Cloning and genetic characterization of the flagellum subunit gene (flaA) of Legionella pneumophila serogroup 1

Abstract

The gene flaA, encoding the flagellum subunit protein of Legionella pneumophila serogroup 1, has been isolated from an expression library of L. pneumophila isolate Corby in Escherichia coli K-12 by using an antiflagellin specific polyclonal antiserum. DNA sequence analysis of the flaA gene revealed the presence of a 1,428-bp open reading frame encoding a protein of 475 amino acids with an apparent molecular mass of 48 kDa that is expressed independently of an E. coli vector promoter. Peptide sequencing of the N terminus of the isolated flagellum subunit protein confirmed that this open reading frame encodes the flagellin. By comparing the FlaA amino acid sequence with those of flagellins of various other bacteria, high degrees of homology in the N-terminal and C-terminal amino acids could be observed. The flaA-specific mRNA was determined to be 1.6 kb in size, the expected size of a monocistronic mRNA. Temperature-dependent expression of flagellin was found to be regulated at the transcriptional level. Sequence analysis and primer extension experiments indicated that the transcription of the gene flaA is directed by a sigma 28-like RpoF-FliA factor. By using fliA and fliA+ E. coli K-12 mutants, it was shown that flaA expression in E. coli required the sigma 28 factor. A flaA-specific DNA probe hybridizes with genomic DNA isolated from L. pneumophila and with most of the genomic DNAs from non-L. pneumophila Legionella strains. Two L. pneumophila strains and isolates of Legionella bozemanii and Legionella feeleii (serogroup 1) carry flaA-specific sequences but were not able to produce flagella.