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.     

Identification of a novel Gsp-related pathway required for secretion of the manganese-oxidizing factor of Pseudomonas putida strain GB-1

The manganese-oxidizing factor of Pseudomonas putida strain GB-1 is associated with the outer membrane. One of the systems of protein transport across the outer membrane is the general secretory pathway (Gsp). The gsp genes are called xcp in Pseudomonas species. In a previous study, it was shown that mutation of the prepilin peptidase XcpA and of a homologue of the pseudopilin XcpT inhibited transport of the factor. In the present study, we describe the genomic region flanking the xcpT homologue (designated xcmT1). We show that xcmT1 is part of a two-gene operon that includes an xcpS homologue (designated xcmS). No other xcp-like genes are present in the regions flanking the xcmT1/xcmS cluster. We also characterized the site of transposon insertion of another transport mutant of P. putida GB-1. This insertion appeared to be located in a gene (designated xcmX) possibly encoding another pseudopilin-related protein. This xcmX is clustered with two other xcpT-related genes (designated xcmT2 and xcmT3) on one side and homologues of three csg genes (designated csmE, csmF and csmG) on the other side. The csg genes are involved in production of aggregative fibres in Escherichia coli and Salmonella typhimurium. A search for XcmX homologues revealed that the recently published genome of Ralstonia solanacearum and the unannotated genome of P. putida KT2440 contain comparable gene clusters with xcmX and xcp homologues that are different from the well-described 'regular'xcp/gsp clusters. They do contain xcpR and xcpQ homologues but, for example, homologues of xcpP, Y and Z are lacking. The results suggest a novel Xcp-related system for the transport of manganese-oxidizing enzymes to the cell surface.     

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.     

Las-like quorum-sensing system negatively regulates both pyoluteorin and phenazine-1-carboxylic acid production in Pseudomonas sp. M18

A las-like quorum-sensing system in Pseudomonas sp. M18 was identified, which consisted of lasI and lasR genes encoding LuxI-LuxR type regulator. Several functions of the las system from strain M18 were investigated in this study. The chromosomal inactivation of either lasI or lasR by recombination increased the production of both pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA) by 4-5 fold and 2-3 fold over that of the wild type strain of M18, respectively. Production of both antibiotics was restored to wild-type levels after in trans complementation with the wild-type lasI or lasR gene. Expression of the translational fusions pltA'-'lacZ and phzA'-'lacZ further confirmed the negative effect of lasI or lasR on both biosynthetic operons, and it was also demonstrated that the las system was related to the ability of swarming motility and the inhibition of cell growth.     

Quorum-sensing and siderophore biosynthesis in Pseudomonas aeruginosa: lasRllasI mutants exhibit reduced pyoverdine biosynthesis

Cell density-dependent gene expression in Pseudomonas aeruginosa is controlled, in part, by the quorum-sensing regulator LasR. lasR null mutants exhibited a reproducible 2-fold decrease in production of the catecholate-hydroxamate siderophore pyoverdine during grown under iron-limiting conditions. Similarly, lasI mutants defective in the biosynthesis of the autoinducer PAI-1 also exhibited a 2-fold decrease in pyoverdine production which could be largely restored upon addition of exogenous PAI-1. lasR mutants were not altered with respect to expression of the pvdD gene involved in the synthesis of the peptide portion of pyoverdine, indicating that some other pyoverdine biosynthetic gene(s) were affected by the LasRI status of the cell. This represents the first report of quorum-sensing regulation of siderophore production in bacteria and highlights the fact that cell density, while not an essential signal for pyoverdine expression, does enhance production of this siderophore.     

Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa.

Xcp proteins constitute the secretory apparatus of Pseudomonas aeruginosa. Deduced amino acid sequence of xcp genes, expression, and subcellular localization revealed unexpected features. Indeed, most Xcp proteins are found in the cytoplasmic membrane although xcp mutations lead to periplasmic accumulation of exoproteins, indicating that the limiting step is translocation across the outer membrane. To understand the mechanism by which the machinery functions and the interactions between its components, it is valuable to know their membrane organization. We report data demonstrating the N(in)-C(out) topologies of three general secretion pathway components, the XcpP, -Y, and -Z proteins.     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model

The human pathogen Pseudomonas aeruginosa has been shown previously to use similar virulence factors when infecting mammalian hosts or Dictyostelium amoebae. Here we randomly mutagenized a clinical isolate of P. aeruginosa , and identified mutants with attenuated virulence towards Dictyostelium . These mutant strains also exhibited a strong decrease in virulence when infecting Drosophila and mice, confirming that P. aeruginosa makes use of similar virulence traits to confront these very different hosts. Further characterization of these bacterial mutants showed that TrpD is important for the induction of the quorum-sensing circuit, while PchH and PchI are involved in the induction of the type III secretion system. These results demonstrate the usefulness and the relevance of the Dictyostelium host model to identify and analyse new virulence genes in P. aeruginosa .     

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).     

SYT09/PI荧光探针标记lasR rhlR基因缺陷对铜绿假单胞菌生物膜形成影响的体外实验研究

目的研究群体感应（QS）系统在铜绿假单胞菌生物膜（BF）形成中的作用。方法体外建立3 d QS系统完整的铜绿假单胞菌野生型PA01菌株与QS系统缺陷（lasRrhlR基因缺陷△lasR△rhlR）型菌株生物模型,通过SYT09/PI荧光探针标记,结合激光共聚焦显微镜摄取BF不同层面的图片,经图像结构分析（ISA）软件分析获得QS系统lasRrhlR缺陷株,PA01菌株BF相关空间结构参数。结果培养第3天PA01菌株可形成较厚、有孔状通道的成熟BF结构,而△lasR△rhlR菌株仅形成明显稀薄的早期BF结构,△lasR△rhlR菌株的3 d BF厚度为（7.36±0.2）μm,PA01菌株为（21.64±0.57）μm（P〈0.05）,区域孔率（areal porosity,AP）分别为：（0.902±0.006）、（0.928±0.002）;平均扩散距离（average diffusion distance,ADD）和结构熵（textural entropy,TE）在△lasR△rhlR菌株分别为：（1.503±0.029）和（5.706±0.190）;在PA01菌株分别为：（1.467±0.015）和（5.213±0.111）,△lasR△rhlR菌株AP较PA01菌株低（P〈0.05）;而ADD、TE较PA01菌株高（P〈0.05）。结论△lasR△rhlR基因缺陷明显影响铜绿假单胞菌的BF形成能力,QS系统lasRrhlR基因在铜绿假单胞菌BF形成中发挥重要作用。