Dataset Information

Prolonged stationary-phase incubation selects for lrp mutations in Escherichia coli K-12.

ABSTRACT: Evolution by natural selection occurs in cultures of Escherichia coli maintained under carbon starvation stress. Mutants of increased fitness express a growth advantage in stationary phase (GASP) phenotype, enabling them to grow and displace the parent as the majority population. The first GASP mutation was identified as a loss-of-function allele of rpoS, encoding the stationary-phase global regulator, sigma(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.

SUBMITTER: Zinser ER

PROVIDER: S-EPMC101964 | biostudies-literature | 2000 Aug

REPOSITORIES: biostudies-literature

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Prolonged stationary-phase incubation selects for lrp mutations in Escherichia coli K-12.

Zinser E R ER Kolter R R

Journal of bacteriology 20000801 15

Evolution by natural selection occurs in cultures of Escherichia coli maintained under carbon starvation stress. Mutants of increased fitness express a growth advantage in stationary phase (GASP) phenotype, enabling them to grow and displace the parent as the majority population. The first GASP mutation was identified as a loss-of-function allele of rpoS, encoding the stationary-phase global regulator, sigma(S) (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1 ...[more]

PMID: 10894750

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Project description:Escherichia coli populations undergo repeated replacement of parental genotypes with fitter variants deep in stationary phase. We isolated one such variant, which emerged after 3 weeks of maintaining an E. coli K-12 population in stationary phase. This variant displayed a small colony phenotype and slow growth and was able to outcompete its ancestor over a narrow time window in stationary phase. The variant also shows tolerance to beta-lactam antibiotics, though not previously exposed to the antibiotic. We show that an RpoC(A494V) mutation confers the slow growth and small colony phenotype on this variant. The ability of this mutation to confer a growth advantage in stationary phase depends on the availability of the stationary-phase sigma factor σS The RpoC(A494V) mutation upregulates the σS regulon. As shown over 20 years ago, early in prolonged stationary phase, σS attenuation, but not complete loss of activity, confers a fitness advantage. Our study shows that later mutations enhance σS activity, either by mutating the gene for σS directly or via mutations such as RpoC(A494V). The balance between the activities of the housekeeping major sigma factor and σS sets up a trade-off between growth and stress tolerance, which is tuned repeatedly during prolonged stationary phase.IMPORTANCE An important general mechanism of a bacterium's adaptation to its environment involves adjusting the balance between growing fast and tolerating stresses. One paradigm where this plays out is in prolonged stationary phase: early studies showed that attenuation, but not complete elimination, of the general stress response enables early adaptation of the bacterium E. coli to the conditions established about 10 days into stationary phase. We show here that this balance is not static and that it is tilted back in favor of the general stress response about 2 weeks later. This can be established by direct mutations in the master regulator of the general stress response or by mutations in the core RNA polymerase enzyme itself. These conditions can support the development of antibiotic tolerance although the bacterium is not exposed to the antibiotic. Further exploration of the growth-stress balance over the course of stationary phase will necessarily require a deeper understanding of the events in the extracellular milieu.

Project description:DNA methylation in prokaryotes is widespread. The most common modification of the genome is the methylation of adenine at the N-6 position. In Escherichia coli K-12 and many gammaproteobacteria, this modification is catalyzed by DNA adenine methyltransferase (Dam) at the GATC consensus sequence and is known to modulate cellular processes including transcriptional regulation of gene expression, initiation of chromosomal replication, and DNA mismatch repair. While studies thus far have focused on the motifs associated with methylated adenine (meA), the frequency of meA across the genome, and temporal dynamics during early periods of incubation, here we conduct the first study on the temporal dynamics of adenine methylation in E. coli by Dam throughout all five phases of the bacterial life cycle in the laboratory. Using single-molecule real-time sequencing, we show that virtually all GATC sites are significantly methylated over time; nearly complete methylation of the chromosome was confirmed by mass spectroscopy analysis. However, we also detect 66 sites whose methylation patterns change significantly over time within a population, including three sites associated with sialic acid transport and catabolism, suggesting a potential role for Dam regulation of these genes; differential expression of this subset of genes was confirmed by quantitative real-time PCR. Further, we show significant growth defects of the dam mutant during long-term stationary phase (LTSP). Together these data suggest that the cell places a high premium on fully methylating the chromosome and that alterations in methylation patterns may have significant impact on patterns of transcription, maintenance of genetic fidelity, and cell survival. IMPORTANCE While it has been shown that methylation remains relatively constant into early stationary phase of E. coli, this study goes further through death phase and long-term stationary phase, a unique time in the bacterial life cycle due to nutrient limitation and strong selection for mutants with increased fitness. The absence of methylation at GATC sites can influence the mutation frequency within a population due to aberrant mismatch repair. Therefore, it is important to investigate the methylation status of GATC sites in an environment where cells may not prioritize methylation of the chromosome. This study demonstrates that chromosome methylation remains a priority even under conditions of nutrient limitation, indicating that continuous methylation at GATC sites could be under positive selection.

Dataset Information

Prolonged stationary-phase incubation selects for lrp mutations in Escherichia coli K-12.

Publications

Prolonged stationary-phase incubation selects for lrp mutations in Escherichia coli K-12.

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