Chauhan Nar Singh, Ranjan Ravi, Purohit Hemant J, Kalia Vipin C, Sharma Rakesh
Microbial Genomics and Biotechnology Group, Institute of Genomics and Integrative Biology, Council of Scientific and Industrial Research (CSIR), Delhi, India.
FEMS Microbiol Ecol. 2009 Jan;67(1):130-9. doi: 10.1111/j.1574-6941.2008.00613.x. Epub 2008 Nov 4.
The majority of bacteria elude culture in the laboratory. A metagenomic approach provides culture-independent access to the gene pool of the whole bacterial community. A metagenomic library was constructed from an industrial effluent treatment plant sludge containing about 1.25 Gb of microbial community DNA. Two arsenic-resistant clones were selected from the metagenomic library. Clones MT3 and MT6 had eight- and 18-fold higher resistance to sodium arsenate in comparison with the parent strain, respectively. The clones also showed increased resistance to arsenite but not to antimony. Sequence analysis of the clones revealed genes encoding for putative arsenate reductases and arsenite efflux pumps. A novel arsenate resistance gene (arsN) encoding a protein with similarity to acetyltransferases was identified from clone MT6. ArsN homologues were found to be closely associated with arsenic resistance genes in many bacterial genomes. ArsN homologues were found fused to putative arsenate reductases in Methylibium petroleiphilum PM1 and Anaeromyxobacter dehalogenans 2CP-C and with a putative arsenite chaperone in Burkholderia vietnamiensis G4. ArsN alone resulted in an approximately sixfold higher resistance to sodium arsenate in wild-type Escherichia coli W3110.
大多数细菌在实验室中无法通过培养获得。宏基因组学方法提供了一种不依赖培养的途径来获取整个细菌群落的基因库。从一个工业废水处理厂的污泥中构建了一个宏基因组文库，该污泥含有约1.25 Gb的微生物群落DNA。从宏基因组文库中筛选出两个抗砷克隆。与亲本菌株相比，克隆MT3和MT6对砷酸钠的抗性分别高8倍和18倍。这些克隆对亚砷酸盐的抗性也有所增加，但对锑没有抗性。对这些克隆的序列分析揭示了编码假定的砷酸盐还原酶和亚砷酸盐外排泵的基因。从克隆MT6中鉴定出一个新的抗砷基因（arsN），其编码的蛋白质与乙酰转移酶相似。在许多细菌基因组中发现，ArsN同源物与抗砷基因密切相关。在嗜油甲基杆菌PM1和脱卤厌氧粘细菌2CP-C中，ArsN同源物与假定的砷酸盐还原酶融合，在越南伯克霍尔德菌G4中与假定的亚砷酸盐伴侣融合。单独的ArsN使野生型大肠杆菌W3110对砷酸钠的抗性提高了约6倍。
