Molecular analysis of the structure and expression of the Aeromonas Salmonicida surface layer protein gene vapA




Chu, Shijian

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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.



Proteins, analysis, Aeromonas