Proteome investigation of the global regulatory role of sigma 54 in response to gentisate induction in Pseudomonas alcaligenes NCIMB 9867

Pseudomonas alcaligenes NCIMB 9867 (strain P25X) utilizes the gentisate pathway for the degradation of aromatic hydrocarbons. The gene encoding the alternative sigma (sigma) factor sigma(54), rpoN, was cloned from strain P25X and a rpoN knock-out strain, designated G54, was constructed by insertional inactivation with a kanamycin resistance gene cassette. The role of sigma(54) in the physiological response of P. alcaligenes P25X to gentisate induction was assessed by comparing the global protein expression profiles of the wild-type P25X with the rpoN mutant strain G54. Analysis of two-dimensional polyacrylamide gel electrophoresis gels showed that 39 out of 355 prominent protein spots exhibited differential expression as a result of the insertional inactivation of rpoN. Identification of the protein spots by matrix-assisted laser desorption/ionization-time of flight/time of flight revealed a wide diversity of proteins that are affected by the sigma(54) mutation, the largest group being proteins that are involved in carbon metabolism. The strictly inducible gentisate 1,2-dioxygenase, one of two isofunctional copies of the key enzyme in the gentisate pathway, and enzymes of the TCA cycle, pyruvate metabolism and gluconeogenesis were part of this group. Other proteins that are part of the sigma(54) regulon include enzymes implicated in nitrogen metabolism, transport proteins, stress-response proteins and proteins involved in cell motility. The results of this study showed that sigma(54) plays a global regulatory role in the expression of a wide variety of genes in P. alcaligenes, including the wild-type response to the presence of the aromatic inducer, gentisate.     

Phytate utilization of maize mediated by different nitrogen forms in a plant–arbuscular mycorrhizal fungus–phosphate-solubilizing bacterium system

A pot experiment was conducted to investigate the organic phosphorus (P) (phytate) utilization of Zea mays L. with different nitrogen (N) forms (NH₄⁺ and NO₃^?) when both arbuscular mycorrhizal (AM) fungus (Funelliformis mosseae) and phosphate-solubilizing bacterium (PSB, Pseudomonas alcaligenes) are present. The soil was supplied with either KNO₃ or (NH₄)₂SO₄ (200 mg kg^?1 N) with or without phytin (75 mg P kg^?1). Results showed that the application of NH₄⁺ to the soil in a plant–AM fungus–PSB system decreased rhizosphere pH and increased phosphatase activity. It also enhanced the mineralization rate of phytin, which resulted in the release of more inorganic P. The application of NO₃^? promoted mycorrhizal colonization and hyphal length density in the soil. The inorganic P in the hyphosphere decreased, but more P was transferred to the plant through the mycorrhizal hyphae. Hence, in addition, the application of the two different N forms did not significantly alter the content of plant P. The plant supplied with different N fertilizers acquired P through different mechanisms associated with other microbes. NH₄⁺ application promoted phytin mineralization by decreasing soil pH, whereas NO₃^? application increased inorganic P uptake by strengthening the mycorrhizal pathway.     

重金属胁迫对Pseudomonas alcaligenes LH7抗生素抗性的影响

从广州某养猪场废水处理系统中筛选出1株优势菌Pseudomonas alcaligenes LH7。为了研究重金属胁迫对细菌抗生素抗性响应的影响,采用琼脂稀释法和K-B纸片扩散法,测定了重金属(Cu2+、Zn2+、Cr6+)的最小抑制浓度(MIC),及不同重金属种类和浓度胁迫下,四种抗生素(红霉素、阿莫西林、头孢拉定、四环素)的抑菌圈直径。结果表明:菌体对Cu2+、Zn2+、Cr6+的MIC分别为125、125、100 mg/L,并且具有四环素、阿莫西林、红霉素和头孢拉定多重抗性。重金属与抗生素之间的交互作用对细菌的抗性有显著影响(P0.05)。重金属和抗生素间的交互作用随重金属种类和浓度的不同而改变,可分为三类:低浓度重金属与抗生素共存时表现为协同抗性,高浓度时则表现为协同杀菌,如Cr6+或Zn2+与红霉素,Cu2+与头孢拉定;低浓度重金属与抗生素共存时表现为协同杀菌,高浓度时则表现为协同抗性,如Cr6+或Zn2+与阿莫西林;只与共存重金属种类相关的抗性组合有Cu2+与四环素或阿莫西林或红霉素,Cr6+与头孢拉定。环境中重金属离子的共存将改变抗生素污染物的生态危害和环境行为,并最终影响对应的污染防治技术的开发和应用。     

Genetic system in Pseudomonas alcaligenes NCIB 9867

Abstract Genetic transfer of both auxotrophic and catabolic markers was detected in filter matings of mutant strains of Pseudomonas alcaligenes NCIB 9867. Bidirectional transfer of auxotrophic markers was demonstrated in most of the crosses. Strains could either act as donors or recipients. Polarized transfer of auxotrophic markers was observed in some crosses. There was low co-inheritance of both 2,5X⁺ catabolic marker and auxotrophic markers. No evidence could be presented indicating the involvement of the indigenous 33-kb plasmid in the genetic transfer process. Partial sensitivity to DNase was observed in some of the crosses. Maximum frequency of recombinant formation obtained with mating cultures from stationary growth phase suggested an influence of physiological states on genetic transfer. As transfer did not appear to be due to classical transformation or to be plasmid-mediated, the likely mechanism could involve the release of DNA upon intimate cell-to-cell contact. The gene transfer system may be useful for linkage analysis of closely linked genes.     

产碱假单胞菌脂肪酶的克隆表达及酶学性质研究

【目的】克隆产碱假单胞菌的脂肪酶基因,实现其在大肠杆菌中异源表达并进行酶学性质研究。【方法】通过基因文库构建和PCR,获得脂肪酶基因,并以pET30a(+)为表达载体、E.coli BL21(DE3)为宿主菌,在大肠杆菌中进行异源表达,表达产物经HisTrapTM亲和层析柱纯化后进行酶学性质研究。【结果】从产碱假单胞菌中克隆得到一个脂肪酶基因,大小为1 575 bp(GenBank登录号为JN674069)。该酶分子量为55 kD,最适底物为p-NPO,最适反应温度和pH分别为35°C、pH 9.0。重组酶经1 mmol/L的Cu2+处理30 min可使酶活提高至156%。在最适反应条件下重组酶的比活力为275 U/mg,Km和Vmax分别为80μmol/L和290 mmol/(min.g protein)。【结论】产碱假单胞菌脂肪酶基因的克隆与表达不仅积累了脂肪酶基因的资源,并为其在手性拆分中的应用奠定基础。     

海洋低温蛋白酶菌株选育及发酵培养基的研究(I)

以渤海和黄海分离出400多株在低温条件下生长良好的菌株为出发菌株,利用常规筛选方法选出2株低温蛋白酶产生菌(Pseudom onas alcaligenes)。经UV、DES、NTG、EMS、L iC l单独及复合诱变,选育出一株(Pa040523)蛋白酶高产突变株。通过单因素实验,确定了Pa040523菌株蛋白酶发酵培养基为:玉米淀粉糖1.8%,尿素0.6%,磷酸氢二钾0.6%,磷酸二氢钾0.3%。该突变株低温蛋白酶产量为940.8 U/mg。     

海洋低温蛋白酶菌株选育及发酵培养基的研究(Ⅰ)

以渤海和黄海分离出400多株在低温条件下生长良好的菌株为出发菌株,利用常规筛选方法选出2株低温蛋白酶产生菌（Pseudorrtortas alcaligenes）。经UV、DES、NTG、EMS、LiCl单独及复合诱变,选育出一株（Pa040523）蛋白酶高产突变株。通过单因素实验,确定了Pa040523菌株蛋白酶发酵培养基为：玉米淀粉糖1．8％,尿素0．6％,磷酸氢二钾0．6％,磷酸二氖钾0．3％。该突变株低温蛋白酶产量为940．8U／mg。     

立体选择性脂肪酶的筛选及其在薄荷醇拆分中的应用

采用双平板透明圈方法,筛选得到1株能够选择性水解L-薄荷醇丙酸酯的微生物。通过16sDNA测序,鉴定为产碱假单胞菌Pseudomonas alcaligenes。该菌所产脂肪酶可水解4对薄荷醇丙酸酯,在35℃时绝对转化率达到50％时,表现出较高的对映体过量值（e.e.p〉99％）与非对映体过量值（d．e．p〉90％）。这一结果表明,产碱假单胞菌脂肪酶是具有工业应用前景的生物催化剂。     

海洋低温蛋白酶菌株发酵条件的研究(Ⅱ)

建立了海洋低温蛋白酶菌株(Pseudomonasalcaligenes简写为Pa040523)发酵最适pH值、温度、时间、接种量、通气量分别为5·5、12℃、72h、7%、170mL;在最适发酵条件下,Pa040523菌株50L发酵罐中低温蛋白酶活性为1,976·2U/mg。