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.

The nucleoside diphosphate kinase of Mycobacterium smegmatis: identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme

“…As indicated in Fig. 6, we found that while the P. aeruginosa Pk modulated the specificity of NTP synthesis to GTP by the 12 kDa form of P. aeruginosa Ndk, as previously demonstrated (Sundin et al, 1996a), the mycobacterial proteins did not distinguish between the 16 kDa ( Fig. 6, lanes 1-5) or the 12 kDa form (Fig.…”

“…4). While we have shown in the case of P. aeruginosa that a truncation of the 16 kDa form of Ndk to a 12 kDa form is required for its complex formation with Pk (Sundin et al, 1996a), we had detected no such modification in the case of the M. The sample (1 ml) was applied directly to a Sephacryl S-300 2 cm × 50 cm column and eluted fractions were assayed for autophosphorylation as well as NTPsynthesizing activity (data not shown).…”

“…Because modulation of NTP synthesis has previously been demonstrated in P. aeruginosa as a result of complex formation between Ndk and other proteins such as Pk or the Ras-like protein Pra (Sundin et al, 1996a;Chopade et al, 1997), it was necessary to dissect the M. smegmatis system in terms of multiple protein species that might be involved in modulating various NTP synthesis as a result of complex formation with Ndk. To delineate the role of putative proteins that might be associated with Ndk, we purified the Ndk protein from M. smegmatis cell extracts and determined the affinity of other proteins to be retained in a column where Ndk was cross-linked to Sepharose beads.…”

“…The fraction eluting at 200 mM NH 4 Cl altered the activity of Ndk towards CTP synthesis, while the 350 mM NH 4 Cl fraction shifted the activity towards UTP synthesis. We have previously shown in the case of P. aeruginosa that a 12 kDa truncated form of Ndk complexes with Pk and specifically synthesizes GTP (Sundin et al, 1996a). We were interested in determining whether any of the proteins that we had recovered from the affinity matrix was indeed Pk.…”

Mammalian heterotrimeric G‐protein‐like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells

“…5, lanes 1-7 and lanes 22-27). That such Ndks were enzymatically active was demonstrated not only by their ability to synthesize all NTPs from [␥-32 P]-ATP and NDPs but also by their ability to specifically synthesize GTP when complexed with Pk (Sundin et al, 1996) or P 50 (Fig. 5, lanes 28 and 29).…”