Abstract
Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na2CO3, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14). Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i) integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries); (ii) peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries); (iii) cytoplasmic or ribosomal membrane-contaminating proteins (80 entries). Thirty-one proteins were experimentally associated with the outer membrane (OM). Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM) proteins with two or more predicted transmembrane domains (TMDs) were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.
Highlights
Yersinia pestis, a Gram-negative bacterium, is the causative agent of the bubonic and pneumonic plague
Experimental approaches to assess protein association with Y. pestis membranes As illustrated in Figure 1, six subcellular fractions were isolated from Y. pestis KIM6+ cell lysates
Fractions derived from cells grown to stationary phase at 26°C were subjected to extensive analyses via differential 2D gel dis
Summary
A Gram-negative bacterium, is the causative agent of the bubonic and pneumonic plague. The genetically unstable chromosomal 102kb pgm locus is important for full virulence of the bubonic plague in mammals and for transmission via blocked fleas [17,18] It encodes the yersiniabactin siderophoredependent iron transport (Ybt) system [19,20] and the Hms-dependent biofilm system. Gene expression and proteomic studies have demonstrated that numerous plasmid- and pgm locus-encoded genes are differentially expressed at 26–30 vs 37°C [2326] and during temperature transition [27] Some of these changes are in agreement with specific functional roles of the encoded proteins in one of the two life stages [24,25,27]
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