Flavobacterium columnare, the etiological agent of columnaris disease, is one of the most important bacterial pathogens of freshwater fish in the world. However, suitable genetic manipulation of this bacterium which has been challenging and producing genetic mutations in this bacterium has not been reported. Therefore, isolation of an effective promoter in F. Columnare may provide tools for the development of a genetic manipulation system in the bacterium. In this study, the acetyl-coenzyme A synthetase gene (acs) and flanking sequences were analyzed to determine promoter function. The acs gene is 2 323 bp long, encoding a protein of 635 amino acids. A TAAAA motif was identified as the conserved sequence for ribosome binding site (RBS) and TATTTTCG and TTG were determined to be -7 and -33 promoter motifs, upstream of the acs start codon. When a plasmid was constructed containing the acs upstream regulation sequence (Pacs) fused upstream of the chloramphenicol ace-tyltransferase (cat) gene and was introduced into F. Columnare G4 strain, the host cells gained chloramphenicol (Cm) resistance. The transcriptional start position (TSP) of acs and cat was both determined to be T locating 46 bp upstream of the start codon. Deletion analysis of the promoter showed that at least 164 bp nucleotides upstream of the start codon were required for promoter activity and for the expression of CAT to sufficiently confer the resistance in F. Columnare. Nucleotide analysis and alignment of the putative RBS for 32 protein-coding genes in F. Columnare G4 revealed the conserved RBS consensus, TAAAA, in Flavobacterium located 10 bp upstream of start codon of acs. The current study described the first successful construction of a plasmid that was able to express cloned genes in F. Columnare, which will allow further studies of the important columnaris disease in fish.%柱状黄杆菌(Flavobacterium columnare)是世界范围内危害淡水鱼类的柱形病的病原.目前对该病原菌遗传操作系统的研究进展较慢,而寻找高效稳定的启动子来调控外源基因在细菌体内的表达,有可能促进该细菌遗传操作系统的构建.本研究获得了柱状黄杆菌乙酰辅酶A合成酶基因(acetyl-coenzyme A synthetase gene,acs)的编码序列及其上游调控序列,该基因全长2 323 bp,编码635个氨基酸.通过序列分析,发现在该基因起始密码子ATG的上游存在核糖体结合位点(ribosome biding site,RBS)序列TAAAA,和启动子-7和-33的保守基序TATTTTCG和TTG.将acs的上游调控序列(promoter sequence,Pacs)置于氯霉素抗性基因(chloramphenicol acetyltransferase,cat)的上游并导入柱状黄杆菌G4株后,cat基因得以表达,并使宿主细胞产生稳定的氯霉素抗性.通过5'RACE技术,确定了外源的cat基因和内源的acs基因的转录起始位点都是位于起始密码子上游46 bp处的T.通过删减分析调控序列Pacs,发现起始密码子上游164 bp的序列是保持启动子活性所必需的.通过分析和比对32个基因的核糖体结合位点区域,在起始密码子上游10 bp处发现了RBS保守基序TAAAA.
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