Influence of culture conditions on expression of the 40-kilodalton porin protein of Vibrio anguillarum serotype O2

Abstract

Vibrio anguillarum serotype O2 strains express a 40-kDa outer membrane porin protein. Immunoblot analysis revealed that antigenic determinants of the V. anguillarum O2 40-kDa porin were conserved within bacterial species of the genus Vibrio. The relative amounts of the V. anguillarum O2 40-kDa porin were enhanced by growth of V. anguillarum O2 in CM9 medium containing 5 to 10% sucrose or 0.1 to 0.5 M NaCl. In contrast, the levels of the porin were significantly reduced when cells were grown at 37 degrees C, and a novel 60-kDa protein was also observed. However, the osmolarity or ionic concentration of the growth medium did not influence expression of the 60-kDa protein. Growth in medium containing greater than 0.6 mM EDTA reduced production of the V. anguillarum O2 40-kDa porin and enhanced levels of a novel 19-kDa protein. Thus, expression of the V. anguillarum O2 40-kDa porin was osmoregulated and possibly coregulated by temperature. The N-terminal amino acid sequence of the V. anguillarum O2 40-kDa protein and the effect of environmental factors on the cellular levels of the porin suggested that the V. anguillarum O2 40-kDa porin was functionally similar to the OmpC porin of Escherichia coli. However, pore conductance assays revealed that the V. anguillarum O2 40-kDa porin was a general diffusion porin with a pore size in the range of that of the OmpF porin of E. coli.

FIG. 1

Effects of growth temperature on expression of the 40-kDa MOMP. V. anguillarum O2 cultures were grown in CM9 for 16 h at the indicated temperatures, and cell suspensions were adjusted to an OD₅₂₀ of 1 (1 × 10⁹ to 3 × 10⁹ CFU/ml). The cell pellet from 1 ml of the suspension was lysed in 60 μl of sample buffer (with 2-ME and SDS) and heated for 10 min at 100°C, and 10-μl samples were loaded in each lane. (A) Coomassie blue-stained gel; (B) blot probed with monospecific MOMP-Ab. The arrowheads indicate the positions of a novel 60-kDa protein and the MOMP. Molecular mass standards are indicated on the left.

FIG. 2

Protein profiles of V. anguillarum O2 cell lysates after growth in EDTA. Cells were grown at 25°C in CM9 containing the indicated concentrations of EDTA. Cell lysates were prepared for SDS-PAGE analysis as described in the legend to Fig. 1. (A) Coomassie blue-stained gel; (B) blot probed with monospecific MOMP-Ab. The arrowheads indicate the positions of the novel 19-kDa protein and the MOMP. Molecular mass standards are indicated on the left.

FIG. 3

Expression of the 40-kDa MOMP is enhanced by increased medium osmolarity and salt concentration. V. anguillarum O2 cell cultures were grown at 25°C in CM9 medium containing increasing concentrations of NaCl (A) and sucrose (B and C). Cell lysates for SDS-PAGE were prepared as described in the legend to Fig. 1. (A and C) Blots probed with monospecific MOMP-Ab. (B) SDS-PAGE of lysates of cells grown in increasing sucrose concentrations. Molecular mass standards are indicated on the left.

FIG. 4

SDS-PAGE of the 40-kDa MOMP of V. anguillarum O2 at different stages of purification. Lanes: 1, sarcosine-insoluble outer membrane protein pellet; 2, SDOC-soluble extract of the outer membrane protein pellet; 3, SDOC-insoluble outer membrane protein; 4 and 5, supernatant and insoluble pellet, respectively, obtained from a second extraction of the insoluble outer membrane protein with SODC buffer; 6, NaCl-SDOC-insoluble outer membrane protein; 7, NaCl-SDOC-soluble 40-kDa MOMP. Protein samples were treated at 100°C for 10 min in sample buffer, and 20 μg of protein was loaded in lanes 1, 3, 5, 6, and 7. Lanes 2 and 4 contained the maximum volume of sample that could be loaded in each lane. Molecular mass standards are indicated on the left. The MOMP is indicated on the right.

FIG. 5

Transition from monomer to trimer of the V. anguillarum O2 MOMP is dependent on temperature and SDS. Purified MOMP (4 μg of protein/lane) was mixed with sample buffer containing the indicated combinations of SDS and 2-ME. SDS-PAGE of MOMP samples treated at 100°C for 10 min (A) and of MOMP samples treated at the indicated temperatures for 10 min in sample buffer containing SDS and 2-ME (B) and 2-ME without added SDS (C) is shown. The apparent molecular masses of the MOMP conformational forms are shown on the right. Molecular mass standards are indicated on the left.