Nucleoside diphosphate kinase from Myxococcus xanthus. I. Cloning and sequencing of the gene

Abstract

By photoaffinity labeling with a photolysable analog of GTP, 8-N3GTP, we were able to find at least five distinct GTP-binding proteins in Myxococcus xanthus; two of them located in the membrane and the other three in the soluble fraction. The amino-terminal sequence of the 16-kDa GTP-binding protein from the soluble fraction was determined, and the gene that encodes this protein was isolated and cloned using degenerate oligonucleotides as a probe. The DNA sequence of the gene was determined, which did not show similarity with other known proteins. The gene product was overexpressed in Escherichia coli, by using the lacZ promoter, to a level of 13% of the soluble protein. Attempts to isolate deletion mutants were unsuccessful, although double crossing-over events leading to a deletion mutation of the gene were detected by Southern blot hybridization. This result indicates that this gene is essential for cell growth. In the following paper (Muñoz-Dorado, J., Inouye, S., and Inouye, M. (1990) J. Biol. Chem. 265, 2707-2712), the gene product was biochemically characterized and identified to be a nucleoside diphosphate kinase.