Construction of ice nucleation active <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> for control of insect pests

Ice nucleation active(INA)bacteria are the most potent heterogeneous ice nuclei in nature,which have become an important biological resource for diverse applications.Many researches have proved that INA bacteria can raise the supercooling points(SCPs)of insect pests,then reduce their cold hardiness.However,INA bacteria‘s inefficient colonization on the surface or in the guts of insects,and the high incidence of frost injury induced by their release hampered the application of INA bacteria in controlling insect pests in agricultural fields.In this study,we constructed a recombinant plasmid mob-Tn5-iceA with the ability of broad-host-range conjugal mobilization and integration of the ina gene of icaA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition.In addition,Ent.cloacae strains stably carrying iceA and expressing high ice nucleation activity(INA),even in the absence of antibiotic pressure,were constructed through conjugal mobilization and Tn5 transposition.Ent,cloacae strains have benn reported to be able to efficently colonize in the guts of insects,but have weak plant epiphytic ability.Therefore,these transgenic Ent.Cloacae may be promising candidates for control of insect pests in agricultural fields.     

Cloning and analysis of the antagonistic related genes of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> B8

To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8,Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonistic character are isolated. Tagging with kanr gene on Tn5,an antagonistic related DNA fragment, the F fragment, right of the Tn5 insertion site is cloned in a plasmid named pTLF,from one of the mutant strains B8F. The 733 bp F fragment is then sequenced after subcloning. Genomic DNA of the original B8 strain is isolated, digested with Pst I and ligated to Pst I cassette. DNA fragments left and right of the F fragment are amplified from the Pst I cassette library using cassette primer and specific primers designed according to known sequence. 1106 bp sequence left of the F fragment and 664bp sequence right of the F fragment are finally obtained. Bioinformatics analysis shows that the contig assembled from the sequences of the cloned antagonistic related DNA fragments of B8 encodes three ORFs and is homogeneous to admM,admN and admO genes of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The ORF, named anrF gene which encodes a polyketide synthase, knocked out by Tn5 insertion, is a homology of admM and the insertion site of Tn5 is at 214 bp upstream of the stop codon. It is concluded that the anrF gene is a gene related to the antagonistic activity of E. cloacae B8, and speculated that the antagonistic substance produced by B8 is an andrimid.     

产超广谱-内酰胺酶和AmpC酶阴沟肠杆菌的表型检测及耐药性分析

目的:监测江汉大学附属医院临床标本中分离出的阴沟肠杆菌产超广谱-内酰胺酶(ESBLs)和AmpC-内酰胺酶的状况及其耐药性,为临床治疗提供依据.方法:用美国临床实验室标准化委员会(NCCLS)推荐的确证实验进行ESBLs的检测,用表型筛选法进行AmpC酶的检测;用纸片扩散法进行药敏试验.结果:从75株阴沟肠杆菌中检出ESBLs阳性9株(12.0%),AmpC酶阳性11株(14.7%);产ESBLs株对亚胺培南、头孢吡肟、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦、丁胺卡那等药物的耐药率较低,产AmpC酶株对亚胺培南、头孢吡肟、丁胺卡那等药物的耐药率较低.结论:本院分离的阴沟肠杆菌产ESBLs和AmpC酶情况尚好;临床治疗应在药敏试验的基础上,针对不同的产酶株,选用亚胺培南、头孢吡肟和丁胺卡那等药物.     

Crystal structure of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> 908R class C β-lactamase bound to iodo-acetamido-phenyl boronic acid,a transition-state analogue

The structures of the class C -lactamase from Enterobacter cloacae 908R alone and in complex with a boronic acid transition-state analogue were determined by X-ray crystallography at 2.1 and 2.3 Å, respectively. The structure of the enzyme resembles those of other class C -lactamases. The structure of the complex with the transition-state analogue, iodo-acetamido-phenyl boronic acid, shows that the inhibitor is covalently bound to the active-site serine (Ser64). Binding of the inhibitor within the active site is compared with previously determined structures of complexes with other class C enzymes. The structure of the boronic acid adduct indicates ways to improve the affinity of this class of inhibitors. This structure of 908R class C -lactamase in complex with a transition-state analogue provides further insights into the mechanism of action of these hydrolases.Received 16 May 2003; accepted 4 June 2003     

一株产生几丁质酶细菌的分离与鉴定

作者从草原毛虫的尸体中，分离到一株能产生较高活性几丁质酶的细菌，该菌所产生的几丁质酶可抑制几种植物病原真菌的生长，对防治蝗虫具增效作用。经鉴定确认，该菌属肠杆菌属，是产气肠杆菌的一个变种。     

医用脲酶菌的分离与选育

从尿毒症病人肠道中分离出一株脲酶菌,经鉴定为Enterobactergergoviae.纯化菌株经驯化后能以尿素为惟一氮源.经紫外线及硫酸二乙酯诱变处理后,其脲酶活性提高约180%.用非降解性高分子材料聚乙烯醇将该菌包埋在具有半透膜性质的微胶囊内,体外模拟尿毒症病人血清试验表明,250mg湿菌在8h内清除尿素约50mg.     

双稠哌啶类生物碱对5个环境细菌菌株的抑制作用

测定了槐定碱、槐胺碱、苦参碱、野靛碱、氧化苦参碱和苦豆草总生物碱对大肠杆菌、产气杆菌、变形杆菌、枯草杆菌、白色葡萄球菌 (即表皮葡萄球菌 )的最低抑菌浓度 (MIC)。结果表明 5种生物碱和苦豆草总生物碱对 5个环境细菌菌株的生长均产生了明显的抑制作用。5种生物碱对 5种环境细菌的最低抑菌浓度除了野靛碱对变形杆菌为 5× 1 0 - 3 mol/L ,其它均在 2× 1 0 - 2 ～ 5× 1 0 - 2 mol/L之间。然而苦豆草总生物碱对 5种环境细菌的最低抑菌浓度却明显较 5种生物碱的为低 ,其中对大肠杆菌和枯草杆菌的最低抑菌浓度达到 5× 1 0 - 3 mol/L。结合双稠哌啶类生物碱在植物中的含量和分布 ,讨论了双稠哌啶类生物碱在开发防治农林细菌病害及医药产品方面的应用价值和在植物化学生态学中的防御作用。     

阴沟肠杆菌(Enterbacter Cloacae)糖蛋白活性组分抑瘤效应的研究

从水体中分离到阴沟肠杆菌（Enterbacter Cloacae)，利用该菌制剂对荷肉瘤S180（实体型）小鼠有抑瘤作用，该菌大量培养后，将菌体超声波破碎，十六烷基三甲基溴化铵（Cetyltrimethylaminonium bromide,CTAB)沉淀，乙醇沉淀得粗提物，再经DEAE纤维素柱层析，分步收集，聚乙二醇浓缩得F1、F2、F3和F4 4种主要组分，形态观察法体外抑瘤实验证明F2、F3组分对肝癌QGY7703细胞有明显杀伤作用，F3作用显著，MTT法检测F3对肝癌QGY7703，肺腺癌A549，胃癌KatoⅢ，肠癌SW1116瘤细胞株均有明显杀伤作用，其IC50分别为0．024，0．0123，0．0035，0.012mg/mL。体外抑瘤试验证明F3对小鼠无急性毒作用，并且对小鼠Lewi肺癌有抑制作用，蛋白电泳，红外和核磁共振表明F3是含木糖成分的糖蛋白，其相对分子质量为38ku.     

产壳聚糖酶的肠杆菌分离鉴定及其培养特性研究

针对筛选的一株产壳聚糖酶菌株进行了鉴定和培养特性研究．首先对产酶菌株进行鉴别培养基培养、显微镜检并结合生理生化与16SrDNA分子鉴定以确定菌属来源,后对菌株产壳聚糖酶的碳源、氮源、金属盐和诱导物进行筛选优化．结果表明：产酶菌株为产气肠杆菌,最佳培养碳源为葡萄糖,最佳氮源为氯化铵和酵母粉,MnSO4,MgSO4,KCl和NaCl对产酶有积极作用．该菌来源的壳聚糖酶为诱导酶,最佳诱导物为粉末壳聚糖,壳聚糖酶活性为1．58U／mL．     

Anaerobic Biodecolorization of Remazol Dye by a Strain of Enterobacter Isolated from Textile Sludge

Enterobacter GY-1 gained from a lab scale anaerobicanoxic-aerobic (A2O) process treating textile effluent can effectively decolorize remazol dye.Under anaerobic condition,91％ of this remazol dye is decolonzed,which is much higher than under aerobic condition.The optimum pH is 7 and the optimum temperature is 35 ℃ for the rcmazol dye decolorization by GY-1.Anthraquinone dyes and monoazo dyes are decolorized more efficiently by GY-1 than other dyes tested decolorized.GY-1 can not decolorize the remazol dye when it is the sole carbon source.Microbial cometabolism and decolorization of dye take place in the presence of some other carbon source called cometabolic substrate.The cometabolic substrate can be glucose,starch,peptone,beef extract,etc.The change of molecular structure of the dye before and after decolorized by GY-1 is studied by UV-Vis absorption spectrum.The results indicate that its molecular structure is changed evidently.