The opportunistic pathogen Pseudomonas aeruginosa is responsible for a wide range of acute and chronic infections. The transition to chronic infections is accompanied by physiological changes in the bacteria favoring formation of biofilm communities. Here we report the identification of LadS, a hybrid sensor kinase that controls the reciprocal expression of genes for type III secretion and biofilm-promoting polysaccharides. Domain organization of LadS and the range of LadS-controlled genes suggest that it counteracts the activities of another sensor kinase, RetS. These two pathways converge by controlling the transcription of a small regulatory RNA, RsmZ. This work identifies a previously undescribed signal transduction network in which the activities of signal-receiving sensor kinases LadS, RetS, and GacS regulate expression of virulence genes associated with acute or chronic infection by transcriptional and posttranscriptional mechanisms.biofilm ͉ pel genes ͉ small RNA ͉ two-component system ͉ type III secretion

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.contact-dependent secretion ͉ pathogenesis ͉ virulence

Pseudomonas aeruginosa, an important opportunistic human pathogen, persists in certain tissues in the form of specialized bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surfaces, resulting in an organized structure. By screening a library of Tn5 insertions in a nonpiliated P. aeruginosa strain, we identified genes involved in early stages of biofilm formation. One class of mutations identified in this study mapped in a cluster of genes specifying the components of a chaperone͞usher pathway that is involved in assembly of fimbrial subunits in other microorganisms. These genes, not previously described in P. aeruginosa, were named cupA1-A5. Additional chaperone͞usher systems (CupB and CupC) have been also identified in the genome of P. aeruginosa PAO1; however, they do not appear to play a role in adhesion under the conditions where the CupA system is expressed and functions in surface adherence. The identification of these putative adhesins on the cell surface of P. aeruginosa suggests that this organism possess a wide range of factors that function in biofilm formation. These structures appear to be differentially regulated and may function at distinct stages of biofilm formation, or in specific environments colonized by this organism.

The human opportunistic pathogen Serratia marcescens is a bacterium with a broad host range, and represents a growing problem for public health. Serratia marcescens kills Caenorhabditis elegans after colonizing the nematode's intestine. We used C.elegans to screen a bank of transposon-induced S.marcescens mutants and isolated 23 clones with an attenuated virulence. Nine of the selected bacterial clones also showed a reduced virulence in an insect model of infection. Of these, three exhibited a reduced cytotoxicity in vitro, and among them one was also markedly attenuated in its virulence in a murine lung infection model. For 21 of the 23 mutants, the transposon insertion site was identified. This revealed that among the genes necessary for full in vivo virulence are those that function in lipopolysaccharide (LPS) biosynthesis, iron uptake and hemolysin production. Using this system we also identified novel conserved virulence factors required for Pseudomonas aeruginosa pathogenicity. This study extends the utility of C.elegans as an in vivo model for the study of bacterial virulence and advances the molecular understanding of S.marcescens pathogenicity.