Development and application of GFP-based monitoring tools to assess conjugative plasmid transfer frequencies in multispecies biofilms (BMBF/ESA Plasmid transfer/MISSEX)

The MISSEX project investigates the distribution, mobility and gene content of MGEs (mobile genetic elements) in a collection of strains isolated from confined environments, such as the CONCORDIA research station in Antarctica and the ISS (International Space Station), by molecular and physiological analyses.

The vast majority of bacteria live in surface-associated communities, so called biofilms, in the environment. Biofilms were also isolated from container systems, like the ISS and the Concordia research station. First aim of the project is detection and isolation of plasmids from bacterial ISS and Concordia isolates, with focus on bacilli and staphylococci and determination of antibiotic resistance profiles of these strains.

The second aim is to study intergramic horizontal plasmid transfer among bacterial ISS and Concordia isolates with gfp-tagged monitoring tools based on the type IV secretion-like transfer system of the broad-host-range conjugative plasmid pIP501. pIP501 is able to self-transfer to many Gram-positive bacteria such as enterococci, staphylococci, multicellular Streptomyces and Gram-negative E. coli (Kurenbach et al., 2003, Plasmid 50, 86-93). The 15 pIP501 transfer genes are organized in an operon which encodes 3 proteins with significant similarity to the type IV secretion system (T4SS) proteins, VirB1, VirB4, and VirD4 from the Agrobacterium T-DNA transfer system responsible for virulence protein and T-DNA transport (Kurenbach et al., 2006, Microbiol. 152, 637-645).

To isolate conjugative plasmids from bacterial ISS and Concordia isolates with a triparental mating system we are constructing a gfp-labelled mobilisable plasmid based upon pIP501. Plasmid transfer to ISS and Concordia isolates will be visualized by gfp-labelled pIP501-based conjugative and mobilisable plasmids. These gfp-tagged monitoring tools will be used in biofilm reactors with ISS and CONCORDIA isolates to assess plasmid transfer frequencies under conditions mimicking nature.

This project is supported by the BMBF.