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

Abstract

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.

Effects of the overexpression of fructose-1,6-bisphosphate aldolase on fermentation pattern and transcription of the genes encoding lactate dehydrogenase and pyruvate formate-lyase in a ruminal bacterium, Streptococcus bovis

“…The source of S. bovis 12U1, a transformable strain, was previously described (Asanuma and Hino, 2002b). The 12U1 p and 12U1 fba strains were constructed as described below.…”

“…The 12U1 p and 12U1 fba strains were constructed as described below. The source of plasmid pSBE11, a shuttle vector between Escherichia coli and S. bovis (Nakamura et al, 2001), was previously described (Asanuma and Hino, 2002b).…”

“…S. bovis was grown in batch culture as previously described (Asanuma and Hino, 2002b). The medium contained (g/L): K 2 HPO 4 , 0.45; KH 2 PO 4 , 0.45; (NH 4 ) 2 SO 4 , 0.9; NaCl, 0.9; CaCl 2 · 2H 2 O, 0.12; MgSO 4 · 7H 2 O, 0.19; Fe(NH 4 ) 2 (SO 4 ) 2 , 0.1; Trypticase (BBL; Becton Dickinson, Cockeysville, MD, USA), 1.0; yeast extract (Difco Laboratories, Detroit, MI, USA), 1.0; and cysteine · HCl, 0.6.…”

“…As a result, the rate of the LDH reaction greatly decreases. Because the LDH reaction is much faster than the PFL reaction, a decrease in the rate of LDH reaction brings about a decrease in the rate of overall glucose fermentation (Asanuma and Hino, 2002b). Consequently, the rate of ATP generation decreases, which leads to a decrease in growth rate.…”

“…Thus, the fermentation pattern in S. bovis is regulated by the activity ratio of LDH to PFL. However, LDH activity is much higher than PFL activity, because the promoter activity of the LDH gene (ldh) is much higher than that of the PFL gene (pfl) (Asanuma and Hino, 2002b). This result may explain why the formateto-lactate ratio in S. bovis is usually low.…”