Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK.
Flagella in procaryotes are complex structures requiring the coordinate expression of over 50 genes, including flagellin, the major repeating structural protein. We have previously shown that a functional RpoN gene product is required for expression of flagellin in Pseudomonas aeruginosa PAK (P. A. Totten and S. Lory, J. Bacteriol. 172:389-396, 1990) and have now cloned, sequenced, and determined the transcriptional start site of the structural gene for this flagellin. The clones containing this gene produced a protein that reacted on Western immunoblots with polyclonal and four different monoclonal antibodies to purified flagella. However, this flagellin protein in Escherichia coli was slightly smaller (41 kDa) than flagellin protein produced in P. aeruginosa PAK (45 kDa), indicating degradation in E. coli or modification in P. aeruginosa. Comparison of the deduced amino acid sequence of this gene with the amino acid sequences of other flagellins revealed a conservation in the N- and C-terminal domains, suggesting conservation of secretion or assembly signals between these organisms. The sequence 5' of the structural gene contained potential RpoN-specific promoters as well as a promoter sequence recognized by RpoF (sigma 28), the alternative sigma factor required for expression of flagellin genes in E. coli (and Bacillus subtilis). Deletion analysis of the promoter region as well as transcriptional start site mapping implicated the RpoF, and not the RpoN, consensus sequences as the functional promoter for the flagellin gene. Models for the involvement of both RpoN and RpoF in the expression of flagellin in P. aeruginosa are presented.
Totten
P A
PA
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195.
Lory
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D005408:Flagellin / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012808:Sigma Factor / Q000502:physiology; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D012333:RNA, Messenger; D012808:Sigma Factor; D005408:Flagellin