Effect of reduced H+-ATPase activity on acid tolerance in Streptococcus bovis mutants

In order to confirm that H+-ATPase plays an important role in the acid tolerance of Streptococcus bovis, two mutants with low activities of H+-ATPase were isolated by use of ethyl methanesulfonate and neomycin resistance. The activity of H+-ATPase per cellular nitrogen was related to the lowest culture pH permitting growth. A mutant with little H+-ATPase activity (Mutant 2) was unable to grow below pH 5.5, which suggests that the intracellular pH should be maintained above 5.5 in S. bovis. Since lactate dehydrogenase activity, which is important for acid tolerance, was similar in parent and mutant strains, H+-ATPase activity is likely to affect acid tolerance. The amount of H+-ATPase protein as determined by Western-blot analysis with polyclonal antibody, was similar in Mutant 2 and its parent, indicating that H+-ATPase activity per enzyme protein is reduced by mutation. Probably, H+-ATPase synthesis was not changed by mutation. The gene encoding H+-ATPase of Mutant 2 had mutations at positions close to the ATP-binding motif A sequence in the beta-subunit, which probably explains the reduced activity of H+-ATPase in this mutant. These results strongly support the assumption that H+-ATPase has a key role in the acid tolerance of S. bovis. (C) 2000 Academic Press.