The cultures were
directly transferred into fresh GCSRF medium (4 ml) under
anaerobic conditions. The cultures were covered with
aluminum foil and incubated in a shaking water bath (39
C) for 6 h. The cultures were centrifuged at 1075 x g,
for 20 min and the supernatant lysate were diluted 10
fold into GCS-RF broth and was tested for presence of
bacteriophages against an indicator strain by the double
agar layer method and by electron microscopy. Lysis of
culture as may occur by UV irradiation was also followed

by 0D reading at 600 nm for 24 h.

4. Thin Sectioning

About 0.1 ml of concentrated phage filtrate
suspension was inoculated into exponentially growing
bacterial cultures and incubate at 39 C for 20 min. The
cultures were centrifuged at 1075 x g for 20 min.
Pellets were placed into warm agar (2%), allowed to
solidify and fixed in coLd 4 % glutaraldehyde in 0.1M
sodium phosphate buffer (pH 7.2) for 1 h. Samples were

washed three times in buffer, then post fixed in 1%

31
osmium tetraoxide in buffer for 1.5 h. at room
temperature, and dehydrated by a graded series of
ethanol. Embedding was done with a mixture of Epon -
Araldite - spurs (Klomparens, 1986). Silver gold (70 -
80 nm) ultrathin sections were cut, stained with uranyl
acetate, followed by lead citrate. Sections were
examined with a JEOL 100 CX11 transmission electron

microscopy at the M.S.U. Electron Optics Center.

4.1. Negative sectioning

Formvar and carbon coated electron microscope grids
were wetted with Sul of the phage sample by the drop
technique (Horne, 1965). The samples were negatively
stained with 1% potassium phosphotungstic acid (pH 6.5),
and grids were examined for phage particles by using a

JEOL 100 CX11 transmission electron microscopy.

4.2. Scanning Electron Microscopy

A suspension of bacterial culture (1 ml) was mixed
with 5% glutaraldehyde (1 ml) in 0.1M phosphate buffer
(PH 7.2). Culture was placed in ice bucket for 30 min. A
drop of 1% poly-L-lysine was dropped on a plastic petri
dish and a glass coverslip layered on the drop. After
standing for 5 min., the coverslip was removed and.rinsed
with drops of water. A drop of cells was placed on the

coverslip to which poly-L-lysine had been exposed and the

32
suspension allowed to settle for 5 min. The coverslip
was washed and placed in a coverslip holder and
transfered through a series of ethanol washes, 25, 50,
75, 90 and 100 % with 5 min in each step. The sample
was then critically dried, mounted to a stub and finally
sputter coat the mounted coverslip. The specimen was
examined by scanning electron microscopy at the M.S.U.

Electron Optics Center.

5. Genomic DNA Preparation

Agarose blocks containing bacterial cells or
supernatant phage lysate were prepared as described by
McClelland et al. (1987) for separation of DNA by pulsed
field gel electrophoresis (PFGE). R. albus culture
infected with the phage and uninfected culture of R.
albus were washed, twice, in L-buffer composed of, EDTA,
0.1 M; Tris-Cl, 0.01 M, NaCL, 0.85 M. The cultures were
centrifuged at 1075 x g for 20 min at 4 C. A pellet (0.5
ml) was added into equal volume of 1% low melting
temperature agarose (Sea-plaque) in Lrbuffer . These
were dispensed into 8 x 3 x 2 mm wells in a mold plate
and were allowed to solidify. The resulting blocks or
’inserts’ were treated insitu with 1mg/ml of lysozyme
(Sigma Co.) and 0.05% sarkosyl at 37 C for 24 h. Inserts
were transferred in L-buffer with 1 mg/ml proteinase K

and 1% SDS, and were incubated at 50 C for 48 h. The

33
inserts were again transferred into Tris- EDTA (TE, pH,

7.5) and stored at 4 C.

5.1. Pulsed Field Gel Electrophoresis

100 ml of 1% agarose (Bio-Rad's Molecular Biology
Certified. Grade) in 0.5 X 'Tris-Borate buffer (TBE)
comprising, Tris-Base, 0.1 M; Boric acid, and EDTA, 0.1
M., was melted and poured on a casting stand. The
agarose was allowed to cool at room temperature for 30
min. The comb was removed. Sample plugs or 'inserts'
were cut into smaller pieces and were added to the wells
using a spatula and pressed to the bottom of the wells.
Each well was sealed with Low Temperature Melt
Preparative Grade Agarose. The gel was placed on a
clamped homogenous electric field (CHEF -DR 11, Bio-Rad
Laboratories) chamber containing 2 liters of 0.5 X TBE
buffer. Electrophoresis was run at 180 volts at 14 C
with alternating pulse 40-80 sec. for 24 h. The gel was
placed into ethidium bromide solution (0.5 ug/ml) and let
stand for 30 min with constant shaking. After destaining
for 1-3 h, the DNA band was photographed by placing the

gel on a ultraviolet transilluminator (254-360 nm).

5.1.2. Hybridization Technique
DNA bands in gel were transferred onto nylon

membrane (BIO-RAD) by the capillary transfer method

34
(Maniatis, 1989) as follows: The gel was placed in a
dish and soaked in several volumes of 1.5 M NaCl and 0.5
N NaOH for 45 min.to denature the DNA" .After the gel was
rinsed in water, it was neutralized by soaking it in a
solution of 1 M Tris (pH 7.4), 1.5 M NaCl at room
temperature with constant agitation. The gel was removed
and placed on a wetted nylon membrane (Bio-Rad) that was
already mounted on a stack of glass plates to form a
support in a dish containing transfer buffer (10 x $80).
A stack of paper towels was put on top of the gel onto
which a glass plate was placed and press it down. The
transfer of DNA from the gel to the nylon membrane was
allowed to proceed for 24 h. The phage DNA was eluted
and purified according to Maniatis (1989). Total or
restriction fragments of the phage DNA was labelled by
random priming using 32p-dCTP (Boehringer Mannheim,
Indianapolis, IN) as follows: Fifty nanogram of phage
DNA in 2 ul was added to 7 ul of water in an Epindorff
tube and boiled for 5 min. After it had been cooled
immediately on ice, 3 ul AGT mixture (dATP, dGTP, dTTP);
2 ul reaction mixture ; 1 ul of klenow enzyme; and 5 ul
of 32p-dCTP (10 uci/ul or 3000 uci/mmole) were added.
The mixture was incubated at 37 C for 30 min. The
reaction was stopped with 5 ul of stop solution and the
probe was boiled for 5 to 10 min. G-50 medium was placed

in a column after which 100 ul of salmon sperm DNA (SSD)

35
was added for binding non specific DNA and the labelled
probe was added. Elution was proceeded with 200 ul
fraction with periodic addition of TE buffer. After
determining the fraction of the labelled DNA fragments
that has the highest count per minute, hybridization was
carried out as follows: Hybridization solution
containing, formamide, 50%; sodium dodecyl sulfate (SDS) ,
1%; NaCl, 1 M, dextran sulfate, 10%, salmon sperm DNA,
100 ug/ml; SSC salt solution, was placed into a tube and
kept at 42 C water bath for 10 to 15 min. NaCl (0.58 g)
was added to the solution and mixed, and the mixture was
further incubated for 15 min. The tube was removed and
the solution was poured into a bag containing nylon
membrane, and the bag was sealed, and placed in water
bath at 42 C for 15 min. with constant agitation. After
incubation, denature SSD (100 ug/mL) and probe (1.2
ng/ml) was added, and the bag was sealed and incubated at
42 C overnight.with constant shaking; iNylon membrane was
washed, and exposed to a film for a week. The film was

developed and examined for presence of hybridization.

36
RESULTS

Isolation of Ruminal Bacteriophages

Before an. attempt. was made to investigate any
interaction between bacteriophages and bacteria of the
rumen, we decided to first ascertain the presence of
bacteriophages and identify as many bacteriophage
morphotypes as possible in the ruminal contents obtained
from a Holstein cow fed an all alfalfa hay diet. Such
identification of ruminal bacteriophages, by negative
staining and electron microscopy, may have provided clues
as to which phage types would associate with which of the
ruminal bacteria under study. Based on this study, five
morphologically distinct bacteriophages were.detected in
ruminal fluid. The bacteriophages varied greatly in size,
shape, and the nature of tail, as illustrated by the
electron micrographs obtained. These included, a phage
possessing an icosahedral shaped head (100 x 100 nm) and
a short, non-contractile, rigid tail measuring 150 x 20
nm in length (fig.1a). The phage had no other peculiar
features, such as a collar connecting the head to the
tail nor a base plate at the tail terminus. .According to
the classification scheme of Bradley (1967), this phage
should be assigned to group B. Another bacteriophage
which was observed, had a cuboidal head (110 nm), and a
short, noncontractile flexible tail, 290 nm in length

(fig. 1b). Collar and base plate were not observed with

37
this phage. Based on its tail length to head diameter
ratio, this phage would.also be classified under group B.
A cluster of phage like-particles with distinctly
hexagonal shaped heads (50 x 50nm) was also observed
(fig. 1c). Rather than a tail, these particles appear to
have thread-like strands interconnecting the particles,
with more than one particle appearing to branch off a
common strand. These particles could be assigned to group
E according to Bradley (1967), but this assignment is
still tenuous because of the presence of the unusual
interconnecting strands. A phage particle with a rather
unique structure, an icosahedral head (40 nm) and a
contractile sheathed tail (120 nm) was also seen in the
rumen fluid (fig; 1d). This.phage‘was classifiable under
group A. The last type of phage like particles observed
in ruminal fluid filtrate were large filamentous forms,
varying in length (100 to 400 nm) and a diameter of about
50 nm (fig. 1e). These filamentous forms were numerous
in the ruminal fluid filtrate, and were seen in every
field examined. The shorter filamentous forms were either
straight or slightly curved, while some of the larger
forms showed sharp bends and right angle branching. BY
their filamentous shape, these phage-like particles could

be assigned to group F, according to Bradley (1967).

38

Figure 1. Transmission electron micrographs of negatively
stained phage-like particles found in ruminal
fluid filtrate. '

a) Particle with icosahedral head, short, non-
contractile rigid tail. Magnification, 72000 X

b) Particle with cuboidal head, short, non-
contractile, flexible tail. Magnification,
108000 X

 

 

40

Figure 1. c) Cluster of particles with hexagonal heads

d)

and thin strands interconnecting the
particles. Magnification, 150000 X

Particles with icosahedral head, short,
contractile sheathed tail. Magnification,
210000 X

 

42

 

Figure 1. e) Filamentous like phage particles in various
sizes. Magnification, 54000 X

43

Bacteriophage - Spirochete Interaction

Once detection of verious morphological types of
phages in the rumen fluid was accopmlished, I decided to
study the physical and physiological interactions between
ruminal bacteriophages and those bacterial species
selected for this study. Inoculation of concentrated
ruminal fluid into an exponential culture of Treponema
saccharophilum isolate with incubation for 20 min
resulted in the attachment of numerous phage like
particles to the cell surface of the ruminal spirochete
(fig. 2a). The phage is distinguished by its cuboidal
head symmetry and the presence of a short tail, which was
observed to penetrate the outer sheath of the spirochete
and terminate in the protoplasmic cylinder (fig 2b). The
head of the phage is about 120 nm in length and 85 nm in
width" The tail of the ‘phage is non-contractile,
measuring about 47 nm in length and 10 nm in width.
There is neither a collar joining the head to the tail
nor other apparent attachments. Because of the constant
motility and swarming behavior of the spirochete,
confluent lawns could not be developed for observing
plaque formation by the phage, which could suggest
possible physiological interaction between the

bacteriophage and the spirochete.

44

Figure 2.a) Transmission electron micrograph of negatively

10)

stained Treponema saccharophilum showing the
attachment of numerous bacteriophage to the cell
surface. Magnification, 28500 X

Tansmission electron micrograph of ultrathin
section of Treponema sacharophilum showing the
penetration of a bacteriophage through the outer
sheath into the protoplasmic cylinder.
Magnification, 285000 X

 

46

Bacteriophage - Butyrivibrio fibrisolvens Interaction

Butyrivibrio fibrisolvens is a major hemicellulose
degrading ruminal bacteria, and from the nutrition stand
point is an important candidate to study in terms of its
interaction with ruminal bacteriophages. Concentrated
rumen fluid was inoculated into exponential cultures of
Butyrivibrio fibrisolvens, strains D1, 49, and A38; and
incubated for 30 min. The presence of numerous spheroidal
phage-like particles were observed within the cytoplasmic
space of both strains D1 and 49 (fig. 3a, 3b). However,
no phage-like particles were found to be associated with
strain A38. The detection of nearly mature phage-like
heads in the cytoplasmic space of both strains 01 and 49,
after 20 to 30 min incubation with ruminal fluid
filtrate, might suggest that the maturation process is
very rapid in this species. However, the length of
exposure was much longer than 30 min since the time
before and during processing of the culture must also be
taken into account. Realistically the total time of
exposure was. more likely between 1 to 2 h. Attachment of
phage-like particles on the outer cell surface of the.B.
fibrisolvens strains were not observed. Because a
recognizable tail could not be clearly discerned within
strain D1 or strain 49, it was unclear whether these were
tailless phages or phages in the process of maturation.

Examination. of control cultures 'which. had not. been

47
inoculated. with. ruminal fluid, indicated. that 'these
laboratory strains of .B. .fibrisolvens had. not been
previously infected with the phage. The double agar
overlay method for maintaining anaerobiosis was
successful in developing confluent lawns of B.
fibrisolvens. However, inoculation with ruminal fluid

filtrate did not result in any plaque formation.

Bacteriophage - Ruminococcus albus Interaction
Ruminococcus albus is one of the three most
predominant cellulolytic bacterial species in the rumen
and therefore a logical ruminal species for study in
terms of relationship with ruminal bacteriophage. When
filter sterilized ruminal fluid was inoculated into a
mid-log phase culture of a wild Ruminococcus albus
strain, and then overlaid with soft agar onto solid agar
media, plaques were produced after 24 h of incubation.
The plaques were clear and smooth and measured 1 to 3 mm
in diameter (fig. 4a). However, control plates, which
had cells only grown in medium containing heat treated
ruminal fluid, did not show plaque formation. When
plates containing plaques were incubated longer, 48 h, a
few of the plaques became turbid and were covered with
surviving cells, suggesting growth of a few, preexisting
bacterial cells which were resistant to infection with

the bacteriophage or growth of cells which had already

48

Figure 3a) Transmission electron micrograph of ultrathin.
section of Butyrivibrio fibrisolvens, strain D1,
showing intracellular phage like particle.
Magnification, 150000 X

b) Transmission electron micrograph of ultrathin
section of Butyrivibrio fibrisolvens, strain 49,
showing intracellular phage like particles.
Magnification, 150000 X

49

 

50

Figure 4a) Plate showing plaque formation by a ruminal
bacteriophage on a lawn of wild isolate,

Ruminococcus albus.

b) Transmission electron micrograph of negatively
stained phage like particles found associated

with R. albus. Magnification, 150000 X

 

 

52
been lysogenized by the phage. Clear plaques were
removed from the plate by picking or by scrapping with a
sterile Pasteur pipette, and crushed into a tube
containing GCSRF medium, centrifuged, and the
bacteriophage isolated from the supernatant. When the
supernatant lysate was examined by electron microscopy,
a small lambda-like phage particle was observed (fig.
4b) . The phage had an icosahedral head, 50 nm in
diameter and 60 nm in length. The tail was non-
contractile and appeared to be flexible and measured 120
nm in length. Neither collar nor base plate was
observed. As turning of clear plaques, which resulted
from the lysis of sensitive bacterial cells, into turbid
plaques is believed to be due to lysogenization of the
bacteria by the phage that infected it, it was necessary
to study the temperate nature of the phage in liquid
culture. Accordingly, the growth and lytic ability of
the phage in its host bacteria was determined by
inoculating the phage, at a multiplicity of infection
(MOI) of 1 to 100, into a mid-log phase culture of R.
albus, and monitor growth by optical density reading (600
nm) at various periods of time. A synchronized step wise
change in turbidity of the infected culture of R. albus
was observed (fig. 5a). Five hours after incubation, a
high number of bacteria were lysed and a considerable

titer of phage, 1.6 x 10‘ pfu per ml were produced. 0n

53
longer incubation, that is 8 h later, the cells increased
in their numbers and reached a cell density almost
similar to early infection. Control culture did not show
any lysis until much later, 48 h, of incubation time.
Because of this phenomenon, the isolated. phage *was
believed to be temperate for R. albus. Major
hemicellulolytic laboratory strains of Butyrivibrio
fibrisolvens D1 and 49 were found to be sensitive to the
temperate phage. Contrary to the growth pattern observed
in infected R. albus, B. fibrisolvens, strain Dl or 49
were significantly lysed by the phage and no
characteristics of lysogenization of the infected D1 or
49 were seen on prolonged incubation (fig. 5b, 5c) .
Electron microscopic examination of supernatant lysate
from infected broth culture of R. albus indicated a phage
particle the same as the one identified in the plaque.
Supernatant lysate from infected culture of D1 indicated
the presence of both the lambda-like phage (fig. 4b) and
a large number of filamentous-like phage particles (fig.

5d), previously not observed.

54

Figure 5a. Growth characteristics of wild R. albus in broth
culture after infection by the R. albus
bacteriophage.

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Figure 5b. Growth characteristics of B. fibrisolvens,
strain D1, after infection by the R. albus
phage.

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Figure 5c. Growth characteristics of B. fibrisolvens,
strain 49, after infection by R. albus
phage.

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Figure 5d. Transmission electron micrograph of negatively
stained filamentous phage like particles found
in supernatant lysates of B. fibrisolvens.
Magnification, 150000 X.

61

Host - Range Specificity of R. albus Phage

In studies to determine the optimal concentration of
host bacteria, strain D1 or strain 49, required to
support phage replication and hence plaque formation, it
was determined that under normal conditions, an optical
density of 0.3 to 0.5 was required. This represented
about 107/ ml to 108/ ml of bacteria incubated with 105 to
10‘5 pfu/ml of phage. At lower concentrations of host
bacteria, the probability of a phage encountering a
susceptible host were reduced and productive phage
infection did not occur. The host range specificity of
the temperate phage was, therefore, determined by placing
0.1 ml of the phage lysate containing 10‘ - 107 pfu/ ml on
an agar overlay inoculated with the bacterial strain to
be tested. Results of tests with ten ruminal
cellulolytic and amylolytic species are shown in Table
(1). The temperate phage was capable of lysing all the
bacteria strains but with differing sensitivity. The
degree of sensitivity, as determined by plaque forming
unit (pfu) varied from one species of bacteria to another
by several degree of magnitude. All these strains
supported plaque formation that differed in numbers (103
to 107pfu/ml). B. amylophilus, H-18 and S. ruminantium,
HD-4 were less sensitive and produced a lower titer of
phage. F. succinogenes, S-85; B. fibrisolvens, D1 and

49; R. flavefaciens, FD1; and laboratory strains of

62

Table 1. Host Range Specificity of Phage
Recoverd From Ruminococcus albus.

 

Bacterial Strains Specificity (pfu/ml)
Ruminococcus albus, 7 10‘
Ruminococcus albus, 8 10’
Ruminococcus albus, Wild -
Ruminococcus flavefaciens, FD1 106
Ruminobacter amylophilus, H18 103
Fibrobacter succinogenes, 885 10‘
Selenomonas ruminantium, HD4 10‘
Butyrivibrio fibrisolvens, D1 10‘

Butyrivibrio fibrisolvens, 49 10‘

63
R. albus, 7and 8 produced a higher titer of phage. None
of the plaques on any of these strains turned turbid when
incubated longer than 24 h. Wild type R. albus did not
produce plaques on its own confluent lawn, suggesting
immunity conferred to superinfection by the same phage.
Although, the phage appears to have a wide host range in
its infectivity, no attempt was made to recover phage

from every infected culture.

Temperature Sensitivity of R. albus Phage
The stability of the phage was determined at various
storage conditions. It was found that about 20 % of the
initial phage titer was lost after 6 months of storage at
-135 C, and almost 50 % of the phage had disappeared when
kept at 4 C for the same period of time. Phages are
generally stable in their own lysate provided it contains
suitable electrolytes and is free from specific
inactivating agents derived from the lysed bacteria which
are often difficult to remove by any means of filtration
or otherwise (Freifelder, 1983). Because of this,
supernatant phage lysate were treated with gelatin (1-2
%) before they were stored and to stabilize the titer.
In a study to determine the temperature tolerance of
R. albus phage showed that the investigated phage
differed in its sensitivity to a high range of

temperatures ( Table 2). Ninety seven percent of the

64

Table 2. Temperature Sensitivity of Phage Recovered

From Ruminococcus albus.

T era re C

39
39
50
50
70
70
90

90

E s

30

6O

3O

60

30

6O

30

60

n) BIBLE—ill.

100
100
100

100

000

Table 3. pH Sensitivity of Phage Recovered From
Ruminococcus albus.

 

 

.21 3.52M e m mun
3 120 20
6.8 120 100
10 120 100

65
phage were inactivated at 70 C for 30 min., but 100
percent of the phage were inactivated when exposed to 70
C for 1h. At 90 C for either 30 min or 1 h., no survival
of phage was observeda IHowever, the phage were resistant

to 50 C for 30 or 60 min and no inactivation was seen.

PH Sensitivity of R. albus Phage

To investigate the resistance of the phage to a range
of pH, phage was exposed to differing pH of GCS-RF media
containing 30 percent ruminal fluid. 'The effect of pH on
R. albus phage is indicated in Table (3). No phage were
affected over a pH range between 6.8 and 10 for 2 h.
However, at pH 3, 80 percent of phage were inactivated.
Such a deleterious effect may not be reflected in the
rumen since a decrease in pH does not usually take place
much below pH 5 even during high intakes of concentrated

diets by the ruminant animal.

Induction 0f R. albus Culture

R. albus phage was inducible by ultra-violet light
irradiation (fig. 6a). Irradiation for at least 45 to 60
sec of mid-log phase culture of R. albus was required to
give inducible phage. Supernatant lysate from the
induced host culture was plated with indicator cells, of
B. fibrisolvens D1, and plaques were produced. Lower (20

sec) or higher (120 sec) UV exposures brought about no

66

Figure 6a) Transmission electron micrograph of negatively
stained phage induced by UV irradiation from
R. albus. Magnification, 72000 X

b) Transmission electron micrograph of negatively
stained phage mutants induced by mitomycin C
from R. albus. Magnification, 72000 X

 

 

68
phage induction, which :might have resulted in less
exposure of cells for those exposed for 20 sec and no
induction, or high exposure which might have resulted in
progressive lysis of cultures. When doses other than the
optimal dose were used, that is either too low or too
high, no plaques were obtained. Mitomycin C (5 ug/ml)
also induced R. albus. Supernatant lysate from either
mitomycin C or ultraviolet irradiation, examined by
electron microscope, showed many incomplete particles,
heads, and ghosts (fig 6b) . In mitomycin C induced
cultures, many more mutant phages were seen than intact
phage particles. Lysate from this culture also did not
produce plaques on indicator cells which suggest that
mitomycin C, unlike UV irradiation, was not a specific
inducing agent for R. albus phage despite the occurrence
of mutant particles in the lysate. Spontaneous induction
of R. albus phage has occasionally been observed in this
study. Phages isolated from such cultures were identical
in their morphology to the temperate phage of R. albus
isolated from the concentrated ruminal fluid. Lysis of
broth cultures.of.R. albus was also brought about by both
mitomycin and UV-irradiation. When followed by optical
density reading at 600 nm, mitomycin C exposed cultures
of the either wild R. albus, or B. fibrisolvens strain D1
or strain 49, slowly for 20 min after which the cultures

lysed gradually within 1 h (fig. 7a, 7b, 7c ). Cultures

69

Figure 7a. Effect of UV irradiation on growth of
Ruminococcus albus culture.

 

 

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Figure 7b. Effect of mitomycin C or UV irradiation on
growth of Butyrivibrio fibrisolvens,
strain D1 culture.

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Figure 7c. Effect of mitomycin C or UV light irradiation
on growth of Butyrivibrio fibrisolvens,
strain 49 culture.

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75
which were not irradiated or not exposed to mitomycin C
showed a growth pattern similar to normal culture. These
results suggest that mitomycin C or UV irradiation exert
their effects on bacteria by induction which requires
intact cells, which lyse eventually leading to disruption

of some of the cells.

Genomic studies and hybridization of R. albus phage

To confirm the true temperate nature of the isolated
R. albus phage, genomic and hybridization studies were
carried out. DNA from both R. albus phage and host
bacteria were isolated by pulsed field gel
electrophoresis. Relative to a lambda phage DNA
standard, the total genomic DNA of the phage was
estimated to be about 48.5 Kb (fig. 8b), and the total
genomic DNA extracted from the host, R. albus was
approximately 970 Kb (fig. 8a). Total genome or
restriction fragments (smaI) of infected and uninfected
cultures of both R. albus and B. fibrisolvens were
isolated and transferred to nylon membranes. The
transfers were hybridized with a probe developed from
restriction fragments (smaI) of the phage. The probe
hybridized with five restriction fragments of DNA from
the infected cultures of R. albus. About 15 percent
homology in the DNA between the temperate phage and its

host, R. albus was demonstrated. Hybridization occurred

76

Figure 8.a) Pulsed field gel electrophoresis of total
and sma I digested DNA from uninfected and
phage infected cultures of R.a1bus and B.

fibrisolvens.

BI, Restriction fragment from infected B.
fibrisolvens DI.

RN, Restriction fragment from uninfected R.
albus.

RI, Restriction fragment from infected R. albus

BN, Total DNA from uninfected B.fibrisolvens,DI

BI, Total DNA from infected B.fibrisolvens,DI

RN, Total DNA from uninfected R.a1bus

RI,

Total DNA from infected R.a1bus

77

 

Figure 8b)

C)

78

Pulsed field gel electrophoresis of total
R.a1bus phage DNA. Electrophoresis was
carried out at 180 volts, 22 h, 14 C,
pulses, 40-80 sec

Autoradiograph showing hybridization of
phage probe to restriction (smaI) fragments
from infected R. albus.

t

79

. 630.5 Kb

a 436.5 Kb

. 242.5 Kb

‘. 97.0 Kb

 

80
with molecular weights, approximately 97.0, 242.5, 436.5
and 630.5 kb of the host DNA fragments (fig. 8c). There
was no hybridization to DNA fragments from infected
cultures of B. fibrisolvens D1 nor to DNA fragments from
the uninfected culture of R. albus. The absence of
hybridization to DNA from the uninfected cultures of R.
albus probably suggests that all R..a1bus were not stably
lysogenic or most probably had been cured from their

prophage early in the experiment.

Effect of phage on sugar fermentation

The effect of the temperate phage on fermentation of
xylan, glucose-cellobiose and cellulose in cultures of
rumen bacteria was investigated. Results of the
fermentations of xylan and glucose- cellobiose by the
phage infected mixed ruminal culture are shown (Tables
4, 5). A mixed culture of ruminal bacteria consisting
of B. fibrisolvens, strains D1 and 49; R. albus, strains
7 and 8 and wild type; R. flavefaciens, strain FD1; and
F. succinogenes, strain 885 was incubated with the phage
for 16 h in a medium containing a soluble xylan (0.5%),
as a sole substrate, and their fermentation products were
determined as described in the methods. There was a
significant decrease in concentrations of lactate,
formate(P <.05 ) in culture infected with the phage.

However, an increase in concentration of butyrate (P

81
<.05 ) was observed. There was no change in
concentrations of succinate, propionate and acetate (P
>.05) between control and treated culture. At 36 h of
incubation, the concentration difference between the
control and treated cultures were consistent. In medium
containing glucose-cellobiose as the main substrate,
there were no significant changes in the concentrations
of succinate, and butyrate (P > .05) between control and
treated culture. A significant increase in
concentrations of formate, acetate and propionate (P
<.05) was seen in treated culture both at 16 and 36 h
incubation. Concentration of lactate decreased
significantly in treated culture at 16 and 36 h

incubation (P <.05).

82

Table 4. Effect of R. albus phage on fermentation‘
of xylan by mixed ruminal bacterial

species‘
WM
990ml limes 9.0.0.2311 Phase SB
Succinate 0.555 0.343 0.537 0.360 0.14
Lactate 2.980b 1.695c 2.933” 1.743° 0.08
Formate 0.715“ 0.378° 0.798" 0.295° 0.02
Acetate 6.725 7.990 6.870 7.847 0.24
Propionate 2.545 3.883 2.313 4.115 0.44
Butyrate 0.628” 0.698° 0.435” 0.890° 0.03

‘each value is the mean mM concentration of
duplicate samples.

bemeans in the same row within one incubation
period with different letters in their
superscript differ (P<.05).

‘represents B. fibrisolvens, strains D1 and 49;
R. albus, strains 7, 8 and wild type; R.
flavefaciens, strain FD1; F. succinogenes,
strain 885.

83

Table 5. Effect of R.a1bus phage on fermentation‘
of Glucose-Cellobiose by mixed ruminal
bacterial species‘

WW
mmmm SE
Succinate 0.718 0.770 0.683 0.805 0.08

Lactate 3.340” 2.365° 5.150” 0.555° 0.09
Formate 1.375” 1,690° 1.475b 1u590° 0.12
Acetate 12 . 430* 14 . 128° 11. 863‘ 14 . 695° 0.23
Propionate 9.050” 11.668° 8.298” 12.420° 0.08

Butyrate 0.690 0.595 0.640 0.645 0.12

'each value is the mean mM concentration of
duplicate samples.

bemeans in the same row within one incubation
period with different letters in their
superscript differ (P<.05).

‘represents B. fibrisolvens, strains D1 and
49; R. albus, strains 7, 8 and wild type; R.
flavefaciens, strain FD1; F. succinogenes,
$85.

84
DISCUSSION

The range in diversity of phage morphotypes observed
in the rumen in this study was much less than what has
previously been observed in other studies. While
differences in sampling procedures and processing methods
can not be discounted. as probable causes for this
difference, there seems to be no a priori reason for
concluding that phage diversity is always high in the
rumen. This discrepancy in phage diversity could be
explained by the fact that ruminal contents of only one
cow receiving an alfalfa hay diet was examined. In
previous studies (Klieve, 1988) greater numbers of both
cattle and sheep on different diets were sampled which
could have resulted in a more diverse phage population.
Under similar conditions to this study, Paynter et al.
(1969) described only six distinct phage types in the
rumen contents of an alfalfa hay fed cow over a one year
sampling period. If most ruminal bacteria harbor phages
in either a stable lysogenic or pseudolysogenic state, as
the induction studies of Klieve et al. (1988) suggest,
then phage diversity could be highly variable in the
rumen depending on the proportions of the bacterial
species present and the ruminal factors which influence
their induction. Several of the phages observed in this
study appear to have been reported in earlier ruminal

studies. The phage with the icosahedral head, short non-

85

contractile tail with no collar and baseplate (Fig 1a)
appears to be similar to a phage previously described by
Paynter et al. (1969). Also, the cluster of particles
with hexagonal heads interconnected by numerous thin
strands (Fig. 1c) very closely resembles the cluster of
polyhedral phage heads, with a common fibrillar
attachment described by Hoogenraad et al. (1967).
Typically, the non-tailed icosahedral head looked like
the RNA coli phage 2J/1 described by Bradley (1964). The
phage with the cuboidal head and flexible tail (Fig. 1b)
somewhat resembles a lambda phage and also the phage
induced from Bacteroides ruminicola AR by Klieve et al.
(1989). The filamentous particles (Fig. 1e) does not
appear to have been previously described in earlier
studies.

The association of a specific phage with
T.saccharophilum is not particularly surprising, since
ruminal spirochetes are found in high numbers in the
rumen of alfalfa hay fed cows. Enumeration by direct
microscopic counts have shown ruminal spirochetes to be
in the order of 10‘ per ml in ruminal contents (Stanton,
1979). Bryant and Burkey (1953) estimated that ruminal
spirochetes constituted 2 to 5% of the culturable
bacteria in the ruminal fluid of an alfalfa hay fed cow.
Although, the ruminal spirochetes are not cellulolytic,

they enhance cellulose digestion by their interactions

86

with cellulolytic species (Kudo, 1986; Stanton, 1979).
T. saccharophilum will ferment pectin, polygalacturonic
acid, soluble starch, dextrin and various monosaccharides
and disaccharides (Paster, 1985). The association of a
phage with T. saccharophilum confirms the earlier report
by Hoogenraad et al. (1967) that ruminal spirochetes are
heavily infected by phages. Morphologically, the phages
attached to T. saccharophilum appears to belong to group
C (e.g. short, non- contractile tail ) under the
classification of Bradley (1967) , and appears to resemble
the phages observed by Hoogenraad et al.(1967) and even
much more to Staphylococcus phage 594n identified by
Bradley (1963). Whether this phage is either temperate
or virulent, or infects other ruminal spirochete strains
remains to be determined.

The infection of B. fibrisolvens by a ruminal phage
is of considerable interest, because B. fibrisolvens is
one of the most predominant species in the rumen of
cattle on a wide variety of diets (Bryant, 1956) .
Because of the high xylanolytic activity of strains,
including D1 and 49, B. fibrisolvens is regarded as the
most important species involved in the ruminal
degradation of hemicellulose (Hespell, 1987 ) . The phage-
like particles which infect D1 and 49 appear to be
morphologically similar. However, since a complete

phage-like particle was not observed in the recovered

87
cells of either strain, this conclusion is still
speculative. Intracellular multiplication has been
examined in comparatively few cases by use of ultrathin
sections. In the T-even phages, changes in the
nucleoplasm have been observed prior to the formation of
mature intracellular phages (Kellenbergen, 1959). With
RNA phages, crystals of virions are formed.prior to lysis
(Schwartz, 1963) . In the case of tailed mycobacteriophage
B-1, the presence of empty phage heads within the host
cell prior to lysis was reported (Tekeya, 1961). Klieve
et al. (1989) were able to detect an unusual filamentous
phage-like particle when B. fibrisolvens A14 was induced
with mitomycin C. However, the phage-like particles we
observed within the cytoplasmic space of strains D1 and
49 were clearly not a filamentous form. Whether this
phage is more adventitious than the filamentous form
remains to be determined, but suggest that B.
fibrisolvens may be infected by two distinctly different
phages. Our inability to develop plaques on confluent
lawns of strains D1 and 49, despite the presence of
phages in our infection studies with these strains is
perplexing. Since the two studies were performed at
different times, it is possible that the phage was not
present.in the ruminal fluid filtrate used for the plaque
studies“ The other' possibility is that the jphage

observed in the infection studies is temperate for B.

88

fibrisolvens. The infection of B. fibrisolvens strains
D1 and 49 by identically appearing phages suggest that
these strains are very likely genetically related.
Similarly, the lack of a phage infection in strain A38,
suggest that strain A38 either is resistant to the phage
or may not be genetically related to strains D1 and 49.
In recent DNA homology studies, Hudson and Gregg (1989)
concluded that, despite similar phenotypic
characteristics, B. fibrisolvens is apparently a very
genetically diverse group. Since phages are highly host
specific and generally do not cross infect genetically
diverse species (Ackerman, 1978), there may be an
opportunity to use the ruminal phages for taxonomic
classification of the indigenous bacterial population.

A close association between a ruminal bacteriophage
and.a major cellulolytic ruminal anaerobe, R. albus, was
established. Of all the bacterial isolates and laboratory
bacterial strains examined by double agar layer method,
only the wild strain of R. albus produced plaques on its
confluent lawn when plated. with filtrate ruminal fluid.
A ruminal phage resembling lambda was isolated. The
lambda- like phage found associated with R. albus has the
same morphology with E. coli lambda phage with respect to
the head and tail, which is flexible. The diameter of
head is the same as lambda head, 0.05 um, from which

projects a tubular tail, 0.12 um long. This phage

89
multiplies in R. albus in the same manner E. coli phage
multiplies in E. coli. The phage was characterized as
being temperate to R. albus because of its ability to
convert turbid plaques on the already clear plaques
formed. Much of the turbidity may be attributed to
surviving lysogenes or possibly due to lysogenization of
host cells. The factors responsible for forming turbid
plaques by the R. albus phage were not investigated in
our study, but three genes called cl, cll, clll, were the
first genes to be identified in lambda (Kaiser, 1957).
Secondary growth, as may occur particularly in long
incubations of infected lawn, is usually indicative of
lysogenization of host cells by temperate phages
(Braksdale, 1974) . Their plaques often had a target like
aspect, consisting of a central colony of surviving
bacteria surrounded by a clear margin, unlike clear
plaques of virulent phage. A culture infected with a
temperate phage does not necessarily always lyse but
could continue to grow and divide normally in its
original host cell. The culture fluid, however, always
contains a low concentration of the phage. The reason
why lysogenic cultures always contains a low level of
phage is because once every several generations or so a
daughter cell suddenly lyses.and liberates several.phages
while the remaining cells carry on dividing. Because of

the stable, hereditary nature of lysogeny and of the

90
immunity which a temperate phage confers, a recognition
of the phage depended on the chance isolation of a
sensitive indicator strain on which the phage will form
plaques. To such effect, after screening several ruminal
anaerobes, B. fibrisolvens, strain D1 and 49, were found
to be sensitive to the phage and were used as indicators
throughout the study. Because of their sensitivity,
plaques always formed on their confluent lawns. Such
occurrence of plaques have not been possible with R.
albus due to immunity to super infection by the same
phage. Moreover, liquid culture of R. albus incubated
with the phage became lysogenized after a large number of
phage were produced (fig. 5a). This phenomenon is in
close agreement to other phages, for example, P2, which
show the Boyd's effect, which is phage lysogenize host
cells more efficiently when the bacterium is multiply
infected or multiplies several fold in most cells before
becoming a stable prophage (Braksdale, 1974). When
growing cells are infected with a temperate phage, the
proportion of infected cells entering the lysogenic
pathway is about the same as the lytiijathway regardless
of the growth medium or the cell division time
(Echolas,1975). Growth of host bacteria above a
threshold value is required for phage replication, which
would lead to an increase in the number of phage. At some

concentration of phage, the numbers would be sufficient

91
to decrease the number of bacteria or the metabolic
activity. These interactions may even be complicated by
either mutations that lead to resistant bacteria. The
multiplicity of infection. is also known ‘to greatly
influence the ratio of the lysogenic to the lytic
response, with.high multiplicities of infection favoring
the lysogenic response and low multiplicities favoring
the lytic response ( Kovirilsky, 1973; Yen, 1980). Since
the lysogeny state is only as stable as the conditions
that favor it, the established prophage is continuously
poised to reverse the prior decision in the light of
changing circumstances and initiate a lytic cycle. Very
likely many factors enter into either the lytic or the
lysogenic process. Physical factors that influence
establishment.of immunity were studied in E. coli P1, and
was found to lysogenize more efficiently at 20 C than at
37 C (Rosner, 1972). The effect of temperature in
lysogenization of R. albus by its phage was not
investigated in this study. However, it appears that
establishment of lysogeny seem to depend on the
multiplicity of infection. Studies involving growth of
R. albus infected with the phage, indicated that a high
number of phage produced prior to lysogenization of the
host (Fig. 5a). Mechanisms by which R. albus perpetuate
its temperate phage in a lysogenic or pseudolysogenic

state have not been investigated in this study but is

92
fairly well established in lambda phage. After
penetrating into the cell, the normally linear lambda
genome circularizes by joining its cohesive ends , and a
few " early genes" are transcribed, including repressor
genes. The lysogenic decision also depends on host
function, i.e, genes ihf (integrase host factor) and hfl
and non-specific factors such as anti-metabolites and
starvation of the host. The circularized lambda genome
integrates linearly into the bacterial chromosome.
Integration is mediated by phage host enzymes, integrase
and/or ihf' and occurs via a cross over at a preferential
site. All phages do not have the same strategy as
lambda. In contrast to phage lambda, P22, the DNA of Mu
phage does not circularize and is integrated at random,
via transposition, into the bacterial genome. Other
phages including P1, certain mutants of lambda, and
phages of Streptomyces, Azomonas, Holobacterium do not
integrate at all, but behave as plasmids. Although,
mechanisms that lead to a lysogenic decision whether or
not to establish a prophage state in R. albus is not
known, hybridization studies have indicated that the
temperate phage could be integrated as part of the host
genome. Such phenomenon was not seen with B.
fibrisolvens, its indicator cell. The site at which the
phage integrates in the host chromosome has not been

determined. Nevertheless, the phage probe hybridized

93
with five restriction DNA fragments of the host suggest
that the provirus was cut into 5 different sizes of DNA
fragments by sma I; .R. albus has also been shown by
induction experiments to be lysogenic and, in some cases
induction occurred spontaneously. For most of these
studies, lysis of the culture was brought about following
UV irradiation, and temperate phage was identified by
formation of plaques on the appropriate indicator strain.
Determination of phage induction by the criterion of
plaque formation following exposure to inducing agents is
known to be poor (Lawrence, 1976) and variable even on a
prophage cured strain (Terzaghi, 1978) . To avoid the
possibility of a misleading result, the lysate from the
induced cultures were examined and confirmed visually for
the presence of phages by electron microscope rather than
relying on plaque formation. Since phage was only
detected in the irradiated cultures but not in the
unirradiated culture, we believe that the phage was
indeed temperate. It should also be recalled that every
bacterium in a culture population is not lysogenic; there
may be a few bacteria.which do not harbor prophagen iMany
bacteria are also known to carry phages which cannot
replicate, but may be visualized in lysate of induced
bacteria, they possess variable morphology, including
complete phages with full or empty heads, headless tails

or tailless heads, and polyheads and polysheaths

94
(Ackermann, 1978) . Results of the induction of the
temperate phage from R. albus exposed to 45 or 60 sec are
consistent with induction studies by Eisensark (1967) , in
which curing of the prophage from the host was achieved
with sublethal UV irradiation. R. albus culture exposed
to 120 sec UV irradiation did not produce lysate which
could form plaques on the indicator cells. This may be
due to the lethal effect of over exposure of UV
irradiation. From all the results, including induction,
lysogenic culture of R. albus must therefore be a source
of lytic phage. However, in the present study, no
evidence could be found that the phage investigated came
directly from induced lysogenic R. albus in the rumen or
from other ruminal species. As the temperate phage also
showed some relationship by hybridization techniques with
infected R.a1bus, would suggest that the phage may have
been derived from lysogenic R. albus. However, since
hybridization study did not extend to determine other
ruminal species for possible lysogenes, the hypothesis
that the temperate phage may have come from R. albus
needs further study with other related species.
Demonstration of the transition of a phage from lysogenic
to lytic in the rumen is difficult to obtain. Only
lysate obtained from induced culture provided sufficient
evidence towards the lysogenic nature of R.a1bus as a

source of lytic phage against B. fibrisolvens, strains D1

95

and 49. These experiments conclusively showed at least
with R. albus, prophage can give rise to virulent phage.

Studies on the effect of temperature on replication
have yielded interesting results. IHigh titer of phage in
B. fibrisolvens, strain D1 was produced after the phage
and bacteria were incubated at 39 C. No phage or very
little was produced when the culture was incubated beyond
50 C. These results indicate that indigenous phages in
the rumen environments are capable of replicating at
temperatures near those of the rumen.

Our preliminary studies involving effect of the
isolated R. albus phage on fermentation of various sugars
by ruminal speciesidid.not indicate consistent changes in
one fermentation by-product or the other. The phage was
shown to have a wide host range in its infectivity and
could be expected to reduce concentration of products
from fermentation of the sugars by way of lysing some of
the bacteria in culture. .As this did not occur, there
must be a different phenomenon by which the phage exerted
its effect on those bacterial species in culture. One of
the possible phenomena may be that the phage could have
lysogenized some of the bacteria instead of lysing them.
Those bacteria that were lysogenized could increase in
number and enhance fermentation products. The increase in
formats concentration in the phage treated culture of B.

fibrisolvens may be explained by lysogenization of B.

96
fibrisolvens albeit this bacteria was found to be
permissive to the phage. B. fibrisolvens is a major
formats producer compared to the other bacterial species
used in the mixed culture. More research is necessary in
this area to elucidate the role of the ruminal phage in
fermentation of various sugars and plant polymers.

The occurrence of two types of bacteriophages in the
supernatant lysate from D1 culture infected with the
temperate R. albus phage is not clear. Nor do we have a
conclusive evidence of its origin. Two possible
explanations that could be thought of may be: The first
is that the filamentous- like phage particle might have
initially existed in the rumen fluid which was used to
infect R. albus and perhaps passed through as a
contaminant during incubation with D1 cells. As a result
of the passage strain D1 may have been sensitive also to
the filamentous like- phage and produced more of those
filamentous like particles. The second possibility may
be that some:D1 cells may spontaneously have been induced
to produce filamentous forms after incubation with the R.
albus phage lysate. This possibility is much more likely
since B. fibrisolvens AR 14 have recently been induced
by mitomycin C to produce filamentous like phage (Klieve,
1989) . However, no conclusive evidence was reported
whether these particles were bacteriophages or

bacteriocins. Although, the origin of the filamentous

97
like form is uncertain, their true forms and existence is
unequivocal because the same forms have also been
described by Bradley (1966).

Host range studies of R. albus phage action have
demonstrated a wide spectrum of interactions, ranging
from highly specific to less specific. Important
cellulolytic and amylolytic rumen bacteria were
permissively infected with differing sensitivity; a
possible explanation for this might be a restriction-
modification system acting in strains. Such systems have
not been studied in the rumen bacteria. The only
bacteria known to have such a system is F. succinogenes
and DNAse from supernatant culture of F. succinogenes
degraded Lambda phage and supercoiled DNA (Forsberg, C.
W., Pers. comm. as cited in P.N. Hobson, 1988). The
broad host specificity of the phage on the rumen bacteria
may be a reflection of the action in the rumen.‘which.may
be to keep the proportion of specific bacterial species
in check when their numbers exceed the ecological

balance.

Conclusion
The occurrence of bacteriophages in important rumen
bacterial species which are functionally significant in
the ruminal degradation and fermentation processes was

demonstrated. A ruminal bacteriophage that infects a

98
ruminal spirochete, Treponsma saccharophilum was
identified. T. saccharophilum is pectinolytic and is
intimately associated with cellulolytic species and
enhances cellulose degradation. This is the first
demonstration of a phage associated with an identified
rumen spirochete in pure culture. Of significance is the
isolation of a temperate phage from ruminal contents
which appear to lysogenize a wild isolate strain of
Ruminococcus albus, a major cellulolytic ruminal
bacteria. The phage is morphologically characterized by
an icosahedral head (50 x 60 nm) and has a flexible , non
contractile tail (120 nm). In contrast to the host, R.
albus, the phage was found to be obligately lytic to
Butyrivibrio fibrisolvens, strain D1 and 49, both of
which are involved in hemicellulose degradation in the
rumen. Studies conducted to determine the host range and
specificity of the temperate phage confirmed that the
phage infects a wide range of predominant rumen bacterial
species involved in many key degradative and metabolic
interactions. The phage infected predominant cellulolytic
species (Ruminococcus flavefaciens, strain FDl,
Fibrobacter succionogsnes, strain S85, Ruminococcus
albus, strains 7 and 8) starch degrading species
(Ruminobacter amylophilus, strain, H18) and a major
propionate producing species (Selenomonas ruminantium,

strain H04).

99
Implications of R. albus phage

The detection and isolation of ruminal bacteriophages
which infect predominant indigenous rumen bacterial
species raises the question as to their role in
influencing rumen bacterial diversity and fermentation
efficiency. The degradation of fibrous feeds in the rumen
involves the concerted interactions of a number of
bacterial species. Changes in the proportions and types
of these bacteria, due to either temperate or lytic phage
infection, could affect the efficiency of degradation and
fermentation of these feeds. Considerable attention is
being directed toward improvement of digestibility of
dietary feedstuffs by physical and chemical treatments.
Unfortunately, there has been little success. Idgnin
digestibility by microorganisms still remains one of the
biggest problems in the rumen. To circumvent these
problems, a new alternative strategy based on recombinant
DNA techniques could be developed. This may be done by
modifying rumen bacteria to produce high activity enzymes
for cellulose and lignin degradation in the rumen. Such
modification of bacteria takes into consideration the
need for an appropriate bacteria to be modified, a
specific genetic material for insertion into the bacteria
by means of a cloning vector. Sophisticated vector
systems have been exploited in the past, including high

number plasmids (Keisen, 1972) and bacteriophages

100
(Harris, 1983). Although these gene cloning tools are
available, obstacles have prevented the rapid full scale
exploitation of the desired genes. Certain plasmids have
been detected in some species of ruminal microorganisms
(Teather, 1982). However, their use as possible vectors
have not been elucidated. An alternate method which will
circumvent the need for efficient replication. of a
recombinant gene will be a lysogenic-phage derived
cloning vector containing a selectable marker, a suitable
cloning site, and a defect in the normal phage
chromosomal integration mechanism. Generally, lysogenic
phages integrate into the host genome by their specific
recombination between a unique phage DNA sequence and a
non—homologous host DNA sequence (Campbell, 1962). The
obvious potential of ruminant phages is then in their
ability to transfer genetic material by transduction and
transfection. Transduction provides a mechanism for
distribution of genetic information during naturally
occurring phage- host interactions. High frequency
transducing ability is associated.with temperate phages,
and Lac+> has been shown to be transduced at high
frequency in S. lactis L2 (McKay, 1973) . Using the recent
genetic engineering, stabilization of lactose and
proteinase genes in the chromosomes of Streptococcus
lactis, 02 by transduction was a most significant

accomplishment for dairy starter cultures (McKay, 1973) .

101
Zinder and Lederberg’s (1952) original observation showed
that many different types of characteristics could be
transduced; nutritional, fermentative, antigenic, and
resistance to antibiotics. It was suggested that this
carriage was due to the accidental incorporation of
genetic units of the host. Phages do offer some
advantages over other vectors because of their high
transfer frequency and the stability of their prophages
within the bacterial genome. If specialized transducing
ruminal phages could be identified, desirable
fermentation traits could be introduced into specific
ruminal bacteria for improving the efficiency of the
ruminal fermentation. R. albus phage was isolated for
its ability to infect and lysogenize R. albus, a major
cellulolytic bacteria. In light of this it can be
employed as a recombinant DNA vector in lieu of plasmids.
The phage has a broad host range and can be propagated in
several species of B. fibrisolvens. Before its use,
however, a physical map which will identify sites needs
to be constructed and several deletion mutants need to be
isolated to determine the regions of the phage containing
non-essential DNA. Such phage deletion mutants can be
used as cloning vectors whereby foreign DNA can be
recombined into the phage in vitro and then introduced
into cell protoplasts by transfection. Since the phage

DNA can integrate, the new genes can be stably maintained

102
and expressed. The temperate phage can provide the
necessary components for recombinant DNA experiments in
organisms lacking plasmids carrying selectable markers.
The ability to use genetic engineering principles to
construct microorganisms that can use growth substrates,
such as lignified cellulose, could be extremely important
to the development of better production strains and to
using new raw materials as growth substances. However,
for construction of microorganisms to take place, a gene
product that degrades lignin must be obtained. Recently,
a lignin degrading gene was described in white rot
fungus, Phanerochaete chrysosporium, by Thompson et al.
(Center for Microbial Ecology research findings, pp. 164,
Fall, 1992). While this organism could offer a potential
source for transferring a lignin gene to R. albus , a
major challenge may be the establishment of the
genetically modified bacteria in the rumen. The
probability of the modified bacteria to survive in the
rumen without being out competed by other ruminal species
may be low. Even if the modified bacteria is fully
competitive with the resident bacteria, its establishment
would be difficult. However, a general strategy could be
employed in which the constructed strain could be made
capable of promoting its own transfer with.high degree of

efficiency so that the desired gene can be introduced

into the resident bacterial population using a very small

103
inoculum.

Another approach where the application of gene
cloning and genetic manipulation to rumen bacteria is of
fundamental interest is in the alleviation of energy
waste by reducing the amount.of methane production in the
inefficient process of rumen fermentation. Methane in the
rumen is produced by the methanogens by carbon dioxide
reduction with hydrogen originating from the ferredoxin
mediated reoxidation of NADH produced during glycolysis.
Specific inhibitors of methanogenesis, such as higher
unsaturated fatty acids and chloroform have caused the
desired fermentation shifts in experiments in vivo and
vitro ( Meyer, 1981). However, they have not been useful
because of undesirable side effects or ineffectiveness in
improving animal performances. The ionophores, monensin
and lasolacid, have showed promise in feeding trials and
now is heavily used as feed supplements for beef cattle.
Monensin increases the production of propionate and
decreases the production of methane in the rumen (Meyer,
1981). Nevertheless, a considerable amount of methane is
still being produced. In circumventing this problem, a
gene product utilizing hydrogen more competitively could
be obtained and cloned into a suitable phage vector
system for transferring the desired gene into a ruminal
bacteria. A non ruminal species, Clostridium aceticum, is

known to utilize hydrogen for the reduction of carbon

104
dioxide to acetate ( Braun et al. 1981) and therefore
provides a potential source for gene isolation. Such
procedures involving gene manipulation of rumen bacteria
provide a more logical approach than others involving
introduction of acetogenic bacteria, as previously
thought in some studies, directly into the rumen which is
not native to the environment.

Research will be conducted in the future for a
better understanding of the bacteriophages in the rumen
ecosystem. Future objectives are:

1. Host range specificity of the temperate
bacteriophage will be extended with other
rumen bacterial species.

2. In vitro studies will be conducted to
determine if the presence of a bacteriophage
in pure mono and mixed bacterial cultures of
ruminal bacteria affect the efficiency of
degradation of various substrates or alter the
fermentation end products.

3. Molecular characterization and restriction
mapping of the phage will be carried out to
determine appropriate sites for cloning

desired genes (traits) into the phage.

1.

2.

4.

5.

6.

8.

9.

105

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