Analysis of the envelope proteins of heat-shocked Vibrio parahaemolyticus cells by immunoblotting and biotin-labeling methods.
Vibrio parahaemolyticus, a common enteropathogen in tropical and subtropical coastal regions, exhibits significant adaptive acid tolerance response and heat-shock response, and the envelope proteins induced by stresses are suggested to be associated with virulence. This work examined the heat-shock proteins located in the envelope of V. parahaemolyticus by two rapid methods; namely, the immunoblotting and biotin-labeling methods. The bacterial cells were cultured at 25 C and heat shocked at 37 or 42 C for 1 or 2 hr. The cells were first lysed, then proteins were separated by gel electrophoresis and probed with antiserum raised against heat-shocked cells. Next, the heat-shocked cells were examined by labeling with water soluble sulfo-NHS-LC-biotin. Proteins of 33, 61, 66, 71, 78, 92 and 101 kDa were induced, while 55, 86, 102, 120 and 160 kDa proteins were markedly enhanced in the envelope of the heat-shocked V. parahaemolyticus cells. The biotin tagged envelope proteins were purified using a monomeric avidin column, and the N-terminal sequence was determined and compared with other high identity protein sequences. The sequence results suggest that Vph1 (55 kDa), Vph2 (46 kDa) and Vph3 (42 kDa) are de novo synthesized heat-shock proteins located in the envelope of this pathogen, and the functions of these proteins in stress protection and virulence have yet to be determined.
Wong
Hin-chung
HC
Department of Microbiology, Soochow University, Taipei, Taiwan 11102, Republic of China. wonghc@mail.scu.edu.tw
Chen
Yu-Chih
YC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D006360:Heat-Shock Proteins / Q000378:metabolism*; D018869:Heat-Shock Response; D006358:Hot Temperature; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D014736:Vibrio parahaemolyticus / Q000378:metabolism / Q000502:physiology*
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D001710:Biotin