The role of S. mutans
in the development of human dental caries has been well defined (20
). Many studies, using a variety of biochemical approaches, have identified several properties of S. mutans
that contribute to the cariogenic potential of this organism, including aciduricity and acidogenicity. The organism is able to persist in the oral cavity by adapting to environmental stresses, particularly the acidification of its milieu (18
). It has been established that the F-ATPase is transcriptionally up-regulated during growth at pH values below 7 (16
). Recently, we have shown that the membrane fatty acid composition of S. mutans
changes dramatically during growth at low pH values (7
). In an effort to contribute to the characterization of additional factors involved with the acid adaptation process, we have looked to identify proteins that allow S. mutans
to survive and grow in an acidified environment. Previously, we identified an AP endonuclease activity that is induced when S. mutans
is grown at acidic pH values (10
). The work presented here describes the initial characterization of an S. mutans
AP endonuclease activity, as well as the role of that enzyme, Smx, in the physiology of the organism.
Based on our earlier work, we presumed that the endonuclease activity observed in cell extracts was similar to Exo III. We identified a gene, termed smx, that possessed a high degree of similarity to the E. coli xth and S. pneumoniae exoA genes. On the basis of this amino acid identity, the AP endonuclease of S. mutans identified here is likely another member of the family of class II AP endonucleases. Promoter analysis of the 5′ untranslated region of smx, determination of the transcriptional start site, and Northern analysis (data not shown) reveal that the smx gene is transcribed as a monocistronic mRNA.
To determine whether the similarities between Smx and the other class II endonucleases extends to biochemical functionality, recombinant Smx protein was expressed and utilized in a cleavage assay with a model abasic substrate. Indeed, the gene cloned and expressed in this study encoded the enzymatic activity that we reported previously. Purified Smx protein was able to catalyze the cleavage of a THF-containing duplex in a manner similar to that of the other class II enzymes (specifically, Exo III), that is, 5′ to the abasic residue. However, Smx differs from Exo III in its efficiency of removal of additional bases. These results indicate that Smx may catalyze auxiliary functions and/or that the kinetic profile of this enzyme may be different from that of Exo III. Studies are under way in the laboratory to extend our knowledge of the biochemical characteristics of Smx.
A hallmark phenotype of E. coli xth
mutant strains is peroxide sensitivity (4
), which proved to be an effective means of determining whether the S. mutans smx
gene would be able to complement an E. coli xth
-deficient strain. In fact, smx
expressed from a plasmid was able to complement the E. coli xth
-deficient strain by alleviating sensitivity to peroxide attack. These findings further demonstrate similar functionalities of the two enzymes. In addition, the smx
gene was able to complement an endA recA
mutant strain of E. coli
. Taken together, these data suggest the possibility that Smx is able to affect other types of DNA repair in S. mutans
Creation of an smx
mutant strain resulted in the inability of cell extracts from pH 5-grown cultures to cleave a THF-containing, model abasic substrate. Some residual AP endonuclease-like activity is still, however, detected in cell extracts from pH 7-grown cultures. We had demonstrated the low likelihood that an endonuclease IV-like activity exists in S. mutans
), and a search of the UA159 genome database (1
) was unsuccessful in locating deduced amino acid sequences with sufficient similarity to endonuclease IV from E. coli
. A candidate to explain the low levels of AP endonuclease activity in the pH 7 extracts is the endonuclease III homologue, which we have located immediately downstream of the smx
gene (Fig. ). Experiments are under way to investigate the role, if any, that this putative nth
-like gene might play in the acid base physiology of S. mutans
The smx-deficient strain UR101 was characterized with respect to sensitivity to environmental and DNA-damaging agents: acid, hydrogen peroxide, and near-UV irradiation. Loss of smx rendered the mutant strain more sensitive to oxidative damage than the wild-type strain when cultures were grown at pH values of 5. Given that peroxide sensitivity is a hallmark phenotype of E. coli strains deficient in xth, this finding further corroborates the conclusion that smx encodes a major, if not the sole, low-pH-inducible AP endonuclease activity expressed in S. mutans. Acid challenge experiments showed that while the mutant strain still acid adapts, the response to acid-mediated damage is not affected by the loss of smx. The difference in sensitivities of the smx strain to acid and hydrogen peroxide suggest the possibility of different mechanisms for the formation of DNA damage by the two agents that we used in this study, at least in S. mutans.
All the strains tested in this study were adversely affected by the presence of iron, confirming earlier results (5
) which have suggested that the mechanism of metal ion toxicity in the absence of oxygen is complex and likely includes mechanisms beyond the involvement of Fenton chemistry. Iron in the form of Fe2+
, plus the addition of hydrogen peroxide to the reaction mixture, significantly altered the survival characteristics and resulted in complete killing of cells. The data strongly suggest that the bulk of iron had probably been converted to Fe3+
via Fenton chemistry (for a review, see reference 36
). Along with the conversion of Fe2+
, the concomitant formation of hydroxyl radical (OH·) likely leads to substantial DNA damage, with the formation of AP sites (37
). Clearly, strains containing a mutation in smx
were far less able than the wild-type or the recA
-deficient strain to cope with oxidative damage. The presence of iron in the reduced state served only to intensify the effects of the damage seen with peroxide alone, confirming our earlier hypothesis that RecA-dependent processes are not involved in the protection of the organism from oxidative damage (27
). The mechanism of iron-mediated killing is still unclear, but the action of iron as a pro-oxidant (iron facilitating hydrogen peroxide killing) was well supported by our observations.
Our results demonstrate that Smx is the major AP endonuclease in S. mutans and that it is capable of removing AP sites in DNA. Moreover, the data show that acid adaptation involves at least some aspects of the oxidative-stress response, in the sense that the smx mutant strain was sensitive to hydrogen peroxide. What remains to be established is how extensively oxidative-stress gene regulation coincides with other attributes of acid adaptation in S. mutans or whether a specific subset of genes, such as smx, overlaps with those products participating in the acid response repertoire of oral streptococci. Experiments designed to provide insights into the regulation of Smx production are being conducted presently. The construction of the expression vector described in this study allows the convenient preparation of purified Smx protein, potentially facilitating the characterization of mutant forms of the enzyme. This will, in turn, aid in a more detailed understanding of the requirements of the Smx enzyme for substrate recognition and cleavage as well as further biochemical characterization. These data will enable us to gain a more complete picture of the overall role of Smx in the survival strategies of S. mutans.