title: Functional analysis of secA homologues from rickettsiae.
abstract: The molecular basis of protein secretion that underlines rickettsial pathogenesis remains unknown. This paper reports the molecular and functional analysis of the putative secA gene, an essential component of the Sec-dependent protein secretion pathway, from Rickettsia rickettsii and Rickettsia typhi, the aetiological agents of Rocky Mountain spotted fever and murine typhus, respectively. The sequence analysis of the cloned secA genes from R. rickettsii and R. typhi show ORFs of 2721 and 2718 nt, respectively. Alignment of the deduced amino acid sequences reveals the presence of highly conserved amino acid residues and motifs considered to be essential for the ATPase activity of SecA in preprotein translocation. Transcription analysis indicates that R. rickettsii secA is expressed monocistronically from the canonical prokaryotic promoter, with a transcriptional start point located 32 nt upstream of the secA initiation codon. Complementation analysis shows that the full-length SecA protein from R. rickettsii and R. typhi fails to restore growth of the temperature-sensitive Escherichia coli strain MM52 secA51(ts) at a non-permissive temperature (42 degrees C), despite the detection of SecA protein expression by Western blotting. However, the chimeric SecA protein carrying the N-terminal 408 aa of R. rickettsii SecA fused with the C-terminal 480 aa of E. coli SecA restores the growth of E. coli strain MM52 secA51(ts) at the non-permissive temperature (42 degrees C). These results suggest that the N-terminal ATPase domain is highly conserved, whereas the C-terminal domain appears to be species specific.
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB: 13-009, Baltimore, MD 21201, USA.
authors.forename: M Sayeedur
authors.initials: MS
authors.lastname: Rahman
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB: 13-009, Baltimore, MD 21201, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Simser
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB: 13-009, Baltimore, MD 21201, USA.
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Macaluso
authors.affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB: 13-009, Baltimore, MD 21201, USA.
authors.forename: Abdu F
authors.initials: AF
authors.lastname: Azad
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012284:Rickettsia rickettsii / Q000235:genetics / Q000378:metabolism*; D012286:Rickettsia typhi / Q000235:genetics / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins