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Appropriate Regulation of the ?E-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli.

ABSTRACT: The alternative sigma factor ?E is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of ?E increases during entry into stationary phase, suggesting an important role for ?E when nutrients are limiting. Elevated ?E activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand ?E-directed cell lysis and the role of ?E in stationary phase, we investigated the effects of elevated ?E activity in cultures grown for 10 days. We demonstrate that high ?E activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced ?E activity, due primarily to point mutations in the region of ?E that binds the -35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High ?E activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg2+ and buffering the growth medium or by deleting the ?E-dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of ?E activity. Our results demonstrate that appropriate regulation of ?E activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability.IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The ?E response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of ?E is tightly regulated to match the production of ?E regulon members to the needs of the cell. In E. coli, loss of ?E results in lethality. Here we demonstrate that excessive ?E activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent.

SUBMITTER: Nicoloff H

PROVIDER: S-EPMC5446625 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Appropriate Regulation of the σE-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli.

Nicoloff Hervé H Gopalkrishnan Saumya S Ades Sarah E SE

Journal of bacteriology 20170525 12

The alternative sigma factor σE is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of σE increases during entry into stationary phase, suggesting an important role for σE when nutrients are limiting. Elevated σE activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase ...[more]

PMID: 28373273

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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

Appropriate Regulation of the ?E-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli.

Publications

Appropriate Regulation of the σ<sup>E</sup>-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli.

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