Aeromonas calmonicida is a Gram negative rod shaped bacterium capable
of causing furunculosis in salmonid fish and other chronic and inflammatory
diseases in goldfish, carp and also salmonids. The surface layer of A. salmonicida, the A-layer, has been demonstrated to be a major virulence factor
for the organism , and its subunit A -protein has been purified and its
Structural gene vapA has been cloned.
The vapA gene from A. salmonicida strain A450 was subcloned (pSC150)
and expressed in Escherichia coli. Its DNA sequence was then determined to
consist of 1,506 bp encoding a 502-amino acid residue protein, containing a 21-
residue signal peptide and a mature protein of 50,778 Dalton. The A-protein
assembled on the cell surface in the form of an S-layer was refractile to trypsin
cleavage while trypsin digestion of the purified mature protein revealed a
highly resistant 39,400 Dalton N-terminal fragment and a 16,700 Dalton C-terminal fragment with moderate resistance, These trypsin-resistant
fragments may form distinct structural domains, consistent with three-dimensional ultrastructural observations.
The plasmid pSC150 contained 62 bp of Aeromonas DNA in front of the
vapA structural gene. A promoter (P2) was predicted in this region which
showed sequence homology to the E. coli c70 promoter. However, prim er
extension in the wild type strain A449 showed a transcriptional start site 181
bp upstream from the gene, and thus, another promoter (P1) was shown to be
the major promoter. The DNA sequence coding for the untranslated leader
mRNA contained two stem-loop structures, a putative small open reading
frame spanning the stem-loop structures, and a palindromic sequence which
overlaps the predicted ribosome binding site. Northern analyses of A449 vapA
mRNA showed that incubation at 15°C produced the highest level of the
transcript, and the transcript half-life was 22 m in in cells grown at 15°C
compared to 11 min in cells grown at 20°C. DNA gyrase inhibitors nalidixic
acid and novobiocin significantly reduced the vapA transcript level.
A. salmcnicida 30°C mutants were found to produce significantly
reduced levels of A-protein and some of them were shown to have the native
insertion elements, ISA1 and ISA2, inserted in the vapA area. These insertion
elements have been cloned and sequenced, and also identified in the wild
type strains A449 and A450. ISA2 was shown to have sequence similarity to
other bacterial insertion elements.
Plasmid encoded vapA expression in E. coli was also affected by a
downstream gene abcA, which, when deleted from the clone, significantly
reduced vapA expression. This reduction could be complemented by the abcA
gene carried on a second plasmid. In addition, the lipopolysaceharide (LPS) O-chain
deficient phenotype of A449 mutant strain TM4, which has the abcA
gene interrupted by ISA1, was also complemented by abcA. DNA sequence
analysis showed that the abcA gene coded for a 308 amino acid residue
protein, which was confirmed by in vivo and in vitro expression and gene
fusion with lacZ, and was localized in the inner membrane fraction of E. coli.
At the N-terminal part of the protein, the predicted sequence of AbcA
displayed high homology with a bacterial transport protein super family,
including a well conserved nucleotide binding sequence. This binding
sequence was shown by site-directed mutagenesis to be required for LPS O-chain
complementation in TM4. ATP binding activity was confirmed in the
purified AbcA-LacZ fusion protein. A leucine zipper-basic region sequence
with predominantly α-helical conformation was predicted further
downstream, with leucine residues in four of the five heptad repeats and a
valine residue in the remaining heptad repeat. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9646 |
| Date | 09 July 2018 |
| Creators | Chu, Shijian |
| Contributors | Trust, Trevor J. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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