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Biosynthesis of a plasmid-encoded outer membrane surface exclusion protein involves processing from a precursor
Abstract:
MRB is a major R222 plasmid-encoded outer membrane protein previously described by this laboratory which is immunologically identical with F plasmid traTp. We have detected an apparent precursor polypeptide of MRB in Escherichia coli minicells which reacts with specific anti-MRB serum. In the presence of proteolytic inhibitors, immunologically reactive precursor MRB polypeptides are found in whole cells and minicells containing R222 or F plasmids. When R222-containing minicells were incubated for short periods of time with [35S]methionine, the precursor was predominantly labeled but was shown to be converted into MRB during subsequent incubation in media containing nonradioactive methionine. This conversion was inhibited in the presence of the proteolytic inhibitor tosyllysylalanyl chloramethyl ketone. These studies show that at least one plasmid-encoded membrane protein is processed in its E. coli host in a way resembling that of host cell membrane proteins.
Insights
Researchers identified a precursor to the R222 plasmid-encoded outer membrane protein MRB in Escherichia coli. This precursor is processed into mature MRB, similar to host cell membrane protein processing.
Area of Science:
- Molecular Biology
- Microbiology
- Protein Biochemistry
Background:
- MRB (Major R222 plasmid-encoded outer membrane protein) is an outer membrane protein encoded by the R222 plasmid.
- MRB is immunologically identical to the traTp protein of the F plasmid.
Purpose of the Study:
- To investigate the processing of the MRB protein in Escherichia coli.
- To determine if plasmid-encoded membrane proteins undergo processing similar to host cell proteins.
Main Methods:
- Detection of MRB precursor using specific anti-MRB serum in Escherichia coli minicells.
- Use of proteolytic inhibitors to stabilize precursor polypeptides.
- Pulse-chase experiments with [35S]methionine to track precursor conversion to mature MRB.
- Inhibition studies using tosyllysylalanyl chloramethyl ketone.
Main Results:
- An apparent precursor polypeptide of MRB was detected in Escherichia coli minicells.
- The precursor was found in cells containing R222 or F plasmids, especially in the presence of proteolytic inhibitors.
- Pulse-chase experiments demonstrated the conversion of the precursor to mature MRB.
- This conversion was inhibited by tosyllysylalanyl chloramethyl ketone, indicating a proteolytic processing step.
Conclusions:
- Plasmid-encoded outer membrane proteins, such as MRB, are processed in Escherichia coli.
- The processing mechanism for MRB resembles that of host cell membrane proteins.
- This suggests a conserved pathway for membrane protein maturation in bacteria.