A PLASMID-ENCODED RFB(O/54) GENE-CLUSTER IS REQUIRED FOR BIOSYNTHESIS OF THE O/54 ANTIGEN IN SALMONELLA-ENTERICA SEROVAR BORREZE
A PLASMID-ENCODED RFB(O/54) GENE-CLUSTER IS REQUIRED FOR BIOSYNTHESIS OF THE O/54 ANTIGEN IN SALMONELLA-ENTERICA SEROVAR BORREZE
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DOI：
10.1111/j.1365-2958.1994.tb00325.x
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发表时间：
1994-02-01
影响因子：
3.6
通讯作者：
WHITFIELD, C
中科院分区：
文献类型：
--
作者：
KEENLEYSIDE, WJ;PERRY, M;WHITFIELD, C
以往的研究表明，7-8kb的表达与肠炎沙门氏菌的内毒素O：54抗原的表达有关。在这项研究中，从肠链球菌Borreze的野外分离株中分离到一个6.7kb的质粒，编码参与O：54多糖合成的酶。治愈该质粒会导致光滑的O-多糖取代的内毒素分子和0：54血清型的同时丢失。对其他O：54菌株的SDS-PAGE分析表明，O：54 O-多糖可以与另一个O-多糖共表达，这可能是由染色体基因编码的。O：54多糖的结构由化学和核磁共振方法相结合确定，是一种不寻常的N-乙酰甘露糖胺(D-ManNAc)残基均聚物。该多糖含有一个二糖重复单位，其结构为：->；4)-beta-D-ManpNAc-(1-->；3)-beta-D-ManpNAc-(1-->；，该结构与肠链霉菌中的其他O-多糖不同。为了研究质粒功能在O：54多糖合成中的作用，将6.7kb的质粒克隆到pGEM-7Zf(+)中，构建了杂合表达载体pWQ800。在大肠杆菌K-12 Delta RFB中，pWQ800指导合成了真正的O：54多糖。小鼠伤寒沙门氏菌RFB和RFC突变株也产生了聚合O：54多糖。根据这些数据，我们得出结论：pWQ800携带RFB(O：54)基因簇，O：54多糖的合成不需要宿主染色体的RFB功能。然而，O：54多糖的合成需要RFE和rffE基因的功能，这两个基因是编码参与肠杆菌共同抗原生物合成的酶的基因簇的一部分。RffE基因产物合成O：54前体尿苷二磷酸-N-乙酰甘露糖胺。这是首次在沙门氏菌中描述了一个由质粒编码的RFB基因簇。
Previous studies demonstrated that the presence of a 7-8kb plasmid is correlated with expression of the lipopolysaccharide (LPS) O:54 antigen in several Salmonella enterica serovars. In this study, a 6.7kb plasmid from a field isolate of S. enterica serovar Borreze was shown to encode enzymes responsible for the synthesis of the O:54 polysaccharide. Curing the plasmid results in simultaneous loss of smooth O-polysaccharide-substituted LPS molecules and 0:54 serotype. SDS-PAGE analysis of other O:54 isolates indicated that the O:54 O-polysaccharide can be co-expressed with an additional O-polysaccharide, likely encoded by chromosomal genes. The structure of the O:54 polysaccharide was determined by a combination of chemical and nuclear magnetic resonance (NMR) methods and was found to be an unusual homopolymer of N-acetylmannosamine (D-ManNAc) residues. The polysaccharide contained a disaccharide repeating unit with the structure: ->4)-beta-D-ManpNAc-(1-->3)-beta-D-ManpNAc-(1--> This structure does not resemble other O-polysaccharides in S. enterica. To examine the role played by plasmid functions in synthesis of the O:54 polysaccharide, the 6.7 kb plasmid was cloned to produce a hybrid plasmid (pWQ800) in pGEM-7Zf(+). In Escherichia coli K-12 Delta rfb, pWQ800 directed the synthesis of authentic O:54 polysaccharide. Polymerized O:54 polysaccharide was also produced in S. entericaserovar Typhimurium rfb and rfc mutants. From these data, we conclude that pWQ800 carries the rfb(O:54) gene cluster and synthesis of the O:54 polysaccharides does not require host chromosomal rfb functions. However, synthesis of the O:54 polysaccharide requires the function of the rfe and rffE genes which are part of the gene cluster encoding enzymes involved in biosynthesis of enterobacterial common antigen. The rffE gene product synthesizes the O:54 precursor, uridine diphospho-N-acetylmannosamine. This is the first description of a plasmid-encoded rfb gene cluster in Salmonella.