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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 Mr 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: +49 2461 612710. e-mail: peterswe@nature.berkeley.edu
Present address: Abteilung für Angewandte Mikrobiologie, Universität Ulm, D-89069 Ulm, Germany
Present address: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94705, USA
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