Tyrosine phosphorylation of the tetragonal paracrystalline array of Aeromonas hydrophila: molecular cloning and high-level expression of the S-layer protein gene.
High virulence strains of the fish pathogenic bacterium Aeromonas hydrophila produce a tetragonally arranged paracrystalline surface protein array (S-layer). The gene (ahsA) encoding the S-protein subunit of A. hydrophila TF7 was cloned into lambda EMBL 3, and sub-cloned into pUC 18. Transformation into Escherichia coli led to stable high-level expression of full-size S-protein under the control of its native promoter. The DNA sequence revealed a 1406 base-pair open reading frame encoding a protein consisting of a 19 amino acid residue signal peptide, and a 448 residue 45,400 Da molecular mass mature protein with a predicted isoelectric point (pI) of 6.72 compared with the measured M(r) of 52,000 and pI of 4.6. This suggested that the S-protein was post-translationally modified and in vivo cell labelling with [32P]orthophosphate, acid phosphatase digestion of S-protein, ascending thin-layer chromatography of partially acid hydrolysed S-protein and Western blot analysis with monoclonal anti-phosphotyrosine antibody showed that the S-protein of strain TF7 contained phosphotyrosine. S-proteins produced by the other strains of motile aeromonads tested also reacted with this anti-phosphotyrosine antibody. Cell fractionation studies employing plasmid-encoded ahsA showed that in A. hydrophila the S-protein subunits were secreted across the outer membrane by the native S-protein secretion pathway, while in E. coli and A. salmonicida the cloned A. hydrophila S-protein inserted into the outer membrane of the foreign host. These findings indicate that the process employed to translocate Aeromonas S-proteins across the outer membrane is highly specific.
Thomas
S R
SR
Department of Biochemistry and Microbiology, University of Victoria, B.C., Canada.
Trust
T J
TJ
D000135:Acid Phosphatase / Q000378:metabolism; D016980:Aeromonas hydrophila / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014443:Tyrosine / Q000276:immunology / Q000378:metabolism*
C091781:AhsA protein, Aeromonas hydrophila; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014443:Tyrosine; D000135:Acid Phosphatase