Partial characterization of a lysU mutant of Escherichia coli K-12

Abstract

The Escherichia coli K-12 strain GNB10181 shows no inducible lysyl-tRNA synthetase (LysRS) activity. Two-dimensional gel electrophoretic analysis of the polypeptides synthesized by this strain indicates that the normal lysU gene product, LysU, is absent. When both GNB10181 and its parent, MC4100, were grown at elevated temperatures (42 to 45 degrees C) no significant difference between their growth rates was observed. The lysU mutation was transferred to other E. coli K-12 backgrounds by using P1 transduction. The lysU transductants behaved comparably to their lysU+ parents at different growth temperatures. Therefore, the LysU proteins does not appear to be essential for growth at high temperatures, at least under the conditions examined here. In addition, lysU transductants were found to be defective for inducible lysine decarboxylase, (LDC), inducible arginine decarboxylase (ADI), and melibiose utilization (Mel), which are all missing in GNB10181. Complementation of the above missing functions was achieved by using the Clarke-Carbon plasmids pLC4-5 (LysU LDC) and pLC17-38 (LysU Mel ADI). From these experiments, it appears that GNB10181 has suffered a chromosomal deletion between 93.4 and 93.7 min, which includes the lysU gene. By using plasmid pLC17-38, the position of ADI on two-dimensional gels was identified. Finally, lysS delta lysU double mutants were constructed which can potentially be used as positive selection agents for the isolation of LysRS genes from other sources.