Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis

Abstract

We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2 and algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild-type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane-associated fractions of P. aeruginosa. The cytosolic Ndk was non-specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction events within the cell and in the production of GDP-mannose, an essential alginate precursor, clearly indicates the importance of Ndk in cellular processes as well as in alginate synthesis.