Molecular characterization and significance of phosphoenolpyruvate carboxykinase in a ruminal bacterium, Streptococcus bovis.

To gain knowledge about the significance of phosphoenolpyruvate (PEP) carboxykinase (PCK) in Streptococcus bovis, the sequence of the gene encoding PCK (pck) was determined. Transcriptional analysis indicated that the pck is transcribed in a monocistronic fashion. The level of pck-mRNA was higher when cells were grown on lactose than on glucose, suggesting that PCK synthesis increases when the growth rate is low. The pck-mRNA level was higher in a mutant lacking ccpA, which encodes the catabolite control protein A (CcpA), than in the parent strain, suggesting that pck transcription is suppressed by CcpA. S. bovis PCK showed oxaloacetate (OAA)-decarboxylating activity, but no PEP-carboxylating activity (reverse reaction). In S. bovis, OAA was speculated to be produced from PEP via pyruvate. Disruption of pck in S. bovis resulted in decreased growth rate and cell yield. When a pck-disrupted mutant was grown in a medium lacking amino acids, the lag phase was longer and the cell yield was lower than the case of the parent strain. These results suggest that pck is involved in the initiation of growth, including the induction of amino acid synthesis and energy metabolism.

Involvement of two-component signal transduction system, ComDE, in the regulation of growth and genetic transformation, in the ruminal bacterium Streptococcus bovis.

In ruminants, Streptococcus bovis is considered to be associated with acute rumen acidosis. To elucidate the regulatory mechanisms of S. bovis growth, we investigated the function of the two components of the peptide pheromone-signaling system, ComD and ComE, which are encoded by comD and comE, respectively, via the competence-stimulating peptide ComC, which is encoded by comC. Deletion of entire comC and two-thirds of comD resulted in decreased growth rate, which may be related to the change in the expression of several proteins, as shown by two-dimensional gel electrophoresis. The transcript level of comED was decreased by the disruption of comCD, suggesting that the transcription of comED might be stimulated by ComC. The transformation frequency was decreased by the disruption of comCD. Addition of recombinant ComC to S. bovis cultures increased the growth rate and transformation frequency. In the cultures of mixed ruminal microbes, addition of mature ComC peptide increased the number of S. bovis per total bacterial counts as estimated by the cDNA amounts of 16SrRNA. Thus, the peptide pheromone-signaling system via ComC, D, and E might be involved in the control of S. bovis growth in addition to competence development. This is the first report suggesting that an autoinducing peptide functions in the ruminal ecosystem.

Molecular and biochemical characterization of phosphoenolpyruvate carboxykinase in the ruminal bacterium Ruminococcus albus.

Molecular properties and transcriptional control of phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.32) in Ruminococcus albus were examined. The putative 537-amino acid PCK polypeptide has a predicted mass of 59.4 kDa and an isoelectric point of 4.82. RT-PCR and Northern blot analyses of pck mRNA suggest that the transcript is monocistronic and that pck transcription is not affected by changes in sugar sources present in growth medium. PCK enzymatic activity requires either Mg(2+) or Mn(2+) and an optimal pH of 7.0. R. albus PCK phosphorylated ADP more readily than GDP. Apparent K ( m ) values of PCK for PEP and ADP were considerably lower than those for OAA and ATP, suggesting that the reaction from PEP to OAA is favored in R. albus. The enzyme properties of PCK in R. albus appear to be more similar to Selenomonas ruminantium PCK than to Ruminococcus flavefacience, although R. albus and R. flavefacience belong to the same genus. The specific activity of PCK, representing the amount of enzyme per cell, in R. albus was much lower than that in S. ruminantium. The amount of succinate produced in R. albus from one unit of cellobiose was also much lower than the sum of succinate and propionate produced in S. ruminantium. Based on these results, we propose enhancement of PCK activity by stimulating PCK transcription as a method to decrease R. albus H(2) production without suppressing growth.

Molecular properties and transcriptional control of the phosphofructokinase and pyruvate kinase genes in a ruminal bacterium, Streptococcus bovis.

Molecular properties of pyruvate kinase (PYK) and phosphofructokinase (PFK) in Streptococcus bovis and transcriptional control of the two enzymes were examined. Sequence analysis indicated that the PYK gene (pyk) clusters with the PFK gene (pfk) and several other genes. It was demonstrated that the pyk and pfk are cotranscribed and their transcription appeared to be regulated at the transcriptional level in response to the sugars supplied. The intracellular pyk-mRNA level was lower in a catabolite control protein A (CcpA)-disrupted mutant than in its parent strain, and a binding site of CcpA was found in the upstream region of pfk. These results suggest that pfk-pyk transcription is enhanced by CcpA. A recombinant pyk-overexpressing strain showed approximately five-fold higher PYK activity, but it did not affect the growth rate or formate-to-lactate ratio significantly, suggesting that the flux in the glycolytic pathway is not altered by an increase in PYK activity.