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.

FIG. 1.

Change in the act mRNA level during growth in batch culture. S. bovis was grown for 2 h (lane 1), 5 h (lane 2), or 6 h (lane 3). Total RNA (10 μg) was added to the gel. The arrow indicates the band that hybridized with an act probe.

FIG. 2.

Western blot analysis of PFL protein. PFL was anaerobically incubated with (lane 1) or without (lane 2) PFL-AE.

FIG. 3.

Primer extension analysis of S. bovis pfl mRNA in S. bovis 12U1 and PF2. The upper and lower arrows indicate the pfl mRNA from the chromosome and the plasmid, respectively. Lanes 1 to 3, 12U1 grown on glucose at pH 7.0 (lane 1), on lactose at pH 7.0 (lane 2), or on glucose at pH 4.5 (lane 3); lanes 4 to 6, PF2 grown on glucose at pH 7.0 (lane 4), on lactose at pH 7.0 (lane 5), or on glucose at pH 4.5 (lane 6).