铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA2072的生物学分析

【背景】铜绿假单胞菌为革兰氏阴性杆菌,是医院感染的常见条件致病菌之一。广泛存在于细菌中的第二信使分子环鸟苷二磷酸(cyclic-di-guanosine monophosphate,c-di-GMP)对细菌生理生化功能具有重要的调节作用。铜绿假单胞菌PAO1中存在参与c-di-GMP代谢的基因PA2072。【目的】探讨铜绿假单胞菌PAO1中c-di-GMP代谢相关基因PA2072的生物学功能。【方法】运用PCR及分子克隆技术构建PA2072基因及各结构域的自杀载体,运用基因敲除方法获取PA2072基因的3个突变株;利用泳动性(swimming)、蜂群运动(swarming)、蹭行运动(twitching)和生物膜定量实验对细菌进行初步的表型分析,进一步通过刚果红染色法对菌株进行分析。【结果】成功构建PA2072基因敲除突变菌株及回补菌株;生物膜定量结果发现基因PA2072的敲除会影响细菌生物膜的形成,PA2072蛋白的不同结构域对生物膜的合成也起到了重要作用;细菌运动能力检测中发现PA2072相关基因的敲除对细菌运动能力也有一定影响。刚果红平板检测结果显示,与野生型PAO1菌株相比,P...     

铜绿假单胞菌中PA0847二鸟苷酸环化酶活性受非催化位点Y700调控

【目的】鉴定铜绿假单胞菌(Pseudomonas aeruginosa)中PA0847胞内结构域及其突变体的二鸟苷酸环化酶活性,并初步探究其催化机制。【方法】利用刚果红平板染色法验证PA0847胞内域的二鸟苷酸环化酶活性;采用PCR技术构建PA0847 PAS-GGDEF结构域及其单点突变体,并经过表达纯化获得相应蛋白;经凝胶分子筛层析分析蛋白在溶液中的聚集状态;通过体外酶促反应鉴定蛋白的二鸟苷酸环化酶活性,基于噻唑橙荧光染色检测蛋白酶促反应后环鸟苷二磷酸(cyclic diguanylate monophosphate, c-di-GMP)的生成量,并筛选与二鸟苷酸环化酶活性密切相关的氨基酸残基;联用结构预测和分子对接获得PA0847 PAS-GGDEF二聚体以及结合三磷酸鸟苷(guanosine triphosphate, GTP)的复合物结构模型。【结果】PA0847 PAS-GGDEF主要以二聚体形式发挥催化活性,PAS结构域促进二聚体形成,有效提高二鸟苷酸环化酶活性;突变筛选发现,在低蛋白浓度(0.6 mg/mL)下,非催化位点突变体Y700A活性较野生型显著提高;凝胶分子筛层析表明,其高活性可能与Y700A突变促进GGDEF (Gly-Gly-Asp-Glu-Phe)结构域二聚化有关;结构模型分析显示,PA0847 PAS-GGDEF与GTP结合位点保守,其中K722的氨基侧链在结合GTP的磷酸基团中发挥着重要作用,而Y700芳香环侧链与K722所在的α螺旋有疏水互作,因此Y700A突变可能改变了K722所在螺旋的空间取向,进而促进K722与底物GTP的结合以及GGDEF二聚化。【结论】铜绿假单胞菌PA0847的非催化位点Y700可间接调控二鸟苷酸环化酶活性。     

PA2580基因与铜绿假单胞菌环境压力耐受性相关

【目的】研究铜绿假单胞菌PAO1 PA2580基因的功能。【方法】构建了PA2580的敲除突变体及突变体互补体,通过最小抑制浓度测定、基因启动子活性检测、蛋白体外表达纯化等方法,对PA2580基因的功能进行了深入的研究。【结果】PA2580突变体对羧苄青霉素、氯霉素、环丙沙星的敏感性增强。PA2580基因的表达还受到不同种类的低于抑制浓度的抗生素的调节。PA2580蛋白产物以NADPH为电子供体,能够高效还原多种醌类物质。此外,PA2580突变体对过氧化氢敏感性增加,过氧化氢酶编码基因在PA2580突变体中的表达降低,表明PA2580与铜绿假单胞菌氧化压力耐受性相关。【结论】PA2580产物是NADPH-醌类的还原酶,其功能与铜绿假单胞菌对环境压力的耐受密切相关。     

铜绿假单胞菌必需基因的研究进展

铜绿假单胞菌为专性需氧非发酵革兰氏阴性杆菌,是医院感染的常见条件致病菌之一,可引起呼吸道、泌尿道、烧伤创面和菌血症等严重感染.铜绿假单胞菌耐药形势日益严峻,给临床治疗带来困难.必需基因是生长过程中必不可少的看家基因,对铜绿假单胞菌必需基因进行深入研究,不仅有助于了解细菌的生长、毒力等基本特性,也有助于筛选新的抗菌药物靶...     

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。     

计算机辅助设计联合斑点杂交筛选铜绿假单胞菌PAO1 motA基因反义寡核苷酸序列

为了筛选出能与铜绿假单胞菌PAO1 motA基因的mRNA结合紧密的反义寡核苷酸序列,采用全基因寻靶技术（full length gene targeting,FLGT）,运用计算机软件（Mfold和RNA Structure4.6）模拟铜绿假单胞菌PAO1 motA基因mRNA的二级结构,根据最小自由能原理设计出8条寡核苷酸探针序列;PCR扩增出全长motA基因,克隆motA基因并进行体外转录,同时用地高辛标记mRNA,以斑点杂交方法筛选出与motA基因mRNA结合紧密、杂交信号较强的寡核苷酸序列。斑点杂交结果显示8条寡核苷酸中的4条有较强的杂交信号,从而成功筛选到了能与motA mRNA牢固结合的反义序列,为进一步研究以motA基因为靶的反义技术抑制生物膜形成打下基础。     

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。     

铜绿假单胞菌耐药性相关基因的筛选及鉴定

铜绿假单胞菌在人体内能引起严重的感染. 由于铜绿假单胞菌高水平的内在性和获得性耐药性, 使其引起的感染难以治愈. 为解决该问题, 弄清病原菌抗生素抗性的分子机制是一个关键. 本实验构建了含有17000个铜绿假单胞菌转座突变株文库, 并在LB固体平板上, 用7种抗生素在最小抑制浓度(MIC)和1/2MIC条件下, 筛选抗生素抗性发生变化的突变株. 结果确定了43株转座突变株, 每个突变株对至少一种抗生素的敏感性表现出增加3倍或降低1/2的表型. 通过随机PCR和DNA测序, 确定了这些铜绿假单胞菌突变体中转座子在基因组上的插入位点, 确定了被破坏的基因. 其中, 9个是已知的与抗生素抗性相关基因, 包括mexI, mexB和mexR; 24个是以前未知与抗性相关的基因, 包括一个菌毛合成基因pilY1, 这个菌毛合成基因的破坏导致菌株对羧苄青霉素的MIC增加了128倍. 此外, 43个基因中有12个是功能完全未知的基因. 这些基因的筛选鉴定有助于了解铜绿假单胞菌中抗生素抗性的产生机制, 这些基因或许可成为控制或扭转抗生素抗性的作用靶点.     

铜绿假单胞菌的双组分系统

双组分系统是存在于原核和少部分真核生物细胞中的信号转导系统,主要由组氨酸蛋白激酶和反应调节蛋白组成,通过感应外界环境信号、信号输入、磷酸基团传递、信号输出等环节调节基因表达,使细胞能更加适应环境变化。铜绿假单胞菌为条件致病菌,其双组分系统构成多样、功能复杂且参与介导耐药性产生,因此铜绿假单胞菌的双组分系统日益引起人们关注。本文对铜绿假单胞菌双组分系统的组成、信号转导机制、种类、研究方法及其临床意义进行了综述。     

c-di-GMP的磷酸二酯酶PA4781在抗菌肽Merecidin抑制铜绿假单胞菌生物被膜中的作用

【背景】抗菌肽Merecidin可抑制临床菌株铜绿假单胞菌PA03生物被膜。PA4781基因是课题组通过生物信息学分析筛选出的差异表达基因,PA4781作为细菌第二信使分子环二鸟苷酸(cyclic diguanylate,c-di-GMP)的磷酸二酯酶具有降解c-di-GMP的作用,其在抗菌肽Merecidin抑制生物被膜中的作用机制尚不清楚。【目的】研究细菌第二信使分子c-di-GMP的磷酸二酯酶PA4781基因在抗菌肽Merecidin抑制铜绿假单胞菌生物被膜中的作用。【方法】利用单碱基突变技术敲除PA4781基因,Sanger测序方法检测敲除的正确性。采用结晶紫染色法观察PA03菌株、PA4781过表达菌株、PA4781敲除菌株24 h生物被膜生长情况,以及在抗菌肽Merecidin 24、48、72μmol/L作用下各菌株生物被膜的生长情况。采用对羟基联苯溶液显色法检测在抗菌肽Merecidin 48、72μmol/L作用下,PA03菌株、PA4781过表达菌株、PA4781敲除菌株生物被膜藻酸盐的变化情况。【结果】Sanger测序结果显示,用pnCasPABEC系统成功实现了...     

The role of c-di-GMP signaling in an Aeromonas veronii biovar sobria strain

Aeromonas is a ubiquitous gram-negative bacterium that persists in the environment. It is shown that all isolates of persistent Aeromonas clones show strong biofilm formation ability. C-di-GMP regulates biofilm formation in many bacteria. To investigate the impact of c-di-GMP signaling, we introduced heterologous GGDEF and EAL domain proteins from Salmonella Typhimurium to an Aeromonas veronii biovar sobria strain. Overexpression of the GGDEF domain protein AdrA increased c-di-GMP concentration and biofilm formation and reduced motility. Production of the quorum-sensing signaling molecule C4-homoserine lactone and adhesion to aquatic plant duckweed and amoeba surfaces were enhanced. On the other hand, overexpression of the EAL domain protein YhjH decreased biofilm formation and increased motility.     

A demonstration of athermal effects of continuous microwave irradiation on the growth and antibiotic sensitivity of Pseudomonas aeruginosa PAO1

Stress, caused by exposure to microwaves (2.45 GHz) at constant temperature (37 ± 0.5°C), alters the growth profile of Pseudomonas aeruginosa PAO1. In the absence of microwave treatment a simple, highly reproducible growth curve was observed over 24 h or more. Microwave treatment caused no reduction in growth during the first 6 h, but at a later stage (>12 h) the growth was markedly different to the controls. Secondary growth, typical of the presence of persisters clearly became apparent, as judged by both the dissolved oxygen and the cell density profiles. These treated cells showed distinct morphological changes, but on regrowth these cells reverted to normal. The microwave induced persisters were subject to antibiotic challenge (tobramycin) and showed increased sensitivity when compared to the unstressed planktonic cells. This is in marked contrast to antibiotic induced persisters which show increased resistance. This provides evidence for both a nonthermal effect of microwaves and a previously undescribed route to a novel form of antibiotic susceptible persister cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:37–44, 2017     

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。     

Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.     

Enhanced biofilm formation and 3-chlorobenzoate degrading activity by the bacterial consortium of Burkholderia sp. NK8 and Pseudomonas aeruginosa PAO1

Aims:  To characterize biofilm formation of a chlorobenzoates (CBs) degrading bacterium, Burkholderia sp. NK8, with another bacterial species, and the biodegradation activity against CBs in the mixed-species biofilm.
Methods and Results:  Burkholderia sp. NK8 was solely or co-cultured with each of five other representative bacteria in microtitre dishes. Biofilm formation involving the strain NK8 was synergistically promoted by co-culturing with only Pseudomonas aeruginosa PAO1. Epifluorescent microscopy revealed that cells of the bacterial strain NK8 were viable and distributed randomly in the mixed-species biofilms. Enumeration of the attached cells on the surface of wells revealed that cells of the strain NK8 increased approx. 10-fold by the co-culture with the strain PAO1 compared to those by monoculture of the strain NK8, and the degradation activity of 3-chlorobenzoate by the dual-species biofilms was more promoted than that by the strain NK8-monocultured biofilms.
Conclusions:  Enhanced biofilm formation of Burkholderia sp. NK8 by the bacterial consortium occurred, but is determined by the partner bacterial species. The mixed-species biofilms have the advantage to degrade CBs on a solid surface.
Significance and Impact of the Study:  This study provides a significance of bacterial consortia on the biofilm formation and the degradation activity of Burkholderia sp. NK8, which contribute for complete degradation of chlorinated aromatics.     

Production of pyoverdine, the fluorescent pigment of Pseudomonas aeruginosa PAO1

Abstract The nutritional requirements for the production of pyoverdine were studied using Pseudomonas aeruginosa PAO₁ in a chemically defined medium, with shaking. Succinic acid and ammonium sulphate were found to be the best sources of carbon and nitrogen for pyoverdine production. The optimum carbon-to-nitrogen ratio was found to be 4:1. Elevated concentrations of phosphate inhibited pyoverdine production.