萘降解质粒pND6-1中萘趋化基因nahY的克隆和核苷酸序列分析

假单胞菌（Pseudomonas）ND6菌株的萘降解基因位于102kb的质粒pND6-1上。以pUC18质粒为载体，制备了含有1.5-3.0kbpND6-1DNA随机片段的基因文库。通过DNA测序和DNA序列的同源性分析，从基因文库中筛选出含有萘趋化基因nahY的克隆。NahY基因的大小为1617bp，编码的萘趋化蛋白由538个氨基酸组成，与假单胞菌G7菌株的nahY基因相比，核苷酸序列的同源性是96.7％，编码的氨基酸序列的同源性是95％。图4参7     

假单胞菌表达载体pYMB03的构建与性能分析

恶臭假单胞菌(Pseudomonas putida)是具有强抗逆性能的环境优势菌,构建其质粒表达载体有着明显的应用潜力.将恶臭假单胞菌AB92019菌株中肽聚糖相关脂蛋白编码基因的启动子PoprL和质粒载体pTrcHis-B的多克隆位点片段插入到质粒载体pUCPl8的EcoRL/HInIII位点,获得了重组载体pYMB03.用绿色荧光基因gfp作为标记进行外源蛋白表达的结果表明,该载体能分别在恶臭假单胞菌AB92019菌株和大肠杆菌DH5a菌株中,由启动子PoprL启动组成型表达GFP蛋白并使细胞产生可见荧光.经SDS-PAGE验证,所产生的GFP蛋白分别占细胞总蛋白的12.5%和5.O%.重组菌株YMB001中GFP表达量与菌体培养时间有关,在稳定期后期其相对荧光强度达到最大值(D600nm=l.0),但与培养温度未见相父性.对携带该载体的2株重组恶臭假单胞菌7次168 h继代培养测定,载体pYMB03的稳定性均为100%.     

苯甲酸类化合物的微生物降解研究

用富集培养法从工业污水中分离到5个能以苯甲酸为唯一碳源和能源而生长的细苗菌株：不动杆菌（Acinetobactersp.）BJ1,无色杆菌（Achromobactersp.）BY1,假单胞苗（Pseudomonasspp.）SJ1、SY1和SH1、测定了这5个菌株的底物特异性和抗菌素抗性其中BJ1菌株含有1个大质粒和2个小质位在最适培养条件下BJ1菌株对苯甲酸的降解率达98％以上．     

恶臭假单胞菌DLL-E4对硝基苯酚降解途径关键基因pnpC的突变分析

通过同源重组成功地敲除了恶臭假单胞菌(Pseudomonas putida)DLL-E4菌株的偏三苯酚1,2-双加氧酶基因(pnpC).pnpC插入失活菌株DLL-△pnpCl失去了降解对硝基苯酚(PNP)和对苯二酚(HQ)的能力,而pnpC敲除菌株DLL-△pnpC恢复了利用PNP和HQ的能力,但是降解速率降低.在不同硫酸铵分级梯度下PNP和邻苯二酚分别诱导的DLL-E4和DLL-△pnpC粗酶液对邻苯二酚的降解活性存在明显差异,表明恶臭假单胞菌DLL-E4中除pnpC参与PNP降解代谢过程外,还存在另一个双加氧酶替代了敲除的pnpC的功能,使得DLL-△pnpC代谢PNP的过程得以继续.     

菲降解菌的筛选鉴定及其降解酶基因的研究

经过富集从活性污泥中筛选出两株菲降解菌WSCII和WSCIII,经16SrDNA鉴定,它们分别属于鞘氨醇单胞菌属(Sphingomonassp. )和假单胞菌属(Pseudomonassp. ),在28℃下4d内对菲( 0. 6g/L)的降解率分别达到了70. 9%和78. 1%;在LB、萘和菲为碳源的培养条件下,测得菌体的双加氧酶比活力不同.在菲的诱导下,两株菌的双加氧酶比活力最高,而以LB和萘为碳源时酶的比活力较低,其中WSCII在萘中不能生长;这表明该酶在两菌中皆为底物诱导表达.以其它菌的萘双加氧酶基因大亚基片段做模板制备异源探针,与两菌总DNA进行斑点杂交;结果表明,这两株菌可能含有不同的菲双加氧酶基因.利用简并引物进行PCR扩增菲双加氧酶,结果仅能从WSCIII的总DNA中扩增得到目的片段,序列分析证明,该基因与已报道的(P. putidaNCIB9816 -4, AF491307 )双加氧酶同源性最高为98%. 图5参10     

紫外线B、臭氧、磁场协同沼泽红假单胞菌降解废水中的苯酚的效果

为了提高沼泽红假单胞菌(Rhodopseudomonas palustris)在降解有机污染物过程中对开放环境的适应性,进一步提高对污染物的降解活性,分别应用紫外线B、臭氧、磁场协同沼泽红假单胞菌对苯酚溶液及炼油厂含酚废水进行降解试验,比较不同协同方式的作用效果.试验得出:在三种方式的协同降解过程中沼泽红假单胞菌的生长量、脱氢酶活性和苯酚的降解率显著高于对照.其中紫外线B协同沼泽红假单胞菌降解苯酚的处理与对照比较,菌体生长量增加了33.53%,在对数生长期的生长速率增加了10%,降解72 h时其脱氢酶活性增强了31.77%.协同处理对人工废水和炼油厂含酚废水的降解效率都有显著提高.结果表明:一定强度的紫外线B、臭氧、磁场能促进沼泽红假单胞菌的生长,增强其脱氢酶活性,提高对苯酚的降解.紫外线B、臭氧、磁场协同沼泽红假单胞菌在降解苯酚过程中,沼泽红假单胞菌对开放环境的适应能力提高.     

生物表面活性剂对铜绿假单胞菌摄取烷烃的强化机制

考察了2株铜绿假单胞菌(Pseudomonasaeruginosa)以正十六烷为底物生长的不同方式,并初步探讨了生物表面活性剂在烃类降解菌摄取烷烃过程中的作用机制.菌株O-2-2在正十六烷中的生长明显快于PS-1,生长过程中O-2-2分泌出鼠李糖脂生物表面活性剂,正十六烷被完全乳化.另外,O-2-2菌细胞表面疏水性高于PS-1,而加入鼠李糖脂使得菌体细胞中脂多糖含量减少,菌细胞表面疏水性明显提高.上述结果表明,鼠李糖脂主要通过乳化疏水性底物和提高降解菌表面疏水性两种机制强化铜绿假单胞菌(P.aeruginosa)对烷烃的摄取.图6表1参14     

荧光假单胞菌(Pseudomonas fluorescens)工程菌株田间残留和扩散的追踪检测

分别在北京和江苏省连云港市对携带Btcry基因的荧光假单胞菌工程菌进行了田间残留与扩散的追踪检测 .对越冬前后的试验地和保护地土壤样品进行抗生素抗性平板分离 ,能检测到极少量的具有与出发菌株相同抗性的荧光假单胞菌菌落 ,但没有发现工程菌株的残留与扩散 .对江苏试验地样品还进行了工程菌株质粒卡那霉素和壮观霉素抗性标记基因的抗性菌落分离 ,绝大多数样品中都能分离到包括荧光假单胞菌在内的抗性菌落 ,土样中菌密度n(cfu) =10 4 ～ 10 5g-1,但进行Btcry基因PCR -RFLP检测时没有从样品中得到特异扩增产物 .研究结果表明 :工程菌株抗性标记基因在自然界广泛存在 ,工程菌在株环境中没有残留和扩散 ,具有良好的生物安全性 .表 4参 8     

一株降解苄嘧磺隆光合细菌的分离鉴定及其降解特性

从农药厂工业废水和污泥中富集分离到一株能降解苄嘧磺隆(Bensulfuron methyl)的光合细菌PSB07-6,根据分离菌株的细胞形态结构、活细胞光吸收特征、生理生化特征以及系统发育分析将该菌初步鉴定为沼泽红假单胞菌(Rhodopseudomonas palustris).高效液相色谱法(HPLC)测定该菌降解光合细菌培养基中苄嘧磺隆的能力,在pH为6.5的光合细菌培养基中培养5 d,对350 mg·L-1苄嘧磺隆降解率达25.03%.添加回收率为105%～112%.降解特性研究结果表明,该菌能以苄嘧磺隆为唯一碳源和氮源,降解最佳条件为30℃、pH6.5.     

紫花苜蓿根际氢氧化细菌的分离与鉴定

利用气体循环培养体系从陕西乾县HUP-豆科植物紫花苜蓿(Medicago sativa)根际土壤中分离获得37株细菌.菌株氧化氢能力测定结果表明,8株菌氧化氢和自养生长能力较强,初步确定为氢氧化细菌类群;根据其形态特征、培养特征和生理生化特性,鉴定为7个不同属:假单胞菌属(Pseudomonas)、邻单胞菌属(Plesiomonas)、脂肪杆菌属(Pimelobacter)、黄色杆菌属(Xanthobacter)、勒米诺氏菌属(Leminorella)、地杆菌属(Terrabacter)和稀有杆菌属(Rarobacter);其中氧化氢能力最强的优势菌株WMQ-7 16S rDNA序列(GenBank登录号为EU807744)长度为1 451bp,GC含量为53.8%,其核苷酸序列与假单胞菌属同源性高于99%,在系统发育树上位于同一分支,将WMQ-7菌株鉴定为假单胞菌属(Pseudomonas).图2表5参16     

Isolation of a strain that degrades 1,2,4-trichlorobenzene and characterization of its degradative plasmid

The genetic information encoding metabolic pathways for xenobiotic compounds in bacteria often resides on catabolic plasmids. The aim of the present work was to know the location of the genes for degrading 1,2,4-trichlorobenzen. In this paper a 1,2,4-trichlorobenzene-degrading strain THSL-1 was isolated from the soil of Tianjin Chemical Plant using 1,2,4-trichlorobenzene as the sole carbon source. The strain was identified as Pseudomonas stutzeri through morphologic survey and 16S rDNA sequence determination. A plasmid was discovered from strain THSL-1 by using the alkali lysis method. When the plasmid was transformed into E. coli. JM109 by the CaCl2 method, the transformant could grow using 1,2,4-trichlorobenzene as the sole carbon source and had the degradation function of 1,2,4-trichlorobenzene. Therefore, it could be deemed that the plasmid carried the degradative genes of 1,2,4-trichlorobenzene. The average size of the plasmid was finally determined to be 40.2 Kb using selectively three kinds of restricted inscribed enzymes (HindIII, BamHI, and XholI) for single cutting and double cutting the plasmid pTHSL-1, respectively.     

多环芳烃降解菌ZL5分离鉴定及其降解质粒

通过选择性富集培养，从辽河油田石油污染土壤中分离到一株多环芳烃(PAHs)降解菌ZL5．它能以菲和芘为唯一碳源生长，但是不能利用萘．16S rDNA核苷酸序列分析结果表明，ZL5属于变形细菌α亚类中的鞘氨醇单胞菌属．该菌株含有一个大小约为60kb的质粒．丝裂霉素C消除实验表明，随着质粒的丢失，菌株利用菲和芘的能力也丧失．用电转化和氯化铷转化法分别将菌株ZL5的质粒导人大肠杆菌JM109和DH5α中，随着质粒的获得，这些转化子获得了降解菲和芘的能力．本研究结果表明，鞘氨醇单胞菌ZL5降解PAHs的功能和质粒有关。     

Isolation of a Pseudomonas Stutzeri strain that degrades 1,2,4-trichlorobenzene and characterization of its degradative plasmid

The genetic information encoding metabolic pathways for xenobiotic compounds in bacteria often resides on catabolic plasmids. The aim of the present work was to know the location of the genes for degrading 1,2,4-trichlorobenzen. In this paper a 1,2,4-trichlorobenzene-degrading strain THSL-1 was isolated from the soil of Tianjin Chemical Plant using 1,2,4-trichlorobenzene as the sole carbon source. The strain was identified as Pseudomonas stutzeri through morphologic survey and 16S rDNA sequence determination. A plasmid was discovered from strain THSL-1 by using the alkali lysis method. When the plasmid was transformed into E. coli. JM109 by the CaCl₂ method, the transformant could grow using 1,2,4-trichlorobenzene as the sole carbon source and had the degradation function of 1,2,4-trichlorobenzene. Therefore, it could be deemed that the plasmid carried the degradative genes of 1,2,4-trichlorobenzene. The average size of the plasmid was finally determined to be 40.2 Kb using selectively three kinds of restricted inscribed enzymes (HindIII, BamHI, and XholI) for single cutting and double cutting the plasmid pTHSL-1, respectively.     

紫云英根瘤菌共生基因分布及其共生效应的多样性

从同一植株不同根瘤分离40株紫云英根瘤菌，所有菌株对10种抗生素的抗药性测定表明，该群体分为22个抗药类群，质粒检测显示所有公离株都含有质粒，质粒数1～4条，用快生型大豆根瘤菌USDA205质粒作参考，估测质粒Mr分布范围为83～226MU．根据图谱分析表明，该菌群可分为6个不同质粒型．各类型质粒通过与Dig-nodABC和Dig-nifHDK杂交，结果显示带有1条质粒的菌株其共生基因定位在染色体上．带有2条或2条以上质粒的菌株各拥有1条共生质粒，共生质粒Mr范围有差异，大约为117～220MU．研究结果也显示，不同质粒型的菌株其共生效应存在明显差异，其中第6质粒型的菌株共生固氮率最强，第1质粒型菌株共生固氮率较低．共生固氮能力最强的第6质粒型菌株，只占总菌数7．5％．     

荧光假单胞菌高效广谱载体的构建及分析

为促进高GC含量基因在荧光假单胞菌(Pseudomonas fluorescens)中表达效果更加理想、操作更加简便,本研究首先采用不依赖基因序列和连接反应的克隆(Sequence and ligation independent cloning,SLIC)方法将载体pCIBhis上与复制相关的序列和标记基因片段构建成克隆载体pCIBS1.然后优化荧光假单胞菌转化方法,用电转化法将pCIBS1导入荧光假单胞菌BL915中,随后又将T7和tac基因启动子分别插入pCIBS1中,成功构建了表达载体pCIBS3和pCIBS2.研究发现载体pCIBS1在大肠杆菌和荧光假单胞菌中均较为稳定,并且将绿色荧光蛋白基因插入表达载体中,在大肠杆菌BL21(DE3)中获得表达,验证了表达载体功能.本研究构建的表达载体和建立的荧光假单胞菌BL915电转化方法,为高GC含量基因在荧光假单胞菌中的表达奠定了基础.     

利用GFP和抗性双类型标记监测联合固氮菌在玉米根际的定殖

将来自质粒ｐＫＲＰ１０、ｐＫＲＰ１１和ｐＫＲＰ１２的氯霉素、卡那霉素和四环素抗性基因分别插入质粒ＧＦＰｍｕｔ２中ｇｆｐ基因下游的ＰｓｔＩ位点,得到ｇｆｐ和不同抗性基因共存的重组质粒,转化Ｅｎｔｅｒｏｂａｃｔｅｒ ｇｅｒｇｏｖｉａｅ ５７－７野生型菌株和耐铵工程菌Ｅ７后,得到既有抗生素抗性又在蓝光下呈现亮绿荧光的菌株。用它们接种玉米后,利用这两种选择标记双重筛选重新分离到的细菌确定了接种菌在玉米幼苗     

Characteristics of two transferable aminoglycoside resistance plasmids in Escherichia coli isolated from pig and chicken manure

pRKZ3 is a non-conjugative IncQ plasmid, while pKANJ7 is a conjugative IncX plasmid. The optimal mating time of pKANJ7 varied under different conditions. Both of the two transferable ARPs had little impact on the growth of their hosts. A relatively high level of fitness cost was observed for pKANJ7. The fitness cost of ARPs depended on their hosts. Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is under reported. In this study, two transferable aminoglycoside resistance plasmids, pRKZ3 and pKANJ7, isolated from pig and chicken manure, were characterized. Results showed that pRKZ3 (8236 bp) is a non-conjugative IncQ plasmid and contains genes encoding for plasmid replication and stabilization (repA, repB and repC), mobilization (mob), and antibiotic resistance (arr-3 and aacA). pKANJ7 (30142 bp) is a conjugative IncX plasmid which codes for a type IV secretion system (T4SS). Conjugative transfer experiments showed that the optimal mating time of pKANJ7 was 8 h under the starvation condition, but the number of tranconjugants increased with time under the nutrient condition. Statistical analysis indicated that the two plasmids had little impact on the growth of their hosts, but a relatively high level of fitness cost due to pKANJ7 was observed. We also found that the fitness cost of plasmids depended on their hosts. Compared with pKANJ7, the relative fitness cost index of pRKZ3 varied within a narrow range during the 10 days of competition. The low level of fitness cost of pRKZ3 might contribute to the persistence of the plasmid in the environment. Our study provides new information for understanding the characterizations of antibiotic resistance plasmids (ARPs) in manure sources and helps to clarify the transfer and persistence of ARPs in the environment following the application of manure.     

两株联合降解甲基一六○五菌的分离及其特性研究

从农药厂污泥中分离得到一个降解甲基一六○五的混合菌群,该菌群由两种菌 Ｍ６ 和 Ｐ３ 组成,初步鉴定均为假单胞菌属（ ｐｓｅｕｄｍｏｎａｓ ．ｓｐ） ． Ｍ６ 菌具有一硫代磷酸酯键水解酶,能够催化甲基一六○五水解为对硝基酚, Ｐ３ 具有对硝基酚降解能力． Ｍ６ 和 Ｐ３ 各自均不能彻底矿化甲基一六○五． Ｍ６ 与 Ｐ３ 混和可以彻底降解甲基一六○五．质粒消除实验表明, Ｓ Ｄ Ｓ、吖啶橙（ Ｏ Ａ） 、丝裂霉素及几种消除剂混和处理均不能使 Ｍ６ 和 Ｐ３ 丧失一六○五水解和对硝基酚降解能力,质粒检测发现在 Ｍ６ 中检测到质粒条带,而 Ｐ３ 中未检测到质粒条带     

Quantification of polyphenols during retting and characterization of bacteria from the Kadinamkulam Backwaters, Kerala

The retting environment which provides a competitive niche for specialized microbes is speculated to harbour a variety of microbes with high biodegradation potential. In this context, an effort has been made to isolate and identify bacterial species having high tolerance to phenol In vitro. Maximum polyphenol (1.897 mg l(-1)) as observed during the initial period of retting, which decreased as retting proceeded. Based on biochemical characterization, the isolated bacterial strains were identified as Micrococcus sp., Moraxella sp. strain MP1, Moraxella sp. strain MP2 and Moraxella sp. strain MP3, Pseudomonas sp. strain PP1 and Pseudomonas sp. strain PP2, Amphibacillus sp., Brucella sp. strain BP1 and Brucella sp. strain BP2, Aquaspirillum sp., Escherichia coli strain EP1 and Escherichia coli strain EP2, Campylobacter sp., Aeromonas sp., Neisseria sp., Vibrio sp., Erwinia sp. and Mesophilobacter sp. These strains were found to tolerate maximum concentration of phenol viz. 200 to 1000 mg l(-1). Plasmid analysis of phenol resistant bacterial isolates showed that almost all the cultures had at least one plasmid of size > 1Kb. Studies on the protein profile of isolated bacterial cultures showed the presence of proteins with molecular sizes ranging from 10 to 85 KDa with exception of Mesophilobacter and Neisseria having still high molecular weight protein (95 KDa). Bacterial strains isolated from coir-ret-liquor showed tolerance to high phenol concentration.