Characterization of the outer membrane proteins of Bordetella avium

Leyh, Randy
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The outer membrane proteins (OMPs) of Bordetella avium were examined by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE). Profiles of Sarkosyl-insoluble OMPs from 50 virulent B. avium isolates were very similar with major 21,000- and 37,000-molecular-weight proteins (21K and 37K proteins) and at least 13 less-intensely stained proteins with molecular weights ranging from 13,500 to 143,000. Both major proteins were heat-modifiable as indicated by increased mobility when solubilized at higher temperatures prior to SDS-PAGE. The major proteins and the 27K and 31K OMPs were noncovalently associated with the underlying peptidoglycan. It was necessary to treat cell envelopes with 2% SDS and temperatures in excess of 60°C for 15 min to release the proteins from the peptidoglycan layer. Cell-surface accessibility to radioiodination was determined by labeling whole cells with [superscript]125I followed by SDS-PAGE. At least 13 bands were present in profiles from surface-labeled cells. Of the surface-labeled bands, eight corresponded to bands in a radiolabeled outer membrane preparation. The most prominent surface-labeled band corresponded to the major 37K OMP band. The OMP profile of B. avium was compared to OMP profiles from other Bordetella spp., including B. avium-like organisms and B. bronchiseptica strains isolated from turkeys. The OMP profile of B. avium was distinct from profiles of the other bordetellae. The effect of variations in growth medium on the expression of OMPs of B. avium was examined. Expression of the 22K, 24K, and 56K OMPs was decreased by addition of either 20 mM MgSO[subscript]4 or 500 ug of nicotinic acid per ml of medium;The amino acid composition and amino-terminal amino acid sequence of the major 21K OMP were determined. Proteins in an OMP-enriched preparation were separated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane. The band containing the 21K OMP was excised and used for amino acid analysis and amino-terminal sequencing. Aspartic acid and asparagine, glutamic acid and glutamine, and alanine were the most prevalent amino acids in the 21K protein. An amino-terminal sequence of QTVDN was obtained.

Veterinary microbiology and preventive medicine, Veterinary microbiology