Development of a genetic transfer system in selenate-respiring bacterium Citrobacter sp. strain JSA which was isolated from natural freshwater sediment

Abstract

Conjugative mating between the selenate-reducing bacterium Citrobacter sp. strain JSA and Escherichia coli S17-1 harboring the broad-host-range plasmid pKT230 or pKT240 (IncQ) allowed genetic transfer to strain JSA at a maximum frequency of 2.5×10(-5) (pKT230) and 5.1×10(-6) (pKT240) per recipient JSA cell. Kanamycin-resistant (selection marker of pKT230 and pKT240) transconjugants were routinely obtained with this method, and we confirmed that both vectors were also successfully transferred and replicated in strain JSA without alteration of the replicon. Furthermore, an electroporation method has also allowed transformation of JSA at a frequency of 10(-7) to 10(-6) transformants per μg vector DNA (per recipient cell), and PCR and hybridization analyses revealed that pKT230 and pKT240 are stably maintained in transformed JSA cells. These results indicated that both InQ plasmids can be used as vectors for gene transfer to selenate-reducing strain JSA. This is the first study to demonstrate an effective method for genetic transfer in a selenate-reducing Citrobacter bacterium and will aid in the elucidation of the selenium oxyanion reduction mechanism in this genus of environmental selenate-respiring isolates.