JOURNAL OF BACTERIOLOGY, Mar. 1996, p. 1394-1400
0021-9193/96/$04.00+0
Copyright 0 1996, American Society for Microbiology

Vol. 178, No. 5

Inorganic Polyphosphate Supports Resistance and Survival

of Stationary-Phase Escherichia coli I

   NARAYANA N. RAO AND ARTHUR KORNBERG*
Department of Biochemistty, Stanford University School of Medicine,
        Stanford, California 94305-5307

     Received 17 July 1995iAccepted 27 December 1995

The Escherichia coli mutant (ppk) lacking the enzyme polyphosphate kinase, which makes long chains of
inorganic polyphosphate (poly P), is deficient in functions expressed in the stationary phase of growth. After
2 days of growth in a medium limited in carbon sources, only 7% of the mutants survived compared with nearly
100% of the wild type; the loss in viability of the mutant was even more pronounced in a rich medium. The
mutant showed a greater sensitivity to heat, to an oxidant (H,O,), to a redox-cycling agent (menadione), and
to an osmotic challenge with 2.5 M NaCI. After a week or so in the stationary phase, mutant survivors were far
fewer in number and were replaced by an outgrowth of a small-colony-size variant with a stable genotype and
with improved viability and resistance to heat and H,O,; neither polyphosphate kinase nor long-chain poly P
was restored. Suppression of the ppk feature of heat sensitivity by extra copies of rpoS, the gene encoding the
RNA polymerase sigma factor that regulates some 50 stationary-phase genes, further implicates poly P in
promoting survival in the stationary phase.

~

Inorganic polyphosphates (poly P), linear polymers of P,
linked by phosphoanhydride bonds, are ubiquitous in nature,
having been found in all organisms examined (31, 32, 57).
Microbes, including bacteria, cyanobacteria, fungi, and algae,
accumulate poly P, which may amount to 10 to 20% of their dry
weight. Chain length may vary from 3 to 1,000 P, residues,
depending on the organism, its growth, and physiological con-
ditions (45, 53, 54). Despite its ubiquity and conservation in
evolution, the physiological roles of poly P remain unknown or
ignored. Poly P provides a substitute for ATP for sugar and
adenylate kinases (8, 13, 22, 42), a pH-stat to counterbalance
alkaline stress (43, 44), a phosphate reservoir with osmotic
advantages (31, 33), an energy source (31, 57), a chelator of
divalent metal ions (4, 14), a membrane component for DNA
entry and transformation (46), and a possible regulator in
transcription. Poly P appears to have a multiplicity of roles in
its many locations (30). To elucidate the functions of poly P in
Escherichia colt, we examined mutants that fail to synthesize
polyphosphate kinase (PPK) and lack the long-chain poly P.
These mutants failed to survive in stationary phase and were
less resistant to heat and oxidants (10). In further studies
reported here, the mutants, after a few days in the stationary
phase, were replaced by a small-colony variant that was genet-
ically stable, more viable, and stress resistant. Still other results
implicate poly P in the complex responses that promote sur-
vival in the stationary phase.

MATERIALS AND METHODS

Reagents. The sources of the reagents used in this study were as follows: ATP,
creatine kinase, catalase, DNase I, and RNase, Boehringer Mannheim; creatine
phosphate. 3-(N-morpholino) propanesulfonic acid (MOPS), menadione, kana-
mycin, ampicillin, and bovine serum albumin, Sigma; H,O,, Fisher Scientific Co.;
Immobilon-N membrane, Millipore; [y-"PIATP, Amersham Corp.; ['H-meth-
yl]thymidinc, ICN Biomedicals Iuc.; and polyethylene imine-cellulose F thin-
layer chromatography plates, Merck. rPPXl used in the poly P assay was pre-
pared and assayed as described previously (59).

* Corresponding author. Mailing address: Department of Biochem-
istry, Beckman Center, Stanford University School of Medicine, Stan-
ford, CA 94305-5307. Phone: (415) 723-6167. Fax: (415) 723-6783.

Bacterial strains and plasmids. The following E. coli K-12 derivatives were
used: JMlOl {h(luc-proAB) supE tht-IF' [fruD36 proAB+ lncl' lncZA M15]};
CA10, the ppk ppx mutant constructed by transfer of a disrupted ppk gene,
insertionally inactivated with a gene for kanamycin resistance, into JMlOl by P1
transduction (10); CAlO pMMkufF3, constructed by transformation of CA38
(CA10 red) with pMmafF3 (40) (obtained from A. Matin, Stanford University,
Stanford, Calif.); and pBC29, a multicopy plasmid bearing the gene ppk, which
was used for poly P overexpression (2). rpoS::TnlO derivatives of CAlO and the
CAlO small-colony phenotype were made by transduction with P1 grown on
MC8301 [MC4100 F- uruD139 A (nrg-luc) u169 rp.sL150 relAl fib E5301 deoC
ptsF25 rpsR rpoS::TnlO].
Survival assays. Long-term survival of E. coli was assayed as described earlier
(10) by growing cells either in MOPS-buffered minimal medium (MOPS medium
[41]) containing glucose (0.1%) as the carbon source and K2HP0, (2 mM) as the
P, source or in a rich medium, Luria-Bertani (LB) liquid medium. Viable cell
counts were determined by plating of cells onto LB agar plates: kanamycin (50
pg/ml) was added to the medium for plating of CA10.
To test the sensitivity of the stationary-phase cells to H202, JMlUl and CAlO
were grown overnight (about 20 h) in LB medium and LB medium with kana-
mycin (50 pg/ml). respectively; the medium for CAlO pBC29 contained 50 kg of
ampicillin per ml. The cells were washed and resuspended in 0.85% NaCl to an
optical density at 540 nm (ODs4") of 1.0; H,O, was added to a final concentra-
tion of 42 mM. At the times indicated, 0.1-ml samples were withdrawn, diluted
immediately in 0.85% NaC1, and plated on LB plates to determine viable cell
counts. For actively growing cells. JMlOl and CAlU were grown aerobically at
37C to the mid-exponential phase (ca. 4 x 10" cells per ml) in MOPS medium
containing glucose (0.4%) and KZHP04 (2 mM). Two-milliliter samples of these
cultures were exposed for 15 min to H,O, (0 to 40 mM) in air at 37C. To study
the lethal effect of H,O,, the exposure was terminated by addition of 2 pg of
catalase per ml, followed by dilution into 0.85% NaCI. Cells were then plated
onto LB plates to determine the viable cell count.
Starvation-induced protection against osmotic challenge in JMlOl and CAlO
cells was assessed by comparison of the viabilities of cultures osmotically chal-
lenged during mid-log growth or at 3 h after entering the stationary phase.
Cultures were grown at 37C in M9 medium (8 mM NaCI) supplemented with
either 0.025% glucose (for starvation at an OD5, of about 1.0 [4 X lo8 cells per
ml]) or in 0.4% glucose for sampling during exponential growth at a similar cell
density. Cultures were osmotically challenged by addition of solid NaCl to the
medium to reach a final concentration of 2.5 M (27). Survival was determined by
colony counts on LB plates compared with those of the cultures just before the
osmotic challenge.
For the heat shock survival assay, cells were grown overnight in LB medium.
Stationary-phase cells were washed once and diluted in 0.85% NaCl to a density
of about 5 X 10' cells per ml. Samples (2 ml) were put into prewarmed glass
tubes (55C); at the times indicated, 0.1-ml portions were plated directly onto LB
plates to determine viable cell numbers.
Growth studies. JM101. CAlO small-colony phenotype, and CAlO big-colony
phenotype cells were grown overnight in MOPS medium containing glucose
(0.4%) and K,HPO, (2 mM). The cultures were then diluted in fresh, pre-

1394

VOL. 178, 1996 INORGANIC POLYPHOSPHATE SUPPORTS SURVIVAL OF E. COLI 1395

012345

Time (min)

FIG 1. Heat resistance. Wild-type (WT), CAl0, and CAlO pMMkatF3 cells
grown overnight (-20 h) at 37C in LB medium were exposed to 55T, and their
viability was determined by plating on LB agar, with antibiotics wherever nec-
essary. Percent survival is determined as the viable cell number at each time
point divided by the viable cell number before exposure to heat.

warmed (37C) medium, and samples were removed at hourly intervals to mon-
itor growth by measurement of the turbidity at 540 nm.
['Hlthjmidine uptake. Cells were grown to the stationary phase in LB me-
dium aerobically at 37C and then incubated further for assay of long-term
survival (10). The number of surviving cells was determined each day by plating
on LB agar. From the 3rd to 10th days, cells were labeled by addition of
['Hlthymidine to a final concentration of 1 pM in 100 kl of culture medium and
incubation for 60 min at 37C with shaking. Cells were collected in a microcen-
trifuge and washed once with 0.85% NaCI. Cold 10% trichloroacetic acid (1 ml)
was added to the pellet; the suspension was vortexed and collected on a What-
man GFK Millipore glass fiber filter, washed first with cold 10% trichloroacetic
acid and then with ethanol. Radioactivity was measured in a liquid scintillation
counter.
Resistance to menadione. For growth-inhibition measurements, 0.1 ml of an
overnight culture grown in LB medium was added to 3 ml of molten F top agar
(39) and poured on a K medium agar plate (12). A solution (100 PI) of mena-
dione (2 mg/100 pl of dimethyl sulfoxide) was allowed to saturate a 13-mm-
diameter filter disc (Whatman no. 1). The disc was then placed in the center of
the solidified top agar plate and allowed to incubate overnight (18 to 24 h). The
zone of inhibition was determined by measurement of the diameter of the zone.
less that of the disc.
Extraction and assay of poly P. Cells were grown in LB medium to the
stationary phase (OD,,,, -4.5). Poly P was extracted, and the concentration was
estimated by a rapid radioassay technique (36).
Other methods. PPK and exopolyphosphatase (PPX) activities in cell extracts
of wild-type and CAlO (small- and big-colony phenotype) cells were assayed as
described previously (1, 2).
Catalase activities were measured in cells grown to the stationary phase in LB
medium. Cells permeabilized by toluene were used in the assay, which measures
the disappearance of peroxide spectrophotometrically at 240 nm (5).
Visualization of catalases (HPI and HPII) on acrylamide gels. Typical hy-
droperoxidase 1 (HPI) and HPII bands were visualized by running whole-cell
extracts on an 8% polyacrylamide gel and then staining them with a 50:SU
solution of 2% K,Fe (CN),, and 2% FeCI, (9,26). Bovine serum catalase (Sigma)
was used as a standard.

RESULTS

Effect of poly P deficiency on long-term survival in the sta-
tionary phase: heat resistance of theppk mutant (CA10). Dur-
ing survival in the stationary phase or upon starvation, cells
acquire ipoS(katF)-mediated resistance to multiple stresses,
including heat and oxidants (3, 6, 16, 19, 27, 33-35, 56). When
poly P-deficient cells (CA10) were held at 55C for 2 min, only
2% survived compared with about 90% of the corresponding
wild-type cells (JM101) (10). The mutant complemented with
multiple copies of the plasmid (pMM katF3) bearing rpoS, the

TABLE 1. Menadione sensitivity of E coli with and without poly P

Strain Genotype Killing zone (rnm?)"

JMlOl PPk +
CAlO PPk


CAlO pBC29 PPk+

CA38 ppk recA
CA38 pUC18 ppk recA

31 5 13
200 5 74
210 2 24
284 t 6

13 t- 6

a The killing zone measurements represent the average of SIX separate exper-

iments (see Matenals and Methods).

stationary-phase RNA polymerase sigma factor, proved to be
as heat resistant as wild-type cells (Fig. 1). Similar results were
obtained when the mutant was complemented with a plasmid
which contained ppk (data not shown).
Resistance to oxidative stress. Preliminary studies had noted
that poly P-deficient cells in the stationary phase were more
sensitive than wild-type cells to 42 mM H,O, (10). When the
viability of exponentially growing cells was determined after
exposure to various levels of H,O, for 15 min, both wild-type
and mutant cells showed similar degrees of sensitivity to low
levels (2.5 to 10 mM) of H,O, (Fig. 2). However, the mutants
were far more sensitive to higher concentrations of H,O, (15
to 45 mM) than the wild type. Complementation ol the mutant
in the stationary phase with a plasmid bearing the ppk gene
restored H,O, resistance to near wild-type levels (data not
presented here).
Exposure of cells to the quinone menadione leads to oxida-
tive stress due to the production of oxygen radicals (51); cells
lacking catalase, peroxidase, and superoxide dismutase show
increased sensitivity (11, 17). Theppk mutant in the stationary
phase also proved to be highly sensitive to menadione toxicity
and could be restored to resistance by complementation with
the ppk gene (Table 1).
Kesistance to osmotic challenge. Stationary-phase cells show
enhanced osmotic resistance compared with cultures in the
exponential growth phase or preadapted to osmotic stress (27).
The levels of osmotic resistance of stationary-phase cells 4 h
after they were depleted of a carbon source (glucose) were
compared with those of cells in the mid-log phase by exposure
to 2.5 M NaC1. Viability in the stationary-phase cultures con-

100

10

1

0.1

0.01 0 F 10 20 30 40

H202 (mM)

FIG. 2. Resistance to HZO,. Wild-type (WT) and CAlO cells were grown
exponentially in MOPS medium. The cells were exposed to various concentra-
tions of HZOz for a period of 15 min at 2O"C, and the viable cell numbers were
determiiied by plating onto LB agar. Survival of 100% corresponds to the viable
cell number determined immediately before the addition of H,O,.

1396 RAO AND KORNBERG J. BACTEKIOL.

.001 -

100

10


1

Fl CAI 0

.001 -01 i

0 5 10 15 20

Time (hours)

FIG. 3. Osmotic resistance to 2.5 M NaCI. Wild-type (WT) and CAlO cells
were examined in the exponential phase (A) or in the stationary phase after 3 h
of starvation for glucose (B).

sistently indicated higher osmotic resistance than did that in
the cultures challenged during the log phase of growth (Fig. 3).
However, the level of protection conferred on the mutant by
carbon starvation was lower than that observed in the wild-type
cells. After exposure of the starving stationary-phase cells to
2.5 M NaCl for 21.5 h, about 10% of the wild-type cells and
only about 1.0% of the mutant cells survived (Fig. 3B). The
sensitivity towards osmotic challenge was more pronounced in
the exponential cultures, and the absence of long-chain poly P
in theppk mutant resulted in even lower levels of viability (Fig.
3A).
Mutants fail to survive in the stationary phase and are
replaced by a small-colony variant. Wild-typc and CAlO mu-
tant cells were grown to the stationary phase in a synthetic
MOPS medium limited in glucose (0.1%) and incubated aer-
obically at 37C for several days. Viable cell counts measured
after 1 day in the stationary phase indicated a 93% loss for the
mutant compared with no loss in the wild type (Fig. 4A). After
the second day, the mutant showed equal numbers of novel
small colonies and the standard larger type. Both the small-
and big-colony phenotypes of the mutant decreased in number
upon prolonged incubation. About 3% of the ppk cells (both
small- and big-colony phenotypes) and 20% of the wild-type
cells survived after 10 days in the stationary phase (Fig. 4A).
Both phenotypes of the mutant exhibited a growth lag when
overnight (-20-h) cultures were diluted and grown in a rich
medium (LB); they were also more heat sensitive (10).

In LB medium, survival of the mutant was 0.001 to 1.0%

during the first 4 days of the stationary phase, dropping to
values as low as 100 CFU/ml of culture. At this point, thc
big-colony phenotype predominated, but when cultures grew
back to about 5 X lo7 CFU/ml, 90% or more of the population
.were of the small-colony phenotype. Despite variations in loss
of viability in several trials (Fig. 4B), over 90% of the mutants
were of the small-colony variety after 5 to 6 days. The viability
of the wild type remained constant after 3 days of incubation
with no visible change in colony morphology.

Thymidine uptake and stationary-phase survival. The dy-
namic nature of stationary-phase cultures incubated for pro-
longed periods is manifested in the appearance of revertants
and suppressors (52, 61). In order to study the rapid takeover
of the mutant population by a small-colony stable phenotype,
both the wild type and the mutant were grown to the stationary
phase in LB medium and incubated aerobically at 37C. The
viability of the cultures was determined daily for 10 days (Fig.
5). Starting from the third day of incubation, when the viability
of both the wild type and the mutant started to decline, thc
cultures were pulse-labeled for 1 h with ['Hlthymidine. A loss
in viability of the wild-type cells was observed until the third
day of incubation, and then viability remained almost un-
changed thereafter; the uptake of [3H]thymidine was not sig-
nificant (Fig. 5A). The viability of CAlO cells decreased con-
siderably with time to about 10" CFU/ml on thc scvcnth day of
incubation (Fig. 5B). The drop in viability of the mutant on the
seventh day coincided with a burst of thymidine uptake and
also coincided with a rapid increase in the number of cells of

100

10

1

n CAI0 (big)
?+?
- .1
E

100


10


1


.I


.01


.001

Y

IIIIIIII

13 5 7 911
     Time (days)

FIG. 4. Long-term survival. Wild-type (WT) and CAlO cells (small and big)
were incuhated in MOPS medium with limited glucose (0.1%). (A) Percent
survival is expressed as the viable cell number at each time point divided by the
viable cell number of the same culture after 24 h after initial inoculation of the

within 4 days of incubation, whereas the wild-type cell number
remained about 20777 of the initial value pig, 4~).   in the
        a small-colony phenotype appeared in the
mutant cultures. Mutants lost about 99% of their viability

culture. Similar results were obtained in three independent experiments. (B)
Wild-type and CAIU cells were kept in LB medium. Curves 1, 2, 3, and 4
represent four different CAlO cultures showing fluctuations in survival due to
time of appearance of the small-colony variant. Percent survival is calculated as
for panel A

VOL. 178, 1996 INORGANIC POLYPHOSPHATE SUPPORTS SURVIVAL OF E. COLf 1397

3H-Thy

100 .1 lL1

10

1

.1

1357911

Time (days)

FIG. 5. Thymidine (Thy) uptake during long-term incubation of stationary-
phase wild-type (W1') (A) and CAlO (B) cells. 'The cells were pulse-labeled with
['Hlthymidine from the 3rd to 10th days. Both survival of the cells and the
incorporation of ['Hlthymidine into cells were measured (Materials and Meth-
ods).

the small-colony phenotype. The viable cell number increased
to about 5 X lo7 CFU/ml after 9 days; most o the cells
(-90%) observed in four separate experiments were the small-
colony variants. Takeover of the cell population by the small-
colony phenotype was invariably accompanied by a burst of
thymidine uptake.
Characteristics of the small-colony variant. (i) Stable phe-
notype. CAlO cells were grown to the stationary phase in LB
medium with kanamycin, incubated aerobically at 37C for 5
days, and plated on LB plates containing kanamycin. The sta-
tionary-phase culture contained small- and big-colony pheno-
types. The big colonies and the more slowly growing small
colonies were isolated and maintained as stable phenotypes.
The colony size was well defined for these phenotypes. When
grown in MOPS-buffered medium for 48 h at 37"C, the colony
diameters were 1.0 ? 0.5 mm for the small-colony phenotype
and 3.0 2 0.5 mm for the big-colony phenotype. A few of the
characteristics of these variants were studied relative to the
role of poly P during survival in the stationary phase.
(ii) Growth lag. When wild-type cells and the small- and
big-colony variants of CAlO were grown in MOPS medium
containing 2 mM K2HP0, and 0.4% glucose, the small-colony
phenotype cells consistently showed a longer growth lag (Fig.
6). Generation times were 56 min for the wild-type and the
CAlO big-colony phenotype cells; the CAlO small-colony cells
grew with a generation time of 60 min.
(iii) Stationary-phase survival. The big- and small-colony
variants of CAlO were grown in LB medium to the stationary
phase and incubated aerobically at 37C for 10 days (Fig. 7).

t

I,IIIIII

2 4 6 8
  Time (hours)

FIG. 6. Growth of wild-type (WT) cells and CAlO cells exhibiting either the
big-colony phenotype or the small-colony phenotype. Prewarmed (37C) MOPS
medium was inoculated with overnight (-20 h) cultures (diluted 1 to 100);
growth was monitored by measuring the turbidity at

The big-colony phenotype, like its parent strain, showed a
considerable decrease in viability; after 5 days, only about 1%
of the original population had survived. On the other hand, the
small-colony phenotype showed an improved viability with
long-term incubation; about 10% of the population survived
until 8 days (Fig. 7).
(iv) Heat resistance. Upon entry into the stationary phase,
wild-type cells develop a pronounced tolerance to heating at 55
to 57C (28). Stationary-phase cells of the wild-type and big-
and small-colony phenotypes of CAlO were exposed to 55C
and their viability was assessed (Fig. 8). The survival profile of
the big-colony phenotype was similar to that of the parent
CAlO strains; less than 1% of the cells survived heating for 2
min compared with over 90% of the wild-type cells (JM101).
The small-colony variant showed a significantly higher level of
resistance towards heat shock than did the big-colony pheno-
type; about 60% of the small-colony cells survived the 2-min

0.1

2 4 6 8 10
    Time (days)

FIG. 7. Comparison of rates of long-term survival of big- and small-colony
phenotypes of CAlO cells in the stationary phase. The cells were grown and
incubated aerobically in LB medium at 37C. Percent survival IS calculated as
described in the legend to Fig. 1.

1398 RAO AND KORNBERG

J. BACTERIOL.

I

12345
   Time (min)

FIG. 8. Heat resistance. Wild-type (WT) cells and big- and small-colony
phenotypes of CAlO were grown overnight (-20 h) at 37C in LB medium and
exposed to 55C; percent survival is calculated as described in the legend to Fig.
1. No viable cells were detected beyond the 3-min timc point for the CAlO
big-colony phenotype.

(v) PPK and PPX activities. To determine whether the en-
hanced survival of the small-colony phenotype was due to a
reversion or suppression of the PPK and PPX mutant pheno-
type, the enzyme activities were compared (Table 2). CAlO
cells still showed very low levels of both PPK (-2% of the
wild-type level) and PPX activity. The residual PPX activity
(about 6% of the wild-type level) in the mutant could be
attributed to the guanosine pentaphosphate poly Pase (29). No
significant difference was observed in the enzyme activities of
the mutant strains showing either the big-colony or the small-
colony phenotype (Table 2). To confirm that the PPX protein
was not being expressed in the mutants, particulate fractions
obtained after lysis of the cells by lysozyme were analyzed by
immunoblotting with an anti-PPX antibody. Neither the big-
colony phenotype nor the small-colony phenotype of CAlO
showed the presence of PPX (data not shown). Similarly, a
Western blot (immunoblot) analysis of a trichloroacetic acid
precipitate of the total cell protein of the mutant phenotypes of
CAlO failed to indicate the presence of PPK (data not shown).
(vi) Poly P content. The poly P content of the stationary-
phase wild-type cultures was 678 ? 19 pmol/mg of protein; the
levels for the CAlO big- and small-colony phenotypes were at
the limit of detection (about 100 pmol/mg of protein). Poly P
levels in an rpoS knockout mutant were compared with those
of a corresponding isogenic wild-type strain, during both the
exponential and stationary phases of growth. No significant
difference in poly P contents was noticed between the wild type

TABLE 2. PPK and PPX activities

Enzyme activity
(Uimg of protein)"

PPK PPX

JMlOl (wild type)             424 248
CA10-s (small colony)             8.0 1.5
CA10-b (big colony)             12 6.0

" The PPK assay measured the production of acid-insoluble [BzP]poly P from
[y3'P]ATP (1). One unit of enzyme is defined as the amount incorporating 1
pmol of phosphate into acid-insoluble poly P per min at 37C. The assay for PPX
measured the loss of acid-insoluble ['zP]poly P. One unit of enzyme is defined as
the amount that decreases the acid-insoluble poly P by 1 pmol of phosphate per
min at 37C.

80

20

CAlO (small)

/

(Small) rpoS::TnlO

CA10 rpoS::TnlO

/I

0

-

024601012
       Time (rnin)

FIG. 9. Heat resistance. CAlO small-colony phenotype, CAlO smallcolony
phenotype rpoS::TnlO, and CAlO rpoS::TnlO cells were grown overnight (-20 h)
at 37C in LB medium and exposed to 55C; percent survival is cdkuhted as
described in the legend to Fig. 1.

and the rpoS::TnlO strain, especially in cells during stationary-
phase growth (data not shown).
(vii) Effect of rpoS on heat resistance. The enhanced heat
resistance acquired by the stationary-phase small-colony phe-
notype might be attributed to a secondary mutation in rpoS. In
order to test this hypothesis, rpoS mutant derivatives of the
small-colony phenotype, as well as that ol the original ppk
strain (CAlO), were constructed. The stationary-phase cells of
these rpoS knockout mutants and the cells of the small-colony
phenotype were tested for their heat resistance (Fig. 9). Intro-
duction of the rpoS mutation to the small-colony ppk mutant
resulted in reduced heat tolerance at 55T, but this effect was
less pronounced than that observed with the rpoS mutant of
the CAlO cells, thus failing to identify an rpoS locus for the
small-colony phenotype; rpoS knockout mutant derivatives of
both the small-colony phenotype and the CAlO cells were
found to be starvation sensitive, and only about 10% of the
stationary-phase cells survived alter 20 h of incubation in LB
medium.
(viii) Catalase activity. Stationary-phase cells exhibit strong
rpoS-dependent resistance to H,O,. Catalase HPII, the rpoS-
controlled katE gene product, is induced in stationary-phase
cells as a defense against H,O, (37). Catalase activities of the
wild type and of the small- and big-colony phenotypes of CAlO
cells were measured in cells grown to the stationary phase in
LB medium (Table 3). The small-colony phenotype contained
about 60% of the activity seen in the wild-type cells; the big-
colony phenotype contained only 35%. When the small-colony
variant was complemented with a plasmid, pBC29, which car-
ries the ppk gene, the catalase activity was restored to the
wild-type level. However, these catalase activity measurements

TABLE 3. Catalase activities of strains used in this study

Strain

Phenotype

  Activity
(Ulmg of protein)"

JMlOl      PPK+ (wild type) 32

CAlO       PPK- (small colony) 19
CAlOpBC29    PPK+++ (small colony) 31

CAlO PPK- (big colony) 11

* The activities of permeabilized stationary-phase cells were measured. One

unit is equal to 1 kmol of hydrogen peroxide decomposed per min at 25C.

VOL. 178, 1996 INORGANIC POLYPHOSPHATE SUPPORTS SURVIVAL OF E. COLI 1399

fail to differentiate between the rpoS-independent HPI and the
tpoS-dependent HPII activities. Therefore, extracts of station-
ary-phase cells, analyzed qualitatively on a nondenaturing
polyacrylamide gel, were stained for catalase activity in the
HPI and HPII bands. No significant difference in HPI activity
was observed between the wild-type and mutant cells (both
small- and big-colony phenotypes). The stationary-phase wild-
type cells possessed a high level of HPII activity; however, the
mutants showed only trace amounts of this enzyme activity.
Complementation of either CAlO cells or the CAlO small-
colony phenotype with the ppk gene (pBC29) or an rpoS plas-
mid (pMMkatF3) elevated synthesis of HPII to the wild-type
level (data not shown).

DISCUSSION

The stationary phase of growth studied in the laboratory can
resemble the stressful and deprived state that characterizes the
natural habitat of most bacteria (47). To cope with adversity,
cells entering the stationary phase undergo drastic changes in
cell physiology and morphology (48). Many genes not required
for exponential growth are induced in order that cells may
survive in the stationary phase (6, 19, 20, 34, 50). Mutations in
these genes in E. coli decrease viability during the prolonged
periods of nutritional deprivation in a stressful environment.
Among them, as the present studies show, isppk, the gene that
encodes PPK, the enzyme responsible for making long-chain

Stresses that E. coli responds to or anticipates include oxi-
dants, heat, and high salt levels (27, 28). Aerobic metabolism
produces reactive byproducts such as superoxide and hydrogen
peroxide (15, 17) that are involved in the generation of cyto-
toxic hydroxyl radicals (7, 23, 24). In log-phase cultures, genes
induced to cope with these oxidants are found in the SOXR and
oxyR regulons (11,17, 18). We report here that theppk mutant,
lacking poly P, is more sensitive to hydrogen peroxide than the
wild type (Fig. 2). Also, in stationary-phase cultures, the
heightened resistance to hydrogen peroxide is diminished in
the ppk mutant (10). This resistance to hydrogen peroxide is
largely restored to the wild-type level when the mutant is
complemented with a plasmid bearing the ppk gene.
Another example of oxidant stress is the toxicity of menadi-
one, the one-electron redox agent that generates cytotoxic
oxidants as in the redox cycling of respiratory chain compo-
nents during aerobic metabolism (58). Catalase activities in-
duced to scavenge oxidants such as hydrogen peroxide include
HPI in log-phase cells and HPII in stationary-phase cells (37).
The ppk mutant proved to be hypersensitive to menadione
(Table l), presumably because of a deficiency in catalase ac-
tivities (Table 3), particularly that of HPII because of the lack
of its induction in the stationary phase (17, 26).
In addition to increased sensitivity to oxidants, responses to
heat and high salt levels were also examined in theppk mutant,
and resistance to these agents was found to be greatly dimin-
ished. After exposure to 55C for 2 min, only 2% of the mutant
cells in the stationary phase survived compared with about
90% of the wild-type cells (Fig. 1). With regard to the chal-
lenge of a medium with 2.5 M NaCl, the mutants lost viability
to a greater extent than wild-type cells in both the log and
stationary phases (Fig. 3).
The most striking phenotype of the ppk mutant is its poor
rate of survival in stationary-phase cultures (10) (Fig. 4). Via-
ble cell counts decreased drastically within a few days upon
aerobic incubation, the more so in a rich medium. Of mutants
in a medium limited in carbon source (i.e., glucose), about 1%
survived after 9 days, whereas in a rich medium only about 1 in

poly P.

lo6 cells survived after 4 days. A similar influence of growth
medium on the long-term survival of wild-type cells has also
been observed (52).
Although the bases for the dramatic poly P effects in pro-
moting resistance to stressful agents and prolonging survival in
the stationary phase are still unexplained, some insight may be
gained from the suppressive influence of rpoS on the ppk mu-
tation. The product of rpoS is the a" subunit of RNA poly-
merase responsible for the expression of nearly 50 genes in-
volved in adjustments in the stationary phase to nutrient
deprivations, high osmolarity (21), and other stressful agents.
When multiple-copy rpoS plasmids were introduced into the
ppk mutant, heat resistance was elevated to the wild-type level
(Fig. 1). RpoS plays a central role in the development of stress
resistance in E. coli. Modulation of the concentration of this
sigma factor brought about either by the exposure of the wild-
type cells to various stresses or by the presence of a plasmid
bearing rpoS in the ppk mutant may be responsible for the
induction of proteins essential lor heat tolerance.
Less direct, but suggestive of a relationship between rpoS
and poly P, is the emergence of a novel small-colony phenotype
inppk mutants (10). After maintenance of theppk mutant for
several days in the stationary phase, the colonies of normal size
are largely replaced by small colonies which are genetically
stable. Their emergence coincides with a burst of DNA syn-
thesis indicated by thymidine incorporation (Fig. 5). The small-
colony variant demonstrates increased resistance to heat (Fig.
8) and an improved rate of survival (Fig. 7). Inasmuch as the
levels of PPK activity and poly P in the variant remain at the
mutant cell levels, some other genetic alteration can be in-
ferred.

The altered gene in the small-colony variant was initially
assumed to be allelic to rpoS, since this locus is involved in
adaptive mutations (49, 52, 61). Besides, rpoS plays a major
regulatory role during survival in the stationary phase (25, 38,
55, 60). The rpoS-negative mutants did not lack poly P during
both exponential and stationary-phase growth. Introduction of
rpos:TnlO into the stable, small-colony variant did not result in
the complete restoration of heat sensitivity in stationary-phase
cells (Fig. 9). It appears that the second site suppressor muta-
tion in the small-colony phenotype may act independently of
tpos.

ACKNOWLEDGMENTS

This work was supported by a grant from the National Institutes of

Health.

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