铜绿假单胞菌PA1550基因对泳动及蹭动的影响

【目的】：研究与铜绿假单胞菌运动能力相关的基因。【方法】：以一株临床分离的铜绿假单胞菌PA68做受体菌,应用人工Mu转座技术建立了库容为2000的突变子文库,从中筛选出泳动能力和蹭动能力丧失或减弱的突变子,通过基因克隆、测序,GenBank BLAST比对测序结果,互补基因表达确定与铜绿假单胞菌运动能力相关的基因。【结果】：突变子Y46在丧失了泳动运动能力的同时,蹭动能力也发生了减弱。在Y46突变子中,Mu转座子插入到功能完全未知的基因PA1550中。对极性效应及PA1550所在操纵子的分析表明,Mu转座子对插入点下游的基因的转录并不造成影响。【结论】：PA1550与铜绿假单胞菌的泳动及蹭动能力有关。     

铜绿假单胞菌PA1550基因对泳动及蹭动的影响

[目的]:研究与铜绿假单胞菌运动能力相关的基因.[方法]:以一株临床分离的铜绿假单胞菌PA68做受体菌,应用人工Mu转座技术建立了库容为2000的突变子文库,从中筛选出泳动能力和蹭动能力丧失或减弱的突变子,通过基因克隆、测序,GenBankBLAST比对测序结果,互补基因表达确定与铜绿假单胞菌运动能力相关的基因.[结果]:突变子Y46在丧失了泳动运动能力的同时,蹭动能力也发生了减弱.在Y46突变子中,Mu转座子插入到功能完全未知的基因PA1550中.对极性效应及PA1550所在操纵子的分析表明,Mu转座子对插入点下游的基因的转录并不造成影响.[结论]:PA1550与铜绿假单胞菌的泳动及蹭动能力有关.     

铜绿假单胞菌泳动能力相关新基因的筛选及鉴定

从Mu转座突变子文库中经过表型筛选,得到12株泳动(Swimming motility)能力缺陷的突变子,经Mu转座子插入位点的确认、基因克隆及测序分析发现其中10个突变子中Mu转座子分别插入到10个不同的与鞭毛运动和功能相关的基因中,2个突变子中Mu转座子插入到功能未知的新基因(PA2950和PA5022)中,电镜观察结果表明这2个突变株均具有完整的鞭毛,初步推测这2个基因可能是参与鞭毛泳动的能量代谢、趋化作用或信息传递的新基因。     

铜绿假单胞菌水解弹性蛋白能力相关基因的筛选与鉴定

【目的】研究铜绿假单胞菌弹性蛋白水解能力相关基因。【方法】应用人工Mu转座技术构建铜绿假单胞菌野生型菌株PA68的转座突变文库,从2000多个突变子中筛选得到4株弹性蛋白水解能力改变的突变子,并通过克隆及测序获得转座子插入位点侧翼的序列。将铜绿假单胞菌弹性蛋白酶结构基因lasB的转录启始区序列整合入载体pDN19lacΩ并将该重组质粒电转化入野生型菌株PA68及4个突变株中,对报告基因在不同菌株中的表达水平进行测定。【结果】发现4个突变株中Mu转座子分别插入lasA、galU、xcpZ和ptsP 4个基因。ptsP基因失活的突变株中,lasB基因的转录水平是野生型菌株的7%,xcpZ和lasA基因的失活使lasB基因的转录水平分别降低为野生株的54%和75%,galU基因的插入失活使lasB基因的转录上升了1倍。【结论】推测ptsP和galU基因很可能直接或间接地调控着弹性蛋白酶的生物合成。     

铜绿假单胞菌色素代谢相关基因的研究北大核心

首次应用Mu转座重组技术研究铜绿假单胞菌色素合成与调控的机制。通过一系列的表型筛选 ,得到 8株色素合成能力改变的突变子。经基因克隆、核苷酸测序研究 ,证明转座子分别插入到hmgA、ptsP、sucC、phzS、phzF1五个基因中。hmgA基因转座失活导致酪氨酸分解代谢中间产物尿黑酸的积累 ,后四种情况转座突变显著地影响了铜绿假单胞菌最重要的色素 绿脓素 (pyocyanin)的合成 ,其中PhzS和phzF1是绿脓素合成过程中的结构基因 ,ptsP基因是 1个磷酸转移酶系统的重要组分 ,sucC基因的产物是三羧酸循环中的琥珀酰辅酶A合成酶 。     

铜绿假单胞菌色素代谢相关基因的研究

首次应用Mu转座重组技术研究铜绿假单胞菌色素合成与调控的机制。通过一系列的表型筛选,得到8株色素合成能力改变的突变子。经基因克隆、核苷酸测序研究,证明转座子分别插入到hmgA、ptsP、sucC、phzS、phzF1五个基因中。hmgA基因转座失活导致酪氨酸分解代谢中间产物尿黑酸的积累,后四种情况转座突变显著地影响了铜绿假单胞菌最重要的色素绿脓素（pyocyanin）的合成,其中PhzS和phzF1是绿脓素合成过程中的结构基因,ptsP基因是1个磷酸转移酶系统的重要组分,sucC基因的产物是三羧酸循环中的琥珀酰辅酶A合成酶,对后两个基因在色素合成的调控方面可能起到重要作用的报道尚属首例。     

铜绿假单胞菌新基因PA0058插入失活对氨基糖苷类抗生素耐药性的影响

【目的】铜绿假单胞菌是一种重要的条件致病菌,临床上常引起难治性和顽固性感染,随着各种抗生素的广泛使用,该菌对多种抗生素呈现耐药性,研究其耐药性机理有着重要意义。【方法】以一株临床分离株Pseudomonas aeruginosa PA68作为出发菌株,应用人工Mu转座技术构建突变文库并从中筛选得到一株对链霉素抗性明显增强的菌株M122,并对突变株M122进行测序分析及表型检测。通过Southern杂交实验证实转座子是否为单拷贝插入,对突变株M122的基因表达谱与野生型PA68菌株进行对比分析。【结果】确定了Mu转座子在M122基因组上为单拷贝插入,插入位点为基因PA0058的第214 bp处。对M122进行表型检测,发现其对多种氨基糖苷类抗生素的耐药性均得到增强,通过转入携带完整基因PA0058的表达质粒可以使突变株M122的耐药性有所降低,利用同源重组的方法,在模式菌株P.aeruginosa PAK中进行PA0058基因敲除,得到的敲除株具有链霉素耐药性升高的表型。基因PA0058的缺失引起多种基因表达水平改变,尤其是katB、ahpC、ahpF等抗氧化酶基因转录表达显著增高。【结论】首次发现铜绿假单胞菌PA0058基因的插入失活提高了细菌对氨基糖苷类抗生素的耐药性,且导致突变株M122中抗氧化酶基因转录表达水平的上调。     

α-氨基苄青霉素抗性转座子Tn2在E.coli K12乳糖操纵子Z基因中的插入区域特异性

转座子Tn2是大肠杆菌质粒RSF 1030上一段带有α-氨基苄青霉素抗性基因的DNA序列。这段序列具有转座能力,它能通过不同于一般的DNA重组机理从一个复制子转座到另一个复制子。已知,噬菌体Mu,插入序列IS1、IS2、IS3和抗药性转座子TnA、Tn5、Tn9、Tn10等均能使被插入的基因发生突变。已有报道,不同的转座子在E.coli K12乳糖操纵子Z基因中的插入模式不同。Mu噬菌体在Z基因     

铜绿假单胞菌最佳电转化条件的研究

以临床分离的一株铜绿假单胞菌 (Pseudomonasaeruginosa)PA68作受体菌 ,将具有卡那霉素抗性标记的质粒pSMC2 8通过电转化导入到受体菌中 ,研究细胞生长状态、电击电压、细胞浓度、感受态细胞的贮备方式对转化效率的影响。结果表明 ,在细胞生长至OD5 40 =0 7～ 0 8时收集菌体 ,在低温 (2℃ )条件下 ,制备浓度为 10 11个细胞 mL的感受态细胞 ,在较高的电压 (2 6kV)电击下 ,能获得较高的转化效率。最高可达 1 68× 10 8个转化子 μgDNA(CFU μgDNA)。用此优化的转化条件 ,在国际上首次成功地将Mu转座复合物导入到P .aeruginosa中 ,并获得 2 4× 10 4 CFU μgDNA的高转化效率。由于Mu转座重组技术具有随机单点插入的优点 ,克服了传统转座子能在染色体上迁移的缺点 ,保证了表型的改变与转座子插入位点所在的基因突变的一一对应关系 ,为进一步研究P .aerugi nosa的基因组功能奠定基础     

铜绿假单胞菌多重耐药基因的筛选及鉴定

[目的]研究铜绿假单胞菌中与耐药性相关的基因.[方法]筛选转座突变体文库中对多种抗菌药物敏感的突变体,通过随机PCR、核苷酸测序及序列比对确定突变体中转座子的插入位点及其破坏的基因.[结果]筛选得到2株对多种抗菌药物敏感的突变体,其中被破坏的基因分别为功能未知的新基因PA2580和PA2800.[结论]PA2580和PA2800可能分别通过参与细胞氧化还原作用和细胞壁合成进而与铜绿假单胞菌耐药性相关.     

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.     

Type IV pilus assembly in Pseudomonas aeruginosa over a broad range of cyclic di-GMP concentrations

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that utilizes polar type IV pili (T4P) for twitching motility and adhesion in the environment and during infection. Pilus assembly requires FimX, a GGDEF/EAL domain protein that binds and hydrolyzes cyclic di-GMP (c-di-GMP). Bacteria lacking FimX are deficient in twitching motility and microcolony formation. We carried out an extragenic suppressor screen in PA103ΔfimX bacteria to identify additional regulators of pilus assembly. Multiple suppressor mutations were mapped to PA0171, PA1121 (yfiR), and PA3703 (wspF), three genes previously associated with small-colony-variant phenotypes. Multiple independent techniques confirmed that suppressors assembled functional surface pili, though at both polar and nonpolar sites. Whole-cell c-di-GMP levels were elevated in suppressor strains, in agreement with previous studies that had shown that the disrupted genes encoded negative regulators of diguanylate cyclases. Overexpression of the regulated diguanylate cyclases was sufficient to suppress the ΔfimX pilus assembly defect, as was overexpression of an unrelated diguanylate cyclase from Caulobacter crescentus. Furthermore, under natural conditions of high c-di-GMP, PA103ΔfimX formed robust biofilms that showed T4P staining and were structurally distinct from those formed by nonpiliated bacteria. These results are the first demonstration that P. aeruginosa assembles a surface organelle, type IV pili, over a broad range of c-di-GMP concentrations. Assembly of pili at low c-di-GMP concentrations requires a polarly localized c-di-GMP binding protein and phosphodiesterase, FimX; this requirement for FimX is bypassed at high c-di-GMP concentrations. Thus, P. aeruginosa can assemble the same surface organelle in distinct ways for motility or adhesion under very different environmental conditions.     

FimX, a multidomain protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa

Twitching motility is a form of surface translocation mediated by the extension, tethering, and retraction of type IV pili. Three independent Tn5-B21 mutations of Pseudomonas aeruginosa with reduced twitching motility were identified in a new locus which encodes a predicted protein of unknown function annotated PA4959 in the P. aeruginosa genome sequence. Complementation of these mutants with the wild-type PA4959 gene, which we designated fimX, restored normal twitching motility. fimX mutants were found to express normal levels of pilin and remained sensitive to pilus-specific bacteriophages, but they exhibited very low levels of surface pili, suggesting that normal pilus function was impaired. The fimX gene product has a molecular weight of 76,000 and contains four predicted domains that are commonly found in signal transduction proteins: a putative response regulator (CheY-like) domain, a PAS-PAC domain (commonly involved in environmental sensing), and DUF1 (or GGDEF) and DUF2 (or EAL) domains, which are thought to be involved in cyclic di-GMP metabolism. Red fluorescent protein fusion experiments showed that FimX is located at one pole of the cell via sequences adjacent to its CheY-like domain. Twitching motility in fimX mutants was found to respond relatively normally to a range of environmental factors but could not be stimulated by tryptone and mucin. These data suggest that fimX is involved in the regulation of twitching motility in response to environmental cues.     

Identification of an operon, Pil-Chp, that controls twitching motility and virulence in Xylella fastidiosa

Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.     

The adsorption of Pseudomonas aeruginosa bacteriophage φKMV is dependent on expression regulation of type IV pili genes

Pseudomonas aeruginosa bacteriophage φKMV requires type IV pili for infection, as observed from the phenotypic characterization and phage adsorption assays on a phage infection-resistant host strain mutant. A cosmid clone library of the host ( P. aeruginosa PAO1) genomic DNA was generated and used to select for a clone that was able to restore φKMV infection in the resistant mutant. This complementing cosmid also re-established type IV pili-dependent twitching motility. The correlation between bacteriophage φKMV infectivity and type IV pili, along with its associated twitching motility, was confirmed by the resistance of a P. aeruginosa PAO1Δ pilA mutant to the phage. Subcloning of the complementing cosmid and further phage infection analysis and motility assays suggests that a common regulatory mechanism and/or interaction between the ponA and pilMNOPQ gene products are essential for bacteriophage φKMV infectivity.     

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa

Virulence 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 C-terminus, 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.     

Pseudomonas aeruginosa D-arabinofuranose biosynthetic pathway and its role in type IV pilus assembly

Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with α1,5-linked D-arabinofuranose (d-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of α1,5-D-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv3791, required for synthesis of a polyprenyl-linked d-ribose precursor and its epimerization to D-Araf. Pa5196 mutants lacking the orthologues of those genes had non-arabinosylated pilins, poor twitching motility, and significantly fewer surface pili than the wild type even in a retraction-deficient (pilT) background. The Pa5196 pilus system assembled heterologous non-glycosylated pilins efficiently, demonstrating that it does not require post-translationally modified subunits. Together the data suggest that pilins of group IV strains need to be glycosylated for productive subunit-subunit interactions. A recombinant P. aeruginosa PAO1 strain co-expressing the genes for d-Araf biosynthesis, the pilin modification enzyme TfpW, and the acceptor PilA(IV) produced arabinosylated pili, confirming that the Pa5196 genes identified are both necessary and sufficient. A P. aeruginosa epimerase knock-out could be complemented with the corresponding Mycobacterium smegmatis gene, demonstrating conservation between the systems of the Corynebacterineae and Pseudomonas. This work describes a novel Gram-negative pathway for biosynthesis of d-Araf, a key therapeutic target in Corynebacterineae.