Isolation and characterization of the outer membrane from Vibrio parahaemolyticus
Related Experiment Videos
Abstract:
The outer membrane of Vibrio parahaemolyticus strain 3283-61 (serotype O2:K3) was isolated from blebs released upon spheroplast formation, in the presence of lysozyme and EDTA, by isopycnic sucrose density gradient centrifugation. SDS-PAGE of the outer membrane fraction prepared from cells grown in nutrient broth containing 3% (w/v) NaCl revealed five major proteins, designated a to e, with apparent approximate molecular weights: a, 44 000; b, 36 000; c, 33 500; d, 26 500; e, 22 000. An increase in NaCl concentration in the growth medium resulted in an increase of proteins b and c, whereas a decrease to 0.5% (w/v) induced two additional major proteins with respective molecular weights of about 35 000 and 32 000. Proteins a and b appeared to be loosely associated with the peptidoglycan layer since they were largely retained after extraction with 2% (w/v) SDS at 50 degrees C for 30 min. Proteins c and/or e may play a role in phage VP1-receptors since phage-resistant mutants derived from strain 3283-61 had significantly diminished amounts of both proteins. The major outer membrane proteins varied in number and molecular weight in strains of V. parahaemolyticus belonging to different K-serotypes.
Insights
Outer membrane proteins of Vibrio parahaemolyticus are influenced by salt concentration and may act as phage receptors. Changes in these proteins were observed in different K-serotypes, impacting phage resistance.
Area of Science:
- Microbiology
- Bacterial Outer Membrane Proteins
- Vibrio parahaemolyticus Research
Background:
- The outer membrane of Vibrio parahaemolyticus plays a crucial role in bacterial structure and interaction.
- Understanding the composition and regulation of outer membrane proteins is vital for comprehending bacterial physiology and pathogenicity.
Purpose of the Study:
- To characterize the major outer membrane proteins of Vibrio parahaemolyticus strain 3283-61 (serotype O2:K3).
- To investigate the effect of varying sodium chloride (NaCl) concentrations on the expression of these proteins.
- To explore the potential role of specific outer membrane proteins as receptors for bacteriophage VP1.
Main Methods:
- Isolation of outer membrane blebs using lysozyme and EDTA, followed by spheroplast formation.
- Purification of outer membrane fractions using isopycnic sucrose density gradient centrifugation.
- Analysis of protein profiles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Main Results:
- Five major outer membrane proteins (a-e) with distinct molecular weights were identified in V. parahaemolyticus strain 3283-61 grown in 3% NaCl.
- Increased NaCl concentration enhanced the levels of proteins b and c, while decreased NaCl induced two new major proteins.
- Proteins a and b showed loose association with the peptidoglycan layer.
- Proteins c and/or e were significantly diminished in phage-resistant mutants, suggesting their role as phage VP1 receptors.
Conclusions:
- The composition and abundance of major outer membrane proteins in Vibrio parahaemolyticus are significantly influenced by environmental NaCl concentrations.
- Specific outer membrane proteins (c and/or e) are implicated as receptors for bacteriophage VP1.
- Variations in outer membrane protein profiles exist among different K-serotypes of V. parahaemolyticus.