Fructose Bisphosphate Aldolase Activity and Glycolytic Intermediate Concentrations in Relation to Lactate Production inStreptococcus bovis

Yoshii

2004

Appl Environ Microbiol

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A ccpA gene that encodes global catabolite control protein A (CcpA) in Streptococcus bovis was identified and characterized, and the involvement of CcpA in transcriptional control of a gene (ldh) encoding lactate dehydrogenase (LDH) and a gene (pfl) encoding pyruvate formate-lyase (PFL) was examined. The ccpA gene was shown to be transcribed as a monocistronic operon. A catabolite-responsive element (cre) was found in the promoter region of ccpA, suggesting that ccpA transcription in S. bovis is autogenously regulated. CcpA required HPr that was phosphorylated at the serine residue at position 46 (HPr-[Ser-P]) for binding to the cre site, but glucose 6-phosphate, fructose 1,6-bisphosphate, and NADP had no effect on binding. Diauxic growth was observed when S. bovis was grown in a medium containing glucose and lactose, but it disappeared when ccpA was disrupted, which indicates that CcpA is involved in catabolite repression in S. bovis. The level of ccpA mRNA was higher when cells were grown on glucose than when they were grown on lactose, which was in line with the level of ldh mRNA. When cells were grown on glucose, the ldh mRNA level was lower but the pfl mRNA level was higher in a ccpA-disrupted mutant than in the parent strain, which suggests that ldh transcription is enhanced and pfl transcription is suppressed by CcpA. The ccpA-disrupted mutant produced less lactate and more formate than the parent, probably because the mutant had reduced LDH activity and elevated PFL activity. In the upper region of both ldh and pfl, a cre-like sequence was found, suggesting that the complex consisting of CcpA and HPr-[Ser-P] binds to the possible cre sites. Thus, CcpA appears to be involved in the global regulation of sugar utilization in S. bovis.

Section: Resultssupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

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Molecular Characterization of CcpA and Involvement of This Protein in Transcriptional Regulation of Lactate Dehydrogenase and Pyruvate Formate-Lyase in the Ruminal Bacterium Streptococcus bovis

Yoshii

2004

Appl Environ Microbiol

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“…Organic acids were analyzed by high-performance liquid chromatography as previously described (11). Cellular nitrogen was determined by the Kjeldahl method as described previously (4).…”

Section: Methodsmentioning

confidence: 99%

“…S. bovis LDH is activated by fructose 1,6-bisphosphate (2,23), and PFL is inhibited by dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate (3). The intracellular concentrations of these glycolytic intermediates change so much that they affect the activities of the enzymes (4). The other level is regulation of LDH and PFL synthesis at the transcriptional level (5,6).…”

mentioning

confidence: 99%

Molecular Characterization and Expression of Pyruvate Formate-Lyase-Activating Enzyme in a Ruminal Bacterium, Streptococcus bovis

2002

Appl Environ Microbiol

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To clarify the significance of the activation of pyruvate formate-lyase (PFL) by PFL-activating enzyme (PFL-AE) in Streptococcus bovis, the molecular properties and gene expression of PFL-AE were investigated. S. bovis PFL-AE was deduced to consist of 261 amino acids with a molecular mass of 29.9 kDa and appeared to be a monomer protein. Similar to Escherichia coli PFL-AE, S. bovis PFL-AE required Fe 2؉ for activity. The gene encoding PFL-AE (act) was found to be polycistronic, and the PFL gene (pfl) was not included. However, the act mRNA level changed in parallel with the pfl mRNA level, responding to growth conditions, and the change was contrary to the change in the lactate dehydrogenase (LDH) mRNA level. PFL-AE synthesis appeared to change in parallel with PFL synthesis. Introduction of a recombinant plasmid containing S. bovis pfl and the pfl promoter into S. bovis did not affect formate and lactate production, which suggests that the activity of the pfl promoter is low. When the pfl promoter was replaced by the S. bovis ldh promoter, PFL was overexpressed, which caused an increase in the formate-to-lactate ratio. However, when PFL-AE was overexpressed, the formate-to-lactate ratio did not change, suggesting that PFL-AE was present at a level that was high enough to activate PFL. When both PFL-AE and PFL were overexpressed, the formate-to-lactate ratio further increased. It is conceivable that LDH activity is much higher than PFL activity, which may explain why the formate-to-lactate ratio is usually low.

Molecular characterization of HPr and related enzymes, and regulation of HPr phosphorylation in the ruminal bacterium Streptococcus bovis

2003

Arch Microbiol

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Molecular properties of HPr, enzyme I, and HPr kinase in Streptococcus bovis, and the regulation of HPr phosphorylation were examined. The genes encoding HPr (ptsH) and enzyme I (ptsI) were found to be cotranscribed. Two transcriptional start sites were detected in a region upstream of the HPr kinase gene (hprK). HPr kinase had both HPr-phosphorylating and HPr-dephosphorylating activities. The importance of phosphorylation of Ser-46 in HPr was shown by using a mutant HPr in which Ser-46 was replaced by Ala. When S. bovis was grown in glucose-limited medium, the amount of seryl-phosphorylated HPr (HPr-[Ser-P]) decreased drastically as the growth rate decreased. In contrast, the amount of histidyl-phosphorylated HPr (HPr-[His-P]) increased gradually as the growth rate decreased. The amount of HPr kinase did not greatly change with the growth phase, whereas the intracellular P(i) concentration increased as the growth rate decreased. HPr-[Ser-P] decreased as the intracellular P(i) increased as a consequence of inhibition of HPr kinase activity by P(i) and simultaneous enhancement of HPr-[Ser-P] phosphatase activity by P(i). Thus, it is conceivable that the ratio of HPr-[Ser-P] to HPr-[His-P] is regulated by the bifunctional activity of HPr kinase in response to intracellular P(i) concentration.