Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the pyc gene

• Published in: Microbiology (Society for General Microbiology) 1998-04, Vol.144 (4), p.915-927
• Main Authors: Peters-Wendisch, Petra G, Kreutzer, Caroline, Kalinowski, Jorn, Patek, Miroslav, Sahm, Hermann, Eikmanns, Bernhard J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence - genetics; Bacterial Proteins - analysis; Bacterial Proteins - genetics; Bacteriology; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Blotting, Northern; Corynebacterium - enzymology; Corynebacterium glutamicum; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Genes, Bacterial - genetics; Genetics; Humans; Microbiology; Mission oriented research; Molecular Sequence Data; Pyruvate Carboxylase - analysis; Pyruvate Carboxylase - genetics; Sequence Alignment
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-144-4-915

Institut für Biotechnologie, Forschungszentrum Jülich, D-52425 Julich, Germany Lehrstuhl für Genetik, Universität Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-14220 Prague, Czech Republic ABSTRACT In addition to phosphoenolpyruvate carboxylase (PEPCx), pyruvate carboxylase (PCx) has recently been found as an anaplerotic enzyme in the amino-acid-producing bacterium Corynebacterium glutamicum . Using oligonucleotides designed according to conserved regions of PCx amino acid sequences from other organisms, a 200 bp fragment central to the C. glutamicum PCx gene ( pyc ) was amplified from genomic DNA by PCR. This fragment was then used to identify and to subclone the entire C. glutamicum pyc gene. The cloned pyc gene was expressed in C. glutamicum , as cells harbouring the gene on plasmid showed four- to fivefold higher specific PCx activities when compared to the wild-type (WT). Moreover, increased PCx protein levels in the pyc -plasmid-carrying strain were readily detected after SDS-PAGE of cell-free extracts. DNA sequence analysis of the pyc gene, including its 5' and 3' flanking regions, and N-terminal sequencing of the pyc gene product predicts a PCx polypeptide of 1140 amino acids with an M r of 123070. The amino acid sequence of this polypeptide shows between 62% and 45% identity when compared to PCx enzymes from other organisms. Transcriptional analyses revealed that the pyc gene from C. glutamicum is monocistronic (3.5 kb mRNA) and that its transcription is initiated at an A residue 55 bp upstream of the translational start. Inactivation of the chromosomal pyc gene in C. glutamicum WT led to the absence of PCx activity and to negligible growth on lactate, indicating that PCx is essential for growth on this carbon source. Inactivation of both the PCx gene and the PEPCx gene in C. glutamicum led additionally to the inability to grow on glucose, indicating that no further anaplerotic enzymes for growth on carbohydrates exist in this organism. * Author for correspondence: Petra G. Peters-Wendisch. Tel: + 49 2461 612544. Fax: [phone]. e-mail: [e-mail] Keywords: Corynebacterium glutamicum , pyruvate carboxylase, pyc gene, anaplerotic reactions, phosphoenolpyruvate carboxylase Present address: Abteilung für Angewandte Mikrobiologie, Universität Ulm, D-89069 Ulm, Germany Present address: Department of Plant and Microbial Biology, Unive