Kato Naoki, Yamazoe Kikuo, Han Chang-Gyun, Ohtsubo Eiichi
Institute of Anaerobic Bacteriology, Gifu University School of Medicine, Japan.
Antimicrob Agents Chemother. 2003 Mar;47(3):979-85. doi: 10.1128/AAC.47.3.979-985.2003.
The 747-bp cfiA gene, which encodes a metallo-beta-lactamase, and the regions flanking cfiA in six imipenem-resistant and four imipenem-susceptible Bacteroides fragilis strains isolated in Japan were analyzed by PCR and DNA sequencing. The nucleotide sequences of the cfiA genes (designated cfiA(1) to cfiA(10)) of all 10 strains tested varied from that of the standard cfiA gene from B. fragilis TAL2480. However, putative proteins encoded by the cfiA variants contained conserved amino acid residues important for zinc binding and hairpin loop formation, suggesting that cfiA variants have the capability of producing metallo-beta-lactamases with full catalytic activities. PCR assay indicated that six metallo-beta-lactamase-producing, imipenem-resistant strains had an insertion mutation in the region immediately upstream of cfiA. Nucleotide sequencing of the PCR-amplified fragments along with the upstream region of cfiA revealed that there were five new kinds of insertion sequence (IS) elements (designated IS612, IS613, IS614, IS615, and IS616, with a size range of 1,594 to 1,691 bp), of which only IS616 was found to be almost identical to IS1188, one of the IS elements previously identified in the upstream region of cfiA. These elements had target site duplications of 4 or 5 bp in length, terminal inverted repeats (14, 15, or 17 bp in size), and a large open reading frame encoding a putative transposase which is required for the transcription of IS elements. Each element was inserted such that the transcriptional direction of the transposase was opposite to that of cfiA. A computer-aided homology search revealed that, based on the homology of their putative transposases, the sizes of their terminal inverted repeat sequences, and their target site duplications, IS612, IS613, IS614, and IS615 belong to the IS4 family, which includes IS942, previously found in some drug-resistant B. fragilis strains, but that IS616 belongs to the IS1380 family. All the IS elements appear to have putative promoter motif sequences (the -7 region's TAnnTTTG motif and the -33 region's TTG or TG) in their end regions, suggesting that the IS elements provide a promoter for the transcription of cfiA upon insertion. These data provide additional proof that various IS elements may exist to provide a promoter to express the cfiA gene.
对在日本分离出的6株耐亚胺培南和4株对亚胺培南敏感的脆弱拟杆菌菌株中，编码金属β-内酰胺酶的747-bp cfiA基因及其侧翼区域进行了PCR和DNA测序分析。所检测的全部10株菌株的cfiA基因（命名为cfiA(1)至cfiA(10)）的核苷酸序列与脆弱拟杆菌TAL2480的标准cfiA基因不同。然而，cfiA变体编码的推定蛋白含有对锌结合和发夹环形成很重要的保守氨基酸残基，这表明cfiA变体有能力产生具有完整催化活性的金属β-内酰胺酶。PCR检测表明，6株产金属β-内酰胺酶的耐亚胺培南菌株在cfiA紧邻上游区域有插入突变。对PCR扩增片段及其cfiA上游区域进行核苷酸测序发现有5种新的插入序列（IS）元件（命名为IS612、IS613、IS614、IS615和IS616，大小范围为1594至1691 bp），其中只有IS616与之前在cfiA上游区域鉴定出的IS元件之一IS1188几乎相同。这些元件有长度为4或5 bp的靶位点重复序列、末端反向重复序列（大小为14、15或17 bp）以及一个编码IS元件转录所需推定转座酶的大开放阅读框。每个元件插入的方式使得转座酶的转录方向与cfiA相反。计算机辅助同源性搜索显示，基于其推定转座酶的同源性、末端反向重复序列的大小及其靶位点重复序列，IS612、IS613、IS614和IS615属于IS4家族，该家族包括之前在一些耐药脆弱拟杆菌菌株中发现的IS942，但IS616属于IS1380家族。所有IS元件在其末端区域似乎都有推定的启动子基序序列（-7区域的TAnnTTTG基序和-33区域的TTG或TG），这表明IS元件在插入时为cfiA的转录提供了一个启动子。这些数据进一步证明可能存在各种IS元件来提供一个启动子以表达cfiA基因。
