Morona J K, Morona R, Paton J C
Molecular Microbiology Unit, Women's and Children's Hospital, North Adelaide, Australia.
Mol Microbiol. 1997 Feb;23(4):751-63. doi: 10.1046/j.1365-2958.1997.2551624.x.
We have previously reported the nucleotide sequence of the first six genes of the Streptococcus pneumoniae type 19F capsular polysaccharide biosynthesis locus (cps19f). In this study we used plasmid insertion/rescue and inverse polymerase chain reaction (PCR) to clone an additional 10 kb downstream region containing the remainder of the cps19f locus, which was then subjected to sequence analysis. The cps19f locus is located in the S. pneumoniae chromosome between dexB and aliA, and consists of 15 open reading frames (ORFs), designated cps19fA to cps19fO, that appear to be arranged as a single transcriptional unit. Insertion-duplication mutants in seven out of the nine new ORFs have been constructed in a smooth type 19F strain, all of which resulted in a rough (nonencapsulated) phenotype, confirming that the operon is essential for capsule production. Comparison with sequence databases has allowed us to propose functions for 12 of the cps19f gene products, and a biosynthetic pathway for type 19F capsular polysaccharide. T7 expression studies confirmed that cps19fH, cps19fK, cps19fL, cps19fM and cps19fN directed the production of polypeptides of the expected size in Escherichia coli. The function of the cps19fK product was confirmed by its ability to complement a mutation in nfrC (rffE) in E. coli, as judged by restoration of sensitivity to bacteriophage N4. Interestingly, the last four genes of the locus (cps19fL-O) exhibit very strong homology (up to 70% amino acid identity) to a portion of the Shigella flexneri rfb gene cluster encoding biosynthesis of dTDP-rhamnose. When expressed in E. coli, cps19fL-O were capable of complementing a mutation deleting the respective Shigella flexneri homologues. Southern hybridization analysis indicated that cps19fA and cps19fB were the only cps genes found in all 16 S. pneumoniae serotypes/groups tested. The region from cps19fG to cps19fK was found only in members of serogroup 19, and, within this, cps19fl was unique to type 19F.
我们之前报道过肺炎链球菌19F型荚膜多糖生物合成位点（cps19f）前六个基因的核苷酸序列。在本研究中，我们使用质粒插入/拯救和反向聚合酶链反应（PCR）克隆了另外一个包含cps19f位点其余部分的10 kb下游区域，随后对其进行序列分析。cps19f位点位于肺炎链球菌染色体上dexB和aliA之间，由15个开放阅读框（ORF）组成，命名为cps19fA至cps19fO，它们似乎排列成一个单一的转录单元。在一株光滑型19F菌株中构建了9个新ORF中7个的插入重复突变体，所有这些突变体均导致粗糙（无荚膜）表型，证实该操纵子对荚膜产生至关重要。与序列数据库的比较使我们能够推测出12个cps19f基因产物的功能以及19F型荚膜多糖的生物合成途径。T7表达研究证实，cps19fH、cps19fK、cps19fL、cps19fM和cps19fN在大肠杆菌中指导产生预期大小的多肽。通过恢复对噬菌体N4的敏感性判断，cps19fK产物能够互补大肠杆菌中nfrC（rffE）的突变，从而证实了其功能。有趣的是，该位点的最后四个基因（cps19fL - O）与编码dTDP - 鼠李糖生物合成的福氏志贺氏菌rfb基因簇的一部分具有非常强的同源性（氨基酸同一性高达70%）。当在大肠杆菌中表达时，cps19fL - O能够互补缺失相应福氏志贺氏菌同源物的突变。Southern杂交分析表明，cps19fA和cps19fB是在所有测试的16种肺炎链球菌血清型/组中发现的仅有的cps基因。从cps19fG到cps19fK的区域仅在血清群19的成员中发现，并且在其中，cps19fl是19F型所特有的。
