Thompson J S, Malamy M H
Department of Molecular Biology and Microbiology, Tufts University Health Sciences Campus, Boston, Massachusetts 02111.
J Bacteriol. 1990 May;172(5):2584-93. doi: 10.1128/jb.172.5.2584-2593.1990.
Using a newly constructed Bacteroides fragilis-Escherichia coli cloning shuttle vector, pJST61, we have cloned the cefoxitin (FOX)-imipenem (IMP) resistance determinant from B. fragilis TAL2480. FOX-IMP resistance in this strain results from the production of a periplasmic, Zn2(+)-containing beta-lactamase which hydrolyzes carbapenems and cephamycins and whose activity is resistant to clavulanic acid but sensitive to Zn2(+)-binding reagents, including EDTA. The pJST61 vector permits efficient library construction in E. coli and allows for the transfer of the library to B. fragilis recipients for the screening or selection of specific phenotypes. The library clone containing the FOX-IMP resistance gene was detected after transfer to B. fragilis TM4000 (Fox-Imps) selecting for Foxr. One of the isolates carrying plasmid pJST241 is resistant to FOX and IMP and synthesizes a periplasmic protein with substrate and inhibitor properties identical to those of strain TAL2480. On the basis of deletion analysis, Tn1000 insertion mutations, and DNA sequencing, we have defined the 747-base cfiA (FOX-IMP resistance) gene within the 3.6-kilobase cloned insert in pJST241. The cfiA gene contains an open reading frame that could code for a precursor protein of 249 amino acids and with a molecular mass of 27,260 daltons. A potential signal sequence has been identified at the N terminus of this protein; cleavage within this sequence would result in a protein of 231 amino acids with a molecular mass of 25,249 daltons. The CfiA protein shows remarkable similarities to the exported, Zn2(+)-requiring, type II beta-lactamase Blm proteins from Bacillus cereus 569/H and 5/B/6. Although overall amino acid identity is only 32%, the Zn ligand-binding His and Cys residues are precisely conserved and the amino acids in the vicinity of these sites show strong similarities (greater than 80%) when the CfiA and Blm proteins are compared.
利用新构建的脆弱拟杆菌-大肠杆菌克隆穿梭载体pJST61，我们从脆弱拟杆菌TAL2480中克隆了头孢西丁（FOX）-亚胺培南（IMP）耐药决定簇。该菌株对FOX-IMP的耐药性源于一种周质含锌β-内酰胺酶的产生，该酶可水解碳青霉烯类和头孢霉素类，其活性对克拉维酸耐药，但对包括EDTA在内的锌结合试剂敏感。pJST61载体允许在大肠杆菌中高效构建文库，并能将文库转移至脆弱拟杆菌受体中以筛选或选择特定表型。将文库克隆转移至脆弱拟杆菌TM4000（Fox-Imps）并选择Foxr后，检测到了含有FOX-IMP耐药基因的文库克隆。携带质粒pJST241的一个分离株对FOX和IMP耐药，并合成一种周质蛋白，其底物和抑制剂特性与菌株TAL2480相同。基于缺失分析、Tn1000插入突变和DNA测序，我们在pJST241中3.6千碱基的克隆插入片段内确定了747碱基的cfiA（FOX-IMP耐药）基因。cfiA基因包含一个开放阅读框，可编码一个249个氨基酸、分子量为27260道尔顿的前体蛋白。在该蛋白的N端已鉴定出一个潜在的信号序列；该序列内的切割将产生一个231个氨基酸、分子量为25249道尔顿的蛋白。CfiA蛋白与蜡样芽孢杆菌569/H和5/B/6分泌的、需要锌的II型β-内酰胺酶Blm蛋白具有显著相似性。尽管总体氨基酸同一性仅为32%，但锌配体结合的组氨酸和半胱氨酸残基精确保守，当比较CfiA和Blm蛋白时，这些位点附近的氨基酸显示出很强的相似性（大于80%）。
