CLONING AND ANALYSIS OF DUPLICATED RFBM AND RFBK GENES INVOLVED IN THE FORMATION OF GDP-MANNOSE IN ESCHERICHIA-COLI O9/K30 AND PARTICIPATION OF RFB GENES IN THE SYNTHESIS OF THE GROUP-I K30 CAPSULAR POLYSACCHARIDE
CLONING AND ANALYSIS OF DUPLICATED RFBM AND RFBK GENES INVOLVED IN THE FORMATION OF GDP-MANNOSE IN ESCHERICHIA-COLI O9/K30 AND PARTICIPATION OF RFB GENES IN THE SYNTHESIS OF THE GROUP-I K30 CAPSULAR POLYSACCHARIDE
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DOI：
10.1128/jb.176.11.3126-3139.1994
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发表时间：
1994-06-01
影响因子：
3.2
通讯作者：
WHITFIELD, C
中科院分区：
文献类型：
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作者：
JAYARATNE, P;BRONNER, D;WHITFIELD, C
rfb(O9) 基因簇负责脂多糖 O9 抗原的合成，是从大肠杆菌 O9:K30 中克隆的。编码 6-磷酸葡萄糖酸脱氢酶的 gnd 基因被鉴定为邻近 rfb(O9) 簇，并通过 DNA 序列分析建立了基因顺序 gnd-rfbM-rfbK。该目与肠杆菌科其他成员的描述不同。使用核苷酸序列分析来鉴定分别编码磷酸甘露糖变位酶和GDP-甘露糖焦磷酸化酶的rfbK和rfbM基因。在肠杆菌科的成员中，这些酶依次作用形成GDP-甘露糖，其作为细胞表面多糖中甘露糖残基的活化糖核苷酸前体。在大肠杆菌 O9:K30 菌株中，在 rfbM(1)-rfbK(1) 下游约 3 kbp 处检测到重复的 rfbM(2)-rfbK(2) 区域，且邻近 rfb(O9) 簇的其余基因。 rfbM 同基因在上游侧翼 DNA 方面有所不同，但在其他方面高度保守。相反，rfbK 同基因在下游侧翼 DNA 和 3' 末端区域有所不同，导致预测的 RfbK 蛋白大小略有差异。 RfbM(O9) 和 RfbK(O9) 分别与 CpsB 和 CpsG 最密切相关。这些分别是 GDP-甘露糖焦磷酸化酶和磷酸甘露糖变位酶的同工酶，被认为参与大肠杆菌 K-12 和鼠伤寒沙门氏菌中粘液多糖豆蔻酸的生物合成。大肠杆菌 O-:K30 突变体（菌株 CWG44）缺乏 rfbM(2)-rbK(2)，并且删除了相邻的必需 rfb(O9) 序列。因此，剩余的染色体基因足以用于 GDP-甘露糖的形成和 K30 荚膜多糖的合成。大肠杆菌 CWG44 的一个突变体，菌株 CWG152，被发现缺乏 GDP-甘露糖焦磷酸化酶，并且失去了合成 K30 荚膜多糖的能力。通过用含有 rfbM(1) 或 rfbM(2) 的质粒转化，可以在 CWG152 中恢复野生型荚膜多糖。将完整的 rfb(O9) 基因簇引入 CWG152 恢复了 O9 和 K30 多糖的合成。因此，rfbM 足以在大肠杆菌 O9:K30 中生物合成 O 抗原和荚膜多糖的 GDP-甘露糖。通过 Southern 杂交和 PCR 扩增实验对血清型 O8 和 O9 分离株进行的分析表明 rfbM-rfbK 区域存在广泛的多态性。
The rfb(O9) gene cluster, which is responsible for the synthesis of the lipopolysaccharide O9 antigen, was cloned from Escherichia coli O9:K30. The gnd gene, encoding 6-phosphogluconate dehydrogenase, was identified adjacent to the rfb(O9) cluster, and by DNA sequence analysis the gene order gnd-rfbM-rfbK was established. This order differs from that described for other members of the family Enterobacteriaceae. Nucleotide sequence analysis was used to identify the rfbK and rfbM genes, encoding phosphomannomutase and GDP-mannose pyrophosphorylase, respectively. In members of the family Enterobacteriaceae, these enzymes act sequentially to form GDP-mannose, which serves as the activated sugar nucleotide precursor for mannose residues in cell surface polysaccharides. In the E. coli O9:K30 strain, a duplicated rfbM(2)-rfbK(2) region was detected approximately 3 kbp downstream of rfbM(1)-rfbK(1) and adjacent to the remaining genes of the rfb(O9) cluster. The rfbM isogenes differed in upstream flanking DNA but were otherwise highly conserved. In contrast, the rfbK isogenes differed in downstream flanking DNA and in 3'-terminal regions, resulting in slight differences in the sizes of the predicted RfbK proteins. RfbM(O9) and RfbK(O9) are most closely related to CpsB and CpsG, respectively. These are isozymes of GDP-mannose pyrophosphorylase and phosphomannomutase, respectively, which are thought to be involved in the biosynthesis of the slime polysaccharide colanic acid in E. coli K-12 and Salmonella enterica serovar Typhimurium. An E. coli O-:K30 mutant, strain CWG44, lacks rfbM(2)-rbK(2) and has adjacent essential rfb(O9) sequences deleted. The remaining chromosomal genes are therefore sufficient for GDP-mannose formation and K30 capsular polysaccharide synthesis. A mutant of E. coli CWG44, strain CWG152, was found to lack GDP-mannose pyrophosphorylase and lost the ability to synthesize K30 capsular polysaccharide. Wild-type capsular polysaccharide could be restored in CWG152, by transformation with plasmids containing either rfbM(1) or rfbM(2). Introduction of a complete rfb(O9) gene cluster into CWG152 restored synthesis of both O9 and K30 polysaccharides. Consequently, rfbM is sufficient for the biosynthesis of GDP-mannose for both O antigen and capsular polysaccharide in E. coli O9:K30. Analysis of a collection of serotype O8 and O9 isolates by Southern hybridization and PCR amplification experiments demonstrated extensive polymorphism in the rfbM-rfbK region.