Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA.

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, analysing 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.

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.

Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria.

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.

Analysis of secretin-induced stress in Pseudomonas aeruginosa suggests prevention rather than response and identifies a novel protein involved in secretin function.

Secretins are bacterial outer membrane proteins that are important for protein export. However, they can also mislocalize and cause stress to the bacterial cell, which is dealt with by the well-conserved phage shock protein (Psp) system in a highly specific manner. Nevertheless, some bacteria have secretins but no Psp system. A notable example is Pseudomonas aeruginosa, a prolific protein secretor with the potential to produce seven different secretins. We were interested in investigating how P. aeruginosa might deal with the potential for secretin-induced stress without a Psp system. Microarray analysis revealed the absence of any transcriptional response to XcpQ secretin overproduction. However, transposon insertions in either rpoN, truB, PA4068, PA4069, or PA0943 rendered P. aeruginosa hypersensitive to XcpQ production. The PA0943 gene was studied further and found to encode a soluble periplasmic protein important for XcpQ localization to the outer membrane. Consistent with this, a PA0943 null mutation reduced the levels of type 2 secretion-dependent proteins in the culture supernatant. Therefore, this work has identified a novel protein required for normal secretin function in P. aeruginosa. Taken together, all of our data suggest that P. aeruginosa lacks a functional equivalent of the Psp stress response system. Rather, null mutations in genes such as PA0943 may cause increased secretin-induced stress to which P. aeruginosa cannot respond. Providing the PA0943 mutant with the ability to respond, in the form of critical Psp proteins from another species, alleviated its secretin sensitivity.

Identification of 17 Pseudomonas aeruginosa sRNAs and prediction of sRNA-encoding genes in 10 diverse pathogens using the bioinformatic tool sRNAPredict2.

sRNAs are small, non-coding RNA species that control numerous cellular processes. Although it is widely 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.

Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence.

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.

Unwounded plants elicit Agrobacterium vir gene induction and T-DNA transfer: transformed plant cells produce opines yet are tumour free.

Agrobacterium tumefaciens is well known to cause crown gall tumours at plant wound sites and to benefit from this plant association by obtaining nutrients called opines that are produced by these tumours. Tumourigenesis requires expression of the vir regulon in response to chemical signals that are thought to be released from wound sites. Here, we examine chemical interactions between A. tumefaciens and unwounded plants. To determine whether unwounded plants can release significant amounts of vir gene inducers, we constructed an A. tumefaciens strain carrying a PvirB-gfp fusion. This fusion was strongly induced by co-culture with tobacco seedlings that have been germinated without any intentional wounding. The release of phenolic vir gene inducers was confirmed by GC/MS analysis. We also constructed a strain containing the gfp reporter located on an artificial T-DNA and expressed from a plant promoter. A. tumefaciens efficiently transferred this T-DNA into cells of unwounded plants in the absence of exogenous vir gene inducers. Many cells of seedlings colonized by the bacteria also produced octopine, which was detected using a Pocc-gfp reporter strain. This indicates transfer of the native T-DNA. However, these transformed plant cells did not form tumours. These results suggest that successful colonization of plants by A. tumefaciens, including T-DNA transfer and opine production, does not require wounding and does not necessarily cause cell proliferation. Transformation of plant cells without inciting tumours may represent a colonization strategy for this pathogen that has largely been overlooked.

Signal quenching, detoxification and mineralization of vir gene-inducing phenolics by the VirH2 protein of Agrobacterium tumefaciens.

Plant tumorigenesis by Agrobacterium tumefaciens requires approximately 20 Vir proteins, transcription of which is induced by a family of phenolic compounds released from plant wound sites. One Vir protein, VirH2, plays a role in the metabolism of at least one phenolic inducer inasmuch as it converts ferulic acid, a potent vir gene inducer, to the non-inducer caffeate by O-demethylation of a methoxyl group. Here, we tested VirH2-dependent O-demethylation of 16 other vir-inducing phenolics, and detected this activity for each compound. However, O-demethylation rates differed enormously, with the strongest vir gene inducers such as acetosyringone being demethylated extremely slowly. Compounds containing two methoxyl groups were demethylated at both positions. In general, phenolic inducers were more toxic than their demethylated counterparts. A virH2 mutant was more sensitive than the wild type to growth inhibition by virtually all phenolic inducers tested, indicating that VirH2 detoxifies these compounds. VirH2 also played a role in mineralization of some phenolics. It converted vanillate to protocatechuate, which was then mineralized via the beta-ketoadipate pathway. Vanillyl alcohol and vanillin were also mineralized after being oxidized to vanillate. All three compounds served as sole sources of carbon, whereas the remaining 13 compounds did not.

VirA of Agrobacterium tumefaciens is an intradimer transphosphorylase and can actively block vir gene expression in the absence of phenolic signals.

The VirA-VirG two-component system regulates the 30-gene vir regulon in response to host-released chemical signals. VirA is a homodimeric membrane-spanning histidine protein kinase. Here, we show that mutations in two essential VirA residues, His-474 and Gly-657, can be complemented by the formation of mixed heterodimers, indicating that each subunit of a VirA dimer transphosphorylates the opposite subunit. VirA contains a receiver domain that inhibits kinase activity. We use the forced heterodimer system to show that the two receiver domains of a VirA dimer act independently and that each inhibits the phosphoacceptor subdomain of the opposite subunit. We also demonstrate that merodiploid strains co-expressing constitutive VirA mutants and wild-type VirA show levels of vir gene expression far lower than haploid strains expressing just the constitutive alleles. The fact that wild-type VirA can actively block vir gene expression in the absence of phenolic signals suggests that it might have a phospho-VirG phosphatase activity. The receiver domain of VirA is essential for this activity, whereas residues H474 and G657 of the kinase domain are not required. Merodiploid strains co-expressing a constitutive VirA allele and an allele that is kinase inactive but proficient in the inhibitory activity show strongly inducible vir gene expression, indicating that the inhibitory activity is modulated by environmental signals.