‘—
‘.,' ' .< 'lx‘. T.
.T .T._ T, ‘H ‘I

w

 

METABOLISM OF ACETATE BY
NITROBACTER AGIUS

Thesis for the Degree of Ph. D.
MiCHIGAN STATE UNIVERSITY
ALINE L. GARRETSON
1971

     
     

LIBRARY
Michigan 39”
Univm. ~

  

This is to certify that the

thesis entitled

Metabolism of Acetate by Nitrobacter agilis

 

presented by a
Aline L. Garretson
has been accepted towards fulfillment

of the requirements for

Ph.D. degree in MiCI'ObiOlOgy

 

(/2? Jet (Amm—

Major professor

Date February 26, 1971

/7 ' ’ ABSTRACT

METABOLISM OF ACETATE BY NITROBACTER AGILIS

 

by Aline L. Garretson

Nitrobacter agilis was incapable of proliferation in a

 

medium containing acetate (5 mM) as sole source of carbon
when various inorganic and organic sources of nitrogen were
substituted for nitrite. Furthermore, culture viability
was not maintained over a 21 day growth period in an acetate
medium supplemented with either glutamate or nitrate. Sig—
nificant increases in cell number were not achieved with
either ammonium or casein hydrolysate additions to a medium
containing acetate. Repeated attempts to grow pure cultur-
es of E. agilis heterotrOphically in a medium containing
acetate and casein hydrolysate were unsuccessful. Replica-
tion in a medium containing both acetate and nitrite approxi-
mated that observed with autotrophic cultures. The dry
weight of cells grown in an autotrophic medium contained 0.3
to O.h% poly-fléhydroxybutyrate (PHB) whereas cells grown in
an autotrophic medium supplemented with acetate contained

as much as 6 to 12% PHB. The increased synthesis of PHB

was accompanied by both a decreased production of protein
and reduced absorbence to dry weight ratios. The distribu-
tion pattern of 1&0 in fractions of cells incorporating
acetate-l-luC remained unchanged when unlabeled bicarbonate
was added but the total amount of 1LLC assimilated was re—

duced 26% in all fractions. In contrast, the addition of

Aline L. Garretson
unlabeled acetate to a medium containing bicarbonate-1&0
resulted in an 80% reduction in isotope assimilated while
the distribution patterns remained the same. In cultures
labeled with acetate-l-luc approximately 30% of the radio—
activity was located in PHB whereas no% was found in the
protein fraction. Intermediates of the tricarboxylic acid
(TCA) cycle and certain amino acids and phosphorylated
compounds located in the ethanol-soluble fraction of a cell
suspension oxidizing nitrite were shown to be labeled within
5 sec after addition of acetate-Z-luC. The greatest amount
of isotope recovered initially was in the carboxylic acids
of the TCA cycle and in glutamate. Citrate and isocitrate
were predominantly labeled while the radioactivity associ-
ated with malate remained at relatively low levels until
nitrite was depleted. The sequential labeling patterns
obtained during nitrite oxidation provided evidence for
the Operation of a TCA cycle, but ruled out the operation
of either a glyoxylate bypass or the oxidative dicarboxylic
acid cycle. However, when nitrite was exhausted. there
were percentage increases in glyoxylate, malate, and succin-
ate and decreases in citrate and isocitrate. It appears
that the TCA cycle no longer is the dominant pathway for
acetate metabolism once nitrite oxidation ceases and that

glyoxylate becomes the principle intermediate labeled.

METABOLISM OF ACETATE BY NITROBACTER AGILIS

 

BY

- :

Aline L. Garretson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Microbiology and Public Health

1971

To members of my family, for their constant encourage-
ment, assistance, and understanding during the course of my
doctoral program.

ACKNOWLEDGMENTS

I wish to thank Dr. Charles L. San Clemente under whose
guidance this dissertation was prepared, for his assistance
and advice during the course of my graduate program.

Sincere appreciation is also extended to Dr. R. N.
Costilow, Dr. A. R. Wolcott, Dr. J. E. Varner, and Dr. C. L.
Winely who contributed helpful advice, constructive criticism

and moral support to my research endeavors.

ii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS.
LIST OF TABLES.
LIST OF FIGURES
INTRODUCTION.
LITERATURE REVIEW .

Structure and Biological Significance of
Nitrobacter . . .
Inorganic Growth Requirements.
Oxidation of nitrite
Metabolism of nitrate.
Fixation of carbon dioxide
Effect of other inorganic ions
Factors Limiting Growth Yields . .
Pure Culture Methods . .
Metabolism of Organic Compounds.
Synthesis of Storage Materials . .
Physiological Classification of Chemolitho—
trophs. . . .
Established Metabolic Pathways in Acetate
Metabolism. . . . .
Tricarboxylic acid cycle
Glyoxylate bypass.

 

MATERIALS AND METHODS

Growth Conditions.
Culture Purity .
Preparation of Cell Suspensions and Chemical

Analysis.

IsotOpic Labeling Procedure for Growing
Cultures.

Isotopic Labeling of Fermentor Grown Cell
Suspensions

Paper Chromatography .
RESULTS . . . . . . .

Growth on Acetate Combined with Various
Sources of Nitrogen . . .

Heterotrophic Growth on Acetate and Casein
Hydrolysate . .

Synthesis of Poly-fl- -hydroxybutyrate (PHB).

Effect of Acetate on Macromolecular Synthesis.

iii

Page

ii

vi

b0

arctipflp

ll
13

15
19

20

22
22

23
2b.

2
27
28

3O
31

3h

31+
37
39
A2

Page

Uptake of Isotopically Labeled Acetate and
Bicarbonate by Growing Cultures . . . . . .
Incorporation of CH lLl-COONa and NathCOB into
Cellular Fractions of Growing Cultures. . . . us
Uptake of lHCH COONa by Cell Suspensions . . . . A9
Distribution of Radioactivity in Cellular

AA

Intermediates During Nitrite Oxidation. . . . 52
Distribution of Radioactivity in Cellular
Intermediates After Nitrite Depletion . . . . 62

DISCUSSION. . . . . . . . . . . . . . . . . . . . . . 69

LITERATURE CITED. . . . . . . . . . . . . . . . . . . 7h

iv

Table

LIST OF TABLES

Influence of various carbon and nitrogen
sources on nitrification and growth of
Nitrobacter agilis. . . . . . . . . . .

 

Occurrence of polyqfl-hydroxybutyrate (PHB)
in Nitrobacter agilis grown on acetate.

 

Relationship of absorbence and nitrite
oxidation to dry weight and protein content

Percentage distribution of 1MC derived from
CH lLLCOONa and NaH +CO3 among cellular
fr ctions . . . . . .

Incorporation of the lbrC of acetate-21uC into
organic acids of the ethanol-soluble fractions
of a growing cell suspension of Nitrobacter

agilis. . . . . . . . . . . . .

Incorporation of the lac of acetate-2-luC into
amino acids of the ethanol—soluble fractions
of a growing cell suspension of Nitrobacter

agilis. . . . . . . . .

Incorporation of the 1“C of acetate-2—1uC into
phosphorylated compounds of the ethanol-
soluble fractions of a growing cell

suspension of Nitrobacter agilis.

 

 

 

Incorporation of the lI‘iC of acetate-Z-luC into
various cellular intermediates located in the
ethanol-soluble fractions of a cell suspension
of Nitrobacter agilis deprived of nitrite-N

 

Page

35

HO

#3

A7

53

SA

55

63

Figure

LIST OF FIGURES
Page

Ultraviolet absorption spectra of crotonic

acid formed from poly-fl-hydroxybutyrate

isolated from cells of Bacillus megaterium

strain KM and Nitrobacter agilis after

heating in concentratengsOu. . . . . . . . . hl

 

Absorbance and uptake of lac-labeled acetate

or bicarbonate in Nitrobacter agilis grow-

ing in a mineral medium containing sodium

nitrite. . . . . . . . . . . . . . . . . . . . MS

 

Comparison of growth of Nitrobacter agilis on
acetate and nitrite with growth on bicarbonate

and nitrite in fermentor cultures in terms of
absorbence at uuo nm and nitrite oxidation

(mg oxidized per ml) . . . . . . . . . . . . . 5O

 

RatIfiCOf nitrite oxidation and incorporation

of into a group of selected cellular
intermediates present in the ethanol-soluble
fraction of a cell suspension f Nitrobacter

agilis growing on acetate~2- MC. . . . . . . . 51

 

The peifientage distribution of 1&0 of acet-

ate-2- C incorporated into carboxylic acids,

amino acids, and phosphorylated compounds

found in the ethanol-soluble fractions of a

growing cell suspension of Nitrobacter agilis. 58

 

The percentage distribution of 1"LC of acet-
ate-2-14C incorporated into various compounds
associated with the tricarboxylic acid cycle

found in the ethanol-soluble fractions of a

growing cell suspension of Nitrobacter agilis. 59

The peifigntage distribution of 1&0 of acet-

ate-2- incorporated into phosphorylated

compounds, C dicarboxylic acids (succinate,
fumarate, anU malate), and C and Cg carboxy-

lic acids (citrate, isocitra e, and glutamate)

found in the ethanol-soluble fractions of a

growing cell suspension of Nitrobacter agilis. 61

 

vi

Figure

10.

The effect of nitrite depletion on the in-
corporation of AC from acetate-2-luC into
phosphorylated compounds and carboxylic
acids of the tricarboxylic acid cycle.

The effect of nitrite depletion onl.he in—
corporation of AC from acetate—2- into
glyoxylate, malate, citrate plus isocitrate,
and succinate plus fumarate.

Possible pathways of acetate metabolism in
Nitrobacter agilis

vii

Page

66

68

72

INTRODUCTION

The accumulated data on the growth of obligate chemo-
autotrophs on organic substrates has not permitted an under-
standing of the failure for growth under heterotrophic condi-
tfiuns. The rapidity of nitrification in organically rich
soils, dung heaps, sewage, and impure cultures has been cited
by Lees (l95h). Consequently, it follows that heterotrophic
growth may be possible under limited and well-defined growth
conditions in which a specific combination of organic sub-
strates may prove essential.

Up to the present time, the supplementation of inorganic
media with a single organic compound has not resulted in
heterotrOphic growth for a number of obligate chemolitho-

trophs, including Nitrobacter. On the other hand, complex

 

supplements, such as yeast extract, have been shown to stim—
ulate growth in an otherwise autotrophic medium. There has

appeared only one report of Nitrobacter agilis growing

 

heterotrOphically (Smith and Hoare, 1968). This organism
reportedly grew, albeit slowly, in the combined presence of
acetate and casein hydrolysate and in the absence of any
other form of nitrogen or carbon. However, heterotrophic

growth of Nitrobacter in a similar medium has not been

 

reported by any other investigators. Furthermore, short-
1

2

term.studies with cells not adapted to heterotrophic growth
.on acetate have shown that acetate as sole source of carbon
and energy does not support the replication of N. agilis
(Delwiche and Finstein, 1965; Ida and Alexander, 1965). In
the previous studies, neither the contribution of acetate as
a precursor of cell metabolites, nor the metabolic function
of casein hydrolysate has been ascertained.

The aim of this study was to elucidate the pathway(s)
of carbon assimilation during growth in a medium containing
acetate and to determine if acetate can serve as a source of
energy for both carbon assimilation and cell growth. This
dissertation reports the inability of N. agilis to grow in
media containing acetate when any one of a variety of organic
or inorganic nitrogenous compounds, including casein hydrol-
ysate, was substituted for nitrite. When nitrite was present
in the medium, growth approximated that of the control auto-
trophic cultures. From isotopic distribution and kinetic
studies, using 1LLC--labeled acetate, I concluded that acetate
was not metabolized by either the glyoxylate bypass or the

oxidative dicarboxylic acid cycle.

LITERATURE REVIEW

Structure and Biological Significance of Nitrobacte;

N. agilis is a gram negative, flagellated bacterium
originating in the soil. Electron microscopy demonstrated
a characteristic peg-shape which is related to the polar
arrangement of the plasma membrane intrusions (Murray and
Watson, 1965). These intrusions consist of at least nine
layers of plasma membrane, closely and regularly apposed
which are arranged in two or three thicknesses over the poles
of the cell. The plasma membrane uniquely possesses an
extremely dense layer applied to the inside of the usual unit
membrane. It is quite probable that this lammellar membrane
system is associated with the cytochrome electron transport
system involved in nitrite oxidation. Aleem and Nason (1959)
have established the presence of this system in the partic-
ulate fraction of cell-free extracts.

The conversion of ammonia to nitrate is effected in
nature by two groups of obligate aerobic, chemolithotrophic
bacteria. Ammonia oxidation is accomplished by Nitrosomonas

while nitrite is oxidized to nitrate by Nitrobacter. Only

 

two species of Nitrobacter are recognized, N. agilis and N.

3

 

u

winogradskyi. The combined activities of the nitrifiers

 

results in the oxidation of the ammonia released during the
mineralization of organic matter. Consequently, in soils
where nitrification is active, nitrate is the principal form
of nitrogen that is available for the growth of plants. The
nitrifiers Which are abundantly found in sewage effluents un-
doubtedly play an ecological role in the reassimilation of

nitrogen in higher forms of life.

Inorganic Growth Requirements

 

Oxidation of nitrite. Nitrite is rapidly metabolized by

 

extracts of Nitrobacter with complete conversion to nitrate

 

and the consumption of oxygen essentially equal to the theo-

retical amount expected according to the following reaction:
1102' + %02 as, NO3- (AF=17.8kcal)

This oxidation provides energy in the form of adenosine
triphosphate (ATP) as well as reduced pyridine nucleotides
for the cellular biosynthetic reactions involving C02 reduc-
tion (Aleem, 1965; Kiesow, 1963, l96u). Nitrite oxidase is
associatedwith the particulate constituents of the cell
(Aleem and Nason, 1959). The minimal size of membrane frag-
ments from sonic homogenates having nitrite oxidizing activ-
ity is estimated to be about 22 nm (Tsien and Laudelout,
1968), but the distribution of cytochrome a appears not to
be affected below this critical size. An oxygen atom obtain—

ed from water, and not molecular oxygen, participates in the

5
oxidation of nitrite to nitrate (Aleem, Hoch, and Varner,
1965: Kiesow, 196u). Water may also act as a hydrogen donor
for the reduction of pyridine nucleotides (Aleem, Hoch, and
Varner, 1965). Nitrite oxidation is mediated by the cyto-
chrome system and is coupled to oxidative phosphorylation
(Aleem and Nason, 1959, 1960).

The theoretical P/O ratio of one has been approached
by Kiesow (196u) and Aleem (1968). Aleem attributed lower
P/O ratios previously obtained to inadequate conditions for
growth and procedures for sonication which resulted in the
destruction of the structural integrity of the phosphoryl-
ating particles without affecting the electron transport
capability. When reduced nicotinamide adenine dinucleotide
(NADH) served as electron donor, a P/O ratio of two was
obtained.

The problem of the transport of electrons from nitrite
to oxygen has been examined independently by the two groups
of investigators led by Kiesow and Aleem. Aleem's studies
have been with N. agilis and other chemolithotrophs, while

Kiesow has limited his studies to N. winogradskyi. Both

 

groups arrived at the concept of reversed electron flow
along the electron transport chain.

Kiesow reported that under anaerobic conditions a
reversal of the normal reaction of nitrite oxidation was
observed in intact cells. NADH, previously found to be
produced during nitrite oxidation in cell homogenates and

purified fractions of submicrOSCOpic particles, acted as

6

the reducing agent for the reduction of nitrate. These
reactions were mediated through two cytochromes and a.
flav0protein and the synthesis of ATP was coupled with

the reduction of nitrate:

NADH2 + No3’ + 2ADP + 2Pi E==;,NAD + 2ATP + H20 + N02-

The same particle-fraction catalyzing the "back reaction"
also catalyzed the oxidation of NADH by 02 using a terminal

oxidase:

NADH2 + %02 + 3ADP + 3P1 é==; NAD + H20 + 3ATP

Kiesow concluded that the "back reaction" occurred in reverse
and that it followed the second reaction in a sequence.

Since all reactants of the second reaction, except oxygen,
are products of the first reaction, it was further concluded
that the sequence was cyclic. The cyclic reaction therefore

proceeds as follows:

NADH + %o

2 + 3ADP + 3P1 ———9 NAD + H20 + 3ATP

2

NO - + 2ATP + NAD + H20 -——9 NO3- + 2ADP + NADH2 + 2P1

2

In this scheme, nitrate serves in place of oxygen as the
terminal electron acceptor. An objection to Kiesow's scheme
lies in the fact that 2 moles of ATP which are required for
the reduction of NAD by nitrite oxidation can not provide

an energy equivalent of 3u,5OO calories, a potential differ-
ence of 750 mv.

The scheme proposed by Aleem's group (Aleem, 1968;

7
Sewell and Aleem, 1969) is thermodynamically more satisfac-

tory. In their system, cytochrome 31 is the site of entry
of nitrite in the electron transport chain and in NAD reduc-
tion electrons are passed along the chain according to the
following sequence:
ATP ATP
N02“ -——9 cytochrome al -J—9 cytochrome c ~é~9
ATP
cytochrome b (Q10) -——9 f1avoproteins ~4h9 NAD
The reduction of each mole of NAD by nitrite requires the
utilization of approximately 5 moles of ATP. This is in
agreement with the calculated 35 kcal free energy needed for
the overall reverse electron flow process. The conservation
of energy in the form of one ATP per nitrite oxidized is
effected by the entry of nitrite at cytochrome a1 and the
subsequent reduction of oxygen by the terminal oxidase of
the electron transport chain. Kiesow's scheme differs in
that in his scheme electron transport from nitrite to oxygen
is not coupled to ATP synthesis and nitrite oxidation con-

sumes energy which is provided by the oxidation of NADH.

Metabolism of nitrate. The presence of a nitrate reductase

 

in N. agilis has been reported by Street and Nason (1965).
This particulate enzyme is energy independent and catalyzes
the reduction of nitrate to nitrite with reduced cytochrome

c as electron donor. Cytochrome a and an unidentified

1
metal component are also implicated in the reaction. Chlor-

ate completely inhibited nitrate reduction. This compound

8

is known to delay growth without affecting nitrite oxidation
in growing cultures of Nitrobacter (Lees and Quastel, l9h5).
These findings suggest that the nitrate reduction may pro—

vide the first step in the acquisition of nutritional nitro-
gen. At present, nothing is known of the origin of nitrogen
used in the synthesis of nitrogenous compounds in Nitrobact-
33. The possibility that nitrate reductase also effects the
recycling of nitrite involved in nitrite oxidation has not

been explored.

Fixation of carbon dioxide. In the chemolithotrOphic bact—

 

eria carbon dioxide fixation is accomplished by a combination
of enzymatic reactions from widely distributed pathways and
specialized reactions which are found only among autotrophic
organisms. The common path of carbon, shared by photolitho-
trophic and chemolithotrophic systems, utilizes reactions
which are also part of glycolysis, pentose phosphate meta-
bolism, and dicarboxylic acid metabolism. The two reactions
which are peculiar to the autotroph are: the phosphorylation
of ribulose 5-phosphate to ribulose 1,5—diphosphate (RuDP)
and the subsequent carboxylation of RuDP resulting in the
formation of 3-phosphoglycerate (3-PGA).

The elucidation of the path of carbon in the photosyn-
thetic carbon reduction cycle, of which RuDP carboxylase is
a part, was made possible through the radiocarbon studies of
Horecker and Racker (Vishniac, Horecker, and Ochoa, 1957).
The pathway as it occurs in higher plants was described by

Bassham.et_alx (195M). Similar mechanisms exist in the

9
obligate and facultative autotrOphic bacteria (Elsden,
1962). Early studies on crude extracts of spinach leaves
(Jakoby, Brummond and Ochoa, 1956) and Thiobacillus thio-
pgggg (Santer and Vishniac, 1955) resulted in the fixation
of 002 in the presence of RuDP with the formation of 3-PGA.
The rapid incorporation of 15002 into 3-PGA and sugar phos-
phates was also accomplished by intact cells. The fixation
of 002 by the RuDP pathway is now known to occur in a number
of other chemolithotrophic bacteria: hydrogen oxidizing
bacteria (Vishniac and Trudinger, 1962), T. denitrificans

(Trudinger, 1955, 1956), N. thiooxidans (Suzuki and Werk—

 

man, 1958a; Iwatsuka, Kuno, and Maruyama, 1962), Ferro-

bacillus ferrooxidans (Maciag and Lundren, 196N),‘N. agilis

 

(Malavolta, Delwiche, and Burge, 1960; Aleem, 1965), and

Nitrosomonas europaea (Delwiche, Burge, and Malavolta,

 

1963).

RuDP carboxylase (EC u.l.l.f) in facultative autotrophic
bacteria such as hydrogen bacteria (Hydrogenomonas species)
is formed adaptively under autotrophic conditions; the high-
est level of activity occurring in organisms grown under
strictly autotrophic conditions (Vishniac and Santer, 1957).
Similar findings have been reported for Thiobacillus novellus
(Vishniac and Trudinger, 1962), N. denitrificans (Kornberg,
Collins, and Bigley, 1960) and Hydrogenomonas facilus
(McFadden and Tu, 1965). Thus, RuDP carboxylase appears to
play an important role in chemosynthesis, and is considered

the critical enzyme involved in autotrophic C02 fixation.

10

The fixation of C02 into oxalacetate is made possible
by the action of two distinct enzymes, phosphoenolpyruvate
(PEP) carboxylase (EC u.1.1.e) and PEP carboxykinase (EC u.
1.1.32). These two enzymatic reactions are readily disting-
uishable by reaction reversibility and nucleotide require—
ments. The reaction catalyzed by PEP carboxylase is irrever-
sible, has no nucleotide requirement, and the phosphate group
of PEP is released as inorganic phosphate. Unlike the pre-
ceding reaction, the reaction utilizing the carboxykinase has
a specific requirement for nucleotides, is readily reversible,
and widely distributed in nature. Both of these enzymes have

been found simultaneously in N. thiooxidans (Suzuki and

 

Werkman, 1957, 1958b). The existence of these carboxylating

enzymes has been established for N. europaea(De1wiche, Burge,

 

and Malavolta, 1963) and N. agilis (Aleem, 1965) but the
predominant type has not been identified.

Malate dehydrogenases (EC 1.1.1.38 or 1.1.1.h0) which
catalyze the reversible formation of L-malate from 002 and
pyruvate have been observed in N. agilis (Smith and Hoare,
1968). The addition of acetate to an autotrophic medium
containing nitrite had no effect on the levels of enzyme in
the cell extracts. A pathway for carbon assimilation involv-
ing C02 has been recently discovered by Evans, Buchanan and
Arnon (1966) in photosynthetic bacteria. It has been describ-
ed as a cyclic process in which enzymes of the TCA cycle
Operate in reverse order and are coupled to pyruvate synthet-
ase ,cx4ketoglutarate synthese, PEP synthetase, and PEP

carboxylase. The

11
first two reactions are the key reactions in this cycle and
are ferredoxin—dependent C02-fixation reactions. These two
COZ-fixation reactions reverse two reactions of the TCA
cycle that in aerobic cells are irreversible. Therefore, it
is highly unlikely that this mechanism of COg-fixation occurs

in Nitrobacter since ferredoxin-dependent COZ-fixation has

 

been demonstrated only in anaerobic bacteria.

Effect of other inorganic ions. The mineral requirements,

 

except for the carbon and energy sources, resemble those
known for heterotrOphs. The nutrient demand in laboratory
studies is low since the number Of cells is relatively small.

The optimal iron concentration for growth of N. winogradskyi

 

is reported to be about 6 pg per ml for the oxidation of 200
pg per ml nitrite-N (Meiklejohn, 1953) but this iron level
exceeds the amount of cell carbon formed. The use of well-
defined culture methods has been recently initiated for
nutritional studies of N. agilis (Aleem and Alexander, 1960).
Using these methods and an alumina purified inorganic medium,
the requirements for various ions were studied by omitting
the nutrient under study and adding graded amounts to flaSks
in a standard series. The Optimal nutrient levels thus
obtained were approximately 5 pg per ml for both phosphorus
amd magnesium and at least 0.005 pg per ml for iron. The
optimal level Of iron established previously by Meiklejohn
may have been necessary to overcome the binding Of iron by

C3003 present in the medium. Cell mass development and

12
concomitant nitrite oxidation is enhanced in freshly inocu-
lated cultures if the medium contains small amounts of molyb-
denum (Finstein and Delwiche, 1965; Zavarzin, 1958). Zavar—
zin postulataithat a molybdo—flavoprotein is concerned in
the energy yielding reaction Of these autotrophs. According
to investigations of Finstein and Delwiche the enhancement
may simply be a response in which greater cell mass occurs
and not a direct molybdenum function in enzymatic nitrite
oxidation. A function of molybdenum.in nitrate reductase
(Nicholas, Nason, and McElroy, 1953) does suggest a function
in the reverse direction. In spite of early reports that Ca
ion was required, Alexander (1965) believes there is no valid
evidence for such a requirement. As would be expected,
potassium and sulfur are required elements (Welch and Scott,
1959) and copper is reported to be stimulatory (Kiesow, 1962,
Zavarzin, 1958). N, agilis is markedly inhibited as are

other Nitrobacter isolates by the presence Of ammonium salts

 

(Bomeke, 1950; Boullanger and Massol, 1903; Meyerhof, 1916).
In recent studies, the presence of 100 ug ammonium.per m1
completely prevented the growth of N; agilis and as little

as 10 pg ammonium per m1 delayed growth (Aleem and Alexander,
1960). In nonproliferating cell suspensions, the amount of
oxygen uptake in the presence Of 70 ng ammonium per ml is
dependent upon pH. At pH 6.0 to 7.0 no inhibition occurs,
but with increasing pH the % inhibition increases until there
is an 82% inhibition at pH 9.5. The ammonium inhibition of

intact cells apparently is unrelated to the nitrite oxidase

13
since the formation of nitrate from nitrite was unaffected

when as much as 700 ng ammonium per ml was incubated with a

cell extract.

Factors Limitinngrowth'Yields

 

A retardation in the initiation of growth when nitrite—
N concentrations exceeded 130 pg per ml was demonstrated by
Aleem and Alexander (1960). Additions of up to 500 pg per
m1 could be made without growth suppression once exponential
growth had commenced. The maximum.amount Of nitrite-N that
could be transformed was found to be NOOO pg per ml. The
soil isolate of Gould and Lees (1960) appeared to exhibit
greater sensitivity to nitrite than did N. ggilis which was
used in the investigations Of Aleem and Alexander. Gould
and Lees found that increments greater than 300’ug nitrite»
N per ml added to growing cultures resulted in a temporary
depression of the oxidation rate, and 600 pg nitrite~N add~
ed at one time delayed oxidation for at least one week.
Oxidation and growth both ceased after 2200 pg nitrite-N
had been oxidized. An important Observation made by Gould
and Less was that the rate of oxidation remained logarithmic
until 33. 200 pg nitrite—N per m1 had been oxidized. Sub-
sequently, the oxidation rate was linear and dependent upon
the rate of air flow through the culture.

Nitrate also exerts toxic effects on these organisms.
Nitrification was completely inhibited at the time of inoc-

ulation by the presence of 5000 pg nitrate—N per m1 and

11;

amounts of 1000 to 2000 pg per m1 prolonged the lag phase.
As much as 500 pg nitrate-N per ml had no effect on the rate
of nitrite oxidation of newly inoculatedcultures. Actively
growing cultures are less sensitive to nitrate-N since amounts
of 2000 to 5000 pg N per ml do not interfere with the process
of nitrite oxidation. Gould and Lees (1960) support the theory
that the accumulation of nitrate is deleterious to growth which
appears to conflict with the data presented by Aleem and Alex—
ander. Finstein and Delwiche (1965) clarified this discrepancy
when they demonstrated that nitrate accumulation did not con-
tribute to a decline in efficiency until a concentration of
0.1 M (lhOO pg N/ml) was attained.

Until very recently it was not possible to prOpagate

Nitrobacter in quantities sufficient for biochemical study.

 

Improved culture methods were first made available by Aleem.
and Alexander (1958) who propagated this bacterium in 8 liters
Of a medium free of chemical precipitate. The cultures were
grown in lO-liter serum bottles aerated with a continuous
stream of sterilized air dispersed with glass spargers.

Using this cultivation procedure, Ida and Alexander (1965)
were able to Obtain only 50 to 60 mg of dry cells per liter
of medium after 6 to 7 days of growth. Gould and Lees (1960)
improved the cell dry weight yield by combining nitrate re—
moval by dialysis and intense aeration of cultures grown in
a custom built, 5-1iter fermentor. Under these conditions,

a cell dry weight of 65 mg per liter was Obtained upon the
oxidation of 2000 pg of nitrite-N per ml. When nitrate re-

Inoval was combined with intense aeration, a 500-ml fermentor

15
culture produced 93. 1.0 g dry weight per liter per 30,000
pg nitrite-N oxidized per ml of culture (within 6.5 to 7
days) without the onset of a stationary phase. In the 500-
ml fermentor, there was a greater amount Of oxygen supplied

to the culture than in the 5-1iter fermentor.
Pure Culture Methods

Considerable difficulty still exists in obtaining cult-
ures free of contaminating microorganisms. These contamin-
ants Often remain undetected because of their morphological

similarity to Nitrobacter and their incapacity to develop on

 

conventional heterotrOphic laboratory media. Common contam-
inants in final enrichment cultures of the nitrifiers Often
include species of Pseudomonas, Hydromicrobium, Mycobacter-

ium, Flavobacterium, and Serratia as well as an occasional

 

 

myxobacterium (Gundersen, 1955). A symbiotic relationship

may exist between Nitrobacter and members Of these species

 

which might explain the growth of the autotroph in unfavor—
able soil environments. In the laboratory, the problem of
contaminants is not ended once the nitrifier is isolated in
pure cultureas recontamination during laboratory manipulations

occurs quite readily (Garretson and San Clemente, 1967).
Metabolism of Organic Compounds

Since WinogradSky (1891) first isolated Nitrobacter in
pure culture, numerous reports have appeared on the influence

of organic compounds on growth and nitrification. Unfortun—

16
ately, the concept of nonutilization of organic compounds
and indeed their inhibitory effect at relatively low con—
centrations which originated with the early work of Wino-
gradsky was carried over in subsequent investigations on the
utilization of organic amendments. The use Of cultures of
mixed flora and unrefined growth conditions has further pro-
pagated the concept of organic inhibition.

During the last decade, a number of attempts have been
made to account for the failure of the nitrifiers, and other
species of chemolithotrophs, to utilize organic compounds as
sole carbon sources. Evidence for the leek Of a permeability
barrier in N. agilis was simultaneaouly presented by Ida and
Alexander (1965) and Delwiche and Finstein (1965). These
investigators found that acetate, glycine, hypoxanthine, and
glycerol were readily incorporated into the cells. However,
neither group of investigators was able to demonstrate growth
of the organism on organic compounds. A lack of stimulation
in nitrite oxidation was Observed with a number of amino acids
and vitamins, including biotin (Aleem and Alexander, 1960).
Krulwich and Funk (1965) reported enhancement of both nitrite
utilization and growth with biotin using four strains of N.
agilis. This stimulation may be artificial since the Optimal
growth yields, as compared to results Obtained by other
investigators, were not obtained from the control cultures.

A slight stimulation of nitrification and growth, as deter—
mined by Optical density, was reported with yeast extract,

Vitamin Free Casamino Acids, and some amino acids (Delwiche

17
and Finstein, 1965). Unfortunately, only Optical density
was used for measuring growth in the presence Of all of the
compounds, except yeast extract. Growth of cultures contain-
ing yeast extract resulted in an increased viable cell count.
The oxidation of formate has been demonstrated in both N.

agilis and N. winogradskyi (Malavolta, Delwiche, and Burge,

 

1962; Van G001 and Laudelout, 1966). The oxidizing enzyme
bears a resemblance to formic dehydrogenase of heterotrophic
bacteria in that it is cytochrome specific, particulate, and
not successfully solubilized. The enzyme shows maximum act—
ivity around pH 7.0 and is stimulated by ATP. However, no
growth was Obtained when formate was the sole energy source
(Van G001 and Laudelout, 1966) and formate contributes only
a small fraction of carbon to resting cells (Delwiche and
Finstein, 1965).

Absence from the cell of the appropriate enzymes to
oxidize permeable organic molecules has not been established
for N, agilis. Cell-free extracts are known to contain
acetyl-coenzyme A (COA) synthetase (EC 6.2.1.1.) and all
of the enzymes of the TCA cycle (Aleem, 1965; Smith and
Hoare, 1968).

In N. agilis, the generation of assimilatory power,
production of ATP and reduced pyridine nucleotides, is
coupled to the oxidation Of nitrite (Aleem, 1965). A
particulate NADH oxidase (EC 1.6.99.3) has also been found
in crude extracts (Smith and Hoare, 1968). Thus it is cer-

tain that Nitrobacter has the assimilatory power which could

 

be coupled to the oxidation of acetate or other organic

18

compounds.

Until Smith and Hoare (1968) suggested that N. agilis
was a facultative autotroph, this microorganism was class-
ified as an obligate autotrOph. This classification had
been previously unchallenged for over 60 years. The reason-
ing of Smith and Hoare was based on the ability of this org-
anism to grow "heterotrophically through seven transfers" on
a medium containing both acetate and casein hydrolysate. If
their results can be confirmed, other obligate autotrophs
should be examined for their growth potential under similar
growth conditions. If presumptive obligate autotrophs prove
to be capable of growth on organic substrates then more
appropriate criteria will have to be applied in the classifi-
cation of autotrOphic microorganisms.

A biochemical basis of obligate autotrOphy in blue—green
algae and thiobacilli was proposed by Smith, London, and
Stanier (1967). The absence of x-ketoglutarate dehydrogenase
(EC 1.2.u.2) and NADH oxidase was reported in the obligate

autotrophs N. thiooxidans and N. thiOparus, but the faculta-

 

 

tive autotrOphs N. intermedius and Hydrogenomonas eutrOpha

 

did not possess these enzymatic deficiences. However, the
presence of NADH oxidase activity has been established by
other investigators in obligate autotrophs including Thio-

bacillus neapolitanus, N. thiOparus, and N. thiooxidans

 

 

 

(Rittenberg, 1969). Among the nitrifiers, both déketo-
glutarate dehydrogenase and NADH oxidase activity have been

detected in Nitrosomonas oceanus (Williams and Watson, 1968)

 

and N. agilis (Smith and Hoare, 1968). In N. europaea both

 

19
these enzymes are lacking (Hooper, 1969). As in the thio-
bacilli, nitrifiers appear to be inconsistent in respect to
the activity of these enzymes, and no common basis for obli-
gate autotrophy has been established.

The metabolic response of autotrophs to growth in a
medium containing acetate may prove to be valid in character-
izing differences between obligate and facultative chemo-
autotrophs. In the facultative chemoautotroph, T. iptgp-
medius, approximately u0% of the carbon in newly synthesized
cellular material is contributed by acetate, whereas in a
number of obligate autotrophs acetate contributes only 10%
of the carbon in newly synthesized cellular material (Smith,

London, and Stanier, 1967).

Synthesis of Storage Materials

 

The presence in Nitrobacter of sudanophilic granules

 

containing poly-fi-hydroxybutyrate (PHB) was first described

by Tobback and Laudelout (1965). A massive accumulation of
this reserve material was reported to occur within N. agilis
cells cultured either in the presence of acetate and casein
hydrolysate, or in the presence of acetate and limited nitrite
over a prolonged period (Smith and Hoare, 1968; POpe, Hoare,
and Smith, 1968). Lesser amounts of PHB were evident when
growth was on a mineral medium supplemented with acetate and
unlimiting nitrite.

Members of the genus Hydrogenomonas, which are faculta-

 

tive chemolithotrophic bacteria, accumulate PHB under condi-

tions where energy and carbon sources are available but

20
growth and multiplication are limited by the lack of some
additional factor, for example, nitrogen or phosphorus

(Schlegel, 1968). In Hydrogenomonas, PHB is known to function

 

as storage material for protein synthesis when the usual con-
ditions for growth are absent. These organisms also store
phosphate primarily as polymerized inorganic metaphosphate
(polyphosphate, volutin). Since the amount of energy thus
fixed is extremely low, the storage of polyphosphate is
significant only for phosphate balance. The storage of

polyphosphate in Nitrobacter has not been investigated.

 

Acetate is incorporated as a total unit during PHB

synthesis in Hydrogenomonas. The pathway proceeds via acetyl-

 

COA, acetoacetyl—COA, K—hydroxy-butyryl-COA, poly16-hydroxy-
butyrate. The synthesis of PBH from 002 by the reductive
pentose phosphate pathway gives uniformly labeled acetate

and PHB. This pathway occurs via 3-PGA, 2-PGA, PEP, pyruvate,

and acetyl-COA.

Physiological Classification of Chemolithotrophs

 

In an excellent review Schlegel (1968) described the
manner in which cells derive cell carbon and energy from
organic matter in terms of our present knowledge of the
chemolithotrophic soil bacteria. The terms "autotrophs"
and "heterotrOphs" are unequivocal and well understood to
mean organisms which can synthesize their cell material from
C02 as the main source of carbon or from preformed organic
substances, respectively. However, it should be emphasized

that autotrophs are those organisms which use 002 as the main

21

source of carbon and that occasionally they can grow with
organic substrates is of lesser importance. The term chemo-
lithotrOphic pertains to organisms which derive metabolically
useful energy from inorganic oxidations and the question of
the derivation of cell carbon is irrelevant.

Rittenberg (1969) uses the term "mixotrophy" to describe
a commingling of alternative modes of energy generation, or
carbon assimilation, or both. A number of variations in the
combinations involved in chemolithotrophic metabolism are
possible and have been observed in a number of soil bacteria.

Mixotrophic metabolism as seen in Desulphovibrio desulph-

 

uricans is the assimilation of organic substrate with the
aid of energy obtained from an inorganic oxidation. These
bacteria do not grow autotrOphically and their lithotrOphy
is not obligatory. Another combination which occurs in

Micrococcus denitrificans is the inability to produce most

 

of its cellular carbon by COg-fixation because of low activ-
ity of the enzymes of the reductive pentose phosphate cycle
and therefore a dependence on supplementation with organic
substrates. A third method is the conversion of either
carbon dioxide or organic substances to cellular carbon using
energy only from the oxidation of inorganic compounds. 0n
the basis of the findings of Smith and Hoare (1968), Ritten—
berg classified N. agilis as a mixotrOph, which uses in-
organic and organic energy sources concurrently, rather than
as an obligate chemolithotrOph.

Historically, the facultative autotrophs are recognized

22
by their ability to grow abundantly in either inorganic or
organic media. In the presence of organic substrates,
members of this group can either express simultaneously
alternate physiologies, or the organic growth substrates
have an inhibitory effect on inorganic oxidations. Smith
and Hoare (1968) classified N. agilis in this group, but as
suggested by Rittenberg the present day usuage of the term
"facultative autotrOph", as well as "obligate autotrOph"

should be eliminated.
Established Metabolic Pathways in Acetate Metabolism

Tricarboxylic acid cycle. The TCA cycle which is responsible

 

for the terminal respiration in both animal tissues and many
organisms is believed to operate in N. agilis as all of the
enzymes are present in sufficient amounts (Smith and Hoare,
1968). One of the functions of this cycle in heterotrophic
organisms is to bring about the oxidation of acetate but it
cannot function on compounds more oxidized than acetate
(Kornberg and Elsden, 1961). In addition to its oxidative
function, the cycle plays a part in the synthesis of many
intermediates, including the precursor of glutamate,
«i-ketoglutarate (Roberts 22 g;., 1955). In order for
synthetic processes to take place at the same time as oxida-
tion, there must be a continual draining off of both d4keto—
glutarate and the h-carbon dicarboxylic acids. The latter
compounds also serve as important precursors of many cell

constituents. For growth to occur on acetate as both the

23
carbon and energy source, ancillary reactions are necessary

for the formation of TCA cycle intermediates.

Glyoxylate bypass. The glyoxylate cycle has been widely

 

observed in bacteria, fungi, algae, protozoa, nematodes,

and plant tissues (Wegener 23 gl., 1968). The glyoxylate
cycle supports the continued Operation of the TCA cycle

and its net effect is the formation of 1 mole of malate from
2 moles of acetate (Kornberg and Krebs, 1957). The key
enzymes whose simultaneous action produces this cyclic
mechanism are isocitrate lyase (EC h.l.3.l) and malate
synthase (EC h.1.3.2). Isocitrate lyase which catalyzes the
reversible aldol cleavage of isocitrate to succinate and
glyoxylate, has been observed in chemoautotrophic bacteria
grown heterotrOphically, including N. facilis (McFadden

and Howes, 1962), N, denitrificans (Kornberg, Collins,

and Bigley, 1960), as well as in N. agilis (Smith and Hoare,
1968). The condensation of glyoxylate with acetyl—COA to
form malate is catalyzed by malate synthase. Enzyme activ-
ity for this reaction has not been reported for N. agilis,
but malate synthase is usually present in microorganisms in
which isocitratase is also present (Kornberg and Elsden,

1961).

MATERIALS AND METHODS

Growth Conditions

 

Nitrobacter agilis (ATCC 1hl23), kindly provided by

 

David Pramer (Rutgers-—The State University) was maintained
by continuous passage in the liquid autotrophic medium of
Smith and Hoare (1968) which was supplemented with 0.15%
KHco3 (w/v) and 0.03% 1131102 (w/v). The pH of the complete
medium after autoclaving was 33. 9.0. Media, however, for
experimental cultures were prepared by supplementation with
various carbon and energy substrates of the liquid autotroph-
ic medium (subsequently referred to as a basal medium). The
basal medium contained 2.55 g NagHPOu, 0.27 g KHgPOM, 20 mg
MgSOu-7H20, 2.5 mg CaClZ-HQO, 10 mg FeSOu-7H20, 11 mg Nag-
ethylenediaminetetraacetic acid, 0.02 mg H3B03, 0.10 mg
CuSOu-5H20, 0.02 mg MnSOu-2H20, 0.02 mg (NHu)6Mo702u-hH20,
0.15 mg ZnSOuo7H20, 0.01 mg 00012 and deionized distilled
water to 1 liter. The following additives were used: sodium
acetate, 5 mM; Vitamin Free Casein Hydrolysate, 0.05% (w/v)
(Nuritional Biochemical Corp.); glutamate, l or 2.7 mM;
KHCOB, 0.15% (w/v); NaN03, 100 pg N/ml; (NHu)2sou, 7.81 pg
N/ml; and NaNOZ, at concentrations up to 1HOO|P8 N/ml.

2A

25

Cell growth was usually followed turbidimetrically in
a Spectronic 20 colorimeter (Bausch and Lomb) at MAO nm
and by chemical analysis of nitrite-N disappearance using
the method of Rider and Mellon (l9h6). The autotrophic
growth medium solidified with 1.5% (w/v) Noble Agar (Difco)
was employed for viable counting by the pour plate method.
The determination of ammonia was by direct nesslerization
using the method of Peach and English (l9hh). Nitrate-N
was estimated according to the procedure of Chase (l9h8).

Experimental shake flask cultures were initiated by a
6.25% (v/v) inoculum from autotrOphically grown cells which
had completely utilized 33. 1000 pg nitrite-N per ml. These
cultures were incubated on a rotary shaker at 32 C in 2—1iter
flasks containing a final culture volume of 800 m1. Station-
ary cultures were inoculated with 2.5 X 10“ organisms per ml
and grown at room temperature in 250 ml Erlenmeyer flasks
containing a final culture volume of 80 ml.

Cell suspensions utilized in the hot alcohol (75%)
extraction of luC-labeled cellular intermediates were obtain-
ed from fermentor cultures. An MF-lh Microferm fermentor
(New Brunswick Scientific CO., Inc.) equipped with two 1h-
liter capacity fermentor jars was operated at 30 C with a
propeller speed of 200 to 3000 r.p.m. and an aeration rate
of 2 to 6 liters of filtered air per min. The fermentor
jars each contained 10 liters of medium identical, except
for a nitrite-N concentration of 200 pg N per ml, to that

previously described for shake flask cultures. Both MgSOu

26

and CaC12 were autoclaved as a separate solution and combined
with the remaining medium constituents which had been auto—
claved in the fermentor assembly. First passage fermentor
cultures were initiated by the transfer of 370 ml of shake
flask culture which had oxidized 1200 pg of nitrite-N per

ml. Second passage fermentor cultures were inoculated with

a 25% (v/v) inoculum taken from a fermentor culture in the
logarithmic growth phase. Exponential growth was maintained
by the addition of 200 to hOO pg N per ml whenever the level

of nitrite-N drOpped below 100 pg per m1.

Culture Purity

 

Cell cultures were continuously monitored for hetero-
trophic contamination by pouring 0.5 m1 of the culture over
the surface of the following agar plates: Nutrient Agar
(Difco), Potato Dextrose Agar (Difco), nitrite-free auto-
trophic medium containing 0.5% yeast extract, and nitrite-
free autotrophic medium containing 0.2% glucose. The inoc-
ulated plates were incubated at room temperature for 10 days.
A further check on culture purity was made by microscopic
examination of cell pellets which were stained by the Gram
stain. A11 heterotrOphic contaminants except pseudomonads
are readily distinguishable from Nitrobacter by this method.
However, the common species of pseudomonads are easily detect—

ed by subculture on agar plates.

27

Preparation of Cell Suspensions and Chemical Analysis

Shake flaSk cultures were harvested by centrifugation
at 9,500 X g for 20 min and the cell pellets rinsed twice
with basal medium and once with distilled water. The rinsed
cells were then suspended in small volumes of deionized
distilled water. Cell cultures grown in the Microferm were
terminated by cooling to 15 C and then harvesting the cells
by continuous flow centrifugation (Sorvall Model SS-l cen-
‘trifuge equipped with the Szent-Gyorgyi and Blum continuous
flow system). The sedimented cells were washed twice in
cold basal medium and suspended in deionized distilled water.
Total protein content was evaluated by digesting the cells
in an equal volume of l M NaOH at no 0 for 2 hr, and then
assaying according to the method of Lowry 23 3;. (1951).
Crystalline bovine serum albumin was used as standard. Poly-
B—hydroxybutyrate (PHB) was extracted from N. agilis using
the procedure of Tobback and Laudelout (1965). Approximately
600 to 800 ml of cell culture fluid from shake flask cultures
of N, agilis containing 20 to hO mg cell dry weight per liter
were used for the extraction of PHB. The cell pellets ob-
tained by centrifugation were washed twice with 20 mM phos-
phate buffer (18 mM NaZHPou and 2 mM KHZPOA, pH 7.65) and
suspended in a final volume of 10 ml with cold distilled
water. Between h to 5 ml of the washed cell suspension was
treated under continuous mixing for 3 hr at 30 C with an
alkaline solution of hypochlorite (Williamson and Wilkinson,

1958). The sediment obtained after centrifugation was

28
washed three times with distilled water and then success-
ively washed with 95% (v/v) ethanol, acetone and ether. The
residue was twice extracted with 6 ml volumes of boiling
chloroform. Assays for PHB content were performed using a
suitable volume of the chloroform extract. The methods of
Williamson and Wilkinson were also employed for the crude

isolation of the standard PHB from Bacillus megaterium strain

 

KM cultures grown in Trypticase Soy Broth (BBL). The stand-
ard PHB was further purified and assayed as described by Law
and Slepecky (1961). Eight 2—liter Erlenmeyer flask cultures
containing 500 ml of medium were grown for 19 hr on a rotary
shaker at 30 C. The cells were sedimented at 9,500 X g for
10 min and washed three times with 0.85% NaCl. The cells
were digested at room temperature in alkaline hypochlorite
for 1 hr and the PHB granules were washed successively with
acetone, alcohol, and ether, and then dried under vacuum to
a constant weight. The polymer was extracted with 100 m1 of
boiling chloroform and then filtered over celite. PHB was
crystallized from 500 ml of acetone cooled to -200 and the
crystals collected by filtration. A yield of 0.981 g of
purified PHB was extracted from 28.8u g wet weight of B.

megaterium cells.

 

Isotopic Labeling Proceduregfor Growinngultures

The incorporation of acetate and bicarbonate by cells
during growth was investigated in 2-1iter Erlenmeyer flasks.

The culture medium was amended before inoculation with

29

sodium acetate-l-luC (lO‘pC) or sodium bicarbonate (1‘pC)
sterilized by passage throughMillipore filters (HA grade).
After all additions were made, the cultures had a final
volume of 300 ml. To determine the rate of uptake of 1&0 by
N. agilis, 5 m1 samples were taken at 12 hr intervals and
drawn through Millipore filters (HA grade). The filters
were rinsed twice with cold 50 mM acetate and twice with
cold water, dried, and cemented to aluminum planchets. The
amount of isotope in the cells retained on the filter was
determined using a model U70 gas-flow detector (Nuclear-
Chicago Corp., Des Plaines, 111.) with an efficiency of 30%.
Samples (0.1 to 0.2 ml) of the whole culture fluid or cell
fractions were mixed with 15 ml of Bray's solution (1960)
and counted in a Nuclear-Chicago (Mark II) liquid scintil—
lation counter with an efficiency of approximately 75%.

Cultures which had been labeled with isotope were
harvested after a minimal absorbence of 0.12 at AHO nm had
been attained and prior to the complete exhaustion of
nitrite—N. Washed cell pellets were prepared as described
previously and then fractionated by the method of Roberts
32 El. (1955). The washed cell pellets obtained from 33.
235 ml of culture fluid were suspended in h m1 of 75% (v/v)
ethanol, heated for 30 min at no to 50 C, centrifuged, and
the supernatant fluid collected as the alcohol-soluble
fraction. This fraction was further extracted by the addi-
tion of equal volumes of ether and water, and then by the
addition of just ether. The ether soluble fractions were

pooled and retained as the alcohol—soluble-ether—solub1e

30
fraction. The alcohol-insoluble precipitate was suspended
in h ml of a mixture containing 2 ml of ether and 2 m1 of
75% ethanol, heated for 15 min at hO to 50 C, centrifuged,
and the supernatant fluid collected as the alcohol-ether-
soluble fraction. The precipitate was further extracted by
adding h m1 of 5% (v/v) trichloroacetic acid (TCA) and boil-
ing for 30 min. The resultant supernatant fluid was desig—
nated as the hot TCA-soluble fraction. The residual material
was then successively washed with h ml volumes of acid alco-
hol ( 3 m1 of HCl added to 100 ml of 70% (v/v) ethanol) and
ether. The insoluble residue was suspended in 2.2 ml of
distilled water and 1 m1 portions analyzed for protein or
PHB content. For the isolation of PHB, carrier PHB (5 mg)
was added to one of the portions and then the polymer iso-
lated according to the method of Tobback and Laudelout
(1965). Another portion of the cell residue was hydrolyzed
in 6 N H01 for 15 hr at 105 0. Samples of the various
fractions were added to Bray's solution, counted by the
liquid scintillation method, and counts per min corrected

for background and quenching using the channels ratio method.
Isotopic Labeling of Fermentor Grown Cell Suspensions

A 12.5 ml volume of washed cells suspended in deionized
distilled water (at a concentration of 100.6 mg wet weight
per ml) was placed in a 125 m1 Erlenmeyer flask containing a
double strength basal medium supplemented with ADD pg nitrite-
N per ml and 10 mM sodium acetate. The diluted cell suspen-

sion was then incubated at 30 C in a gyrotory water bath

31
shaker (Model G 76, New Brunswick Scientific Co., Inc.) and
equilibrated for 55 min prior to the addition of sodium
acetate-2-lu0 (200 pC). The addition of the labeled acetate,
which was suspended in 2 ml of sterile deionized distilled
water, resulted in a final cell suspension volume of 29 m1.
At various time intervals 8 2 ml sample was removed from the
cell suspension and immediately transferred to a centrifuge
tube containing 6 ml of absolute ethanol at 80 C. The alco-
holic mixture was subsequently heated at 50 C for 30 min and
the insoluble precipitate removed by centrifugation. The
alcohol-insoluble fraction was rinsed with 2 m1 of 20% (v/v)
ethanol heated to 80 C and the supernatant fluid combined
with the initial alcohol-soluble fraction. The alcohol-
soluble extract was then evaporated to dryness at 50 C and

the residue suspended in 2 m1 of 20% ethanol.

Paper Chromatography

 

Whatman no. 1 paper (A6 by 57 cm) was employed for the
2 dimensional separation of labeled cellular intermediates
in the ethanol-soluble fraction of cell suspensions. For
the chromatography of the organic acids and organic phosph-
ates, the papers were prewashed by either descending flow
or soaking in a trough for 30 min. Washing with 0.1 N HCl
was followed by washing with distilled water. For the re-
solution of the organic phosphates, the above washes were
preceded by a washing with 0.2% (w/v) ethylenediaminetetra-
acetic acid (EDTA), pH 8.5, to remove metallic ions. Each

of the ethanolic extracts was applied to chromatograms

32

spotted with all of the amino acid standards. The chromato-
graphic separation of the phosphorylated compounds was done
in a similar manner. However, this procedure was not used
with most of the organic acids as larger concentrations of
the reference compounds were required for identification and
certain of the acids were not well separated. The latter
compounds were separated into 2 groups: one containing malate,
succinate, fumarate, and pyruvate, and the other containing
citrate, isocitrate, and glyoxylate. All of the ethanolic
extracts were run individually with each group of standard
compounds. Control chromatograms were prepared simultaneously
using the same reference compounds which were located using
various indicator sprays. Amino acids were identified using
0.25% (w/v) ninhydrin in acetone. Organic acids were detect-
ed by spraying an 0.5% (w/v) ethanolic solution of bromo-
cresol green. Organic phosphates were identified primarily
by spraying with an ammonium molybdate solution (Hanes and
Isherwood, 1949) which was modified by the addition of 0.1%
EDTA and followed by reduction of the phosphomolybdate com-
1exes with ultraviolet light. An aniline hydrogen phthalate
spray (Block, Durrum, and Zweig, 1958) was used for the de-
tection of ribose-5-phosphate.

Different solvent systems were employed for each of the
classes of compounds investigated. For amino acids, the
chromatograms were first developed with butanol—acetic acid-
water and then with phenol-ammonia-water (Smith, 1960).

Organic acids were separated using an ethanol-ammonia-water

33

solvent system followed by a butanol-acetic acid—water sol-
vent system.(Nordmann and Nordmann, 1960). The method of
TysZkiewicz (1962) which employs isobutyric acid-ammonia—
EDTA for the first direction and butanol—propionic acid—
water for the second direction was employed for the separa-
tion of the organic phsophates.

After the location of the various compounds had been
established by chemical tests, the areas suspected of con-
taining the isotopic label were scanned for radioactivity
with a Model 108 mica end-window detector tube (Nuclear—
Chicago Corp., Des Plaines, 111.). Radioactive areas
corresponding with the sites of the compounds identified on
the control chromatograms were cut out within M X 6 cm rec-
tangles and counted in 15 ml of a toluene scintillation sol-
ution with a liquid scintillation counter. The scintilla-
tion solution was prepared by dissolving h g of 2,5—diphenyl-
oxazole and 0.05 g of 1,h-bis-2(5—phenyl-oxazoyl)—benzene in

1 liter of toluene.

RESULTS

Growth on Acetate Combined with Various Sources of Nitrogen

 

Stationary cultures of N. agilis were grown at room
temperature for 21 days in the basal medium supplemented
with various combinations of nitrogen and carbon substrates.
Only the control autotrophic cultures contained sodium bi-
carbonate but carbon dioxide of atmospheric origin was avail-
able to the cultures. The results of this growth experiment
(Table 1) clearly demonstrate that acetate in the absence of
nitrite as an energy source cannot adequately support the
growth of N. agilis. Neither glutamate nor nitrate additions
to the acetate medium could maintain culture viability let
alone support meaningful cell multiplication. Assays for
nitrate-N content made periodically during the growth period
shown no change in nitrate content. Ammonium sulfate, added
at nontoxic levels to the acetate medium, maintained culture
viability but a significant increase in plate count was not
Observed. The consumption of ammoniumrN was slight but of
sufficient magnitude for the maintenance of a viable cell
population. Growth in flasks containing both acetate and
casein hydrolysate was evident but the low cell yields

offered little promise for metabolic study.
311

35

TABLE 1. Influence of various carbon and nitrogen sources on.

nitrification and growth of Nitrobacter agilis

 

 

 

 

Nitrogen b
a Viable cell count
Additions consumed
(cells/ml) (% of control)
(pa/ml)
NaNOg + KHCO3 900 1.57 x 107 100
NaNo2 919 n.9h x 107 315
NaNo2 + CHBCOONa 886 2.67 x 107 170
CH3COONa +
Casein hydrolysate 5.3h X 105 3
CHBCOONa + NaNo3 0 3.09 x 102 -—
h
CHBCOONa + (NHu)280u 2 8.80 x 10 <1
CHBCOONa + Glutamate ——d
CHBCOONa 1.20 x 10“ -—0

 

 

 

 

a

Concentrations of the carbon and nitrogen

described in the text.

sources are

All cultures were initiated with 2.5 X 10h cells per m1 and

then grown for a period of 21 days without forced aeration

in a basal medium supplemented with carbon and nitrogen as

indicated under Additions.

 

four replicate flaSk cultures.

The results are mean values of

The number of viable cells after 21 days was reduced to a

level below that contained in the culture at zero time.

36
TABLE 1. continued.

 

d
Plate counts were not made as there was no increase in

absorbence.

37

A comparison of the results obtained with the auto-
trophic cultures in the presence and absence of bicarbonate
indicated that the requirement for bicarbonate ions can be
met with atmospheric carbon dioxide, even in the absence of
forced aeration. Apparently the lowering of the pH of the
medium to 33. 7.6 by the omission of bicarbonate also had no

effect on nitrite oxidation.
HeterotrOphic Growth on Acetate and Casein Hydrolysate

As indicated in Table 1, growth on a nitrite—free medium
containing acetate and casein hydrolysate was barely percept-
ible. The small increase in cell number most likely was sup-
ported early in the growth period by nutrients and cellular
intermediates carried over from the previous cell passage in
an autotrOphic medium. The effect on growth by the combina—
tion of acetate and casein hydrolysate, however, was exhaus—
tively pursued in numerous experiments in both stationary
and shake flaSk cultures. In preliminary investigations
growth was consistently noted in the heterotrophic medium as
evidenced by increased absorbence. Occasionally these cul-
tures was overtly contaminated and were discarded, but in the
remaining cultures, which appeared to be contaminant-free,
growth rates mimicked those reported by Smith and Hoare (1968).
In,the latter cultures, heterotrophic contamination was not
readily detectable by inoculation into either the media rou—
tinely used in testing for culture purity, or in the follow-

ing media: Brain Heart Infusion (Difco), Nutrient Broth

38

(Difco), and agar supplemented with nitrite-free autotrophic
medium containing acetate and casein hydrolysate at the same
concentrations employed in shake flaSk culture. Frequently,
growth of one to four colonies was noted on Nutrient Agar

and less often on yeast extract agar. Initially, the appear-
ance of these colonies was interpreted as arising from acci-
dental exposure to environmental contaminants. After two
successive passages of 17 and 28 days, one of these hetero—
trOphic cultures was subcultured into the autotrophic medium.
After a greatly prolonged period of 18 days nitrite oxida-
tion was observed, indicating the survival of N. agilis in
an organic medium. Subsequent microscopic investigations of
cell pellets indicated the presence of gram-positive cocci
in grape-like clusters in these cultures as well as in all

of our stock lines. Since Staphylococcus aureus is widely

 

used in other studies carried out in this laboratory, it is
assumed to have been the contaminating organism. The growth
of this contaminant on conventional media showed little re-
semblance tO S. aureus insofar as growth rates were concerned.
This phenomenon suggests the possibility of a biochemical
mutation or selection having occurred during prolonged growth
in association with N. agilis. Growth of this contaminant

was suppressed in the autotrophic cultures, but in the medium
supplemented with acetate and casein hydrolysate growth was
greatly stimulated. No colonies of N. agilis were observed

on any of the organic agars. This was confirmed by staining
smears of representative colonies using the Gram stain. Those

colonies which did appear on Nutrient Agar were made up en-

39

tirely of the coccal forms.

Growth studies were later renewed using a newly receiv-
ed culture Of N, agilis which was scrutinized for contamina-
tion and found to be entirely free of heterotrophic micro-
organisms. Repeatedly, the inoculation of the heterotrOphic
medium with pure cultures of N. agilis resulted in either
contaminated cultures or no growth at all even after 30 to
hO days incubation. The simultaneous inoculation into auto—
trophic media resulted in detectable nitrite oxidation with-

in h days in all cases.

Synthesis of Poly154hydroxybutyrate (PHB)

 

Growth in the presence of acetate was evaluated at 32
C in 2-liter shake flaSks containing the basal medium supple-
mented with acetate and nitrite and in some cases bicarbonate.
Cultures were maintained for 89 to 10h hr with a continuous
supply of nitrite. Prior to the induction of the stationary
phase of growth, samples of these experimental cultures were
analyzed for protein and PHB content (Table 2). In the ab-
sence of acetate, 0.3 to 0.h% PHB (on a dry weight basis) was
obtained whereas cultures grown in the presence of acetate
had polymer concentrations of 5.9 to 12.2%. The absence of
cellular material which might interfere with the accuracy of
the PHB assay was established by spectral analysis at wave
lengths of 220 to 280 nm. The profiles obtained were identi-

cal to those of purified polymer isolated from B. megaterium

 

(Fig. 1.).

hO

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oopooHHoo mHHoo one opflmpws wsflpflsflasd flpfiz moHSpHSO oxmnm mm QBOnw onoz mfimflsmmmo
m
e.oa H.0m o.:m mmm :oa moome.+
m.s e.eH m.eH ewe om aeooommo + mozsz
o.m s.sm :.mm mme :oa
N.NH 3.3m m.mm 0mm ow szoommu + moZmz
:.ov e.om m.om fieHH mo
m.ov s.bm 0.0m mmHH we moons +.mozsz
DARV DARV AQ\mSV AHE\mlq Ahflv moonSOm sopsmo
mmm sfiopong .pz hep pegbmsoo oowpom one Gomonpflz
fleece deco acmoapaz cheese

 

 

 

oompoom no

QBOAm madame sopompompfiz Ga Ammmv oaaphpfiphxonphnnqrhaom Mo

oesosnfiooo .N mqm<e

FIG. 1. Ultraviolet absorption spectra of crotonic acid
formed from polyjfi-hydroxybutyrate isolated from cells of

Bacillus megaterium strain KM and Nitrobacter agilis after

 

heating in concentrated H280“. Symbols: I, purified poly-

mer from B. megaterium cells; a», polymer extracted from N.

 

agilis cells grown on basal medium plus acetate, nitrite,
and bicarbonate;.t, polymer extracted from N. agilis cells

grown in the same medium lacking bicarbonate.

 

w ‘_ ‘i ~—
—— .

LLl

 

 

 

 

 

 

280

260

lEIIlI.

24o
'I'E

220

172
Effect of Acetate on Macromolecular Synthesis

From calculations made using the data of Table 2, it
‘was possible to identify various relationships existing be-
'tween macromolecular synthesis, culture density, and nitrite
‘oxidized (Table 3). It was immediately evident that the
synthesis of PHB during nitrite oxidation was not directly
correlated to increased cell density as had been suggested
'by previous investigators (Smith and Hoare, 1968). The
addition of acetate to the medium stimulated PHB synthesis
greater than lO-fold but decreased the absorbance to dry
“weight ratios. If the presence of this lipid polymer were
solely responsible for greater optical density, then cells
containing the larger amounts of PHB would have had higher
AMHO nm per dry weight value . However, this was not the
case as higher absorbance readflngs were obtained with the
autotrOphically grown cells containing the least amount of
PHB and the most protein.

In order to gain insight into the effect of acetate on
cell physiology, nitrite oxidation was compared with the
production of cell mass and protein. On a dry weight basis,
cultures containing acetate oxidized less nitrite than did
the autotrophic cultures. However, on the basis of protein
synthesized per nitrite oxidized, the values were higher
for both the autotrOphic cultures and the acetate cultures
lacking bicarbonate than for the cultures containing the
combination of acetate plus bicarbonate. It is concluded

that less of the energy made available through nitrite oxi-

M3

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m

 

 

   

ms

 

 

 

 

 

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mo.b Hmo.o ::.m mod sZOOommo + woesz
H:.e :mo.o em.m awe
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awesome \\MHM\N::< . ash oena eds cowosaaz

 

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Au
dation is coupled with the mechanism for protein synthesis

during growth in a medium containing both acetate and bi-

carbonate.

Uptake of Isotopically Labeled Acetate and Bicarbonate

by Growing Cultures

The rates of incOrporation of acetate—l-luC (7.7M X lOLL
dpm per ml) and sodium bicarbonate (7.79 X 103 dpm per ml)
were followed in 2-liter shake flaSks containing a final
culture volume of 300 m1. Except for the addition of the
labeled substrates and a reduced culture volume, growth
conditions were identical to those of the previous experi-
ment. A duplicate series of cultures in which isotopes were
omitted was included in this study for following nitrite
oxidation and absorbance changes during the course of this
experiment. The use of duplicate cultures minimized the

lb.

frequency and amount of sampling of the C-containing cul-
tures. The uptake of luC and absorbance are plotted against
nitrite oxidation in Fig. 2.

As in the previous experiment, the addition of bicarbon-
ate to cells growing on acetate resulted in a decrease in
growth rate per unit of nitrite oxidized. This was evident
by comparison of the rates of increase in absorbance shown
in Fig. 2B and 2D with 2 A. The rates of uptake of acetate-
l-luC approximated the absorbance rates. In addition, un-
labeled bicarbonate does not appear to compete with labeled

acetate for entry into the cells. That acetate competes with

labeled NaHlI‘i’CO3 for uptake by N. agilis is shown in Fig. 2C

FIG. 2. Absorbance (O) and uptake of lLl-C-labeled acetate
or bicarbonate (O) in Nitrobacter agilis growing in a miner—
al medium containing sodium nitrite. (A) acetate-l-luC.

(B) acetate-l—luC and unlabeled bicarbonate. (C) bicarbonate-

18'0. (D) bicarbonate-180 and unlabeled acetate.

 

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he
and 2D.

Incorporation of CquuCOONa and NaHluCOq into Cellular

Fractions of Growing Cultures

The addition of unlabeled bicarbonate to cultures con-
taining acetate-l-luC resulted in a 25.1 % reduction in the
total radioactivity (disintegrations per min per ug cell dry
wt.) recovered in all of the combined fractions (Table A).
Therefore, it appears that approximately 25% of the radio-
active carbons incorporated from acetate-l-luC were replaced
by unlabeled carbon atoms. However, when unlabeled acetate
competed with labeled bicarbonate, a calculated reduction in
radioactivity of 80% was attributed to the replacement of
labeled carbon atoms with unlabeled carbon.

The percent distributions of 1MC among cellular frac-
tions from cultures grown on labeled acetate in the presence
and absence of bicarbonate were also compared with each other
and those Of autotrOphic cultures grown on labeled bicarbon-
ate. The distribution pattern obtained with lLiC—acetate as
a tracer was little affected by the presence of unlabeled
bicarbonate. When the contribution of PHB was omitted in
the recalculation of the percent distributions, the percent-
ages of both the alcohol and protein fractions of the two
cultures differed from their former values by only 2%. The
values for the alcohol—soluble fractions were increased to
23.6% for the acetate culture and to 21.6% for the acetate
plus bicarbonate culture, while the protein fractions in-

increased to 55.h and 58.u%, respectively. When these

A7

 

 

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szOoommo m moomsz m

mouammsz ooefimsz szOoomHmmo ezOooaa mo nonsense

 

 

 

 

wepwmpmnsm Gopnmo

 

 

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L19

values were compared with those obtained from cultures pro-
vided with labeled bicarbonate, the percent distributions
for all four cultures differed only in respect to the hot-
TCA-soluble and protein fraction. Approximately 10% less
radioactivity was observed in the protein fractions from
the bicarbonate labeled cultures, than in the acetate
labeled cultures. However, the hot-TCA-soluble fraction
from.the bicarbonate labeled cultures contained twice the

amount of isotope.
. 1h .
Uptake of CHQCOONa by Cell Suspen31ons

Washed cell suspensions which were preadapted to growth
on acetate during a second passage in fermentor cultures
(Fig. 3) were utilized for the incorporation of acetate-2-
luC into ethanol-soluble fractions. Adaptation to growth
in a medium containing acetate and no bicarbonate required
a transition period between first passage autotrOphically
grown cultures and acetate-adapted cultures. The genera-
tion time for the transition period was increased from 27.5
hr (generation time of autotrophic culture) to 65.0 hr.

Once the cell culture became adapted to growth in the pre-
sence of acetate the generation time was reduced to 35.0 hr.

The washed cell suspension which was.preincubated with
nitrite for 55 min before the addition of acetate-2-luC con-
tinued to oxidize nitrite at a rate linear with time until
the nitrite-N was exhausted at 1200 sec (Fig. h). The rate
of incorporation of isotope into the ethanol-soluble frac—

tion of the cells slowly decreased after 200 sec, but the

FIG. 3. Comparison of growth of Nitrobacter agilis on

 

acetate and nitrite with growth on bicarbonate and nitrite
in fermentor cultures in terms of absorbance at th nm and
nitrite oxidation (mg oxidized per m1). Curves A and D,
growth of a first passage culture in the basal medium plus
nitrite and bicarbonate; curves B and E, growth of a first
passage culture in the basal medium plus acetate and nitrite;
curves C and F, growth of a second passage culture in the
basal medium plus acetate and nitrite. The logarithms of
the measured values are transposed to make the curves coin-
cide and the distance between horizontal lines corresponds
to one doubling. The vertical lines indicate time zero for

the individual curves.

 

 

 

 

 

 

 

 

 

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II... 1' JDIVIIOOI' I01

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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IIZIEIXO SUN.“ .01

FIG. h. Rates of nitrite oxidation (0) and incorporation
of luC (0) into a group of selected cellular intermediates
present in the ethanol-soluble fraction of a cell suspension

of Nitrobacter agilis growing on acetate-2-luC. The incor-

 

poration of 1&0 is expressed as disintegrations per min per
0.05 ml of the culture and represent the sum total of radio-
activity detected chromatographically in the various cellular

intermediates studied.

 

2.}: 2:: = :2:-

 

—80
6

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4
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32-
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52
isotope continued to be incorporated for the duration of
the experiment (210 min). During the course of this experi-
:ment the protein concentration increased from 2.07 mg per

ml to 3.0 mg per m1 of cell suspension.

Distribution of Radioactivity in Cellular Intermediates

 

During Nitrite Oxidation

Within 5 sec radioactiviy was detected in many of the
intermediates studies. Particularly high amounts were loc-
ated in the carboxylic acids of the TCA cycle and in glu-
tamate directly derived by the amination of d—ketoglutarate
(Tables 5 and 6). The compounds predominately labeled init-
ially were citrate plus isocitrate and succinate plus fum-
arate. These same compounds retained the greatest amount
of label for the first 180 sec. Since the TCA cycle un-
doubtedly functions in N. agilis (Smith and Hoare, 1968)
when acetate is present in the medium, the high amounts of
incorporation of label into citrate indicates that acetate
was incorporated into the TCA cycle by condensation with
oxalacetate. Since malate had relatively little label
initially, there was no evidence for additional incorpora-
tion of acetate into malate as would have been the case had
the glyoxylate bypass been Operative. Among the phosphor-
ylated compounds studied, phosphoenolpyruvate (PEP) and
adenosine monophosphate were the most highly labeled com-
pounds initially (Table 7). The label accumulated more

slowly in 3-PGA than in PEP which may have been derived

53

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psomoamoh mesam> HH4

m

 

 

 

 

 

 

 

oso.s .mmw.a eom.H emw.: em:.m oom.H

wee.: map mp: mmp.m eme.fi ooe

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pea ems ob :eH.H ema.a om

mm emH me see ems mm

as mmm we one one m

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opmfihxohaw oom>sshm opmamz one one Aoomv
assesses escapee ease
sepaaaeesoaesm

 

 

 

madame nepowoonpwz ho Cowmsoamdm HHoo mstOam e no msowpomnu oHQdHOmnHoswflpo

one mo mofiom oasmmmo Opsfi odamuopmpoow Mo 03H cup mo sowpwnomnoosH .m mgm<e

5h

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w

 

 

 

 

 

 

 

 

 

es pm mmH :: and amp :Hm.e oom.a

em om pea m: as com emm.b ooc

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s m NH pH as me. be m

see ass sea was was can see Aces:
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55

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m

 

 

 

 

 

 

 

 

 

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: seeH>HaonHOsm , osHe

 

 

 

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quo oouwfihnonamosm Opsfi odaumnopmpoom wo 03H esp mo nowpwhomnoosH .N mqm¢e

56

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57
from oxalacetate not synthesized by the autotrophic 002-
fixation mechanism. Aspartate which has been shown to be
labeled initially to a greater extent than glutamate dur—
ing both the fixation of HlbrCO3 (Aleem, 1965) and the meta-
bolism of acetate by the glyoxylate cycle (Kornberg, 1958)
was poorly labeled throughout the growth period in our
investigation.

Curves obtained by plotting the percentage of the tot-
al radioactivity isolated chromatographically from each
samle against time indicate the variation in the distri-
bution of label from acetate—2-lu0 incorporated into the
various classes of cellular intermediates present in the
ethanol-soluble fraction. Early labeling occurred in the
carboxylic acids (Fig. 5). For the first 120 sec, 73 to
82% of the label was located in the carboxylic acids, where—
as the amino acids contained 6 to 12%, and the phosphory-
lated compounds contained 10 to 18%. During the steady
state (600 to 1200 sec) in which nitrite continued to be
oxidized linearly, the radioactivity associated with cit-
rate plus isocitrate and succinate plus fumarate declined
to 7 and 1h% respectively (Fig. 6). However, steady state
values for the organic acids (Fig. 5) were maintained by
increased concentrations of glyoxylate and pyruvate. With
the exception of citrate, isocitrate, glyoxylate, and pyru-
vate, the authentic compounds were chromatographically well
separated. (The chromatographic areas designated as glyoxy-
late and pyruvate overlapped each other as well as the cit-

rate plus isocitrate areas. The ethanol extracts were run

 

‘23s.

.Iiul‘ljtlr tee. to. .
t. .e.
N

 

 

 

P2

Qt

. ._ - Tuiififiss‘
_

Earl‘LP It

 

v"
fi.s:.......—.'._e_ "

 

 

FIG. 5. The percentage distribution of 180 of acetate-2-

luC incorporated into carboxylic acids (A), amino acids
(0), and phosphorylated compounds (0) found in the ethanol-

soluble fractions of a growing cell suspension of Nitrobacter

 

agilis. The data are expressed as the % of the total radio-

activity (disintegrations per min) isolated chromatographic-
ally.

 

58

 

 

 

T T ° “J
—.‘3
—-co

.-

._ -

-

-

u

an

I.

 

 

 

 

 

 

L
O
V

(x) Immmn a”

 

u”; PEI '

agar

FIG. 6. The percentage distribution of lbrC of acetate-2-
l[4’0 incorporated into various compounds associated with the
tricarboxylic acid cycle found in the ethanol-soluble fract-
ions of a growing cell suspension of Nitrobacter agilis.
The data are expressed as % of the total radioactivity
(disintegrations per min) isolated chromatographically.
Symbols: 0, glutamate; o, aspartate; A, malate; A, citrate

and isocitrate; o, succinate and fumarate.

59

 

 

 

 

 

 

A

12

IO

 

 

 

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v -
l ‘_ l l l i
O O O O
‘ n N l-

(as) mmmm a“

 

 

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‘.'.e u

60

on separate sets of chromatograms, one set was cochromato-
graphed with authentic citrate, isocitrate, and glyoxylate,
while a second set was cochromatographed with authentic
‘pyruvate and the remaining organic acids. After the iso-
citrate plus citrate area was cut out, the remaining activity
located adjacent to'this area was designated as glyoxylate.
'Therefore, some of the activity attributable to isocitrate
plus citrate probably also contained glyoxylate, or pyruvate, ,;
or both. This mixed activity was certainly the case for

the values obtained for the 120 and 180 sec samples. L11

 

After the establishment of steady state conditions, h
the radioactivity associated with the CS amd C6 organic
acids (citrate, isocitrate, and glutamate) represented
approximately 35 to 38% of the total radioactivity. On the
other hand, the activity of the CH carboxylic acids (fum—
arate, succinate, and malate) contained only 18 to 26% of
the label (Fig. 7). Approximately 12 to 17% of the radio—
activity was accounted for in the phosphorylated compounds
which were formed, either by the complete oxidation of
acetate to 002 followed by the assimilation of the releas-
ed C02, or by the conversion of oxalacetate to PEP. The
reduction of 002 by the autotrOphic mechanism would result
in the labeling of RuDP and 3-PGA, as well as the other
sugar phosphates involved in the interconversion of sugars.
These compounds did become labeled after relatively high

levels of radioactivity were reached in the carboxylic acids.

 

 

FIG. 7. The percentage distribution of 1&0 of acetate-
2-luC incorporated into phosphorylated compounds (I), Ch
dicarboxylic acids (succinate, fumarate, and malate) (A),
and CS and C6 carboxylic acids (citrate, isocitrate, and
glutamate) (0) found in the ethanol—soluble fractions of a
growing cell suspension of Nitrobacter agilis. The data
are expressed as the % of the total radioactivity (disinte-

grations per min) isolated chromatographically.

 

 

61

 

 

 

 

   

12

IO

 

 

 

 

50"—

l%) Illlllllflll I

l
2
I

low—
0

 

62

Distribution of Radioactivity in Cellular

Intermediates After Nitrite Depletion

After 75 min of incubation at 30 C, the cell suspen-
sion had exhausted all of the nitrite-N in the medium. At
this time the cells had been exposed to acetate-2-luC for
20 min. At the 10 min sampling only 27 pg nitrite—N per ml
remained, an amount which was probably inadequate for the
continued generation of energy. Three additional samples
were then taken at 30, 60, and 210 min and the ethanolic
extracts treated as before. The cells contined to incor-
porate 1AC from acetate-2-luC (Table 8). However, the
percentage distribution patterns were altered considerably
for many of the metabolites.) The relationship between the
phosphorylated compounds and the carboxylic acids formerly
existing during the nitrite-oxidizing period(Fig. 7) showed
a reversal after nitrite depletion (Fig. 8). The CM and
the 05 plus C6 groups of carboxylic acids, together with
the phosphorylated compounds represented 66 to 73% of the
total label isolated. The percentage of 1LLC appearing in
the phosphorylated compounds increased concomitantly with
decreases in the CS and C6 acids. Interestingly, the Cu
acids remained at a fairly constant level. For the most
part, the increased radioactivity among the phosphorylated
compounds was attributable to increased incorporation into
3-PGA, the various sugars present in the "sugar phosphate
mixture", and two unknown areas which migrated either with

orthophosphate or slightly ahead of the orthophosphate area.

 

63

 

 

 

 

 

 

 

 

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FIG. 8. The effect of nitrite depletion on the incor-

poration of 1’40 from acetate-2-lu0 into phosphorylated

compounds and carboxylic acids of the tricarboxylic acid

cycle. Symbols: .A, Cu_dicarboxylic acids (succinate, fun-

arate, and malate); I, C5 and C6 carboxylic acids (citrate,

isocitrate, and glutamate); o, phosphorylated compounds.

The data are expressed as the % of the total radioactivity

(disintegrations per min) isolated chromatographically.

 

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67

The percentage levelsof the carboxylic acids was also
altered (Fig. 9). The radioactivity in the combinations,
succinate plus fumarate and citrate plus isocitrate, pro—
ressively decreased, while malate showed an upward trend.
The increased percentage levels of malate were too high to
merely reflect a lag in the passage of label through the
TCA cycle. Fumarate plus succinate contained similarly
high amounts of radioactivity. Glyoxylate concentrations
rose sharply and quickly surpassed the levels of the acids
in the TCA cycle. Although glyoxylate was poorly resolved
chromatographically, it was expected that the amount of
isotope actually present would have been underestimated as
some of its activity may have been ascribed to citrate plus
isocitrate.

The overall trend of an initial lowering in isotope
incorporated into components of the TCA cycle and a slower
decrease in labeling in both the phosphorylated compounds
and in the amino acids proline and aspartate, which are
derived from the TCA cycle may indicate a cessation of the
introduction of acetate into the TCA cycle at citrate after
nitrite depletion. However, the sudden attainment of high
levels of glyoxylate particularly after nitrite exhaustion
is not compatible with the concept that acetate utilization
is limited merely to oxidation via the TCA cycle and the

synthesis of PHB.

 

FIG. 9. The effect of nitrite depletion on the incorpora-

tion of 1&0 from acetate-2-luC into glyoxylate (o), malate

(O), citrate plus isocitrate (a), and succinate plus fum-

arate (I). The data are expressed as the % of the total

radioactivity (disintegrations per min) isolated chromato-

graphically.

 

 

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DISCUSSION

The data reported in this paper confirm the earlier
claims of previous investigators (Delwiche and Finstein,
1965; Ida and Alexander, 1965) that N. agilis, when active-
ly replicating, relies upon nitrite as the sole source of
energy and upon carbon dioxide as the principal source of
carbon. The inability of acetate to serve as an energy
source for the growth of N, agilis was first cited by Ida
and Alexander (1965). We extended their investigations on
the use of acetate, but added 002 and all forms of nitrogen
which are usually utilized by bacteria for protein synthe-
sis. A nitrite-free basal salts medium amended with other
forms of nitrogen together with acetate and ambient 002 did
not support meaningful growth of pure cultures of N. ggilig.
Clearly the utilization of acetate did not generate suffi—
cient energy for significant cell replication.

The synthesis of PHB from acetate would appear to be
of ecological value. The PHB stored under conditions where
energy and substrates are readily available could be oxid-
ized when environmental conditions become adverse. Approxi-
nmtely one-third of the label appearing in the insoluble
residue fraction of growing cells was in PBH. However, it

is concluded that during nitrite oxidation the incorporation
69

 

70
of acetate into protein plays the major role in the util-
ization of acetate.

Since both the oxidation of acetate and the presence
of acetyl-COA synthetase and all of the enzymes of the TCA
cycle have been reported for N, agilis (Smith and Hoare,
1968), the labeling observed not only in the intermediates
of the TCA cycle, but in the phosphorylated compounds during
nitrite oxidation could have been initiated solely by this
oxidative process. However, in order for growth to occur
using acetate as a carbon source, acetate would have to be
metabolized by some biosynthetic pathway such as the gly—
oxylate bypass or the glycerate pathway.
11+

The percentage distribution patterns Of C from
acetate-2-luC incorporated into components of the ethanol—

soluble fraction of Pseudomonas and other microorganisms

 

which possess a glyoxylate cycle (Kornberg, 1958; Kornberg
and Elsden, 1961) differ markedly from the patterns observed
in N, agilis. The most significant difference is in the
incorporation of lhc into malate. In microorganisms having
a functional glyoxylate cycle, malate initially bears more
1H0 than does citrate plus isocitrate, or glutamate, since
in the glyoxylate cycle two molecules of labeled acetate,
rather than just one, contribute to the formation of malate.
For the reasons just cited, a glyoxylate cycle in the pre-
sence of nitrite oxidation is believed to be nonfunctional
in N, agilis. The pathways in the utilization of acetate
for PHB synthesis and protein synthesis via the TCA cycle

 

71
and the glyoxylate bypass are presented in Fig. 10.

In microorganisms having an oxidative dicarboxylic acid
cycle, malate again is the predominantly labeled component
of the early ethanolic fractions (Kornberg and Sadler, 1961).
Therefore, the dicarboxylic acid cycle also appears to be
nonfunctional in N. agilis when nitrite oxidation takes
place.

It is evident from the changes in the percentage dis-
tribution patterns which occurred at the time of nitrite
depletion that the mode of incorporation of th was also

altered. Since the data shows an accumulation of glyoxylate

 

just prior to and after nitrite-N was depleted and Smith
and Hoare (1968) reported a considerably increased level of
isocitrate lyase (lOO—fold) when acetate was present in
the medium, it is concluded that isocitrate is split to
form glyoxylate in N. agilis. The metabolism of glyoxy-
late initially may be prevented by low activity of malate
synthase. The presence of glyoxylate in sufficient quant-
ity may be necessary to activate enzyme formation or act-
ivity. Although malate synthase has not been found in
extracts of N. ggilig grown in an autotrophic medium con-
taining both nitrite and acetate (Smith and Hoare, 1968),
the absence of nitrite oxidation may be required for
enzyme activation. This possibility is worthy of future
investigation. The increased levels of malate and succin-
ate in conjunction with decreased levels of citrate and
isocitrate offer evidence that glyoxylate is metabolized

in the absence of nitrite oxidation.

 

 

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73

The data obtained from.the investigation on the growth
of pure cultures of N. agilis in media containing acetate
does not support the reclassification Of this microorganism
as a "facultative autotroph" as proposed by Smith and Hoare
(1968). While the isotopic kinetic studies indicate that
acetate is metabolized via glyoxylate and certain intermedi-
ates Of the TCA cycle in the absence of nitrite oxidation,
supportive data involving enzyme assays with cell-free ex-
tracts are needed to determine if glyoxylate can be metabol-

ized by an energy generating and biosynthetic pathway.

 

Since our growth studies indicate that growth is not support—
ed by acetate it appears that either the combination of an
energy yielding and biosynthetic pathway is nonexistent or
the energy generated from such a pathway can not be coupled

to other necessary biosynthetic processes.

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