title: The pilE gene of Neisseria gonorrhoeae MS11 is transcribed from a sigma 70 promoter during growth in vitro.
abstract: Type 4 pili are essential for virulence in Neisseria gonorrhoeae. The gonococcal pilin subunit is encoded by pilE, upstream of which three putative promoter sequences (P1, P2, and P3) have been identified. P1 and P2 are sigma 70-like promoters and are functional when a PpiE::cat transcriptional fusion is expressed in Escherichia coli DH5 alpha. P3 is sigma 54 dependent and overlaps the P1 sequence. Site-directed mutagenesis of the pilE promoters followed by transcriptional analysis in E. coli indicated that in the absence of an appropriate activator protein, binding of RNA polymerase-sigma 54 to P3 inhibits transcription from P1 on the order of 30-fold. Transcription from P3 was undetectable in E. coli. However, PilR-dependent, P3-associated expression was detected in Pseudomonas aeruginosa PAK containing a PpilE::cat fusion, with P3 the only intact promoter. A similar analysis was performed on gonococcal reporter strains containing wild-type and mutated PpilE::cat cassettes recombined into the chromosome. In such piliated gonococcal recombinants cultured in vitro, P1 was responsible for cat expression and almost certainly for transcription of pilE. Transcription from P2 and P3 was not detectable under these conditions. Inhibition of transcription from P1 by sigma 54 binding to P3 was not apparent in N. gonorrhoeae MS11-A, suggesting that sigma 54 was either absent or unable to bind to P3 in these cells.
authors.affiliation: Department of Microbiology, Monash University, Clayton, Victoria, Australia.
authors.forename: J A
authors.initials: JA
authors.lastname: Fyfe
authors.forename: C S
authors.initials: CS
authors.lastname: Carrick
authors.forename: J K
authors.initials: JK
authors.lastname: Davies
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003470:Culture Media; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D007070:Immunoglobulin A / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009344:Neisseria gonorrhoeae / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D018933:Pili, Sex / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011550:Pseudomonas aeruginosa / Q000235:genetics; D051877:RNA Polymerase Sigma 54; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D007070:Immunoglobulin A; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; C074063:pilE protein, Neisseria gonorrhoeae; C497751:rpoN protein, E coli; D036781:Fimbriae Proteins; D015500:Chloramphenicol O-Acetyltransferase; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54