CHARACTERIZATION OF MEMBRANE-ASSOCIATED PSEUDOMONAS-AERUGINOSA RAS-LIKE PROTEIN PRA, A GTP-BINDING PROTEIN THAT FORMS COMPLEXES WITH TRUNCATED NUCLEOSIDE DIPHOSPHATE KINASE AND PYRUVATE-KINASE TO MODULATE GTP SYNTHESIS

We report the purification and characterization of a protein from the membrane fraction of Pseudomonas aeruginosa showing intrinsic guanosin e triphosphatase (GTPase) activity. The protein was purified as a 48-k Da polypeptide capable of binding and hydrolyzing GTP. The N-terminal sequence of the purified protein revealed its similarity to the Escher ichia coli Ras-Like protein (Era), and the protein cross-reacted with anti-Era antibodies. This protein was named Pseudomonas Ras-like prote in (Pra). Anti-Pra antibodies also crossreacted with E. coli Era prote in. Pra is autophosphorylated in vitro, with phosphotransfer of the te rminal phosphate from [gamma-P-32]GTP but not [gamma-P-32]ATP. Pra is capable of complex formation with the truncated 12-kDa form of nucleos ide diphosphate kinase (Ndk) but not with the 16-kDa form. Purified Pr a was also shown to physically interact with pyruvate kinase (Pk); Pk and Pra can form a complex, but when the 12-kDa Ndk, Pk, and Pra are a ll present, Pk has a higher affinity than Pra for forming a complex wi th the 12-kDa Ndk. The 12-kDa Ndk-Pra complex catalyzed increased synt hesis of GTP and dGTP and diminished synthesis of CTP and UTP or dCTP and dTTP relative to their synthesis by uncomplexed Ndk. Moreover, the complex of Pra with Pk resulted in the specific synthesis of GTP as w ell when Pra was present in concentrations in excess of that of Pk. Me mbrane fractions from cells harvested in the mid-log phase demonstrate d very little nucleoside triphosphate (NTP)-synthesizing activity and no detectable Ndk. Membranes from cells harvested at late exponential phase showed NTP-synthesizing activity and the physical presence of Nd k but not of Pk or Pra. In contrast, membrane fractions of cells harve sted at early to late stationary phase showed predominant GTP synthesi s and the presence of increasing amounts of Pk and Pra. It is likely t hat the association of Pra with Ndk and/or Pk restricts its intrinsic GTPase activity, which may modulate stationary-phase gene expression a nd the survival of P. aeruginosa by modulating the level of GTP.