Cloning, expression, and purification of the K5 capsular polysaccharide lyase (kflA) from coliphage K5A: Evidence for two distinct K5 lyase enzymes
Cloning, expression, and purification of the K5 capsular polysaccharide lyase (kflA) from coliphage K5A: Evidence for two distinct K5 lyase enzymes
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DOI：
10.1128/jb.182.13.3761-3766.2000
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发表时间：
2000-07-01
影响因子：
3.2
通讯作者：
Roberts, IS
中科院分区：
文献类型：
--
作者：
Clarke, BR;Esumeh, F;Roberts, IS
大肠杆菌K5荚膜多糖[-4）-β GlcA-（1，4）-α GlcNAc-（1-]是荚膜特异性噬菌体K5 A的受体，与噬菌体K5 A的结构相关的是多糖裂解酶，其降解K5荚膜以暴露下面的细菌细胞表面。克隆了噬菌体K5 A裂解酶基因（klfA），并进行了序列测定。kflA基因编码预测分子量为66.9kDa的多肽，该多肽与大肠杆菌染色体上编码的K5多糖裂解酶ElmA具有氨基酸同源性。coli SEER 3282中，kflA和elmA基因之间只有有限的核苷酸同源性，表明这两个基因是不同的，并且要么来自不同的祖先，要么已经从一个共同的祖先分化了相当长的时间。Southern印迹分析显示，kflA不存在于B的染色体上。检查大肠杆菌菌株。相反，elmA存在于E.大肠杆菌菌株。K5 A噬菌体kfLA基因侧翼的DNA与E. coli K1荚膜特异性噬菌体K1 E。这些非编码序列之间的DNA同源性表明噬菌体K5 A和K1 E是相关的。推导的噬菌体K1 E的ORFL多肽序列与噬菌体K5 A的KflA的N端同源，表明ORFL是KflA的截短残基。这种截短的kflA基因的存在意味着噬菌体K1 E通过获得内唾液酸酶基因和随后的功能性kflA的丧失而从噬菌体K5 A进化而来。A（His）（6）-KflA融合蛋白在E.大肠杆菌，并纯化至均一，产量为4.8mg/L细菌培养物。该重组酶在较宽的pH范围和NaCl浓度下都有活性，能够将K5多糖降解为低分子量产物。
The Escherichia coli K5 capsular polysaccharide [-4)-beta GlcA-(1,4)-alpha GlcNAc-(1-] is a receptor for the capsule-specific bacteriophage K5A, Associated with the structure of bacteriophage K5A is a polysaccharide lyase which degrades the K5 capsule to expose the underlying bacterial cell surface. The bacteriophage K5A lyase gene (klfA) was cloned and sequenced. The kflA gene encodes a polypeptide with a predicted molecular mass of 66.9 kDa and which exhibits amino acid homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of E. coli SEER 3282, There was only limited nucleotide homology between the kflA and elmA genes, suggesting that these two genes are distinct and either have been derived from separate progenitors or have diverged from a common progenitor for a considerable length of time. Southern blot analysis revealed that kflA was not present on the chromosome of the B. coli strains examined. In contrast, elmA was present in a subset of E. coli strains. Homology was observed between DNA flanking the kfLA gene of bacteriophage K5A and DNA flanking a small open reading frame (ORFL) located 5' of the endosialidase gene of the E. coli K1 capsule-specific bacteriophage K1E. The DNA homology between these noncoding sequences indicated that bacteriophages K5A and K1E were related. The deduced polypeptide sequence of ORFL in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORFL is a truncated remnant of KflA. The presence of this truncated kflA gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional kflA. A (His)(6)-KflA fusion protein was overexpressed in E. coli and purified to homogeneity with a yield of 4.8 mg per liter of bacterial culture. The recombinant enzyme was active over a broad pH range and NaCl concentration and was capable of degrading K5 polysaccharide into a low-molecular-weight product.