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 periplasm 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.     

Protein secretion in Pseudomonas aeruginosa.

The Gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into the extracellular medium. At least two distinct secretion pathways can be discerned. The majority of the exoproteins are secreted via a two-step mechanism. These proteins are first translocated across the inner membrane in a signal sequence-dependent fashion. The subsequent translocation across the outer membrane requires the products of at least 12 distinct xcp genes. The exact role of one of these proteins, the XcpA protein, has been resolved. It is a peptidase that is required for the processing of the precursors of four other Xcp proteins, thus allowing their assembly into the secretion apparatus. This peptidase is also required for the processing of the precursors of type IV pili subunits. Two other Xcp proteins, XcpR and XcpS, display extensive homology to proteins involved in pili biogenesis, which suggests that the assembly of the secretion apparatus and the biogenesis of type IV pili are related processes. The secretion of alkaline protease does not require the xcp gene products. This enzyme, which is encoded by the aprA gene, is not synthesized in a precursor form with an N-terminal signal sequence. Secretion across the two membranes probably takes place in one step at adhesion zones that may be constituted by three accessory proteins, designated AprD, AprE and AprF. The two secretion pathways found in P. aeruginosa appear to have disseminated widely among Gram-negative bacteria.     

Protein secretion in Pseudomonas aeruginosa

Abstract The Gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into the extracellular medium. At least two distinct secretion pathways can be discerned. The majority of the exoproteins are secreted via a two-step mechanism. These proteins are first translocated across the inner membrane in a signal sequence-dependent fashion. The subsequent translocation across the outer membrane requires the products of at least 12 distinct xcp genes. The exact role of one of these proteins, the XcpA protein, has been resolved. It is a peptidase that is required for the processing of the precursors of four other Xcp proteins, thus allowing their assembly into the secretion apparatus. This peptidase is also required for the processing of the precursors of type IV pili subunits. Two other Xcp proteins, XcpR and XcpS, display extensive homology to proteins involved in pili biogenesis, which suggests that the assembly of the secretion apparatus and the biogenesis of type IV pili are related processes. The secretion of alkaline protease does not require the xcp gene products. This enzyme, which is encoded by the aprA gene, is not synthesized in a precursor form with an N-terminal signal sequence. Secretion across the two membranes probably takes place in one step at adhesion zones that may be constituted by three accessory proteins, designated AprD, AprE and AprF. The two secretion pathways found in P. aeruginosa appear to habe disseminate widely among Gram-negative bacteria.     

Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins.

We isolated 15 mutants of Pseudomonas aeruginosa PAO which were defective in the formation of certain extracellular proteins, such as elastase, staphylolytic enzyme, and lipase ( Xcp mutants). The mutations were mapped on the chromosome by conjugation and transduction. The locations were xcp -1 near 0', with the gene order cys-59- xcp -1- proB , and loci xcp -2, xcp -3, and xcp -31 at 35', with the gene order trpC , D- xcp -3/ xcp -31- xcp -2- argC . Loci xcp -4 and xcp -41 through xcp -44 were cotransducible with proA at 40'; loci xcp -5, xcp -51, xcp -52, and xcp53 were located at 55', with the gene order leu-10- trpF -met-9010- xcp -53- xcp -5/ xcp -51/ xcp+ ++-52, and xcp -6 was located at 65' to 70', between catA and mtu-9002. Nine mutations ( xcp -2, xcp -3, xcp -31, xcp -4, and xcp -41 through xcp -45) caused decreased production of extracellular enzymes. Six strains with mutations xcp -1, xcp -5, xcp -51, xcp -52, xcp -53, and xcp -6 produced cell-bound exoproteins and had defective release mechanisms. The regulation of production of alkaline phosphatase and phospholipase C is different from other exoproteins , such as elastase, but they all seem to share a common release mechanism. Alkaline protease had separate mechanisms for regulation and release, since this protease was found in culture supernatants of all but one of the mutants, and none of the strains had cell-bound enzyme.     

The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family)

The xcp gene products in Pseudomonas aeruginosa are required for the secretion of proteins across the outer membrane. Four of the Xcp proteins, XcpT, U, V and W, present sequence homology to the subunits of type IV pili at their N-termini, and they were therefore designated pseudopilins. In this study, we characterized the xcpX gene product, a bitopic cytoplasmic membrane protein. Remarkably, amino acid sequence comparisons also suggested that the XcpX protein resembles the pilins and pseudopilins at the N-terminus. We show that XcpX could be processed by the prepilin peptidase, PilD/XcpA, and that the highly conserved glycine residue preceding the hydrophobic segment could not be mutated without loss of the XcpX function. We, therefore, classified XcpX (GspK) as the fifth pseudopilin of the system.     

A novel type II secretion system in Pseudomonas aeruginosa

The genome sequence of Pseudomonas aeruginosa strain PAO1 has been determined to facilitate postgenomic studies aimed at understanding the capacity of adaptation of this ubiquitous opportunistic pathogen. P. aeruginosa produces toxins and hydrolytic enzymes that are secreted via the type II secretory pathway using the Xcp machinery or 'secreton'. In this study, we characterized a novel gene cluster, called hxc for homologous to xcp. Characterization of an hxcR mutant, grown in phosphate-limiting medium, revealed the absence of a 40 kDa protein found in the culture supernatant of wild-type or xcp derivative mutant strains. The protein corresponded to the alkaline phosphatase L-AP, renamed LapA, which is secreted in an xcp-independent but hxc-dependent manner. Finally, we showed that expression of the hxc gene cluster is under phosphate regulation. This is the first report of the existence of two functional type II secretory pathways within the same organism, which could be related to the high adaptation potential of P. aeruginosa.     

Structure-Function Analysis of XcpP, a Component Involved in General Secretory Pathway-Dependent Protein Secretion in Pseudomonas aeruginosa

The general secretory pathway of Pseudomonas aeruginosa is required for the transport of signal peptide-containing exoproteins across the cell envelope. After completion of the Sec-dependent translocation of exoproteins across the inner membrane and cleavage of the signal peptide, the Xcp machinery mediates translocation across the outer membrane. This machinery consists of 12 components, of which XcpQ (GspD) is the sole outer membrane protein. XcpQ forms a multimeric ring-shaped structure, with a central opening through which exoproteins could pass to reach the medium. Surprisingly, all of the other Xcp proteins are located in or are associated with the cytoplasmic membrane. This study is focused on the characteristics of one such cytoplasmic membrane protein, XcpP. An xcpP mutant demonstrated that the product of this gene is indeed an essential element of the P. aeruginosa secretion machinery. Construction and analysis of truncated forms of XcpP made it possible to define essential domains for the function of the protein. Some of these domains, such as the N-terminal transmembrane domain and a coiled-coil structure identified at the C terminus of XcpP, may be involved in protein-protein interaction during the assembly of the secretory apparatus.     

Protein secretion in gram-negative bacteria: transport across the outer membrane involves common mechanisms in different bacteria.

The xcp genes are required for protein secretion by Pseudomonas aeruginosa. They are involved in the second step of the process, i.e. the translocation across the outer membrane, after the exoproteins have reached the periplasm in a signal peptide dependent fashion. The nucleotide sequence of a 2.5 kb DNA fragment containing xcp genes showed at least two complete open reading frames, potentially encoding proteins with molecular weights of 41 and 19 kd. Products with these apparent molecular weights were identified after expression of the DNA fragment in vitro and in vivo. Subcloning and complementation experiments showed that both proteins are required for secretion. The two products are located in the inner membrane and share highly significant homologies with the PulL and PulM proteins which are required for the specific secretion of pullulanase in Klebsiella pneumoniae. These homologies reveal the existence of a common mechanism for protein secretion in Pseudomonas aeruginosa and Klebsiella pneumoniae.     

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 (Tn1725) insertion mutagenesis demonstrated that: (i) the overall size of DNA necessary for xcp expression was 9kb, (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 4kb EcoRI fragment.     

Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition

Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).     

Secretion of extracellular proteins by Pseudomonas aeruginosa

Pseudomonas aeruginosa is a bacterial species of commercial value secreting numerous extracellular proteins, involved in pathogenesis. Most strains produce at least a lipase, a phospholipase, an alkaline phosphatase, an exotoxin and 2 proteases (elastase and alkaline protease). Various mechanisms for secretion of exoproteins appear to exist in P aeruginosa. Genetic analysis has led to the identification of 2 secretion pathways: i) a "general" secretion pathway, defined by the xcp mutations, which mediates secretion of most extracellular proteins, and; ii) an independent secretion pathway specific for alkaline protease. Our present knowledge on the pathways and components of the secretion machinery in P aeruginosa is reviewed in this article.     

Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during xylogenesis in Arabidopsis roots

Establishing the mechanisms regulating the autolysis of xylem tracheary elements (TEs) is important for understanding this programmed cell death process. These data demonstrate that two paralogous Arabidopsis thaliana proteases, XYLEM CYSTEINE PROTEASE1 (XCP1) and XCP2, participated in micro-autolysis within the intact central vacuole before mega-autolysis was initiated by tonoplast implosion. The data acquisition was aided by the predictable pattern of seedling root xylogenesis, the availability of single and double total knock-out T-DNA lines, anti-sera that recognized XCP1 and XCP2, and the microwave-assisted processing of whole seedlings prior to immunolabeling and observation in the transmission electron microscope. During secondary wall thickening, XCP1 and XCP2 (in wild type), XCP1 (in xcp2 seedlings) or XCP2 (in xcp1 seedlings) were imported into the TE central vacuole. Both XCP1 and XCP2 heavily labeled dense aggregates of material within the vacuole. However, because of XCP1 deficiency in xcp1 and xcp1 xcp2 TEs, non-degraded cellular remnants first accumulated in the vacuole and then persisted in the TE lumen (longer than in the wild type) after the final mega-autolysis was otherwise complete. This delayed TE clearing phenotype in xcp1 was rescued by complementation with wild-type XCP1. Although TEs in the xcp2 single knock-out cleared comparably with wild type, the non-degraded remnants in xcp1 xcp2 TEs were more densely packed than in xcp1 TEs. Therefore, XCP2 has a minor but distinct role in micro-autolysis. After tonoplast implosion, XCP1 and XCP2 remained associated with disintegrating cellular material as mega-autolysis, aided by additional lytic enzymes, destroyed the bulk of the cellular contents.     

XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus

Pseudomonas aeruginosa is an opportunistic gram-negative pathogen equipped with multiple secretion systems. The type II secretion machinery (Xcp secreton) is involved in the release of toxins and enzymes. The Xcp secreton is a multiprotein complex, and most of its components share homology with proteins involved in type IV pili biogenesis. Among them, the XcpT-X pseudopilins possess characteristics of the major constituent of the type IV pili, the pilin PilA. We have shown previously that XcpT can be assembled in a multifibrillar structure that was called the pseudopilus. By using two different microscopic approaches, we show here that the pseudopili are preferentially isolated fibers rather than tight bundles. Moreover, none of the other four pseudopilins are able to form a pseudopilus, suggesting that the assembly of such a structure is a unique property of XcpT. Moreover, we show that 5 of the 12 Xcp proteins are not required for pseudopilus biogenesis, whereas they are for type II secretion. Most interestingly, we showed that one pseudopilin, XcpX, controls the assembly of XcpT into a pseudopilus. Indeed, when the number of XcpX subunits increases, the length of the pseudopilus decreases. Conversely, in the absence of XcpX, the pseudopilus length is abnormally long. Our results indicate that XcpT and XcpX directly interact with each other. Furthermore, this interaction induces a clear destabilization of XcpT. The interaction between XcpT and XcpX could be part of the molecular mechanism underlying the dynamic control of pseudopilus elongation, which could be crucial for type II-dependent protein secretion.     

Assembly of XcpR in the Cytoplasmic Membrane Is Required for Extracellular Protein Secretion in Pseudomonas aeruginosa

A broad range of extracellular proteins secreted by Pseudomonas aeruginosa use the type II or general secretory pathway (GSP) to reach the medium. This pathway requires the expression of at least 12 xcp gene products. XcpR, a putative nucleotide-binding protein, is essential for the secretion process across the outer membrane even though the protein contains no hydrophobic sequence that could target or anchor it to the bacterial envelope. For a better understanding of the relationship between XcpR and the other Xcp proteins which are located in the envelope, we have studied its subcellular localization. In a wild-type P. aeruginosa strain, XcpR was found associated with the cytoplasmic membrane. This association depends on the presence of the XcpY protein, which also appears to be necessary for XcpR stability. Functional complementation of an xcpY mutant required the XcpY protein to be expressed at a low level. Higher expression precluded the complementing activity of XcpY, although membrane association of XcpR was restored. This behavior suggested that an excess of free XcpY might interfere with the secretion by formation of inactive XcpR-XcpY complexes which cannot properly interact with their natural partners in the secretion machinery. These data show that a precise stoichiometric ratio between several components may be crucial for the functioning of the GSP.     

Protein secretion in Pseudomonas aeruginosa: the xcpA gene encodes an integral inner membrane protein homologous to Klebsiella pneumoniae secretion function protein PulO.

xcp mutations have pleiotropic effects on the secretion of proteins in Pseudomonas aeruginosa PAO. The nucleotide sequence of a 1.2-kb DNA fragment that complements the xcp-1 mutation has been determined. Sequence analysis shows the xcpA gene product to be a 31.8-kDa polypeptide, with a highly hydrophobic character. This is consistent with a localization in the cytoplasmic membrane in P. aeruginosa, determined after specific expression of the xcpA gene under control of the T7 phi 10 promoter. A very strong homology was found between XcpA and PulO, a membrane protein required for pullulanase secretion in Klebsiella pneumoniae. This suggests the existence of a signal sequence-dependent secretion process common to these two unrelated gram-negative bacteria.     

Export of the pseudopilin XcpT of the Pseudomonas aeruginosa type II secretion system via the signal recognition particle-Sec pathway

Type IV pilins and pseudopilins are found in various prokaryotic envelope protein complexes, including type IV pili and type II secretion machineries of gram-negative bacteria, competence systems of gram-positive bacteria, and flagella and sugar-binding structures in members of the archaeal kingdom. The precursors of these proteins have highly conserved N termini, consisting of a short, positively charged leader peptide, which is cleaved off by a dedicated peptidase during maturation, and a hydrophobic stretch of approximately 20 amino acid residues. Which pathway is involved in the inner membrane translocation of these proteins is unknown. We used XcpT, the major pseudopilin from the type II secretion machinery of Pseudomonas aeruginosa, as a model to study this process. Transport of an XcpT-PhoA hybrid was shown to occur in the absence of other Xcp components in P. aeruginosa and in Escherichia coli. Experiments with conditional sec mutants and reporter-protein fusions showed that this transport process involves the cotranslational signal recognition particle targeting route and is dependent on a functional Sec translocon.     

Differentiation of Xanthomonas campestris pvs. pelargonii and hederae by Polymerase Chain Reaction

Geranium isolates of Xanthomonas campestris pv. pelargonii ( Xcp ) and English ivy isolates of X. campestris pv. hederae ( Xch ) were tested by polymerase chain reaction (PCR) to determine whether the two pathovars could be discriminated using amplification conditions developed to identify and detect Xcp in infected geraniums. Using PCR, other workers reported that the genomes of Xcp and Xch were indistinguishable. The objective of this study was to determine whether the two pathovars have sufficient sequence diversity to allow them to be distinguished by molecular means. Three primer pairs were used for PCR amplification. Two of the primer pairs (REP and XcpM1/XcpM2) were able to distinguish between Xch and Xcp , whereas amplification with the third primer pair (ERIC) did not allow discrimination between the two pathovars. Based on PCR amplification, Xcp and Xch are distinctly different pathovars. Additionally, all three primer pairs showed discrimination between Xcp and Acidovorax , a bacterial pathogen that induces leaf spot on geranium.