JOURNAL OF BACTERIOLOGY, Sept. 2004, p. 5614–5620
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 186, No. 17
A Regulatory Trade-Off as a Source of Strain Variation in
the Species Escherichia coli†
Thea King,1Akira Ishihama,2Ayako Kori,2and Thomas Ferenci1*
School of Molecular and Microbial Biosciences G08, The University of Sydney, Sydney, Australia,1
and Nippon Institute for Biological Science, Ome, Tokyo, Japan2
Received 30 March 2004/Accepted 18 May 2004
There are few existing indications that strain variation in prokaryotic gene regulation is common or has
evolutionary advantage. In this study, we report on isolates of Escherichia coli with distinct ratios of sigma
factors (RpoD, ?D, or ?70and RpoS or ?S) that affect transcription initiated by RNA polymerase. Both
laboratory E. coli K-12 lineages and nondomesticated isolates exhibit strain-specific endogenous levels of RpoS
protein. We demonstrate that variation in genome usage underpins intraspecific variability in transcription
patterns, resistance to external stresses, and the choice of beneficial mutations under nutrient limitation. Most
unexpectedly, RpoS also controlled strain variation with respect to the metabolic capability of bacteria with
more than a dozen carbon sources. Strains with higher ?Slevels were more resistant to external stress but
metabolized fewer substrates and poorly competed for low concentrations of nutrients. On the other hand,
strains with lower ?Slevels had broader nutritional capabilities and better competitive ability with low
nutrient concentrations but low resistance to external stress. In other words, RpoS influenced both r and K
strategist functions of bacteria simultaneously. The evolutionary principle driving strain variation is proposed
to be a conceptually novel trade-off that we term SPANC (for “self-preservation and nutritional competence”).
The availability of multiple SPANC settings potentially broadens the niche occupied by a species consisting of
individuals with narrow specialization and reveals an evolutionary advantage offered by polymorphic regula-
tion. Regulatory diversity is likely to be a significant contributor to complexity in a bacterial world in which
multiple sigma factors are a universal feature.
The major source of variation in prokaryotes is thought to be
the loss or gain of functional genes or elements, such as patho-
genicity islands (14, 33). Members of a bacterial species such as
Escherichia coli have common properties and similar chromo-
somal organizations, but the species is phenotypically diverse
(44). Isolates of E. coli exhibit many distinct properties, includ-
ing distinct growth rates (28) and stress sensitivities (1, 43).
Some of the differences are undoubtedly due to loss or gain of
genes, but is there also a difference in gene usage or expression
between strains? The gene regulatory consistency of bacteria is
relatively poorly studied, but it needs to be understood if the
full range of bacterial variation is to be established. In this
study, we investigated whether strain-specific gene usage is a
source of bacterial variation in E. coli.
Our starting point for examining this question arose from
recent studies of the polymorphism of the RpoS sigma factor in
isolates of E. coli and Salmonella (11, 31). If a central regulator
of stress resistance genes (RpoS or ?S[24, 40]) is not con-
served, then how constant is gene usage on a global scale? It is
evident from both laboratory studies and the occurrence of
rpoS mutations in natural populations that regulatory diver-
gence can arise and flourish in particular environments (11). In
this study, we found that natural regulatory settings are far
from uniform within a species and include a wide range of
A significant level of control over expression of multiple
genes in bacteria involves RNA polymerase sigma factors,
which partition transcription to different bacterial promoters
(13, 17). The concentration of a sigma factor, such as ?S,
controls general stress resistance, starvation survival (16), and
gene expression under nutrient limitation (10). In addition,
because ?Scompetes for a fixed amount of RNA polymerase,
the level of ?Salso inversely influences the expression of other
? factor-controlled genes, including housekeeping genes (8,
26). Within this expanding model of cellular control through ?
factor competition (20, 21), we investigated whether RpoS
protein levels also influenced additional phenotypic and nutri-
tional abilities of various E. coli strains. As shown below, an
unexpected inverse relationship between stress resistance and
nutritional capabilities was found in different strains. Further-
more, a molecular explanation of strain variation can now be
offered on the basis of the equally unexpected variation in the
endogenous concentration of sigma factors within a species.
The numerous implications of these findings for understanding
bacterial diversity and evolution are discussed below.
MATERIALS AND METHODS
Strains and strain construction. All bacterial strains used in this study are
shown in Table 1. P1 transduction (29) with P1 cml clr1000 grown on ZK1171 was
used to introduce rpoS::Tn10 into BW2952 and MG1655. lac?derivatives of
BW2952, BW3709, ZK126, and ZK1171 were made by P1 transduction with P1
cml clr1000 grown on MG1655.
To study nondomesticated E. coli strains, the extensive collection of P. Reeves
(Sydney, Australia) was surveyed for rpoS-related properties. Forty-one patho-
genic and EcoR isolates were screened (34). Of these, only 16 strains were
RpoS?as determined by the glycogen screening test described below. In further
phenotypic screening, isolates EcoR38 and EcoR10 and O157:H7 isolate M534
were found to exhibit the range of properties shown by K-12 strains MG1655,
* Corresponding author. Mailing address: School of Molecular and
Microbial Biosciences G08, The University of Sydney, Sydney, NSW
2006, Australia. Phone: (61) (2) 9351 4277. Fax: (61) (2) 9351 4571.
† Supplemental material for this article may be found at http://jb
ZK126, and BW2952 and were used for further experiments. An rpoS mutation
could not be introduced into the P1-resistant non-K-12 strains by transduction,
so rpoS null mutants of M534 and EcoR38 were isolated directly from chemostat
cultures as previously described (31) to obtain strains BW3737 and BW3736,
Growth medium and culture conditions. The medium used in chemostat
cultures was minimal medium A (29). The carbon source in all cases was glucose,
which was present at a concentration of 0.02 or 0.04% (wt/vol) in the feed
medium in glucose-limiting experiments. For batch cultures and agar plates,
glucose or acetate was included at a concentration of 0.2% (wt/vol). Eighty-
milliliter chemostat cultures were set up as described previously (31). The dilu-
tion rates were set to 0.1 h?1(doubling time, 6.9 h). The culture densities were
between 1.9 ? 108and 2.1 ? 108bacteria ml?1.
To assess the metabolism of 95 substrates by the strains in a Biolog GN2
MicroPlate (Oxoid Ltd., Sydney, Australia) (3), the manufacturer’s instructions
were followed. Positive readings were defined as optical densities at 600 nm of
?0.2 after 24 h of incubation.
Detection of rpoS status. rpoS mutants were distinguished from wild-type
strains by staining glycogen in colonies on Luria agar plates. The plates were
incubated overnight at 37°C and then left at 4°C for 24 h before they were
flooded with concentrated iodine as previously described (31).
rpoS amplification and DNA sequencing. A 1,302-bp fragment containing the
rpoS gene was amplified from chemostat isolates by PCR by using two external
primers, RpoSF1 (5?-CGGACCTTTTATTGTGCACA-3?) and RpoSR1 (5?-TG
ATTACCTGAGTGCCTACG-3?), and an internal primer, RpoSI (5?-CTGTTA
ACGGCCGAAGAAGA-3?), as previously described (31).
?-Galactosidase and catalase assays. Five-milliliter samples were removed
from chemostat cultures, and ?-galactosidase activity was measured as described
by Miller (29) by using sodium dodecyl sulfate and chloroform-treated cells.
KatE/hydroperoxidase II catalase activity was assayed as described by Visick and
Quantitation of RNA polymerase subunits. Bacteria were harvested from
1-day-old chemostats, extracted, and analyzed by using the standard quantitative
immunoblot system (19). Probing was performed with antibodies against purified
RpoA, RpoD, or RpoS in parallel with known amounts of purified RNA poly-
merase subunits. The data presented below are means from three blots of each
of two independent samples.
Tolerance to external stress. Assays were conducted with 1-day-old chemostat
cultures (31) of each strain. To test acid resistance in rich media, the percentage
of survivors was measured after 30 min of exposure to Luria broth acidified to pH
1 with HCl. Bacteria were plated directly onto nutrient agar plates, and dilutions
were counted after overnight incubation at 37°C. Survival of bacteria in water
was assessed after 15 h of incubation at 25°C.
Strain variation in metabolism and stress resistance. We
compared six E. coli strains, all rpoS?, for metabolism of 95
substrates in a Biolog assay (3). Several strains utilized 47 to 50
substrates, but BW2952 and M534 metabolized only 31 and 24
substrates, respectively (see Table S2 in the supplemental ma-
terial). To test the possible role of ?Sin metabolism, rpoS-
defective derivatives of the strains were also assayed. Strik-
ingly, the number of substrates metabolized by M534 and
BW2952 greatly increased upon introduction of an rpoS mu-
tation (Fig. 1A). The nutritional profiles of the rpoS disruption
mutants were generally similar. Some individual metabolic dif-
ferences were found and were probably due to structural gene
differences between strains (35), but the results in Fig. 1 sug-
gest that RpoS has a pleiotropic effect on the metabolic capa-
bility of certain bacteria. The substrates that were poorly uti-
lized by both BW2952 and M534, whose metabolism was
stimulated by an rpoS disruption, included
?-methyl-D-glucoside, L-rhamnose, D-sorbitol, acetic acid, D-
galacturonic acid, succinic acid, bromosuccinic acid, L-alanine,
L-alanyl-glycine, L-asparagine, L-aspartic acid, and DL-?-glyc-
erol phosphate. The complete Biolog results are shown in
Table S2 in the supplemental material.
Interestingly, the metabolic capabilities were inversely re-
lated to the stress resistance properties of the six strains. Con-
sistent with previous surveys, E. coli isolates are not uniformly
stress resistant (1, 43) and as shown in Fig. 1B and C, the
nutritionally versatile strains, such as MG1655 and EcoR10,
were the strains that were most sensitive to stress. Conversely,
the nutritionally restricted strains were the most stress resis-
tant. An rpoS mutation disrupted resistance to starvation and
the osmotic shock that would be experienced during incubation
in water, as expected from the established role of RpoS (16).
Similarly, resistance to acid was also low in rpoS mutants.
Acetate was one of the substrates whose metabolism was
TABLE 1. Strains used in this study
StrainRelevant genotypeReference or origin
K-12 E. coli strains
Non-K-12 E. coli strains
F?araD139 ?(argF-lac)U169 rpsL150 deoCl relA1 thiA ptsF25 flb5301 rbsR
Supposed fully wild-type strain fully sequenced
W3110 ?lacU169 tna-2
EcoR38 rpoS chemostat isolate
M534 rpoS chemostat isolate
Enterohemorrhagic E. coli isolate from the
State Health Laboratory, Perth, Australiaa
aObtained from the culture collection of Peter Reeves (Sydney, Australia).
VOL. 186, 2004SIGMA FACTOR VARIATION IN E. COLI5615
stimulated by an rpoS disruption. A further indication of the
role of ?Sin nutrition came from prolonged incubation of the
E. coli K-12 isolates on acetate plates (Fig. 2). BW2952 showed
much poorer growth than MG1655, which is consistent with
the Biolog data. Growth of ZK126 was partially impaired on
acetate plates. However, after 5 days, individual colonies that
grew faster appeared in the BW2952 streak lines on acetate
medium. All of these colonies proved to be rpoS mutants (data
not shown). Growth of a defined rpoS derivative of the
BW2952 strain, as well as ZK126 (Fig. 2), on acetate was much
faster, so the suppression of metabolic capacity by RpoS could
be overcome by rpoS mutations.
Sigma factor levels in strains of E. coli. To test the basis of
the differences in metabolic and stress properties among the
RpoS?strains, the endogenous levels of the RNA polymerase
components and ? factors (40) were measured in the strains, as
shown in Fig. 2 and 3. In quantitating the concentration of the
?Sfactor relative to the concentration of a core subunit
(RpoA) or the housekeeping-metabolic ? factor (RpoD), it
was clear that the RpoD/RpoA ratio was relatively constant
(Fig. 3). In contrast, the amount of ?Svaried, and the organ-
isms with a low RpoS/RpoD ratio were more proficient in
acetate utilization and metabolism generally. Unexpectedly,
the three K-12 strains shown in Fig. 2 differed in the propor-
tion of the sigma factor over a sixfold range during growth on
acetate despite having identical rpoS sequences (results not
shown). The difference in RpoS levels was also not confined to
acetate medium, and the concentrations of RpoS protein were
markedly different in isolates at identical steady-state growth
rates in a glucose-limited chemostat (Fig. 3). Especially inter-
esting was the relationship among stress sensitivity, metabolic
capacity, and the endogenous level of RpoS.
Transcriptional effects of distinct RpoS/RpoD ratios. The
most likely way that RpoS levels influenced metabolic and
stress capabilities was through altered patterns of transcrip-
tion. The effect of having distinct steady-state RpoS levels in
the six isolates was revealed by comparing the expression of
housekeeping genes transcribed by using RpoD (?Dor ?70)
with the expression of genes expressed through RpoS or ?S
(Fig. 4). Consistent with the ?S/?Dratios in Fig. 3, quantita-
tion of expression of a ?D-dependent gene, lacZ, showed that
there was a trend towards increasing lacZ expression with
decreasing ?Sin strains, and the highest levels of LacZ were in
rpoS mutants (Fig. 4A). Conversely, when katE, an rpoS-de-
pendent gene (30), was examined, the levels of expression were
FIG. 1. Strain variation in substrate utilization and the role of
RpoS. (A) Numbers of carbon sources metabolized by the strains. A
total of 95 substrates were examined with a Biolog GN2 MicroPlate
(Oxoid Ltd.) (3). (B) Resistance to exposure to pH 1 for 30 min. (C)
Starvation survival after 15 h of incubation in water at 25°C. Assays
were conducted with 1-day-old chemostat (31) samples of each strain,
and survival was measured by determining viable counts. The error
bars indicate the standard deviations based on two replicate experi-
ments. The rpoS status of each strain is indicated by a plus sign or a
minus sign. The designations of the rpoS derivatives of the parental
strains are as follows: BW3737 (M534), BW3709 (BW2952), ZK1171
(ZK126), BW3736 (EcoR38), and BW3708 (MG1655).
FIG. 2. Strain variation in the growth of E. coli K-12 with acetate as
the sole carbon source. The rpoS status of each E. coli K-12 strain is
indicated by a plus sign or a minus sign. The designations of the rpoS
derivatives of the parental strains are as follows: BW3709 (BW2952),
ZK1171 (ZK126), and BW3708 (MG1655). The RpoS/RpoD protein
ratio in acetate-grown bacteria is indicated for each of the rpoS?
strains; the ratios for the rpoS strains were less 0.03. For quantitation
of RpoS and RpoD levels in acetate cultures we used the standard
quantitative immunoblot system (19).
5616 KING ET AL.J. BACTERIOL.
highest in the high RpoS strains (Fig. 4B) (EcoR38 was anom-
alous in not having KatE activity). There was a good correla-
tion between the expression patterns and the stress and me-
tabolism capabilities of the six strains.
Strain variation in mutational adaptation and competitive
ability. RpoS levels in different strains of E. coli influenced two
other bacterial characteristics. First, the mutational adaptation
pathway of strains growing under nutrient limitation (10) was
initiated differently. Under experimental evolution conditions
(32), as shown in Fig. 5, some strains, including strain BW2952
studied previously (11, 31), rapidly accumulated rpoS muta-
tions in chemostats under glucose limitation. ZK126 accumu-
lated rpoS mutations more slowly, whereas populations of
MG1655 did not acquire rpoS mutations. Again, there was a
good correlation between RpoS and ?S-dependent transcrip-
tional patterns and the rate of mutation accumulation; the
strains with high ?Slevels were under stronger pressure to lose
RpoS in a nutrient-stressed situation. These results parallel the
acetate mutation selection results shown in Fig. 2.
An important ecological characteristic of bacteria is the abil-
ity to compete for low levels of nutrients (9). As shown in Fig.
6, the RpoS status is a major determinant of fitness in a low-
nutrient environment. The BW2952 strain with a high level of
?Swas initially outcompeted in a glucose-limited environment
compared to MG1655 (Fig. 6A), so not only was the BW2952
strain more restricted in terms of nutritional range, but it also
had a lower fitness for glucose. After further growth, the ap-
pearance of rpoS derivatives in the BW2952 subpopulation
increased the competitiveness of the clone, whereas no rpoS
mutants of the MG1655 bacteria appeared. The proportion of
the BW2952 clone continued to increase due to the accumu-
lation of further mutations described elsewhere (32). When
competition experiments were started with rpoS derivatives of
BW2952 and MG1655, there was no initial difference in fitness,
suggesting that the two strains had similar metabolic potentials
once the constraint imposed by RpoS was removed (Fig. 6).
The distinct levels of RpoS in different strains were a major
source of phenotypic differences in six strains of E. coli. Our
results show that even the metabolic profile of bacteria is
subject to regulatory variation. This has major implications for
microbiology, in which nutrition is often used to type organ-
isms. Our results indicate that the ability to use or not use
groups of substrates may be simply a question of global regu-
Another unexpected conclusion from this study is that a
regulatory setting affects both the competitiveness of a bacte-
rium for specific substrates and also its range of substrates.
Strains such as EcoR10 and MG1655 are the best specialists
for using glucose and also have the broadest nutritional profile.
This finding is novel in ecological terms, as generalist and
specialist strategies are considered mutually exclusive in ecol-
These results also have an impact on our molecular under-
standing of trade-offs in evolution, which are characterized by
the inability of an organism to optimize different traits simul-
taneously (7, 38). The inverse relationship between nutrition
and stress resistance exhibited by bacteria with low and high
levels of ?Sis not a nutrition-nutrition trade-off like that be-
tween R and k strategists (25) or a specialist-generalist balance
(22), but it is a novel stress protection-nutrition SPANC (“self-
preservation and nutritional competence”) trade-off. Our re-
sults are also consistent with the conclusion that there is no
expected trade-off in fitness between adapting to low concen-
trations of nutrients and adapting to high concentrations of
nutrients (41). Transcriptional competition between ? factors
(8, 26) and the different RpoS/RpoD levels provide a molec-
ular explanation for the set SPANC balance for different iso-
Historically, it is important that in gene expression studies
FIG. 3. Strain variation in levels of sigma factors. (A) Quantitation
of RpoD relative to core subunit RpoA. (B and C) RpoS/RpoA
(B) and RpoS/RpoD (C) ratios of 1-day chemostat samples deter-
mined as described by Jishage and Ishihama (19) by using antibodies
against purified RpoA, RpoD, or RpoS in parallel with known
amounts of purified RNA polymerase subunits. Error bars represent
the standard deviations from three blots of each of two independent
VOL. 186, 2004SIGMA FACTOR VARIATION IN E. COLI5617
with E. coli K-12 workers have used numerous genetic back-
grounds, including the MG1655, MC4100, and W3110 lineages
used here, but our results suggest that RNA polymerase dif-
ferences need to be considered before strains are interchanged
or compared. Indeed, there was a previously noted discrepancy
in sigma factor content even within the W3110 lineage (19). It
is also relevant that recent results showed that underproduc-
tion of RpoD mimics a stringent response (27), which may also
partially be the situation in the strains with high ?Slevels. In
turn, this may be relevant to the finding that growth rate
variation is due to differences in ribosomal function (28), which
is in turn subject to stringent control (6). Even more intrigu-
ingly, the ratios of other sigma factors may also be subject to
trade-offs, because the ?54content of some W3110 strains was
also not constant (19).
From our survey, there is insufficient evidence to suggest that
particular ?Slevels are associated with particular taxonomic
groups or virotypes of E. coli. If anything, the evidence points the
other way, with a wide range of settings found even within the
taxonomic A subgroup (36), including EcoR10 and the three
K-12 strains. Still, a more systematic study is needed to test this
point. More speculatively, the variation in ? factor levels is likely
to be variation that can arise frequently, and it occurred indepen-
dently in the three K-12 lineages, as can happen during prolonged
laboratory storage (19, 39). Adaptation of the SPANC balance is
therefore likely to be common in nature.
FIG. 4. Strain variation in gene expression. (A) Expression of lacZ as determined by quantitating ?-galactosidase activity. (B) Specific activity
of KatE/hydroperoxidase II (42) of chemostat samples of each strain. The rpoS status of each strain is indicated by a plus sign or a minus sign. The
designations of the rpoS derivatives of the parental strains are as follows: BW3737 (M534), BW3709 (BW2952), ZK1171 (ZK126), BW3736
(EcoR38), and BW3708 (MG1655). The error bars indicate the standard deviations based on two replicates.
5618KING ET AL. J. BACTERIOL.
So far, no explanation for what fixes the discrete but distinct
RpoS levels in the different strains is available. At least in the
three K-12 strains with identical rpoS sequences, the influence
on RpoS levels must be extragenic. Complicating matters is the
finding that more than one regulatory element may differen-
tiate the strains with low and high RpoS levels because there
are numerous, complex inputs for controlling the level of this
? factor in the cell (15). Several regulators control each stage
of rpoS transcription and translation and ?Sprotein stability
(18). Detailed investigation of each input is needed to identify
the causes of RpoS variation. Intracellular ppGpp was a po-
tential source of variation in RpoS levels, particularly as
BW2952 (an MC4100 derivative) has a known relA1 mutation.
However, when ppGpp levels were compared by the method of
Rudd et al. (37), there was no correlation between ppGpp
levels and RpoS levels. BW2952 had low ppGpp levels but high
RpoS levels, whereas M534 had high levels of both. Likewise,
the ppGpp level in EcoR10 was lower than the ppGpp level in
MG1655, but both strains had low RpoS levels (results not
shown). Hence, ppGpp levels are nonuniform in different
strains but do not solely explain the RpoS differences observed.
Nevertheless, it is also clear that intragenic changes in rpoS
can influence all the properties discussed above. Leaky rpoS
mutations that exhibit partial stress resistance are also known
to be selected in particular environments (12, 31); these iso-
lates also show altered transcription patterns and partial in-
creases in metabolic versatility (results not shown). The rpoS
isolates in population samples (11) also add to the SPANC
diversity of bacteria, and rpoS mutants are the best-adapted
organisms nutritionally (Fig. 1 and 2). Hence, the SPANC
setting of members of E. coli can be adjusted by both extra-
genic and intragenic rpoS polymorphisms.
In summary, a ? factor protein that is associated with RNA
polymerase and central to global gene expression is present at
various endogenous levels in a species. Given that multiple ?
factors are universal in bacteria, it is highly likely that such
variations are common in the prokaryotic world and that vari-
ation in genome usage extends to bacteria, as well as to higher
organisms (4). The regulatory variation resulting from set lev-
els of RpoS provides a means of broadening the ecological and
phenotypic properties of a species. These results suggest that
polymorphic regulation is central to understanding the pheno-
typic properties of bacteria, bacterial strain variation, and the
trade-offs between environmentally useful characteristics. Fi-
nally, the SPANC trade-off may be a more general kind of
evolutionary adaptation that may be important for free-living
organisms that encounter nonconstant environments. Specula-
tively, the availability of multiple SPANC settings can be a
considerable advantage to a species by broadening its niche, so
individuals with narrow SPANC specialization may fill envi-
ronments with particular stress-nutrition combinations.
We thank Etsuko Koshio for some of the assays and Paul Rainey,
Mike Cashel, and Andy Holmes for constructive comments.
We also thank the Australian Research Council for funding support.
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