Molecular biology of oxygen tolerance in lactic acid bacteria: Functions of NADH oxidases and Dpr in oxidative stress

JOURNAL

OF BIOSCIENCE

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Vol. 90, No. 5, 484-493. 2000

REVIEW Molecular Biology of Oxygen Tolerance in Lactic Acid Bacteria: Functions of NADH Oxidases and Dpr in Oxidative Stress MASAKO HIGUCHI, YUJI YAMAMOTO, AND YOSHIYUKI KAMIO* Laboratory of Applied Microbiology, Department of Molecular and Cell Biology, Graduate School of Agriculture, Tohoku University, Aoba-ku, Sendai 981-8555, Japan Received 4 September 20OO/Accepted 11 September 2000

Lactic acid bacteria including Streptococcus mutans lack cytochromes and heme-containing proteins. Most lactic acid bacteria also lack catalase. However, they can grow in the presence of air. In view of the defense against oxygen toxicity, the lack of catalase in lactic acid bacteria is not always consistent with its aerotolerance. Mechanisms, by which lactic acid bacteria establish their growth in air, are therefore an active area of investigation. We identified two kinds of NADH oxidase genes, nox-2 and nox-2 for HzOz-forming NADH oxidase (Nox-1) and HzO-forming NADH oxidase (Nox-2), respectively, in S. mutuns and found that Nox-1 is homologous with flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase (AhpR), consisting of AhpF and AhpC. We also identified ahpC which is homologous with ahpC of S. typhimurium, upstream of nox-2 in S. mutans. In the first and second parts of this article, we will refer to the role of Nox-1 which acts together with AhpC as bi-component peroxidase system in S. mutuns, catalyzing the NADH-dependent reduction of organic hydroperoxides or Hz02 to their respective alcohol and/or HzO. We will also refer to the role of Nox-2 in carbohydrate metabolism of S. mutuns in its aerobic life. Nox-2 was found to be involved in regenerating NAD+, which is required for glycolysis in S. mutans. While studying nox-l and ahpC double deletion mutant of S. mutans, we found that the mutant still showed the same level peroxide tolerance as did the wild-type strain. The finding suggested the existence of another antioxidant system in addition of Nox-1 and AhpC in S. mutans. We identified a new gene, dpr (for Dps-lie Peroxide Resistance gene) and its product, Dpr, as an iron-binding protein which is responsible for oxygen tolerance in S. mutans. In the third part of this article, we will refer to the current status of knowledge of molecular cloning of dpr, the characteristics of dpr-disruption mutants, and a mechanism by which Dpr confers aerotolerance to S. mutans. [Key words: oxygen tolerance, Streptococcusmutans, lactic acid bacteria, NADH oxidase, peroxidase, Dpr] Most biological electron flow is conducted to oxygen by a chain of carriers terminating in cytochrome oxidase system which accomplishes the tetravalent reduction of oxygen to water, without the release of intermediates (1). However, reactive oxygen species, including the superoxide anion (O*-), hydrogen peroxide (H202), and the hydroxyl radical (HO.), can be generated by the incomplete reduction of oxygen to water during respiration, by exposure to radiation, light, metals or oxidation-reduction (redox) active drugs such as paraquat, or by release from stimulated macrophages (2, 3). Aerobes and facultative anaerobes such as Escherichia coli and Salmonella typhimurium have developed an efficient mechanism for protection against the reactive oxygen species during aerobic respiration or during encounters with oxidative stress (4-6). Although physiological adaptation to oxidative stress is typically complex, E. coli possesses enzymatic defenses against oxidative stress including two hemoprotein catalases, three types of superoxide dismutases (SOD), glutathione reductase, thiol peroxidase (7), bacterioferritin comigratory protein (BCP) (8), nonspecific DNA-binding protein, Dps (9), and alkyl hydroperoxide reductase, consisting of AhpF and AhpC

(10). In contrast, facultative anaerobes such as lactic acid bacteria including Streptococcus, Enterococcus, and Lactococcus, cannot synthesize heme (ll), and therefore lack catalase, and cytochrome oxidases required for energy-linked oxygen metabolism. In addition, streptococci lack the moderate-to-high levels of intracellular glutathione found in gram-negative bacteria (12). As a matter of course lactic acid bacteria have a preference for anaerobiosis, and depend strictly on fermentation which involves the production of endogenous electron acceptors in a redox-balanced dismutation of utilizable energy by substrate-level phosphorylation (13). However, lactic acid bacteria show a greater metabolic potential when the reduced cofactors, from which NADH is the most important, are regenerated by exogenous electron acceptors (14-16). The simplest way to oxidize NADH is by the reduction of molecular oxygen (02) via the activity of NADH oxidase. Actually most aerotolerant strains in many lactic acid bacteria showed the activity of NADH oxidase when they grew under aerobic conditions (14, 15). Previously we have shown that O2 affected the growth on mannitol with a variation dependent on strains of mutans streptococci, and the growth response to 02 was correlated with the ability of strains to induce NADH oxidase and superoxide dismutase (SOD) activity under aerobic conditions (14, 17). These findings suggested that NADH oxidase plays an important role in the regulation of the aerobic metabolism in lactic acid

* Corresponding author. Mailing address: Laboratory of Applied Microbiology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, l-l Tsutsumi-dori Amamiya-machi, Aoba-ku, Sendai 981-85.55, Japan.

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bacteria. Interestingly, the NADH oxidase induced by 02 in an OTtolerant strain of Streptococcus mutans was shown to contain two types of enzyme activity, one forming HzOz and the other Hz0 (17). In the following studies, we purified the two NADH oxidases from S. mutans and identified as two distinct NADH oxidases that correspond to HzOz-forming oxidase (Nox-I) and HzOforming oxidase (Nox-2) (18-20). Recent advances in molecular genetics have been disclosing the real functions of the two NADH oxidases, Nox-1 and Nox-2, and the mechanisms of defense against oxidative stress in lactic acid bacteria (21-24). This review focuses on the contribution of NADH oxidases to aerobic metabolism, the function of Nox-I combination with AhpC homologue as alkyl hydroperoxide reductase, and the role of the new gene, dpr, coding for iron-binding protein, Dpr, which was discovered as a potential peroxide tolerance gene from S. mutans chromosome, in oxidative stress in S. mutans. CONTRIBUTION OF NADH OXIDASES TO AEROBIC METABOLISM Diversity of NADH oxidizing reaction in lactic acid bacteria NADH oxidizing enzymes catalyze the one-,

two-, or four-electron of O2 to 02-, HzOz, or Hz0 (2527). NADH + 202”-NAD+ + H+ +202- (reaction 1) NADH + H+ + O2 b NAD+ + H202 (reaction 2) 2NADH + 2H + + O2 _f 2NAD + + 2Hz0 (reaction 3) Production of 02- accounted for 17% of NADH-dependent O2 uptake by extracts from Enterococcus faecalis (28). Some lactic acid bacteria release H202 to the extracellular medium, and cell-free extracts produce HzOz (29), which may be due to reduction of 02 to HzOz. Aerotolerant bacteria, including S. mutans (14, 30), contain SOD activities (30, 31). 202 - + 2H +=O,

+ Hz02

(reaction 4)

Hydrogen peroxide may arise from spontaneous or SOD-catalyzed dismutation of OZ. Some lactic acid bacteria have NADH peroxidase that eliminate H202 (32). NADH + H + + HzOzPepNAD+

+ 2Hz0 (reaction 5)

The combination of oxidase and peroxidase activities (reactions 2 and 5) can reduce O2 to water at the expense of 2 NADH (11). However, certain bacteria contain a flavoprotein NADH oxidase (reaction 3) that reduces 02 directly to water without producing Hz02 as an intermediate (18, 33, 34). Existence of two types of NADH oxidases in lactic On further investigation, two types of acid bacteria

NADH oxidase activity found in aerobically grown cells of 02-tolerant S. mutans were purified and identified as two distinct NADH oxidases corresponding to HzOzforming oxidase (Nox-1) and HzO-forming oxidase (Nox-2) (18). Characteristics of these two enzymes were remarkably different each other (18). Nox-I catalyzed the two-electron reduction of O2 by

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NADH, whereas Nox-2 catalyzed the four-electron reduction of O2 by NADH. The oxidase activity of Nox-1 was stimulated on addition of free FAD, but that of Nox-2 was independent of free FAD. The subunit molecular mass was 55 kDa for Nox-1 and 50 kDa for Nox-2, estimated at first on the basis of mobility in SDS-polyacrylamide gel and later based on the deduced amino acid sequence of each structural gene (18-20). Moreover, antibodies raised against Nox-1 or Nox-2 reacted with their corresponding antigen, but did not cross-react (18). Flavin-containing NADH oxidases have been purified and characterized from several bacteria (33-41). With the exception of the NADH oxidase from Acholeplasma laidlawii, which is a metallo-flavoprotein containing FMN, all enzymes purified so far are flavoproteins containing FAD (41). The HzO-forming NADH oxidases were derived from lactic acid bacteria, including S. mutans (18), E. faecalis (33) and Leuconostoc mesenteroides (34) and from an anaerobic spirochete, Serpulina hyodysenteriae (35), with similarity in size of their subunits and in substrate specificity for NADH. In contrast, the H202-forming NADH oxidase from lactic acid bacteria except of S. mutans has not been well characterized. Nevertheless, recently the occurrence of the H202forming NADH oxidase was demonstrated in Lactobacillus delbrueckii subsp. bulgaricus, which could reduce OZ into HzOz with an NADH oxidase (42). Furthermore, based on sequence information Streptococcus pyogenes has been demonstrated to have both types of NADH oxidase homologues that correspond to the HZOz-forming oxidase and the HzO-forming oxidase (Roe, B. A., Clifton, S., McShan, M., and Ferretti, J., Streptococcal Genome Sequencing Project at the University of Oklahoma). Genetic characterization of two types of NADH oxidases To discover the molecular properties of two dis-

tinct NADH oxidases, corresponding to Nox-1 and Nox2 induced in S. mutans, each structural gene, nox-I and nox-2, has been analyzed (19, 20). The nox-Z consisted of 1530 base-pairs, which encode a polypeptide consisting of 510 amino acids with a predicted molecular mass of 55.2 kDa. In contrast, the nox-2 comprised 1371 basepairs, encoding a polypeptide of 457 amino acid residues. The deduced relative molecular mass of 49.9 kDa agreed with the previous value obtained from the purified Nox-2 protein. Sequence analyses have confirmed that these two oxidase proteins are a quite distant homology of the deduced amino acid sequence between these enzymes and their separate positions on genomic DNA (19, 20). Alignment of the amino acid sequence of Nox-I with those of NADH oxidases from other microorganisms showed identities of 75.6% for S. pyogenes (Roe, B. A., Clifton, S., McShan, M., and Ferretti, J., Streptococcal Genome Sequencing Project at the University of Oklahoma), and 55.6% for Amphibacillus xylanus EpOl (36), but those of 20.8%, 20.3%, and 7.3% for E. faecalis lOC1 (44), Thermoanaerobium brockii Rt8.G4 (45), and Thermus thermophilus HB8 (46), respectively. Comparisons of Nox-2 with the NADH oxidase from E. faecalis lOC1 yield identities of 41% (44). The HzO-forming NADH oxidase from Streptococcus pneumoniae (47) and S. pyogenes (43) also showed 74.8 and 74.7% identities to S. mutans Nox-2. Functional analysis by Nox-1 and Nox-2 mutants of S.

mutans

A role for these flavoproteins in facilitating

486

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Glucose

Mannitol (or Sorbitol)

mutans (Fig. 1).

2H 1 FBP Nox-2

4H Pyruvate

2H

BIOENG.,

H20

Lactate Acetate FIG. 1. Aerobic metabolism of glucose, mannitol, and sorbitol in S. mutans. Black arrows indicate the flow of electron in the catabolic pathway. FBP, fructose 1,6-bisphosphate; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase.

the aerobic metabolism of lactic acid bacteria may require the regeneration of NAD+ by oxidases for glycolysis. Especially for fermentation of sugar alcohol, such as mannitol and sorbitol, which contains two more hydrogen atoms compared to glucose, the regeneration of NAD+ by NADH oxidases must be important. In sugar alcohol fermentation, S. mutans degrades 1 mol of mannitol or sorbitol to 2 mol of pyruvate with a concomitant generation of 3 mol of NADH by the metabolic steps of mannitol l-phosphate (or sorbitol 6phosphate) dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase (Fig. 1). For smooth operation of glycolysis, NADH has to be oxidized to NAD+, but lactate dehydrogenase (LDH) can oxidize only 2 mol of NADH (Fig. 1). Thus, NADH has to be oxidized by another pathway, such as NADH oxidases, Nox-1 and/or Nox-2. To address the possible functions of the two types oxidase, we have constructed Anox-l or Anox- single mutant and Anox-l Anox- double mutant. S. rnutans wild-type strain GS-5 and the Anox-2, Anox-I, and Anox-l Anox- mutants grew well on glucose or mannitol under anaerobic conditions (21). However, under aerobic conditions, the Anox- and Anox-l Anoxmutants were severely hampered in the ability to grow on mannitol. The aerobic conditions had no significant effect on the mannitol growth of the Anox-I mutant or on the glucose growth of any strain. Under aerobic conditions, the Anox-2, and Anox-l Anox- mutants also demonstrated poor growth on sorbitol, while these mutants grew well on sorbitol anaerobically (21). We also demonstrated that the Anox- mutant possessedlow NADH oxidizing activity (less than 1 to 5% of that of wild-type strain GS-5 and Anox-l mutant) (21). The low activity of NADH oxidase in the Anox- mutant was consistent with the poor growth of this mutant on mannitol or sorbitol. These finding clearly indicated that Nox-2 corresponding to the H20-forming NADH oxidase is an essential enzyme for the regeneration of NAD+ during aerobic sugar alcohol metabolism in S.

Although no significant difference was observed in the aerobic growth on glucose between wild-type GS-5 and Nox-Zdeficient mutant strains, it was conceivable that the NADH oxidase activity in GS-5 affected the fermentation end products through a change in the ratio of NADH to NAD+. Analysis of fermentation end product under aerobic conditions revealed that the Nox-2deficient mutant produced large amounts of lactate (more than 90%) and small amounts of acetate (5X), in contrast to GS-5, which produced less lactate (78%) and more acetate (17%) (21). This shift in the fermentation end products indicates that the Nox-Zdeficient mutant could not convert pyruvate into acetate during aerobic metabolism. S. mutans has an additional branch in the pathway involving pyruvate dehydrogenase (PDH) (48), where pyruvate is oxidized to acetate along with the generation of NADH (Fig. 1). The NADH derived from PDH also has to be oxidized by NADH oxidase. That is, the Nox-2 deficient mutant cannot operate the PDH pathway. Recently, it has been reported that NADH oxidaseoverproducing Lactococcus lactis strains constructed by cloning the S. mutans nox-2 showed a shift from homo-lactic to mixed-acid fermentation along with a decreased NADH/NAD+ ratio during aerobic glucose catabolism (49). These results also indicated that in the presence of Nox-2 pyruvate was converted to acetate by PDH (48), whereas in the absence of Nox-2 pyruvate was mostly converted to lactate during aerobic glucose catabolism (Fig. 1). On the other hand, the contribution of NADH oxidase activity by Nox-1 to mannitol growth seems negligible, since Nox-1 could not support aerobic mannitol growth in the absence of Nox-2 and the lack of Nox-1 enzyme had no effect on the mannitol growth. Thus we suggest that Nox-1 is another NADH-oxidizing enzyme functionally distinct from Nox-2 and not important in energy metabolism. Contribution of H20-forming NADH oxidase to aerobic metabolism of S. pyogenes and S. pneumoniae

As mentioned above, NADH oxidases corresponding to Nox-2 from two human pathogens S. pyogenes and S. pneumoniae were found and the nucleotide sequences of nox encoding the enzymes have been determined (43, 47). To elucidate a putative role for NADH oxidases in aerobic metabolism, NADH oxidase (NOXase)-deficient mutants were constructed. Characterization of the resulting mutans from S. pyogenes revealed that growth in rich medium under low-O2 conditions was indistinguishable from that of the wild-type. However, the mutants were unable to grow under high-O2 conditions and demonstrated enhanced sensitivity to the superoxidegenerating agent paraquat. Mutants cultured in liquid medium under conditions of carbohydrate limitation and high O2 tension were characterized by an extended lag phase, reduction in growth, and a greater accumulation of H202 in the growth medium compared to the wild-type strain. All of these mutant phenotypes could be overcome by the addition of glucose (43). Characterization of the resulting mutans from S. pneumoniae revealed that the growth rate and yield of the NOXase-deficient strains were not changed under aerobic or anaerobic conditions, but the efficiency of development

of competence

for

genetic

transformation

during

aerobic growth was markedly altered. The presence of

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NOXase may be relevant to the metabolic regulation of competence for genetic exchange (47). FUNCTION OF THE H202-FORMING NADH OXIDASE AS AN ALKYL HYDROPEROXIDE REDUCTASE SYSTEM Sequence-based searches for proteins homologous with AhpF and AhpC, the two components of alkyl hydroperoxide reductase from S. typhimurium, have indicated a very wide distribution of the peroxide-reducing protein, AhpC, in organisms ranging from archaebacteria to plants and mammals, whereas close homologues of the flavoenzyme, AhpF, have been found only in bacteria (50, 51). Identification of the Nox-1 as an aikyl hydroperoxide Unexpectedly, sequence searches ulreductase system timately confirmed that the 55 kDa Nox-1 protein is a homologue of S. typhimurium AhpF (the flavoprotein component of alkyl hydroperoxide reductase) and a member of the thioredoxin reductase-like group of pyridine nucleotide : disulfide oxidoreductase (50, 52). Furthermore, analysis of the DNA sequence upstream of the nox-l gene clearly indicated the presence of a open reading frame homologous with the structural gene of S. typhimurium AhpC (the non-flavoprotein component of alkyl hydroperoxide reductase) (52, 53). This finding implies that H202 produced by Nox-I can be reduced to HZ0 by the AhpC, as follows: 2NADH+2H+ +02 -----f 2NAD+ +2Hz0 (Nox-1 + AhpC) Alignments of bacterial proteins related to Nox-1 and AhpF indicate a high degree of sequence conservation surrounding motifs for NADH and FAD binding and for the redox-active half-cystine residues of AhpF (22). AhpC homologues corresponding to those bacteria that possess AhpF homologues as described above have been aligned. These bacterial AhpC proteins exhibit a very high degree of sequence conservation, particularly surrounding the catalytic half-cystines (at positions 46 and 165/164 in S. typhimurium and S. mutans protein). S. pyogenes just like S. mutans was demonstrated to have both ahpC and ahpF genes, whereas E. faecaks has only ahpC gene. The pairwise comparisons of deduced amino acid sequences for S. mutans AhpC protein to S. pyogenes AhpC homologue showed 88.2% identity, instead of lower homology to E. faecaks AhpC homologue and to S. typhimurium AhpC, 54.8% and 62.2%, respectively (22). Nox-1 catalytic activities in the presence and absence of AhpC Nox-1 was previously identified as an HzOz-forming NADH oxidase, an activity also known to be exhibited by related flavoproteins from A. xylanus and S. typhimurium (18, 36, 54). In the presence of excess S. typhimurium AhpC, S. typhimurium AhpF or A. xylanus NADH oxidase also supported the full four-electron reduction of oxygen to water (54): [2NADH+2H+ + O2 + 2NAD + + 2Hz0]. A direct comparison of the oxidase activities of S. typhimurium AhpF and S. mutans Nox-1 determined spectroscopically in the presence of 260 PM oxygen indicated approximately 5-fold higher activity for the S. mutans flavoprotein relative to that of S. typhimurium. In both cases the oxidase activity is increased on addition of free FAD. This additional activity results from the

I

%

8 9

+catalase

156 206

+c&lase 260

I I 02468

I

I

I

II ,I I 024

I

I

Time (min) FIG. 2. Oxygen consumption during oxidase assays of Nox-1 in the presence or absence of AhpC using limiting NADH. Panel A, The reaction mixture contained Nox-1 alone (1 nmol) and initiated by the addition of 200 yM NADH; catalase (80 ,ug) was added after the consumption of oxygen ceased. Panel B, The reaction was carried out as in Panel A, but included 1 nmol AhpC.

ability of these flavoproteins to catalytically reduce free FAD; oxygen then reacts with the free FADH* nonenzymatically (22). Oxidase assays were performed using an oxygen electrode and limiting NADH to demonstrate the conversion of Nox-1 from an HzOz-producing oxidase system to an HzO-producing oxidase system on addition of the AhpC compound, as indicated by the presence or absence of O2 regeneration at l/2 eq on addition of catalase at the end of the reaction (Fig. 2). Owing to the assay conditions included NADH in limiting concentrations relative to 02, the conversion was demonstrated to be accompanied by the expected change in stoichiometry of NADH: O2 consumed from 1 : 1 to 2 : 1 on addition of AhpC (Figs. 2 and 3). On varying the AhpC concentration, intermediate ratios of NADH: O2 are obtained because of the competition between the two activities (oxidative activity of Nox-1 alone or peroxidase activity of Nox-I plus AhpC) for the limited supply of NADH. Any change in conditions favoring one activity over the other shifts the amount of AhpC need to achieve an intermediate ratio (e.g., 1.5 NADH : 02). Peroxidase activities assessed by anaerobic assays with AhpC in excess over flavoprotein indicated very similar kinetic parameters for the S. mutans Nox-l/AhpC system compared with AhpF/AhpC from S. typhimurium, although the V,, for the reaction was a little lower in the former case (22). As shown previously for the S. typhimurium peroxidase system (51), these assays produced identical results when carried out aerobically in spite of the higher oxidase activity of Nox-1. Reactions catalyzed by Nox-l/AhpC system which differ from NADH peroxidase Some lactic acid bacteria have one or more enzymes that eliminate HzOz by reduction (55). The NADH peroxidase from E. faecalis (5659) and the alkyl hydroperoxide reductase from S. typhimurium (51, 53) represent the two known flavin-dependent hydroperoxidase, or peroxide reductases. More recent studies with the purified NADH peroxidase have revealed the presence of one FAD per 50 kDa subunit and have shown that the enzyme exist in solution as a stable homotetramer (58). Sequence analyses of the E.

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Reactions

catalyzed

02tNADH+H+ O2 t PNADH ROOH

t NADH

t 2H’ + H’

H202

H20

(ROOH)

(ROW

by Nox-l/AhpC: -

Hz02

t NAD

-

2H20

t 2NAD’

__)

ROH t H20 + NAD’

FIG. 3. Scheme of reactions catalized by Nox-1 and AhpC.

faecalis NADH peroxidase and the flavoprotein component (AhpF) of the S. typhimurium alkyl hydroperoxide reductase indicated clear evolutionary links with members of the flavoprotein disulfide reductase family. However, chemical and spectroscopic evidence demonstrated that the non-flavin redox center in NADH peroxidase is an unusual stabilized cysteine-sulfenic acid (Cys-SOH) derivative that cycles between oxidized and reduced states (60), and not a cystine disulfide as found in the disulfide reductases. As mentioned before, comparisons of Nox-2 with the NADH oxidase from E. faecalis lOC1 yield identities of 41% (44). Besides, the sequence of amino acid of the NADH peroxidase from E. faecalis lOC1 has 44% identical to the NADH oxidase from E. faecalis lOC1, with the most highly conserved segments containing the non flavin redox center and the FADand NADH-binding regions, indicating structural similarity between the two enzymes (66). The NADH oxidase protein thus appears to achieve full 4-electron oxidation of oxygen to water by reaction of fully reduced (EH4) protein, with oxygen converted to hydrogen peroxide by the reduced flavin and hydrogen peroxide reduced to water by the cysteine thiol. The cysteine sulfenic acid that is proposed to form in the oxidase is the same one that has been proven to exist in the peroxidase (62). With Nox-1 or AhpF, on the other hand, the reaction of oxygen with the flavoprotein obviously only occurs

with enzyme with reduced flavin reducing the oxygen to hydrogen peroxide. There is no peroxidatic center as in the NADH peroxidase or oxidase (above). Rather, the protein also acts as a flavoprotein disulfide reductase, reducing the AhpC protein to catalyze the reduction of any hydrogen peroxide reduced as well as other organic hydroperoxides. The Nox-1 and AhpF protein functions are related to the well-characterized reactions of thioredoxin reductase, except that there is an additional 200 amino acids at the N-terminus and an additional redoxactive disulfide center (63, 64). Functions of Nox-1 and AhpC as alkyl hydroperoxide reductase in oxidative stress The alkyl hydroperoxide

reductase which is composed of AhpC and AhpF in 5’. typhimurium has been identified as an antioxidant enzyme system capable of reducing organic hydroperoxides and hydrogen peroxide (51, 53). The AahpCF mutants obtained in S. typhimurium and E. coli were hypersensitive to killing by cumene hydroperoxide (10). To determine whether or not Nox-1 actually functions as AhpF in vivo in combination with AhpC, the sensitivity to killing by cumene hydroperoxide of E. coli TA4315 (AahpCF) transformed with either or both nox-I and ahpC genes was analyzed. Compared with the zone of inhibition for E. coli TA4315 by cumene hydroperoxide, this strain harboring pAN119 containing both nox-Z and ahpC demonstrated a striking reduction in diameter,

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In vitro Nox-I functions as alkyl hydroperoxide reductase when combined with AhpC. Furthermore, the bicomponent system restored the resistance of E. coli TA4315 (ahpCF-defective mutant) (10) to cumene hydroperoxide (21). Particularly for S. mutans lacking catalase and heme-containing peroxidases, the peroxidase activity of Nox-1 combined with AhpC should be important at least in defense against peroxide-mediated stress. However, the Anox-l ahpC double mutant of S. mutans still showed the same level of peroxide tolerance as did the wild-type strain (21). These results suggested the existence of other antioxidant system(s) in addition to Nox-I and AhpC in S. mutans. Identification gen tolerance

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Primary and secondary structure comparison Dpr, Dps, and Dps homologues suggested that Dpr was a member of Dps family of proteins (24). Dps was found to be a nonspecific DNA-binding protein which accumulates in stationaryphase cells of E. coli (9). Dps family of proteins form spherical complexes like ferritin (9, 66-70) and some of them bind iron (67, 68). To date, three family member including E. coli Dps were shown to bind to DNA for protection from oxidative stress (9, 69-71). On the other hand, functional divergency of other Dps family proteins was also reported, i.e., the non-heme ferritin of Listeria innocua (67), the fine tangled pilus major subunit of Haemophilus ducreyi (72), and the neutrophil activating protein of Helicobacter pylori (68). The dpr product, Dpr, was identified as a protein band of 20 kDa detected in crude extracts of wild-type but not in dpr mutant. Dpr is induced by exposure of S.

Cloning of potential peroxide tolerance gene(s) from the S. mutans chromosome was done by its ability to complement an AahpCF mutant of E. coli with tert-butyl hydroperoxide (tBHP) hypersensitive phenotype (10, 23, 24, 65). An ORF which consisted of

Organism and protein

LACTIC

(24). Dpr is an iron-binding protein

of a new gene responsible for the oxy-

TABLE

IN

525 bp encoding a protein of 19,617 Da (20 kDa protein) with 175 amino acid residues was identified as tBHP resistance gene (23, 24). The deduced amino acid sequence of the 20 kDa protein showed low homology (2040% identical) with that of the Dps (DNA binding protein from starved cells) (9) family of proteins (Table 1). The predicted secondary structure of the 20 kDa protein is similar to that of Dps (24). Accordingly, the gene was designated as dpr (dpr for Dps-like peroxide resistance gene) (accession number AB036428). To address the function of dpr in S. mutans, three dpr-disruption mutants of S. mutans, Adpr, Adpr AahpC Anox-I, and Adpr Asod, were constructed and their ability to grow under both anaerobic and aerobic conditions was examined. All mutants grew as well as the wild-type strain under anaerobic conditions. Under aerobic conditions, the Adpr mutant grew as well as the wild-type S. mutans in liquid medium. However, the Adpr mutant could not form colonies on agar plate under air. In addition, neither the Adpr AahpC Anox-l triple mutant nor the Adpr Asod double mutant was able to grow aerobically in liquid medium (24). It was revealed that dpr is an essential gene for colony formation in S. mutans in the presence of air. Functional significance of dpr in oxygen tolerance was also demonstrated in liquid medium. The addition of catalase or deferoxamine, which is cell-permeable iron chelator, in the growth medium compensated for all these growth defects caused by the absence of dpr

even smaller than that for an E. coli wild-type (K-12) strain. Furthermore, strain TA4315 harboring pMS1 containing only ahpC also exhibited a reduction in diameter equal to that of K-12. In contrast, TA4315 harboring only the vector, pUC119, or pNox-1H containing nox-Z did not exhibit augmented resistance toward cumene hydroperoxide (21). To identify the in vivo function of S. mutans AhpR in defense against peroxide stress, the same sensitivity assays of strains AahpC, Anox-I, AahpC Anox-I, and wildtype GS-5 were performed under the conditions as above except for the use of THB medium (21). Unexpectedly, the sensitivities of these three mutants to killing by HzOz and cumene hydroperoxide showed almost the same levels as those of wild-type strains. Furthermore, even in the absence of ahpC, strain Bl showed increased tolerance toward oxidants following treatment by sub-lethal doses of H202 and cumene hydroperoxide to approximately the same level as that of the wild-type strain (21). These results indicated that defense systems of S. mutans for alkyl hydroperoxide differ from those of E. coli. Presumably, the function of Nox-1 as AhpR is masked by overlapping effects of other antioxidant system(s) in S. mutans. ROLE OF THE IRON BINDING PROTEIN, IN OXYGEN TOLERANCE

TOLERANCE

1. Pairwise identities of the amino acid sequences among S. mutans Dpr and Dps family proteins % Sequence identity with: S. mutans Dpr

L. innocua Fera B. subtilis MrgAb

E. coli Dps’

L. innocua Fera 40.0 B. subtilis MrgAb 30.9 36.0 E. co/i Dpsc 27.6 25.6 22.4 45.5 20.0 B. subtilis Dpsd 26.3 43.0 29.5 22.4 H. pylori NapAe 26.1 32.1 23.0 21.5 21.2 35.1 H. ducreyi Pilin’ Synechococcus DpsAs 21.5 26.3 26.8 22.6 a Nonheme ferritin in Listeria innocua (accession no. P80725). b MrgA of Bacillus subtilis (accession no. P37960). c Dps of Escherichia coii (accession no. P27430). d Dps of Bacillus subtilis (accession no. AFO08220). e Neutrophil-activating protein of Helicobacterpylori (accession no. AE000543). f Fine-tangled pilus major subunit of Haemophilus ducreyi (accession no. 447953).

B. subtilis Dpsd H. pylori NapAe

34.2 21.6 22.7

23.6 20.5

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Fenton reaction

Fe*’

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FIG. 4. Proposed linkage among iron, Dpr, and oxygen metabolism in S. mufuns.

mutans to air and accumulated abundantly in S. mutans (24). Iron binding ability of Dpr was also demonstrated by staining with iron-specific staining reagent Ferene S (24). Details of the molecular properties of Dpr including DNA binding activity was not determined, but a possible function of Dpr may be a ferritin like iron-binding protein. The model for aerotorelance mechanism by Dpr

Iron has toxic properties in the presence of oxygen. Iron ions stimulate the generation of highly reactive and toxic oxygen species such as hydroxyl radicals. In vitro experiments have shown that Fe2+ catalyzes the non-enzymatic synthesis of hydroxyl radical from H202 via the Fenton reaction (73), but H202 remained intact in the absence of iron ions at physiological pH (73). Therefore, tight regulation of intercellular free iron ion concentration is believed to be a significant factor for organisms to survive under aerobic conditions, Dpr was found to be a member of the Dps family of proteins which form spherical oligomers like ferritin. In fact, Dpr was found to have iron-binding ability. Thus, a possible role of protection by Dpr might be to sequester iron, thereby protecting cells from peroxides and conferring oxygen tolerance. Figure 4 shows the possible linkage between iron, Dpr, and oxygen metabolism in S. mutuns. An important feature of the model shown in Fig. 4 for oxygen tolerance mechanism by Dpr is that the existence of a high concentration of Dpr in S. mutans might enable intracellular free iron ion concentrations to be kept low. The ability of the AahpC Anox-l double mutant of S. mutans to grow aerobically might be due to the titration of the intracellular free iron ions by Dpr, resulting in non-conversion of H202 to hydroxyl radical (Fig. 4). Neither the Adpr AahpC Anox-l triple mutant nor the Adpr Asod double mutant could grow under air. In the former case, the high concentrations of intracellular H202 and of free intracellular iron ions might prompt the conversion of H202 to hydroxyl radicals via the Fenton reaction and lead to cell death. Addition of catalase and deferoxamine, to the growth medium complemented the growth defect of not only the former mutant but also the latter mutant, indicating that the growth defect of the latter mutant may also have been caused by hydroxyl radical formation. It has been reported that 02-, derived from SOD deficiency, enhances the Fenton reaction by releasing Fe2+ from ironcontaining proteins (74, 75). In Adpr Asod mutant, increased amount of hydroxyl radicals by Fe2+ would be enough to kill the cell (Fig. 4). Wai et al. reported that a

ferritin-deficient mutant of Campylobacter jejuni shows H202 and 0~~ sensitivity, indicating that sequestration of iron by ferritin contributes to oxygen tolerance (76). Touati et al. reported that non-heme ferritin, FtnA, which is over-expressed by a multicopy plasmid carrying ftnA, suppressed iron-mediated oxygen sensitivity of E. coli (Pfur ArecA) (77). These findings agree well with our results from S. mutans. One more significant role of protection by Dps and Dps family proteins was considered to be DNA binding ability (71). Wolf et al. has shown that E. coli Dps forms an extensive crystalline lattice in the presence of DNA in vitro and in vivo proposing that DNA-Dps cocrystallization mode provides protection of DNA by sequestration (78). On the other hand, it was reported that a member of Dps family proteins, L. innocua ferritin and I-Z. pylori HP-NAP, function as authentic ferritin and cannot bind DNA (67, 68). Molecular properties of Dpr including DNA binding ability are not elucidated yet. All three mutants of S. mutans, Adpr, Adpr AahpC Anox-1, and Adpr Asod, are unable to form colonies on solid media under air. Unlike the results obtained from liquid cultures, the ability to form colonies was absolutely dependent on Dpr. Addition of catalase to agar plates compensated for the growth defect, indicating that there exists a significant amount of H202 in the cell despite the presence of ahpC and nox-1. Our previous study indicated that the expression level of AhpC, which is the peroxidatic protein of the bi-component peroxidase system, was low during the early growth phase under air (21). At an early growth phase of S. mutuns on solid medium, the cells would also contain a small amount of AhpC protein. Therefore, hydroxyl radical could be generated from the remaining H202 to cause cell death in the absence of Dpr on the solid medium. In contrast, the Adpr mutant could grow aerobically in liquid medium. As Hz02 is a highly diffusible oxidant in the liquid medium, endogenously synthesized H202 might permeate the cell membrane and be released easily out of the cells until its intracellular and extracellular concentrations reached equilibrium (79). Consequently, at early exponential phase when both cell density and AhpC levels are low, the concentration of the intracellular H202 might be kept low enough to enable the Adpr mutants to grow. From mid log phase, the increase in cell mass would lead to increased production of H202, causing accumulation of both internal and external H202. At this stage, the Adpr AahpC &ox-l mutant could not grow as mentioned above. Dpr homologues were also present in other streptococci, including S. pyogenes (Roe, B. A., Clifton, S., McShan, M., and Ferretti, J., Streptococcal Genome Sequencing Project at the University of Oklahoma) and S. pneumoniae (80), and their deduced amino acid sequences showed 75.4 and 57.1% identity, respectively, with that of S. mutans Dpr. To date, non-heme peroxidases (22, 58), pseudocatalase (81), and oxidases (18, 33, 43, 47) of lactic acid bacteria including streptococci have been reported to substitute for catalase which is present in other aerotolerant bacteria but not lactic acid bacteria, and some of them, including SOD, were proven to play a role in aerotolerance (43, 82-86). Unlike them, Dpr did not directly react with oxygen and reactive oxygen species. Iron binding ability of Dpr might indirectly contribute oxygen tolerance in S. mutans. This mecha-

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nism of aerotolerance by Dpr is rational for lactic acid bacteria that are known to lack catalase and to require only low levels of iron for growth. 20. ACKNOWLEDGMENTS We are grateful to Dr. Nobuo Kato, Kyoto University, for enabling us to write this article. Our works described in this article were supported in part by Grants-in Aid from the Ministry of Education, Science, Sports, and Culture of Japan. REFERENCES 1. Chance, B.: Spectra and reaction kinetics of respiratory pigment of homogenized and intact cells. Nature, 169, 215-221 (1952). 2. Hassen, H. M. and Fridvich, I.: Intracellular production of superoxide radical and of hydrogen peroxide by redox active compounds. Arch. Biochem. Biophys., 196, 385-395 (1979). 3. Antonini, E., Brunori, M., Greenwood, C., and MaImstrom, B. G.: Catalytic mechanism of cytochrome oxidase. Nature, 228, 936-937 (1970). 4. Demple, B.: Regulation of bacterial oxidative stress genes. Ann. Rev. Genet., 25, 315-337 (1991). 5. Farr, S. and Kogoma, T.: Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbial. Rev., 55, 561-585 (1991). 6. Storz, G., TartagIia, A., Farr, S. B., and Ames, B. N.: BacteriaI defenses against oxidative stress. Trends Genet., 6, 365-368 (1990). 7. Cha, M. K., Kim, H. K., and Kim, I. H.: Thioredoxin-linked “thiol peroxidase” from periplasmic space of Escherichia coli. J. Biol. Chem., 270, 28635-28641 (1995). 8. Jeong, W, Cha, M. K., and Kim, I. H.: Thioredoxin-dependent hydroperoxide peroxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thiol-specific antioxidant protein (TSA)/Alkyl hydroperoxide peroxidase C (AhpC) family. J. Biol. Chem., 275, 29242930 (2000). 9. Abniron, M., Link, A. J., Furlong, D., and Kolter, R.: A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli. Genes Dev., 6, 2646-2654 (1992). 10. Storz, G., Jacobson, F. S., Tartaglia, L. A., Morgan, R. W., SiIveira, L. A., and Ames, B. N.: An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp. J. Bacterial., 171, 2049-2055 (1989). 11. Dolin, M. I.: Cytochrome-independent electron transport enzymes of bacteria, p. 425-460. In Gunsalus, I. C. and Starrier, R. Y. (ed.), The bacteria, vol. 2. Academic Press Inc., New York, N. Y. (1961). 12. Fahey, R. C., Brown, W. C., Adams, W. B., and Worsham, M. B.: Occurrence of glutathione in bacteria. J. Bacterial., 133, 1126-1129 (1978). 13. Clark, D. P.: The fermentation pathways of Escherichia coli. FEMS Microbial. Rev., 63, 223-234 (1989). 14. Higuchi, M.: Effect of oxygen on the growth and mannitol metabolism of Streptococcus mutans. J. Gen. Microbial., 130, 1819-1826 (1984). 15. Smart, J. B. and Thomas, T. D.: Effect of oxygen on lactose metabolism in kactic streptococci. Appl. Environ. Microbial., 53, 533-541 (1987). 16. Condon, S.: Responses of lactic acid bacteria to oxygen. FEMS Microbial. Rev.. 46. 269-280 (1987). 17. Higuchi, M.: Reduced’ nicotinamide‘adenine dinucleotide oxidase involvement in defense against oxygen toxicity of Streptococcus mutans. Oral Microbial. Immun., 7, 309-314 (1992). 18. Higuchi, M., Shtmada, M., Yamamoto, Y., Hayashi, T., Koga, T., and Kamio, Y.: Identification of two distinct NADH oxidases corresponding to HzOz-forming oxidase and HzOforming oxidase induced in Streptococcus mutans. J. Gen. Microbial., 139, 2343-2351 (1993). 19. Higuchi, M., Shimada, M., Matsumoto, J., Yamamoto, Y.,

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