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 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 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.

The expression of the flagellum of Legionella pneumophila is modulated by different environmental factors

“…Growth temperature has recently been shown to regulate the £aA expression which is repressed at temperatures higher than 37³C [4,5]. These results could be con¢rmed by determining the light production as shown in Fig.…”

“…During growth of L. pneumophila in YEB, L-galactosidase activity was measured by quantifying the hydrolysis of ONPG as described by Miller [11] and is given in Miller units (MU). Isolation of bacterial RNA and Northern blot analysis using an internal £aA-speci¢c probe was carried out as described previously [4].…”

“…L. pneumophila suspensions were adjusted to an optical density at 600 nm of 1 and 300 Wl of the cell suspension was pelleted by centrifugation then suspended in 50 Wl of SDS sample lysis bu¡er and loaded onto a SDS-13% polyacrylamide gel. Western blots were carried out as described elsewhere [13] using a polyclonal monospeci¢c antibody against L. pneumophila Corby £agellin [4].…”

“…It was shown that L. pneumophila is exclusively £agellated in the ¢nal phase of its intracellular life cycle [1,3]. The £agellin is the major subunit of the £agella of L. pneumophila, and it has recently been shown that various L. pneumophila strains and isolates of species other than L. pneumophila are able to produce £agella [4]. Previous reports from this labo-ratory have shown that the temperature-dependent expression [5] of the gene £aA, encoding the £agellum subunit, is regulated at the transcriptional level [4], possibly directed by the alternative c PV factor [6].…”

“…The £agellin is the major subunit of the £agella of L. pneumophila, and it has recently been shown that various L. pneumophila strains and isolates of species other than L. pneumophila are able to produce £agella [4]. Previous reports from this labo-ratory have shown that the temperature-dependent expression [5] of the gene £aA, encoding the £agellum subunit, is regulated at the transcriptional level [4], possibly directed by the alternative c PV factor [6]. Many bacteria modulate their repertoires of gene expression to cope with various changes in the environment such as nutrients, temperature, cell density and osmolarity.…”