Kelly R F, Whitfield C
Department of Microbiology, University of Guelph, Ontario, Canada.
J Bacteriol. 1996 Sep;178(17):5205-14. doi: 10.1128/jb.178.17.5205-5214.1996.
Klebsiella species express a family of structurally related lipopolysaccharide O antigens which share a common backbone known as D-galactan I. Serotype specificity results from modification of D-galactan I by addition of domains of altered structure or by substitution with O-acetyl and/or alpha-D-Galp side groups with various linkages and stoichiometries. In the prototype, Klebsiella serotype O1, the his-linked rfb gene cluster is required for synthesis of D-galactan I, but genes conferring serotype specificity are unlinked. The D-galactan I part of the O polysaccharide is O acetylated in Klebsiella serotype O8. By cloning the rfb region from Klebsiella serotype O8 and analyzing the O polysaccharide synthesized in Escherichia coli K-12 hosts, we show that, like rfbO1, the rfbO8 region directs formation of unmodified D-galactan I. The rfbAB genes encode an ATP-binding cassette transporter required for export of polymeric D-galactan I across the plasma membrane prior to completion of the lipopolysaccharide molecule by ligation of the O polysaccharide to lipid A-core. Complementation experiments show that the rfbAB gene products in serotypes O1 and O8 are functionally equivalent and interchangeable. Hybridization experiments and physical mapping of the rfb regions in related Klebsiella serotypes suggest the existence of shared rfb genes with a common organization. However, despite the functional equivalence of these rfb gene clusters, at least three distinct clonal groups were detected in different Klebsiella species and subspecies, on the basis of Southern hybridization experiments carried out under high-stringency conditions. The clonal groups cannot be predicted by features of the O-antigen structure. To examine the relationships in more detail, the complete nucleotide sequence of the serotype O8 rfb cluster was determined and compared with that of the serotype O1 prototype. The nucleotide sequences for the six rfb genes showed variations in moles percent G+C values and in the values for nucleotide sequence identity, which ranged from 66.9 to 79.7%. The predicted polypeptides ranged from 64.3% identity (78.4% total similarity) to 94.3% identity (98.0% similarity). The results presented here are not consistent with dissemination of the Klebsiella D-galactan I rfb genes through recent lateral transfer events.
克雷伯氏菌属表达了一族结构相关的脂多糖O抗原，它们共享一个被称为D-半乳聚糖I的共同主链。血清型特异性源于D-半乳聚糖I通过添加结构改变的结构域或用具有不同连接方式和化学计量的O-乙酰基和/或α-D-半乳糖侧链取代而发生的修饰。在原型克雷伯氏菌血清型O1中，与组氨酸相连的rfb基因簇是合成D-半乳聚糖I所必需的，但赋予血清型特异性的基因是不连锁的。在克雷伯氏菌血清型O8中，O多糖的D-半乳聚糖I部分被O-乙酰化。通过从克雷伯氏菌血清型O8克隆rfb区域并分析在大肠杆菌K-12宿主中合成的O多糖，我们表明，与rfbO1一样，rfbO8区域指导未修饰的D-半乳聚糖I的形成。rfbAB基因编码一种ATP结合盒转运蛋白，该转运蛋白是聚合D-半乳聚糖I在通过将O多糖连接到脂多糖A核心完成脂多糖分子之前跨质膜输出所必需的。互补实验表明，血清型O1和O8中的rfbAB基因产物在功能上是等效的且可互换。相关克雷伯氏菌血清型中rfb区域的杂交实验和物理图谱表明存在具有共同组织的共享rfb基因。然而，尽管这些rfb基因簇在功能上等效，但基于在高严谨条件下进行的Southern杂交实验，在不同的克雷伯氏菌物种和亚种中检测到至少三个不同的克隆群。这些克隆群无法通过O抗原结构的特征来预测。为了更详细地研究这些关系，测定了血清型O8 rfb簇的完整核苷酸序列并与血清型O1原型的序列进行了比较。六个rfb基因的核苷酸序列在鸟嘌呤与胞嘧啶摩尔百分比值以及核苷酸序列同一性值方面存在差异，范围从66.9%到79.7%。预测的多肽同一性范围从64.3%（总相似性为78.4%）到94.3%（相似性为98.0%）。此处呈现的结果与克雷伯氏菌D-半乳聚糖I rfb基因通过近期横向转移事件传播不一致。
