Effects of growth temperature, 47-megadalton plasmid, and calcium deficiency on the outer membrane protein porin and lipopolysaccharide composition of Yersinia pestis EV76

Abstract

The expression of several virulence determinants of Yersinia pestis is known to be dependent of the in vitro growth temperature. One of these, calcium dependence, is associated with the presence of a 47-megadalton plasmid. We have examined the effects of incubation temperature, calcium in the growth medium, the presence of the 47-megadalton plasmid on the outer membrane protein, and the lipopolysaccharide composition of Y. pestis EV76. When cells were grown at 37°C as opposed to 26° C, a change in lipopolysaccharide composition and a decrease in the amount of an outer membrane protein (protein E) were observed. The lipopolysaccharide obtained from cells incubated at 37° C had a lower proportion of 2 keto-3-deoxyoctanate, a lower phosphate to 2-keto-3-deoxyoctanate ratio, and an increased gel mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis when compared with lipopolysaccharide obtained from cells grown at 26°C. Because of its growth temperature-related abundance, we investigated the nature of protein E. This protein had physical properties similar to those of other enterobacterial porins, including apparent formation of an oligomer on sodium dodecyl sulfate-polyacrylamide gels when solubilized at low temperature, acidic isoelectric point, and strong noncovalent association with the peptidoglycan. Protein E was purified and shown to form an aqueous channel in planar lipid membranes with a conductance of 1.1 nS in 1 M KCl. In addition to growth temperature-related alterations in the lipopolysaccharide and porin components of the outer membrane, the amount of three spots in two-dimensional polyacrylamide gels was shown to be related to the temperature or the presence of calcium during growth. One of these spots was shown to contain residual unmodified portions of two major heat-modifiable proteins which failed to shift to their heat-modified positions on gels, despite solubilization at 100° C for 10 min before electrophoresis. The other two spots were the heat-modifie d and unmodified forms of another outer membrane protein (J) which did not appear in the isoelectric focusing gel of cells grown at 37°C. It is proposed that the appearance of these spots in two-dimensional analyses is related to the lipopolysaccharide composition of the cells from which the outer membrane is derived and reflects lipopolysaccharide-protein interactions or calcium-protein interactions.