AlgR2 positively regulates nucleoside diphosphate kinase (NdK) synthesis and, as a result, may modulate the levels of GTP, ppGpp, alginate and poly P. Alginate synthesis requires a supply of GTP, as does (p)ppGpp; the latter promotes the accumulation of poly P (Kuroda et al., 1997). The conversion of the 16 kDa cytoplasmic form of Ndk to the 12 kDa membrane-associated form at the onset of stationary phase (Shankar et al., 1996) promotes the synthesis of GTP and, in turn, ppGpp (Cashel, 1994). The latter induces expression of the stationary phase sigma factor, σS, in E. coli (Gentry et al., 1993), which controls over 40 genes essential for stationary phase resistance and survival (Loewen and Hengge-Aronis, 1994). However, the effect of ppGpp on the level of RpoS in P. aeruginosa is unknown at present. As the membrane-associated 12 kDa Ndk appears at the onset of the stationary phase (Shankar et al., 1996; Chopade et al., 1997) and makes GTP preferentially and very little dNTPs (Figs 8 and 9), the precursor pools of RNA and DNA are probably reduced with a consequent slowdown in RNA and DNA synthesis. In addition to Pk, a Ras-like protein, Pra, also appears in the membrane at the stationary phase and forms a complex with 12 kDa Ndk, directing its specificity towards GTP synthesis (Chopade et al., 1997). Apart from 12 kDa Ndk, Pra can also form a complex with Pk, modulating its specificity from NTP synthesis to GTP (Chopade et al., 1997). In the presence of 12 kDa Ndk, however, Pk has a higher affinity for 12 kDa Ndk, so that the Pk–Pra complex dissociates. In the algR2 mutant, which has very little Ndk, it is Pk that allows NTP synthesis, and it is likely that the Pk–Pra complex generates the GTP and very little dNTP for transition to stationary phase (Chopade et al., 1997).
The co-regulation of two polymers, alginate and poly P, by a common regulator, such as AlgR2, is interesting. The mucoid Alg+ cells are rich in poly P, while the non-mucoid segregant or the non-mucoid laboratory isolate produces much less poly P. Thus, the genetic switch that turns on alginate synthesis in P. aeruginosa also turns on poly P synthesis, implying a common mode of genetic regulation. As hyperexpression of the ndk gene largely restores alginate, GTP, ppGpp and poly P synthesis in the algR2 mutant (Table 1, Figs 3 and 4[link]), Ndk plays a critical role in the synthesis of these two polymers. As AlgR2 positively regulates Ndk and Scs formation and as hyperexpression of the ndk gene can compensate for AlgR2 deficiency, it is likely that the phenotypes demonstrated by the algR2 mutant are largely caused by the reduced formation of Ndk. Although P. aeruginosa is normally non-mucoid, it is converted to a mucoid form under several conditions: in the lungs of CF patients (Shankar et al., 1995), during growth under starvation conditions and in the presence of inhibitors of energy metabolism (Speert et al., 1990; Woods et al., 1991; Terry et al., 1992). Starvation conditions, particularly for phosphate, allow an activation of the algR2 promoter, leading to enhanced Ndk synthesis and, presumably, an enhanced formation of GTP, pppGpp and ppGpp. High concentrations of pppGpp or ppGpp are known to lead to the accumulation of poly P (Kuroda et al., 1997). Indeed, the poly P accumulation in P. aeruginosa is maximal in the stationary phase (Fig. 2) when the membrane-associated complexes of Pk and Pra with the 12 kDa Ndk generate large amounts of GTP. Alginate is also known to accumulate predominantly in the late exponential–early stationary phase cells (Tatnell et al., 1993; Hassett, 1996). As alginate synthesis requires both phosphorylated sugars and GTP (Fig. 1) and as poly P may substitute for ATP (Kornberg, 1995), it is possible that poly P can contribute to alginate synthesis. Recent efforts in our laboratory to isolate polyphosphate kinase-negative (ppk ) mutants of stably mucoid P. aeruginosa strain 8830 have produced presumptive mutants, many of which are non-mucoid with little PPK activity (A. Zago, unpublished observations). When the cloned ppk gene of P. aeruginosa becomes available, its capacity to restore alginate synthesis in the presumptive ppk non-mucoid mutants can be determined. Given the ubiquitous nature of poly P and its accumulation in the stationary phase (Kornberg, 1995), it can be imagined that poly P, among its many functions, can be used by bacteria to produce capsular polysaccharides for their protection in stressful situations, as for P. aeruginosa in the CF lung. Similarly, isolation of a relA mutant, incapable of converting GTP to pppGpp or ppGpp, in the mucoid strain 8830 may provide insights into the role of these regulatory nucleotides in alginate synthesis.