RpoS, an alternative sigma factor, is critical for stress response in Escherichia coli. The RpoS regulon expression has been well characterized in rich media that support fast growth and high growth yields. In contrast, though RpoS levels are high in minimal media, how RpoS functions under such conditions has not been clearly resolved. In this study, we compared the global transcriptional profiles of wild type and an rpoS mutant of E. coli grown in glucose minimal media using microarray analyses. The expression of over 200 genes was altered by loss of RpoS in exponential and stationary phases, with only 48 genes common to both conditions. The nature of the RpoS-controlled regulon in minimal media was substantially different from that expressed in rich media. Specifically, the expression of many genes encoding regulatory factors (e.g., hfq, csrA, and rpoE) and genes in metabolic pathways (e.g., lysA, lysC, and hisD) were regulated by RpoS in minimal media. In early exponential phase, protein levels of RpoS in minimal media were much higher than that in Luria-Bertani media, which may at least partly account for the observed difference in the expression of RpoS-controlled genes. Expression of genes required for flagellar function and chemotaxis was elevated in the rpoS mutant. Western blot analyses show that the flagella sigma factor FliA was expressed much higher in rpoS mutants than in WT in all phase of growth. Consistent with this, the motility of rpoS mutants was enhanced relative to WT. In conclusion, RpoS and its controlled regulators form a complex regulatory network that mediates the expression of a large regulon in minimal media.
"For all experiments cells should be in the stationary growth phase, so that σ 38 is expressed and occupies a significant amount of Eσ , , , allowing the observation of transcription activity with a mixture of Eσ 70 and Eσ 38 (P(Eσ 70 ) WT <1). In the mutant strain lacking σ 38 , most E will be bound by σ 70 , ,  (P(Eσ 70 ) MT =1). The strategy to induce the stationary growth is described in . "
[Show abstract][Hide abstract] ABSTRACT: One of the global regulators of transcription dynamics in Escherichia coli is the intracellular population
of σ factors, due to their role in gene selection for transcription. It is unknown to which degree σ factors affect
the dynamics of transcription initiation, following the binding between the RNAP holoenzyme (Eσ) and the
promoter, and the closed complex formation. Proposed here is a new method to study the kinetics of the underlying steps in transcription initiation from time-lapse imaging of transcription events at the single RNA level in live cells. Namely, assuming a promoter that can be transcribed by Eσ70 or Eσ38, the researchers make use of in silico data from a stochastic model of transcription dynamics of that promoter, to show that the method estimates consistently and effectively the kinetics rates of closed and open complex formation by Eσ70 and Eσ38. In the end, the necessary measurement procedures for acquiring the data needed to apply this new methodology are described.
International Conference on Biological and Medical Sciences, Shanghai, China; 09/2015
"The −35 region within the σ S promoter family is varying as well, with only a small set of σ S promoters known to be induced by osmolarity (Lee and Gralla, 2004). Many E. coli genes are under the control of both σ factors, including those for stress response (e.g., compatible solute synthesis, DNA repair or protein folding), iron acquisition or the transport, biosynthesis, and metabolism of sugars, amino acids, and fatty acids, among others (Weber et al., 2005; Dong and Schellhorn, 2009). "
[Show abstract][Hide abstract] ABSTRACT: Chromohalobacter salexigens is a halophilic γ-proteobacterium that responds to osmotic and heat stresses by accumulating ectoine and hydroxyectoine, respectively. Evolution has optimized its metabolism to support high production of ectoines. We analyzed the effect of an rpoS mutation in C. salexigens metabolism and ectoines synthesis. In long-term adapted cells, the rpoS strain was osmosensitive but not thermosensitive and showed unaltered ectoines content, suggesting that RpoS regulates ectoine(s)-independent osmoadaptive mechanisms. RpoS is involved in the regulation of C. salexigens metabolic adaptation to stress, as early steps of glucose oxidation through the Entner-Doudoroff pathway were de-regulated in the rpoS mutant, leading to improved metabolic efficiency at low salinity. Moreover, a reduced pyruvate (but not acetate) overflow was displayed by the rpoS strain at low salt, probably linked to a slowdown in gluconate production and/or subsequent metabolism. Interestingly, RpoS does not seem to be the main regulator triggering the immediate transcriptional response of ectoine synthesis to osmotic or thermal upshifts. However, it contributed to the expression of the ect genes in cells previously adapted to low or high salinity.
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"ppGpp bound to DksA also induces the expression of the stationary phase sigma factor (RpoS or sσ) , however, the induction of the RpoS regulon requires high ppGpp concentrations, namely famine conditions . RpoS controls the expression of many stationary phase genes including the genes encoding transport proteins for better nutrient scavenging [73,74] and degradation of less favorable substrates [75-79] as well as genes of the oxidative stress response  and other genes related to general cell protection  (Additional file 1: Table S4). RpoS also exerts positive control on the expression of metabolic pathway genes, namely the genes of the lower and upper glycolytic pathway [56,81] and, importantly, also on acs[52,81,82] and poxB[56,81,83] (Figure 6, Additional file 1: Table S4). "
[Show abstract][Hide abstract] ABSTRACT: The proteome reflects the available cellular machinery to deal with nutrients and environmental challenges. The most common E. coli strain BL21 growing in different, commonly employed media was evaluated using a detailed quantitative proteome analysis.
The presence of preformed biomass precursor molecules in rich media such as Luria Bertani supported rapid growth concomitant to acetate formation and apparently unbalanced abundances of central metabolic pathway enzymes, e.g. high levels of lower glycolytic pathway enzymes as well as pyruvate dehydrogenase, and low levels of TCA cycle and high levels of the acetate forming enzymes Pta and AckA. The proteome of cells growing exponentially in glucose-supplemented mineral salt medium was dominated by enzymes of amino acid synthesis pathways, contained more balanced abundances of central metabolic pathway enzymes, and a lower portion of ribosomal and other translational proteins. Entry into stationary phase led to a reconstruction of the bacterial proteome by increasing e.g. the portion of proteins required for scavenging rare nutrients and general cell protection. This proteomic reconstruction during entry into stationary phase was more noticeable in cells growing in rich medium as they have a greater reservoir of recyclable proteins from the translational machinery.
The proteomic comparison of cells growing exponentially in different media reflected the antagonistic and competitive regulation of central metabolic pathways through the global transcriptional regulators Cra, Crp, and ArcA. For example, the proteome of cells growing exponentially in rich medium was consistent with a dominating role of phosphorylated ArcA most likely a result from limitations in reoxidizing reduced quinones in the respiratory chain under these growth conditions. The proteomic alterations of exponentially growing cells into stationary phase cells were consistent with stringent-like and stationary phase responses and a dominating control through DksA-ppGpp and RpoS.
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