Simon L. Dove

The outer membrane protein, Antigen 43, mediates cell‐to‐cell interactions within Escherichia coli biofilms

Transcription of the agn43 locus, which specifies an outer membrane protein of Escherichia coli, is regulated in a phase‐variable fashion by the OxyR–DNA binding protein and Dam methylase. Despite its well‐characterized regulation, the function of Ag43 has remained elusive until now. Previous studies indicated that Ag43 mediates autoaggregation of certain strains of E. coli in liquid culture. Given this phenotype, we examined the role of Ag43 in biofilm formation. Here, we report that Ag43 contributes to E. coli biofilm formation in glucose‐minimal medium, but not in Luria–Bertani broth. In addition, we show that flagellar‐mediated motility is required for biofilm formation in both rich and minimal environments. Altogether, our results suggest that E. coli uses both common and specific gene sets for the development of biofilms under various growth conditions.

ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa

Type III secretion systems are toxin delivery systems that are present in a large number of pathogens. A hallmark of all type III secretion systems studied to date is that expression of one or more of their components is induced upon cell contact. It has been proposed that this induction is controlled by a negative regulator that is itself secreted by means of the type III secretion machinery. Although candidate proteins for this negative regulator have been proposed in a number of systems, for the most part, a direct demonstration of their role in regulation is lacking. Here, we report the discovery of ExsE, a negative regulator of type III secretion gene expression in Pseudomonas aeruginosa. Deletion of exsE deregulates expression of the type III secretion genes. We provide evidence that ExsE is itself secreted by means of the type III secretion machinery and physically interacts with ExsC, a positive regulator of the type III secretion regulon. Taken together, these data demonstrate that ExsE is the secreted negative regulator that couples triggering of the type III secretion machinery to induction of the type III secretion genes.

The GacS/GacA signal transduction system of Pseudomonas aeruginosa acts exclusively through its control over the transcription of the RsmY and RsmZ regulatory small RNAs

We report here the results of an analysis of the regulatory range of the GacS/GacA two‐component system in Pseudomonas aeruginosa. Using microarrays, we identified a large number of genes that are regulated by the system, and detected a near complete overlap of these genes with those regulated by two small RNAs (sRNAs), RsmY and RsmZ, suggesting that the expression of all GacA‐regulated genes is RsmY/Z‐dependent. Using genome‐wide DNA–protein interaction analyses, we identified only two genomic regions that associated specifically with GacA, located upstream of the rsmY and rsmZ genes. These results demonstrate that in P. aeruginosa, the GacS/GacA system transduces the regulatory signals to downstream genes exclusively by directly controlling the expression of only two genes rsmY and rsmZ. These two sRNAs serve as intermediates between the input signals and the output at the level of mRNA stability, although additional regulatory inputs can influence the levels of these two riboregulators. We show that the A+T‐rich DNA segment upstream of rsmZ is bound and silenced by MvaT and MvaU, the global gene regulators of the H‐NS family. This work highlights the importance of post‐transcriptional mechanisms involving sRNAs in controlling gene expression during bacterial adaptation to different environments.

Protein–Protein Contacts that Activate and Repress Prokaryotic Transcription

Recently the structures of two of the DNA-binding domains of RNAP, the α-CTD (7xJeon, Y.H, Negishi, T, Shirakawa, M, Yamazaki, T, Fujita, N, Ishihama, A, and Kyogoku, Y. Science. 1995; 270: 1495–1497Crossref | PubMedSee all References, 6xGaal, T, Ross, W, Blatter, E.E, Tang, H, Jia, X, Krishnan, V.V, Assa-Munt, N, Ebright, R.H, and Gourse, R.L. Genes Dev. 1996; 10: 16–26Crossref | PubMedSee all References), and a portion of σ70 containing the −10 region recognition motif (Malhotra et al. 1996xMalhotra, A, Severinova, E, and Darst, S.A. Cell. 1996; 87: 127–136Abstract | Full Text | Full Text PDF | PubMed | Scopus (239)See all ReferencesMalhotra et al. 1996), have been determined. Such advances in the understanding of RNAP structure should facilitate elucidation of the more complex activation and repression mechanisms.In the case of σ70, this structural information in conjunction with the finding that σ plays an important role in directing and stabilizing promoter melting is likely to shed light on the mechanism of action of at least some activators that mediate their effects through σ. Since −10 region recognition involves base-specific contacts between the σ subunit and the melted nontemplate strand (Roberts and Roberts 1996xRoberts, C.W and Roberts, J.W. Cell. 1996; 86: 495–501Abstract | Full Text | Full Text PDF | PubMed | Scopus (107)See all ReferencesRoberts and Roberts 1996), it is possible that regulators that interact with σ may, in some cases, stabilize a conformation that favors the formation of these contacts, rather than merely stabilizing the binding of the −35 region recognition domain. Unfortunately, there is as of yet no high resolution structural information about the portion of σ that binds the promoter −35 region, the apparent target of a number of activators that bind in the immediate vicinity of the −35 box. Whether or not the effects of such activator–σ interactions can be transmitted through the structure of σ to the −10 region recognition motif remains to be learned.As the structural analysis of RNAP proceeds, it will be particularly informative to study complexes containing a DNA-bound regulator together with a relevant portion of RNAP. Finally, structural information about the catalytic subunits of RNAP are likely to enhance our understanding of how some activators that contact these subunits work.

Nucleoid occlusion factor SlmA is a DNA-activated FtsZ polymerization antagonist

The tubulin-like FtsZ protein initiates assembly of the bacterial cytokinetic machinery by polymerizing into a ring structure, the Z ring, at the prospective site of division. To block Z-ring formation over the nucleoid and help coordinate cell division with chromosome segregation, Escherichia coli employs the nucleoid-associated division inhibitor, SlmA. Here, we investigate the mechanism by which SlmA regulates FtsZ assembly. We show that SlmA disassembles FtsZ polymers in vitro. In addition, using chromatin immunoprecipitation (ChIP), we identified 24 SlmA-binding sequences (SBSs) on the chromosome. Remarkably, SlmA binding to SBSs dramatically enhanced its ability to interfere with FtsZ polymerization, and ChIP studies indicate that SlmA regulates FtsZ assembly at these sites in vivo. Because of the dynamic and highly organized nature of the chromosome, coupling SlmA activation to specific DNA binding provides a mechanism for the precise spatiotemporal control of its anti-FtsZ activity within the cell.

H-NS family members function coordinately in an opportunistic pathogen

The histone-like nucleoid structuring protein, H-NS, is a prominent global regulator of gene expression. Many Gram-negative bacteria contain multiple members of the H-NS family of proteins. Thus, a key question is whether H-NS family members have overlapping or distinct functions. To address this question we performed genome-wide location analyses with MvaT and MvaU, the two H-NS family members present in Pseudomonas aeruginosa. We show that MvaT and MvaU bind the same chromosomal regions, coregulating the expression of ≈350 target genes. We show further that like H-NS in enteric bacteria, which functions as a transcriptional silencer of foreign DNA by binding to AT-rich elements, MvaT and MvaU bind preferentially to AT-rich regions of the chromosome. Our findings establish that H-NS paralogs can function coordinately to regulate expression of the same set of target genes, and suggest that MvaT and MvaU are involved in silencing foreign DNA elements in P. aeruginosa.

Region 4 of σ as a target for transcription regulation

Bacterial σ factors play a key role in promoter recognition, making direct contact with conserved promoter elements. Most σ factors belong to the σ70 family, named for the primary σ factor in Escherichia coli. Members of the σ70 family typically share four conserved regions and, here, we focus on region 4, which is directly involved in promoter recognition and serves as a target for a variety of regulators of transcription initiation. We review recent advances in the understanding of the mechanism of action of regulators that target region 4 of σ.

A Bacterial Two-Hybrid System Based on Transcription Activation

We describe the use of a bacterial two-hybrid system for the study of protein-protein interactions in Escherichia coli. This system is based on transcription activation and involves the synthesis of two fusion proteins within the bacterial cell whose interaction stimulates transcription of a reporter gene. Specifically, one of the fusion proteins can function as a transcription activator when its interaction partner is fused to a subunit of the bacterial RNA polymerase. This bacterial two-hybrid system has been used to study a number of interacting proteins from both prokaryotes and eukaryotes, and can be used to find interacting proteins from complex protein libraries.

RshA, an anti-sigma factor that regulates the activity of the mycobacterial stress response sigma factor SigH

SigH, an alternative sigma factor of Mycobacterium tuberculosis,  is  a  central regulator  of  the  response to oxidative and heat stress. Exposure to these stresses results in increased expression of sigH itself, and of genes encoding additional regulators and effectors of the bacterial response to these stresses. In this work we show that RshA, a protein encoded by a gene in the sigH operon, is an anti‐sigma factor of SigH. We demonstrate that RshA binds to SigH in vitro and in vivo. This protein–protein interaction, as  well  as the  ability  of RshA to inhibit SigH‐dependent transcription, is redox‐dependent, with RshA functioning as a negative regulator of SigH activity only under reducing conditions. The interaction of SigH and RshA is also disrupted in vitro by elevated temperature. RshA, a protein of 101 amino acids, contains five conserved cysteine residues of which two appear to be essential for RshA to inhibit SigH activity, suggesting that these cysteines may be important for the redox state dependence of RshA function. Our results indicate that RshA is a sensor that responds to oxidative stress, and also to heat stress, resulting in activation of SigH and expression of the SigH‐dependent genes that allow M. tuberculosis to adapt to these stresses.

Protein–Protein and Protein–DNA Interactions of σ70 Region 4 Involved in Transcription Activation by λcI

Abstract The cI protein of bacteriophage λ (λcI) activates transcription from promoter PRM through an acidic patch on the surface of its DNA-binding domain. Genetic evidence suggests that this acidic patch stimulates transcription from PRM through contact with the C-terminal domain (region 4) of the σ70 subunit of Escherichia coli RNA polymerase. Here, we identify two basic residues in region 4 of σ70 that are critical for λcI-mediated activation of transcription from PRM. On the basis of structural modeling, we propose that one of these σ70 residues, K593, facilitates the interaction between λcI and region 4 of σ70 by inducing a bend in the DNA upstream of the −35 element, whereas the other, R588, interacts directly with a critical acidic residue within the activating patch of λcI. Residue R588 of σ70 has been shown to play an important role in promoter recognition; our findings suggest that the R588 side-chain has a dual function at PRM, facilitating the interaction of region 4 with the promoter −35 element and participating directly in the protein–protein interaction with λcI.