Regulating expression of virulence determinants in enterotoxigenic escherichia coli H10407

Enterotoxigenic $Escherichia$ $coli$ (ETEC) is a major cause of traveller’s and infantile diarrhoea in developing countries, and results in considerable mortality in under 5 year olds. Disease is mediated through adhesion of ETEC cells to the intestinal brush border and the secretion of the heat-stable and/or heat-labile toxin. Whilst the toxins have been well studied it is still not clear how their expression is regulated. This work has defined binding of CRP, H-NS and σ$^7$$^0$ across the genome of the prototypical ETEC strain H10407. We demonstrate a central role for all three factors in regulating pathogenicity in ETEC H10407. Hence, we show that CRP directly regulates expression of both $estA2$ and $estA1$, which encode the heat-stable toxins. Furthermore, CRP indirectly represses expression of the heat-labile toxin. This work also identifies a role for CRP in controlling transcription of a small open reading frame, imbedded within a gene, at the 3’ end of an operon encoding a type I secretion system

Unusually Situated Binding Sites for Bacterial Transcription Factors Can Have Hidden Functionality

A commonly accepted paradigm of molecular biology is that transcription factors control gene expression by binding sites at the 5' end of a gene. However, there is growing evidence that transcription factor targets can occur within genes or between convergent genes. In this work, we have investigated one such target for the cyclic AMP receptor protein (CRP) of enterotoxigenic Escherichia coli. We show that CRP binds between two convergent genes. When bound, CRP regulates transcription of a small open reading frame, which we term aatS, embedded within one of the adjacent genes. Our work demonstrates that non-canonical sites of transcription factor binding can have hidden functionality

The quorum sensing transcription factor AphA directly regulates natural competence in Vibrio cholerae

Many bacteria use population density to control gene expression via quorum sensing. In Vibrio cholerae, quorum sensing coordinates virulence, biofilm formation, and DNA uptake by natural competence. The transcription factors AphA and HapR, expressed at low and high cell density respectively, play a key role. In particular, AphA triggers the entire virulence cascade upon host colonisation. In this work we have mapped genome-wide DNA binding by AphA. We show that AphA is versatile, exhibiting distinct modes of DNA binding and promoter regulation. Unexpectedly, whilst HapR is known to induce natural competence, we demonstrate that AphA also intervenes. Most notably, AphA is a direct repressor of tfoX, the master activator of competence. Hence, production of AphA markedly suppressed DNA uptake; an effect largely circumvented by ectopic expression of tfoX. Our observations suggest dual regulation of competence. At low cell density AphA is a master repressor whilst HapR activates the process at high cell density. Thus, we provide deep mechanistic insight into the role of AphA and highlight how V. cholerae utilises this regulator for diverse purposes

The multiple antibiotic resistance operon of enteric bacteria controls DNA repair and outer membrane integrity

Transcription factors MarR and MarA confer multidrug resistance in enteric bacteria by modulating efflux pump and porin expression. Here, Sharma et al. show that MarA also upregulates genes required for lipid trafficking and DNA repair, thus reducing antibiotic entry and quinolone-induced DNA damage

cAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization

The bacterium Vibrio cholerae is native to aquatic environments and can switch lifestyles to cause disease in humans. Lifestyle switching requires modulation of genetic systems for quorum sensing, intestinal colonization, and toxin production. Much of this regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, we have mapped the binding of cAMP receptor protein (CRP) and RNA polymerase across the V. cholerae genome. We show that CRP is an integral component of the regulatory network that controls lifestyle switching. Focusing on a locus necessary for toxin transport, we demonstrate CRP-dependent regulation of gene expression in response to host colonization. Examination of further CRP-targeted genes reveals that this behavior is commonplace. Hence, CRP is a key regulator of many V. cholerae genes in response to lifestyle changes.Cholera is an infectious disease that is caused by the bacterium Vibrio cholerae. Best known for causing disease in humans, the bacterium is most commonly found in aquatic ecosystems. Hence, humans acquire cholera following ingestion of food or water contaminated with V. cholerae. Transition between an aquatic environment and a human host triggers a lifestyle switch that involves reprogramming of V. cholerae gene expression patterns. This process is controlled by a network of transcription factors. In this paper, we show that the cAMP receptor protein (CRP) is a key regulator of V. cholerae gene expression in response to lifestyle changes

The <i>aatS</i> mRNA contains a functional ribosome binding site.

<p>The graph shows activity of different <i>aatS</i>:<i>lacZ</i> translational fusions. The wildtype ribosome binding site (5'-AAGAAG-3') in the <i>aatS</i>1 fragment was mutated to (5'-TTCTTC-3') in <i>aatS</i>2. LacZ activites was determined using the lysates of stationary phase M182 or M182<i>Δcrp</i>. In M182 cells <i>crp</i> was supplied in trans by plasmid pCRP that encodes <i>crp</i> under the control of its own promoter. Values shown are percentages of activity observed in strain M182 (5 Miller units). We obtained 0.25 and 0.26 Miller units of activity from lysates of M182 or M182Δ<i>crp</i>, carrying promoterless pRW225, respectively. Error bars represent the standard deviation of three independent experiments.</p