Demonstration of a peptidoglycan-linked lipoprotein and characterization of its trypsin fragment in the outer membrane of Brucella spp.
The sodium dodecyl sulfate (SDS) extraction-trypsin digestion protocol used by Braun and Sieglin (V. Braun and U. Sieglin, Eur. J. Biochem. 13:336-346, 1970) to show the peptidoglycan-linked lipoprotein of Escherichia coli was applied to both Brucella abortus and E. coli. Whereas a single polypeptide of 8,000 molecular weight was obtained from E. coli, several proteins of apparent molecular weight lower than 35,000 were demonstrated by SDS-polyacrylamide gel electrophoresis in B. abortus. These results did not change when the trypsin digestion conditions were modified. On the other hand, when the SDS extractions were performed under conditions more stringent than those used for other gram-negative bacteria, only a polypeptide fragment of apparent molecular weight of 8,000 was obtained from B. abortus. This polypeptide was similar to the trypsin fragment of the E. coli lipoprotein with respect to its behavior in SDS-polyacrylamide gels, isoelectric point in urea, molecular weight, and presence of both ester- and amide-linked fatty acids. Moreover, the amino acid analysis showed an overall similarity with respect to the amino acid composition of E. coli lipoprotein. A polypeptide of the same molecular weight, isoelectric point, and amino acid composition was also obtained from Brucella ovis by the same method. These results demonstrated that B. abortus and B. ovis cell envelopes contain a lipoprotein and strongly support the hypothesis that it is the only major protein covalently linked to the peptidoglycan.
D000596:Amino Acids / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D002002:Brucella / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D008074:Lipoproteins / Q000032:analysis*; D008970:Molecular Weight; D010457:Peptidoglycan / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D008074:Lipoproteins; D010457:Peptidoglycan; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin