WAS-EMF! 0%: PASTEURELLA PESTIS
T0 UTEE...EZE AMMGNIW {OMS AS A
F‘REWJEY SOURCE OF NKWOGEN

Thesis for the Degree of M. S.
MICHtGAN STATE UNIVERSITY.
ANDREW SULEN JR.
1959

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ABSTRACT

INABILITY OF PASTEURELLA PESTIS TO UTILIZE

 

AMMONIUM IONS AS A PRIMARY SOURCE OF NITROGEN

BY
Andrew Sulen Jr.

Wild-type Pasteurella pestis is unable to grow in

 

a synthetic medium containing NH4+ as a primary source of
nitrogen. Comparative studies between a mutant that can
utilize NH4+ for growth and the wild type showed no dif-
ferences in synthesis of primary a-amino groups, excretion
products, organic acid pools, and permease activities.
Assays of the primary nicotinamide adenine dinucleotide
phosphate generating systems revealed no differences be-
tween wild type and mutant cells; however, the possibility
that reductive amination of a-keto groups is limited in

wild-type cells is suggested by these results.

INABILITY OF PASTEURELLA PESTIS TO.UTILIZE

 

AMMONIUM IONS AS A PRIMARY SOURCE OF NITROGEN

BY

Andrew Sulen Jr.

A THESIS

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

MASTER OF SCIENCE
Department of Microbiology and Public Health

1969

6.53583-

M}

2/27

ACKNOWLEDGEMENTS

I would like to express my sincere thanks to
Dr. Robert R. Brubaker for his excellent advice and
guidance during the course of this study.

I also wish to extend my appreciation to
Dr. Ralph N. Costilow for his assistance and the gen-
erous use of his laboratory facilities.

The author would also like to thank Dr. David
Bing and Dr. Bernard J. Abbot for their technical

assistance in this investigation.

ii

TABLE OF CONTENTS.

ACKNOWLEDGEWNTS O O O O O O O O O O O O O O O 0
INTRODUCTION 0 I O O O O O O O I O O O O O O O O
Uptake of Ammonia into Organic Linkages. . .

Metabolic Reactions for the Production of

Reduced NADPH O O O O O O O O O O I O O 0

EXPERIMENTAL METHODS. . . . . . . . . . . . . .

Organisms . . . . . . . . . . . . . . . .
Media . . . . . . . . . . . . . . . . . .
Isolation of Mutants. . . . . . . . . . .
Cultivation of Organisms. . . . . . . . .
Growth Studies. . . . . . . . . . . . . .
Preparation of Extracts . . . . . . . . .
Enzyme Determinations . . . . . . . . . .
Organic Acid Production by Resting Cells.
Estimation of Protein . . . . . . . . . .
NH4+ Uptake by Resting Cells. . . . . . .

Organic and Fatty Acid Excretions by
Resting cells. 0 I O O O O O O O O O 0

Amino Acid Excretion by Resting Cells . .

RESULTS 0 O O O O O O O O O O O O O O O O O O 0

Growth Studies. . . . . . . . . . . . . .

Page

ii

10
10
11
l3
13

14

14
15
17

17

TABLE OF CONTENTS (cont.)

Page
Studies of the Synthesis of a-amino Groups. . 22
Studies of Organic Acid Production by

Resting Cells. . . . . . . . . . . . . . . 23

NH4+ Uptake by Resting Cells. . . . . . . . . 23
Studies of Organic Acid, Amino Acid, and

Lipid Excretions by Resting Cells. . . . . 23

DISCUSSION. . . . . . . . . . . . . . . . . . . . . 26

SUMMARY . . . . . . . . . . . . . . . . . . . . . . 30

LITERATURE CITED. . . . . . . . . . . . . . . . . . 31

iv

LIST OF TABLES

Table Page
1. Virulence Properties of g. pestis . . . . . . 7
2. Composition of Nitrogen-Deficient Medium. . . 8

3. Comparison of the Enzymatic Activities of
N+ and N‘ Cells. . . . . . . . . . . . . . 22

LIST OF FIGURES

Figure

1. Growth patterns of N+ and N‘ organisms in
minimal media plus glutamine. . . . .

2. Growth patterns of N+ and N' organisms in
minimal media plus ammonium chloride. . . .

3. Assay for pyridine nucleotide transhydrogenase
in NI cells . . . . . . . . . . . . . . . .

4. Comparison of ammonia uptake by resting cells
of N+ versus N' . . . . . . . . . . . . .

vi

Page

18

19

21

24

INTRODUCTION

Pasteurella pestis, the causative agent of bubonic

 

plague, is a gram negative, short, plump.rod that exhibits
a characteristic bipolar staining reaction with aniline
dyes (1). These bacilli are nonmotile, and when grown on
nutrient agar produce colonies that have a round granular
center and thin uneven edges. On heminrcontaining media
the colonies are dark brown, the pigmentation being de-
rived from hemin absorbed from the agar (2).

Virulence is related to five factors; namely,

(i) the ability to produce V and W antigens.(VW+), (ii)
fraction I antigens (FI+), (iii) pigmented colonies on
hemin agar (P+), (iv) endogenous purine (pu+), and (v) the
associated pesticin (PI+), coagulase (C+), and fibriolytic
factor (3+) (3, 26). Loss of any one of these virulence
determinants results in.a decrease of virulence (3).

P. pestis_possesses a functional Emden-Meyerhof
pathway and utilizes this pathway almost exclusively when
grown under aerobic or anerobic conditions on hexoses (4%.
Under anerobic conditions, Douderoff (5) reported that
P. pestis_will utilize glucose to produce lactic, acetic,
formic, and succinic acids as well as ethanol, C02, and

1

small amounts of pyruvate. When grown on-glucose under
aerobic conditions Levine (6) reported these organisms will
oxidatively decarboxylate pyruvate to acetate (7). Although
P. pestis possesses all of the enzymes of the citric acid
cycle (under aerobic conditions), the organisms lack the
ability to oxidize acetate during growth. However, acetate
is oxidized by resting cells (8). This failure to oxidize
acetate was considered by Englesberg (8) to be a result of
the destruction of oxalacetate by an oxalacetate decarbo-
xylase, Which inhibits or prevents the condensation reac-
tion, and to be due also to the large concentration of
acetate required to initiate its phosphorylation.

Another unique and limiting feature.of the meta-
bolism of P. pestis is its lack of glucose-6-phosphate
dehydrogenase (9, 10). When grown on glucose, this de-
ficiency results in the inability of P. pestis to utilize
the hexose—monOphosphate or the Entner-Douderoff pathways.
The organisms grow readily in the presence of gluconate,
and the existence of transketolases and transaldolase of
the hexose-monOphosphate pathway, and gluconate dehydrase
and 2-keto-3-deoxy-6-phosphogluconate aldolase of the

Entner-Doudoroff pathway have been demonstrated (10).

Uptake of Ammonia into
Organic‘Linkages

 

 

There are three principle reactions in bacteria
which result in primary synthesis of a-amino groups from
NH4+. The first is that catalyzed by.glutamic dehydro-
genase which, in the presence of a-ketoglutarate and re-
duced nicotinamide adenine dinucleotide.phosphate (NADPH),
will produce glutamic acid. This system.is considered to
be the main NH4+ assimilation system in gram negative
bacteria; glutamic acid, a product of this enzyme, serves
as the amino donor in most transaminase reactions. A
second well documented reaction is that involving the
enzyme aspartase. In the presence of NH4+ and.fumaric
acid this enzyme catalyzes the reversable formation of
aspartic acid (11). The third reaction involves alanine
dehydrogenase which, in the presence of pyruvate and re-
duced nicotinamide adenine dinucleotide (NADH), produces
alanine (ll). Other mechanisms for the assimilation of
NH4+ have been reported such as aSparagine synthetase,
B-alanyl-Co-A—synthetase, and B-methyl aspartase, but
their presence has been reported in only a few organisms
therefore their ubiquitous nature is questionable.

Metabolic Reactions.for the
Production of Reduced NADPH

 

 

As described by Hollmann (27), there are 3 pre-

dominant enzymes for the reduction of NADP. The first,

pyridine nucleotide transhydrogenase,.is an enzyme that
can reversibly transfer electrons from NADH and NADP. A
second, isocitrate dehydrogenase, oxidizes isocitrate
with the resultant production of NADPH.. The third and
probably most important enzyme for NADPH production is
glucose-6-phosphate dehydrogenase. This enzyme functions
not only in the direct production of NADPH, but also pro-
vides substrate for the enzyme 6-ph08phogluconate dehy-
drogenase which itself catalyzes a second oxidation with
the production of NADPH.

P. pestis is unable.to utilize NH4+ as a primary
source of nitrogen (R. R. Brubaker, personal communica-
tion). To study this inability, mutants were obtained by
plating organisms on minimal media supplemented with
NH4C1 as its primary source of nitrogen.. These mutants
(N+) were then used in comparative studies with wild-type
organisms (N') to determine the physiological change that
permitted the N+ cells to use NH4+.

Four approaches were used to investigate this
problem. The first involved the fact that_g. pestis lacks
glucose-6-phosphate dehydrogenase (10), and thus lacks an
important mechanism for the generation of NADPH. Since
NADPH is an obligatory cofactor for glutamic acid dehy-
drogenase (a primary enzyme for the assimilation of NH4+

into a-amino groups) remaining enzymes that produce NADPH

were assayed to determine if differences existed between
N+ and N" cells. A second approach involved a search for
an alternative mechanism of NH4+--assimilation that might
in the N+ but be missing in the N" cells. A third ap-
proach considered the study of products excreted by rest-
ing cells. This study was undertaken in the hOpe of find-
ing possible differences in the general metabolism of N+
versus N_ cells. The fourth approach was concerned with

the disappearance of NH + in suspensions of resting N+

4
and N' cells. This investigation was designed to elim-
inate the possibility that permease activity might account

for the differences between the two types of cells.

EXPERIMENTAL METHODS

Organisms.--The organisms used were obtained from

 

stock cultures supplied by R. R. Brubaker. .Table l (26)
lists the strains used and the virulence factors that they
possess (3). Ten N+ organisms were.isolated.from these
strains by the procedure outlined below; strain A4x was
used in most investigations.

Mgdia.--Table 2 gives the composition.of the ni-
trogen deficient medium used. The basal medium.is a
modified version of those described by Higuchi and Carlin
(12) and Englesberg (13). The small concentrations (10
ug per ml) of L-phenylalanine, L-isoleucine, L-valine, and
L-methionine were added in order to satisfy the nutrih
tional requirements of g. pestis. Sodium thiosulfate was
also added as a secondary source of sulfur. To prepare
one liter of this medium 10 mg of each of the above amino
acids was dissolved in 850 ml of distilled water to which
100 ml of a concentrated Salt solution had been added
(see Table 2). Phenyl red (lOppm) was added as a pH in-
dicator, and the medium was then autoclaved. After cool-
ing, the pH was adjusted to 7 with 5 N NaOH, and 10 ml of
a 100 fold concentrated solution of glucose, sodium

6

Table l

Virulence Properties of P. pestis*

 

 

Strain VW P FI Pu PI F
OX/DODSON 0 + + 0 0
A1122 0 0 + _ + + +
G32 + 0 + + 0 0
G35 0 0 0 + 0 0
OX/M23 0 + 0 + + +
EV76(M.R.) + 0 + + + +
EV76(EISLER) + 0 .+ + + , +
EV76(DETRICK) + o + + + +
JAVA 0 0 0 + 0 0
A4x O 0 + + 0 0
G25 + 0 + + o o

 

*Symbols refer to prOperties described in the text.

Table 2

Composition of Nitrogen-Deficient.Medium

 

 

 

Component Concentration per Liter
TE
KZHPO4a 25
Citric acida 10
Sodium lactateb 10
D-glucoseb ' 10
Mgc12a 2.5
CaClzb 2.5
Na28203b 2.5
FeClza 0.1
MnClza 0.01
g_
(Agar) (15)
L-phenylalanine 0.01
L-isoleucine . 0.01
L-valine 0.01
L-methionine 0.01

 

aPrepared as a single concentrated (x10) stock salt solu-
tion.

bPrepared as individual concentrated (x100) stock salt
solutions.

thiosulfate, sodium lactate, and calcium chloride was
added aseptically.

Isolation of Mutants.--Cells from the stock cul-

 

tures were grown on slants of blood agar base (Difco) and
incubated at 26°C. The organisms were washed with 0.033
M phosphate buffer and samples of 0.1 ml were spread on
plates of nitrogen deficient medium supplemented with

agar (1.5%) and NH Cl (.01 M). The plates were allowed

4
to incubate at 26°C for 4 to 5 days at.which.time a few
colonies of mutant cells began to appear. These N+ cells
were again streaked out on the same medium and, after
growth, organisms from a single colony were picked and
transferred to a BAB slant. Following incubation, the
cells were suspended in sterile 0.033 M phosphate buffer
and added to a screw-cap tube along with.an equal volume
of sterile glycerol. After mixing, these stocks were

then stored at -20°C.

Cultivation of Organisms.--Following incubation

 

on BAB at 269C for 48 hours, the cells were washed with
sterile 0.033 M phosphate buffer and then inoculated into
100 ml of nitrogen deficient medium containing .01 M
glutamine. These cultures were incubated statically for
16 hours at 26°C. The cells were harvested by centrifu-
gation at 20,000 g for 10 minutes in a RC2-B Sorval

Centrifuge, washed, and reinoculated into the standard

10

medium supplemented with .01 M NH4C1. .The cultures were
then allowed to incubate at 26°C for 16 hours without
aeration.

Growth Studies.--N+ and N- cells were inoculated

 

into minimal media containing either glutamine or NH4C1
as the primary source of nitrogen. .Growth in both cases
was measured by determining the optical density changes
at 620 nm with a Bausch and Lomb Spectronic 20 using the
uninoculated medium in both cases as a blank.

To eliminate the possibility that.the mutation
did not involve a change to a prototrophic state for the
four required amino acids, N' organisms were streaked on
minimal medium that contained 0.01 M NH4C1. The plates
were incubated until background growth appeared, and
drops of 0.001 M solutions of the trace.amino acids used
in the minimal medium (see Table 2) were placed on the
plates. Incubation was continued at 26°C and frequent

checks for growth were made.

Preparation of Extracts.—~Following cultivation

 

of the organisms, the cells were harvested by centrifuga-
tion at 20,000 x g for 15 minutes, washed in 0.033 M
phosphate buffer, centrifuged again and resuspended in
cold 0.1 M tris (hydroxymethyl) amino-methane (tris)-HC1
pH 7.75. Extracts were prepared by ultrasonic oscilla-

tion of 2-4 ml of cell suspension for 3, 30-second

11

intervals in a lOO-W ultrasonic disintegrator (Measuring
and Scientific Equipment Ltd., London). The cell debris
was removed by centrifugation at 20,000 x g for 15 min.
in the cold, and the supernatant was.dialyzed overnight
against cold 0.033 M phosphate buffer containing 0.001 M
mercaptoethanol.

Enzyme Determinations.--In most cases.a volume of

 

3 ml was used for spectrophotometric assays. Reactions.
linked to pyridine nucleotides were read at 340 nm with a
Beckman DU spectrophotometer (Beckman Instruments, Inc.,
Fullerton, California) and were corrected for nonspecific
oxidase or reductase activity. In all reactions one unit
of activity is expressed as the amount of enzyme neces-
sary to convert l umole of substrate to product per
minute at 25°C. These values were then expressed in terms
of specific activity (units per mg of protein).

The transhydrogenase reaction was measured, ac-
cording to a modified procedure outlined by Kaplan (14),
in 300 tris-HCl buffer pH 7.0, 3.3 mM reduced glutathione,
0.16 mM NADH, and 0.16 mM NADP. The reaction was started
with the addition of the enzyme and readings were taken
every 15 seconds. Glutathione Reductase activity was
found not to be rate limiting in this assay system.

Glucose-G-phosphate dehydrogenase was measured,

according to the method of Kornberg and Horecker (15), in

12

100 mM tris-HCl buffer pH 7.0, 3.3 mM glucose-Gephosphate,
and 0.16 mM NADP. Any increase in absorbance was recorded
at lS-second intervals.

Aspartase was measured, according to Virtanen and
Ellfork (16), by the formation of aspartic acid in an
80 mM potassium phosphate buffer pH 7.0, 16 mM potassium

fumarate, and 16 mM NH Cl. After 0, 5, 10, 20, and 30

4
minute incubation periods at 25°C, the.reaction was stopped
with an equal volume of 1.0 M perchloric.acid and the tubes
were refrigerated for 30 minutes at 5°C.. After removal of
precipitated material by centrifugation the samples were
treated with 5 N KOH and then centrifuged at 15,000 rpms
for 10 minutes. The samples were then spotted on Whatman
number 1 paper in a decending system with nebutanol:
ethanol: water (13:8:4 v/v) as the solvent. After dry-
ing, the chromatograms were sprayed with ninhydrin.
According to the method of Strecker (l7), glutamic
dehydrogenase was measured in a 300 mM solution of tris-
HCl buffer pH 8.5, 3.33 mM potassium a-ketoglutarate,
3.33 mM NH4C1, and 0.16 mM NADPH. The decrease in absor-
bance was determined at every 15-second interval of time.
Isocitrate dehydrogenase was assayed, according

to the method of Plaut (18), in a reaction mixture of 100

IMM tris-HCl buffer pH 7.5, 0.16 mM NADP, and 3.33 mM

13

potassium isocitrate. Readings were taken after time
intervals of 15 seconds.

Organic Acid Production.byResting.Cells.--To
study the production of organic acids, the.cells were
taken from the minimal medium supplemented with NH4C1,
washed, and then reinoculated into a 0.033 M potassium
phosphate buffer that contained only 0.01.M glucose.
This step was performed to eliminate the citrate present
in the minimal medium and also to deprive the cells of
necessary growth requirements. The organisms were in-
cubated at 26°C until acid production ceased and the pH
was then adjusted to l with sulfuric acid; the cultures
were refrigerated for 12 hours. The suSpension was then
centrifuged to remove cell debris and extracted with
ether for 24 hours. The ether was evaporated and the re-
maining residue resuspended in a small volume of distilled
water. The samples were then chromatogramed along with
standards on Whatman number 1 paper with an ethanol:
NH4OH: distilled water (160:10:30 v/v) solvent. The
chromatograms were dried and the resulting organic acids
were visualized with bromo cresol purple.

Estimation of Protein.--Estimation of protein was

 

performed spectrOphotometrically by the method of Lowry

(19).

14

NH4+ Uptake by Resting Cells.~-Both NI and N-

 

cells were cultivated as described in minimal medium plus
glutamine and transferred to the minimal medium plus
NH4C1. After the cells had reached log phase they were
havested, washed, and reinoculated in equal numbers into
a medium composed of 0.075 M phosphate buffer, 0.0018 M
sodium citrate, 0.00041 M magnesium sulfate, 0.001 M
NH4C1, and 0.01 M glucose (20). The cultures were allowed
to incubate at 26°C; 2 m1 samples were taken every hour-
and filtered through a membrane filter (Millipore 0.45 H)
to remove the cells. The samples were then assayed for
the amount of NH4+ remaining in solution by using
Nesslers reagent (21); the color was estimated at 480 nm
on a Beckman DU spectrophotometer. A standard curve was

made by preparing various dilutions of the uninoculated

medium in which the molarity of NH4C1 was known.

 

Organic and Fatty Acid Excretions by Resting Cells.--
The standard cultivation procedure was performed and the
cells were removed and reinoculated into minimal medium plus
0.01 M NH4C1 lacking the trace amounts of the 4 amino acids
needed for growth. The organisms were incubated until acid
production ceased, and the cells were spun out and dis-
carded. The medium was then filtered to further remove
any remaining cells. Next, the pH was adjusted to l with

sulfuric acid, and a continuous ether extraction was

15

performed for 24 hours. The ether was separated from the
medium by means of a separatory funnel and evaporated by
steam. The residue was washed with distilled water and
the soluble fraction was chromatogramed on Whatman number
1 paper using ethanol: NH4OH: distilled water (160:10:
30: v/v) as a solvent and organic acids were visualized
as previously described. The remaining water insoluable
residue was dissolved in a 95% solution of ethyl alcohol
containing on equal volume of 1.0 M KOH and autoclaved
for 24 hours in sealed glass tubes (22). The hydrolysate
was then methylated (23), and gas chromatography was per-

formed using a 810 F and M Research Chromatogram (Avon—

dale, Pennsylvania).

 

Amino Acid Excretion by Resting Cells.--The media
was frozen in an acetone dry ice bath, and 1ypholyzed by
a New Brunswick Freeze Dryer (New Brunswick Scientific
Co.). The precipitate was then resuspended in 15 ml of
water and centrifuged to remove insoluble debri. After
neutralization with 5 N NaOH, centrifugation was again
required to remove precipitate. The solution was evapor-
ated under partial vacuum to 5 ml using a Rotary Evapo-
Mix Dryer.(Buchler Instruments). The pH was then adjusted
to 2 with sulfuric acid, and the solution passed through
a column of Dowex 50 W x 4,I200-400 mesh, in H+ form
equilibrated with water. The amino acids were then eluted

with l M NH4OH, dried, and resuspended in 0.5 ml distilled

16

water. Samples were placed on Whatman number 1 paper and
chromatogramed with n-butanol: acetic acid: distilled
water (100:22:50 v/v). A phenol: water (4/1 w/w) system
in a HCN atmosphere also was used to identify certain

amino acids.

RESULTS

Growth Studies.--To demonstrate the difference in

 

ability to utilize NH4+ that exists between N+ and N'
cells, two determinations of growth were conducted. One
experiment compared the rates of growth of the organisms

when NH + was supplied in the form of a a-amino and 6-

4
amino groups of glutamine (Figure 1). The results showed
both organisms grew well with the N+ phenotype showing
somewhat more prolonged growth. Glutamine was used in-
stead of glutamic acid due to the apparent inability of
P. pestis to effectively transport this amino acid when
cultivated in minimal media (R. R. Brubaker, personal
communication). However, enormous differences in growth
were observed (Figure 2) when the organisms were grown in
minimal medium containing NH4C1 as the primary source of
nitrogen. The N+ cells grew with the same ability as
they demonstrated in the presence of glutamine, but the
N' cells showed little if any sustained growth. They
instead demonstrated a slight lag followed by what~ap-

peared to be a single division of cells after which no

growth was observed for 10 hours.

17

18

OD

 

   

.01

 

 

NE) 1 11 L, l ‘ihS_—-'

HOURS

Fig. l.--Growth patterns of N+ and N— organisms in
minimal media plus glutamine. The medium was prepared and
dispersed in 250 ml flasks and incubated at 26°C. Readings

were taken at the designated time intervals as described in
Experimental Methods. 0 = W", O = N”.

19

f T Trrl

OD
I I
o

 

 

 

HOURS

Fig. 2.--Growth patterns of N+ and N_ organisms in minimal
media plus ammonium chloride. The medium was prepared and dispersed
in 250 ml flasks and incubated at 26°C. Readings were taken at the
designated time intervals as described in Experimental Methods.
O=N,.=N-.

20

A determination was also conducted to determine
whether the mutation that occurred was a change to a
new phenotype rather than a suppressor mutation involv-
ing acquisition of independence for the 4 required amino
acids. The study involved growing N‘ cells on minimal
agar plus 0.01 M NH4C1 and adding drOps of .001 M solu—
tions of the 4 amino acids. The results showed growth
was not enhanced by the addition of these 4 amino acids
either alone or in combination; however threonine, gly-
cine, and glutamine were able to support the growth of
N" cells in this experimental system.1

Since P. pestis lacks glucose-G-phosphate dehydro-
genase and can not oxidize acetate (see Introduction),
NADPH could be the rate limiting step for the incorpora-
tion of NH4+ into a-keto groups. Thus, an investigation
was conducted to see if the N+ cells possessed an enzyme
system that would produce the reduced pyridine nucleotide
in greater amounts than exist in the N' cells. The re-
sults demonstrate an absence of pyridine nucleotide trans-
hydrogenase (Figure 3) and glucose-6-phosphate dehydro-
genase (Table 3) in both the N+ and N‘ cells.‘ Although
the existence of isocitrate dehydrogenase in g. pestis
has been documented by Englesberg (8), its activity in

producing a-ketoglutarate in N+ versus N‘ cells needed

1__

1Similar results were observed by Brubaker (per-
sonal communication).

21

 

 

l 1 1 l 1 41—
30 60 90

Seconds

Fig. 3.--Assay for pyridine nucleotide transhydrogenase in
N cells. The complete system contained a cell free extract (.347
mgs of protein), 300nmmflartmis buffer, 3.3 mmolar reduced glutath-
ione, .16 mmolar NADH, and .16 mmolar NADP. (3 with reduced glu-
tathione, O without reduced glutathione.

22

Table 3

Comparison of the Enzymatic Activities”
of N+ and N' Cells

 

 

umoles of product per min per mg of protein

 

 

Enzyme
N+ extract NI extract

Isocitrate de-

hydrogenase .057 .049
Glutamic de-

hydrogenase .029 .017
G-6-P D 0 0
Aspartase 0 0

 

to be compared. Table 3 shows that only a very insignifi—
cant difference in specific activity exists between N+ and
N‘ organisms.

Studies of the Synthesis of seaminoiGroups.--To

 

determine if any difference existed in the activity of
glutamic dehydrogenase, an assay was performed as described
in Experimental Methods. As is shown in Table 3, the dif—
ference between N+ and N" cells is negligible.

Aspartase was determined by the production of aspar-
tic acid in the presence of fumarate and NH4C1. Chroma-
tography revealed no aspartic acid production by either
N+ or N' cells. Attempts to detect release of NH4+ from

asparate by direct Nesslerization were also unsuccessful.

23

Studies of Organic Acid Production by Resting

 

gell§.--By studying the organic acids that an organism
produces it is often possible to obtain information con-
cerning the metabolism of that organism. The cells were
grown in minimal medium and organic acids were extracted
as described in Experimental Methods. The residue from
the ether extraction was chromatogramed; the results re-
vealed an identical accumulation of Lactate. The presence
of equal concentrations of unknown minor compounds was
also noted in both organisms.

NH4+ Uptake by Resting Cells.--To determine

 

whether N+ cells have a permeability advantage over N‘
cells an uptake experiment was performed. The cells

were inoculated in equal numbers (108 cells/ml) into a
medium lacking the trace amounts of amino acids needed
for growth. Samples were taken every hour for 9 hours,
filtered, and assayed for the disappearance of NH4+. As
shown in Figure 4, NH4+ disappeared at an equal rate in
suspensions of N+ and N' cells, with the activity of the
N+ organisms lasting for a slightly longer period of time.
Boiled cells that were handled under-the same experimental
conditions showed no disappearance over a 9-hour period.

Studies of Organic Acid, Amino Acid, and Lipid

 

Excretions by Resting-Cells.--Organisms growing in a medium

 

which lacks a necessary growth requirement often continue

24

ll Moles of Nitrogen
w

 

 

 

 

,__L1114|L14_
I 5 9

HOURS

Fig. 4.--Comparison of ammonia uptake by resting
cells of N+ versus N’. Samples were taken every hour and
assayed for the disappearance of ammonia as described in
Experimental Methods. 0 = N+, O = N”.

25

to metabolize, but instead of multiplying may pile up and
excrete metabolic intermediates. Studies of these excre-
tion products may thus lead to an understanding of the
metabolism in the cell. An investigation was undertaken
to determine if differences in excretion of amino acids,
organic acids, and lipids exist between the N+ and N'
organisms. Analysis of the spent medium by paper chroma-
tography and paper electrophoresis revealed that both N+
and N' cells produce and excrete glutamic acid, alanine,
and arginine. No other significant differences were ob-
served.

Analysis of the methylated ether-soluble residue
by gas chromatography demonstrated no significant excre-
tion of fatty acids by either the N+ or N’ cells. Paper
chromatography of aqueous extracts of the acid-ether frac-
tion revealed no differences in organic acids excreted by

the N+ and N” organisms.

DISCUSSION

During a study of growth requirements of P. pestis
in minimal media, Brubaker (personal communication) ob-
served that this organism was unable to grow when supplied
with NH4+ as a primary source of nitrogen. To further
substantiate this observation nutritional studies were
conducted. This investigation conclusively showed that
when glutamine was used as a source of nitrogen, both
types of organisms grew at approximately the same rate.
However, when NH4+ was the primary source of nitrogen
large differences in rates of growth were seen. The N+
cells grew at a rate equivalent to that observed when the
medium was supplemented with glutamine. However, the N"
cells showed little if any significant growth except that
resembling a single division of cells. This slight in-
crease may have been due to the availability of an endo-
genous nitrogen source. The only subsequent growth that
occurred was a gradual increase observed 20 hours after
inoculation. This increase would be expected if N+ mutants
were present in the culture of N“ cells.

Because 3. pestis lacks glucose-G-phosphate dehydro-
genase, a mutation permitting expression of the N+ phenotype

26

27

might involve a suppressor mutation resulting in an exces-
sive production of NADPH. A search for an NADPH generat-
ing enzyme system exclusively characteristic of N+ cells
was not successful. However, the existence of such a dis-
tinction cannot be eliminated on the basis of available
evidence. Even though no differences in specific activity
of isocitrate dehydrogenase or glucose-6-phosphate dehydro-
genase were detected, the availability of substrate for
the former enzyme may differ between N+ and N' organisms.
For example a mutation resulting in loss of the oxalace-
tate decarboxylase activity should result in the accumula-
tion of oxalacetate Which could be eventually converted

to isocitrate, a substrate that can be oxidized to produce
NADPH. The possibility of this system functioning could
be answered by cultivating N+ and N- cells with radioac-
tive glutamine as the primary source of nitrogen. If the
N+, as compared to the N' organisms, produced large amounts
of radioactive C02, while N" cells instead excreted radio-
active acetate, then more positive conclusions could be
drawn regarding the generation of NADPH in 2112. However,
negative evidence in this case could not rule out the pos-
sibility that the generation of NADPH in N- cells is lim-
ited. Growth data have shown that both threonine and gly-
cine also promote growth of N- cells in the presence of
NH4C1. This phenomena seems to give additional credita-

bility to this hypothesis when evaluated in light of the

28

findings of Sagers and Gusalus (24).‘ These workers demon—
strated that Diplococcus glycinophilus can oxidize glycine

by converting its number 2 carbon to methenyl-tetrahydrofalate
by an enzymatic step that produces NADPH. Furthermore,
L-threonine may yield glycine for this pathway by the un-

resolved mechanism described in Escherichia coli by Van

 

Lenten and Simmons (25). Studies of amino acid excretion
by resting cells showed that N+ and N' organism accumulated
and excreted alanine, glutamic acid, and arginine. Since
glutamic acid and arginine require NADPH for their synthe-
sis, one might suspect that the assimulation of NH4+ is not
NADPH-linked. However, Englesberg (8) has shown that a
stimulation of the citric acid cycle can be initiated by

a large concentration of acetate. This situation might be
expected to occur in resting cells where acetate accumu-
lates from glucose. Thus, NADPH might be available via
isocitric dehydrogenase, to produce arginine, by way of
ornithine, and glutamic acid.

The results also demonstrate that no difference
exists in the ability to synthesize a-amino groups. Glu-
tamic acid dehydrogenase activity was found to be approx-
imately the same for the N+ and N‘ cells, while aspartase
activity was lacking in both. Work by Brubaker (personal
communication) also demonstrated a common deficiency of

alanine dehydrogenase. Investigation of the relationship

29

between permeability and the ability to utilize NH4+ again
revealed no significant differences between N+ and N‘
cells.

Available data suggests that the answer to why N-
cells of P. pestis cannot utilize NH4+ as a primary source
of nitrogen will lie in the identification of an enzyme
system in N+ but not N' cells which provides substrate
for the subsequent generation of NADPH. Alternatively,
the N+ cells may have acquired a metabolic block which
prevents the metabolism of a potential substrate of an

NADP-linked dehydrogenase by some alternative pathway.

SUMMARY

The results of nutritional experiments have con-
firmed previous findings that P. pestis lacks the ability
to utilize NH4+ as a primary source of nitrogen. Compara-
tive studies between the wild-type and a mutant capable
of using NH4+ eliminted the possibility that differences
in growth reflect differences in the permeation of NH4+.
Similarly, no distinction in the ability to assimilate

NH4+

into a-keto groups was noted. Investigations of ex-
cretion products and organic acid pools again revealed

no difference between the wild-type and its mutant. The
possibility of an NADPH deficiency, resulting in the ina-
bility of glutamic dehydrogenase to function in N' cells
was not effectively eliminated. Assays of NADPH-generating
systems showed no differences between the N+ and N” organ-
isms. However, the possibility of a deficiency of avail-
able substrate for the NADPH-generating systems of the N-

cells was not excluded, and further work in this area is

needed.

30

10.

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