Response of Guanosine 5′-Triphosphate Concentration to Nutritional Changes and Its Significance for Bacillus subtilis Sporulation

Abstract

The changes in the GTP and P-ribosyl-PP [phosphoribosyl pyrophosphate] pools in stringent and relaxed strains of B. subtilis were investigated under conditions frequently used to initiate sporulation. After a shiftdown from a Casamino Acid-glutamate to a glutamate medium (Sterlini-Mandelstam shiftdown), the pools of ATP and P-ribosyl-PP increased in both strains; in the stringent strain, ppGpp [guanosine 3''-diphosphate 5''-diphosphate] and pppGpp [guanosine 3''-diphosphate 5''-triphosphate] increased and GTP decreased rapidly, whereas in the relaxed strain, ppGpp and pppGpp increased only slightly and GTP decreased only slowly and less extensively. The stringent strain sporulated well; the relaxed strain sporulated late and poorly. Addition of decoyinine, an inhibitor of GMP synthetase, caused a further decrease of GTP and initiated good sporulation of the relaxed strain. After a shiftdown from a glucose-lactate to a lactate medium (Ramaley-Burden shiftdown), the pool of P-ribosyl-PP (and GTP) decreased in both strains, indicating a shortage of purine precursors. This shiftdown also caused a stringent response which prevented the consumption of nucleotides, as shown by the maintenance of ATP at a high concentration in the stringent strain but not in the relaxed strain. After a delay, the relaxed strain, in which GTP decreased as fast as in the stringent strain, sporulated also as efficiently. In nutrient sporulation medium the stringent strain and, less effectively, the relaxed strain accumulated ppGpp and pppGpp transiently towards the end of exponential growth. Eventually, the P-ribosyl-PP pool decreased drastically in both strains. In all cases the initiation of sporulation was correlated with a significant decrease of GTP. Granaticin, an antibiotic which prevents the charging of leucyl-tRNA was used to show that the stringent response inhibited the formation of XMP from IMP. It prevented the accumulation of XMP in decoyinine-treated cultures of the stringent strain but not in those of the relaxed strain.