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The filA (rpoF) gene of Pseudomonas aeruginosa encodes an alternative sigma factor required for flagellin synthesis
Author(s) -
Starnbach M. N.,
Lory S.
Publication year - 1992
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1992.tb01490.x
Subject(s) - biology , sigma factor , flagellin , rpon , gene , genetics , microbiology and biotechnology , gene expression , promoter
Summary In order to better understand the regulation of Pseudomonas aeruginosa flagellin expression we cloned the sigma factor of RNA polymerase used to transcribe the flagellin gene. It is a member of the σ 28 class of alternative sigma factors described in several bacterial genera. Using the published sequence of the filA gene encoding the σ 28 from Salmonella typhimurium , we designed two oligonucleotides and, using the polymerase chain reaction, isolated the fliA gene from S. typhimurium chromosomal DNA. This heterologous probe was used in the DNA blot analysis of restriction digests of P. aeruginosa DNA. A 1.7 kb SalI‐EcoRI fragment reacted with the probe and this fragment was cloned into the pBluescript vectors. The P. aeruginosa fliA gene was able to complement the motility defect of an Escherichia coli fliA mutant, but only when transcription was driven from the vector promoter. Insertional inactivation of the fliA gene with a gentamicin gene cassette rendered P. aeruginosa non‐motile and unable to express the flagellin gene. The 1.7 kb cloned fragment was sequenced and shown to contain the entire fliA gene. P. aeruginosa FliA shares 67% amino acid similarity with the homologous S. typhimurium sequence. Transcriptional analysis of the fliA gene showed that its expression was not dependent on RpoN, a sigma factor shown also to be required for flagellin synthesis. A reading frame downstream of fliA was found to encode the P. aeruginosa homologue of the enterobacterial cheY gene.