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

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Abstract
The expression of several virulence determinants of Y. pestis is known to be dependent on the in vitro growth temperature. One of these, Ca dependence is associated with the presence of a 47 megadalton plasmid. The effects of incubation temperature, Ca in the growth medium and the presence of the 47 megadalton plasmid on the outer membrane protein and the lipopolysaccharide [LPS] composition of Y. pestis EV76 were examined. When cells were grown at 37.degree. C as opposed to 26.degree. C, a change in LPS composition and a decrease in the amount of an outer membrane protein (protein E) were observed. The LPS obtained from cells incubated at 37.degree. C had a lower proportion of 2-keto-3-deoxyoctanate, a lower PO43-:2-keto-3-deoxyoctanate ratio and an increased gel mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] when compared with LPS obtained from cells grown at 26.degree. C. Because of its growth temperature-related abundance, the nature of protein E was investigated. This protein had physical properties similar to those of other enterobacterial porins, including apparent formation of an oligomer in SDS-PAGE 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 LPS and porin components of the outer membrane, the amount of 3 spots in 2-dimensional PAGE was shown to be related to the temperature or the presence of Ca during growth. One of these spots contained residual unmodified portions of 2 major heat-modifiable proteins which failed to shift to their heat-modified positions on gels, despite solubilization at 100.degree. C for 10 min before electrophoresis. The other 2 spots were the heat-modified and unmodified forms of another outer membrane protein (J) which did not appear in the isoelectric focusing gel of cells grown at 37.degree. C. Evidently, the appearance of these spots in 2-dimensional analyses is related to LPS composition of the cells from which the outer membrane is derived and reflects LPS-protein interactions or Ca-protein interactions.