Expression, Characterization, And Mutant Construction Of Oligopeptide Permease From Vibrio Harveyi

Vibrio harveyi is a Gram-negative bacterium, which widely existed in marine environments and marine animals such as fish and crustaceans. The bacterium has been reported as major causal agent of luminous vibriosis, which affected a diverse range of marine invertebrates. Mass mortalities of shrimp larvae due to V. harveyi infection have been reported from a number of countries. V. harveyi has also been associated with fish diseases in China and South Asian countries.Some Gram-negative bacteria such as E.coli have three peptide uptake systems with varying substrate specificities, allowing them to utilize a broader range of peptides for nutrition purpose: dipeptide permease (Dpp), tripeptide permease (Tpp) and oligopeptide permease (Opp), which handles peptides of two to five residues. Opp is ATP-binding cassette transporter, and composed of five subunits: two transmembrane protein (OppB, OppC), two ATP binding proteins (OppD, OppE) and an oligopeptide binding protein (oligopeptide permease A, OppA). Studies have revealed that bacterial oppA is a solute-binding protein (SBP) and plays an important role in transport of oligopeptides into the cell and in various signaling processes. Increasing evidence indicated that the ABC type transporter proteins take part in the bacterial pathogenic processes.An oligopeptide permease A (oppA) gene was cloned from the genome of V. harveyi SF-1.A. Sequence analysis showed that the cloned gene consisted of 1665 base pairs and encoded a 554 amino acid protein. It showed high similarity to the OppA of V.vulnificus and the ABC-type oligopeptide transporter sequences from V.parahaemolyticus and V.fluvialis. The gene was subcloned into pET-28a (+) and expressed in Escherichia coli. The expressed recombinant protein was purified by Ni-NTA metal affinity chromatography and showed a 58 kDa band on SDS-PAGE. The protein exhibited phospholipase C activity. The optimum temperature and pH was 55 degree centigrade and 8.0, respectively. It was stable at pH 6-9 and below temperature of 55 degree centigrade. In addition, the recombinant protein exhibited lecithin activity and hemolytic activity on the TSB plates adding lecithin and LB plates coated with sheep erythrocytes. A oligopeptide permease mutant of V. harveyi SF-1 was constructed by homologous recombination. Virulence of the oppA mutant strain was also decreased when they were intraperitoneally inoculated into zebra fish, the LD50 of the wild-type and the mutant were 3.11×104 and 5.46×105 CFU per fish respectively. These results indicated that the oppA of V. harveyi played important roles in bacterial pathogenicity.