Function of the pyruvate oxidase-lactate oxidase cascade in interspecies competition between Streptococcus oligofermentans and Streptococcus mutans

Complex interspecies interactions occur constantly between oral commensals and the opportunistic pathogen Streptococcus mutans in dental plaque. Previously, we showed that oral commensal Streptococcus oligofermentans possesses multiple enzymes for H(2)O(2) production, especially lactate oxidase (Lox), allowing it to out-compete S. mutans. In this study, through extensive biochemical and genetic studies, we identified a pyruvate oxidase (pox) gene in S. oligofermentans. A pox deletion mutant completely lost Pox activity, while ectopically expressed pox restored activity. Pox was determined to produce most of the H(2)O(2) in the earlier growth phase and log phase, while Lox mainly contributed to H(2)O(2) production in stationary phase. Both pox and lox were expressed throughout the growth phase, while expression of the lox gene increased by about 2.5-fold when cells entered stationary phase. Since lactate accumulation occurred to a large degree in stationary phase, the differential Pox- and Lox-generated H(2)O(2) can be attributed to differential gene expression and substrate availability. Interestingly, inactivation of pox causes a dramatic reduction in H(2)O(2) production from lactate, suggesting a synergistic action of the two oxidases in converting lactate into H(2)O(2). In an in vitro two-species biofilm experiment, the pox mutant of S. oligofermentans failed to inhibit S. mutans even though lox was active. In summary, S. oligofermentans develops a Pox-Lox synergy strategy to maximize its H(2)O(2) formation so as to win the interspecies competition.     

L-氨基酸氧化酶的研究进展

L-氨基酸氧化酶(L-amino acid oxidase, LAAO)能特异性催化L-氨基酸氧化脱氨,生成α-酮酸、氨和H₂O₂。该酶分布较广,其中蛇毒源LAAO是该类酶中研究最为深入的一类,近年来,越来越多的非蛇毒源LAAO被发现和报道,现对蛇毒源和非蛇毒源LAAO的研究进展进行了综述。现有研究表明,不同物种来源的LAAO,其底物选择性、等电点、稳定性等理化性质不尽相同;虽对其结构的研究还较少,但现有的研究表明蛇毒源和非蛇毒源LAAO的结构都含有FAD结合结构域、底物结构域和螺旋结构域;研究已发现不同来源的LAAO体外具有多种不同的生物学功能,而这些生物学功能多数是由于其产物H₂O₂作用的结果;对LAAO异源表达的研究较少且都不甚成功,可能是由于其需要进行翻译后修饰。     

Streptococcus oligofermentans inhibits Streptococcus mutans through conversion of lactic acid into inhibitory H2O2: a possible counteroffensive strategy for interspecies competition

The oral microbial flora contains over 500 different microbial species that often interact as a means to compete for limited space and nutritional resources. Streptococcus mutans, a major caries-causing pathogen, is a species which tends to interact competitively with other species in the oral cavity, largely due to its ability to generate copious quantities of the toxic metabolite, lactic acid. However, during a recent clinical study, we discovered a novel oral streptococcal species, Streptococcus oligofermentans, whose abundance appeared to be inversely correlated with that of S. mutans within dental plaque samples and thus suggested a possible antagonistic relationship with S. mutans. In this study, we used a defined in vitro interspecies interaction assay to confirm that S. oligofermentans was indeed able to inhibit the growth of S. mutans. Interestingly, this inhibitory effect was relatively specific to S. mutans and was actually enhanced by the presence of lactic acid. Biochemical analyses revealed that S. oligofermentans inhibited the growth of S. mutans via the production of hydrogen peroxide with lactic acid as the substrate. Further genetic and molecular analysis led to the discovery of the lactate oxidase (lox) gene of S. oligofermentans as responsible for this biological activity. Consequently, the lox mutant of S. oligofermentans also showed dramatically reduced inhibitory effects against S. mutans and also exhibited greatly impaired growth in the presence of the lactate produced by S. mutans. These data indicate that S. oligofermentans possesses the capacity to convert its competitor's main 'weapon' (lactic acid) into an inhibitory chemical (H(2)O(2)) in order to gain a competitive growth advantage. This fascinating ability may be an example of a counteroffensive strategy used during chemical warfare within the oral microbial community.     

Establishing a genetic system for ecological studies of Streptococcus oligofermentans

Streptococcus oligofermentans is a newly characterized species belonging to the mitis group of oral streptococci. So far no correlation has been demonstrated between S. oligofermentans and dental caries. Furthermore, a reverse correlation has been observed between the number of S. oligofermentans and the number of Streptococcus mutans, a major cariogenic pathogen, in the oral cavity. These properties suggest that S. oligofermentans may have a potential to be used as a 'probiotics' for caries prevention. In this study, we aim to establish a genetic system in S. oligofermentans to further study the biology of this new species. Using homologous regions of the comCDE locus in other streptococci, the comC gene was isolated and sequenced. A synthetic competence-stimulating peptide (CSP) was synthesized and shown to be able to effectively induce competence in S. oligofermentans. This CSP-induced transformation system in S. oligofermentans was used to construct green fluorescent protein (gfp) and luciferase (luc) reporter systems, both of which are driven by the lactate dehydrogenase (ldh) promoter. These reporter systems were further shown to be highly expressed in planktonic and biofilm cells, suggesting that these reporter systems can be used in future ecological studies of S. oligofermentans.     

Dpr蛋白在寡发酵链球菌抗过氧化氢中的作用

摘要：【目的】寡发酵链球菌（Streptococcus oligofermentans）是从无龋人的口腔中分离到的一株链球菌,好氧条件下产生、同时也耐受高浓度（4.4 mmol/L）的过氧化氢。本研究探讨dpr基因对寡发酵链球菌抗过氧化氢的贡献。【方法】克隆和表达寡发酵链球菌dpr基因,分析Dpr蛋白的功能;构建寡发酵链球菌的dpr基因突变株,比较野生株和突变株对不同浓度过氧化氢的耐受程度;并将寡发酵链球菌dpr基因克隆到对过氧化氢耐受力低的变形链球菌中,分析其对变形链球菌过氧化氢耐受能力的影响。【结果】     

Streptococcus mutans H2O2-forming NADH oxidase is an alkyl hydroperoxide reductase protein

Nox-1 from Streptococcus mutans, the bacteria which cause dental caries, was previously identified as an H2O2-forming reduced nicotinamide adenine dinucleotide (NADH) oxidase. Nox-1 is homologous with the flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase. A partial open reading frame upstream of nox1, homologous with the other (peroxidase) component, ahpC, from the S. typhimurium system, was also identified. We report here the complete sequence of S. mutans ahpC. Analyses of purified AhpC together with Nox-1 have verified that these proteins act as a cysteine-based peroxidase system in S. mutans, catalyzing the NADH-dependent reduction of organic hydroperoxides or H2O2 to their respective alcohols and/or H2O. These proteins also catalyze the four-electron reduction of O2 to H2O2, clarifying the role of Nox-1 as a protective protein against oxygen toxicity. Major differences between Nox-1 and AhpF include: (i) the absolute specificity of Nox-1 for NADH; (ii) lower amounts of flavin semiquinone and a more prominent FADH2 to NAD+ charge transfer absorbance band stabilized by Nox-1; and (iii) even higher redox potentials of disulfide centers relative to flavin for Nox-1. Although Nox-1 and AhpC from S. mutans were shown to play a protective role against oxidative stress in vitro and in vivo in Escherichia coli, the lack of a significant effect on deletion of these genes from S. mutans suggests the presence of additional antioxidant proteins in these bacteria.     

Functions of Two Types of NADH Oxidases in Energy Metabolism and Oxidative Stress of Streptococcus mutans

We have previously identified two distinct NADH oxidases corresponding to H(2)O(2)-forming oxidase (Nox-1) and H(2)O-forming oxidase (Nox-2) induced in Streptococcus mutans. Sequence analyses indicated a strong similarity between Nox-1 and AhpF, the flavoprotein component of Salmonella typhimurium alkyl hydroperoxide reductase; an open reading frame upstream of nox-1 also showed homology to AhpC, the direct peroxide-reducing component of S. typhimurium alkyl hydroperoxide reductase. To determine their physiological functions in S. mutans, we constructed knockout mutants of Nox-1, Nox-2, and/or the AhpC homologue; we verified that Nox-2 plays an important role in energy metabolism through the regeneration of NAD(+) but Nox-1 contributes negligibly. The Nox-2 mutant exhibited greatly reduced aerobic growth on mannitol, whereas there was no significant effect of aerobiosis on the growth on mannitol of the other strains or growth on glucose of any of the strains. Although the Nox-2 mutants grew well on glucose aerobically, the end products of glucose fermentation by the Nox-2 mutant were substantially shifted to higher ratios of lactic acid to acetic acid compared with wild-type cells. The resistance to cumene hydroperoxide of Escherichia coli TA4315 (ahpCF-defective mutant) transformed with pAN119 containing both nox-1 and ahpC genes was not only restored but enhanced relative to that of E. coli K-12 (parent strain), indicating a clear function for Nox-1 as part of an alkyl hydroperoxide reductase system in vivo in combination with AhpC. Surprisingly, the Nox-1 and/or AhpC deficiency had no effect on the sensitivity of S. mutans to cumene hydroperoxide and H(2)O(2), implying that the existence of some other antioxidant system(s) independent of Nox-1 in S. mutans compensates for the deficiency.     

Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species, leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species, Streptococcus gordonii, Streptococcus sanguinis, and cariogenic Streptococcus mutans. S. sanguinis and S. gordonii used oxygen availability and the differential production of hydrogen peroxide (H(2)O(2)) to compete effectively against S. mutans. Interspecies antagonism was influenced by glucose with reduced production of H(2)O(2). Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV of S. mutans, as well as the H(2)O(2)-dependent release of heterologous DNA from mixed cultures of S. sanguinis and S. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms of S. mutans in the oral biofilm.     

Cloning, characterization and mutagenesis of Russell's viper venom L-amino acid oxidase: Insights into its catalytic mechanism

To investigate the structure-function relationships and geographic variations of L-amino acid oxidase (LAAO) from Daboia venoms, a single LAAO (designated as DrLAO) was purified from eastern Indian Daboia russelii venom and characterized. The purified DrLAO showed subunit molecular mass of 60-64kDa; its N-terminal sequence (1-20) was identical to those of several true viper LAAOs. Its preferred substrates were hydrophobic l-amino acids and the kinetic specificities were ordered as follows: Phe, Tyr, Met, Leu, and Trp. Enzyme assay and Western blotting showed that the venom LAAO contents of D. russelii were higher than those of Daboia siamensis. DrLAO dose-dependently inhibited ADP- and collagen-induced platelet aggregation with IC(50) values of 0.27 and 0.82μM, respectively. Apparently, DrLAO may synergize with other venom components to prolong and enhance bleeding symptoms after Daboia envenoming. The full sequence of DrLAO was deduced from its cDNA sequence and then confirmed by peptide mass fingerprinting. Molecular phylogenetic analysis revealed that SV-LAAO family members could be differentiated not only by snake taxonomy but also by the variations at position 223, and they divided into H223, S223, N223, and D223 subclasses. We have further prepared recombinant DrLAO and mutants by the Pichia expression system. Mutagenic analyses of DrLAO His223 revealed that this residue bound substrates instead of serving as an essential base in the catalytic steps. Our results suggest a direct hydride transfer from substrate to FAD as the mechanism for SV-LAAOs.     

Identification of antibacterial mechanism of L-amino acid oxidase derived from Trichoderma harzianum ETS 323

Although L-amino oxidase (LAAO; EC 1.4.3.2) has been reported to be a potent antibacterial agent, the mechanism responsible for its antibacterial activity has not been identified. The present study aimed to identify the mechanism responsible for the antibacterial activity of Th-LAAO, an LAAO recently isolated from the extracellular proteins of Trichoderma harzianum ETS 323, at the same time as elucidating the nature of this enzyme. The results obtained indicate that the enzyme activity and structure of Th-LAAO are stable at pH 6-8 and less stable at both pH 4-5.5 and pH 9. At pH 7.0, the optimum temperature for Th-LAAO was found to be 40 °C, comprising the temperature at which enzymatic activity is greatest, with enzymatic activity deceasing with further increases in temperature as a result of thermal denaturation of the enzyme, leading to partial denaturation at 50 °C. The results obtained by confocal microscopy and flow cytometry indicate that Th-LAAO interacts with bacteria to cause membrane permeabilization, and this interaction may be promoted by the amphipathic sequence in Th-LAAO and other cytotoxic LAAOs located at the N-terminus. The findings of increased exogenous H(2) O(2) production and reactive oxidative species accumulation in Th-LAAO-treated bacteria indicate that reactive oxidative species accumulation may trigger forms of cell damage, including lipid peroxidation and DNA strand breakage that results in bacterial growth inhibition. Taken together, the results indicate that the processes of bacterial interaction, membrane permeabilization and H(2)O(2) production are involved in the mechanism responsible for the antibacterial activity of Th-LAAO.     

Environmental influences on competitive hydrogen peroxide production in Streptococcus gordonii

Streptococcus gordonii is an important member of the oral biofilm. One of its phenotypic traits is the production of hydrogen peroxide (H2O2). H2O2 is an antimicrobial component produced by S. gordonii that is able to antagonize the growth of cariogenic Streptococcus mutans. Strategies that modulate H2O2 production in the oral cavity may be useful as a simple therapeutic mechanism to improve oral health, but little is known about the regulation of H2O2 production. The enzyme responsible for H2O2 production is pyruvate oxidase, encoded by spxB. The functional studies of spxB expression and SpxB abundance presented in this report demonstrate a strong dependence on environmental oxygen tension and carbohydrate availability. Carbon catabolite repression (CCR) modulates spxB expression carbohydrate dependently. Catabolite control protein A (CcpA) represses spxB expression by direct binding to the spxB promoter, as shown by electrophoretic mobility shift assays (EMSA). Promoter mutation studies revealed the requirement of two catabolite-responsive elements (CRE) for CcpA-dependent spxB regulation, as evaluated by spxB expression and phenotypic H2O2 production assays. Thus, molecular mechanisms for the control of S. gordonii spxB expression are presented for the first time, demonstrating the possibility of manipulating H2O2 production for increased competitive fitness.     

Role of the dpr product in oxygen tolerance in Streptococcus mutans

We have previously identified and characterized the alkyl hydroperoxide reductase of Streptococcus mutans, which consists of two components, Nox-1 and AhpC. Deletion of both nox-1 and ahpC had no effect on the sensitivity of S. mutans to cumene hydroperoxide or H(2)O(2), implying that the existence of another antioxidant system(s) independent of the Nox-1-AhpC system compensates for the deficiency. Here, a new antioxidant gene (dpr for Dps-like peroxide resistance gene) was isolated from the S. mutans chromosome by its ability to complement an ahpCF deletion mutant of Escherichia coli with a tert-butyl hydroperoxide-hypersensitive phenotype. The dpr gene complemented the defect in peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. Under aerobic conditions, the dpr disruption mutant carrying a spectinomycin resistance gene (dpr::Spc(r) mutant) grew as well as wild-type S. mutans in liquid medium. However, the dpr::Spc(r) mutant could not form colonies on an agar plate under air. In addition, neither the dpr::Spc(r) ahpC::Em(r)::nox-1 triple mutant nor the dpr::Spc(r) sod::Em(r) double mutant was able to grow aerobically in liquid medium. The 20-kDa dpr gene product Dpr is an iron-binding protein. Synthesis of Dpr was induced by exposure of S. mutans cells to air. We propose a mechanism by which Dpr confers aerotolerance on S. mutans.     

A thermostable L-aspartate oxidase: a new tool for biotechnological applications

L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0–10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.     

L-amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma. Comparative sequence analysis and characterization of active and inactive forms of the enzyme.

Here we report the cDNA-deduced amino-acid sequence of L-amino-acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FAD-dependent ophidian LAAOs to FAD-dependent oxidases such as monoamine oxidases, D-amino-acid oxidases and tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine oxidases, tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl(-), prevent pH-induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.     

L-氨基酸氧化酶的结构、底物谱、家族进化及应用研究进展

L-氨基酸氧化酶(LAAO)是一类生物体内参与氨基酸氧化代谢的重要氧化还原酶,能够以氧分子为电子受体催化L-氨基酸氧化脱氨,生成相应的酮酸、氨(NH₃)和过氧化氢(H₂O₂).近期发现有些LAAO能够专一性识别特定氨基酸,而不受其他种类氨基酸的干扰,因而在手性胺类化合物拆分、α-酮酸生物合成、临床样本、食品及氨基酸发酵过程中氨基酸含量检测等领域都发挥着重要作用.本文重点综述目前研究报道的底物专一性LAAO,总结并比较这些酶的酶学性质、结构功能,以及家族进化规律等,并进一步讨论这些酶在生物催化及氨基酸检测中的应用.本综述将为底物特异性LAAO的分子机制研究及产业应用研究提供重要的素材和指导.     

l-Amino acid oxidase as biocatalyst: a dream too far?

l-Amino acid oxidase (LAAO) is a flavoenzyme containing non-covalently bound flavin adenine dinucleotide, which catalyzes the stereospecific oxidative deamination of l-amino acids to α-keto acids and also produces ammonia and hydrogen peroxide via an imino acid intermediate. LAAOs purified from snake venoms are the best-studied members of this family of enzymes, although a number of LAAOs from bacterial and fungal sources have been also reported. From a biochemical point of view, LAAOs from different sources are distinguished by molecular mass, substrate specificity, post-translational modifications and regulation. In analogy to the well-known biotechnological applications of d-amino acid oxidase, important results are expected from the availability of suitable LAAOs; however, these expectations have not been fulfilled yet because none of the “true” LAAOs has successfully been expressed as a recombinant protein in prokaryotic hosts, such as Escherichia coli. In enzyme biotechnology, recombinant production of a protein is mandatory both for the production of large amounts of the catalyst and to improve its biochemical properties by protein engineering. As an alternative, flavoenzymes active on specific l-amino acids have been identified, e.g., l-aspartate oxidase, l-lysine oxidase, l-phenylalanine oxidase, etc. According to presently available information, amino acid oxidases with “narrow” or “strict” substrate specificity represent as good candidates to obtain an enzyme more suitable for biotechnological applications by enlarging their substrate specificity by means of protein engineering.     

Heterologous expression of Rhodococcus opacus L-amino acid oxidase in Streptomyces lividans

L-amino acid oxidase (L-AAO) from Rhodococcus opacus is a highly enantioselective enzyme with a broad substrate specificity that catalyses the oxidation of L-amino acids to keto acids. The lao-gene (AY053450) from R. opacus was cloned into different Escherichia coli and Streptomyces lividans expression vectors. Expression in E. coli resulted in the accumulation of insoluble protein, but S. lividans was a suitable host for the heterologous production of L-AAO. When using the thiostrepton-inducible vector pIlaao, a specific activity of 0.18 Umg(-1) was obtained in the crude extract of S. lividans 1326. For the vector pUlaao, which contains the constitutive ermEp(*) promoter, a specific activity of 0.05 Umg(-1) was reached. Both the wild type and the recombinant L-AAO were purified to homogeneity. The expression systems described here now allow the structural and biochemical analysis of the L-AAO using genetic engineering methods.