Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase

• Published in: Microbiology (Society for General Microbiology) Vol. 140; no. 8; pp. 1817 - 1828
• Main Authors: Eikmanns, Bernhard J, Thum-Schmitz, Natalie, Eggeling, Lothar, Ludtke, Kai-Ulf, Sahm, Hermann
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.08.1994; Society for General Microbiology
• Subjects: Amino Acid Sequence; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Bacteriology; Base Sequence; Biological and medical sciences; Citrate (si)-Synthase - antagonists & inhibitors; Citrate (si)-Synthase - biosynthesis; Citrate (si)-Synthase - genetics; Cloning, Molecular; Corynebacterium - enzymology; Corynebacterium - genetics; Corynebacterium glutamicum; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetics; Microbiology; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Fusion Proteins - biosynthesis; RNA, Bacterial - genetics; RNA, Messenger - genetics; Sequence Alignment; Sequence Homology, Amino Acid; Transcription, Genetic; Nucleotide sequence; Transcription; Enzyme; Lyases; Gene expression; Oxo-acid-lyases; Carbon-carbon lyases; Regulation(control); Enzymatic activity; Citrate (si)-synthase; Corynebacterium glutamicum; Bacteria; Actinomycetes; Corynebacteriaceae; Aminoacid sequence
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/13500872-140-8-1817

Institut für Biotechnologie, 1 des Forschungszentrums Jülich, D-52425 Jülich, Germany ABSTRACT Citrate synthase catalyses the initial reaction of the citric acid cycle 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 affected by NADH or 2-oxoglutarate and was only weakly inhibited by ATP (apparent K i = 10 mM). These results suggest that in C. glutamicum neither the formation nor the activity of citrate synthase is subject to significant regulation. The citrate synthase gene, gltA , was isolated, subcloned on plasmid pJC1 and introduced into C. glutamicum. Relative to the wild-type the recombinant strains showed six- to eightfold higher specific citrate synthase activity. The nucleotide sequence of a 3007 bp DNA fragment containing the gltA gene and its flanking regions was determined. The predicted gltA gene product consists of 437 amino acids ( M r 48936) and shows up to 49.7% identity with citrate synthase polypeptides from other organisms. Inactivation of the chromosomal gltA gene by gene-directed mutagenesis led to absence of detectable citrate synthase activity and to citrate (or glutamate) auxotrophy, indicating that only one citrate synthase is present in C. glutamicum. Transcriptional analysis by Northern (RNA) hybridization and primer extension experiments revealed that the gltA gene is monocistronic (1.45 kb mRNA) and that its transcription initiates at two consecutive G residues located 121 and 120 bp upstream of the translational start. Author for correspondence: Bernhard J. Eikmanns. Tel: +49 2461 61 3967. Fax: +49 2461 61 2710. Keywords: Corynebacterium glutamicum , gltA gene, citrate synthase The EMBL/GenBank/DDBJ accession number for the sequence reported in this paper is X66112 .