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Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation
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TrfA-dependent inner membrane-associated plasmid RK2 DNA synthesis and association of TrfA with membranes of

T Banack1, P D Kim, W Firshein

  • 1Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, USA.

Journal of Bacteriology
|July 27, 2000
PubMed

Insights

The replication initiator protein TrfA binds to bacterial cell membranes across multiple gram-negative species. DNA replication initiated by TrfA primarily occurs within the inner membrane, crucial for plasmid maintenance.

Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • Broad-host-range plasmid RK2 relies on the replication initiator protein TrfA for its propagation.
  • Understanding TrfA's host interactions is key to plasmid stability and transfer.
  • Previous studies focused on Escherichia coli, necessitating broader host investigation.

Purpose of the Study:

  • To investigate the membrane binding capabilities of TrfA in diverse Gram-negative bacteria.
  • To determine the specific cellular fraction responsible for TrfA-mediated DNA synthesis.
  • To assess the functional role of TrfA in endogenous plasmid DNA replication across species.

Main Methods:

  • Fractionation of bacterial cells (E. coli, P. aeruginosa, P. putida, S. Typhimurium, R. sphaeroides) into soluble, inner membrane, and outer membrane components.
  • Western blotting using anti-TrfA antibodies to detect TrfA protein localization.
  • Assay of endogenous DNA synthesis in cellular fractions, assessing inhibition by anti-TrfA antibody.

Main Results:

  • TrfA was consistently found associated with cell membranes in all tested Gram-negative species.
  • Significant endogenous plasmid DNA synthesis occurred exclusively in the inner membrane fraction, inhibited by anti-TrfA antibody.
  • Outer membrane DNA synthesis was minimal and unaffected by the antibody, while soluble fraction synthesis in R. sphaeroides was non-specific.

Conclusions:

  • TrfA's membrane association is conserved across diverse Gram-negative hosts.
  • The inner membrane is the primary site for TrfA-dependent plasmid DNA replication.
  • These findings highlight the conserved mechanism of plasmid replication initiation involving bacterial membranes.

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