Commensal bacteria, redox stress, and colorectal cancer: mechanisms and models

The potential role for commensal bacteria in colorectal carcinogenesis is explored in this review. Most colorectal cancers (CRCs) occur sporadically and arise from the gradual accumulation of mutations in genes regulating cell growth and DNA repair. Genetic mutations followed by clonal selection result in the transformation of normal cells into malignant derivatives. Numerous toxicological effects of colonic bacteria have been reported. However, those recognized as damaging epithelial cell DNA are most easily reconciled with the currently understood genetic basis for sporadic CRC. Thus, we focus on mechanisms by which particular commensal bacteria may convert dietary procarcinogens into DNA damaging agents (e.g., ethanol and heterocyclic amines) or directly generate carcinogens (e.g., fecapentaenes). Although these and other metabolic activities have yet to be linked directly to sporadic CRC, several lines of investigation are reviewed to highlight difficulties and progress in the area. Particular focus is given to commensal bacteria that alter the epithelial redox environment, such as production of oxygen radicals by Enterococcus faecalis or production of hydrogen sulfide by sulfate-reducing bacteria (SRB). Super-oxide-producing E. faecalis has conclusively been shown to cause colonic epithelial cell DNA damage. Though SRB-derived hydrogen sulfide (H(2)S) has not been reported thus far to induce DNA damage or function as a carcinogen, recent data demonstrate that this reductant activates molecular pathways implicated in CRC. These observations combined with evidence that SRB carriage may be genetically encoded evoke a working model that incorporates multifactorial gene-environment interactions that appear to underlie the development of sporadic CRC.     

Proteome analyses of hydrogen-producing hyperthermophilic archaeon Thermococcus onnurineus NA1 in different one-carbon substrate culture conditions

Thermococcus onnurineus NA1, a sulfur-reducing hyperthermophilic archaeon, is capable of H(2)-producing growth, considered to be hydrogenogenic carboxydotrophy. Utilization of formate as a sole energy source has been well studied in T. onnurineus NA1. However, whether formate can be used as its carbon source remains unknown. To obtain a global view of the metabolic characteristics of H(2)-producing growth, a quantitative proteome analysis of T. onnurineus NA1 grown on formate, CO, and starch was performed by combining one-dimensional SDS-PAGE with nano UPLC-MS(E). A total of 587 proteins corresponding to 29.7% of the encoding genes were identified, and the major metabolic pathways (especially energy metabolism) were characterized at the protein level. Expression of glycolytic enzymes was common but more highly induced in starch-grown cells. In contrast, enzymes involved in key steps of the gluconeogenesis and pentose phosphate pathways were strongly up-regulated in formate-grown cells, suggesting that formate could be utilized as a carbon source by T. onnurineus NA1. In accordance with the genomic analysis, comprehensive proteomic analysis also revealed a number of hydrogenase clusters apparently associated with formate metabolism. On the other hand, CODH and CO-induced hydrogenases belonging to the Hyg4-II cluster, as well as sulfhydrogenase-I and Mbx, were prominently expressed during CO culture. Our data suggest that CO can be utilized as a sole energy source for H(2) production via an electron transport mechanism and that CO(2) produced from catabolism or CO oxidation by CODH and CO-induced hydrogenases may subsequently be assimilated into the organic carbon. Overall, proteomic comparison of formate- and CO-grown cells with starch-grown cells revealed that a single carbon compound, such as formate and CO, can be utilized as an efficient substrate to provide cellular carbon and/or energy by T. onnurineus NA1.     

Carbon allocation in wild-type and Glc+ Rhodobacter sphaeroides under photoheterotrophic conditions.

The photosynthetic bacterium Rhodobacter sphaeroides is capable of producing H2 via nitrogenase when grown photoheterotrophically in the absence of N2. By using 14C-labeled malate, it was found that greater than 95% of this substrate was catabolized completely to CO2 during H2 production. About 60% of this catabolism was associated with H2 biosynthesis, while almost 40% provided reductant for other cellular purposes. Thus, only a small fraction of malate provided carbon skeletons. The addition of ammonium, which inhibited nitrogenase activity, increased substrate conversion into carbon skeletons threefold. Catabolism of malate occurred primarily via the tricarboxylic acid cycle, but gluconeogenesis was also observed. The wild-type organism grew poorly on glucose, accumulated gluconate and 2-keto-3-deoxygluconate, and did not produce H2. More than 50% of metabolized glucose appeared in carbon skeletons or in storage compounds. A glucose-utilizing mutant was five times more effective in utilizing this substrate. This mutant produced H2 from glucose, using 74% of metabolized substrate for this purpose. Glucose converted to storage products or to other carbon skeletons was reduced to 8%. Fixation of CO2 competed directly with H2 production for reducing equivalents and ATP. Refixation of CO2 released from these substrates under H2-producing conditions was, at most, 10 to 12%. Addition of ammonium increased refixation of respired CO2 to 83%. Patterns of carbon flow of fixation products were associated with the particular strains and culture conditions.     

Carbon allocation in wild-type and Glc+ Rhodobacter sphaeroides under photoheterotrophic conditions

The photosynthetic bacterium Rhodobacter sphaeroides is capable of producing H2 via nitrogenase when grown photoheterotrophically in the absence of N2. By using 14C-labeled malate, it was found that greater than 95% of this substrate was catabolized completely to CO2 during H2 production. About 60% of this catabolism was associated with H2 biosynthesis, while almost 40% provided reductant for other cellular purposes. Thus, only a small fraction of malate provided carbon skeletons. The addition of ammonium, which inhibited nitrogenase activity, increased substrate conversion into carbon skeletons threefold. Catabolism of malate occurred primarily via the tricarboxylic acid cycle, but gluconeogenesis was also observed. The wild-type organism grew poorly on glucose, accumulated gluconate and 2-keto-3-deoxygluconate, and did not produce H2. More than 50% of metabolized glucose appeared in carbon skeletons or in storage compounds. A glucose-utilizing mutant was five times more effective in utilizing this substrate. This mutant produced H2 from glucose, using 74% of metabolized substrate for this purpose. Glucose converted to storage products or to other carbon skeletons was reduced to 8%. Fixation of CO2 competed directly with H2 production for reducing equivalents and ATP. Refixation of CO2 released from these substrates under H2-producing conditions was, at most, 10 to 12%. Addition of ammonium increased refixation of respired CO2 to 83%. Patterns of carbon flow of fixation products were associated with the particular strains and culture conditions.     

Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate, pH and the F(0)F(1)-ATPase

Molecular hydrogen (H(2)) can be produced via hydrogenases during mixed-acid fermentation by bacteria. Escherichia coli possesses multiple (four) hydrogenases. Hydrogenase 3 (Hyd-3) and probably 4 (Hyd-4) with formate dehydrogenase H (Fdh-H) form two different H(2)-evolving formate hydrogen lyase (FHL) pathways during glucose fermentation. For both FHL forms, the hycB gene coding small subunit of Hyd-3 is required. Formation and activity of FHL also depends on the external pH ([pH](out)) and the presence of formate. FHL is related with the F(0)F(1)-ATPase by supplying reducing equivalents and depending on proton-motive force. Two other hydrogenases, 1 (Hyd-1) and 2 (Hyd-2), are H(2)-oxidizing enzymes during glucose fermentation at neutral and low [pH](out). They operate in a reverse, H(2)-producing mode during glycerol fermentation at neutral [pH](out). Hyd-1 and Hyd-2 activity depends on F(0)F(1). Moreover, Hyd-3 can also work in a reverse mode. Therefore, the operation direction and activity of all Hyd enzymes might determine H(2) production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating of different Hyd enzymes activity is an effective way to enhance H(2) production by bacteria in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H(2) production, reduced fuels and other chemicals with higher yields than those obtained by common sugars.     

Mutagenesis and phenotypic selection as a strategy toward domestication of Chlamydomonas reinhardtii strains for improved performance in photobioreactors

Microalgae have a valuable potential for biofuels production. As a matter of fact, algae can produce different molecules with high energy content, including molecular hydrogen (H(2)) by the activity of a chloroplastic hydrogenase fueled by reducing power derived from water and light energy. The efficiency of this reaction, however, is limited and depends from an intricate relationships between oxygenic photosynthesis and mitochondrial respiration. The way toward obtaining algal strains with high productivity in photobioreactors requires engineering of their metabolism at multiple levels in a process comparable to domestication of crops that were derived from their wild ancestors through accumulation of genetic traits providing improved productivity under conditions of intensive cultivation as well as improved nutritional/industrial properties. This holds true for the production of any biofuels from algae: there is the need to isolate multiple traits to be combined and produce organisms with increased performances. Among the different limitations in H(2) productivity, we identified three with a major relevance, namely: (i) the light distribution through the mass culture; (ii) the strong sensitivity of the hydrogenase to even very low oxygen concentrations; and (iii) the presence of alternative pathways, such as the cyclic electron transport, competing for reducing equivalents with hydrogenase and H(2) production. In order to identify potentially favorable mutations, we generated a collection of random mutants in Chlamydomonas reinhardtii which were selected through phenotype analysis for: (i) a reduced photosynthetic antenna size, and thus a lower culture optical density; (ii) an altered photosystem II activity as a tool to manipulate the oxygen concentration within the culture; and (iii) State 1-State 2 transition mutants, for a reduced cyclic electron flow and maximized electrons flow toward the hydrogenase. Such a broad approach has been possible thanks to the high throughput application of absorption/fluorescence optical spectroscopy methods. Strong and weak points of this approach are discussed.     

Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate:ferredoxin oxidoreductase

The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry (F(v)/F(m)). During autofermentation, the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H(2) production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H(2) production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H(2) revealed two temporally resolved phases of H(2) production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD(+) ratio revealed that the reductant generated by PFOR contributing to the first phase of H(2) production was not in equilibrium with bulk NADH/NAD(+) and that the second phase corresponded to the equilibrium NADH-mediated process.     

Inorganic Phosphate (Pi) Enhancement of Dark Respiration in the Pi-Limited Green Alga Selenastrum minutum (Interactions between H+/Pi Cotransport,the Plasmalemma H+-ATPase,and Dark Respiratory Carbon Flow)

Inorganic phosphate (Pi) enrichment of the Pi-limited green alga Selenastrum minutum in the dark caused a 2.5-fold increase in the rate of O2 consumption. Alkalization of the media during Pi assimilation was consistent with a H+/Pi cotransport mechanism with a stoichiometry of at least 2 H+ cotransported per Pi. Dark O2 consumption remained enhanced beyond the period of Pi assimilation and did not recover until the medium was reacidified. This result, coupled with an immediate decrease in adenylate energy charge following Pi enrichment, suggested that respiration is regulated by the ATP requirements of a plasmalemma H+-ATPase that is activated to maintain intracellular pH and provide proton motive force to power Pi uptake. Concentrations of tricarboxylic acid cycle intermediates decreased following Pi enrichment and respiratory CO2 efflux increased, indicating that the tricarboxylic acid cycle was activated to supply reductant to the mitochondrial electron transport chain. These results are consistent with direct inhibition of electron transport by ADP limitation. Enhanced rates of starch breakdown and increases in glycolytic metabolites indicated that respiratory carbon flow was activated to supply reductant to the electron transport chain and to rapidly assimilate Pi into metabolic intermediates. The mechanism that initiates glycolytic carbon flow could not be clearly identified by product:substrate ratios due to the complex nature of Pi assimilation. High levels of triose-P and low levels of phosphoenolpyruvate were the primary regulators of pyruvate kinase and phosphofructokinase, respectively.     

Isolation of key methanogens for global methane emission from rice paddy fields: a novel isolate affiliated with the clone cluster rice cluster I

Despite the fact that rice paddy fields (RPFs) are contributing 10 to 25% of global methane emissions, the organisms responsible for methane production in RPFs have remained uncultivated and thus uncharacterized. Here we report the isolation of a methanogen (strain SANAE) belonging to an abundant and ubiquitous group of methanogens called rice cluster I (RC-I) previously identified as an ecologically important microbial component via culture-independent analyses. To enrich the RC-I methanogens from rice paddy samples, we attempted to mimic the in situ conditions of RC-I on the basis of the idea that methanogens in such ecosystems should thrive by receiving low concentrations of substrate (H(2)) continuously provided by heterotrophic H(2)-producing bacteria. For this purpose, we developed a coculture method using an indirect substrate (propionate) in defined medium and a propionate-oxidizing, H(2)-producing syntroph, Syntrophobacter fumaroxidans, as the H(2) supplier. By doing so, we significantly enriched the RC-I methanogens and eventually obtained a methanogen within the RC-I group in pure culture. This is the first report on the isolation of a methanogen within RC-I.     

三株新型发酵产氢菌生物学特征鉴定与比较

比较了三株发酵产氢细菌的形态和生理生化特征,分析了它们的16S rDNA序列系统发育关系,并测定了它们的产氢量和液相发酵产物。认为它们同属于一类新的发酵产氢菌属,其液相产物以乙醇和乙酸为主,在一定的培养条件下可获得较高产氢量。此外,对该类细菌的某些共同的生物学特征也作了描述。     

Effects of extracellular pH on the metabolic pathways in sulfur-deprived,H2-producing Chlamydomonas reinhardtii cultures

Sustained photoproduction of H(2) by the green alga, Chlamydomonas reinhardtii, can be obtained by incubating cells in sulfur-deprived medium [Ghirardi et al. (2000b) Trends Biotechnol. 18: 506; Melis et al. (2000) Plant Physiol. 122: 127]. The current work focuses on (a) the effects of different initial extracellular pHs on the inactivation of photosystem II (PSII) and O(2)-sensitive H(2)-production activity in sulfur-deprived algal cells and (b) the relationships among H(2)-production, photosynthetic, aerobic and anaerobic metabolisms under different pH regimens. The maximum rate and yield of H(2) production occur when the pH at the start of the sulfur deprivation period is 7.7 and decrease when the initial pH is lowered to 6.5 or increased to 8.2. The pH profile of hydrogen photoproduction correlates with that of the residual PSII activity (optimum pH 7.3-7.9), but not with the pH profiles of photosynthetic electron transport through photosystem I or of starch and protein degradation. In vitro hydrogenase activity over this pH range is much higher than the actual in situ rates of H(2) production, indicating that hydrogenase activity per se is not limiting. Starch and protein catabolisms generate formate, acetate and ethanol; contribute some reductant for H(2) photoproduction, as indicated by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone inhibition results; and are the primary sources of reductant for respiratory processes that remove photosynthetically generated O(2). Carbon balances demonstrate that alternative metabolic pathways predominate at different pHs, and these depend on whether residual photosynthetic activity is present or not.     

Microbial community structure of ethanol type fermentation in bio-hydrogen production

Three continuous stirred-tank reactors (CSTRs) were used for H(2) production from molasses wastewater at influent pH of 6.0-6.5 (reactor A), 5.5-6.0 (reactor B), or 4.0-4.5 (reactor C). After operation for 28 days, the microbial community formed ethanol type (C), propionate type (A) and ethanol-butyrate-mixed type (B) fermentation. The H(2) production rate was the highest for ethanol type fermentation, 0.40 l (g VSS)(-1) day(-1) or 0.45 l H(2) (g COD removed)(-1). Microbial community dynamics and diversity were analysed using double-gradient denaturing gradient gel electrophoresis (DG-DGGE). Denaturing gradient gel electrophoresis profiles indicated that the community structures changed quickly in the first 14 days. Phylogenetic analysis indicated that the dominant bacterial groups were low G+C Gram-positive bacteria, Bacteroides, gamma-Proteobacteria and Actinobacteria; alpha-Proteobacteria, beta-Proteobacteria, delta-Proteobacteria and Spirochaetes were also presented as minor groups in the three reactors. H(2)-producing bacteria were affiliated with Ethanoligenens, Acetanaerobacterium, Clostridium, Megasphaera, Citrobacter and Bacteroides. An ethanol-based H(2)-producing bacterium, Ethanoligenens harbinense CGMCC1152, was isolated from reactor C and visualized using fluorescence in situ hybridization (FISH) to be 19% of the eubacteria in reactor C. In addition, isoenzyme activity staining for alcohol dehydrogenase (ADH) supported that the majority of ethanol-producing bacteria were affiliated with Ethanoligenens in the microbial community.     

Fermentative H2 production in an upflow anaerobic sludge blanket reactor at various pH values

Fermentative H(2) production in an upflow anaerobic sludge blanket reactor (UASB) at various pH values was investigated in this study. Experimental results show that the H(2) partial pressure in biogas, H(2) production rate and H(2) yield were all pH-dependent, in the range of 0.25-0.52 atm, 42-145 ml-H(2) l(-1) h(-1) and 0.47 to 1.61 mol-H(2)mol-glucose(-1), respectively. The maximum pH for the H(2) partial pressure was observed at pH 7.50. However, the optimum H(2) production rate and H(2) yield were observed at pH 6.50-7.50. In this UASB reactor, acetate, propionate, butyrate, i-butyrate, valerate, caporate and ethanol were present in the effluent as main aqueous products, and the dominant fermentation was butyrate-type at various pHs. The metabolic pathways and thermodynamics of H(2) production were also analyzed. Both H(2) production performance and fermentation pathways in this H(2)-producing UASB reactor were significantly affected by the pH value.     

Metabolism of oxygen radicals in peroxisomes and cellular implications.

Peroxisomes are subcellular respiratory organelles which contain catalase and H2O2-producing flavin oxidases as basic enzymatic constituents. These organelles have an essentially oxidative type of metabolism and have the potential to carry out different important metabolic pathways. In recent years the presence of different types of superoxide dismutase (SOD) have been demonstrated in peroxisomes from several plant species, and more recently the occurrence of SOD has been extended to peroxisomes from human and transformed yeast cells. A copper,zinc-containing SOD from plant peroxisomes has been purified and partially characterized. The production of hydroxyl and superoxide radicals has been studied in peroxisomes. There are two sites of O2- production in peroxisomes: (1) in the matrix, the generating system being xanthine oxidase; and (2) in peroxisomal membranes, dependent on reduced nicotinamide adenine dinucleotide (NADH), and the electron transport components of the peroxisomal membrane are possibly responsible. The generation of oxygen radicals in peroxisomes could have important effects on cellular metabolism. Diverse cellular implications of oxyradical metabolism in peroxisomes are discussed in relation to phenomena such as cell injury, peroxisomal genetic diseases, peroxisome proliferation and oxidative stress, metal and salt stress, catabolism of nucleic acids, senescence, and plant pathogenic processes.     

Oxygen-tolerant hydrogenases in hydrogen-based technologies

To develop a viable H2 technology, production of H2 has to be significantly enlarged by using renewable resources. One option of generating H2 is the photosynthetic conversion of sunlight and water directly to H2 and O2. Photosystems and hydrogenases are currently being exploited for the design of efficient H2-producing systems that require highly active and O2-tolerant biocatalysts. This communication focuses on two challenging features: hydrogenases that produce H2 in the presence of O2, and direct electron transfer between photosystem I (PS I) and hydrogenase. The latter is accomplished by connecting both modules through a protein fusion or a synthetic molecular wire. These are first steps toward a photosynthetic microbial cell or a semi-synthetic system that may be employed in future H2-based technologies.     

The E3 ubiquitin ligase adaptor Ndfip1 regulates Th17 differentiation by limiting the production of proinflammatory cytokines

Ndfip1 is an adaptor for the E3 ubiquitin ligase Itch. Both Ndfip1- and Itch-deficient T cells are biased toward Th2 cytokine production. In this study, we demonstrate that lungs from Ndfip1(-/-) mice showed increased numbers of neutrophils and Th17 cells. This was not because Ndfip1(-/-) T cells are biased toward Th17 differentiation. In fact, fewer Ndfip1(-/-) T cells differentiated into Th17 cells in vitro due to high IL-4 production. Rather, Th17 differentiation was increased in Ndfip1(-/-) mice due to increased numbers of IL-6-producing eosinophils. IL-6 levels in mice that lacked both Ndfip1 and IL-4 were similar to wild-type controls, and these mice had fewer Th17 cells in their lungs. These results indicate that Th2 inflammation, such as that observed in Ndfip1(-/-) mice, can increase Th17 differentiation by recruiting IL-6-producing eosinophils into secondary lymphoid organs and tissues. This may explain why Th17 cells develop within an ongoing Th2 inflammatory response.     

Influence of unsaturated fatty acids in chloroplasts. Shift of the pH optimum of electron flow and relations to deltapH, thylakoid internal pH and proton uptake.

Linolenic acid (C18:3) is the main endogenous unsaturated fatty acid of thylakoid membrane lipids, and seems in its free form to exert significant effects on the structure and function of photosynthetic membranes. In this investigation the effect of linolenic acid was studied at various pH values on the electron flow rate in isolated spinach chloroplasts and related to deltapH, the proton pump and the pH of the inner thylakoid space (pHi). The deltapH and pHi were estimated from the extent of the fluorescence quenching of 9-aminoacridine. Linolenic acid caused a shift (approximately one unit) of the pH optimum for electron flow toward acidity in the following systems: (a) photosystems II + I (from H2O to NADP+ or to 2,6-dichlorophenolindophenol) coupled or non-coupled; (b) photosystem II (from H2O to 2,6-dichlorophenolindophenol in the presence of dibromothymoquinone). In photosystem I conditions (phenazine methosulphate), the deltapH of the control increased as a function of external pHo with a maximum around pH 8.8. When linolenic acid was added, the deltapH dropped, but its optimum was shifted toward more acidic pHo. The same phenomena were also observed in photosytems II + I (from H2O to ferricyanide) and in photosystem II conditions (from H2O to ferricyanide in the presence of dibromothymoquinone). However, the deltapH was smaller and the sensitivity of the proton gradient toward linolenic acid was eventually higher than for photosystem I electron flow activity. The proton pump which might be considered as a measure of the internal buffering capacity of thylakoids was optimum at pHo, 6.7 in the controls. An addition of linolenic acid diminished the proton pump and shifted its optimum toward higher pHo. As a consequence, pHi increased when pHo was raised. At the optimal pHo 8.6 to 9, pHi were 5 to 5.5. Additions of increasing concentrations of linolenic acid displaced the curves toward higher pHi. A decrease of pHo was therefore required to maintain the pHi in the range of 5-5.5 for maximum electron flow. In conclusion, the electron flow activity seems to be delicately controlled by the proton pump (buffer capacity), deltapH, pHi and pHo. Fatty acids damage the membrane integrity in such a way that the subtile equilibrium between the factors is disturbed.     

A novel agar medium to detect hydrogen peroxide-producing bacteria based on the prussian blue-forming reaction

The classic method for H(2)O(2) detection involving Prussian blue formation was adapted to formulate a novel agar medium that makes possible in situ detection of H(2)O(2) produced by bacteria. Using this medium, colonies of H(2)O(2)-producing species including Streptococcus pyogenes were easily identified by the appearance of blue halos. The utility of the medium was further illustrated by its successful application to the isolation of H(2)O(2)-producing mutants from a non-H(2)O(2)-producing stain of S. pyogenes.     

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.     

Microbial ecosystem and methanogenesis in ruminants

Ruminant production is under increased public scrutiny in terms of the importance of cattle and other ruminants as major producers of the greenhouse gas methane. Methanogenesis is performed by methanogenic archaea, a specialised group of microbes present in several anaerobic environments including the rumen. In the rumen, methanogens utilise predominantly H2 and CO2 as substrates to produce methane, filling an important functional niche in the ecosystem. However, in addition to methanogens, other microbes also have an influence on methane production either because they are involved in hydrogen (H2) metabolism or because they affect the numbers of methanogens or other members of the microbiota. This study explores the relationship between some of these microbes and methanogenesis and highlights some functional groups that could play a role in decreasing methane emissions. Dihydrogen ('H2' from this point on) is the key element that drives methane production in the rumen. Among H2 producers, protozoa have a prominent position, which is strengthened by their close physical association with methanogens, which favours H2 transfer from one to the other. A strong positive interaction was found between protozoal numbers and methane emissions, and because this group is possibly not essential for rumen function, protozoa might be a target for methane mitigation. An important function that is associated with production of H2 is the degradation of fibrous plant material. However, not all members of the rumen fibrolytic community produce H2. Increasing the proportion of non-H2 producing fibrolytic microorganisms might decrease methane production without affecting forage degradability. Alternative pathways that use electron acceptors other than CO2 to oxidise H2 also exist in the rumen. Bacteria with this type of metabolism normally occupy a distinct ecological niche and are not dominant members of the microbiota; however, their numbers can increase if the right potential electron acceptor is present in the diet. Nitrate is an alternative electron sinks that can promote the growth of particular bacteria able to compete with methanogens. Because of the toxicity of the intermediate product, nitrite, the use of nitrate has not been fully explored, but in adapted animals, nitrite does not accumulate and nitrate supplementation may be an alternative under some dietary conditions that deserves to be further studied. In conclusion, methanogens in the rumen co-exist with other microbes, which have contrasting activities. A better understanding of these populations and the pathways that compete with methanogenesis may provide novel targets for emissions abatement in ruminant production.