Corynebacterium glutamicum is able to accumulate up to 600 mM cytosolic phosphorus in the form of polyphosphate (poly P). Granular poly P (volutin) can make up to 37% of the internal cell volume. This bacterium lacks the classic enzyme of poly P synthesis, class I polyphosphate kinase (PPK1), but it possesses two genes, ppk2A (corresponds to NCgl0880) and ppk2B (corresponds to NCgl2620), for putative class II (PPK2) PPKs. Deletion of ppk2B decreased PPK activity and cellular poly P content, while overexpression of ppk2B increased both PPK activity and cellular poly P content. Neither deletion nor overexpression of ppk2A changed specific activity of PPK or cellular poly P content significantly. Purified PPK2B of C. glutamicum is active as a homotetramer and formed poly P with an average chain length of about 125, as determined with 31 P nuclear magnetic resonance. The catalytic efficiency of C. glutamicum PPK2B was higher in the poly P-forming direction than for nucleoside triphosphate formation from poly P. The ppk2B deletion mutant, which accumulated very little poly P and grew as C. glutamicum wild type under phosphate-sufficient conditions, showed a growth defect under phosphate-limiting conditions.Inorganic polyphosphate (poly P), a linear polymer consisting of three to hundreds of orthophosphate residues linked by phosphoanhydride bonds (20, 37), has been found in all living cells examined: archaea, bacteria, and eukarya (7,20,37). Poly P is synthesized by polyphosphate kinases (PPKs), using the terminal phosphate of ATP as the substrate, and is degraded to inorganic phosphate (P i ) by both endo-and exopolyphosphatases (19,20,37). Among the many functions poly P performs, the most prominent are the responses to many stresses. A variety of defects in responses to environmental stresses and/or virulence were observed in PPK gene mutants of Escherichia coli, Pseudomonas aeruginosa, Shigella, Salmonella, Vibrio cholerae, and Helicobacter pylori (5,14,17,32). In E. coli, it could be demonstrated that under conditions of amino acid starvation, poly P accumulates and is bound by Lon protease (21). The Lon-poly P complex degrades ribosomal proteins supplying amino acids in response to starvation (21).PPKs, which can be grouped into two classes, catalyze the reversible transfer of the gamma phosphoryl group of ATP or GTP to poly P. Genetic and biochemical studies have established the role of PPKs for biosynthesis of poly P, e.g., in the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa (2, 3, 44). The archetypes of PPKs are class I PPK, PPK1, from E. coli, and class II PPK, PPK2, from P. aeruginosa. PPK2 of P. aeruginosa differs from ATP-specific PPK1, as it accepts GTP as well as ATP (12). Moreover, while the rate of GTP synthesis catalyzed by P. aeruginosa PPK2 is 75-fold higher than the rate of poly P synthesis, E. coli PPK1 shows a preference for poly P synthesis (12). Based on the presence or absence of PPK1 and/or PPK2 genes within complete genome sequences, Zhang et al. (44) distinguished fo...