The physical and gentic map of the A. salmonicida A449 chromosome : molecular characterization of recA and a novel fla operon
Date
2017-08-10
Authors
Umelo, Elizabeth Rhoda Osondu
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Abstract
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.
Description
Keywords
Gene mapping, Operons, Salmonidae