恶臭假单胞菌ND6菌株catA基因的克隆和表达及其儿茶酚裂解途径探讨

恶臭假单胞菌ND6菌株的萘降解质粒pND6-1中编码儿茶酚1,2-双加氧酶的catA基因在大肠杆菌中进行了克隆和表达,并研究表达产物的酶学性质。结果表明:酶的Km为0.019μmol/L,Vmax为1.434μmol/(min.mg);具有很好的耐热性,在50℃保温45min后仍能够保留酶活力的93.7%;Fe2 对酶活性有显著的促进作用,其比活力是对照反应的292%;酶对4-氯儿茶酚的催化活性非常低,属于Ⅰ型儿茶酚1,2-双加氧酶。以萘为底物生长时,ND6菌株的细胞提取液中既存在催化邻位裂解途径的儿茶酚1,2-双加氧酶活性,也存在催化间位裂解途径的儿茶酚2,3-双加氧酶活性。以苯甲酸、对羟基苯甲酸和苯乙酸为唯一碳源生长时,ND6菌株细胞提取液的儿茶酚1,2-双加氧酶活性远远大于儿茶酚2,3-双加氧酶活性。表明ND6菌株既能通过儿茶酚间位裂解途径降解萘,也能通过儿茶酚邻位裂解途径降解萘,而以苯甲酸、对羟基苯甲酸和苯乙酸为诱导物时只利用儿茶酚邻位裂解途径。     

恶臭假单胞菌ND6菌株catA基因的克隆和表达及其儿茶酚裂解途径探讨

恶臭假单胞菌ND6菌株的萘降解质粒pND6-1中编码儿茶酚1,2-双加氧酶的catA基因在大肠杆菌中进行了克隆和表达,并研究表达产物的酶学性质。结果表明:酶的K_m为0.019μmol/L,V_max为1.434μmol/(min.mg);具有很好的耐热性,在50℃保温45min后仍能够保留酶活力的93.7%;Fe²⁺对酶活性有显著的促进作用,其比活力是对照反应的292%;酶对4-氯儿茶酚的催化活性非常低,属于Ⅰ型儿茶酚1,2-双加氧酶。以萘为底物生长时,ND6菌株的细胞提取液中既存在催化邻位裂解途径的儿茶酚1,2-双加氧酶活性,也存在催化间位裂解途径的儿茶酚2,3-双加氧酶活性。以苯甲酸、对羟基苯甲酸和苯乙酸为唯一碳源生长时,ND6菌株细胞提取液的儿茶酚1,2-双加氧酶活性远远大于儿茶酚2,3-双加氧酶活性。表明ND6菌株既能通过儿茶酚间位裂解途径降解萘,也能通过儿茶酚邻位裂解途径降解萘,而以苯甲酸、对羟基苯甲酸和苯乙酸为诱导物时只利用儿茶酚邻位裂解途径。     

4株苯系物降解菌菌株的筛选鉴定、降解特性及其降解基因研究

分别以苯、甲苯为碳源,从厦门污水处理厂活性污泥中富集筛选获得了2株苯降解菌B1、B2和2株甲苯降解菌J2、J6。16S rRNA基因鉴定结果表明B1、J2属于假单胞菌属(Pseudomonassp.),B2、J6属于不动杆菌属(Acinetobactersp.)。研究表明,这些菌在pH7~10的碱性范围内能很好生长。在以0.1%(V/V)苯或甲苯为唯一碳源的无机盐培养基中,B1、B2菌在72小时内对苯的降解率分别为67.7%、94.2%,J2、J6菌对甲苯的降解率分别为92.4%、84.8%。简并PCR扩增、序列分析表明,这些菌含有相同的苯双加氧酶基因,表明苯降解基因在这些降解菌中可能存在水平转移。此外,J2,J6两株菌还含有甲苯双加氧酶基因,而且J2能在甲苯浓度为70%(V/V)的LB培养基中生长。这些降解菌在苯、甲苯污染的生物治理中有应用前景。     

1株高效菲降解不动杆菌的筛选、鉴定及性能研究

从兰州某化工厂石油废水中分离筛选出1株高效降解菲的细菌F-1并对其菌种进行鉴定,结合紫外分光光度法及气相色谱-质谱联用(GC-MS)对菌株生长特性、不同烃类化合物降解特性及菲降解动力学等进行了研究,利用PCR技术检测了芳香烃代谢相关基因。结果表明,菌株F-1属于约翰逊不动杆菌(Acinetobacter johnsonii),可在终浓度为50～800 mg/L的含菲基础培养基中正常生长。在温度30℃、pH 7. 0、盐度0. 3%(质量分数)、转速180 r/min条件下培养5 d后菲(终浓度为100 mg/L)降解率为43. 57%,降解过程符合一级动力学特征。菌株F-1也能利用联苯、萘、蒽、芘为唯一碳源生长。GC-MS分析显示菌株对C10-C28部分直链烷烃具有较强的降解能力。PCR扩增结果表明,菌株F-1基因组中存在邻苯二酚-1,2-双加氧酶、苯甲酸盐双加氧酶、铁氧化还原蛋白还原酶、乙醇脱氢酶、二羟酸脱水酶、醛缩酶和氧化还原蛋白基因。研究结果为该菌株应用到含菲废水及多环芳烃污染土壤的处理和深度修复研究提供参考。     

一株深海热液环境来源的PAHs降解菌TVG9-Ⅶ的系统发育与降解基因

【目的】对一株深海热液环境来源的多环芳烃(PAHs)降解菌进行系统发育分析并对其降解特性和降解机制进行研究。【方法】对16S rRNA基因进行扩增和测序,进行基于16S rRNA基因序列的系统发育分析;利用GC-MS测定其对PAHs的降解率;通过构建基因组Fosmid文库,克隆PAHs降解基因簇;并利用RT-PCR和qPCR研究关键降解酶基因在不同PAHs诱导下的表达情况。【结果】从西南太平洋劳盆地热液沉积物中分离到一株PAHs降解菌株TVG9-Ⅶ,系统发育分析结果表明,该菌株属于新鞘氨醇杆菌属(Novosphingobium),与该属的Novosphingobium indicum H25T系统发育关系最为密切,它们的16S rRNA基因序列相似性高达99.7%。该菌株在21 d内对菲、荧蒽和芘的降解率分别为95.2%,57.3%和69.6%。从Fosmid文库中筛选得到一个负责PAHs降解的上游基因簇,包含了PAHs起始降解双加氧酶大小亚基(pheA1a/b)基因和一个脱氢酶基因;RT-PCR和qPCR实验表明,双加氧酶大亚基基因pheA1a在菲的诱导下上调表达4.2倍,而在萘及高环荧蒽和芘的诱导下无上调。【结论】菌株TVG9-Ⅶ是Novosphingobium属深海热液来源的PAHs降解菌,具有良好的降解特性,特别是对高环PAHs的降解效果较好。     

一株芘降解菌的分离鉴定及其降解效果

以芘为唯一碳源,采用平板升华法,从徐州市卧牛山焦化厂周围污染土壤中分离得到一株芘降解菌SE12.经形态观察、生理生化试验和16S rDNA鉴定,该菌株属于分枝杆菌属(Mycobacterium sp.)菌株,与快速生长型非致病性南非分枝杆菌(M.austroa fricanum ATCC33464)的同源性达到98%.SE12降解芘的最适pH和温度为pH9和30℃.当土壤芘初始含量为100和200mg.kg-1,SE12接种量为107CFU.g-1时,30℃培养28d后土壤芘降解率分别达到97%和99%.利用双加氧酶基因的同源序列引物nidAF/nidAR和nidBF/nidBR进行扩增,得出了该菌株编码双加氧酶大亚基和小亚基的基因片段,它们与已知降解芘的分枝杆菌的双加氧酶基因具有高度同源性.     

一株菲降解细菌的分离鉴定及其特性

通过选择性富集培养,从沈抚灌区石油污染土壤中分离到1株菲降解细菌.试验证明该菌株能以菲为唯一碳源和能源生长.经形态学、生理生化鉴定和16S rRNA基因序列比对分析,确定该菌株属于不动杆菌属,命名为Acinetobacter sp. L2. 系统发育进化分析发现,L2菌株与Acinetobacter sp. DG880［AY258108］亲源关系最近.L2菌株培养7 d后对菲的降解率达96.3%.邻苯二酚2,3-双加氧酶活力测定表明,L2菌株可能含有菲降解基因.     

陶厄氏菌Thauera sp.K11对酚类化合物降解作用及途径研究

旨在分析陶厄氏菌属(Genus Thuaera)中的一株菌株Thauera sp.K11对含酚废水中酚类化合物的降解作用和途径。以石化污水厂分离菌株K11为研究对象,克隆其16S r RNA基因和关键酶基因,并进行系统发育分析,在基因水平探究苯酚降解机理;利用气相色谱技术检测酚类化合物降解效果和苯酚降解机理。结果显示,利用16S r RNA系统学分析发现K11是陶厄氏菌属的一株细菌。该菌对11种酚类化合物具有降解作用,其中5种酚类化合物72 h的降解率90%。克隆并获得了K11的苯酚羟化酶和邻苯二酚双加氧酶基因。酶活性测定表明,K11通过苯酚羟化酶催化苯酚转化为邻苯二酚,然后利用邻苯二酚-2,3-双加氧酶催化产生2-HMSA。陶厄氏菌Thauera sp.K11是一株能够降解多种酚类化合物的菌株,具有较强的酚类污染物降解能力,其通过苯酚→邻苯二酚→2-HMSA途径进行苯酚降解。     

对硝基苯酚降解菌Pseudomonas sp. PDS-7的降解特性及 其降解相关基因的克隆

[目的]本研究的目的是分离对硝基苯酚(PNP)降解菌,研究其对PNP的降解特性;克隆其降解相关基因,并进行表达.[方法]本研究通过富集培养法和系列稀释平板涂布法分离PNP降解菌株;采用形态观察、生理生化特征测定和16S rDNA分析对菌株进行初步鉴定;通过摇瓶试验研究菌株降解特性;利用SEFA-PCR技术克隆降解相关基因,并亚克隆到表达载体pET29a中,构建重组表达质粒pETpnpC,再转入受体菌E.coli BL21(DE3)中进行诱导表达;通过分光光度法测定表达产物的酶活力.[结果]分离到一株PNP降解菌PDS-7,将该菌株鉴定为假单胞菌属(Pseudomonassp.);该菌株能够以PNP作为唯一碳源、氮源和能源生长,菌株对PNP的最高耐受浓度为80 mg/L,最适降解温度为30℃,偏碱性条件有利于菌株对PNP的降解;克隆了PNP降解过程中的偏苯三酚1,2-双加氧酶基因pnpC及马来酰醋酸还原酶基因pnpD(GenBank登陆号EU233791);将pnpC在E.coli BL21(DE3)菌株进行了诱导表达,表达产物对偏苯三酚和邻苯二酚均有邻位开环活性,比活力分别为0.45 U/mg protein和0.37 U/mg protein,表明偏苯三酚1,2-双加氧酶基因pnpC得到了活性表达.[结论]分离鉴定了一株PNP降解菌Pseudomonas sp.PDS-7,研究了该菌株的降解特性,克隆和表达了降解相关基因.     

DndB蛋白负调控变铅青链霉菌dndA基因转录

为了探究变铅青链霉菌dndA基因转录调控机制,本研究利用LC-MS分析了野生型变铅青链霉菌1326和dndB基因同框缺失菌株HXY2的DNA硫修饰丰度差异。通过半定量RT-PCR比较dndA基因在1326与HXY2中的表达差异。用儿茶酚2, 3-双加氧酶活力检测实验和对dndA启动子区域进行删除或突变来确定负责dndA基因表达调控的顺式作用元件。研究发现,HXY2的DNA硫修饰丰度高于1326,dndA基因在HXY2中的表达高于1326。儿茶酚2,3-双加氧酶活力检测实验显示,删除或突变r重复序列能使1326的dndA启动子活力明显升高。突变r重复序列后,HXY2的dndA启动子活力没有明显变化。结果显示,DndB蛋白抑制dndA基因转录进而下调DNA硫修饰丰度,以及DndB蛋白通过结合到r重复序列而抑制dndA基因转录。这是首次对dndA基因的转录调控机制进行研究,并且本研究进一步丰富了DndB蛋白的调控机理。     

Diversity of genetic systems responsible for biodegradation of naphthalene in Pseudomonas fluorescens strains

The genetic systems that are responsible for naphthalene catabolism were analyzed in 18 naphthalene-degrading Pseudomonas fluorescens strains isolated from oil-contaminated soils in different regions of Russia. It was found that thirteen strains contain plasmids, from 20 to 120 kb in size, at least five of which are conjugative and bear the catabolic genes responsible for the complete utilization of naphthalene and salicylate. Five plasmids belong to the P-7 incompatibility group, and two plasmids belong to the P-9 incompatibility group. The naphthalene biodegradation genes of P. fluorescens are highly homologous to each other. The study revealed a new group of the nahAc genes and two new variants of the nahG gene. The suggestion is made that the key genes of naphthalene biodegradation, nahAc and nahG, evolve independently and occur in P. fluorescens strains in different combinations.     

Nucleotide sequence analysis of the Pseudomonas putida PpG7 salicylate hydroxylase gene (nahG) and its 3'-flanking region

Gene nahG of naphthalene/salicylate catabolic plasmid NAH7 encodes a protein of molecular weight 45,000, salicylate hydroxylase. This enzyme catalyzes the formation of catechol from salicylate, a key intermediate in naphthalene catabolism. DNA sequence analysis of the 3.1-kilobase HindIII fragment containing the nahG locus reveals an open reading frame (ORF) of 1305 base pairs that corresponds to a protein of 434 amino acid residues. The predicted amino acid sequence of salicylate hydroxylase is in agreement with the molecular weight, NH2-terminal amino acid sequence, and total amino acid composition of the purified salicylate hydroxylase [You, I.-S., Murray, R. I., Jollie, D., & Gunsalus, I. C. (1990) Biochem. Biophys. Res. Commun. 169, 1049-1054]. The amino acid sequence between positions 8 and 37 of salicylate hydroxylase shows homology with known ADP binding sites of other FAD-containing oxidoreductases, thus confirming its biochemical function. The sequence of the Pseudomonas putida salicylate hydroxylase was compared with those of other similar flavoproteins. A small DNA segment (831 base pairs) disrupts the continuity of the known gene order nahG and nahH, the latter encoding catechol 2,3-dioxygenase. The complete nucleotide sequence of the intergenic region spanning genes nahG and nahH has been determined and its biological role proposed.     

Natural horizontal transfer of a naphthalene dioxygenase gene between bacteria native to a coal tar-contaminated field site.

Horizontal transfer of genes responsible for pollutant biodegradation may play a key role in the evolution of bacterial populations and the adaptation of microbial communities to environmental contaminants. However, field evidence for horizontal gene transfer between microorganisms has traditionally been very difficult to obtain. In this study, the sequences of the 16S rRNA and naphthalene dioxygenase iron-sulfur protein (nahAc) genes of nine naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site, as well as a naphthalene-degrading bacterium from a contaminated site in Washington state and two archetypal naphthalene-degrading strains, were compared. Seven strains from the study site had a single nahAc allele, whereas the 16S rRNA gene sequences of the strains differed by as much as 7.9%. No nahAc alleles from the site were identical to those of the archetypal strains, although the predominant allele was closely related to that of Pseudomonas putida NCIB 9816-4, isolated in the British Isles. However, one site-derived nahAc allele was identical to that of the Washington state strain. Lack of phylogenetic congruence of the nahAc and 16S rRNA genes indicates that relatively recent in situ horizontal transfer of the nahAc gene has occurred, possibly as a direct or indirect consequence of pollutant contamination. Alkaline lysis plasmid preparations and pulsed-field gel electrophoresis have revealed the presence of plasmids ranging in size from 70 to 88 kb in all site isolates. Southern hybridizations with a 407-bp nahAc probe have suggested that the nahAc gene is plasmid borne in all the site isolates but one, a strain isolated from subsurface sediment 400 m upstream from the source of the other site isolates. In this strain and in the naphthalene-degrading strain from Washington state, nahAc appears to be chromosomally located. In addition, one site isolate may carry nahAc on both chromosome and plasmid. Within the group of bacteria with identical nahAc sequences the Southern hybridizations showed that the gene was distributed between plasmids of different sizes and a chromosome. This suggests that plasmid modification after transfer may have been effected by transposons. Horizontal transfer of catabolic genes may play a significant role in the acclimation of microbial communities to pollutants.     

Physiological role of the novel salicylaldehyde dehydrogenase NahV in mineralization of naphthalene by Pseudomonas putida ND6

The classical salicylaldehyde dehydrogenases found in naphthalene-degrading bacteria are denoted as NahF. In addition to NahF, NahV, and its corresponding gene nahV, were found here in multiple naphthalene-degrading bacteria isolated from industrial wastewater polluted with polycyclic aromatic hydrocarbons (PAHs). In this study, we described for the first time the biological function and regulation model of NahV for the mineralization of naphthalene by P. putida ND6 via the construction of nahF-, nahV- and regulatory gene nahR-deficient strains. The two mutants of salicylaldehyde dehydrogenase genes and wild-type Pseudomonas ND6 were compared with respect to growth rate, naphthalene degradation efficiency, protein expression level, and salicylaldehyde dehydrogenase activity. The data showed that the presence of NahV conferred a physiological advantage on P. putida ND6 for the catabolism of naphthalene in the presence of NahF. NahV could facilitate naphthalene degradation by increasing total salicylaldehyde dehydrogenase activity when both dehydrogenases are present and it could replace the function of NahF when nahF gene is deleted or mutated, thus ensuring mutants could survive in naphthalene-containing environments. To investigate regulation model of NahV, we detected the expression levels and salicylaldehyde dehydrogenase activity in the wild-type and the nahR mutant strains following cultivation in the presence of glucose±salicylate. The data demonstrated that just like the classical salicylaldehyde dehydrogenases, NahF, NahV was induced by salicylate in the presence of NahR.     

Effects of the inoculant strain Pseudomonas putida KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community

The naphthalene-degrading activity of a Pseudomonas sp. strain isolated from a creosote-contaminated soil was shown to be encoded by the IncP9 plasmid pNF142 by transfer to Pseudomonas putida KT2442. The effects of the inoculant strain KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community were studied in microcosms with the following treatments: (I) soil, (II) soil with naphthalene, (III) soil with naphthalene and inoculated with KT2442 (pNF142). The inoculant became the dominant bacterial population in treatment (III) as evidenced by cultivation and denaturing gradient gel electrophoresis (DGGE) analysis. The bacterial DGGE profiles revealed drastically reduced complexity due to the numerical dominance of the inoculant. However, group-specific fingerprints (beta-proteobacteria, actinobacteria) that excluded KT2442 (pNF142) showed less severe changes in the bacterial community patterns. A major effect of naphthalene on the soil bacterial community was observed in treatment (II) after 21 days. Two dominant bands appeared whose sequences showed the highest similarity to those of Burkholderia sp. RP007 and Nocardia vinaceae based on 16S rRNA gene sequencing. These bands were less intense in treatment (III). The increased abundance of RP007-like populations due to naphthalene contamination was also confirmed by PCR amplification of the phnAc gene. The nahAc and nahH genes were detected in DNA and cDNA only in treatment III. Although the inoculant strain KT2442 (pNF142) showed good survival and expression of genes involved in naphthalene degradation, this study suggests that KT2442 (pNF142) suppressed the enrichment of indigenous naphthalene degraders.     

Horizontal transfer of phnAc dioxygenase genes within one of two phenotypically and genotypically distinctive naphthalene-degrading guilds from adjacent soil environments

Several distinct naphthalene dioxygenases have been characterized to date, which provides the opportunity to investigate the ecological significance, relative distribution, and transmission modes of the different analogs. In this study, we showed that a group of naphthalene-degrading isolates from a polycyclic aromatic hydrocarbon (PAH)-contaminated hillside soil were phenotypically and genotypically distinct from naphthalene-degrading organisms isolated from adjacent, more highly contaminated seep sediments. Mineralization of (14)C-labeled naphthalene by soil slurries suggested that the in situ seep community was more acclimated to PAHs than was the in situ hillside community. phnAc-like genes were present in diverse naphthalene-degrading isolates cultured from the hillside soil, while nahAc-like genes were found only among isolates cultured from the seep sediments. The presence of a highly conserved nahAc allele among gram-negative isolates from the coal tar-contaminated seep area provided evidence for in situ horizontal gene transfer and was reported previously (J. B. Herrick, K. G. Stuart-Keil, W. C. Ghiorse, and E. L. Madsen, Appl. Environ. Microbiol. 63:2330-2337, 1997). Natural horizontal transfer of the phnAc sequence was also suggested by a comparison of the phnAc and 16S ribosomal DNA sequences of the hillside isolates. Analysis of metabolites produced by cell suspensions and patterns of amplicons produced by PCR analysis suggested both genetic and metabolic diversity among the naphthalene-degrading isolates of the contaminated hillside. These results provide new insights into the distribution, diversity, and transfer of phnAc alleles and increase our understanding of the acclimation of microbial communities to pollutants.     

Identification of the key genes of naphthalene catabolism in soil DNA

The key genes nahAc and xylE of the naphthalene catabolism of fluorescent Pseudomonas spp. in the total soil DNA samples were detected by the polymerase chain reaction (PCR) technique. The collection of fluorescent Pseudomonas spp. was screened for the occurrence of these genes. The results obtained show the possibility of using this approach in the goal-directed search for plasmid-containing naphthalene-degrading fluorescent pseudomonads in soil. The distribution of the naphthalene catabolism genes in soils contaminated with creosote and petroleum products was also studied.     

Bacterial community dynamics during <Emphasis Type="Italic">in-situ</Emphasis> bioremediation of petroleum waste sludge in landfarming sites

In-situ bioremediation of petroleum waste sludge in landfarming sites of Motor Oil Hellas (petroleum refinery) was studied by monitoring the changes of the petroleum composition of the waste sludge, as well as the changes in the structure of the microbial community, for a time period of 14 months. The analyses indicated an enhanced degradation of the petroleum hydrocarbons in the landfarming areas. A depletion of n-alkanes of approximately 75–100% was obtained. Marked changes of the microbial communities of the landfarms occurred concomitantly with the degradation of the petroleum hydrocarbons. The results obtained from terminal restriction fragment length polymorphism (T-RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rRNA genes demonstrated that bacteria originating from the refinery waste sludge and newly selected bacteria dominated the soil bacterial community during the period of the highest degradation activity. However, the diversity of the microbial community was decreased with increased degradation of the petroleum hydrocarbons contained in the landfarms. T-RFLP fingerprints of bacteria of the genera Enterobacter and Ochrobactrum were detected in the landfarmed soil over the entire treatment period of 14 months. In contrast, the genus Alcaligenes appeared in significant numbers only within the 10 month old landfarmed soil. Genes encoding catechol 2,3-dioxygenase (subfamily I.2.A) were detected only in DNA of the untreated refinery waste sludge. However, none of the genes known to encode the enzymes alkane hydroxylase AlkB, catechol 2,3-dioxygenase (subfamily I.2.A) and naphthalene dioxygenase nahAc could be detected in DNA of the landfarmed soils.