Yamamoto S, Miyake K, Koike Y, Watanabe M, Machida Y, Ohta M, Iijima S
Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan.
J Bacteriol. 1999 Sep;181(17):5176-84. doi: 10.1128/JB.181.17.5176-5184.1999.
The type-specific capsular polysaccharide (CP) of a group B streptococcus, Streptococcus agalactiae type Ia, is a high-molecular-weight polymer consisting of the pentasaccharide repeating unit 4)-[alpha-D-NeupNAc-(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1- ->3 )]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1. Here, cloning, sequencing, and transcription of the type Ia-specific capsular polysaccharide synthesis (cps) genes and functional analysis of these gene products are described. A 26-kb DNA fragment containing 18 complete open reading frames (ORFs) was cloned. These ORFs were designated cpsIaA to cpsIaL, neu (neuraminic acid synthesis gene) A to D, orf1 and ung (uracil DNA glycosylase). The cps gene products of S. agalactiae type Ia were homologous to proteins involved in CP synthesis of S. agalactiae type III and S. pneumoniae serotype 14. Unlike the cps gene cluster of S. pneumoniae serotype 14, transcription of this operon may start from cpsIaA, cpsIaE, and orf1 because putative promoter sequences were found in front of these genes. Northern hybridization, reverse transcription-PCR, and primer extension analyses supported this hypothesis. DNA sequence analysis showed that there were two transcriptional terminators in the 3' end of this operon (downstream of orf1 and ung). The functions of CpsIaE, CpsIaG, CpsIaI, and CpsIaJ were examined by glycosyltransferase assay by using the gene products expressed in Escherichia coli JM109 harboring plasmids containing various S. agalactiae type Ia cps gene fragments. Enzyme assays suggested that the gene products of cpsIaE, cpsIaG, cpsIaI, and cpsIaJ are putative glucosyltransferase, beta-1, 4-galactosyltransferase, beta-1,3-N-acetylglucosaminyltransferase, and beta-1,4-galactosyltransferase, respectively.
B族链球菌Ia型无乳链球菌的型特异性荚膜多糖（CP）是一种高分子量聚合物，由五糖重复单元4）-[α-D-唾液酸-N-乙酰神经氨酸-（2→3）-β-D-半乳糖-（1→4）-β-D-N-乙酰葡糖胺-（1→3）]-β-D-半乳糖-（1→4）-β-D-葡萄糖-（1组成。本文描述了Ia型特异性荚膜多糖合成（cps）基因的克隆、测序和转录以及这些基因产物的功能分析。克隆了一个包含18个完整开放阅读框（ORF）的26 kb DNA片段。这些ORF被命名为cpsIaA至cpsIaL、neu（神经氨酸合成基因）A至D、orf1和ung（尿嘧啶DNA糖基化酶）。Ia型无乳链球菌的cps基因产物与参与III型无乳链球菌和肺炎链球菌14型CP合成的蛋白质同源。与肺炎链球菌14型的cps基因簇不同，该操纵子的转录可能起始于cpsIaA、cpsIaE和orf1，因为在这些基因前面发现了推定的启动子序列。Northern杂交、逆转录PCR和引物延伸分析支持了这一假设。DNA序列分析表明，该操纵子的3'端（orf1和ung的下游）有两个转录终止子。通过使用在携带含有各种Ia型无乳链球菌cps基因片段的质粒的大肠杆菌JM109中表达的基因产物进行糖基转移酶测定，研究了CpsIaE、CpsIaG、CpsIaI和CpsIaJ的功能。酶分析表明，cpsIaE、cpsIaG、cpsIaI和cpsIaJ的基因产物分别是推定的葡糖基转移酶、β-1,4-半乳糖基转移酶、β-1,3-N-乙酰葡糖胺基转移酶和β-1,4-半乳糖基转移酶。
