Wacharotayankun R, Arakawa Y, Ohta M, Tanaka K, Akashi T, Mori M, Kato N
Department of Bacteriology, Nagoya University School of Medicine, Japan.
Infect Immun. 1993 Aug;61(8):3164-74. doi: 10.1128/iai.61.8.3164-3174.1993.
We determined the complete nucleotide sequence of a 2.1-kb HindIII-EcoRI fragment that was cloned from a resident large plasmid of Klebsiella pneumoniae Chedid, a highly virulent and mucoviscous strain of the O1:K2 serotype. This fragment encoded an ability to enhance K2 capsular polysaccharide synthesis in K. pneumoniae, and a 636-bp open reading frame (rmpA2) was found. The 411-bp rmpA reported to be involved in the virulence and mucoid phenotypes of K. pneumoniae by Nassif et al. (Mol. Microbiol. 3:1349-1359, 1989) was a part of rmpA2. Eighty percent homology in nucleotide sequence was found between rmpA2 and rmpA in the corresponding regions. The central domain of the deduced amino acid sequence of RmpA2 showed considerable homology to the central domains of NtrC of K. pneumoniae and Escherichia coli, to which the sigma factor of RNA polymerase binds. The C-terminal domain of RmpA2 also demonstrated considerable homology with the putative helix-turn-helix motifs of LuxR of Vibrio fischeri and FixJ of Rhizobium meliloti. Moreover, RmpA2 also showed some homology in its N- and C-terminal regions to those of RcsA, a transcriptional activator for colanic acid synthesis in E. coli. On the other hand, a sequence upstream of rmpA2 was found to be highly homologous to insertion sequence 3 of members of the family Enterobacteriaceae. Southern hybridization analysis suggested that rmpA2 exists on the large plasmids of all mucoviscous virulent K2 strains but not on those of the slightly mucoviscous avirulent strains. Freeze substitution electron microscopy and fluorescent-antibody staining with anti-K2 serum revealed that K. pneumoniae Chedid has a dense and thick capsule (180 nm) with dense extracapsular substance, whereas K. pneumoniae K2-215, one of the slightly mucoviscous and avirulent strains, has a capsule which is looser and thinner (120 nm) than that of strain Chedid and no extracapsular substance. Introduction of rmpA2 into K2-215 as well as reference strains K. pneumoniae K9 and K72 resulted in a change of the colony phenotype to highly mucoviscous through abundant production of extracapsular substance which reacted with anti-K2, -K9, or -K72, respectively, as did their parental strains. From these results, it is suggested that RmpA2 belongs to the family of transcriptional regulators and confers a highly mucoviscous phenotype on cells of various serotypes of K. pneumoniae by enhancing extracapsular polysaccharide synthesis.
我们测定了一个2.1kb HindIII - EcoRI片段的完整核苷酸序列，该片段是从肺炎克雷伯菌Chedid（一种O1:K2血清型的高毒力且黏液样菌株）的一个常驻大质粒中克隆得到的。这个片段编码了增强肺炎克雷伯菌中K2荚膜多糖合成的能力，并且发现了一个636bp的开放阅读框（rmpA2）。据Nassif等人（《分子微生物学》3:1349 - 1359，1989年）报道，参与肺炎克雷伯菌毒力和黏液样表型的411bp的rmpA是rmpA2的一部分。在相应区域，rmpA2与rmpA在核苷酸序列上有80%的同源性。RmpA2推导的氨基酸序列的中央结构域与肺炎克雷伯菌和大肠杆菌的NtrC的中央结构域有相当高的同源性，RNA聚合酶的sigma因子与之结合。RmpA2的C末端结构域也与费氏弧菌的LuxR和苜蓿根瘤菌的FixJ的假定螺旋 - 转角 - 螺旋基序有相当高的同源性。此外，RmpA2在其N末端和C末端区域与大肠杆菌中参与合成柯氏酸的转录激活因子RcsA也有一些同源性。另一方面，发现rmpA2上游的一个序列与肠杆菌科成员的插入序列3高度同源。Southern杂交分析表明，rmpA2存在于所有黏液样高毒力K2菌株的大质粒上，但不存在于黏液样低毒力菌株的大质粒上。冷冻置换电子显微镜和用抗K2血清进行的荧光抗体染色显示，肺炎克雷伯菌Chedid有一个致密且厚的荚膜（180nm），带有致密的荚膜外物质，而肺炎克雷伯菌K2 - 215（一种黏液样低毒力菌株）的荚膜比菌株Chedid的更疏松、更薄（120nm），且没有荚膜外物质。将rmpA2导入K
