Dissipation of Proton Motive Force is not Sufficient to Induce the Phage Shock Protein Response in Escherichia coli

Phage shock proteins (Psp) and their homologues are found in species from the three domains of life: Bacteria, Archaea and Eukarya (e.g. higher plants). In enterobacteria, the Psp response helps to maintain the proton motive force (PMF) of the cell when the inner membrane integrity is impaired. The presumed ability of ArcB to sense redox changes in the cellular quinone pool and the strong decrease of psp induction in ΔubiG or ΔarcAB backgrounds suggest a link between the Psp response and the quinone pool. The authors now provide evidence indicating that the physiological signal for inducing psp by secretin-induced stress is neither the quinone redox state nor a drop in PMF. Neither the loss of the H+-gradient nor the dissipation of the electrical potential alone is sufficient to induce the Psp response. A set of electron transport mutants differing in their redox states due to the lack of a NADH dehydrogenase and a quinol oxidase, but retaining a normal PMF displayed low levels of psp induction inversely related to oxidised ubiquinone levels under microaerobic growth and independent of PMF. In contrast, cells displaying higher secretin induced psp expression showed increased levels of ubiquinone. Taken together, this study suggests that not a single but likely multiple signals are needed to be integrated to induce the Psp response

Transcription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors

Poly(A) polymerase I (PAP I), encoded by the pcnB gene, is a major enzyme responsible for RNA polyadenylation in Escherichia coli, a process involved in the global control of gene expression in this bacterium through influencing the rate of transcript degradation. Recent studies have suggested a complicated regulation of pcnB expression, including a complex promoter region, a control at the level of translation initiation and dependence on bacterial growth rate. In this report, studies on transcription regulation of the pcnB gene are described. Results of in vivo and in vitro experiments indicated that (a) there are three σ70-dependent (p1, pB, and p2) and two σS-dependent (pS1 and pS2) promoters of the pcnB gene, (b) guanosine tetraphosphate (ppGpp) and DksA directly inhibit transcription from pB, pS1 and pS2, and (c) pB activity is drastically impaired at the stationary phase of growth. These results indicate that regulation of the pcnB gene transcription is a complex process, which involves several factors acting to ensure precise control of PAP I production. Moreover, inhibition of activities of pS1 and pS2 by ppGpp and DksA suggests that regulation of transcription from promoters requiring alternative σ factors by these effectors of the stringent response might occur according to both passive and active models

BosR (BB0647) Controls the RpoN-RpoS Regulatory Pathway and Virulence Expression in Borrelia burgdorferi by a Novel DNA-Binding Mechanism

In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ54 (RpoN) directly activates transcription of another alternative σ factor, σS (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ54-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ54-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ54–dependent gene regulation in bacteria

Comparative analyses imply that the enigmatic sigma factor 54 is a central controller of the bacterial exterior

Contains fulltext : 95738.pdf (publisher's version ) (Open Access)BACKGROUND: Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. RESULTS: We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. CONCLUSION: Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm

A novel regulatory factor affecting the transcription of methionine biosynthesis genes in Escherichia coli experiencing sustained nitrogen starvation

The initial adaptive transcriptional response to nitrogen (N) starvation in Escherichia coli involves large-scale alterations to the transcriptome mediated by the transcriptional activator, NtrC. One of these NtrC-activated genes is yeaG, which encodes a conserved bacterial kinase. Although it is known that YeaG is required for optimal survival under sustained N starvation, the molecular basis by which YeaG benefits N starved E. coli remains elusive. By combining transcriptomics with targeted metabolomics analyses, we demonstrate that the methionine biosynthesis pathway becomes transcriptionally dysregulated in ΔyeaG bacteria experiencing sustained N starvation. It appears the ability of MetJ, the master transcriptional repressor of methionine biosynthesis genes, to effectively repress transcription of genes under its control is compromised in ΔyeaG bacteria under sustained N starvation, resulting in transcriptional derepression of MetJ-regulated genes. Although the aberrant biosynthesis does not appear to be a contributing factor for the compromised viability of ΔyeaG bacteria experiencing sustained N starvation, this study identifies YeaG as a novel regulatory factor in E. coli affecting the transcription of methionine biosynthesis genes under sustained N starvation

Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies.

Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ(70) or σ(54), that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ(54) version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ(70) and σ(54), the domain movements of the latter have evolved to require an activator ATPase

Regulation of Type VI Secretion Gene Clusters by σ54 and Cognate Enhancer Binding Proteins▿†

Type VI secretion systems (T6SS) are bacteriophage-derived macromolecular machines responsible for the release of at least two proteins in the milieu, which are thought to form an extracellular appendage. Although several T6SS have been shown to be involved in the virulence of animal and plant pathogens, clusters encoding these machines are found in the genomes of most species of Gram-negative bacteria, including soil, marine, and environmental isolates. T6SS have been associated with several phenotypes, ranging from virulence to biofilm formation or stress sensing. Their various environmental niches and large diversity of functions are correlated with their broad variety of regulatory mechanisms. Using a bioinformatic approach, we identified several clusters, including those of Vibrio cholerae, Aeromonas hydrophila, Pectobacterium atrosepticum, Pseudomonas aeruginosa, Pseudomonas syringae pv. tomato, and a Marinomonas sp., which possess typical −24/−12 sequences, recognized by the alternate sigma factor sigma 54 (σ54 or σN). σ54, which directs the RNA polymerase to these promoters, requires the action of a bacterial enhancer binding protein (bEBP), which binds to cis-acting upstream activating sequences. Putative bEBPs are encoded within the T6SS gene clusters possessing σ54 boxes. Using in vitro binding experiments and in vivo reporter fusion assays, we showed that the expression of these clusters is dependent on both σ54 and bEBPs