PMID: 8955392Dec 1, 1996Paper

Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis

Journal of Bacteriology
G W SundinA M Chakrabarty

Abstract

We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not compleme...Continue Reading

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Citations

Sep 28, 2016·World Journal of Gastroenterology : WJG·Anand KumarGireesh Rajashekara
Apr 7, 2020·Current Genetics·Indu Kapoor, Umesh Varshney

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