Related Experiment Videos
Subcellular localization and processing of the lytic transglycosylase of the conjugative plasmid R1
1Institut für Molekularbiologie, Biochemie und Mikrobiologie, Karl-Franzens-Universität Graz, Austria.
Insights
Protein P19, crucial for DNA transfer and phage R17 infection, is processed by Escherichia coli signal peptidase I. This lysozyme-like protein localizes to the periplasm, potentially linking to DNA transport complexes.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Biochemistry
Background:
- Protein P19 from plasmid R1 is vital for conjugative DNA transfer and R17 phage infection.
- Sequence analysis reveals P19 belongs to a lysozyme-like virulence factor family, common in secretion systems.
Purpose of the Study:
- To investigate the processing and subcellular localization of Protein P19.
- To elucidate the role of P19 in bacterial conjugation and phage interactions.
Main Methods:
- Pulse-chase experiments to track protein processing.
- Gene fusions (19-phoA) to assess membrane translocation.
- Cell fractionation and Sarkosyl solubilization for localization.
- Sucrose density gradient centrifugation to separate membrane fractions.
Main Results:
- P19 processing is mediated by Escherichia coli signal peptidase I.
- Translocation across the inner membrane was confirmed.
- P19 was detected in both inner and outer membrane fractions.
- Mature P19 is localized to the periplasmic space.
Conclusions:
- Mature P19 is a periplasmic protein.
- P19 may associate with the membrane-spanning DNA transport complex.
- These findings contribute to understanding plasmid conjugation and phage R17 biology.
Abstract:
Protein P19 encoded by the conjugative resistance plasmid R1, is essential for efficient conjugative DNA transfer and infection by the pilus-specific RNA phage R17. Based on sequence homologies P19 belongs to a family of lysozyme-like virulence factors which are found in type III and type IV secretion systems. In this report we describe the processing and subcellular localization of P19. Pulse-chase experiments were used to demonstrate the processing of P19 by the signal peptidase I of Escherichia coli. Translocation of P19 across the inner membrane was shown by gene 19-phoA fusions. Cell fractionation studies of P19 expressing cells showed the presence of P19 in the membrane compartment. P19 was solubilized with the detergent Sarkosyl indicating an inner membrane localization. Using sucrose density gradient centrifugation to separate inner and outer membranes, P19 was found in both membrane fractions. Taken together, our data suggest that mature P19 is a periplasmic protein which may be attached to the proposed membrane-spanning DNA transport complex.