Abu Kwaik Y, Engleberg N C
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109-0620.
Mol Microbiol. 1994 Jul;13(2):243-51. doi: 10.1111/j.1365-2958.1994.tb00419.x.
The synthesis of a global stress protein (GspA) of Legionella pneumophila is induced in the intracellular environment of the phagocytic cell and by various in vitro stress stimuli. We used techniques of reverse genetics to isolate the gspA gene from a genomic library of L. pneumophila. Sequence analysis of approximately 1700 bp of a representative clone (pBSP1) showed the presence of two open reading frames (ORFs). ORF1 encoded for a polypeptide with an inferred molecular mass of 19 kDa and an isoelectric point of 6.1. These predictions correlated with the migration of the GspA protein on two-dimensional SDS-polyacrylamide gels. The predicted amino acid sequence of the GspA protein was identical to 22/23 residues of the N-terminal amino acid sequence derived by Edman degradation of the purified protein. The GspA protein was 41.3% and 36.5% identical to the 16 kDa IbpA and IbpB heat-shock proteins, respectively, of Escherichia coli. Primer extension from mRNA isolated from L. pneumophila showed that transcription of the gspA gene was controlled by two overlapping promoters. One of the promoters was a sigma 70 promoter, while the other was a heat-shock promoter and was regulated by the sigma 32 transcription factor in E. coli. Northern blot analysis showed that the level of gspA mRNA was elevated 3.4-, 5.0-, and 6.7-fold after exposure of L. pneumophila to heat shock, oxidative stress and osmotic shock, respectively. The gspA gene was conserved among 13 serogroups of L. pneumophila. Our data showed that the gspA gene of L. pneumophila, which is induced by intracellular infection and by various stress stimuli, is controlled transcriptionally by two overlapping and separately regulated promoters.
嗜肺军团菌的一种全局应激蛋白（GspA）的合成在吞噬细胞的细胞内环境以及各种体外应激刺激下被诱导。我们使用反向遗传学技术从嗜肺军团菌的基因组文库中分离出gspA基因。对一个代表性克隆（pBSP1）约1700 bp的序列分析显示存在两个开放阅读框（ORF）。ORF1编码一种推断分子量为19 kDa且等电点为6.1的多肽。这些预测与GspA蛋白在二维SDS - 聚丙烯酰胺凝胶上的迁移情况相关。GspA蛋白的预测氨基酸序列与通过对纯化蛋白进行埃德曼降解得到的N端氨基酸序列的22/23个残基相同。GspA蛋白分别与大肠杆菌的16 kDa IbpA和IbpB热休克蛋白有41.3%和36.5%的同源性。从嗜肺军团菌分离的mRNA进行引物延伸表明，gspA基因的转录受两个重叠启动子控制。其中一个启动子是σ70启动子，另一个是热休克启动子，在大肠杆菌中受σ32转录因子调控。Northern印迹分析表明，嗜肺军团菌分别在热休克、氧化应激和渗透休克后，gspA mRNA水平分别升高3.4倍、5.0倍和6.7倍。gspA基因在嗜肺军团菌的13个血清群中保守。我们的数据表明，嗜肺军团菌的gspA基因在细胞内感染和各种应激刺激下被诱导，其转录受两个重叠且分别调控的启动子控制。
