Acid adaptation is a major factor in the ability of S. mutans
to thrive in dental plaque and to outcompete commensal organisms found within the oral cavity. Many of the adaptive mechanisms that allow the organism to survive in dental plaque have been studied in detail (9
). Proteomic studies of S. mutans
grown under acid stress conditions suggested that the bacterium may use biosynthesis of amino acids as a way to reroute pyruvate away from lactic acid and also as a way to maintain proper intracellular pH conditions (40
). Modulation of the intermediates of glycolysis, as well as removal of protons from the cytoplasm through degradation/biosynthesis of amino acids, can be important for virulence. IlvE was among the proteins identified by Len et al. as being upregulated following growth at low pH (39
). Protein sequence homology of IlvE with other known ATases supported its role as a branched-chain amino acid aminotransferase.
Initial characterization of the ilvE mutant strain created in this study revealed a requirement for branched-chain amino acids, which could be restored by supplementing the medium with amino acids or amino acid-derived products such as isovaleric acid. Due to the catabolic and biosynthetic enzymatic activities inherent to ATases, it is possible that the loss of IlvE also had an effect on the end products of amino acid degradation and, for this reason, an exogenous supply of isovaleric acid also aided its growth.
The role of ilvE during growth at low pH was also explored. Growth of the ilvE mutant strain at pH 5.4 showed a lag during exponential phase, with a lower final yield than that of UA159. The defect was specific for acidic growth conditions, since growth at neutral pH was unaffected unless nutritional limitation was encountered.
Since acid adaptation is a key virulence component of S. mutans, the loss of ilvE could alter the ability of the organism to form reducing equivalents from ammonia or to reroute end products such as pyruvate for amino acid biosynthesis. Hence, the acid tolerance of the mutant strain was of interest. We found that the ilvE mutant strain was defective in its acid tolerance compared to the parental strain, in stark contrast to our previous reports concerning acid-adaptive attributes of the parental strain. Our data demonstrate that the loss of ilvE is sufficient to cause a change in acid adaptation and support the contribution of IlvE to acid tolerance.
To clarify the mechanism(s) underlying the acid-sensitive phenotype of the ilvE
mutant strain, we first analyzed the membrane fatty acid content by GC-FAME analysis to determine if changes in composition could account for the acid sensitivity. Previous studies have shown that the inability to elevate unsaturated fatty acid content in response to external acidification leads to acid sensitivity (21
). GC-FAME analysis of the ilvE
mutant strain grown at steady-state culture pHs of 5 and 7 revealed a fatty acid composition similar to that of the parent strain. In agreement with our previous reports (19
), the ilvE
mutant produced elevated levels of unsaturated membrane fatty acids when it was grown at pH 5. Thus, the mutant exhibited membrane composition changes in response to acid exposure. In unison with the membrane composition data, proton permeability was also relatively unaffected in the mutant. Even though these characteristics are not interchangeable, membrane composition can contribute to and partly explain the permeability characteristics of the mutant. The acid sensitivity displayed by the ilvE
mutant strain was not due to an inability to modulate its membrane fatty acid composition, suggesting that the loss of ilvE
causes a defect in acid adaptation that is independent of membrane composition. The data also show that the acid-adaptive changes occurring in the membrane are insufficient to compensate for the loss of ilvE
Results from minimum glycolytic pH assays demonstrated that even though ilvE was absent, the amount of acid produced by the mutant was comparable to that of the parent strain. This was unexpected, because we had hypothesized that the loss of ilvE could potentially have an effect on acid production. Redundancy within the genome could account for the glycolytic profile of the mutant if other ATases are induced in the absence of IlvE.
In experiments with the ilvE mutant and parent strains, we established that IlvE exhibits enzymatic activity on isoleucine and valine. Furthermore, AspC, an aromatic amino acid aminotransferase, does not participate in branched-chain amino acid catabolism, since deletion of the gene had no significant effect on ATase activity compared to that of the parent strain. The data also support an argument that there is another, as yet undefined ATase with activity for leucine.
The biosynthetic role of branched-chain amino acid aminotransferases is vital for the synthesis of important signaling molecules, i.e., the branched-chain amino acids. Under conditions of acid stress, S. mutans
requires bcAAs to signal known acid stress responses reliant upon branched-chain amino acids as sensors of overall fitness (38
). CodY, a global regulator of S. mutans
, uses intracellular branched-chain amino acid pools as coeffectors for regulation of its target genes (53
). Loss of ilvE
as a branched-chain amino acid aminotransferase could have global effects on the regulatory network of S. mutans
. A recent study has shown that CodY activity is affected by ATases that modulate the intracellular pools of amino acids (12
). The loss of ilvE
could have a much larger effect on the regulatory capabilities of CodY and its targets, which could partly explain the acid sensitivity displayed by the ilvE
mutant, particularly if important acid-adaptive processes are disregulated due to inadequate CodY function in the absence of branched-chain amino acids.
The proton-pumping ATPase is essential for maintenance of intracellular pH conditions that are favorable for S. mutans. Levels of ATPase activity were diminished in the ilvE mutant compared to those in the parent strain, reflecting the impact of the mutation on another key adaptive mechanism that was altered in the absence of ilvE.
A mechanism to explain the downregulation of F-ATPase activity in the ilvE
mutant strain is not yet clear at this point. We have shown previously that F-ATPase operon transcription is upregulated during growth at low pH (34
) and that if the ΔpH is disrupted via the fabM
mutation, the cytoplasm becomes acidified and ATPase production is elevated in response (21
). Because of the presence of a cre
site for potential CcpA binding at position −160 in the F-ATPase operon, it is appealing to interpret reduced F-ATPase activity in the ilvE
mutant strain in light of potential CodY and CcpA interactions. Previous work with B. subtilis
has shown that the global regulators of transcription CcpA, CodY, and TnrA can participate in regulating branched-chain amino acid biosynthesis (57
). Studies with other bacterial model systems, including S. mutans
) and L. lactis
), have shown that CodY regulates ilvE
in addition to many other genes and that, for S. mutans
, a codY
strain is acid sensitive (38
). Moreover, CodY and CcpA can act as negative and positive regulators of the same gene(s), or cooperatively as positive regulators, in controlling carbon flow through acetate kinase in B. subtilis
). Experiments are under way to determine potential relationships between CcpA, CodY, and isoleucine and their effects on the expression of the F-ATPase.
The acid-sensitive phenotype of the ilvE mutant strain suggested that the ilvE gene could be under external pH regulation. ilvE promoter fusions showed that transcription of the gene is increased under low-pH conditions. Currently, we are studying the transcriptional regulation of ilvE and its interaction with CodY with respect to its effects on membrane composition and acid tolerance in S. mutans. Transcriptional as well as physiological data indicate that ilvE is part of the acid tolerance response of S. mutans.