Summary auto-generated
This study characterizes pyruvate carboxylase (PCx), an anaplerotic enzyme in the amino acid-producing bacterium Corynebacterium glutamicum. Using PCR with primers designed from conserved regions of PCx genes in other organisms, researchers isolated and sequenced the complete pyc gene from C. glutamicum. The gene encodes a 1140-amino-acid protein with a molecular weight of 123,070 Da, showing 62% identity to PCx from Mycobacterium tuberculosis and 45-47% identity to other bacterial PCx enzymes. Transcriptional analysis revealed the pyc gene is monocistronic, producing a 3.5 kb mRNA transcript. When researchers inactivated the chromosomal pyc gene, cells lost PCx activity and could not grow on lactate as the sole carbon source. Double mutants lacking both PCx and phosphoenolpyruvate carboxylase (PEPCx) could not grow on glucose, demonstrating these are the only two anaplerotic enzymes in C. glutamicum. Overexpression of the pyc gene on a plasmid increased PCx activity four- to fivefold compared to wild-type cells, confirming successful gene expression and revealing PCx's important role in carbon source utilization alongside PEPCx.
Key findings
- The C. glutamicum pyc gene encodes a 1140-amino-acid pyruvate carboxylase protein (Mr 123,070) that shares 62% amino acid identity with M. tuberculosis PCx
- PCx and PEPCx are the only two anaplerotic enzymes in C. glutamicum; cells lacking both cannot grow on glucose or lactate
- The pyc gene is monocistronic with a 3.5 kb mRNA transcript and transcription initiates 55 bp upstream of the translation start site
- PCx-deficient mutants cannot utilize lactate but retain wild-type growth rates on glucose, indicating PEPCx can compensate on carbohydrates but not organic acids
This summary was generated automatically from the article PDF and is not part of the original publication. Refer to the PDF for the authoritative text.
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.