趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷(MP)高效降解菌株,同时对MP具有趋化性。cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异。通过土壤盆钵试验(MP浓度为50mg/kg),发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%~30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用。     

水稻条斑病菌gacA基因的克隆及功能初析

根据黄单胞菌gacA基因的同源性设计简并引物,采用PCR方法从水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)中克隆了gacA同源基因,命名为gacA_Xooc。序列比较显示,该基因在黄单胞菌中是相对保守的。通过同源重组的方法,构建了gacA_Xooc的插入突变株。对0.1% Tryptone的趋化应答能力检测发现,gacA突变株的趋化能力明显降低,证明gacA与Xooc的趋化性相关。     

Pseudomonas mosselii E1铁载体合成相关基因cysI的克隆与功能初步分析

从棉花根际分离的铁载体产生菌E1,其16SrDNA与Pseudomonas mosselii ATCCBAA-99的同源性为100%。采用三亲本杂交方法将携带转座子Tn5-1063的质粒pRL1063a导入E1中进行转座子插入诱变。利用CAS法,从1000个突变株中,筛选到一株铁载体合成缺失突变株E1-185。利用TAIL-PCR方法,扩增位于Tn5-1063两端的侧翼序列。测序结果表明,转座子插入到E1的cysI基因内。该基因与Pseudomonas entomophila L48的cysI同源性为96%,其CysI氨基酸序列相似性为97%。该基因与半胱氨酸的合成密切相关,而在加有半胱氨酸的CAS平板上,突变株恢复了铁载体产生能力,证明cysI在E1铁载体合成过程中具有重要作用。据推测,cysI可能与铁载体合成途径中关键蛋白acyl-S-PCPs的形成有关。     

苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26Aa和cry28Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26Aa和cry28Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26Aa在仅消除cry26Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26Aa所在质粒有关。     

苏云金芽胞杆菌G03 spoIVF操纵子敲除对芽胞和晶体形成的影响

spoIVF是一个普遍存在于芽胞杆菌中的操纵子。在枯草芽胞杆菌中,它编码的两个蛋白是芽胞形成所必需的。采用基因重组技术敲除了苏云金芽胞杆菌G03菌株中的spoIVF操纵子,构建了spoIVF缺失株G03(spoIVF-)。研究表明:该突变株丧失了形成芽胞和晶体的能力。lacZ基因与cry1Aa基因的启动子融合表达分析发现:突变株中的cry1Aa基因的活性严重降低。利用载体pSTK携带spoIVF操纵子在突变株中的表达,使突变株部分恢复了产胞和形成杀虫晶体蛋白的能力。这说明spoIVF操纵子是所必需的,同时该操纵子还影响σ^E因子控制的cry1Aa基因表达。     

毒力岛基因ibeT有助于大肠杆菌抵抗人脑微血管内皮细胞溶酶体的降解

大肠杆菌是导致新生儿细菌性脑膜炎最常见的革兰氏阴性致病菌．为探讨毒力岛基因ibeT在大肠杆菌K1株致病过程中的作用,构建了ibeT基因缺失的大肠杆菌K1株,细菌在细胞内存活试验结果显示,ibeT基因缺失抑制了大肠杆菌K1株在人脑微血管内皮细胞中的生长．利用激光共聚焦扫描显微镜观察到,在细菌侵袭进入人脑微血管内皮细胞后,与野生型相比,ibeT基因缺失突变株较多地滞留在溶酶体内;透射电镜结果进一步显示,ibeT基因缺失使大肠杆菌K1株逃逸ECV(含有大肠杆菌的囊泡)的能力发生了下降,继而使其在细胞浆内的复制减少．利用体外模拟的弱酸性环境,检测大肠杆菌菌体胞内的缓冲容量,发现ibeT基因缺失突变株菌体胞内的缓冲能力较野生型低．这些结果提示,在大肠杆菌K1株侵袭进入人脑微血管内皮细胞后,ibeT基因有利于大肠杆菌降解ECV膜,避免与溶酶体融合,进而促使大肠杆菌逃逸进入细胞浆并进行复制．     

金黄色葡萄球菌tst-1基因敲除菌株的构建

抽提金黄色葡萄球菌834菌株的基因组DNA,PCR克隆扩增tst-1及tst-1的上、下游基因,通过将tst-1上、下游基因分别重组到载体质粒pAULA中,形成同源重组质粒pAULA Δtst-1,将pAULA-Δtst-1电转入细菌内,进行同源重组,以PCR、Western blot鉴定tst-1基因敲除菌株无tst-1基因片段,且无TSST-1蛋白表达,表明已成功构建金黄色葡萄球菌tst-1基因的敲除菌株。     

苏云金芽胞杆菌杀线虫伴胞晶体蛋白基因cry6Aa的克隆与表达

通过对晶体蛋白N-末端氨基酸测序,设计简并探针,从对根结线虫高毒力苏云金芽胞杆菌YBT-1518菌株中克隆到1个含有杀线虫晶体蛋白基因的片段。序列测定表明该序列含有两个ORF(orf1和orf2),其中orf1与基因cry6Aa1同源性为98%,已在GenBank上登录(Acc.NO.AF499736),并被命名为cry6Aa2。将克隆的该片段克隆到穿梭载体pHT304上,并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株可形成米粒状伴胞晶体。生物测定表明,表达的毒素蛋白对北方根结线虫的LC₅₀为9.47μg/mL,毒力与出发菌株(10.74μg/mL)相当。     

核桃细菌性黑斑病菌rpfG基因的功能分析

[目的] 本研究旨在揭示核桃细菌性黑斑病菌（Xanthomonas arboricola pv.juglandis,Xaj） DW3F3中rpfG基因的生物学功能,从而为核桃细菌性黑斑病防治药剂的开发提供作用靶点。[方法] 以野油菜黄单胞菌（Xanthomonas campestris pv.campestris,Xcc）8004菌株以及水稻白叶枯病菌（Xanthomonas oryzae pv.oryzae,Xoo） PXO99^A的rpfG基因为模板序列,对Xaj野生型菌株DW3F3的基因组序列进行检索。利用同源重组技术,对Xaj中rpfG基因进行敲除,并用生物化学方法对基因缺失菌株的相关毒力因子、抗逆性进行检测。[结果] 通过同源比对,在XajDW3F3的基因组中发现了与XccrpfG、XoorpfG同源的基因,并成功获得rpfG的缺失突变株ΔrpfG。与野生型相比,突变株ΔrpfG的生物被膜形成能力仅为野生型XajDW3F3的44.58%;胞外多糖产量也由野生型的8.47 mg/mL降为5.23 mg/mL;ΔrpfG的絮凝活性增加,能使菌液变澄清;运动性实验显示ΔrpfG的运动直径比野生型增加了12.38%;胞外酶的分泌也发生了不同程度的改变,突变株分泌纤维素酶的能力极显著降低,淀粉酶活性有所提高,而分泌蛋白酶的能力未发生变化;此外rpfG缺失后,Xaj对逆境（盐、酸、SDS、硫酸铜）的耐受力降低。[结论] 结果表明rpfG基因能影响核桃细菌性黑斑病菌的致病相关性状,并赋予了细菌一定的抗逆性。     

阿特拉津降解菌Arthrobacter sp. AG1降解基因研究

菌株Arthrobacter sp. AG1能以4000 mg/L的阿特拉津（AT）为唯一碳源、氮源和能源生长。通过设计特异引物从AG1中扩增出阿特拉津氯水解酶基因trzN的全序列,该基因与已报道的trzN基因序列相似性为99%。AG1菌株中含有两个大于100kb的质粒,Southern杂交结果显示trzN和atzB基因均位于其中较大的一个质粒pAG1上。将AG1菌株在LB液体培养基中转接三代后,发现34%的细菌细胞丢失了降解活性,但却未发现丢失质粒,PCR扩增结果表明突变子丢失了trzN基因,但atzB和atzC基因未丢失,说明降解活性的缺失是trzN基因片段从质粒上丢失的结果,表明trzN基因在环境中存在水平转移现象,暗示菌株AG1中的阿特拉津降解基因是基因的水平转移重组的结果。     

Comparison of in-situ biodegrading abilities of Pseudomonas putida mutants: leuB− auxotroph,fliC− non-motility,and cheA− non-chemotaxis

Bioremediation of pollutants in natural environments is affected by many factors, such as bacterial survival, motility, and chemotaxis. However, these roles in in-situ biodegradation of organophosphorus pesticides have not been examined extensively. In this paper, a highly effective methyl-parathion (MP) degrading strain, Pseudomonas putida DLL-1, which also demonstrates motile ability and chemotactic response toward MP, was selected as the research material. A leuB^? auxotroph mutant A3-27 and fliC^? non-motility mutant a4-8 were first constructed by random insertion of the kanamycin gene into the chromosome of P. putida DLL-1 with the mini-transposon system. Biodegradation of MP in liquid medium and soil microcosms by A3-27, a4-8 and a previously constructed cheA^? non-chemotaxis mutant P. putida DAK were compared. The kinetic parameters for MP degradation were all similar in the well-mixed liquid systems. However, in soil microcosms, all the three mutants had lower degrading rates compared with wild-type P. putida DLL-1. The auxotroph mutant A3-27 had the lowest degrading rate and could only degrade 25.7–34.2% MP in 5 days, and the non-motility mutant a4-8 and non-chemotaxis mutant DAK could only degrade 53.5–68.1% and 64.3–85.7% MP, respectively. This paper emphasizes, for the first time, the use of non-auxotroph bacteria for efficient removal of organophosphorus pesticides in contaminated sites, and also points out the importance of select microorganisms with specific motile or chemotactic affinities in optimizing pesticide bioremediation.     

A histidine-kinase cheA gene of Pseudomonas pseudoalcaligens KF707 not only has a key role in chemotaxis but also affects biofilm formation and cell metabolism

A histidine-kinase cheA gene in Pseudomonas pseudoalcaligenes KF707 plays a central role in the regulation of metabolic responses as well as in chemotaxis. Non-chemotactic mutants harboring insertions into the cheA gene were screened for their ability to form biofilms in the Calgary biofilm device. Notably, ≥95% decrease in the number of cells attached to the polystyrene surface was observed in cheA mutants compared to the KF707 wild-type biofilm phenotype. The ability to form mature biofilms was restored to wild-type levels, providing functional copies of the KF707 cheA gene to the mutants. In addition, phenotype micro-arrays and proteomic analyses revealed that several basic metabolic activities and a few periplasmic binding proteins of cheA mutant cells differed compared to those of wild-type cells. These results are interpreted as evidence of a strong integration between chemotactic and metabolic pathways in the process of biofilm development by P. pseudoalcaligenes KF707.     

Bacterial chemotaxis to naphthalene desorbing from a nonaqueous liquid

Bacterial chemotaxis has the potential to increase the rate of degradation of chemoattractants, but its influence on degradation of hydrophobic attractants initially dissolved in a non-aqueous-phase liquid (NAPL) has not been examined. We studied the effect of chemotaxis by Pseudomonas putida G7 on naphthalene mass transfer and degradation in a system in which the naphthalene was dissolved in a model NAPL. Chemotaxis by wild-type P. putida G7 increased the rates of naphthalene desorption and degradation relative to rates observed with nonchemotactic and nonmotile mutant strains. While biodegradation alone influenced the rate of substrate desorption by increasing the concentration gradient against which desorption occurred, chemotaxis created an even steeper gradient as the cells accumulated near the NAPL source. The extent to which chemotaxis affected naphthalene desorption and degradation depended on the initial bacterial and naphthalene concentrations, reflecting the influences of these variables on concentration gradients and on the relative rates of mass transfer and biodegradation. The results of this study suggest that chemotaxis can substantially increase the rates of mass transfer and degradation of NAPL-associated hydrophobic pollutants.     

The smaller of two overlapping cheA gene products is not essential for chemotaxis in Escherichia coli.

The cheA locus of Escherichia coli encodes two similar proteins, CheAL (654 amino acids) and CheAS (557 amino acids), which are made by initiating translation from different in-frame start sites [start(L) and start(S)]. CheAL plays an essential role in chemotactic signaling. It autophosphorylates at a histidine residue (His-48) and then donates this phosphate to response regulator proteins that modulate flagellar rotation and sensory adaptation. CheAS lacks the first 97 amino acids of CheAL, including the phosphorylation site at His-48. Although it is unable to autophosphorylate, CheAS can form heterodimers with mutant CheAL subunits to restore kinase function and chemoreceptor control of autophosphorylation activity. To determine whether these or other activities of CheAS are important for chemotaxis, we constructed cheA lesions that abrogated CheAS expression. Mutants in which the CheAS start codon was changed from methionine to isoleucine (M98I) or glutamine (M98Q) retained chemotactic ability, ranging from 50% (M98Q) to 80% (M98I) of wild-type function. These partial defects could not be alleviated by supplying CheAS from a specialized transducing phage, indicating that the lesions in CheAL--not the lack of CheAS--were responsible for the reduced chemotactic ability. In other respects, the behavior of the M98I mutant was essentially normal. Its flagellar rotation pattern was indistinguishable from wild type, and it exhibited wild-type detection thresholds and peak positions in capillary chemotaxis assays. The lack of any substantive defect in this start(S) mutant argues that CheAS makes a negligible contribution to chemotactic ability in the laboratory. Whether it has functional significance in other settings remains to be seen.     

Evidence for plasmid-mediated chemotaxis of Pseudomonas putida towards naphthalene and salicylate

A naphthalene (Nap) and salicylate (Sal) degrading microorganism, Pseudomonas putida RKJ1, is chemotactic towards these compounds. This strain carries a 83 kb plasmid. A 25 kb EcoRI fragment of the plasmid contains the genes responsible for Nap degradation through Sal. RKJ5, the plasmid-cured derivative of RKJ1, is neither capable of degradation nor is chemotactic towards Nap or Sal. The recombinant plasmid pRKJ3, which contained a 25 kb EcoRI fragment, was transferred back into the plasmid-free wild-type strain RKJ5, and the transconjugant showed both degradation and chemotaxis. The recombinant plasmid pRKJ3 was also transferred into motile, plasmid-free P. putida KT2442. The resulting transconjugant (RKJ15) showed chemotaxis towards both Nap and Sal. Two mutant strains carrying deletions in pRKJ3 (in KT2442) with phenotypes Nap- Sal+ and Nap- Sal-, were also tested for chemotaxis. It was found that the Nap- Sal+ mutant strain showed chemotaxis towards Sal only, whereas the Nap- Sal- mutant strain is non-chemotactic towards both the compounds. These results suggest that the metabolism of Nap and Sal may be required for the chemotactic activity.     

茎瘤固氮根瘤菌ORS571中motA基因的功能分析

[目的] MotA是细菌的鞭毛马达蛋白,是跨膜质子通道的重要组成结构之一,在调控鞭毛运动中具有至关重要的作用。本研究探究了Azorhizobium caulinodans ORS571中鞭毛马达基因motA对菌株表型和植物互作的影响。[方法] 通过同源重组原理和三亲接合转移方法构建突变菌株∆motA,测定野生型与突变体在菌体生长、运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力的差异。[结果] 与野生型相比,突变体菌体生长没有明显差异,但其运动能力完全丧失,固氮、胞外多糖合成、生物膜形成及根系定殖能力减弱。[结论] MotA鞭毛马达蛋白对A.caulinodans ORS571的运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力均有调控作用。     

Diversity and chemotaxis of soil bacteria with antifungal activity against Fusarium wilt of banana

The chemotactic response of bacteria to root exudates plays an important role in the colonization of bacteria in the rhizosphere. In this study, 420 strains of antifungal bacteria against Fusarium oxysporum f. sp. cubense (Foc) were screened for chemotaxis based on a cheA molecular diagnostic method. A total of 124 strains with antifungal efficiencies of 27.26-67.14?% generated a characteristic band of cheA. The chemotaxis of 97 bacterial strains producing a cheA band was confirmed using the drop assay and swarm plate assay using catechol, p-hydroxybenzoic acid, salicylic acid, and asparagine as the attractants. A phylogenetic analysis based on restriction fragment length polymorphisms (RFLPs) and 16S rDNA sequences indicated that the 124 chemotactic antagonists of Foc were affiliated with 18 species of Paenibacillaceae, Bacillaceae, Streptomycineae, Enterobacteriaceae, and Pseudomonadaceae. The chemical composition of banana root exudates were analyzed by GC-MS, and 62 compounds, including alkanes, alkenes, naphthalenes, benzenes, and alcohols, were evaluated. Five representative antagonists of Foc showed 1.76- to 7.75-fold higher chemotactic responses than the control to seven compounds in banana root exudates, as determination by capillary assays.     

Mutants defective in bacterial chemotaxis show modified protein phosphorylation

To examine the correlation between CheA phosphorylation and bacterial chemotaxis, cheA mutations leading to defects in chemotaxis were mapped and characterized. Mutant CheA proteins were tested in vitro for phosphorylation and were grouped into four classes: nonphosphorylated, partially phosphorylated, phosphorylated but not dephosphorylated by CheB and CheY, and phosphorylated and dephosphorylated. Nearly all the mutants were found to be defective in an aspect of phosphorylation. Furthermore, the mutant phenotypes were found to cluster in different regions of the cheA gene. We suggest that the CheA protein has three functional domains: one for interaction with CheB and CheY, a second for regulating phosphorylation and controlling the stability of the protein, and a third for receiving input signals regulating CheA activity.     

细菌对环境污染物的趋化性及其在生物修复中的作用

细菌对有机化合物的降解能力是一种利用碳源和能源的优势,这种能力可以用来设计安全、有效和无二次污染的污染物的生物修复系统。趋化性是细菌适应外界化学环境变化而作出的行为反应,是一种寻找碳源和能源的优势。细菌的趋化性能够增强细菌在自然环境中的降解污染物的效果,细菌的趋化性与降解性之间的关系研究已经成为热点。介绍了细菌的趋化性的基本概念和趋化信号转导的机制,重点讨论了细菌对环境污染化合物的趋化性,从基因水平揭示了趋化性与降解性之间的紧密联系,认为趋化性可以有效地促进降解性细菌对污染物的生物修复作用。     

Bacterial chemotaxis to naphthalene

The chemotaxis of two pseudomonads, P. putida AZ (Naph+) and P. putida AZ (Naph-), differing in the ability to metabolize naphthalene was studied by the known capillary method of Adler and the densitometric method devised in our laboratory. The migration of P. putida AZ (Naph+) cells toward increasing levels of naphthalene was accompanied by the formation of a migrating front of converted naphthalene. P. putida AZ (Naph-) cells, too, exhibited positive chemotaxis to naphthalene, but they did not form the front of converted naphthalene. The analysis of experimental data in terms of a kinetic model of bacterial chemotaxis showed that the densitometric method is a potential tool for studying bacterial chemotaxis to hydrophobic organic substances.