Department of Microbiology and Molecular
Genetics, Harvard Medical School, Boston, Massachusetts 02115
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TEXT |
Natural microbial populations spend
the majority of their lives under nutrient deprivation, due to intense
competition for available resources (31, 32). The
chemoorganotroph Escherichia coli can survive extended
periods of carbon starvation; cells isolated from batch cultures
starved for several years can still grow when supplied with exogenous
carbon (11, 12). Prolonged starvation is a condition in
which microbes such as E. coli undergo rapid evolution by
natural selection: mutants with an increased fitness, termed the growth
advantage in stationary phase (GASP) phenotype, grow and displace their
wild-type parents as the majority (12, 41, 42, 43). The GASP
phenomenon is continuous throughout the starvation period, as multiple
rounds of population takeover have been observed (11, 43,
44). The first GASP mutation identified was an allele of
rpoS (rpoS819) (43). Mutants with the
rpoS819 allele are referred to as GI
(GASPI) strains, as they have a GASP phenotype
when competed against their wild-type (G0)
parent (43, 44). Strain ZK1141, isolated from an aged
culture of the rpoS819 GI strain
(ZK819), expresses the GASP phenotype when competed against its
GI parent and was thus designated a
GII strain (41, 44). The
GII GASP phenotype of ZK1141 is due to three
mutations, designated sgaA, sgaB, and
sgaC (44). In the work reported here, we
identified the sgaB mutation as an allele of lrp,
encoding the leucine-responsive regulatory protein, and demonstrate
that a published lrp null allele also confers a GASP phenotype.
The sgaB GASP mutation is an allele of lrp.
Lrp is a dimeric DNA-binding protein that can act as either an
activator or a repressor, depending on the promoter (6, 9,
33). Also depending on the promoter, Lrp's activity can be
modulated positively or negatively by intracellular leucine levels. The
Lrp regulon is extensive and includes many genes involved in amino acid
metabolism and transport. In general, Lrp regulates amino acid
metabolism by increasing anabolism and decreasing catabolism. Lrp
appears to be a protein widely conserved in microbes, as lrp homologs have been detected in other proteobacteria such as
Bradyrhizobium japonicum and Klebsiella
aerogenes, in the gram-positive bacterium Bacillus
subtilis, and even in the archaea Pyrococcus furiosus and Sulfolobus solfataricus (3, 7, 14, 21, 24).
The sgaB locus was mapped roughly to min 20 on the E. coli chromosome (44), and further analysis by P1
transduction (29) showed that the sgaB locus was
closely (89%) linked to the cydC locus (Table
1) at min 20.0. The lrp locus
mapped nearby at min 20.1, and like sgaB mutants
(44), lrp null mutants are more sensitive than
the wild type to L-serine during growth on glucose (2). For these reasons, we considered lrp a
candidate for sgaB and determined the sequence of the
lrp allele of ZK1141 and the alleles of its parents, ZK819
(GI) and ZK126 (G0). The
lrp genes (including 69 upstream bases that contains the
minimal promoter [25]) were PCR amplified, and both
strands were sequenced. Both alleles from the parental strains ZK126
and ZK819 were identical to the published wild-type allele (Genbank
accession number M35869) (40). However, we found a single
lesion in the coding region of the lrp gene of ZK1141: an
in-frame, 3-bp deletion of the bases 5'-GGA-3', resulting in a protein
that lacks the Gly-39 residue of wild-type Lrp (40).
Platko and Calvo (34) identified a putative helix-turn-helix
(HTH) DNA-binding motif in the N-terminal region of Lrp
(34). Most HTH proteins identified thus far have a glycine
at position 9 in the HTH domain (4, 16), corresponding to
the Gly-39 of Lrp absent in Lrp-1141. This glycine residue is thought
to play a critical role in creating the turn structure, as its lack of
a 
carbon reduces any steric interference in creating the left-handed 
helix of the turn (4, 16, 30). However, in some HTH proteins other amino acids can be substituted for glycine at
position 9 of the motif and still allow proper folding (16, 17). To our knowledge this is the first characterization of an
HTH protein deleted for the glycine at position 9. We predicted that
the mutant protein has a misfolded DNA-binding motif and would behave
as a loss-of-function protein.
The lrp-1141 GASP allele is a loss-of-function
allele.
To determine if the lrp-1141 allele was a
loss-of-function mutation, we compared several of its phenotypes with
those of the wild-type allele and a published null allele,
lrp-35 (Table 1). All three alleles were assayed in the
ZK126 (G0) genetic background. To control for
possible effects of expression from the tetR or
tetA gene of the mini-Tn10 insertion of the
lrp-35 allele (22, 23), we introduced into the
lrp+ and lrp-1141 strains by P1
transduction a mini-Tn10 transposon linked (22%) to the
lrp locus. This mini-Tn10 insertion did not affect the outcome of the assays described below, nor did it affect the
GASP phenotype of the GI lrp-1141
strain (data not shown).
Strains with lrp null alleles are reported to grow more
slowly than wild type on glucose and have an increased sensitivity to
serine during growth on glucose (2). We confirmed these results and observed that the lrp-1141 mutant behaves
identically to the lrp-35 null mutant in both assays. As a
more sensitive assay for Lrp activity, we measured L-serine
deaminase (L-SD) activity in these strains (18). The major
L-SD of E. coli is encoded by sdaA, whose
expression is repressed directly by Lrp and activated by leucine and
glycine; the leucine activation is due in part to relief of
Lrp-mediated repression (26, 37). We assayed L-SD activity
as previously described (18) for the wild type and the two
lrp mutants grown in the presence or absence of the L-SD
inducers glycine and leucine. In both conditions, the
lrp-1141 mutant showed L-SD activities (milligrams of
pyruvate formed per minute per unit of optical density at 600 nm
[OD600]) indistinguishable from the lrp null
allele activity (Fig. 1). Similar results
were observed in assays of an Lrp-activated gene, gltB (see
below). These results indicate that lrp-1141 is a null allele, as it shows no residual repression or activation activity at
these promoters.
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FIG. 1.
The lrp-1141 mutant has the same L-SD
activity as the lrp-35 mutant. L-SD activity was assayed in
mid-exponential-phase cells (OD600 of 0.3) grown in M63
glucose (0.2%)-valine (0.005%)-isoleucine (0.005%) medium, with
(open bars) or without (hatched bars) the L-SD inducers glycine
(0.0075%) and leucine (0.005%). Bars indicate standard deviations
(n  4).
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The 2-fold-higher L-SD activity in the lrp mutants grown in
the presence of leucine and glycine is interesting, considering previous reports demonstrating that leucine alone increased L-SD activity only 1.2-fold in lrp mutants (26). As
glycine and leucine increase L-SD activity in an additive manner
(18), these results indicate that unlike leucine, glycine
may induce L-SD in an Lrp-independent manner. Alternatively, the
different findings may be due differences in strain backgrounds.
The lrp-1141 allele is dominant negative when
overexpressed.
Mutations in the DNA-binding domain of
lrp that disrupt the DNA-binding activity but do not affect
its stability or its ability to dimerize act in a dominant-negative
manner, as their protein products are stable and can form heterodimers
with the wild-type Lrp monomers that cannot bind DNA (34).
To determine if the lrp-1141 allele is dominant negative, we
cloned both the mutant and wild-type genes, including 288 bases of
upstream sequence, and introduced these clones by transformation into
strains with different chromosomal lrp alleles. The
lrp+ and lrp-1141 alleles of ZK126
and ZK1141 were amplified by PCR with primers engineered with 5' ends
recognizable by the restriction enzyme EcoRI, and these
EcoRI-digested PCR products were ligated into the
low-copy-number plasmid pSU2718 (27), creating plasmids pEZ1
(lrp+) and pEZ2 (lrp-1141). We
sequenced both strands of the cloned inserts and confirmed that they
were the correct alleles. For both constructs, pEZ1 and pEZ2, the
lrp gene was inserted in an orientation opposite that of the
lac promoter of the pSU2718 vector.
To determine the dominant or recessive nature of the
lrp-1141 allele, three assays were performed on the strains
carrying various combinations of lrp alleles on the
chromosome (single copy) and on a plasmid (multicopy). We assayed two
growth phenotypes, colony size and serine sensitivity, on M63 glucose
plates as described above. We also assayed transcription of the
gltBDF operon, encoding glutamate synthase, which is
positively regulated by Lrp (10). The
gltB::lacZ transcriptional fusion
(Table 1) was crossed into our strains by P1 transduction. The results
of all three assays were the same: when the lrp-1141 allele
is in multicopy, it is dominant to the single-copy wild-type allele
(Table 2). These results indicate that
the Lrp-1141 protein is stable and can form inactive heterodimers with
the wild-type Lrp monomers. However, the wild-type allele in multicopy
is dominant to the lrp-1141 allele in single copy.
Overexpression of the wild-type allele most likely restores
Lrp-dependent activity by increasing the total concentration of
wild-type monomers such that, in addition to the inactive heterodimers,
a sufficient amount of active wild-type homodimers form.
The lrp-35 null mutation also confers a GASP
phenotype.
If lrp-1141 is a null allele, then the
lrp-35 null allele should exhibit the same GASP phenotype as
lrp-1141. We therefore determined the stationary-phase
fitness of the lrp-35 null allele relative to the
lrp+ allele by competition assays in the
GI (rpoS819) background, as described
previously (44). We eliminated any possible fitness losses
of the lrp-35 strains due to expression from the
mini-Tn10 insertion by competing the lrp-35
mutants against two different lrp+ strains
carrying either the zbi-29::Tn10 or
zfd-1::Tn10 intergenic insertion (Table
1). Competing strains carried one of two neutral markers to distinguish
them: the ability to grow on -glucosides (Bgl+) or the
ability to grow on glucose in the presence of valine (Valr)
(44). These markers were switched between strains to confirm that they did not affect fitness.
Unlike the lrp+ control strain (Fig.
2A), the lrp-1141 strain
expresses a GASP phenotype when inoculated as a 1,000-fold minority into a culture of the lrp+ strain (Fig. 2B)
(44). As predicted, the lrp-35 null mutant exhibited a GASP phenotype essentially identical to that of the lrp-1141 mutant (Fig. 2C). This result confirmed that
loss-of-function mutations in lrp confer a GASP phenotype on
E. coli. This indicates that the Lrp regulon is induced
during prolonged carbon starvation, and that activity of this regulon
under these conditions results in a fitness loss relative to those
cells unable to express this regulon.
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FIG. 2.
The lrp-35 mutation confers a competitive
advantage to GI cells. Into a 1-day-old LB
culture of a Valr GI mutant ( )
was inoculated as a 1,000-fold minority a 1-day-old LB culture of a
Bgl+ GI mutant (A;  ),
GI lrp-1141 mutant (B;  ), or
GI lrp-35 mutant (C;  ). Viable
counts of the two competing populations were determined by titering the
mixed culture onto M63-glucose-valine or M63-salicin plates. Asterisks
indicate that viable counts fell below detectable levels
(<103 CFU/ml). Similar patterns were observed in six
replicate mixtures per strain pairing, including ones when the
selectable markers were switched between strains. Data for panel B are
from reference 44.
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The lrp-1141 and lrp-35 alleles compete
with equal fitness.
The possibility remained that there is some
undetected activity of the Lrp-1141 protein that plays a role in GASP.
In fact, lrp-1141 mutants, unlike lrp-35 null
mutants, display mucoid growth on glucose at 30°C (44).
However, the lrp-1141 mutants displayed no fitness advantage
relative to the lrp-35 null mutants when competed in
stationary phase (data not shown). Hence, the mucoidy phenotype of
lrp-1141 plays no role in stationary-phase competitions, and
we conclude that it is the loss-of-function nature of the lrp-1141 allele that is responsible for the GASP phenotype.
Since there was no detectable fitness difference between the
lrp-1141 and insertion (null) allele, we found it surprising that the GASP allele we isolated from a starved culture was
lrp-1141. Small, in-frame deletions such as
lrp-1141 should be rare among the total array of spontaneous
null mutations possible, as only one-third of all spontaneous deletions
are in frame, and deletions themselves would occupy only a small
percentage of the null mutations possible. At least two factors
independent of relative fitness may have increased the likelihood of
isolating a strain with such an unexpected GASP allele.
One possible factor is that lrp contains a potential
deletion hot spot. The 3-bp deletion of the lrp-1141 allele
overlaps the sequence 5'-GTGG-3' of the wild-type allele, which has
been identified as a hot spot for spontaneous mutation in E. coli: a high frequency of mutations was found at or in close
proximity to this sequence in wild-type strains (13), and
even higher frequencies were found in PolA
strains
(13, 19). Among the classes of spontaneous mutations associated with this sequence was a high frequency of deletion mutations, many of which were small. It is therefore possible that this
same (unknown) mechanism acted at the 5'-GTGG-3' hot spot of
lrp and facilitated the lrp-1141 deletion. If so,
this may indicate a lowered level of DNA polymerase I activity during prolonged starvation, that is, that polA+ cells
may be phenotypically PolA in stationary phase.
A second possible factor leading to the selection of
lrp-1141 is its dominant nature. While recessive mutations
do not change the cell's physiology until the wild-type product
disappears (by degradation or dilution resulting from cell division),
dominant mutations can potentially change the cell's physiology as
soon as its mutant product is made. Hence, dominant GASP mutations (dominant negative or gain of function) may predominate in starved populations because they can change the physiology of the cell more
rapidly than recessive mutations. This may be of critical importance to
starved cells, if dilution of wild-type protein by cell division is
rare or absent and the cells have only a limited time to acquire GASP
mutations before they are outcompeted for nutrients and die. Of
particular relevance to this model is the finding that starved E. coli cells can have multiple copies of their chromosome
(1), which would make it even more difficult for a recessive
loss-of-function GASP phenotype to be expressed. It would therefore be
of great interest to isolate and characterize the nature of
lrp and other GASP mutations of other starvation survivors
to determine the frequency of dominant versus recessive GASP alleles.
Mutations that disrupt alanine, serine, and threonine catabolism do
not prevent the lrp GASP phenotype.
Previously, we
demonstrated that the sgaB GASP allele (lrp-1141)
conferred faster growth when serine, threonine, or alanine was used as
the sole carbon and energy source (44). Lrp represses the
genes encoding the primary catabolic enzymes for these amino acids:
sdaA (L-SD), glyA (serine
hydroxymethyltransferase), dadAX (D-alanine
deaminase and alanine racemase), and kbl-tdh
(2-amino-3-ketobutyrate coenzyme A lyase and threonine dehydrogenase)
(28, 33). Thus, the most likely reason why lrp
mutants grow more rapidly than the wild type on these amino acids is
that the quantity of these enzymes in the wild type is growth rate
limiting, and lrp mutants produce more of these enzymes.
We proposed a model that the increased catabolism of these amino acids
is responsible for the GASP phenotype of the sgaB
(lrp-1141) mutant (44). To address this model, we
constructed sdaA, glyA, and dadA
insertion mutants of the GI lrp-1141
strain by P1 transduction (Table 1) and assayed for a loss of the GASP phenotype. Significantly, GI lrp-1141
strains with mutations in any of the three genes, or any combination of
the three, were still able to outcompete the GI
strain. These results indicate that enhanced catabolism of alanine,
serine, and threonine is dispensable for the lrp GASP
phenotype. However, while not essential, these catabolic activities may
still play a significant role stationary-phase competition, as
sdaA mutants in the G0,
GI, and GI
lrp-1141 backgrounds were outcompeted by their respective sdaA+ parents (data not shown).
It is of particular note that the two known GASP loci, lrp
and rpoS, are both regulators of many genes. Mutations in
regulators such as these make global shifts in metabolism and
physiology, often with a coordinated effect (for instance, enhanced
catabolism of multiple amino acids in lrp and
rpoS mutants [44]). Therefore, while
altering a single activity may increase fitness, altering many
activities simultaneously by altering the function of a global regulator may result in an even higher fitness gain. Hence, beneficial mutations in global regulators may be selected over other mutations when bacteria are exposed to new environments. In support of this hypothesis are reports that clinical and soil isolates of E. coli and salmonellae have extensive allele variation of
rpoS (15, 20, 38; E. R. Zinser and
R. Kolter, unpublished data) indicating that there is considerable
selective pressure acting on the rpoS locus in the natural world.
We thank M. Freundlich and R. Matthews for providing strains. We
thank G. O'Toole for technical assistance. We thank S. E. Finkel
for critical reading of the manuscript and members of the Kolter lab
for helpful comments.
This work was supported by grants from the National Science Foundation
(MCB9728936), the National Institutes of Health (GM55199), and the
Department of Health and Human Services (ES07155-15) (E.R.Z.).
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Abstract
Text
S (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757-1760, 1993). We now report that a second
global regulator, Lrp, can also play a role in stationary-phase competition. We found that a mutant that took over an aged culture of
an rpoS strain had acquired a GASP mutation in
lrp. This GASP allele, lrp-1141, encodes a
mutant protein lacking the critical glycine in the turn of the
helix-turn-helix DNA-binding domain. The lrp-1141 allele
behaves as a null mutation when in single copy and is dominant negative
when overexpressed. Hence, the mutant protein appears to retain
stability and the ability to dimerize but lacks DNA-binding activity.
We also demonstrated that a lrp null allele generated by a
transposon insertion has a fitness gain identical to that of the
lrp-1141 allele, verifying that cells lacking Lrp activity
have a competitive advantage during prolonged starvation. Finally, we
tested by genetic analysis the hypothesis that the lrp-1141
GASP mutation confers a fitness gain by enhancing amino acid catabolism
during carbon starvation. We found that while amino acid catabolism may
play a role, it is not necessary for the lrp GASP
phenotype, and hence the lrp GASP phenotype is due to more
global physiological changes.
