NUCLEOTIDE-SEQUENCE, EXPRESSION AND TRANSCRIPTIONAL ANALYSIS OF THE CORYNEBACTERIUM-GLUTAMICUM GLTA GENE ENCODING CITRATE SYNTHASE

Citrate synthase catalyses the initial reaction of the citric acid cyc le and can therefore be considered as the rate-controlling enzyme for the entry of substrates into the cycle. In Corynebacterium glutamicum, the specific activity of citrate synthase was found to be independent of the growth substrate and of the growth phase. The enzyme was not a ffected by NADH or 2-oxoglutarate and was only weakly inhibited by ATP (apparent K-i = 10 mM). These results suggest that in C. glutamicum n either the formation nor the activity of citrate synthase is subject t o significant regulation. The citrate synthase gene, gltA, was isolate d, subcloned on plasmid pJC1 and introduced into C. glutamicum. Relati ve to the wild-type the recombinant strains showed six- to eightfold h igher specific citrate synthase activity. The nucleotide sequence of a 3007 bp DNA fragment containing the gltA gene and its flanking region s was determined. The predicted gltA gene product consists of 437 amin o acids (M(r) 48936) and shows up to 49.7 % identity with citrate synt hase polypeptides from other organisms. Inactivation of the chromosoma l gltA gene by gene-directed mutagenesis led to absence of detectable citrate synthase activity and to citrate (or glutamate) auxotrophy, in dicating that only one citrate synthase is present in C. glutamicum. T ranscriptional analysis by Northern (RNA) hybridization and primer ext ension experiments revealed that the gltA gene is monocistronic (1.45 kb mRNA) and that its transcription initiates at two consecutive C res idues located 121 and 120 bp upstream of the translational start.