Characterization of plasmids in Gluconobacter

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Virginia Tech

The gluconobacters are well known for their plasma membrane-bound dehydrogenases that rapidly oxidize compounds and release the products into the medium. In 1989, Qazi et al. proposed that genes coding for membranebound glucose dehydrogenase are on a plasmid in G. oxydans strain ATCC 9937. The only other known report of gluconobacter plasmids was by Fukaya, et al. who reported that 23 of 36 strains examined contained plasmids. I wish to learn more about the presence and significance of plasmids in the gluconobacters. In this study, I selected 14 strains representing the three gluconobacter species to determine possible similarities and differences in their plasmid profiles. To date, our most successful method for extracting plasmids involves alkaline lysis of cells at 60°C followed by phenol-chloroform extraction which leaves the nucleic acids in the aqueous phase. These nucleic acids are then precipitated in a LiCl-ethanol solution using glycogen as a carrier, and plasmids are separated on agarose gels. We found that 11 of the 14 gluconobacter strains surveyed contain plasmids ranging in size from 2.7 to greater than 200 kb. The type strain of the genus (G. oxydans strain ATCC 19357) contained 6 plasmids ranging in size from 4 to 120 kb. Strains from G. frateurii and G. asaii also contained a wide range of plasmid sizes. Hybridization techniques were utilized to determine if plasmids of similar size are genetically similar. The 2.7 and 16.2 kb plasmids of G. oxydans strain ATCC 621 and IFO 12528 were shown to share homology. The 100 and 120 kb plasmids of G. oxydans strain ATCC 19357 hybridized with the 250 kb plasmids of ATCC 621 and the 210 kb plasmid of ATCC 9937. Also showing homology were the 6.4 kb plasmids of G. oxydans strains ATCC 9937 and IFO 12467.