The uncertain consequences of transferring bacterial strains between laboratories - rpoS instability as an example

Spira, Beny; Toledo, Rodrigo de Almeida; Maharjan, Ram; Ferenci, Thomas

doi:10.1186/1471-2180-11-248

Cited by 47 publications

(32 citation statements)

References 39 publications

(73 reference statements)

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“…Different environmental stresses are known to upregulate RpoS to varied levels (8), suggesting that some insensitive genes may only be expressed under certain stresses. In addition to the effects on gene expression, sensitivity to RpoS may also impact the effects of mutations in rpoS, which have been seen to evolve in the lab (52,53). We expect mutations attenuating RpoS have the strongest effect on insensitive genes and the weakest effect on sensitive genes.…”

Section: Discussionmentioning

confidence: 99%

Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12

et al. 2017

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The alternative sigma factor RpoS is a central regulator of many stress responses in Escherichia coli. The level of functional RpoS differs depending on the stress. The effect of these differing concentrations of RpoS on global transcriptional responses remains unclear. We investigated the effect of RpoS concentration on the transcriptome during stationary phase in rich media. We found that 23% of genes in the E. coli genome are regulated by RpoS, and we identified many RpoS-transcribed genes and promoters. We observed three distinct classes of response to RpoS by genes in the regulon: genes whose expression changes linearly with increasing RpoS level, genes whose expression changes dramatically with the production of only a little RpoS ("sensitive" genes), and genes whose expression changes very little with the production of a little RpoS ("insensitive"). We show that sequences outside the core promoter region determine whether an RpoS-regulated gene is sensitive or insensitive. Moreover, we show that sensitive and insensitive genes are enriched for specific functional classes and that the sensitivity of a gene to RpoS corresponds to the timing of induction as cells enter stationary phase. Thus, promoter sensitivity to RpoS is a mechanism to coordinate specific cellular processes with growth phase and may also contribute to the diversity of stress responses directed by RpoS. IMPORTANCEThe sigma factor RpoS is a global regulator that controls the response to many stresses in Escherichia coli. Different stresses result in different levels of RpoS production, but the consequences of this variation are unknown. We describe how changing the level of RpoS does not influence all RpoS-regulated genes equally. The cause of this variation is likely the action of transcription factors that bind the promoters of the genes. We show that the sensitivity of a gene to RpoS levels explains the timing of expression as cells enter stationary phase and that genes with different RpoS sensitivities are enriched for specific functional groups. Thus, promoter sensitivity to RpoS is a mechanism that coordinates specific cellular processes in response to stresses. KEYWORDS RpoS, promoters, S , stress response, transcriptional regulation, transcriptome G enome-wide measurements of RNA levels have revolutionized our understanding of how cells organize their patterns of transcription. These studies have given us snapshots of how patterns of gene expression change in response to changes in the external environment. They have also allowed us to define the regulons controlled by specific transcription factors (TFs). A major weakness of the vast majority of these

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Section: Discussionmentioning

confidence: 99%

Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12

et al. 2017

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“…Although the strains had suffered few subcultures (about 5) and were stored at Ϫ80°C, they had been passaged twice in stabs for short periods. It has recently been reported that storage in stabs selects for partial or complete loss-of-function mutations in rpoS (59,60). The high level of rpoS mutations could be related to the storage conditions.…”

Section: Discussionmentioning

confidence: 99%

Fitness, Stress Resistance, and Extraintestinal Virulence in Escherichia coli

et al. 2013

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eThe extraintestinal virulence of Escherichia coli is dependent on numerous virulence genes. However, there is growing evidence for a role of the metabolic properties and stress responses of strains in pathogenesis. We assessed the respective roles of these factors in strain virulence by developing phenotypic assays for measuring in vitro individual and competitive fitness and the general stress response, which we applied to 82 commensal and extraintestinal pathogenic E. coli strains previously tested in a mouse model of sepsis. Individual fitness properties, in terms of maximum growth rates in various media (Luria-Bertani broth with and without iron chelator, minimal medium supplemented with gluconate, and human urine) and competitive fitness properties, estimated as the mean relative growth rate per generation in mixed cultures with a reference fluorescent E. coli strain, were highly diverse between strains. The activity of the main general stress response regulator, RpoS, as determined by iodine staining of the colonies, H 2 O 2 resistance, and rpoS sequencing, was also highly variable. No correlation between strain fitness and stress resistance and virulence in the mouse model was found, except that the maximum growth rate in urine was higher for virulent strains. Multivariate analysis showed that the number of virulence factors was the only independent factor explaining the virulence in mice. At the species level, growth capacity and stress resistance are heterogeneous properties that do not contribute significantly to the intrinsic virulence of the strains.

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“…RpoS is an alternative sigma factor of RNA polymerase that regulates more than 10% of E. coli genes and plays a critical role in survival under conditions of exposure to several types of stress, including acid, heat, and oxidative stress (14,15). A particular case of in vitro evolution is the inactivation of crl (which modulates the balance between different sigma factors in the RNA polymerase holoenzyme, particularly RpoS [16][17][18]) or rpoS (19)(20)(21), during stab storage of E. coli or Salmonella strains or during nutrient starvation, which favors the inactivation of the general stress response. Regulatory networks are also dynamic in vivo.…”

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The rpoS Gene Is Predominantly Inactivated during Laboratory Storage and Undergoes Source-Sink Evolution in Escherichia coli Species

et al. 2014

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The rpoS gene codes for an alternative RNA polymerase sigma factor, which acts as a general regulator of the stress response. Inactivating alleles of rpoS in collections of natural Escherichia coli isolates have been observed at very variable frequencies, from less than 1% to more than 70% of strains. rpoS is easily inactivated in nutrient-deprived environments such as stab storage, which makes it difficult to determine the true frequency of rpoS inactivation in nature. We studied the evolutionary history of rpoS and compared it to the phylogenetic history of bacteria in two collections of 82 human commensal and extraintestinal E. coli strains. These strains were representative of the phylogenetic diversity of the species and differed only by their storage conditions. In both collections, the phylogenetic histories of rpoS and of the strains were congruent, indicating that horizontal gene transfer had not occurred at the rpoS locus, and rpoS was under strong purifying selection, with a ratio of the nonsynonymous mutation rate (Ka) to the synonymous substitution rate (Ks) substantially smaller than 1. Stab storage was associated with a high frequency of inactivating alleles, whereas almost no amino acid sequence variation was observed in RpoS in the collection studied directly after isolation of the strains from the host. Furthermore, the accumulation of variations in rpoS was typical of source-sink dynamics. In conclusion, rpoS is rarely inactivated in natural E. coli isolates within their mammalian hosts, probably because such strains rapidly become evolutionary dead ends. Our data should encourage bacteriologists to freeze isolates immediately and to avoid the use of stab storage.T he rates of recombination and mutation in a genome are critical issues because the diversity generated by these mechanisms is the substrate for selection. Some regions of bacterial chromosomes are more prone to mutation (1-4) or recombination (5) than other regions, due to physical or physiological constraints. Then, selection can favor diversification, for example, that of cell surface proteins such as beta barrel porins (6), and recombination events, which can restore particular functions, as in the case of the mismatch repair (MR) genes (7,8).There is growing evidence that selection also acts on central regulators to allow adaptation by rewiring of networks. In vitro experimental models of Escherichia coli evolution have shown that adaptation occurs through modifications of global regulatory networks, which are often interconnected (9-13). These networks control DNA superhelicity (12), the stringent response (12), and the general stress response, via RpoS (9, 10, 13). RpoS is an alternative sigma factor of RNA polymerase that regulates more than 10% of E. coli genes and plays a critical role in survival under conditions of exposure to several types of stress, including acid, heat, and oxidative stress (14,15). A particular case of in vitro evolution is the inactivation of crl (which modulates the balance between different sigma f...

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The uncertain consequences of transferring bacterial strains between laboratories - rpoS instability as an example

Cited by 47 publications

References 39 publications

Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12

Genome-Wide Transcriptional Response to Varying RpoS Levels in Escherichia coli K-12

Fitness, Stress Resistance, and Extraintestinal Virulence in Escherichia coli

The rpoS Gene Is Predominantly Inactivated during Laboratory Storage and Undergoes Source-Sink Evolution in Escherichia coli Species

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