Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida.
The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.
Chu
S
S
Department of Biochemistry, University of Victoria, British Columbia, Canada.
Cavaignac
S
S
Feutrier
J
J
Phipps
B M
BM
Kostrzynska
M
M
Kay
W W
WW
Trust
T J
TJ
D000333:Aeromonas / Q000235:genetics*; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D015183:Restriction Mapping; D037521:Virulence Factors
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008562:Membrane Glycoproteins; C055764:S-layer proteins; D037521:Virulence Factors