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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, need to develop genetic systems to mutagenize and manipulate the genomes of our permafrost isolates. Several factors that were hampering genetic manipulation of Psychrobacter arcticus were identified. It was found that P. arcticus has a methylation sensitive restriction system that destroys “foreign” methylated deoxyribonucleic acid (DNA); that successful electroporation requires freshly prepared competent cells and long recovery times after electroporation (between 16-20 h); and that plasmids anticipated to serve as “suicide vectors” (RSF1010, p15a, and ColE1), in fact replicate in P. arcticus. However, we have now had success with P. arcticus using the allelic exchange vector pJK100 (with a Pir-dependent origin of replication) to create targeted gene mutations and the mariner transposon system to create random mutations (Figure 1 and 2).

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The function of a TRAP transporter, encoded by orf746, was explored by targeted gene deletion. Deletion of orf746 decreased the efficiency of P. arcticus growth, particularly at low temperatures. Growth of mutants was inhibited more significantly at 4°C than at 22°C when grown on acetate, butyrate, fumarate or glutamate (Figure 3). These data suggest that orf746 transports these substrates at low temperatures and that other enzymes transport these substrates at high temperatures; i.e., that isofunctional enzymes (isozymes) provide the same function (transport) at different temperatures. Isozymes with specific adaptations to temperature are commonly found in animals, but few examples have been documented in bacteria.

A library of 3,000 random transposon mutants of P. arcticus was created and screened to identify genes required for biofilm formation. Twenty-six of 900 mutants screened were found to be impaired in biofilm formation (results for mutant 9H are presented in Figure 4). Efforts are now in progress to determine the gene(s) affected in these mutants and whether they contribute to the ability of P. arcitcus to inhabit the permafrost environment.