Development and use of genetic system to identify genes required for efficient low-temperature growth of Psychrobacter arcticus 273-4

Abstract

We describe the development of genetic tools (electroporation, conjugation, vector for targeted gene replacement) for use in the psychrophile Psychrobacter arcticus 273-4 to test hypotheses about cold adaptation. Successful electroporation only occurred with nonstandard parameters, such as: electrocompetent cells freshly prepared from stationary-phase cultures, high field strengths (25 kV cm(-1)), long recovery times (16-24 h), and selection with low concentrations of antibiotics. Transformation frequencies were greatly affected by a methylation-dependent restriction barrier homologous to DpnI. The vector pJK100 (which was self-transmissible and contained a Pir-dependent R6K origin of replication) proved effective as a suicide plasmid that could be used to recombine mutations into the P. arcticus 273-4 genome. We used this vector for targeted replacement of dctT, the substrate-binding periplasmic subunit of a TRAP (tripartite ATP-independent periplasmic) transporter (which we have named dctTUF), as it was more highly expressed at cold temperatures. The replacement of dctT (with kan) decreased the rate of growth at low temperatures in mineral medium with glutamate, acetate, butyrate, and fumarate, but not with pyruvate suggesting that DctTUF participates in the transport of glutamate, acetate, butyrate, and fumarate at cold temperatures. This is the first report to demonstrate the creation of site-specific mutants in the genus Psychrobacter, their affect on low-temperature growth, and a substrate range for TAXI proteins of TRAP transporters.