Takaku C, Sekizuka T, Tazumi A, Moore J E, Millar B C, Matsuda M
Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Fuchinobe 1-17-71, Sagamihara 229-8501, Japan.
Br J Biomed Sci. 2009;66(2):85-92. doi: 10.1080/09674845.2009.11730250.
Following TA cloning and sequencing with a novel in silico-designed polymerase chain reaction (PCR) primer pair (f-ClvacJ/r-ClvacJ), approximately 750 base pairs (bp) of promoter and structural gene regions for vacJ and its adjacent genetic loci (approximately 1.14 kbp) were identified in 20 isolates of Campylobacter lari (urease-negative C. lari [n=7]; urease-positive thermophilic Campylobacter [n=13]). The nucleotide sequences of an approximately 70-bp non-coding region, including the typical promoter structure, showed sequence differences at 12 loci among 21 isolates including C. lari RM2100. The putative sigma70 promoter region upstream of the putative open reading frame (ORF), a start codon TTG and a probable ribosome binding site, AGGA, for the vacJ gene were also identified in all 21 C. lari isolates examined. Each ORF for the vacJ terminated with a TAA stop codon. No hypothetical transcriptional terminators were identified within the amplicons. The putative ORFs of the vacJ gene from 21 C. lari isolates consisted of 684 bases, similarly differing from those of the other thermophilic campylobacters (696 bases for C. jejuni RM1221 and NCTC11168 and C. coli RM2228; 690 for C. upsaliensis RM3195). Reverse transcription PCR analysis confirmed the transcription of the vacJ gene in the C. lari cells. A neighbour joining tree suggested a strong molecular discrimination efficacy between UPTC and UN C. lari employing vacJ nucleotide sequence information. The vacJ gene homologue from C. lari organisms appears not to be a lipoprotein signal peptide or a signal peptide in silico.
使用一对新的计算机设计的聚合酶链反应（PCR）引物（f-ClvacJ/r-ClvacJ）进行TA克隆和测序后，在20株空肠弯曲菌分离株（脲酶阴性空肠弯曲菌[n = 7]；脲酶阳性嗜热弯曲菌[n = 13]）中鉴定出约750个碱基对（bp）的vacJ启动子和结构基因区域及其相邻基因座（约1.14 kbp）。包括典型启动子结构在内的约70 bp非编码区的核苷酸序列在包括空肠弯曲菌RM2100在内的21株分离株中的12个位点显示出序列差异。在所有21株检测的空肠弯曲菌分离株中还鉴定出推定开放阅读框（ORF）上游的推定σ70启动子区域、起始密码子TTG和vacJ基因可能的核糖体结合位点AGGA。vacJ的每个ORF均以TAA终止密码子结束。在扩增子内未鉴定出假定的转录终止子。21株空肠弯曲菌分离株的vacJ基因推定ORF由684个碱基组成，与其他嗜热弯曲菌的ORF（空肠弯曲菌RM1221和NCTC11168以及大肠弯曲菌RM2228为696个碱基；乌普萨拉弯曲菌RM3195为690个碱基）不同。逆转录PCR分析证实了vacJ基因在空肠弯曲菌细胞中的转录。邻接法树状图表明，利用vacJ核苷酸序列信息，UPTC和UN空肠弯曲菌之间具有很强的分子鉴别效力。空肠弯曲菌生物体的vacJ基因同源物在计算机模拟中似乎不是脂蛋白信号肽或信号肽。
