【作者】 蔡林；

【导师】 王革娇；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 砷是一种剧毒类金属,在自然环境中主要以亚砷酸盐（As（Ⅲ））和砷酸盐（As（Ⅴ））的无机形式存在并通过微生物的氧化和还原相互转化。As（Ⅲ）不带电荷且移动性强,其毒性和危害性比As（Ⅴ）大很多,因而,微生物可以通过对砷的氧化还原转化危害人类的健康和安全。本论文围绕砷抗性细菌对As（Ⅲ）的氧化和细胞质As（Ⅴ）的还原开展了以下两方面的研究工作,主要结果如下:在我国砷高污染TS区、中污染SY区、低污染LY区和YC区分别采集了4个土壤样品,从这4个代表3种不同砷污染水平土壤样品中共分离到58株砷抗性菌（TS1-45、SY1-8、LY1-4和YC1）,其中共有12株As（Ⅲ）高抗菌株（MICs＞20mM）,并且都是从高污染TS区中分离得到,分布在Acinetobacter、Agrobacterium、Arthrobacter、Comamonas、Rhodococcus、Stenotrophomonas和Pseudomonas等7个属。利用KMnO₄法从58株菌中检测出5株As（Ⅲ）氧化菌（Achromobacter sp.SY8、Pseudomonas sp.TS44和Agrobacterium spp.TS43,TS45,LY4）,它们的平均As（Ⅲ）抗性水平高于其余53株非As（Ⅲ）氧化菌。采用简并PCR方法成功扩增出5个编码As（Ⅲ）氧化酶大亚基aoxB片段和51个As（Ⅲ）通道蛋白基因片段（18个arsB、12个ACR3（1）和21个ACR3（2））,发现aoxB特异性存在于As（Ⅲ）氧化菌中,而arsB、ACR3（1）和ACR3（2）在As（Ⅲ）氧化菌和非As（Ⅲ）氧化菌中都存在。采用邻近距离法构建了58株菌16S rRNA基因进化树、5个aoxB和51个As（Ⅲ）通道蛋白基因的蛋白进化树,详细分析并比较了它们之间的进化关系,发现5个aoxB与其16S rRNA基因的系统发生完全吻合,但有8个ACR3（2）和1个arsB与其16S rRNA基因的进化矛盾,很可能是水平基因转移所致。研究还发现长期受砷污染的环境中砷抗性菌的多样性丰富而且As（Ⅲ）抗性水平高,进一步分析发现水平基因转移加快了砷抗性菌的侧向进化。本研究是对我国不同砷污染土壤中砷抗性菌及其抗性基因比较研究的首次报道,也是检测As（Ⅲ）通道蛋白基因发生水平转移的首次报道。以上述其中两株As（Ⅲ）氧化菌新种Achromobacter sp.SY8和Pseudomonas sp.TS44为研究对象,利用Fosmid文库方法从SY8菌株中分离到一个17.5kb的DNA序列,覆盖了完整的As（Ⅲ）氧化酶基因簇（aoxX-aoxS-aoxR和aoxA-aoxB-aoxC-aoxD）,利用反向PCR方法从TS44菌株中克隆到一个14.6kb的DNA序列,包含了完整的As（Ⅲ）氧化酶基因簇（arsD-arsA-aoxA-aoxB）和砷抗性基因簇（arsC1-arsR-arsC2-ACR3-arsH-DSP-GAPDH-MFS）。通过RT-PCR研究发现:SY8的aoxXSR和aoxABCD分别共转录,属于两个操纵子;TS44的两个基因簇各自共转录,每个基因簇属于一个操纵子:除了TS44的arsC1-arsR组成型表达外,上述所有基因在加入As（Ⅲ）、As（Ⅴ）或Sb（Ⅲ）诱导后表达水平均上调。生物信息学分析表明SY8 As（Ⅲ）氧化酶基因簇受双组分信号传导系统调控,但是与已报道的Agrobacterium tumefaciens 5A具有较大的差异性,因此推测在SY8中存在一套新型细菌As（Ⅲ）感应和信号传导的双组分调控模型。TS44的As（Ⅲ）氧化酶基因簇结构不典型,因为它只含有结构基因并在其上游出现了arsDA,但是除了TS44外,在GenBank数据库中没有检索到其它只含有结构基因的As（Ⅲ）氧化酶基因簇。TS44的砷抗性基因簇的前5个基因被广泛报道与砷抗性相关,但是后3个基因则没有被报道与砷抗性有关。上述三点分别揭示了SY8和TS44的3个砷解毒基因簇结构的复杂性和新颖性。本论文的结果在抗砷性微生物资源的开发、砷污染环境的微生物生态、细菌砷代谢和细菌砷抗性的分子机制等方面都具有理论价值和创新性。更多还原

【Abstract】 Arsenic is known as a toxic metalloid,which primarily exists in inorganic forms of arsenite[As（Ⅲ）]and arsenate[As（Ⅴ）]in the natural environment and can be transformed by microbial redox processes.As（Ⅲ） is much more toxic and mobile than As（Ⅴ）,hence microorganisms can greatly influence the human health and safety by the processes of arsenic redox transformation.This dissertation focused on the studies of As（Ⅲ） oxidation and cytoplasmic As（Ⅴ） reduction of arsenite-resistant bacteria and performed two research subjects.The main results were as follows:Four soil samples were collected in China from regions with high（TS）, intermediate（SY）,and low（LY and YC） arsenic contamination.A total of 58 arsenite-resistant bacteria（TS1-45,SY1-8,LY1-4,and YC1） were isolated from these four samples with three different arsenic-contaminated levels.12 high As（Ⅲ） resistance bacteria（MICs>20 mM） were only isolated from the highly arsenic-contaminated TS site including Acinetobacter,Agrobacterium,Arthrobacter, Comamonas,Rhodococcus,Stenotrophomonas and Pseudomonas.Using the KMnO₄ screening method,5 As（Ⅲ）-oxidizing bacteria（Achromobacter sp.SY8, Pseudomonas sp.TS44,Agrobacterium spp.TS43,TS45 and LY4） were identified from the 58 strains that displayed a higher average As（Ⅲ） resistance level than the 53 non-As（Ⅲ） oxidizers.Five aoxB fragments encoding the large subunit of As（Ⅲ） oxidase and 51 As（Ⅲ） transporter genes[18 arsB,12 ACR3（1） and 21 ACR3（2）]were successfully amplified from these strains using PCR with degenerate primers.The aoxB particularly existed in the As（Ⅲ）-oxidizers whereas arsB,A CR3（1） and A CR3（2） could occur in both As（Ⅲ）-oxidizers and non-As（Ⅲ） oxidizers.Phylogenetic trees of 16S rRNA gene,deduced AoxB,and deduced ArsB/Acr3（1）p/Arc3（2）p were constructed by the neighbor-joining method and analyzed in detail.According to the comparison of these trees,5 aoxBs had the similar phylogeny with 16S rRNA gene, but the discrepancies could be detected in 8 ACR3（2） and 1 arsB which were probably acquired by horizontal gene transfer（HGT）.These results indicated that soils with long-term arsenic contamination may result in the evolution of highly diverse arsenic-resistant bacteria and elevated As（Ⅲ） resistance level.Further analyses revealed that HGT promoted the lateral evolution of arsenic-resistant bacteria.This study is the first report of comparative study on arsenic-resistant bacteria and their resistance genes from different arsenic contaminated soils in China,also the first report of detecting HGT of As（Ⅲ） transporters.Two of the above As（Ⅲ）-oxidizing bacteria（Achromobacter sp.SY8 and Pseudomonas sp.TS44） were used as the second research subject.A 17.5 kb DNA sequence containing the complete arsenite oxidase（aox） gene cluster （aoxX-aoxS-aoxR and aoxA-aoxB-aoxC-aoxD） was isolated from SY8 using a Fosmid library approach.Similarly,a 14.6 kb DNA sequence including the complete aox cluster（arsD-arsA-aoxA-aoxB） and the arsenic resistance（ars） gene cluster （arsC1-arsR-arsC2-ACR3-arsH-DSP-GAPDH-MFS） was obtained from TS44 by inverse PCR method.According to RT-PCR experiments,SY8 aoxXSR and aoxABCD transcribed as two different transcripts and belonged to two operons;TS44 aox and ars clusters transcribed as a single transcript in their respective cluster and each of them belonged to one operon.All of these genes were found to be up-regulated by the addition of As（Ⅲ）,As（Ⅴ） or antimonite[Sb（Ⅲ）],except TS44 arsC1-arsR appeared to be expressed constitutively.Bioinformatic analyses revealed that the SY8 aox cluster was regulated by a two-component signal transduction system which was different from the reported work of Agrobacterium tumefaciens 5A.Hence,we predicted that a novel two-component regulatory model of bacterial As（Ⅲ） sensing and signal transduction could exist in SY8.The organization of TS44 aox cluster was unusual since it contained structural genes only and arsDA in its upstream.However, except TS44,no aox cluster with structural genes only was searched in GenBank database.The first five genes of TS44 ars cluster were widely reported to be associated with arsenic resistance,but the last three genes were not.The above three points indicated that these arsenic detoxification gene clusters of SY8 and TS44 were complex and had interesting and novel arrangements.The results of this dissertation have made great contributions to the exploitation of arsenic-resistant microorganisms,the microbial ecology of arsenic-contaminated environments,the bacterial arsenic metabolisms,and the molecular mechanisms of bacterial arsenic resistance.更多还原