The <Emphasis Type="Italic">flhDC</Emphasis> gene affects motility and biofilm formation in <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

The flagella master regulatory gene flhDC of Yersinia pseudotuberculosis serotype III (YPIII) was mutated by deleting the middle region and replaced by a tetracycline resistant gene, and the subsequent mutant strain named YPIIIΔflhDC was obtained. Swimming assay showed that the swimming motility of the mutant strain was completely abolished. The promoter region of the flagella second-class regulatory gene fliA was fused with the lux box, and was conjugated with the mutant and the parent strains respectively for the first cross. LUCY assay result demonstrated that flhDC regulated the expression of fliA in YPIII as reported in E. coli. Biofilm formation of the mutant strain on abiotic and biotic surfaces was observed and quantified. The results showed that mutation of flhDC decreased biofilm formation on both abiotic and biotic surfaces, and abated the infection on Caenorhabdtis elegans. Our results suggest that mutation of the flagella master regulatory gene flhDC not only abolished the swimming motility, but also affected biofilm formation of YPIII on different surfaces. The new function of flhDC identified in this study provides a novel viewpoint for the control of bacterial biofilm formation.     

A Highly Specific One-step PCR Assay for the Rapid Discrimination of Enteropathogenic Yersinia enterocolitica from Pathogenic Yersinia pseudotuberculosis and Yersinia pestis

Based on differences within the yopT-coding region of Yersinia. enterocolitica, Y. pseudotuberculosis and Y. pestis, a rapid and sensitive one-step polymerase chain reaction assay with high specificity for pathogenic Y. enterocolitica was developed. By this method pathogenic isolates of Y. enterocolitica can be easily identified and discriminated from other members of this genus. The entire coding sequence of the yopT effector gene of Y. pseudotuberculosis Y36 was determined.     

假结核耶尔森氏菌中Phd-Doc毒素-抗毒素系统的功能鉴定

【背景】毒素-抗毒素系统在微生物体内广泛存在,在微生物对抗外界不良环境方面发挥重要作用。【目的】以模式细菌假结核耶尔森氏菌(Yersinia pseudotuberculosis,Yptb)为材料,探究其编码的Phd-Doc毒素-抗毒素系统的作用机制和生物学功能。【方法】通过生物信息学方法预测Yptb中编码的Phd-Doc毒素-抗毒素系统,通过毒性分析、基因表达分析及蛋白相互作用对其进行鉴定;通过抗生素胁迫、氧胁迫、生物被膜形成等实验研究Phd-Doc在体内发挥的生物学功能。【结果】生物信息学分析鉴定出一对Phd-Doc毒素-抗毒素系统,发现二者共转录且相互作用;毒素蛋白Doc能够引起大肠杆菌形态发生变化并抑制其生长,抗毒素蛋白Phd能中和Doc的毒性;Phd-Doc毒素-抗毒素系统具有自调控抑制效应;phd-doc的缺失对Yptb自身的生长无影响,而且毒素蛋白Doc在野生型Yptb内过表达并未显示毒性;phd-doc在转录水平上响应了抗生素胁迫和氧胁迫,其中,对氯霉素胁迫最为敏感,但并不影响Yptb的生长;同时,Phd-Doc能够影响Yptb的生物被膜形成能力。【结论】Yptb中Phd-Doc毒素-抗毒素系统的功能鉴定对于更好地了解在多变的外部环境下微生物的定殖和响应机制具有重要意义。     

Identification of a new gene PA5017 involved in flagella-mediated motility, chemotaxis and biofilm formation in Pseudomonas aeruginosa

Flagella-mediated motility is recognized as one of the major factors contributing to virulence in Pseudomonas aeruginosa. During a screening of a mini-Mu transposon mutant library of P. aeruginosa PA68, a mutant partially deficient in swimming and swarming motility was identified in a new locus that encodes a predicted protein of unknown function annotated PA5017 in the P. aeruginosa PAO1 genome sequence. Chemotaxis plate assay indicated that inactivation of the PA5017 gene led to a decreased chemotactic response. Complementation of the PA5017 mutant with the wild-type PA5017 gene restored normal motility and chemotaxis phenotype. A promoter-lacZ reporter activity assay of the cheYZAB operon from chemotaxis gene cluster 1 showed that there was almost a twofold difference in expression levels of the wild-type PA68 and the PA5017 mutant. This suggested that the PA5017 affected expression of the cheYZAB operon negatively. Further study showed that inactivation of the PA5017 gene in PA68 led to increased biofilm formation in a static system and to the formation of a heterogeneous biofilm in a flow-chamber system. These results suggested that PA5017 possibly affected flagellum-dependent motility and in turn biofilm formation via the chemotaxis signal transduction pathway.     

The O-specific polysaccharide structure and biosynthetic gene cluster of Yersinia pseudotuberculosis serotype O:11

In the Yersinia pseudotuberculosis serotyping scheme, 21 serotypes are present originating from about 30 different O-factors distributed within the species. With regard to the chemical structures of lipopolysaccharides (LPSs) and the genetic basis of their biosynthesis, a number, but not all, of Y. pseudotuberculosis strains representing different serotypes have been investigated. In order to present an overall picture of the relationship between genetics and structures, we have been working on the genetics and structures of various Y. pseudotuberculosis O-specific polysaccharides (OPSs). Here, we present a structural and genetic analysis of the Y. pseudotuberculosis serotype O:11 OPS. Our results showed that this OPS structure has the same backbone as that of Y. pseudotuberculosis O:1b, but with a 6d-l-Altf side-branch instead of Parf. The 3′ end of the gene cluster is the same as that for O:1b and has the genes for synthesis of the backbone and for processing the completed repeat unit. The 5′ end has genes for synthesis of 6d-l-Altf and its transfer to the repeating unit backbone. The pathway for the synthesis of the 6d-l-Altf appears to be different from that for 6d-l-Altp in Y. enterocolitica O:3. The chemical structure of the O:11 repeating unit is

Full-size image (5K)

     

肽酶PepT参与副溶血弧菌的浮游运动和生物被膜形成

pepT基因编码一种金属依赖性肽酶T (peptidase T,PepT),能特异性催化三肽N端氨基酸,因此也称为氨肽酶T。研究发现大多数氨肽酶参与细菌蛋白质新陈代谢和调节三肽活性,但关于PepT在细菌毒力及致病性方面的报道较少。[目的]本文选取PepT为研究对象,研究其对副溶血弧菌生物学特性及致病性的影响。[方法]通过构建缺失株ΔpepT和回补株CΔpepT,比较菌株在运动性、生物被膜、环境耐受、细胞毒性等方面的差异。[结果]与野生株相比,ΔpepT缺失株的极性鞭毛转录水平极显著下降,浮游运动能力降低;同时生物被膜形成能力减弱,而细菌群集运动及环境耐受能力无显著差异。此外,缺失pepT基因会导致副溶血弧菌的细胞毒性和小鼠毒力作用显著下降。[结论]pepT基因与副溶血弧菌浮游运动和生物被膜形成能力相关,并且影响其致病性。     

AI-2通过调节c-di-GMP代谢酶DosC影响类志贺邻单胞菌生物膜形成及运动性

细菌中广泛分布的群体感应信号分子autoinducer-2 (AI-2)会影响细菌的生物膜形成及运动性等生理过程,然而该信号对类志贺邻单胞菌相关表型的调控作用及其分子机制尚未有所报道。【目的】揭示AI-2通过影响胞内环二鸟苷单磷酸(c-di-GMP)水平调控类志贺邻单胞菌生物膜形成及运动性的内在机制,为类志贺邻单胞菌感染的防治提供新思路。【方法】首先利用同源重组方法构建luxS基因敲除菌株(ΔluxS),通过软琼脂平板法和结晶紫染色法分别检测其与野生型泳动能力和生物膜形成水平的差异;之后通过序列比对找到AI-2的潜在受体蛋白DosC(SAMEA2665130＿2180),利用哈维氏弧菌生物发光实验及等温滴定量热实验(ITC)研究DosC的配体结合结构域(ligand-binding domain,LBD)与AI-2的结合能力;通过体外酶活实验、胞内c-di-GMP定量分析研究AI-2对DosC受体活性的影响;最后参照前述方法构建受体DosC编码基因敲除菌株(ΔdosC)并检测其与野生型相比泳动能力和生物膜形成水平的变化。【结果】AI-2与DosC-LBD显示出高亲和作用力;通过高效液相...     

Tannin derived materials can block swarming motility and enhance biofilm formation in Pseudomonas aeruginosa

Surface-associated swarming motility is implicated in enhanced bacterial spreading and virulence, hence it follows that anti-swarming effectors could have clinical benefits. When investigating potential applications of anti-swarming materials it is important to consider whether the lack of swarming corresponds with an enhanced sessile biofilm lifestyle and resistance to antibiotics. In this study, well-defined tannins present in multiple plant materials (tannic acid (TA) and epigallocathecin gallate (EGCG)) and undefined cranberry powder (CP) were found to block swarming motility and enhance biofilm formation and resistance to tobramycin in Pseudomonas aeruginosa. In contrast, gallic acid (GA) did not completely block swarming motility and did not affect biofilm formation or tobramycin resistance. These data support the theory that nutritional conditions can elicit an inverse relationship between swarming motility and biofilm formation capacities. Although anti-swarmers exhibit the potential to yield clinical benefits, it is important to be aware of possible implications regarding biofilm formation and antibiotic resistance.     

Mycobacterium smegmatis biofilm formation and sliding motility are affected by the serine/threonine protein kinase PknF

Eighteen 'eukaryotic-like' serine/threonine kinases are present in the Mycobacterium smegmatis genome. One of them encoded by the ORF 3677 demonstrates high similarity to the Mycobacterium tuberculosis protein kinase PknF. A merodiploid strain was generated, which showed reduced growth associated with irregular cell structure. The merodiploid strain displayed altered colony morphology, defective sliding motility and biofilm formation. These data indicate a role for PknF in biofilm formation, possibly associated with alterations in glycopeptidolipid composition.     

鞭毛介导的运动性与细菌生物膜的相互关系

摘要:由于运动缺陷型细菌形成生物膜的能力会下降,长期以来细菌的运动性都被认为与生物膜的形成呈正相关,但这一理论现在证明还有待商榷,而且运动性不是影响膜形成的绝对因素。本文详细介绍了细菌的生物膜和运动性,并重新定义了两者的相互关系。     

鼠疫减毒活疫苗研究现状与展望

鼠疫(plague)是由鼠疫耶尔森氏菌(Yersinia pesits)引起的烈性传染病,在人类历史上曾造成约2亿人的死亡,在我国被列为甲类传染病。由于鼠疫菌具有高度致病性、传染性,被列为最具潜力的生物战剂和生物恐怖剂。在面临鼠疫威胁时,疫苗是最为有力的武器。鼠疫疫苗研究中,减毒活疫苗是重要的研究方向,现就鼠疫减毒活疫苗的研究现状进行综述,为新疫苗的研制提供参考。     

大肠埃希菌生物被膜基因调控研究进展

大肠埃希菌(Escherichia coli)是一种兼性厌氧、有鞭毛的革兰氏阴性短杆菌,常寄生于人和动物肠道内,是常见的人畜共患病病原之一。大肠埃希菌易形成生物被膜,这是一种由细菌群落分泌能够包裹自身的胞外基质与细菌结合形成的特殊聚集体,也是临床细菌感染疾病难以治愈的主要原因。生物被膜的形成不仅帮助细菌逃避宿主的防御系统,还可以降低或阻止药物发挥作用,从而诱发生物被膜相关感染(biofilm-associated infections, BAI)。本文从生物被膜形成的基因调控系统和相关调控蛋白等角度,归纳总结调控大肠埃希菌生物被膜形成的分子机制,并对防治BAI的策略进行了概述,为寻找合适的药物靶点以及防治BAI提供参考。     

鼠疫耶尔森氏菌环二鸟苷酸代谢及生物被膜形成调控研究进展

鼠疫耶尔森氏菌(Yersinia pestis,以下简称"鼠疫菌")是烈性传染病鼠疫的病原菌,以鼠蚤作为传播媒介。鼠疫菌在其传播媒介鼠蚤的前胃中形成生物被膜从而促进其在宿主间传播。鼠疫菌生物被膜的形成受第二信使分子环二鸟苷酸(c-di-GMP)的正向调控。鼠疫菌中c-di-GMP由二鸟苷酸环化酶(DGC)HmsT和HmsD合成,由磷酸二酯酶(PDE)HmsP降解。文中主要介绍影响鼠疫菌环二鸟苷酸代谢及生物被膜形成的调控因子,并对其作用机制进行讨论和总结。     

Influence of temperature on biofilm formation by Listeria monocytogenes on various food-contact surfaces: relationship with motility and cell surface hydrophobicity

Aims:  To assess the ability of Listeria monocytogenes to form biofilm on different food-contact surfaces with regard to different temperatures, cellular hydrophobicity and motility.
Methods and Results:  Forty-four L. monocytogenes strains from food and food environment were tested for biofilm formation by crystal violet staining. Biofilm levels were significantly higher on glass at 4, 12 and 22°C, as compared with polystyrene and stainless steel. At 37°C, L. monocytogenes produced biofilm at significantly higher levels on glass and stainless steel, as compared with polystyrene. Hydrophobicity was significantly ( P  < 0·05) higher at 37°C than at 4, 12 and 22°C. Thirty (68·2%) of 44 strains tested showed swimming at 22°C and 4 (9·1%) of those were also motile at 12°C. No correlation was observed between swimming and biofilm production.
Conclusions:  L. monocytogenes can adhere to and form biofilms on food-processing surfaces. Biofilm formation is significantly influenced by temperature, probably modifying cell surface hydrophobicity.
Significance and Impacts of the Study:  Biofilm formation creates major problems in the food industry because it may represent an important source of food contamination. Our results are therefore important in finding ways to prevent contamination because they contribute to a better understanding on how L. monocytogenes can establish biofilms in food industry and therefore survive in the processing environment.     

A role for TonB1 in biofilm formation and quorum sensing in Pseudomonas aeruginosa

This study revealed that a Pseudomonas aeruginosa tonB1 mutant was unable to produce a mature biofilm and showed reduced swarming and twitching motilities compared with the parent strain. The tonB1 mutant was also found to produce significantly lower cell-free and cell-associated levels of the quorum sensing (QS) signal molecule 3-oxo-C12-AHL. Altered biofilm and motility phenotypes were restored to wildtype with the addition of exogenous N-acylhomoserine lactones. These functions were independent of the role of TonB1 in iron uptake. This is the first time that a link has been established between TonB1 activity and QS.