SummaryVirulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine-and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its Cterminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA . The Chp system is also required for full virulence in a mouse model of acute pneumonia.

SummaryA bioassay was developed to identify stimuli that promote the transcriptional induction of the algD operon for alginate biosynthesis in Pseudomonas aeruginosa. Strain PAO1 carried the algD promoter fused to a chloramphenicol acetyl-transferase cartridge (PalgD-cat), and Ͼ 50 compounds were tested for promoting chloramphenicol resistance. Most compounds showing PalgD-cat induction were cell wall-active antibiotics that blocked peptidoglycan synthesis. PalgD-cat induction was blocked by mutations in the genes for s 22 (algT/algU) or regulators AlgB and AlgR. Anti-sigma factor MucA was the primary regulator of s 22 activity. A transcriptome analysis using microarrays verified that the algD operon undergoes high induction by D-cycloserine. A similar s E -RseAB complex in Escherichia coli responds to envelope stress, which requires DegS protease in a regulated intramembrane proteolysis (RIP) cascade to derepress the sigma. Mutant phenotypic studies in P. aeruginosa showed that AlgW (PA4446) is likely to be the DegS functional homologue. A mutation in algW resulted in a complete lack of PalgD-cat induction by D-cycloserine. Overexpression of algW in PAO1 resulted in a mucoid phenotype and alginate production, even in the absence of cell wall stress, suggesting that AlgW protease plays a role in s 22 activation. In addition, a mutation in gene PA3257 (prc), encoding a Prc-like protease, resulted in poor induction of PalgD-cat by D-cycloserine, suggesting that it also plays a role in the response to cell wall stress.

Transposon mutagenesis was used to identify a new locus required for twitching motility in Pseudomonas aeruginosa. Four Tn5-B21 mutants which lacked twitching motility and a fifth which exhibited impaired motility were found to map to the same KpnI restriction fragment at approximately 40 min on the P. aeruginosa genome. Cloning and sequencing studies showed that all five transposon insertions occurred within the same 28 kb ORF, which was termed fimV. The product of this gene has a putative peptidoglycan-binding domain, predicted transmembrane domains, a highly acidic C terminus and anomalous electrophoretic migration, indicating unusual primary or secondary structure. The P. aeruginosa genome also possesses a paralogue of fimV. Homologues of fimV were also found in the sequenced genomes of the other type-IV-fimbriated bacteria Neisseria gonorrhoeae, Neisseria meningitidis, Legionella pneumophila and Vibrio cholerae, but not in those of other bacteria which lack type IV fimbriae. A fimV homologue was also found in the genome sequence of Shewanella putrefaciens, along with many other homologues of type IV fimbrial genes, indicating that this bacterium is also likely to produce type IV fimbriae. Wild-type twitching motility was restored to fimV mutants by complementation in a dosage-dependent manner. Overexpression of fimV resulted in an unusual phenotype where the cells were massively elongated and migrated in large convoys at the periphery of the colony. It is suggested that FimV may be involved in remodelling of the peptidoglycan layer to enable assembly of the type IV fimbrial structure and machinery.

Alginate production in mucoid (MucA-defective) Pseudomonas aeruginosa is dependent upon several transcriptional regulators, including AlgB, a two-component response regulator belonging to the NtrC family. This role of AlgB was apparently independent of its sensor kinase, KinB, and even the N-terminal phosphorylation domain of AlgB was dispensable for alginate biosynthetic gene (i.e., algD operon) activation. However, it remained unclear whether AlgB stimulated algD transcription directly or indirectly. In this study, microarray analyses were used to examine a set of potential AlgB-dependent, KinB-independent genes in a PAO1 mucA background that overlapped with genes induced by D-cycloserine, which is known to activate algD expression. This set contained only the algD operon plus one other gene that was shown to be uninvolved in alginate production. This suggested that AlgB promotes alginate production by directly binding to the algD promoter (PalgD). Chromosome immunoprecipitation revealed that AlgB bound in vivo to PalgD but did not bind when AlgB had an R442E substitution that disrupted the DNA binding domain. AlgB also showed binding to PalgD fragments in an electrophoretic mobility shift assay at pH 4.5 but not at pH 8.0. A direct systematic evolution of ligands by exponential enrichment approach showed AlgB binding to a 50-bp fragment located at bp ؊224 to ؊274 relative to the start of PalgD transcription. Thus, AlgB belongs to a subclass of NtrC family proteins that can activate promoters which utilize a sigma factor other than 54

The virulence of Pseudomonas aeruginosa and other surface pathogens involves the coordinate expression of a wide range of virulence determinants, including type IV pili. These surface filaments are important for the colonization of host epithelial tissues and mediate bacterial attachment to, and translocation across, surfaces by a process known as twitching motility. This process is controlled in part by a complex signal transduction system whose central component, ChpA, possesses nine potential sites of phosphorylation, including six histidine-containing phosphotransfer (HPt) domains, one serine-containing phosphotransfer domain, one threonine-containing phosphotransfer domain, and one CheY-like receiver domain. Here, using site-directed mutagenesis, we show that normal twitching motility is entirely dependent on the CheY-like receiver domain and partially dependent on two of the HPt domains. Moreover, under different assay conditions, point mutations in several of the phosphotransfer domains of ChpA give rise to unusual "swarming" phenotypes, possibly reflecting more subtle perturbations in the control of P. aeruginosa motility that are not evident from the conventional twitching stab assay. Together, these results suggest that ChpA plays a central role in the complex regulation of type IV pilus-mediated motility in P. aeruginosa.Pseudomonas aeruginosa is a rod-shaped bacterium that is an opportunistic pathogen of plants and animals, including immunocompromised patients such as cystic fibrosis sufferers (14). The bacterium is equipped with a large arsenal of secreted and cell-associated virulence factors, including type IV pili, which are filamentous appendages located at the poles of the bacterium that facilitate attachment to host epithelial cells, and a form of surface translocation called twitching motility (TM) (16).Twitching motility occurs on wet surfaces and is an important factor for host infection and colonization as well as other forms of complex colonial behavior (16). Twitching motility occurs by the extension and retraction of type IV pili and is distinct from swimming motility (as in Escherichia coli and P. aeruginosa), which is mediated by the rotation of polar flagella, and from orthodox "swarming motility" (as in Proteus mirabilis), which is mediated by the coordinated action of peritrichous flagella (23)."Swarming motility" is, however, a loosely defined term that has been used somewhat indiscriminately to describe various forms of organized bacterial motility. A form of "swarming motility" in P. aeruginosa has been reported but differs from that described for other bacteria in that it appears to demonstrate a reliance on type IV pili rather than flagella (11). However, there is contradictory evidence, with another report indicating that flagella are essential for swarming (22), which is consistent with the usual basis of swarming in other species.In P. aeruginosa, around 40 genes whose products are required for TM, 16 of which code for proteins that are involved in transcriptional regulatio...