The physical and genetic map of the A. salmonicida A449 chromosome, molecular characterization of reca and a novel fla operon

by Umelo, Elizabeth Rhoda

Aeromonas salmonicida is a Gram negative pathogenic fish bacterium. To facilitate the construction of the chromosomal map of A. salmonicida A449, several previously uncharacterized genes, including recA and four fZa genes were identified. While the location of al1 the genes îdentified as a result of this study were mapped on the chromosome, the recA and fln genes were further characterized at the molecular Ievel. The A. salmonicida A449 recA was cloned, sequenced and expressed in vitro. The 1059 bp recA open reading frame encoded a 353 amino acid protein with predicted molecular weight (Y) of 37,900. Southern blot analysis was performed to demonstrate the high degree of conservation between the A449 recA and those of the other typical and atypical strains of A. salmonicida examined. The predicted amino aad sequence of A. salmonicida A449 RecA was found to possess a number of domains identical to those characterized in Escherichia col i RecA. binding, DNA binding These included domains for adenosine triphosphate and protein-protein interactions. The A. sa1 mo n icida A449 recA was mobilized into an E. coli recA strain and was shown to allow increased sumival in the presence of the chernical mutagen methyl methane sulfonate and ultra violet (UV) irradiation. The rate of the A. snlmonicidn A449 recA-mediated recombination in E. coli was increased by exposure to UV light, which suggested that SOS induction in A. salmonicida paralleled that of E. coli. The A. salrnonicida A449 recA also possessed a potential regulatory SOSbox in the DNA 5' of the gene. A novel flagellin operon was identified in A. snlmonicida A449, characterization of which revealed the presence of two tandemly linked flagelh structural genes fZaA and flaB. The JaA and flaB genes were in turn tandemly linked to flaG encoding a protein of unknown hction, and flaH encoding a protein homologous to the Hook Associated Protein II of other bacteria. The f[aA and flaB genes with 79% nucleotide sequence identity, were consewed in typical and atypical strains of A. salmonicida, and displayed significant divergence at the nucleotide level from the f7a genes of the motile speàes Aeromonas hydrophila and Aeromonas veronii biotype sobria. fIaA, PaE3 andf7aG encode unprocessed proteins with predicted of 32,351, 32,056 and 15,965 respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in E. coli DH5a, and FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when unuiduced and poorly expressed on induction. The A. salmonicida flagellins are antigenically crossreactive with A. hydrophila TF7 flageh(s), and evolutionally closely related to the flagellins of Pseudomonas aeruginosa and Vibrio angzr illariîrn. Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at extremely low frequency. Finally, the physical and genetic map of the chromosome of A. salmonicida A449 was constructed using pulsed-field gel electrophoresis and Southem blot analysis. The three restriction enzymes used in the map construction were CeuI, PmeI and PacI. The chromosome of A. salmonicida A449, with an estimated size of 4,658 + 29.75 kb, was determined to be circular in structure. Several genes of A. salmonicida, including those which encoded proteins implicated in virulence, were localized on the chromosome map. The chromosomal locations of the recA andfla genes were also identified. The global genomic relationship between the typical and atypical strains of A. salmonicida was investigated by comparing the CeuI cleavage fingerprint of the respective genomes. The results showed that the typical strains were indeed very homogenous as had been previously reported. The atypical strains expressed extensive variation both in the number of DNA fragments obtained with CeuI and also in the digestion fingerprint. The cornparison of the CeuI digestion fingerprint of atypical strains revealed a dustering of some strainç whidi suggested that this could be a powerful taxonomie tool for better classification of the atypical group. The two A. sobrin strains analyzed with CeicI were ako homogenous and showed significant similarities to the A. salmon icidn typical strains Ceri1 genomic fingerprints. In contrast, four A. hydrophila strains yielded Cezdderived fragments which !ike the atypical strain varied both in number and patterns. There was also minimal observed similarities between the genome of A. hydrophila strains and the A. sahonicida strains.