Cloning and molecular characterization of a Legionella pneumophila gene induced by intracellular infection and by various in vitro stress conditions.
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.
Abu Kwaik
Y
Y
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109-0620.
Engleberg
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NC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D016952:Legionella pneumophila / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010587:Phagocytosis; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000502:physiology; D014157:Transcription Factors
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C090745:global stress protein A, bacteria; C069774:heat-shock sigma factor 32