Smx Nuclease Is the Major, Low-pH-Inducible Apurinic/Apyrimidinic Endonuclease in Streptococcus mutans

Abstract

The causative agent of dental caries in humans, Streptococcus mutans, outcompetes other bacterial species in the oral cavity and causes disease by surviving acidic conditions in dental plaque. We have previously reported that the low-pH survival strategy of S. mutans includes the ability to induce a DNA repair system that appears to involve an enzyme with exonuclease functions (K. Hahn, R. C. Faustoferri, and R. G. Quivey, Jr., Mol. Microbiol 31:1489-1498, 1999). Here, we report overexpression of the S. mutans apurinic/apyrimidinic (AP) endonuclease, Smx, in Escherichia coli; initial characterization of its enzymatic activity; and analysis of an smx mutant strain of S. mutans. Insertional inactivation of the smx gene eliminates the low-pH-inducible exonuclease activity previously reported. In addition, loss of Smx activity renders the mutant strain sensitive to hydrogen peroxide treatment but relatively unaffected by acid-mediated damage or near-UV irradiation. The smx strain of S. mutans was highly sensitive to the combination of iron and hydrogen peroxide, indicating the likely production of hydroxyl radical by Fenton chemistry with concomitant formation of AP sites that are normally processed by the wild-type allele. Smx activity was sufficiently expressed in E. coli to protect an xth mutant strain from the effects of hydrogen peroxide treatment. The data indicate that S. mutans expresses an inducible, class II-like AP endonuclease, encoded by the smx gene, that exhibits exonucleolytic activity and is regulated as part of the acid-adaptive response of the organism. Smx is likely the primary, if not the sole, AP endonuclease induced during growth at low pH values.Streptococcus mutans inhabits dental plaque, where, as a causative agent of dental caries, it creates and survives in an acidic milieu (20). Acidification of plaque is a direct result of the secretion of organic acids, by-products of carbohydrate metabolism by S. mutans and other bacterial inhabitants of the oral cavity. The survival of the microbe in the oral cavity is predicated on its ability to elaborate an acid-adaptive response (30). Since pH values in dental plaque have been reported to be as low as 4.0, the intracellular environment is potentially exposed to acidic conditions (11,12,15), which likely leads to an increased formation of abasic sites in DNA (19). This idea is indirectly supported by our previous observation that the process of acid adaptation in S. mutans also includes expression of a low-pH-inducible apurinic/apyrimidinic (AP) endonuclease (10, 27). These studies suggested that the AP endonuclease activity in S. mutans more closely resembles exonuclease III (Exo III) of Escherichia coli than endonuclease IV (10).Based on this hypothesis, we expected that the gene encoding the AP endonuclease activity in S. mutans would show homology to exoA from Streptococcus pneumoniae and xth from E. coli. In the present study, we have identified and cloned a homologue of the E. coli class II AP endonuclease, exonuclease III, which we have nam...

Mutation of the NADH Oxidase Gene ( nox ) Reveals an Overlap of the Oxygen- and Acid-Mediated Stress Responses in Streptococcus mutans

“…The fluctuating carbon availability (3), oxygen tension changes (37), and the necessity of competing with hundreds of other microbial species in dental plaque, some of which are net producers of ROS (37), suggest that it is not surprising that adaptation to changes in acidic conditions and oxygen levels would be important to the survival of the organism. In previous studies, we have shown that the acid stress response includes upregulation of the membrane-bound F-ATPase (32, 33) and a recA-independent DNA repair system (18,22) and, more recently, that S. mutans acts to increase the proportion of unsaturated membrane fatty acids in response to external acidification (19,20) and, further, that loss of unsaturated fatty acids results in a substantial loss of virulence in the rat model of dental caries (21).…”

“…An insertional mutation was made in the S. mutans UA159 gene SMU.1117 (presumptively designated nox by NCBI [http://www.ncbi.nlm.nih.gov /nuccore/AE014133.2?reportϭgenbank&fromϭ1058760&toϭ1060133]) (7) using splice overlap extension (SOE) PCR as previously described (18,20,26,27). To create a unique BglII site at position ϩ450 of the predicted coding region, the primer pair NoxRevEco and NoxBglUp (Table 2) was designed to amplify the 5= portion of the coding region.…”

Transcriptome analysis of LuxS‐deficient Streptococcus mutans grown in biofilms

“…Like Smx and UvrA (Hanna et al. , 2001; Faustoferri et al. , 2005), SMU.44‐deficiency was found to affect the ability of the deficient mutants to endure acid and hydrogen peroxide killing.…”

Stress Physiology of Lactic Acid Bacteria

“…An S. mutans uvrA mutant, in addition to being sensitive to DNA damage, was also sensitive to acidic conditions, indicating that UvrA plays a role in the repair of acid-induced DNA damage (353). Other DNA repair activities that have been implicated in the resistance of S. mutans to acidic pH include those of the Smx nuclease (355,356). The S. pyogenes C family DNA polymerase DnaE was shown to be highly error-prone and produced frameshift and point mutations during replication of both damaged and undamaged DNAs in vitro (357).…”