Marc Bally

Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase

The xcp genes are required for the secretion of most extracellular proteins by Pseudomonas aeruginosa. The products of these genes are essential for the transport of exoproteins across the outer membrane after they have reached the periptasm via a signal sequence‐dependent pathway. To date, analysis of three xcp genes has suggested the conservation of this secretion pathway in many Gram‐negative bacteria. Furthermore, the xcpA gene was shown to be identical to pilD, which encodes a peptidase involved in the processing of fimbrial (pili) subunits, suggesting a connection between pili biogenesis and protein secretion. Here the nucleotide sequences of seven other xcp genes, designated xcpR to ‐X, are presented. The N termini of four of the encoded Xcp proteins display similarity to the N‐termini of type IV pili, suggesting that XcpA is involved in the processing of these Xcp proteins. This could indeed be demonstrated in vivo. Furthermore, two other proteins, XcpR and XcpS, show similarity to the PilB and PilC proteins required for fimbriae assembly. Since XcpR and PilB display a canonical nucleotide‐binding site, ATP hydrolysis may provide energy for both systems.

Regulation of the xcp secretion pathway by multiple quorum-sensing modulons in Pseudomonas aeruginosa.

The virulence of the opportunistic pathogen Pseudomonas aeruginosa is largely dependent upon the extracellular production of a number of secreted proteins with toxic or degradative activities. The synthesis of several exoenzymes is controlled in a cell‐density‐dependent manner by two interlinked quorum‐sensing systems. Their secretion across the outer membrane occurs through the Xcp translocation machinery. The xcp locus located at 40 min on the chromosome consists of two divergently transcribed operons, namely xcpPQ and xcpR to xcpZ. In this study, transcriptional fusions were constructed between the xcpP and xcpR genes and the lacZ reporter. Transcriptional activation of the xcpP and xcpR genes in P. aeruginosa is growth‐phase dependent and the lasR–lasI autoinduction system is required for this control. In the heterologous host Escherichia coli, the lasR gene product, together with its cognate autoinducer N‐(3‐oxododecanoyl)‐l‐homoserine lactone (OdDHL), activates both the xcpP–lacZ and the xcpR–lacZ gene fusion. The second P. aeruginosa quorum‐sensing modulon rhlR–rhlI (vsmR–vsmI ) is also involved in the control of the xcp genes. Expression of the lacZ fusions is strongly reduced in PANO67, a pleiotropic mutant defective in the production of N‐acyl‐homoserine lactones responsible for the activation of RhlR. Furthermore, introduction of the lasR mutation in PANO67 results in additional diminution of xcpR transcription, indicating that the two systems can regulate their target genes independently. These data demonstrate that expression of the xcp secretion system depends on a complex regulatory network involving cell–cell signalling which controls production and secretion of virulence‐associated factors.

RpoS-dependent stress tolerance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is able to persist during feast and famine in many different environments including soil, water, plants, animals and humans. The alternative sigma factor encoded by the rpoS gene is known to be important for survival under stressful conditions in several other bacterial species. To determine if the P. aeruginosa RpoS protein plays a similar role in stationary-phase-mediated resistance, an rpoS mutant was constructed and survival during exposure to hydrogen peroxide, high temperature, hyperosmolarity, low pH and ethanol was investigated. Disruption of the rpoS gene resulted in a two- to threefold increase in the rate of kill of stationary-phase cells. The rpoS mutant also survived less well than the parental strain during the initial phase of carbon or phosphate-carbon starvation. However, after 25 d starvation the remaining population of culturable cells was not significantly different. Stationary-phase cells of the RpoS-negative strain were much more stress resistant than exponentially growing RpoS-positive cells, suggesting that factors other than the RpoS protein must be associated with stationary-phase stress tolerance in P. aeruginosa. Comparison of two-dimensional PAGE of the rpoS mutant and the parental strain showed four major modifications of protein patterns associated with the rpoS mutation.

Xcp‐mediated protein secretion in Pseudomonas aeruginosa: identification of two additional genes and evidence for regulation of xcp gene expression

In Pseudomonas aeruginosa, several exoproteins synthesized with a signal sequence (elastase, lipase, phospholipases, alkaline phosphatase and exotoxin A) are secreted by a two‐step mechanism. They first cross the inner membrane in a signal sequence‐dependent way, and are further translocated across the outer membrane in a second step requiring secretion functions encoded by several xcp genes. Ten xcp genes have already been characterized (Bally et al., 1992a). In this study, two additional xcp genes, xcpP and xcpQ, are described. They are located in the 40 min region of the chromosome where they probably define an operon, divergent from the xcpR–Z operon previously characterized in this region. These two genes encode two proteins, XcpP and XcpQ, similar to PulC and PulD of the pul system of Klebsiella oxytoca. Moreover, the two divergent operons share a common regulation which is growth‐phase dependent.

A direct sulfhydrylation pathway is used for methionine biosynthesis in Pseudomonas aeruginosa.

The relationship between genes and enzymes in the methionine biosynthetic pathway has been studied in Pseudomonas aeruginosa. The first step is catalysed by an O-succinylhomoserine synthase, the product of the metA gene mapped at 20 min on the chromosome. The second step is achieved by direct sulfhydrylation, involving the enzyme encoded by a metZ gene that we have identified and sequenced, located at 40 min. Thus Pseudomonas appears to be the only organism so far described that uses O-succinylhomoserine as substrate for a direct sulfhydrylation. As in yeast, the two transsulfuration pathways between cysteine and homocysteine, with cystathionine as an intermediate, probably exist in parallel in this organism.

Phosphate regulation in Pseudomonas aeruginosa: Cloning of the alkaline phosphatase gene and identification of phoB- and phoR-like genes

SummaryIn Pseudomonas aeruginosa, phosphate limitation results in the synthesis of several protein species. We report the cloning of the P. aeruginosa alkaline phosphatase structural gene, phoA, and we show that this gene is regulated normally in Escherichia coli. We have also identified and cloned two P. aeruginosa genes which can complement phoB and phoR mutations in E. coli. This suggests that a pho regulon system similar to that in E. coli may exist in P. aeruginosa, using at least two similar regulatory factors.

Cloning of xcp genes located at the 55 min region of the chromosome and involved in protein secretion in Pseudomonas aeruginosa

Pleiotropic mutations (xcp) affecting secretion of proteins in Pseudomonas aeruginosa have been previously characterized and mapped at 0 min, 55 min and 65 min. Genomic libraries of this organism have been constructed and the genes xcp‐5 and xcp‐54, located at the 55 min region, were cloned using the adjacent met allele as a marker, and complementation of xcp strains. From our linkage and cloning analysis, the most probable gene order in this region appears to be pyrD., xcp‐5/xcp‐54/met‐9011/oru‐314/trpF/leu‐10. Restriction mapping and transposon (Tn 1725) insertion mutagenesis demonstrated that: (i) the overall size of DNA necessary for xcp expression was 9 kb, (ii) the two loci are not adjacent on the chromosome, and (iii) the two loci are expressed independently. The xcp‐5 gene has been subcloned on a 4 kb Eco RI fragment.

Cloning and orientation of the gene encoding aminopeptidase N in Escherichia coli

SummaryThe pepN gene, that encodes aminopeptidase N in Escherichia coli, has been cloned into the multicopy plasmid pBR322. Expression of the cloned pepN gene results in overproduction of the enzyme. The restriction map of the 6.7 Kb insert was established and the gene was further localized by analysis of the in vitro constructed deletion plasmid and mutant plasmids generated by Tn5 insertions. Chromosome mobilization experiments, using pepN-lac fusion strains allowed us to infer a clockwise direction of transcription for the pepN gene.

ThrH, a homoserine kinase isozyme with in vivo phosphoserine phosphatase activity in Pseudomonas aeruginosa.

Homoserine kinase, the product of the thrB gene, catalyses an obligatory step of threonine biosynthesis. In Pseudomonas aeruginosa, unlike Escherichia coli, inactivation of the previously identified thrB gene does not result in threonine auxotrophy. A new gene, named thrH, was isolated that, when expressed in E. coli thrB mutant strains, results in complementation of the mutant phenotype. In P. aeruginosa, threonine auxotrophy is observed only when both thrB and thrH are simultaneously inactivated. Thus, thrH encodes a protein with an in vivo homoserine-kinase-like activity. Surprisingly, thrH overexpression allows complementation of serine auxotrophy of E. coli and P. aeruginosa serB mutants. These mutants are affected in the phosphoserine phosphatase protein, an enzyme involved in serine biosynthesis. Comparison analysis revealed sequence homology between ThrH and the SerB proteins from different organisms. This could explain the in vivo phosphoserine phosphatase activity of ThrH when overproduced. ThrH differs from the protein encoded by the serB gene which was identified in P. aeruginosa. Thus, two SerB-like proteins co-exist in P. aeruginosa, a situation also found in Mycobacterium tuberculosis.

Physical mapping of the gene for aminopeptidase N in Escherichia coli K12

SummaryThe pepN gene has been cloned into the multicopy plasmid pBR322. The restriction map of the insert was established and the gene was localized. By comparison with the restriction map of the plasmid pJP30 bearing the ompF region, it has been possible to order the ompF, asnS, and pepN genes. The ompF and asnS genes are contiguous and pepN is separated from asnS by a DNA fragment of about 1.6 kb.