Hoffman P S, Ripley M, Weeratna R
Department of Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada.
J Bacteriol. 1992 Feb;174(3):914-20. doi: 10.1128/jb.174.3.914-920.1992.
The major outer membrane protein of Legionella pneumophila is composed of 28- and 31-kDa subunits cross-linked by interchain disulfide bonds. The oligomer is covalently anchored to the underlying peptidoglycan via the 31-kDa subunit. We have cloned the structural gene ompS encoding both proteins. Oligonucleotide probes synthesized from the codons of the N-terminal amino acid sequence of purified 28- and 31-kDa subunits were used to identify cloned sequences. A 2.9-kb HindIII fragment cloned into pBluescript (clone H151) contained the ompS gene. Nucleotide sequence analysis revealed an open reading frame of 891 bp encoding a polypeptide of 297 amino acids. A leader sequence of 21 amino acids was identified, and the mature protein contained 276 amino acids. The deduced amino acid sequence of OmpS matched the experimentally determined amino acid sequence (32 amino acids), with the exception of two cysteine residues. The deduced amino acid sequence was rich in glycine and aromatic amino acids and contained four cysteine residues, two in the amino terminus and two in the carboxy region. Primer extension analysis (total RNA from L. pneumophila) identified the transcription start at 96 to 98 bp upstream of the translation start, but no Escherichia coli-like promoter sequences were evident. While an mRNA transcript from clone H151 was detected, no cross-reactive protein was detected by immunoblotting with either monoclonal or polyclonal antibody. Attempts to subclone the gene in the absence of the putative promoter region (i.e., under the control of the lac promoter) proved unsuccessful, possibly because of overproduction lethality in E. coli. The ompS DNA sequence was highly conserved among the serogroups of L. pneumophila, and related species also exhibited homology in Southern blot analysis at a moderately high stringency. Evidence is presented to suggest that this gene may be environmentally regulated in L. pneumophila.
嗜肺军团菌的主要外膜蛋白由通过链间二硫键交联的28 kDa和31 kDa亚基组成。该寡聚体通过31 kDa亚基共价锚定在下层肽聚糖上。我们克隆了编码这两种蛋白的结构基因ompS。根据纯化的28 kDa和31 kDa亚基N端氨基酸序列的密码子合成的寡核苷酸探针用于鉴定克隆序列。克隆到pBluescript中的一个2.9 kb HindIII片段（克隆H151）包含ompS基因。核苷酸序列分析揭示了一个891 bp的开放阅读框，编码一个297个氨基酸的多肽。鉴定出一个21个氨基酸的前导序列，成熟蛋白包含276个氨基酸。OmpS推导的氨基酸序列与实验确定的氨基酸序列（32个氨基酸）匹配，但有两个半胱氨酸残基除外。推导的氨基酸序列富含甘氨酸和芳香族氨基酸，包含四个半胱氨酸残基，两个在氨基末端，两个在羧基区域。引物延伸分析（来自嗜肺军团菌的总RNA）确定转录起始于翻译起始上游96至98 bp处，但未发现类似大肠杆菌的启动子序列。虽然检测到来自克隆H151的mRNA转录本，但用单克隆或多克隆抗体进行免疫印迹未检测到交叉反应蛋白。在没有假定启动子区域的情况下（即在lac启动子的控制下）亚克隆该基因的尝试未成功，可能是因为在大肠杆菌中过量表达致死。ompS DNA序列在嗜肺军团菌的血清群中高度保守，相关物种在Southern印迹分析中也在中等严格条件下表现出同源性。有证据表明该基因在嗜肺军团菌中可能受环境调控。
