Regulation of H + -ATPase Synthesis in Response to Reduced pH in Ruminal Bacteria

Abstract

The capacity of ruminal bacteria to regulate H(+)-ATPase synthesis in response to reduced pH was investigated to explain acid tolerance. The activity of H(+)-ATPase in Streptococcus bovis, an acid-tolerant bacterium, was 2.2-fold higher at pH 4.5 than at pH 5.5. The increase in the amount of H(+)-ATPase protein was similar, suggesting that the increase in H(+)-ATPase activity is owing to the increase in H(+)-ATPase synthesis. The level of atp-mRNA at pH 4.5 was 2.5-fold higher than at pH 5.5, indicating that H(+)-ATPase synthesis is regulated at the transcriptional level, responding to low pH. In Ruminococcus albus, an acid-sensitive bacterium, H(+)-ATPase activity, the amount of H(+)-ATPase protein, and the level of atp-mRNA at pH 7.0 were similar to the values at pH 6.0, the lowest pH permitting growth. This result suggests that R. albus is incapable of enhancing H(+)-ATPase synthesis at low pH. Thus, acid tolerance appeared to be related to the capacity to augment the synthesis of H(+)-ATPase responding to low pH.

Purification and gene sequencing of conjugated linoleic acid reductase from a gastrointestinal bacterium, Butyrivibrio fibrisolvens

“…Antiserum against the purified CLA‐R was prepared using rats as previously described (Miwa et al. 2001), and then purified by affinity chromatography with antigen‐bound CNBr‐activated Sepharose 4B (GE Healthcare Bio‐Sciences Corp.).…”

“…2001), and then purified by affinity chromatography with antigen‐bound CNBr‐activated Sepharose 4B (GE Healthcare Bio‐Sciences Corp.). Western blotting was performed as previously described (Miwa et al. 2001).…”

“…Total RNA was extracted from B. fibrisolvens at the late‐log stage, and purified as described previously (Miwa et al. 2001).…”

“…The probe specific to the cla‐r mRNA was labelled by PCR with Digoxigenin‐dUTP (Dig DNA Labeling Kit; Roche Diagnostic GmbH, Mannheim, Germany) according to the manufacturer's instructions. Hybridization (42°C, over night) was performed as described (Miwa et al. 2001).…”

“…Antiserum against the purified CLA-R was prepared using rats as previously described (Miwa et al 2001), and then purified by affinity chromatography with antigen-bound CNBr-activated Sepharose 4B (GE Healthcare Bio-Sciences Corp.). Western blotting was performed as previously described (Miwa et al 2001). Briefly, MDT-5 and MDT-10 cells harvested at the mid-log growth stage were immediately cooled on ice, and disrupted by ultrasonication at 4°C for 3 min.…”