The opportunistic pathogen Pseudomonas aeruginosa is responsible for systemic infections in immunocompromised individuals and chronic respiratory disease in patients with cystic fibrosis. Cyclic nucleotides are known to play a variety of roles in the regulation of virulence-related factors in pathogenic bacteria. A set of P. aeruginosa genes, encoding proteins that contain putative domains characteristic of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that are responsible for the maintenance of cellular levels of the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) was identified in the annotated genomes of P. aeruginosa strains PAO1 and PA14. Although the majority of these genes are components of the P. aeruginosa core genome, several are located on presumptive horizontally acquired genomic islands. A comprehensive analysis of P. aeruginosa genes encoding the enzymes of c-di-GMP metabolism (DGC- and PDE-encoding genes) was carried out to analyze the function of c-di-GMP in two disease-related phenomena, cytotoxicity and biofilm formation. Analysis of the phenotypes of DGC and PDE mutants and overexpressing clones revealed that certain virulence-associated traits are controlled by multiple DGCs and PDEs through alterations in c-di-GMP levels. A set of mutants in selected DGC- and PDE-encoding genes exhibited attenuated virulence in a mouse infection model. Given that insertions in different DGC and PDE genes result in distinct phenotypes, it seems likely that the formation or degradation of c-di-GMP by these enzymes is in highly localized and intimately linked to particular targets of c-di-GMP action.

Summary One of the prokaryotic post-transcriptional regulatory mechanisms involves the CsrA/RsmA family of proteins that act by modulating translation initiation at target mRNAs. In this study, we identified the regulon of RsmA of the Pseudomonas aeruginosa PAK strain by using cultures in the stationary phase of growth. The RsmA regulon includes over 500 genes, of which approximately one third were affected by an rsmA mutation negatively, while the rest were affected positively. By isolating RsmA/mRNA complexes, analyzing transcriptional and translational fusions, and performing gel-shift analyses, we identified 40 genes in six operons that are regulated by RsmA directly at the level of translation. All of these genes were affected by RsmA negatively and include genes encoding the type VI secretion system HSI-I, which has been implicated in the P. aeruginosa chronic infections. On the other hand, we were unable to demonstrate a direct interaction of RsmA with transcripts that are positively affected by this protein, including mRNAs encoding the type III secretion system and the type IV pili genes. Our work supports a model in which RsmA acts as a negative translational regulator, and where its positive effects are achieved indirectly by RsmA-mediated interference with translation of specific regulatory factors.

SummaryWe 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.

Diverse interactions between hosts and microbes are initiated by the detection of host-released chemical signals. Detection of these signals leads to altered patterns of gene expression that culminate in specific and adaptive changes in bacterial physiology that are required for these associations. This concept was first demonstrated for the members of the family Rhizobiaceae and was later found to apply to many other plant-associated bacteria as well as to microbes that colonize human and animal hosts. The family Rhizobiaceae includes various genera of rhizobia as well as species of Agrobacterium. Rhizobia are symbionts of legumes, which fix nitrogen within root nodules, while Agrobacterium tumefaciens is a pathogen that causes crown gall tumors on a wide variety of plants. The plant-released signals that are recognized by these bacteria are low-molecular-weight, diffusible molecules and are detected by the bacteria through specific receptor proteins. Similar phenomena are observed with other plant pathogens, including Pseudomonas syringae, Ralstonia solanacearum, and Erwinia spp., although here the signals and signal receptors are not as well defined. In some cases, nutritional conditions such as iron limitation or the lack of nitrogen sources seem to provide a significant cue. While much has been learned about the process of host detection over the past 20 years, our knowledge is far from being complete. The complex nature of the plant-microbe interactions makes it extremely challenging to gain a comprehensive picture of host detection in natural environments, and thus many signals and signal recognition systems remain to be described

sRNAs are small, non-coding RNA species that control numerous cellular processes. Although it iswidely accepted that sRNAs are encoded by most if not all bacteria, genome-wide annotations for sRNA-encoding genes have been conducted in only a few of the nearly 300 bacterial species sequenced to date. To facilitate the efficient annotation of bacterial genomes for sRNA-encoding genes, we developed a program, sRNAPredict2, that identifies putative sRNAs by searching for co-localization of genetic features commonly associated with sRNA-encoding genes. Using sRNAPredict2, we conducted genome-wide annotations for putative sRNA-encoding genes in the intergenic regions of 11 diverse pathogens. In total, 2759 previously unannotated candidate sRNA loci were predicted. There was considerable range in the number of sRNAs predicted in the different pathogens analyzed, raising the possibility that there are species-specific differences in the reliance on sRNA-mediated regulation. Of 34 previously unannotated sRNAs predicted in the opportunistic pathogen Pseudomonas aeruginosa, 31 were experimentally tested and 17 were found to encode sRNA transcripts. Our findings suggest that numerous genes have been missed in the current annotations of bacterial genomes and that, by using improved bioinformatic approaches and tools, much remains to be discovered in ‘intergenic’ sequences.