Host Range, Stability and Compatibility of Broad Host-Range-Plasmids and a Shuttle Vector in Moderately Halophilic Bacteria. Evidence of Intrageneric and Intergeneric Conjugation in Moderate Halophiles

Abstract

Broad-host-range plasmids, belonging to the incompatibility groups IncP (pVK102), IncQ (pKT230), IncW (pGV1124), and IncN (pCU1), as well as pHS15 (a shuttle vector based on an endogenous plasmid of the moderate halophile Halomonas elongata) were transferred by conjugation from E. coli to moderately halophilic strains of the genera Chromohalobacter, Deleya, Halomonas, Vibrio and Volcaniella, to test their utility as cloning vectors for these extremophiles. Several factors affecting the efficiency of conjugation (cell growth phase, mating time, ratio donor:recipient, and composition and salinity of the mating medium) were evaluated. The highest frequencies (from 10-1 to 10-4 transconjugants/recipient cell) were achieved when exponentially grown donor and recipient cells were mixed and incubated for 16 h at 30 °C on SW-2 saline medium at a ratio 1:1 (for Deleya, Halomonas and Volcaniella strains) or SW-5 saline medium at a ratio 2:1 (for Chromohalobacter and Vibrio strains). Whereas IncP, IncQ, and pHS15 plasmids were able to replicate in all strains tested, the IncW plasmid pGV1124 was only maintained in H. elongata, and pCU1 could not be established in any of the moderate halophiles tested. The IncQ plasmid pKT230 was the most stably maintained in the majority of the moderate halophiles. The shuttle vector pHS15 revealed to be compatible with each of the broad-host-range plasmids assayed. Finally, both intrageneric and intergeneric conjugation between moderate halophiles have been demonstrated by using the self-transmissible IncP plasmid RK2. Transfer of RK2 between Halomonas spp. was observed at 2–7% total salts, yielding the highest transfer frequencies (from 1.2×10-3 to 2.8×10-4) on SW-2 medium. Intergeneric conjugation on SW-2 between Halomonas and other moderate halophiles such as Volcaniella (transfer frequency of 2×010-5) and Deleya (5×10-5) were also found. This is the first report on the existence of conjugation between moderately halophilic bacteria.