Response of the microaerophilic Bifidobacterium species, B. boum and B. thermophilum, to oxygen

We investigated the effects of O2 on Bifidobacterium species using liquid shaking cultures under various O2 concentrations. Although most of the Bifidobacterium species we selected showed O2 sensitivity, two species, B. boum and B. thermophilum, demonstrated microaerophilic profiles. The growth of B. bifidum and B. longum was inhibited under high-O2 conditions accompanied by the accumulation of H2O2 in the medium, and growth was restored by adding catalase to the medium. B. boum and B. thermophilum grew well even under 20% O2 conditions without H2O2 accumulation, and growth was stimulated compared to anoxic growth. H2O-forming NADH oxidase activities were detected dominantly in cell extracts of B. boum and B. thermophilum under acidic reaction conditions (pH 5.0 to 6.0).     

Plasma membrane-stimulated vanadate-dependent NADH oxidation is not the primary mediator of vanadate toxicity in Saccharomyces cerevisiae

Interactions of oxyvanadium compounds with cellular metabolism have recently been demonstrated. Membrane-stimulated vanadate-dependent NADH oxidation has been hypothesized to involve the cellular accumulation of H2O2, which may cause the vanadate sensitivity of animals and microbes. This report shows that the vanadate-dependent NADH oxidation activity of the yeast plasma membrane requires oxygen and is present in vanadate-resistant mutants of Saccharomyces cerevisiae. In addition, the vanadate sensitivity of growth in S. cerevisiae is the same during aerobic and anaerobic growth. These results imply that neither plasma membrane-mediated vanadate-stimulated NADH oxidation, nor any other oxidative process, is the primary cause of vanadate sensitivity in yeast cells.     

Hydrogen peroxide production in Streptococcus pyogenes: involvement of lactate oxidase and coupling with aerobic utilization of lactate

Streptococcus pyogenes strains can be divided into two classes, one capable and the other incapable of producing H2O2 (M. Saito, S. Ohga, M. Endoh, H. Nakayama, Y. Mizunoe, T. Hara, and S. Yoshida, Microbiology 147:2469-2477, 2001). In the present study, this dichotomy was shown to parallel the presence or absence of H2O2-producing lactate oxidase activity in permeabilized cells. Both lactate oxidase activity and H2O2 production under aerobic conditions were detectable only after glucose in the medium was exhausted. Thus, the glucose-repressible lactate oxidase is likely responsible for H2O2 production in S. pyogenes. Of the other two potential H2O2-producing enzymes of this bacterium, NADH and alpha-glycerophosphate oxidase, only the former exhibited low but significant activity in either class of strains. This activity was independent of the growth phase, suggesting that the protein may serve in vivo as a subunit of the H2O2-scavenging enzyme NAD(P)H-linked alkylhydroperoxide reductase. The activity of lactate oxidase was associated with the membrane while that of NADH oxidase was in the soluble fraction, findings consistent with their respective physiological roles, i.e., the production and scavenging of H2O2. Analyses of fermentation end products revealed that the concentration of lactate initially increased with time and decreased on glucose exhaustion, while that of acetate increased during the culture. These results suggest that the lactate oxidase activity of H2O2-producing cells oxidizes lactate to pyruvate, which is in turn converted to acetate. This latter process proceeds presumably via acetyl coenzyme A and acetyl phosphate with formation of extra ATP.     

Formation and conversion of oxygen metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under different growth conditions

A semidefined medium based on Casamino Acids allowed Lactococcus lactis ATCC 19435 to grow in the presence of oxygen at a slow rate (0.015 h(-1)). Accumulation of H(2)O(2) in the culture prevented a higher growth rate. Addition of asparagine to the medium increased the growth rate, whereby H(2)O(2) accumulated only temporarily during the lag phase. H(2)O(2) is an inhibitor for several glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase being the most sensitive. Strain ATCC 19435 contained NADH oxidase (maximum specific rate under aerobic conditions, 426 nmol of NADH min(-1) mg of protein(-1)), which reduced oxygen to water, whereby superoxide was formed as a by-product. H(2)O(2) originated from the dismutation of superoxide by superoxide dismutase. Although H(2)O(2) was rapidly destroyed under high metabolic fluxes, neither NADH peroxidase nor any other enzymatic H(2)O(2)-reducing activity was detected. However, pyruvate, the end product of glycolysis, reacted nonenzymatically and rapidly with H(2)O(2) and hence was a potential alternative for scavenging of this oxygen metabolite intracellularly. Indeed, intracellular concentrations of up to 93 mM pyruvate were detected in aerobic cultures growing at high rates. It is hypothesized that self-generated pyruvate may serve to protect L. lactis strain ATCC 19435 from H(2)O(2).     

Diversion of the metabolic flux from pyruvate dehydrogenase to pyruvate oxidase decreases oxidative stress during glucose metabolism in nongrowing Escherichia coli cells incubated under aerobic, phosphate starvation conditions

Ongoing aerobic metabolism in nongrowing cells may generate oxidative stress. It is shown here that the levels of thiobarbituric acid-reactive substances (TBARSs), which measure fragmentation products of oxidized molecules, increased strongly at the onset of starvation for phosphate (P(i)). This increase in TBARS levels required the activity of the histone-like nucleoid-structuring (H-NS) protein. TBARS levels weakly increased further in DeltaahpCF mutants deficient in alkyl hydroperoxide reductase (AHP) activity during prolonged metabolism of glucose to acetate. Inactivation of pyruvate oxidase (PoxB) activity decreased the production of acetate by half and significantly increased the production of TBARS. Overall, these data suggest that during incubation under aerobic, P(i) starvation conditions, metabolic flux is diverted from the pyruvate dehydrogenase (PDH) complex (NAD dependent) to PoxB (NAD independent). This shift may decrease the production of NADH and in turn the adventitious production of H(2)O(2) by NADH dehydrogenase in the respiratory chain. The residual low levels of H(2)O(2) produced during prolonged incubation can be scavenged efficiently by AHP. However, high levels of H(2)O(2) may be reached transiently at the onset of stationary phase, primarily because H-NS may delay the metabolic shift from PDH to PoxB.     

Cyanide-resistant respiration in Taenia crassiceps metacestode (cysticerci) is explained by the H2O2-producing side-reaction of respiratory complex I with O2

The nature of the cyanide-resistant respiration of Taenia crassiceps metacestode was studied. Mitochondrial respiration with NADH as substrate was partially inhibited by rotenone, cyanide and antimycin in decreasing order of effectiveness. In contrast, respiration with succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was more sensitive to antimycin and cyanide. The saturation kinetics for O2 with NADH as substrate showed two components, which exhibited different oxygen affinities. The high-O2-affinity system (Km app=1.5 microM) was abolished by low cyanide concentration; it corresponded to cytochrome aa3. The low-O2-affinity system (Km app=120 microM) was resistant to cyanide. Similar O2 saturation kinetics, using succinate or ascorbate-TMPD as electron donor, showed only the high-O2-affinity cyanide-sensitive component. Horse cytochrome c increased 2-3 times the rate of electron flow across the cyanide-sensitive pathway and the contribution of the cyanide-resistant route became negligible. Mitochondrial NADH respiration produced significant amounts of H2O2 (at least 10% of the total O2 uptake). Bovine catalase and horse heart cytochrome c prevented the production and/or accumulation of H2O2. Production of H2O2 by endogenous respiration was detected in whole cysticerci using rhodamine as fluorescent sensor. Thus, the CN-resistant and low-O2-affinity respiration results mainly from a spurious reaction of the respiratory complex I with O2, producing H2O2. The meaning of this reaction in the microaerobic habitat of the parasite is discussed.     

Molecular cloning and analysis of the gene encoding the NADH oxidase from Streptococcus faecalis 10C1. Comparison with NADH peroxidase and the flavoprotein disulfide reductases.

The gene encoding the streptococcal flavoprotein NADH oxidase (NOXase), which catalyzes the four-electron reduction of O2-->2H2O, has been cloned and sequenced from the genome of Streptococcus (Enterococcus) faecalis 10C1 (ATCC 11700). The deduced NOXase protein sequence corresponds to a molecular mass of 48.9 kDa and contains three previously sequenced cysteinyl peptides obtained with the purified enzyme. In Escherichia coli, the expressed nox gene produced a catalytically active product, which retained its immunoreactivity to affinity-purified NOXase antisera. Alignment of the NOXase protein sequence with that of streptococcal NADH peroxidase (NPXase) revealed that the proteins are 44% identical. Among the most highly conserved segments is a sequence containing Cys42; this residue is known to exist as a stabilized cysteine-sulfenic acid (Cys-SOH) in NPXase and serves as the non-flavin redox center. In addition, three previously identified NPXase segments, known to be involved in FAD and NAD(P)-binding in other pyridine nucleotide-linked flavoprotein oxidoreductases, are strongly conserved in NOXase. Overall, the extensive homology observed between NOXase and NPXase suggests that the monomer chain fold of the oxidase closely resembles that of the peroxidase. Both sequences share limited but significant homology to those of glutathione reductase and other members of the flavoprotein disulfide reductase family. These and other considerations suggest that these two unusual streptococcal flavoproteins constitute a distinct class of FAD-dependent oxidoreductases, the flavoprotein peroxide reductases, easily contrasted with enzymes such as glutathione reductase and thioredoxin reductase.     

Properties and synthesis regulation of NAD(P)H dehydrogenases from Rhodobacter capsulatus

Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome-c reductase activity, which was stimulated by menadione and ubiquinone Q₁. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant.     

The Group B Streptococcus NADH oxidase Nox-2 is involved in fatty acid biosynthesis during aerobic growth and contributes to virulence

Numerous Streptococcaceae produce an H2O-forming NADH oxidase, Nox-2, which has been generally implicated in aerobic survival. We examined the roles of Nox-2 in Group B Streptococcus (GBS), a leading agent of neonatal infections. While nox2 inactivation caused an aerobic growth arrest, no improvement was seen by addition of antioxidants to cultures, suggesting that this defect was not due to accumulation of toxic oxygen species. Using several approaches, we show that the observed inability of the nox2 mutant to grow aerobically is mainly due to an underlying defect in fatty acid (FA) biosynthesis: (i) the nox2 aerobic growth defect is fully and rapidly complemented by adding oleic acid to culture medium, and (ii) direct assimilation of this unsaturated FA in both wild type (WT) and nox2 GBS membranes is demonstrated and correlated with mutant growth rescue. We propose that NAD+ depletion in the nox2 mutant results in reduced acetyl-CoA production, which perturbs FA biosynthesis and hence blocks growth in aerobiosis. The nox2 aerobic growth defect was also complemented when GBS respiration metabolism was activated by exogenous haem and menaquinone. The membrane NADH oxidase activity generated by the functional respiratory chain thus compensates the cytoplasmic NADH oxidase deficiency. The nox2 mutant was attenuated for virulence, as assessed in lung, intraperitoneal and intravenous murine infection models. As the nox2 defect seems only to affect aerobic growth of GBS, its reduced virulence supports the suggestion that aerobic conditions and NADH oxidase activities are relevant to the GBS infection process.     

Examination of Lactobacillus plantarum lactate metabolism side effects in relation to the modulation of aeration parameters

AIMS: The characterization of global aerobic metabolism of Lactobacillus plantarum LP652 under different aeration levels, in order to optimize acetate production kinetics and to suppress H2O2 toxicity. METHODS AND RESULTS: Cultures of L. plantarum were grown on different aeration conditions. After sugar exhaustion and in the presence of oxygen, lactate was converted to acetate, H2O2 and carbon dioxide with concomitant ATP production. Physiological assays were performed at selected intervals in order to assess enzyme activity and vitality of the strain during lactic acid conversion. The maximal aerated condition led to fast lactate-to-acetate conversion kinetics between 8 and 12 h, but H2O2 immediately accumulated, thus affecting cell metabolism. Pyruvate oxidase activity was highly enhanced by oxygen tension and was responsible for H2O2 production after 12 h of culture, whereas lactate oxidase and NADH-dependent lactate dehydrogenase activities were not correlated to metabolite production. Limited NADH oxidase (NOX) and NADH peroxidase (NPR) activities were probably responsible for toxic H2O2 levels in over-aerated cultures. CONCLUSION: Modulating initial airflow led to the maximal specific activity of NOX and NPR observed after 24 h of culture, thus promoting H2O2 destruction and strain vitality at the end of the process. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimal aeration conditions were determined to minimize H2O2 concentration level during growth on lactate.     

Alkyl Hydroperoxide Reductase Is the Primary Scavenger of Endogenous Hydrogen Peroxide in Escherichia coli

Hydrogen peroxide is generated during aerobic metabolism and is capable of damaging critical biomolecules. However, mutants of Escherichia coli that are devoid of catalase typically exhibit no adverse phenotypes during growth in aerobic media. We discovered that catalase mutants retain the ability to rapidly scavenge H(2)O(2) whether it is formed internally or provided exogenously. Analysis of candidate genes revealed that the residual activity is due to alkyl hydroperoxide reductase (Ahp). Mutants that lack both Ahp and catalase could not scavenge H(2)O(2). These mutants excreted substantial amounts of H(2)O(2), and they grew poorly in air. Ahp is kinetically a more efficient scavenger of trace H(2)O(2) than is catalase and therefore is likely to be the primary scavenger of endogenous H(2)O(2). Accordingly, mutants that lack Ahp accumulated sufficient hydrogen peroxide to induce the OxyR regulon, whereas the OxyR regulon remained off in catalase mutants. Catalase still has an important role in wild-type cells, because the activity of Ahp is saturated at a low (10(-5) M) concentration of H(2)O(2). In contrast, catalase has a high K(m), and it therefore becomes the predominant scavenger when H(2)O(2) concentrations are high. This arrangement is reasonable because the cell cannot provide enough NADH for Ahp to rapidly degrade large amounts of H(2)O(2). In sum, E. coli does indeed generate substantial H(2)O(2), but damage is averted by the scavenging activity of Ahp.     

Enzymatic and physiological study of d-xylose metabolism by Candida shehatae

Summary Candida shehatae carbon metabolic pathways were correlated with fermentative activity under different growth conditions. Reduced nicotine adenine dinucleotide (NADPH) is the coenzyme preferred for xylose reductase by C. shehatae under in vitro anoxic cell culture conditions. To prevent a redox imbalance derived from intracellular accumulation of NADH in the second enzymatic step of xylose metabolism, the operation of phosphoketolase via in addition the classic pentose phosphate pathway essential for NADH dissimilation is suggested. Variation in cultivation conditions showed a different NADH/NADPH ratio coupled to xylose reductase activity. The existence of two xylose reductases is discussed. Like ethanol, xylitol accumulates only under oxygen-limited or anaerobic conditions. Xylitol accumulaiton under unaerobic conditions was higher when using respiring cells than respirofermentative cells. This fact suggests that cells pregrown under oxygen limitation are better adapted to starting alcoholic fermentation than cells previously grown under aerobic conditions.Offprint requests to: M. T. Amaral-Collaço     

NADH-peroxidase activity and H2O2 decomposition in a peroxidogenic strain of Streptococcus durans

Streptococcus durans S-76 can accumulate hydrogen peroxide to high concentrations under aerobic conditions when it is previously grown anaerobically. An NADH-peroxidase enzyme protects this bacterium from the bactericidal effect of H2O2. The relationship between oxygen uptake and H2O2 excretion into the medium has been investigated in cultures containing or lacking glucose under various conditions of incubation. The results obtained suggest that neither oxygen nor H2O2 regulate the cellular levels of NADH-peroxidase whose activity seems to be controlled exclusively by the availability of reduced NADH.     

Effect of glucose on glycerol bioconversion by Lactobacillus reuteri

The impact of glucose on glycerol metabolism, especially on 3-hydroxypropionaldehyde (3-HPA) accumulation by resting cells of Lactobacillus reuteri has been investigated. Two systems were used in the study: MRS(-) (modified MRS - omitting glucose, acetate and Tween 80) and distilled water (H(2)O). In MRS(-), addition of glucose enhanced glycerol metabolism in resting cells of L. reuteri, consequently increasing the accumulation of 3-HPA by regulating the NAD/NADH ratio. Enhanced glycerol metabolism correlated positively with the concentration of glucose. NADH produced during glucose metabolism was preferentially reoxidized to NAD by the reduction of 3-HPA to 1,3-propanediol; an adequate supply of glycerol therefore outweighed the repression of glucose on the accumulation of 3-HPA. At a molar ratio of glucose to glycerol no greater than 0.33, accumulation of 3-HPA was favored. In non-growing medium (H(2)O), addition of glucose seemed to be counter-productive with respect to 3-HPA accumulation. Lactate had a positive impact on glycerol metabolism, presumably by altering the redox flux, resulting in enhanced 3-HPA accumulation in both MRS(-) and H(2)O systems.     

Influence of dilution rate on NAD(P) and NAD(P)H concentrations and ratios in a Pseudomonas sp. grown in continuous culture.

A freshwater Pseudomonas sp. was grown in continuous culture under steady-state conditions in L-lactate-, succinate-, glucose- or ammonium-limited media. Under carbon limitation, the NAD(H) (i.e. NAD + NADH) concentration of the organisms increased exponentially from approximately 2 to 7 mumol/g dry wt as the culture dilution rate (D) was decreased from 0.5 to 0.02 h-1. Organisms grown at a given D in any of the carbon-limited media possessed very similar levels of NAD(H). Therefore, under these conditions, cellular NAD(H) was only a function of the culture O and was independent of the nature of the culture carbon source. D had no influence on the NAD(H) content of cells grown under ammonium limitation. In contrast, cellular NADH concentration was not influenced by D in carbon- or ammonium-limited media. In L-lactate-limited medium, bacteria possessed 0.14 mumol NADH/g dry wt; very similar levels were found in organisms grown in the other media. The results are consistent with those of Wimpenny & Firth (1972) that bacteria rigidly maintain a constant NADH level rather than a constant constant NADH: NAD ratio. NADP(H) (i.e. NADP + NADPH) and NADPH levels were also not influenced by changes in the culture carbon source or in D; in L-lactate-limited medium these concentrations were 0.97 and 0.53 mumol/g cell dry wt, respectively. The NADPH:NADP(H) ratio was much higher than the NADH:NAD(H) ratio, averaging 55% in carbon-limited cells.     

Oxidative stress and antioxidant cell protection systems in the microaerophilic bacterium Spirillum winogradskii

The influence of oxygen availability during cultivation on the biosynthetic processes and enzymatic activities in the microaerophilic bacterium Spirillum winogradskii D-427 was studied, and the roles played by different systems of the defense against oxidation stress were determined. The metabolic adjustments caused by transition from microaerobic (2% O2) aerobic conditions (21% O2 of the gas phase) were found to slow down constructive metabolism and increase synthesis of exopolysaccharides as a means of external protection of cells from excess oxygen. This resulted in a twofold decline of the growth yield coefficient. Even though the low activity of catalase is compensated for by a multifold increase in the activities of other cytoplasmic enzymes protecting from toxic forms of O2--peroxidase and enzymes of the redox system of glutathione (glutathione peroxidase and glutathione reductase)--massive lysis of cells starts in the mid-exponential phase and leads to culture death in the stationary phase because of H2O2 accumulation in the periplasm (up to 10 micrograms/mg protein). The absence in cells of cytochrome-c-peroxidase, a periplasmic enzyme eliminating H2O2, was shown. It follows that the major cause of oxidative stress in cells is that active antioxidant defenses are located in the cytoplasm, whereas H2O2 accumulates in the periplasm due to the lack of cytochrome-c-peroxidase. The addition to the medium of thiosulfate promotes elimination of H2O2, stops cell lysis under aerobic conditions, lends stability to cultures, and results in a threefold increase in the growth yield.     

Are respiratory enzymes the primary sources of intracellular hydrogen peroxide?

Endogenous H2O2 is believed to be a source of chronic damage in aerobic organisms. To quantify H2O2 formation, we have generated strains of Escherichia coli that lack intracellular scavenging enzymes. The H2O2 that is formed within these mutants diffuses out into the medium, where it can be measured. We sought to test the prevailing hypothesis that this H2O2 is primarily generated by the autoxidation of redox enzymes within the respiratory chain. The rate of H2O2 production increased when oxygen levels were raised, confirming that H2O2 is formed by an adventitious chemical process. However, mutants that lacked NADH dehydrogenase II and fumarate reductase, the most oxidizable components of the respiratory chain in vitro, continued to form H2O2 at normal rates. NADH dehydrogenase II did generate substantial H2O2 when it was when overproduced or quinones were absent, forcing electrons to accumulate on the enzyme. Mutants that lacked both NADH dehydrogenases respired very slowly, as expected; however, these mutants showed no diminution of H2O2 excretion, suggesting that H2O2 is primarily formed by a source outside the respiratory chain. That source has not yet been identified. In respiring cells the rate of H2O2 production was approximately 0.5% the rate of total oxygen consumption, with only modest changes when cells used different carbon sources.     

Effect of beta-lapachone on superoxide anion and hydrogen peroxide production in Trypanosoma cruzi.

Addition of beta-lapachone, an o-naphthoquinone endowed with trypanocidal properties to respiring Trypanosoma cruzi epimastigotes induced the release of O2- and H2O2 from the whole cells to the suspending medium. The same beta-lapachone concentration (4 micron) that released H2O2 at maximal rate completely inhibited T. cruzi growth in a liquid medium. The position isomer, alpha-lapachone, did not stimulate O2- and H2O2 release, and did not inhibit epimastigote growth. beta-Lapachone was able to stimulate H2O2 production by the epimastigote homogenate in the presence of NADH as reductant. The same effect was observed with the mitochondrial fraction supplemented with NADH, where beta-lapachone enhanced the generation of O2- and H2O2 4.5- and 2.5-fold respectively. beta-Lapachone also increased O2- and H2O2 production (2.5 and 2-fold respectively) by the microsomal fraction with NADPH as reductant. Cyanide-insensitive NADH and NADPH oxidation by the mitochondrial and microsomal fractions (quinone reductase activity) was stimulated to about the same extent by beta-lapachone. alpha-Lapachone was unable to increase O2- and H2O2 production and quinone reductase activity of the mitochondrial and microsomal fractions.     

Impact of aeration and heme-activated respiration on Lactococcus lactis gene expression: identification of a heme-responsive operon

Lactococcus lactis is a widely used food bacterium mainly characterized for its fermentation metabolism. However, this species undergoes a metabolic shift to respiration when heme is added to an aerobic medium. Respiration results in markedly improved biomass and survival compared to fermentation. Whole-genome microarrays were used to assess changes in L. lactis expression under aerobic and respiratory conditions compared to static growth, i.e., nonaerated. We observed the following. (i) Stress response genes were affected mainly by aerobic fermentation. This result underscores the differences between aerobic fermentation and respiration environments and confirms that respiration growth alleviates oxidative stress. (ii) Functions essential for respiratory metabolism, e.g., genes encoding cytochrome bd oxidase, menaquinone biosynthesis, and heme uptake, are similarly expressed under the three conditions. This indicates that cells are prepared for respiration once O(2) and heme become available. (iii) Expression of only 11 genes distinguishes respiration from both aerobic and static fermentation cultures. Among them, the genes comprising the putative ygfCBA operon are strongly induced by heme regardless of respiration, thus identifying the first heme-responsive operon in lactococci. We give experimental evidence that the ygfCBA genes are involved in heme homeostasis.     

Comparative study of the physiological roles of three peroxidases (NADH peroxidase, Alkyl hydroperoxide reductase and Thiol peroxidase) in oxidative stress response, survival inside macrophages and virulence of Enterococcus faecalis

The opportunistic pathogen Enterococcus faecalis is well equipped with peroxidatic activities. It harbours three loci encoding a NADH peroxidase, an alkyl hydroperoxide reductase and a protein (EF2932) belonging to the AhpC/TSA family. We present results demonstrating that ef2932 does encode a thiol peroxidase (Tpx) and show that it is part of the regulon of the hydrogen peroxide regulator HypR. Characterization of unmarked deletion mutants showed that all three peroxidases are important for the defence against externally provided H(2)O(2). Exposure to internal generated H(2)O(2) by aerobic growth on glycerol, lactose, galactose or ribose showed that Npr was absolutely required for aerobic growth on glycerol and optimal growth on the other substrates. Growth on glycerol was also dependent on Ahp. Addition of catalase restored growth of the mutants, and therefore, extracellular H(2)O(2) concentrations have been determined. This showed that the time point of growth arrest of the Deltanpr mutant correlated with the highest H(2)O(2) concentration measured. Analysis of the survival of the different strains inside peritoneal macrophages revealed that Tpx was the most important antioxidant activity for protecting the cells against the hostile phagocyte environment. Finally, the Deltatpx and the triple mutant showed attenuated virulence in a mouse peritonitis model.