Regulation of nucleoside diphosphate kinase and secretable virulence factors in Pseudomonas aeruginosa: roles of algR2 and algH

Abstract

Alginate is an important virulence factor for Pseudomonas aeruginosa during infection of the lungs of cystic fibrosis patients. The genes encoding enzymes for alginate production by P. aeruginosa are normally silent. They are activated in response to several environmental conditions, including high osmolarity, exposure to ethanol, or long-term growth under conditions of nutrient deprivation. Several genes which participate in the activation of alginate gene promoters have been identified; among these is the algR2 (algQ) gene. AlgR2 is an 18-kDa protein which has been shown to regulate the critical algD gene encoding GDP-mannose dehydrogenase as well as to regulate the levels of a tricarboxylic acid cycle enzyme, i.e., succinyl coenzyme A synthetase, and nucleoside diphosphate kinase (Ndk), an enzyme involved in nucleoside triphosphate synthesis. Succinyl coenzyme A synthetase and Ndk form a complex in P. aeruginosa. While algR2 is required for alginate synthesis at 37؇C, an algR2 insertion mutant was still able to make alginate slowly at 37 or at 30؇C. We used this observation to identify and clone a gene, termed algH. A strain with mutations in both algR2 and algH is unable to produce alginate at either 37 or 30؇C, and it is fully defective in Ndk production.

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

“…Wild-type P. aeruginosa 8830 cell extract supernatant contains Ndk activity and the activity of an alternative kinase which can substitute for Ndk (33). The alternate kinase, which was not inhibited by anti-Ndk antibodies but was inhibited by Tween 20, was subsequently identified as Pk (41).…”

“…We have previously determined that an autophosphorylation assay using [␥- 32 P]ATP provides a sensitive means to assess Ndk levels and corresponds well to the measurement of Ndk levels by Western blotting (32,33). In this study, each of the 12 mutant ndk genes were hyperexpressed in 8830R2::Cm, which has very little chromosomally encoded Ndk, and the Ndk levels in supernatant extracts were examined by autophosphorylation and by Western blotting.…”

“…In Pseudomonas aeruginosa, the expression of ndk is under the dual control of the algR2 and algH genes (33). The levels of Ndk are drastically reduced in a P. aeruginosa algR2 algH double mutant; however, NTP synthesis is still evident, indicating that an alternate kinase is present in these cells.…”

“…The algR2 mutant of P. aeruginosa was defective in alginate synthesis, had greatly reduced Ndk levels, and contained less than 10% of wild-type levels of intracellular pools of GTP and ppGpp (14,33,41). We recently demonstrated that hyperexpression of the ndk gene in the P. aeruginosa algR2 mutant could largely restore the production of the exopolysaccharide alginate to this mutant and restored the levels of Ndk, GTP, and ppGpp to the levels of the wild type (14,41).…”

Involvement of AlgQ in Transcriptional Regulation of Pyoverdine Genes in Pseudomonas aeruginosa PAO1

“…Interestingly, reduced siderophore levels were observed in the stable alginate-producing P. aeruginosa 8830 strain carrying a mutation in the algQ gene, also known as algR2 (49,60). Alginate is a critical virulence factor in chronic lung infections sustained by P. aeruginosa since it confers a typical mucoid phenotype which insulates bacteria from the host defenses (16,18).…”