Aerotolerance and peroxide resistance in peroxidase and PerR mutants of Streptococcus pyogenes

Survival in aerobic conditions is critical to the pathogenicity of many bacteria. To investigate the means of aerotolerance and resistance to oxidative stress in the catalase-negative organism Streptococcus pyogenes, we used a genomics-based approach to identify and inactivate homologues of two peroxidase genes, encoding alkyl hydroperoxidase (ahpC) and glutathione peroxidase (gpoA). Single and double mutants survived as well as the wild type under aerobic conditions. However, they were more susceptible than the wild type to growth suppression by paraquat and cumene hydroperoxide. In addition, we show that S. pyogenes demonstrates an inducible peroxide resistance response when treated with sublethal doses of peroxide. This resistance response was intact in ahpC and gpoA mutants but not in mutants lacking PerR, a repressor of several genes including ahpC and catalase (katA) in Bacillus subtilis. Because our data indicate that these peroxidase genes are not essential for aerotolerance or induced resistance to peroxide stress in S. pyogenes, genes for a novel mechanism of managing peroxide stress may be regulated by PerR in streptococci.     

Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: a review

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.     

Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: A review

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.     

Aerobic-type ribonucleotide reductase in the anaerobe Bacteroides fragilis.

Bacteroides fragilis, a component of the normal intestinal flora, is an obligate anaerobe capable of long-term survival in the presence of air. Survival is attributed to an elaborate oxidative stress response that controls the induction of more than 28 peptides, but there is limited knowledge concerning the identities of these peptides. In this report, RNA fingerprinting by arbitrarily primed PCR identified five new genes whose expression increased following exposure to O2. Nucleotide sequence analysis of the cloned genes indicated that they encoded an outer membrane protein, an aspartate decarboxylase, an efflux pump, heat shock protein HtpG, and an NrdA ortholog constituting the large subunit of a class Ia ribonucleotide reductase (RRase). Attention was focused on the nrdA gene since class I RRases are obligate aerobic enzymes catalyzing the reduction of ribonucleoside 5'-diphosphates by a mechanism that requires molecular oxygen for activity. Sequence analysis of the nrd locus showed that two genes, nrdA and nrdB, are located in the same orientation in a 4.5-kb region. Northern hybridization and primer extension experiments confirmed induction of the genes by O2 and suggested they are an operon. The B. fragilis nrdA and nrdB genes were overexpressed in Escherichia coli, and CDP reductase assays confirmed that they encoded an active enzyme. The enzyme activity was inhibited by hydroxyurea, and ATP was shown to be a positive effector of CDP reductase activity, while dATP was an inhibitor, indicating that the enzyme was a class Ia RRase. A nrdA mutant was viable under anaerobic conditions but had decreased survival following exposure to O2, and it could not rapidly resume growth after O2 treatment. The results presented indicate that during aerobic conditions B. fragilis NrdAB may have a role in maintaining deoxyribonucleotide pools for DNA repair and growth recovery.     

Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria

Oxidative stress, through the production of reactive oxygen species, is a natural consequence of aerobic metabolism. Escherichia coli has several major regulators activated during oxidative stress, including OxyR, SoxRS, and RpoS. OxyR and SoxR undergo conformation changes when oxidized in the presence of hydrogen peroxide and superoxide radicals, respectively, and subsequently control the expression of cognate genes. In contrast, the RpoS regulon is induced by an increase in RpoS levels. Current knowledge regarding the activation and function of these regulators and their dependent genes in E. coli during oxidative stress forms the scope of this review. Despite the enormous genomic diversity of bacteria, oxidative stress response regulators in E. coli are functionally conserved in a wide range of bacterial groups, possibly reflecting positive selection of these regulators. SoxRS and RpoS homologs are present and respond to oxidative stress in Proteobacteria, and OxyR homologs are present and function in H(2)O(2) resistance in a range of bacteria, from gammaproteobacteria to Actinobacteria. Bacteria have developed complex, adapted gene regulatory responses to oxidative stress, perhaps due to the prevalence of reactive oxygen species produced endogenously through metabolism or due to the necessity of aerotolerance mechanisms in anaerobic bacteria exposed to oxygen.     

Biochemical characterization of the structural Zn2+ site in the Bacillus subtilis peroxide sensor PerR

In Bacillus subtilis most peroxide-inducible oxidative stress genes are regulated by a metal-dependent repressor, PerR. PerR is a dimeric, Zn2+-containing metalloprotein with a regulatory metal-binding site that binds Fe2+ (PerR:Zn,Fe) or Mn2+ (PerR: Zn,Mn). Reaction of PerR:Zn,Fe with low levels of hydrogen peroxide (H2O2) leads to oxidation of two His residues thereby leading to derepression. When bound to Mn2+, the resulting PerR:Zn,Mn is much less sensitive to oxidative inactivation. Here we demonstrate that the structural Zn2+ is coordinated in a highly stable, intrasubunit Cys4:Zn2+ site. Oxidation of this Cys4:Zn2+ site by H2O2 leads to the formation of intrasubunit disulfide bonds. The rate of oxidation is too slow to account for induction of the peroxide stress response by micromolar levels of H2O2 but could contribute to induction under severe oxidative stress conditions. In vivo studies demonstrated that inactivation of PerR:Zn,Mn required 10 mM H2O2, a level at least 1000 times greater than that needed for inactivation of PerR:Zn,Fe. Surprisingly even under these severe oxidation conditions there was little if any detectable oxidation of cysteine residues in vivo: derepression was correlated with oxidation of the regulatory site. Because oxidation at this site required bound Fe2+ in vitro, we suggest that treatment of cells with 10 mM H2O2 released sufficient Fe2+ into the cytosol to effect a transition of PerR from the PerR:Zn,Mn form to the peroxide-sensitive PerR: Zn,Fe form. This model is supported by metal ion affinity measurements demonstrating that PerR bound Fe2+ with higher affinity than Mn2+.     

Rapid viability loss on exposure to air in a superoxide dismutase-deficient mutant of Porphyromonas gingivalis.

Porphyromonas gingivalis, an obligate anaerobe, exhibits a relatively high degree of aerotolerance and possesses superoxide dismutase (SOD) which is induced by exposure to air. To clarify roles for SOD in this organism, the gene encoding SOD (sod) on the P. gingivalis chromosome was disrupted in a gene-directed way by use of a suicide plasmid containing a mutated sod. A sod mutant thus obtained showed no SOD activity in crude extracts and exhibited a rapid viability loss immediately after exposure to air, whereas the wild-type parent showed no decrease in viability for at least 5 h under aerobic conditions. These results clearly indicate that SOD is essential for aerotolerance in P. gingivalis.     

Glutathione and catalase provide overlapping defenses for protection against respiration-generated hydrogen peroxide in Haemophilus influenzae

Glutathione is an abundant and ubiquitous low-molecular-weight thiol that may play a role in many cellular processes, including protection against the deleterious effects of reactive oxygen species. We address here the role of glutathione in protection against hydrogen peroxide (H2O2) in Haemophilus influenzae and show that glutathione and catalase provide overlapping defense systems. H. influenzae is naturally glutathione deficient and imports glutathione from the growth medium. Mutant H. influenzae lacking catalase and cultured in glutathione-deficient minimal medium is completely devoid of H2O2 scavenging activity and, accordingly, substantial amounts of H2O2 accumulate in the growth medium. H. influenzae generates H2O2 at rates similar to those reported for Escherichia coli, but the toxicity of this harmful metabolite is averted by glutathione-based H2O2 removal, which appears to be the primary system for protection against H2O2 endogenously generated during aerobic respiration. When H2O2 concentrations exceed low micromolar levels, the hktE gene-encoded catalase becomes the predominant scavenger. The requirement for glutathione in protection against oxidative stress is analogous to that in higher and lower eukaryotes but is unlike the situation in other bacteria in which glutathione is dispensable for aerobic growth during both normal and oxidative stress conditions.     

Oxidative pathway of choline to betaine in the soluble fraction prepared from Arthrobacter globiformis.

One strain of bacteria which showed high H2O2-generating activity was isolated from soil and characterized as Arthrobacter globiformis based on its morphological, nutritional, and physiological characteristics. The activities of H2O2 generation, NAD reduction and oxygen consumption in the bacterial cells were examined using choline, betaine aldehyde or betaine as substrate. Choline was oxidized to betaine aldehyde under aerobic conditions in a reaction coupled with H2O2 generation and oxygen consumption. On the other hand, betaine aldehyde seemed to be oxidized to betaine through two distinct oxidative reactions, H2O2 generation (oxygen consumption) under aerobic conditions and NAD reduction under either aerobic or anaerobic conditions. These enzyme activities were found in the supernatant fraction of the sonicated cell preparation.     

Persistence and multiplication of obligate anaerobe bacteria in amebae under aerobic conditions

After co-cultivation of Mobiluncus curtisii, an obligate non-sporeforming anaerobe, with free living amebae from the Acanthamoeba spp. under aerobic conditions, internalization, multiplication and persistence of bacterial cells were established for at least 4-6 weeks. Under the same conditions and media without viable amebae, the cells of M. curtisii did not replicate and died in 4-7 days. The infection of amebae occurred with 10 to 100 bacteria per ml of co-cultivation media. In 7-14 days the amount of bacterial cells increased to 1x10(5)-1x10(6) CFU/mL. Electron microscopic examinations revealed bacteria within vacuoles in the amebae and intracellular replication. These results suggest a previously undescribed mechanism for spread, replication and persistence of obligately anaerobe bacteria in the environment and new possible sources, reservoirs and transfer mechanisms of infections caused by obligate anaerobe bacteria.     

Proteomics-based analysis of a counter-oxidative stress system in Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative anaerobic pathogen associated with chronic periodontitis. Although anaerobic, P. gingivalis exhibits a high degree of aerotolerance, which enables it to survive within periodontal pockets. The aim of the present study was to examine the effect of oxidative stress on protein expression in P. gingivalis to obtain a better understanding of the mechanism underlying its aerotolerance. To accomplish this, P. gingivalis cells were grown under conditions of hemin limitation (0.01 microg/mL) to avoid the oxygen protective effect of hemin on oxidative stress. The proteins were then extracted from cultures either left untreated or subjected to oxidative stress and separated by 2-DE. The resultant protein expression profiles were examined by image scanning, and those found to differ depending on the presence or absence of aeration were subjected to MALDI-MS and then analyzed using the ORF database of P. gingivalis W83 from The Institute of Genomic Research. Oxidative stress was found to affect the expression of numerous proteins in P. gingivalis cells. In particular, the levels of HtpG, GroEL, DnaK, AhpC, TPR domain protein, and trigger factor were substantially increased.     

Identification of an oxidative stress-sensitive protein from Campylobacter jejuni, homologous to rubredoxin oxidoreductase/rubrerythrin

An oxidative stress-sensitive protein was found in the microaerophile Campylobacter jejuni. A novel 27-kDa protein was found to decrease concomitantly with a decrease in viability from either exogenous H(2)O(2) stress or endogenous oxidative stresses in aerobic conditions. Sequence analyses revealed that the 27-kDa protein was identical to Cj0012c in C. jejuni NCTC11168 and its deduced 215 amino acid sequence has similarity to two non-heme iron proteins found in other bacteria, rubredoxin oxidoreductase (Rbo) and rubrerythrin (Rbr). Thus, we designated the protein as Rrc (Rbo/Rbr-like protein of C. jejuni). In H(2)O(2)-treated cells, Western blot analysis showed some bands smaller than Rrc, and RT-PCR showed similar expression of Rrc mRNA to the control without treatment, suggesting that the sensitive response of Rrc to oxidative stress is due to degradation of the protein.     

Use of 2-D electrophoresis and ESI mass spectrometry techniques to characterize Fusobacterium nucleatum proteins up-regulated after oxidative stress

The genus Fusobacterium belongs to the Fusobacteriaceae family and is a Gram-negative obligate anaerobic bacterium found in the human oral microbiota. Even that Fusobacterium nucleatum cannot grow under aerobic conditions, they may exhibit aerotolerance as an adaptive response which could figure as an important virulence factor, during the stages of infection, when these anaerobes are shifted to aerobic conditions. In this regard, little is known about bacterial oxidative stress adaptive response and the influence of this adaptation on the host-bacteria relationship. We aimed to use both techniques 2-DE and Electrospray Ionization Mass Spectrometry (ESI-MS) to characterize proteins in F. nucleatum, after oxidative stress. We related three different proteins which were up-regulated by oxidative stress. As its genome is already sequenced, these proteins were found in data base search, by homology. Thus, by using techniques as ESI-Q/TOF-MS, in addition to 2-DE, the opportunity exists to gain a more holistic view of the bacterial proteome of human pathogens, to achieve a better understanding of species diversity and to elucidate the role of specific proteins in disease. This work represents one of the first studies using genetic and physiological approaches to understand the phenomenon of oxidative stress in F. nucleatum.     

Global Metabolic Response of Enterococcus faecalis to Oxygen

Oxygen and oxidative stress have become relevant components in clarifying the mechanism that weakens bacterial cells in parallel to the mode of action of bactericidal antibiotics. Given the importance of oxidative stress in the overall defense mechanism of bacteria and their apparent role in the antimicrobial mode of action, it is important to understand how bacteria respond to this stress at a metabolic level. The aim of this study was to determine the impact of oxygen on the metabolism of the facultative anaerobe Enterococcus faecalis using continuous culture, metabolomics, and ¹³C enrichment of metabolic intermediates. When E. faecalis was rapidly transitioned from anaerobic to aerobic growth, cellular metabolism was directed toward intracellular glutathione production and glycolysis was upregulated 2-fold, which increased the supply of critical metabolite precursors (e.g., glycine and glutamate) for sulfur metabolism and glutathione biosynthesis as well as reducing power for cellular respiration in the presence of hemin. The ultimate metabolic response of E. faecalis to an aerobic environment was the upregulation of fatty acid metabolism and benzoate degradation, which was linked to important changes in the bacterial membrane composition as evidenced by changes in membrane fatty acid composition and the reduction of membrane-associated demethylmenaquinone. These key metabolic pathways associated with the response of E. faecalis to oxygen may represent potential new targets to increase the susceptibility of this bacterium to bactericidal drugs.     

Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus

Oxidative-stress resistance in Staphylococcus aureus is linked to metal ion homeostasis via several interacting regulators. In particular, PerR controls the expression of a regulon of genes, many of which encode antioxidants. Two PerR regulon members, ahpC (alkylhydroperoxide reductase) and katA (catalase), show compensatory regulation, with independent and linked functions. An ahpC mutation leads to increased H2O2 resistance due to greater katA expression via relief of PerR repression. Moreover, AhpC provides residual catalase activity present in a katA mutant. Mutation of both katA and ahpC leads to a severe growth defect under aerobic conditions in defined media (attributable to lack of catalase activity). This results in the inability to scavenge exogenous or endogenously produced H2O2, resulting in accumulation of H2O2 in the medium. This leads to DNA damage, the likely cause of the growth defect. Surprisingly, the katA ahpC mutant is not attenuated in two independent models of infection, which implies reduced oxygen availability during infection. In contrast, both AhpC and KatA are required for environmental persistence (desiccation) and nasal colonization. Thus, oxidative-stress resistance is an important factor in the ability of S. aureus to persist in the hospital environment and so contribute to the spread of human disease.