Identification and characterization of the pckA gene from Staphylococcus aureus.
ABSTRACT: The Staphylococcus aureus pckA gene was identified and characterized. A pckA mutant lacked detectable phosphoenolpyruvate carboxykinase activity and grew poorly in the absence of glucose. Both enzymatic activity and pckA promoter activity in wild-type cells grown in the absence of glucose were at least 22-fold greater than activities in cells grown in the presence of glucose.
Project description:A Planctomyces limnophilus mutant generated using the EZ-Tn5 transposome was found to possess an insertion within pckA, encoding phosphoenolpyruvate carboxykinase. Disruption of pckA expression and elimination of enzymatic activity resulted in poor growth in glucose-free medium, demonstrating a gluconeogenic role for pckA in P. limnophilus.
Project description:The pckA gene of Rhizobium meliloti, encoding phosphoenolpyruvate carboxykinase, was isolated from a genomic cosmid library by complementation of the succinate growth phenotype of a Pck- mutant. The gene region was mapped by subcloning and Tn5 insertion mutagenesis. The DNA sequence for a 2-kb region containing the structural gene and its promoter was determined. The pckA gene encodes as 536-amino-acid protein that shows homology with other ATP-dependent Pck enzymes. The promoter was identified following primer extension analysis and is similar to sigma 70-like promoters. Expression analysis with a pckA::lacZ gene fusion indicated that the pckA gene was strongly induced at the onset of stationary phase in complex medium. When defined carbon sources were tested, the expression level of the pckA gene was found to be high when cells were grown in minimal media with succinate or arabinose as the sole carbon source but almost absent when glucose, sucrose, or glycerol was the sole carbon source. Glucose and sucrose were not found to strongly repress pckA induction by succinate.
Project description:The sequence of the pckA gene coding for phosphoenolpyruvate carboxykinase in Escherichia coli K-12 and previous molecular weight determinations indicate that this allosteric enzyme is a monomer of Mr 51,316. The protein is homologous to ATP-dependent phosphoenolpyruvate carboxykinases from Trypanosoma brucei and Saccharomyces cerevisiae. A potential ATP binding site was conserved in all three sequences. A potential binding site for the allosteric activator, calcium, identified in the E. coli enzyme, was only partially conserved in T. brucei and S. cerevisiae, consistent with the observation that the enzymes from the latter organisms were not activated by calcium. The published sequence of the ompR and envZ genes from Salmonella typhimurium is followed by a partial sequence that is highly homologous to pckA from E. coli. The order of these genes and the direction of transcription of the presumptive S. typhimurium pckA gene are the same as those in E. coli. The potential calcium binding site of the E. coli enzyme is conserved in the partial predicted sequence of the S. typhimurium phosphoenolpyruvate carboxykinase, consistent with the observation that calcium activation of the S. typhimurium phosphoenolpyruvate carboxykinase is very similar to that observed for the E. coli enzyme. A pckA mRNA transcript was observed in stationary-phase cells but not in logarithmically growing cells. The mRNA start site was mapped relative to the sequence of the pckA structural gene.
Project description:The phosphoenolpyruvate (PEP) carboxykinase-encoding gene from the anaerobic, CO2-fixing, succinate-producing bacterium Anaerobiospirillum succiniciproducens was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a 532-residue polypeptide with a calculated molecular mass of 58.7 kDa. The sequence of the A. succiniciproducens PEP carboxykinase was similar to those of all known ATP/ADP-dependent PEP carboxykinases. In particular, the A. succiniciproducens enzyme was 67.3% identical and 79.2% similar to the E. coli enzyme. The A. succiniciproducens pckA transcription start site was determined, and putative promoter regions were identified. The recombinant enzyme was overexpressed in E. coli. The purified enzyme was indiscernible from the native enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had the same activity as the native enzyme.
Project description:Phosphoenolpyruvate carboxykinase (PCK) catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate in the gluconeogenic pathway in most organisms. A pckA gene encoding PCK was cloned and sequenced from strain Rhizobium pusense NRCPB100, a spontaneous rifampicin resistant mutant of R. pusense NRCPB10T (JCM16209T) of a recently described new species of a non-nodulating and non-tumorigenic bacterium. The mapping of the pck gene region following Tn5 mutagenesis located the gene downstream of a transcriptional regulatory protein gene (chvI) and upstream of a conserved hypothetical protein gene. The pck of 1,611 bp was deduced to encode 536 amino acids and showed high homology to the genes of known ATP-dependent PCK enzymes. Phylogenetic analysis of the gene placed it in a cluster with pck of other known members of Rhizobiales. Amino acid sequences of the putative functional regions of the deduced enzyme were found to be conserved.
Project description:The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes.
Project description:The pckA gene, encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), was cloned by PCR amplification from the purple nonsulfur bacterium Rhodopseudomonas palustris No. 7. Sequencing of a 2.5-kb chromosomal SmaI-PstI fragment containing the structural gene revealed an open reading frame encoding 537 amino acids, homologous to known pckA genes. Primer extension analysis identified a transcriptional start site 72 bp upstream of the pckA initiation codon and an upstream sequence similar to sigma70 promoters. Studies of a pckA-lacZ gene fusion indicated that when cells were grown in minimal media with various carbon sources, such as succinate, malate, pyruvate, lactate, or ethanol, under both anaerobic light and aerobic dark conditions, the pckA gene was induced in log phase, irrespective of the carbon source. A R. palustris No. 7 PEPCK-deficient strain showed growth characteristics identical to those of the wild-type strain either anaerobically in the light or aerobically in the dark when a C4-dicarboxylic acid, such as succinate or malate, was used as a carbon source. These results indicate that in R. palustris No. 7, an alternative gluconeogenic pathway may exist in addition to PEPCK.
Project description:1. After nicotinic acid treatment, rat liver glycogen is depleted and phosphoenolpyruvate carboxykinase activity increased, to about twice the initial value. 2. The increase in phosphoenolpyruvate carboxykinase activity promoted by nicotinic acid is prevented by cycloheximide or actinomycin D, suggesting that this effect is produced by synthesis of the enzyme de novo. 3. Despite the enhancement of phosphoenolpyruvate carboxykinase activity and glycogen depletion, which occurs 5h after the injection of nicotinic acid, the gluconeogenic capacity of liver is low and considerably less than the values found in rats starved for 48h. 4. When the livers of well-fed rats are perfused in the presence of low concentrations of glucose, the activity of phosphoenolpyruvate carboxykinase significantly increases compared with the control. 5. This increase is not related to the glycogen content, but seems to be also the result of synthesis of the enzyme de novo, since this effect is counteracted by previous treatment with cycloheximide or actinomycin D. 6. Phosphoenolpyruvate carboxykinase activity is not increased in the presence of low concentrations of circulating glucose when 40 mM-imidazole (an activator of phosphodiesterase) is added to the perfusion medium. 7. Addition of dibutyryl cyclic AMP to the perfusion medium results in an increase in phosphoenolpyruvate carboxykinase activity, in spite of the presence of normal concentrations of circulating glucose. On the other hand, the concentration of cyclic AMP in the liver increases when that of glucose in the medium is low. 8. These results suggest that, in the absence of hormonal factors, the regulation of phosphoenolpyruvate carboxykinase can be accomplished by glucose itself, inadequate concentrations of it resulting in the induction of the enzyme. The mediator in this regulation, as in hormonal regulation, seems to be cyclic AMP.
Project description:1. Gluconeogenesis in developing rat kidney cortex was studied by assaying the activities of two enzymes, glucose 6-phosphatase and phosphoenolpyruvate carboxykinase, and by measuring glucose formation in tissue slices. 2. Glucose 6-phosphatase and phosphoenolpyruvate carboxykinase are present in late foetal (21-22-day-old) tissue and increase rapidly postnatally. Maximum activity of phosphoenolpyruvate carboxykinase occurs at 7 days of age, followed by a decline to the adult level. Glucose 6-phosphatase activity rises during the first 2 postnatal weeks and then declines. 3. Late foetuses synthesize glucose from both pyruvate and l-glutamate. The rate increases during the first 2 weeks to above adult levels. Synthesis is always higher from pyruvate than from glutamate. 4. The effect of 24hr. starvation was studied in perinatal animals. The results indicate that the ability to increase the rate of glucose synthesis as a result of starvation is not present at birth, but develops some time after the second postnatal day.
Project description:The activities of phosphoenolpyruvate carboxykinase, ;malic enzyme', citrate-cleavage enzyme and glucose 6-phosphate dehydrogenase were assayed in homogenates of rumen mucosa, liver and adipose tissue of cattle. Rumen mucosa cytoplasm contained activities of ;malic enzyme' approximately sevenfold those of phosphoenolpyruvate carboxykinase, suggesting that the conversion of propionate into lactate by rumen mucosa involves ;malic enzyme'. Neither starvation for 8 days nor feeding with a concentrate diet for at least 3 months before slaughter produced enzyme patterns in the tissues different from those in cattle given only hay, except that the all-concentrate diet caused increased activities of glucose 6-phosphate dehydrogenase and ;malic enzyme' in adipose tissues. Rumen mucosa, liver and adipose tissue contained phosphoenolpyruvate carboxykinase activity. ;Malic enzyme' was absent in liver. Citrate-cleavage enzyme activity was present in liver and adipose tissue but was quite low in rumen mucosa. Liver contained much less glucose 6-phosphate dehydrogenase activity than rumen mucosa or adipose tissue.