An ffh mutant of Streptococcus mutans is viable and able to physiologically adapt to low pH in continuous culture

Abstract

Previously, we described in Streptococcus mutans strain NG8 a 5-gene operon (sat) that includes ffh, the bacterial homologue of the eukaryotic signal recognition particle (SRP) protein, SR54. A mutation in ffh resulted in acid sensitivity but not loss of viability. In the present study, chemostat-grown cells of the ffh mutant were shown to possess only 26% and 39% of the parental membrane F-ATPase activity and 55% and 75% of parental glucose-phosphotransferase (PTS) activity when pH-7 and pH-5-grown cells, respectively, were assayed. Two-dimensional-gel electrophoretic analyses revealed significant differences in protein profiles between parent and ffh-mutant strains at both pH 5 and pH 7. It appears that the loss of active SRP (Ffh) function, while not lethal, results in substantial alterations in cellular physiology that includes acid tolerance.

Streptococcal viability and diminished stress tolerance in mutants lacking the signal recognition particle pathway or YidC2

“…As expected, the enzymatic activity of membrane fractions from acid-shocked (pH 5.0) wild-type cells was significantly increased relative to the pH 7.0-maintained culture and reflects the ability of S. mutans to rapidly adapt to this environmental challenge; this was not the case for the mutant strains in which the specific activities remained unchanged or decreased significantly in response to acid-shock. Expression of the enzymatically active F 1 component was not likely affected by the ffh mutation because we showed that ATPase activity from whole decryptified cells was identical in chemostat-grown wild-type and ffh-mutant MK4 cultures (39). However, differences in activities of membrane fractions were similar to those shown here.…”

“…Taken together, the results indicate that complete removal of Ffh from S. mutans is not lethal, but does render the organism less able to contend with several environmental stressors. Interestingly, mutant MK4 was shown previously to adapt to acid stress and grow when pH was allowed to drop gradually from normal metabolism, indicating that given time compensatory mechanisms are engaged upon environmental challenge in the absence of an intact SRP pathway (39).…”

“…Preparation of bacterial membranes and measurement of H ϩ ͞ATPase enzymatic activity were performed as described (39). ATPasespecific activities of membrane fractions prepared from cultures grown at pH 7.0 or 5.0 (acid shock) of the wild-type and mutant strains were calculated and expressed as nmol of Pi released per min per mg of total protein.…”

YidC1 and YidC2 are functionally distinct proteins involved in protein secretion, biofilm formation and cariogenicity of Streptococcus mutans

“…The mechanisms of insertion of the F 1 F 0 ATPase in S. mutans membranes are not known; however, mutations of ffh or yidC2 decrease acid tolerance and membraneassociated ATPase activity (Crowley et al, 2004;Hasona et al, 2005). In previous work, a deletion of yidC1 had no apparent effect on acid tolerance or membrane-associated ATPase activity (Hasona et al, 2005), leading to the hypothesis that, in S. mutans, YidC2 and the SRP pathways are involved in the assembly of the F-ATPase, while YidC1 is not.…”

Membrane Composition Changes and Physiological Adaptation byStreptococcus mutansSignal Recognition Particle Pathway Mutants

“…Surprisingly, interruption of ffh was not lethal, in contrast to reports for Escherichia coli (41) and Bacillus subtilis (19), but did render S. mutans sensitive to acid shock (pH 5.0). Mutants subsequently engineered to lack ffh, scRNA, or ftsY, singly or in combination, remained viable in complex growth media under nonstress conditions but were unable to survive at pH 5.0 or with 3.5% NaCl, and growth in the presence of 0.3 mM H 2 O 2 was impaired (10,17). Acid tolerance in S. mutans is mediated in large part by an F 1 F 0 ATPase proton pump (4), and all SRP pathway mutants have decreased membrane-associated ATPase activity, which partially explains their acid sensitivity (10,17).…”