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

Abstract

NADH oxidase (Nox) is a flavin-containing enzyme used by Streptococcus mutans to reduce dissolved oxygen encountered during growth in the oral cavity. In this study, we characterized the role of the NADH oxidase in the oxidative and acid stress responses of S. mutans. A nox-defective mutant strain of S. mutans and its parental strain, the genomic type strain UA159, were exposed to various oxygen concentrations at pH values of 5 and 7 to better understand the adaptive mechanisms used by the organism to withstand environmental pressures. With the loss of nox, the activities of oxygen stress response enzymes such as superoxide dismutase and glutathione oxidoreductase were elevated compared to those in controls, resulting in a greater adaptation to oxygen stress. In contrast, the loss of nox led to a decreased ability to grow in a low-pH environment despite an increased resistance to severe acid challenge. Analysis of the membrane fatty acid composition revealed that for both the nox mutant and UA159 parent strain, growth in an oxygen-rich environment resulted in high proportions of unsaturated membrane fatty acids, independent of external pH. The data indicate that S. mutans membrane fatty acid composition is responsive to oxidative stress, as well as changes in environmental pH, as previously reported (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70: 929 -936, 2004). The heightened ability of the nox strain to survive acidic and oxidative environmental stress suggests a multifaceted response system that is partially dependent on oxygen metabolites.T he ability to metabolize oxygen is a nearly universal trait among bacteria. In many species, oxygen serves as an electron acceptor in the electron transport chain for production of ATP via oxidative phosphorylation, which prevents the formation of potentially dangerous metabolites (28). However, cellular respiration itself can lead to the production of reactive oxygen species (ROS), including superoxide radical (O 2 Ϫ ), hydroxyl anion (HO Ϫ ), and hydrogen peroxide (H 2 O 2 ) (29). The accumulation of ROS in cells can lead to protein, DNA, and membrane lipid damage, along with enzyme inactivation, ultimately resulting in cell death. Bacteria have evolved various means of coping with the deleterious effects of respiration, including detoxification mechanisms such as catalase, superoxide dismutase, and various dehydrogenases and peroxidases (58).The oral bacterium Streptococcus mutans is a facultative anaerobe found primarily on the human tooth surface in a multispecies biofilm known as dental plaque and to a lesser extent in saliva (2, 37, 41). Current models of dental plaque architecture are consistent with biofilm models of microbial environments, in that channels exist in biofilms that allow fluid movement, delivery of nutrients, and potential chemical challenges (31,39,61). Given the estimated numbers of bacterial species present in dental plaque (1, 2), it is perhaps contrary to expectation that oxygen tensions are not zero in much of plaque (37). I...

Streptococcus mutans NADH Oxidase Lies at the Intersection of Overlapping Regulons Controlled by Oxygen and NAD + Levels

“…Two homologs of Spx, SpxA (SMU.1142) and SpxB (SMU.2084), have been described in S. mutans (32,37). Mounting evidence suggests that both proteins are able to positively regulate genes encoding oxygen stress response enzymes, although SpxB appears to play a secondary role in this regulation (21,32). In addition to Spx, the Rex regulatory protein (SMU.1053) has been implicated in the repression of energy metabolism, maintenance of oxygen homeostasis, and biofilm formation in response to changes in the redox potential of S. mutans and several other species of Gram-positive bacteria (31,(38)(39)(40)(41).…”

“…Two proteins that have been implicated in nox regulation are the transcription factors Spx, the suppressor of ClpP and ClpX, and Rex, the redox-sensing regulator (21,31,32). The Spx regulator senses the redox state through a CXXC motif and has been described previously in many firmicutes, including Bacillus subtilis (33)(34)(35)(36).…”

“…Our laboratory has previously shown that Nox is the major oxygen-metabolizing enzyme used by S. mutans, responsible for reduction of approximately 40% of the dissolved oxygen encountered in dental plaque, and that the Nox enzyme shows elevated activity under conditions of both oxidative stress and low pH (21). Mutation of nox in the genomic type strain, S. mutans UA159, has demonstrated that NADH oxidase plays a role in the acid stress response, as inactivation of nox invokes changes in membrane fatty acid composition and metabolic output reminiscent of those seen during exposure to low pH (21,(23)(24)(25). In addition to reducing oxygen, Nox is also critical in the regeneration of NAD ϩ , a key molecule in the production of pyruvate during carbon metabolism in streptococci (26)(27)(28)(29).…”

“…S. mutans encodes several enzymes important in eliminating potentially dangerous reactive oxygen species (ROS) (20). The nox gene (SMU.1117) encodes the flavin-based NAD (NADH) oxidase (Nox), which acts to reduce oxygen, one electron at a time, to H 2 O, through the oxidation of NADH to NAD ϩ (21,22). Our laboratory has previously shown that Nox is the major oxygen-metabolizing enzyme used by S. mutans, responsible for reduction of approximately 40% of the dissolved oxygen encountered in dental plaque, and that the Nox enzyme shows elevated activity under conditions of both oxidative stress and low pH (21).…”

“…The nox gene (SMU.1117) encodes the flavin-based NAD (NADH) oxidase (Nox), which acts to reduce oxygen, one electron at a time, to H 2 O, through the oxidation of NADH to NAD ϩ (21,22). Our laboratory has previously shown that Nox is the major oxygen-metabolizing enzyme used by S. mutans, responsible for reduction of approximately 40% of the dissolved oxygen encountered in dental plaque, and that the Nox enzyme shows elevated activity under conditions of both oxidative stress and low pH (21). Mutation of nox in the genomic type strain, S. mutans UA159, has demonstrated that NADH oxidase plays a role in the acid stress response, as inactivation of nox invokes changes in membrane fatty acid composition and metabolic output reminiscent of those seen during exposure to low pH (21,(23)(24)(25).…”