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。     

云南滇池富磷区趋化性细菌的系统多样性

从云南滇池富磷区100份土样中筛选解磷细菌(PSB),通过组氨酸激酶编码基因(cheA)筛选趋化性PSB,并通过软琼脂平板法验证其趋化性;利用钼蓝比色法测定PSB对磷酸三钙的溶解能力;基于16S rRNA基因序列分析趋化性PSB的系统亲缘关系.结果表明： 分离到的145株PSB的溶磷圈直径在0.5～2 cm,其中37株为趋化性PSB.该37株PSB对供试的4种趋化底物均具有趋化性,而且对磷酸三钙均具有解磷活性.基于16S rRNA基因序列的系统发育分析显示,这37株趋化性PSB分属于10属,共17种细菌,其中假单胞菌属种类最多(5种9株),肠杆菌属次之(3种8株),芽孢杆菌属尽管只分离到1个种(Bacillus aryabhattai),但共分离到9个菌株.     

空肠弯曲菌cheA基因插入突变及其对小鼠空肠定植能力的影响

摘要：【目的】构建空肠弯曲菌（Campylobacter jejuni）cheA基因插入突变株,了解CheA与空肠弯曲菌小鼠体内定植的相关性。【方法】运用同源重组的原理构建空肠弯曲菌cheA基因突变株,采用PCR技术检测cheA突变株的构建情况。通过基因回补试验构建cheA基因回补株。空肠弯曲菌感染小鼠,运用小鼠空肠内容物涂板计数的方法检测cheA突变株、cheA基因回补株和野生株定植小鼠能力的差异。【结果】PCR检测显示成功构建cheA基因突变株。空肠弯曲菌cheA基因突变株定植小鼠空肠的数量明显减少（P<0.05）;cheA基因回补株定植小鼠空肠的数量跟野生株相比无明显差异（P>0.05）。【结论】本研究成功构建cheA基因突变株及其回补株。cheA基因可能参与空肠弯曲菌在小鼠体内定植的过程。     

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gacAXooc。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gacAXooc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。     

茎瘤固氮根瘤菌ORS571鞭毛马达基因fliN与fliM的功能分析

【目的】考察茎瘤固氮根瘤菌ORS571中鞭毛马达蛋白FliN、FliM的编码基因分别缺失的突变体表型,初步探究其功能机理。【方法】本研究采用同源重组和三亲本接合转移的方法构建突变体,测定野生型及突变株的生长曲线、趋化性、胞外多糖的分泌、生物膜的形成及细胞絮凝等表型。【结果】三种菌株的生长速率基本无差,与野生型菌株相比突变株鞭毛结构丧失,趋化能力、分泌的胞外多糖和生物膜形成能力均下降,但相同时间内细胞絮凝程度比野生型明显。【结论】实验表明,鞭毛基因fliN、fliM对茎瘤固氮根瘤菌ORS571鞭毛的形成、趋化运动、胞外多糖的分泌、生物膜的形成及细胞絮凝能力等均有调控作用。     

几株新鞘氨醇杆菌的趋化性及其相关基因组成特点

【目的】选择了几株隶属于新鞘氨醇杆菌属(Novosphingobium)的有和没有多环芳烃(PAHs)降解能力的细菌,在基因组水平比较分析它们的趋化通路,并探究其中一些菌株对芳香化合物和三羧酸(TCA)循环中间代谢产物的趋化性。【方法】通过基因组的比较分析,研究了几株新鞘氨醇杆菌的趋化蛋白组成和趋化基因分布,并采用滴定法和游动平板法检测了相关菌对芳香族化合物和TCA循环中间代谢产物的趋化性。【结果】N.pentaromativorans US6-1、N.pentaromativorans F2、Novosphingobium indicum K13、Novosphingobium stygium DSM 12445、Novosphingobium sp.C2AC等5株新鞘氨醇杆菌对芳香族化合物和TCA循环中间代谢物具有不同程度的趋化性;所选的7株已完成基因组测序的新鞘氨醇杆菌N.pentaromativorans F2、N.pentaromativorans US6-1、Novosphingobium sp.PP1Y、Novosphingobium sp.AP12、Novosphingobium sp.Rr 2-17,Novosphingobium sp.B-7和Novosphingobium sp.DSM 19370均含有趋化蛋白MCP、CheW、CheA、CheB、CheR和CheY,且亲缘关系最近的US6-1、F2和PP1Y 3株菌的che基因簇中的基因排列一致,为che W-Y-D-B-R-A-(X);新鞘氨醇杆菌的趋化系统属Fla类型。【结论】几株新鞘氨醇杆菌都具有较为完整的趋化通路,且对多种芳烃及其代谢物的趋化性各不相同,其中菌株US6-1趋化现象最明显。     

茎瘤固氮根瘤菌甲基化受体蛋白Tlp1的功能

【目的】考察茎瘤固氮根瘤菌中趋化基因簇上游的受体蛋白Tlp1编码基因的突变表型,初步探究其功能机理。【方法】利用同源重组和三亲本接合转移的方法构建突变株,在TY培养基中测定生长情况,半固体平板法观察趋化圈,刚果红固体培养基观察胞外多糖和次生代谢产物的分泌,乙炔还原法测定菌株的固氮酶活性。【结果】与野生型菌株相比,tlp1突变株的生长速率没有影响。在以甘油为碳源的L3半固体平板上突变株的趋化圈变小,其回补菌株能部分回补趋化能力。突变株的胞外多糖分泌与野生型没有区别,但其次生代谢产物黑色素出现的时间比野生型稍早。在固氮酶活性测定中,发现突变株酶活性明显比野生型降低,回补菌株能够部分回补。【结论】茎瘤固氮根瘤菌Tlp1蛋白对甘油表现出一定的趋化能力,并且影响细菌的次生代谢产物和固氮能力。     

代谢工程法强化恶臭假单胞菌利用木质素积累PHA的能力

采用代谢工程方法强化恶臭假单胞菌利用木质素积累PHA的能力,为提高木质素转化效率和PHA的高效、低成本生产提供研究思路和技术基础。将PHA解旋酶编码基因phaZ的上下游片段插入pK18mobsacB,构建了敲除载体pK18PHAC1C2;将来源于R. jostii RHA1的过氧化物酶编码基因dypB插入pVLT33,构建了表达载体pVLTpelBDYPB。以P. putida QSR1为出发菌株,利用同源重组的方法敲除了木质素解旋酶编码基因phaZ;进而通过异源表达的方法在该突变株中引入过氧化物酶DyPB;分析各基因修饰菌株的生长情况和PHA积累量。成功构建了phaZ基因缺失突变株P. putida QSRZ6和DyPB表达菌株P. putida QSRZ6B。在限制氮源的情况下,以葡萄糖为碳源培养48 h,与出发菌株QSR1相比,QSRZ6的生长量和PHA积累量分别提高了29%和80%。在以木质素为碳源积累PHA的过程中,与QSR1相比,QSRZ6的生长量和PHA积累量分别提高了48%和182%;与QSRZ6和QSR1相比,QSRZ6B的PHA积累量分别提高了13%和218%,达到了140 mg/L。phaZ基因的缺失和异源表达过氧化物酶使菌株利用木质素积累PHA的能力得到增强,表明代谢工程是一种有效的调控木质素转化和PHA积累的有效方法。     

尼古丁降解菌SCUEC1菌株的分离及其降解基因

【目的】分离并鉴定1株具有尼古丁降解能力的细菌,研究其尼古丁降解特性并对其降解基因进行分析,为尼古丁微生物降解提供基础。【方法】从烟草种植地土壤中分离1株具有尼古丁降解能力的细菌,通过16S r RNA基因和生理生化特性对该菌株进行鉴定,检测该菌株尼古丁降解率与生长量的关系,并进一步对该菌株进行尼古丁浓度耐受性测定,采用高通量测序技术对菌株进行全基因组测序,BLAST比对分析尼古丁降解相关基因。【结果】筛选到1株具有尼古丁降解能力的细菌,经鉴定命名为根癌土壤杆菌(Agrobacterium tumerficience)SCUEC1菌株,根癌土壤杆菌SCUEC1菌株尼古丁降解率可达到94.81%,该菌株在尼古丁浓度为0.50–5.00 g/L范围内生长良好且有较高的尼古丁降解能力。对根癌土壤杆菌SCUEC1菌株全基因组序列进行BLAST比对分析,推测该菌株的尼古丁降解代谢途径与中间苍白杆菌SYJ1菌株的尼古丁降解途径相似。【结论】本研究揭示了Agrobacterium tumerficienceSCUEC1菌株具备尼古丁降解特性,初步推测出尼古丁降解相关基因和降解代谢途径,为尼古丁微生物降解提供基础。     

假单胞菌菌株CTN-3对百菌清污染土壤的生物修复

百菌清被美国环境保护署列为优先控制污染物,利用微生物的降解作用修复被污染的土壤、清除环境中的污染物等具有重要的现实意义.假单胞菌(Pseudomonas sp.)菌株CTN-3是一株从污染土壤中分离得到的百菌清降解菌,考察了其在实验室条件下对百菌清污染土壤的生物修复能力及其影响因素.结果表明:降解菌株在灭菌土壤中的降解效果略好于未灭菌土壤;在外源添加降解菌106 CFU·g-1、温度15 ～ 30℃和pH5.8～8.3条件下,该菌株能有效降解土壤中10 ～200 mg·kg-1的百菌清.菌株CTN-3在百菌清污染土壤的生物修复中具有良好的应用前景.     

Control of bacterial chemotaxis

Bacterial chemotaxis, which has been extensively studied for three decades, is the most prominent model system for signal transduction in bacteria. Chemotaxis is achieved by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein, CheY. This protein is activated by a stimulus-dependent phosphorylation mediated by an autophosphorylatable kinase (CheA) whose activity is controlled by chemoreceptors. Upon phosphorylation, CheY dissociates from its kinase, binds to the switch at the base of the flagellar motor, and changes the motor rotation from the default direction (counter-clockwise) to clockwise. Phosphorylation may also be involved in terminating the response. Phosphorylated CheY binds to the phosphatase CheZ and modulates its oligomeric state and thereby its dephosphorylating activity. Thus CheY phosphorylation appears to be involved in controlling both the excitation and adaptation mechanisms of bacterial chemotaxis. Additional control sites might be involved in bacterial chemotaxis, e.g. lateral control at the receptor level, control at the motor level, or control by metabolites that link central metabolism with chemotaxis.     

Inhibition of oyster drill chemotaxis

A bioassay was developed by Rittschof el al. (1983) to examinedistance chemoreception in the predatory marine gastropod, Urosalpinxcinerea. This bioassay was used to test the effect of a senesof low mol. wt. organics on the ability of newly hatched oysterdrills to detect a prey odor released from barnacles, Balanusbalanoides. Two series of low mol. wt. organics were testedusing methanol as the reference compound. In one series, R-OH,the carbon chain length was varied from 1 to 4. In the secondseries, CH³-R, the chain length was held constant while thefunctional group, R, was varied. When these compounds were presentin the rnM range, they inhibited the creeping response of oysterdrills towards barnacle prey odor. In the CH₃-R series, inhibitionincreased in the following order: sodium acetate > ethylacetate > acetonitnle > methanol; and, in the alcoholseries C₁ to C₄, inhibition increased with increasing chainlength. No creeping response was observed when these compoundswere tested in the absence of prey odor.     

Systems biology of bacterial chemotaxis

Motile bacteria regulate chemotaxis through a highly conserved chemosensory signal-transduction system. System-wide analyses and mathematical modeling are facilitated by extensive experimental observations regarding bacterial chemotaxis proteins, including biochemical parameters, protein structures and protein-protein interaction maps. Thousands of signaling and regulatory chemotaxis proteins within a bacteria cell form a highly interconnected network through distinct protein-protein interactions. A bacterial cell is able to respond to multiple stimuli through a collection of chemoreceptors with different sensory modalities, which interact to affect the cooperativity and sensitivity of the chemotaxis response. The robustness or insensitivity of the chemotaxis system to perturbations in biochemical parameters is a product of the system's hierarchical network architecture.     

Bleb-driven chemotaxis of Dictyostelium cells

Blebs and F-actin–driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up-gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function is impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but involving PI3-kinase and downstream PH domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase.     

Three-dimensional simulation of obstacle-mediated chemotaxis

Amoeboid cells exhibit a highly dynamic motion that can be directed by external chemical signals, through the process of chemotaxis. Here, we propose a three-dimensional model for chemotactic motion of amoeboid cells. We account for the interactions between the extracellular substances, the membrane-bound proteins, and the cytosolic components involved in the signaling pathway that originates cell motility. We show two- and three-dimensional simulations of cell migration on planar substrates, flat surfaces with obstacles, and fibrous networks. The results show that our model reproduces the main features of chemotactic amoeboid motion. Our simulations unveil a complicated interplay between the geometry of the cell’s environment and the chemoattractant dynamics that tightly regulates cell motion. The model opens new opportunities to simulate the interactions between extra- and intra-cellular compounds mediated by the matrix geometry.     

Bioavailability of pollutants and chemotaxis

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Highlights► The exposure of bacteria to pollutants induces chemoattraction or chemorepellence. ► Pollutant chemoreceptors so far identified are present on plasmids. ► Chemotaxis increases pollutant bioavailability and enhances biodegradation rates. ► Chemotaxis may improve microbial inoculants designed just on catabolic potential.     

Two mechanisms of chemotaxis inParamecium

Summary Paramecia show chemotaxis, that is, they accumulate in or disperse from the vicinity of chemicals. This study examines both the avoiding reactions (abrupt random changes of swimming direction) and velocities of normal and mutant paramecia in attractants and repellents and shows that the animals accumulate or disperse either by changing the frequency of avoiding reactions or by changing swimming velocity. Mutations or conditions that eliminate avoiding reactions abolish the chemotaxis response to chemicals that cause accumulation or dispersal by modulation of frequency of avoiding reactions but not the response to chemicals that cause chemotaxis by modulation of velocity.The current knowledge of the bioelectric control of the swimming behavior inParamecium and observations of mutants defective in bioelectric control and in chemotaxis are used to develop a hypothesis for membrane potential control of chemotaxis: attractants that require the avoiding reaction slightly hyperpolarize the membrane; repellents that require the avoiding reaction slightly depolarize the membrane; repellents that cause chemitaxis by modulation of velocity strongly hyperpolarize the membrane.I am grateful to D. Kusher and P. Foletta for their technical assistance, to C. Kung and E. Orias for support and discussion of this work, to H. Machemer and M. Levandowsky for stimulating discussions, and to B. Diehn for suggestion of the modified assay. This work was supported in part by Public Health Service Grant F32 NSO5587 to JVH and NSF GB-3164X and PHS GM-19406 to C. Kung.     

Methyl group turnover on methyl-accepting chemotaxis proteins during chemotaxis by Bacillus subtilis

The addition of attractant to Bacillus subtilis briefly exposed to radioactive methionine causes an increase of labeling of the methyl-accepting chemotaxis proteins. The addition of attractant to cells radiolabeled for longer times shows no change in the extent of methylation. Therefore, the increase in labeling for the briefly labeled cells is due to an increased turnover of methyl groups caused by attractant. All amino acids gave enhanced turnover. This turnover lasted for a prolonged time, probably spanning the period of smooth swimming caused by the attractant addition. Repellent did not affect the turnover when added alone or simultaneously with attractant. Thus, for amino acid attractants, the turnover is probably the excitatory signal, which is seen to extend long into or throughout the adaptation period, not just at the start of it.