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

被引:229
作者
EIKMANNS, BJ
THUMSCHMITZ, N
EGGELING, L
LUDTKE, KU
SAHM, H
机构
[1] Institut fur Biotechnologie, 1 des Forschungszentrums Julich
来源
MICROBIOLOGY-SGM | 1994年 / 140卷
关键词
CORYNEBACTERIUM GLUTAMICUM; GLT A GENE; CITRATE SYNTHASE;
D O I
10.1099/13500872-140-8-1817
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 C residues located 121 and 120 bp upstream of the translational start.
引用
收藏
页码:1817 / 1828
页数:12
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