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TrbK, a small cytoplasmic membrane lipoprotein, functions in entry exclusion of the IncP alpha plasmid RP4
Abstract:
TrbK is the only plasmid-encoded gene product involved in entry exclusion of the broad-host-range plasmid RP4. The corresponding gene, trbK, coding for a protein of 69 amino acid residues maps in the Tra2 region within the mating pair formation genes. TrbK carries a lipid moiety at the N-terminal cysteine of the mature 47-residue polypeptide. The mutant protein TrbKC23G cannot be modified or proteolytically processed but still acts in entry exclusion with reduced efficiency. An 8-amino-acid truncation at the C terminus of TrbK results in a complete loss of the entry exclusion activity but still allows the protein to be processed. TrbK localizes predominately to the cytoplasmic membrane. Its function depends on presence in the recipient cell but not in the donor cell. TrbK excludes plasmids of homologous systems of the P complex; it is inert towards the IncI system. The likely target for TrbK action is the mating pair formation system, because DNA or any of the components of the relaxosome were excluded as possible targets.
Insights
TrbK protein prevents other plasmids from entering recipient cells by targeting mating pair formation systems. Its function depends on the recipient cell, not the donor cell.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- TrbK is the sole plasmid-encoded protein responsible for entry exclusion of the broad-host-range plasmid RP4.
- The trbK gene is located in the Tra2 region, within the mating pair formation genes.
Purpose of the Study:
- To investigate the molecular mechanisms and requirements of TrbK's entry exclusion activity.
- To determine the role of TrbK modification, processing, and localization in its function.
Main Methods:
- Site-directed mutagenesis to create TrbK mutants (TrbKC23G, C-terminal truncation).
- Analysis of protein modification, processing, and localization.
- Assays to evaluate entry exclusion activity against different plasmid systems.
Main Results:
- TrbK is a 47-residue polypeptide with an N-terminal lipid modification.
- Mutant TrbKC23G, lacking modification and processing, showed reduced entry exclusion efficiency.
- C-terminal truncation abolished entry exclusion activity, though processing occurred.
- TrbK localizes to the cytoplasmic membrane and requires the recipient cell for function.
- TrbK excludes homologous P complex plasmids but not IncI plasmids.
Conclusions:
- TrbK's entry exclusion activity is dependent on its lipid modification and C-terminal region.
- The mating pair formation system is the likely target of TrbK action.
- TrbK's function is recipient-specific, highlighting its role in plasmid competition.