The physical and gentic map of the A. salmonicida A449 chromosome : molecular characterization of recA and a novel fla operon




Umelo, Elizabeth Rhoda Osondu

Journal Title

Journal ISSN

Volume Title



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 fla genes were identified. While the location of all the genes identified as a result of this study were mapped on the chromosome, the recA and fla genes were further characterized at the molecular level. 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 (Mᵣ) 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 acid sequence of A.salmonicida A449 RecA was found to possess a number of domains identical to those characterized in Escherichia coli RecA.These included domains for adenosine triphosphate binding, DNA binding and protein-protein interactions. The A.salmonicida A449 recA was mobilized into an E.coli recA strain and was shown to allow increased survival in the presence of the chemical mutagen methyl methane sulfonate and ultra violet (uv) irradiation. The rate of the A.salmonicida 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.salmonicida A449 recA also possessed a potential regulatory SOS-box in the DNA 5' of the gene. A novel flagellin operon was identified in A.salmonicida A449, characterization of which revealed the presence of two tandemly linked flagellin structural genes flaA and flaB. The flaA and flaB genes were in turn tandemly linked to flaG encoding a protein of unknown function, and flaH encoding a protein homologous to the Hook Associated Protein II of other bacteria. The flaA and flaB genes with 79% nucleotide sequence identity, were conserved in typical and atypical strains of A.salmonicida, and displayed significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA, flaB and flaG encode unprocessed proteins with predicted Ms 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 DH5α, and FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A.salmonicida flagellins are antigenically cross-reactive with A. hydrophila TF7 flagellin(s), and evolutionally closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum.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 Southern blot analysis. The three restriction enzymes used in the map construction were CeuI, Pmel 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 and fla 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 comparison of the CeuI digestion fingerprint of atypical strains revealed a clustering of some strains which suggested that this could be a powerful taxonomic tool for better classification of the atypical group. The two A. sobria strains analyzed with CeuI were also homogenous and showed significant similarities to the A. salmonicida typical strains CeuI genomic fingerprints. In contrast, four A. hydrophila strains yielded CeuI-derived fragments which like 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. salmonicida strains.



Gene mapping, Operons, Salmonidae