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Project Progress

A major goal of our research team is to identify genes that enable bacteria to inhabit the permafrost environment. To accomplish this, we need to develop genetic systems to manipulate (including mutagenize) the genomes of the permafrost isolates. Progress has been slow, but recent results are encouraging. Several factors that had been hampering transformation of Psychrobacter 273-4 have been identified. First, a methylation sensitive restriction system has been shown to destroy foreign deoxyribonucleic acid ( DNA) that has been methylated. To overcome this, we now propagate plasmids for transformation in Escherichia coli GM2163 ( dam - dcm - ) which does not methylate critical sites recognized by the Psychrobacter 273-4 restriction system. Second, we have found that successful electroporation of Psychrobacter 273-4 requires high voltages of 20-25kV/cm (normal E. coli conditions are 10-12.5kV/cm) and long recovery times after electroporation (between 8-24 h). Third, antibiotic resistance markers for kanamycin and chloramphenicol have been found to be poorly expressed. But taking these factors into account, we can now transform Psychrobacter 273-4 by electroporation. In addition, we have identified conditions whereby we can introduce wide host range plasmids, including pRL412, an RSF1010, into Psychrobacter 273-4 by conjugation. Finally, we have examined natural competence of Psychrobacter 273-4 and Psychrobacter cryopegella . Transformation frequencies (using Psychrobacter DNA) of about 10^-6 to 10^-7 and 10^-8 to 10^-9 transformants per recipient per µg of DNA have been obtained with Psychrobacter 273-4 and Psychrobacter cryopegella , respectively. The bacteria are transformable in the stationary phase of growth if cultures are initially grown in tryptic soy broth and subsequently incubated with DNA on marine agar (other combinations of growth stage and media have been tried and have not yielded transformants).