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.

Infection with mucoid, alginate-producing strains of Pseudomonas aeruginosa is the leading cause of mortality among patients with cystic fibrosis. Alginate production by P. aeruginosa is not constitutive but is triggered by stresses such as starvation. The algR2 (also termed algQ) gene has been previously identified as being necessary for mucoidy; an algR2 mutant strain is unable to produce alginate when grown at 370C. We show here that the levels of phosphorylated succinyl coenzyme A synthetase (Scs) and nucleoside diphosphate kinase (Ndk), which form a complex in P. aeruginosa, are reduced in the algR2 mutant. We were able to correlate the lower level of phosphorylated Scs with a decrease in Scs activity. Western blots (immunoblots) also showed a decreased level of Ndk in the algR2 mutant, but the presence of another kinase activity sensitive to Tween 20 provides the missing Ndk function. The elect of AlgR2 on tricarboxylic acid (TCA) cycle enzymes appears to be specific for Scs, since none of the other TCA cycle enzymes measured showed a significant decrease in activity. Furthermore, the ability of the algR2 mutant to grow on TCA cycle intermediates, but not glucose, is impaired. These data indicate that AlgR2 is responsible for maintaining proper operation of the TCA cycle and energy metabolism.

The enzyme nucleoside-diphosphate kinase (Ndk), responsible for the conversion of (deoxy)ribonucleoside diphosphates to their corresponding triphosphates, has been purified from Pseudomonas aeruginosa. The N-terminal 12 amino acid sequence of P. aeruginosa Ndk shows s nt homology with that of Myxococcus xanthus and that of Eschernchia colt. Ndk enzyme activity is also associated with succinyl-CoA synthetase activity in P. aeruginosa, whose a and 13 subunits show extensive sequence homology with those of E.coli and Dictyosteium discoideum. 7). Expression of nm23 from a constitutive promoter in highly metastatic murine tumor cells was found to actually suppress tumor metastasis (8). Null mutations in the Ndk gene of Drosophila, named awd, cause abnormalities in development of the larvae leading to tissue necrosis and death at the prepupal stage (7, 9). In the slime mold Dictyostelium discoideum, the Ndk gene is developmentally regulated with a sharp decrease in Ndk transcript levels coinciding with the onset ofthe starvation induced developmental cycle (10, 11). A gene encoding a DNA-binding protein, PuF, which is required for transcription of c-myc in vitro, is highly homologous to the human Ndk gene nm23-H2 (12). This implies that nm23-H2 may be involved in the regulation of c-myc.Pseudomonas aeruginosa is a ubiquitous soil and waterborne bacterium which undergoes a transition to mucoidy under stress and/or starvation conditions (13,14). We have purified Ndk from P. aeruginosa in order to study its involvement in these developmental changes and have found that Ndk is associated and copurifies with the ATP-utilizing prokaryotic succinyl-CoA synthetase (Scs) complex. These results indicate that aside from its role in DNA and RNA precursor synthesis, Ndk may be involved in regulating intracellular energy metabolism and that understanding its role in energy metabolism may be critical to understanding its involvement in regulation of development. MATERIALS AND METHODS Detection of [y-32P]ATP-Autophosphorylated Proteins.Samples were added to TMD buffer (50 mM Tris'HCl, pH 7.5/1 mM MgCl2/0.2 mM dithiothreitol) in <19 t4 and brought to a final volume of 20 id with TMD buffer.[y-32P]ATP (1G4; 74 MBq/ml) was added to the sample (final ATP concentration, 20 nM) and reactions were allowed to proceed for at least 6 sec. Reactions were terminated by addition of 4x SDS gel running buffer [0.2 M Tris HCl, pH 6.8/0.62% dithiothreitol/8% SDS/0.01% bromophenol blue/ 40o (vol/vol) glycerol] and analyzed by electrophoresis in an SDS/polyacrylamide gel (3% stacking gel, 15% separating gel).Purification of Ndk from P. aeruginosa strain 8822. Strain 8822 was grown overnight in Luria broth containing 0.1% glucose, and a crude cell extract was prepared from 4 liters of culture by French press disruption and ultracentrifugation at 300,000 x g for 1 hr. The supernatant was subjected to (NH4)2S04 fractionation at 45% and 65% saturation (2), and precipitates at 65% were suspended in 9.6 ml of TMD buffer containing 1 M (NH4)2SO4 (pH ...