Succinyl coenzyme A synthetase of Pseudomonas aeruginosa with a broad specificity for nucleoside triphosphate (NTP) synthesis modulates specificity for NTP synthesis by the 12-kilodalton form of nucleoside diphosphate kinase

Pseudomonas aeruginosa secretes copious amounts of an exopolysaccharide cal led alginate during infection in the lungs of cystic fibrosis patients. A m utation in the algR2 gene of mucoid P. aeruginosa is known to exhibit a non mucoid (nonalginate-producing) phenotype and showed reduced activities of s uccinyl-coenzyme A (CoA) synthetase (Scs) and nucleoside diphosphate kinase (Ndk), implying coregulation of Ndk and Scs in alginate synthesis, We have cloned and characterized the sucCD operon encoding the alpha and beta subu nits of Scs from P. aeruginosa and have studied the role of Scs in generati ng GTP, an important precursor in alginate synthesis. We demonstrate that, in the presence of GDP, Scs synthesizes GTP using ATP as the phosphodonor a nd, in the presence of ADP, Scs synthesizes ATP using GTP as a phosphodonor . In the presence of inorganic orthophosphate, succinyl-CoA, and an equimol ar amount of ADP and GDP, Scs synthesizes essentially an equimolar amount o f ATP and GTP, Such a mechanism of GTP synthesis can be an alternate source for the synthesis of alginate as well as for the synthesis of other macrom olecules requiring GTP such as RNA and protein. Scs from P. aeruginosa is a lso shown to exhibit a broad NDP kinase activity. In the presence of inorga nic orthophosphate (P-i), succinyl-CoA, and either GDP, ADP, UDP or CDP, it synthesizes GTP, ATP, UTP, or CTP, Scs was previously shown to copurify,vi th Ndk, presumably as a complex. In mucoid cells of P. aeruginosa, Ndk is a lso known to exist in two forms, a 16-kDa cytoplasmic form predominant in t he log phase and a 12-kDa membrane-associated form predominant in the stati onary phase. We have observed that the 16-kDa Ndk-Scs complex present in no nmucoid cells, synthesizes all three of the nucleoside triphosphates from a mixture of GDP, UDP, and CDP, whereas the 12-kDa Ndk-Scs complex specifica lly present in mucoid cell predominantly synthesizes GTP and UTP but not CT P, Such regulation may promote GTP synthesis in the stationary phase when t he bulk of alginate is synthesized by mucoid P. aeruginosa.