1887

Abstract

In addition to phosphoenolpyruvate carboxylase (PEPCx), pyruvate carboxylase (PCx) has recently been found as an anaplerotic enzyme in the amino-acid-producing bacterium . Using oligonucleotides designed according to conserved regions of PCx amino acid sequences from other organisms, a 200 bp fragment central to the PCx gene () was amplified from genomic DNA by PCR. This fragment was then used to identify and to subclone the entire gene. The cloned gene was expressed in , 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 -plasmid-carrying strain were readily detected after SDS-PAGE of cell-free extracts. DNA sequence analysis of the gene, including its 5’ and 3’ flanking regions, and N-terminal sequencing of the gene product predicts a PCx polypeptide of 1140 amino acids with an 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 gene from 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 gene in 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 led additionally to the inability to grow on glucose, indicating that no further anaplerotic enzymes for growth on carbohydrates exist in this organism.

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1998-04-01
2021-08-05
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