Factors affecting competence in a high frequency of transformation marine Vibrio

Abstract

Natural plasmid transformation may be a mechanism for the horizontal transfer of non-conjugative plasmids in the marine environment, yet there are few marine model systems available for the study of this process. Using multimers of IncQ/P4 plasmids and a filter transformation assay, we have measured the effects of nutrients, salinity, temperature, as well as the development and maintenance of competence for genetic transformation in the high frequency of transformation (HFT) marine Vibrio strain WJT-1C. Transformation frequency was proportional to the amount of DNA used from 0.1 to 1.0 pg DNA and was saturated at concentrations greater than 1-0 pg. Competence began in the early-exponential phase and reached a maximum at the onset of stationary phase. Once attained, competence was maintained in both spent and nutrient-free media for at least 10 d. Thus, the establishment and maintenance of competence was unique compared to previously described transformation systems. Temperatures ranging from 4 to 33 "C had no significant effect on the maximal transformation frequency of WJT-lC, but at 37 "C the transformation frequency was reduced. However, temperature did affect the rate of the transformation process. Salinities in the range 12 to 50%, had no significant effect on the transformation frequency but transformation frequencies were lower at 6%, and 63%,. Cells were transformed equally well in nutrient-free media or rich media. The ability of this marine HF'T Vibrio strain to develop competence under a wide spectrum of conditions and to maintain the competent state indicates that natural plasmid transformation could occur in conditions found in tropical and subtropical estuaries.

Plasmid transfer to indigenous marine bacterial populations by natural transformation

“…pQSR50 is a derivative of Rl162 [24] which contains transposon Tn5 and encodes resistance to the antibiotics kanamycin and streptomycin [25]. pQSR50 was used in earlier transformation studies [20][21][22][23]. Plasmid DNA was purified and multimerized as previously described [20].…”

“…Recently we have developed a marine model system for the investigation of natural plasmid transformation in the environment [20,21]. Using this system we have demonstrated the potential for natural transformation in the marine environment [22] and the potential for inter-and intraspecific plasmid transfer to occur between marine Vibrio strains or between E. coli and Vibrio strains [23].…”

Plasmids isolated from the sugar beet phyllosphere show little or no homology to molecular probes currently available for plasmid typing

“…After incubation, cells were lysed by placing membranes, colony side up, on Whatman 3MM filter paper soaked with 2 x SSC, 5 YO (w/v) SDS for 2 min, followed by heating at maximum power in a 650 W microwave oven for 2.5 min, as described by Buluwela e t al. (1989) and Frischer et al (1990). The membranes were transferred to filter paper soaked with denaturing solution (0.5 M NaOH, 1-5 M NaCl) and incubated for a further 7 min.…”

Gene transfer among bacteria under conditions of nutrient depletion in simulated and natural aquatic environments

“…Addition of low nutrient levels (100 mg l-1 of peptone plus 20 mg 1-1 of yeast extract) to any of the water column microcosms, resulted in increased transformation frequencies, whereas the addition of 10-times higher nutrient levels appeared to depress transformation frequencies. In a further study, it was found that non-growing WJT-1C cells were able to maintain competence for at least 10 days when held either in spent rich medium or artificial seawater with no added nutrients [19]. Also, WJT-1C showed no significant difference in transformation frequency by plasmid pQSR50 when the cells were grown in artificial seawater containing yeast extract and peptone concentrations ranging from 10 to 2000 and 50 to 10000 mg 1 -a, respectively, in contrast to results from earlier studies [18].…”