Synthesis of alpha-ketoglutarate by reductive carboxylation of succinate in Veillonella, Selenomonas, and Bacteriodes species.

Evidence for reductive carboxylation of succinate to synthesize alpha-ketoglutarate was sought in anaerobic heterotrophs from the rumen and from other anaerobic habitats. Cultures were grown in media containing unlabeled energy substrates plus [14C]succinate, and synthesis of cellular glutamate with a much higher specific activity than that of cellular asparate was taken as evidence for alpha-ketoglutarate synthase activity. Our results indicate alpha-ketoglutarate synthase functions in Selenomonas ruminantium, Veillonella alcalescens, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides uniformis, Bacteroides distasonis, and Bacteroides multiacidus. Evidence for this carboxylation was not found in strains representative of 10 other species.     

A Simple Bile-Disk Method for the Identification of Bacteroides fragilis

A simple method for the separation and identification of Bacteroides fragilis from other Bacteroides species through the use of oxgall-impregnated filter paper disks is described. As in the antibiotic sensitivity test, filter paper disks containing defined amounts of bile salt were placed on a lawn of bacterial cells on Gifu anaerobic medium agar plates. Bile-resistant bacteria were identified as strains of Bacteroides fragilis after incubation in anaerobic jars containing hydrogen and carbon dioxide generators at 35 C for 24 hr. The optimum concentration of bile salt, yielding results very similar to those of the conventional tube method, was determined to be 25 mg per disk, empirically. Since this method is easy to perform, time saving, economical and gives clear results, it may be readily used as a daily routine test in the clinical microbiology laboratory.     

Effects of Energy Substrates on Nitrate Reduction and Nitrate Reductase Activity in a Ruminal Bacterium, Selenomonas ruminantium

The factors affecting the rate of nitrate reduction and the nitrate reductase content in Selenomonas ruminantium were examined. The rate of nitrate reduction per cell mass was higher when S. ruminantium was grown on lactate than when grown on glucose, and the rate was further enhanced when grown on succinate. The nitrate reduction rate was parallel to the nitrate reductase content in cells, suggesting that the amount of nitrate reductase limits the rate of nitrate reduction. The amount of nitrate reductase was inversely related to growth rate. The growth rate was related to the level of intracellular ATP, which was inversely related to the levels of ADP and AMP. The ratio of NADH to NAD⁺was related to the rate of nitrate reduction and to the amount of nitrate reductase. From these results, it is conceivable that the synthesis of nitrate reductase is regulated in response to the sufficiency of energy and electron supply. Intracellular concentrations of adenine nucleotides and pyridine nucleotides may be the regulating factors. The amount of nitrate reductase was increased by the presence of nitrate, suggesting that the synthesis of nitrate reductase is enhanced by nitrate. In addition, nitrate reduction altered the fermentation pattern as a result of electron consumption.     

Vitamin K composition of anaerobic gut bacteria

The menaquinone (vitamin K₂) composition of a wide range of obligately anaerobic bacteria was investigated using chromatographic and physicochemical techniques. Members of the genera Bifidobacterium, Butyrivibrio, Clostridium, Fusobacterium, Lachnospira, Megasphaera, Propionispira, Ruminococcus, Selenomonas, Succinovibrio and Succinomonas were found to lack menaquinones, In contrast, all species of the genera Actinomyces, Arachnia, Bacteroides, Propionibacterium, Veillonella and Wolinella examined possessed menaquinones. Considerable variation in length and degree of saturation of the C-3 isoprenyl side-chains of the menaquinones isolated from these taxa were evident. In addition to menaquinone, strains of Wolinella and Eubacterium lentum contained methylated menaquinones. The possible contribution of bacterial menaquinones to the total pool of vitamin K in the human gut is discussed.     

Enrichment and Isolation of Rumen Bacteria That Reduce trans- Aconitic Acid to Tricarballylic Acid

Bacteria from the bovine rumen capable of reducing trans-aconitate to tricarballylate were enriched in an anaerobic chemostat containing rumen fluid medium and aconitate. After 9 days at a dilution rate of 0.07 h⁻¹, the medium was diluted and plated in an anaerobic glove box. Three types of isolates were obtained from the plates (a crescent-shaped organism, a pleomorphic rod, and a spiral-shaped organism), and all three produced tricarballylate in batch cultures that contained glucose and trans-aconitate. In glucose-limited chemostats (0.10 h⁻¹), trans-aconitate reduction was associated with a decrease in the amount of reduced products formed from glucose. The crescent-shaped organism produced less propionate, the pleomorphic rod produced less ethanol, and the spiral made less succinate and possibly H₂. Aconitate reduction by the pleomorphic rod and the spiral organism was associated with a significant increase in cellular dry matter. Experiments with stock cultures of predominant rumen bacteria indicated that Selenomonas ruminantium, a species taxonomically similar to the crescent-shaped isolate, was an active reducer of trans-aconitate. Strains of Bacteroides ruminicola, Butyrivibrio fibrisolvens, and Megasphaera elsdenii produced little if any tricarballylate. Wolinella succinogenes produced some tricarballylate. Based on its stability constant for magnesium (K_eq = 115), tricarballylate could be a factor in the hypomagnesemia that leads to grass tetany.     

Influence of NO3 - and NH4 + nutrition on fermentation,nitrate reductase activity and adenylate energy charge of roots of Carex pseudocyperus L. and Carex sylvatica Huds. exposed to anaerobic nutrient solutions

The adenylate energy charge, production of ethanol and lactate, and nitrate reductase activity were determined in order to study the influence of different nitrogen sources on the metabolic responses of roots of Carex pseudocyperus L. and Carex sylvatica HUDS. exposed to anaerobic nutrient solutions. Determination of adenylates was carried out by means of a modified HPLC technique. Total quantity of adenylates was higher in Carex pseudocyperus than in Carex sylvatica under all conditions. In contrast, the adenylate energy charge was only slightly different between the species and decreased more or less in relation to the applied nitrogen source under oxygen deficiency. The adenylate energy charge in roots of plants under nitrate nutrition showed a smaller decrease under anaerobic environmental conditions than plants grown with ammonium or nitrate/ammonium. Roots of nitrate-fed plants showed a lower ethanol and lactate production than ammonium/nitrate- and ammonium-fed plants. Ethanol production was higher in C. pseudocyperus, formation of lactate was lower compared to that in Carex sylvatica. The activity of enzymes involved in fermentation processes (ADH, LDH and PDC) was enhanced significantly after 24 hours of exposure to anaerobic nutrient solutions in roots of both species. The induction of these enzymes was only slightly influenced by different nitrogen supply. In vivo nitrate reductase activity increased almost 3-fold compared to the aerobic treatment in both species and overcompensated loss of NADH reoxidation capacity caused by decrease of ethanol and lactate development. Induction of in vitro nitrate reductase activity was enhanced 313% in C. pseudocyperus and 349% in C. sylvatica under anaerobic environmental conditions and nitrate supply. These results indicate that nitrate may serve as an alternative electron acceptor in anaerobic plant root metabolism and that the nitrate-supported energy charge may be due to an accelerated glycolytic flux resulting from a more effective NADH reoxidation capacity by nitrate reduction plus fermentation than by fermentation alone.Abbreviations ADH alcohol dehydrogenase - AEC adenylate energy charge - DMSO dimethyl sulfoxide - EDTA ethylen diamine tetraacetic acid - HPLC high performance liquid chromatography - LDH lactate dehydrogenase - NRA nitrate reductase activity - PCA perchloric acid - PDC pyruvate decarboxylase - PVP polyvinylpyrrolidone - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid, Tris-tris(hydroxymethyl)aminomethane     

Phylogenetic characterization of Centipeda periodontii, Selenomonas sputigena and Selenomonas species by 16S rRNA gene sequence analysis.

The nearly complete 16S rRNA gene sequences for oral Gram-negative anaerobic motile bacteria, Centipeda periodontii, Selenomonas sputigena and Selenomonas species (formerly S. sputigena type strain), were determined in order to unveil their relationship to other oral motile bacteria. To determine the phylogenetic characterization of these bacteria, their 16S rRNA gene sequences were obtained and compared with those from the ribosomal sequence databases previously reported. The 16S rRNA gene sequences of these bacteria were similar to those of Selenomonas ruminantium and Schwartzia succinivorans isolated from rumens, and to Pectinatus cerevisiiphilus isolated from spoiled beer. Among oral bacteria, the nucleotide sequence analysis of these bacteria revealed high nucleotide similarity to Veillonella species, whereas low similarity to oral motile bacteria such as Campylobacter species. Phylogenetic analysis clearly confirmed that C. periodontii and two Selenomonas species were classified as relatives of a group besides Selenomonas, Schwartzia, and Pectinatus species, and not as close relatives to oral motile bacteria, such as Campylobacter species. These results suggest that such oral Gram-negative anaerobic motile bacteria are close relatives of oral bacteria.     

Transformations of inorganic nitrogen by Azospirillum spp.

Azospirillum spp. participate in all steps of the nitrogen cycle except nitrification. They can fix molecular nitrogen and perform assimilatory nitrate reduction and nitrate respiration. Culture conditions have been defined under which nitrate is used both as terminal respiratory electron acceptor and as nitrogen source for growth. Nitrate and, possibly to a very limited extent, nitrite, but not sulfate, iron or fumarate support anaerobic respiration. Under anaerobic conditions, nitrate can also supply energy for nitrogen fixation but without supporting growth. Nitrate-dependent nitrogenase activity lasts only for 3–4 h until the enzymes of assimilatory nitrate reduction are synthesized. Nitrite accumulates during this period and inhibits nitrogenase activity at concentrations of about 1 mM.     

Ability of Selenomonas ruminantium,Veillonella parvula,and Wolinella succinogenes to Reduce Nitrate and Nitrite with Special Reference to the Suppression of Ruminal Methanogenesis

When mixed ruminal microbes were grown in a medium containing ground hay and concentrate, cell numbers of nitrate-reducing bacteria, Veillonella parvula and Wolinella succinogenes, drastically decreased as estimated by competitive PCR. However, decrease in their numbers was prevented by the addition of nitrate, suggesting that energy acquisition by electron transport phosphorylation (ETP) coupled with nitrate and/or nitrite reduction is important for the survival of these bacteria in the rumen. On the other hand, the number of Selenomonas ruminantium increased, and addition of nitrate did not affect the number, suggesting that the numbers of nitrate-reducing strains of S. ruminantium are low. A nitrate-reducing strain of S. ruminantium subsp. lactilytica was found to have the ETP system coupled with both nitrate and nitrite reduction. V. parvula, W. succinogenes, S. ruminantium, and Streptococcus bovis were more tolerant to nitrite toxicity than other representative ruminal bacteria. Methanogens were particularly sensitive to nitrite. W. succinogenes reduced nitrate and nitrite at higher rates than V. parvula and the subsp.lactilytica , but growth of W. succinogenes on nitrate and H₂ was slower than the growth of V. parvula and the subsp. lactilytica. Methane production by unidentified methanogen mixture was markedly reduced by the coexistence of W. succinogenes, V. parvula or the subsp. lactilytica in the presence of nitrate and H₂. W. succinogenes was shown to be most effective to augment nitrate and nitrite reduction, and to reduce methanogenesis.     

Microbiota of Initial Periodontitis in Adults

This paper reviews our recent studies of the microbiota and host response of initial periodontitis. Understanding the initial stages of periodontitis will allow appropriate early treatment and prevention strategies. Out studies aimed to determine the major bacterial species that differentiated initial periodontitis from health, and evaluate whether subjects with initial periodontitis differed in serum IgG reactivity to putative initial periodontitis pathogens compared with healthy subjects. Initial periodontitis was characterized clinically using longitudinal periodontial attachment level measurements. Progressing periodontal loss was detected at interproximal (initial periodontitis), and buccal (progressing recession) locations from the study population of minimally periodontally diseased subjects. Initial periodontitis was characterized microbiologically by elevated proportions of Bacteroides forsythus, Selenomonas noxia and Campylobacter rectus when compared with non-periodontitis sites. The immunological checkerboard assay did not detect differences in serum IgG reactivity among healthy, gingivitis or initial periodontitis subjects, or changes in reactivity co-incident with detection of initial peridontitis. Clinical, microbiological and immunological characterization of initial periodontitis was consistent with infection-associated Gram-negative anaerobic periodontal species. Progressing recession sites were colonized byActinomyces and Streptococcus species, as were healthy sites. Progressing recession sites demonstrated periodontal loss that appeared unrelated to infection and appeared to be consistent with a traumatic tooth brushing etiology. Different types of lesions will require different approaches to therapy and prevention.     

Effects of a Chlorhexidine Gluconate-Containing Mouthwash on the Vitality and Antimicrobial Susceptibility of In Vitro Oral Bacterial Ecosystems

Oral bacterial microcosms, established using saliva inocula from three individuals, were maintained under a feast-famine regime within constant-depth film fermenters. Steady-state communities were exposed four times daily, postfeeding, to a chlorhexidine (CHX) gluconate-containing mouthwash (CHXM) diluted to 0.06% (wt/vol) antimicrobial content. The microcosms were characterized by heterotrophic plate counts and PCR-denaturing gradient gel electrophoresis (DGGE). CHXM caused significant decreases in both total anaerobe and total aerobe/facultative anaerobe counts (P < 0.05), together with lesser decreases in gram-negative anaerobes. The degree of streptococcal and actinomycete inhibition varied considerably among individuals. DGGE showed that CHXM exposure caused considerable decreases in microbial diversity, including marked reductions in Prevotella sp. and Selenomonas infelix. Pure-culture studies of 10 oral bacteria (eight genera) showed that Actinomyces naeslundii, Veillonella dispar, Prevotella nigrescens, and the streptococci were highly susceptible to CHX, while Lactobacillus rhamnosus, Fusobacterium nucleatum, and Neisseria subflava were the least susceptible. Determination of the MICs of triclosan, CHX, erythromycin, penicillin V, vancomycin, and metronidazole for microcosm isolates, before and after 5 days of CHXM exposure, showed that CHXM exposure altered the distribution of isolates toward those that were less susceptible to CHX (P < 0.05). Changes in susceptibility distributions for the other test agents were not statistically significant. In conclusion, population changes in plaque microcosms following repeated exposure to CHXM represented an inhibition of the most susceptible flora with a clonal expansion of less susceptible species.     

5-Nitroimidazole-derived Schiff bases and their copper(II) complexes exhibit potent antimicrobial activity against pathogenic anaerobic bacteria

In the present work a family of novel secnidazole-derived Schiff base compounds and their copper(II) complexes were synthesized. The antimicrobial activities of the compounds were evaluated against clinically important anaerobic bacterial strains. The compounds exhibited in vitro antibacterial activity against Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides ovatus, Parabacteroides distasonis and Fusubacterium nucleatum pathogenic anaerobic bacteria. Upon coordination to copper(II) the antibacterial activity significantly increased in several cases. Some derivatives were even more active than the antimicrobial drugs secnidazole and metronidazole. Therefore, the compounds under study are suitable for in vivo evaluation and the microorganisms should be classified as susceptible to them. Electrochemical studies on the reduction of the nitro group revealed that the compounds show comparable reduction potentials, which are in the same range of the bio-reducible drugs secnidazole and benznidazole. The nitro group reduction potential is more favorable for the copper(II) complexes than for the starting ligands. Hence, the antimicrobial activities of the compounds under study might in part be related to intracellular bio-reduction activation. Considering the increasing resistance rates of anaerobic bacteria against a wide range of antimicrobial drugs, the present work constitutes an important contribution to the development of new antibacterial drug candidates.     

Patterns and drivers of anaerobic nitrogen transformations in sediments of thermokarst lakes

Significant attention has been given to the way in which the soil nitrogen (N) cycle responds to permafrost thaw in recent years, yet little is known about anaerobic N transformations in thermokarst lakes, which account for more than one-third of thermokarst landforms across permafrost regions. Based on the N isotope dilution and tracing technique, combined with qPCR and high-throughput sequencing, we presented large-scale measurements of anaerobic N transformations of sediments across 30 thermokarst lakes over the Tibetan alpine permafrost region. Our results showed that gross N mineralization, ammonium immobilization, and dissimilatory nitrate reduction rates in thermokarst lakes were higher in the eastern part of our study area than in the west. Denitrification dominated in the dissimilatory nitrate reduction processes, being two and one orders of magnitude higher than anaerobic ammonium oxidation (anammox) and dissimilatory nitrate reduction to ammonium (DNRA), respectively. The abundances of the dissimilatory nitrate reduction genes (nirK, nirS, hzsB, and nrfA) exhibited patterns consistent with sediment N transformation rates, while α diversity did not. The inter-lake variability in gross N mineralization and ammonium immobilization was dominantly driven by microbial biomass, while the variability in anammox and DNRA was driven by substrate supply and organic carbon content, respectively. Denitrification was jointly affected by nirS abundance and organic carbon content. Overall, the patterns and drivers of anaerobic N transformation rates detected in this study provide a new perspective on potential N release, retention, and removal upon the formation and development of thermokarst lakes.     

Changes in rumen bacterial community composition in steers in response to dietary nitrate

The effect of dietary nitrate supplementation on rumen bacterial community composition was examined in beef steers fed either a nitrate-N diet or urea-N diet. An automated method of ribosomal intergenic spacer analysis was applied to solid and liquid fractions of ruminal contents to allow comparison of bacterial communities. Supplemental N source affected relative population size of four amplicon lengths (ALs) in the liquid fraction and three ALs in the solid fraction. Five ALs were more prevalent after adaptation to nitrate. Correspondence analysis indicated that feeding the steers the nitrate-N diet versus urea-N diet changed the bacterial community composition in the liquid but not in the solid fraction. This led to an investigation of the relative sizes of potential nitrate-reducing populations. Mannheimia succiniciproducens, Veillonella parvula, and Campylobacter fetus were obtained from nitrate enrichment culture and quantified by real-time PCR based on 16S rRNA sequence. Nitrate supplementation increased the percentage of C. fetus in the liquid and solid phases, and in solid phase, the percentage of M. succiniciproducens increased. No change in species prevalence was observed for V. parvula. However, even after adaptation to dietary nitrate, the relative population sizes for all three putative nitrate-reducing species were very low (<0.06 % of 16S rRNA gene copy number). The liquid-associated bacterial community composition changed due to nitrate supplementation, and at least part of this change reflects an increase in the species prevalence of C. fetus, a species which is not typically regarded as a ruminal inhabitant.     

Oxygen and nitrate reduction kinetics of a nonflocculating strain of Zoogloea ramigera

The oxygen and nitrate reduction kinetics of a nonflocculating strain of Zoogloea ramigera were determined. Axenic, nitrate-reducing bacterial suspensions were acclimated to various oxygen levels in a chemostat while measuring nitrate reduction in the presence of high ammonium nitrogen concentrations. Significant nitrate reduction was observed at oxygen concentrations up to 8 mg L^–1. Oxygen consumption was inhibited by oxygen concentrations in excess of 2 mg L^–1.     

Anaerobic bacteria cultured from the tongue dorsum of subjects with oral malodor

The bacteria on the dorsum of the tongue are the most frequent cause of oral malodor; however, the bacterial flora of the tongue has not been well defined. Although recent studies have used DNA probes to detect the presence of certain periodontal pathogens, cultural studies have been limited because of the complexity of the flora of the tongue dorsum. The purpose of this study was to grow and to identify maximum numbers of capnophylic Gram-negative bacilli and anaerobic micro-organisms by culturing tongue samples on to several selective and non-selective media. The most frequently isolated species included Peptostreptococcus anaerobius, Collinsella aerofaciens, Eubacterium group, Actinomyces spp., Eikenella corrodens, Veillonella spp., Fusobacterium nucleatum, pigmented Prevotella spp. and Selenomonas spp. Reported for the first time are Actinomyces turicensis, Collinsella aerofaciens, Eubacterium saburreum, E. timidum, Prevotella tannerae, Campylobacter concisus, Campylobacter mucosalis, Leptotrichia buccalis, Selenomonas flueggei, and Centipeda periodontii. Species not previously reported in studies that used only molecular techniques were identified in the present study.     

Nitrate Reduction and the Growth of Veillonella alcalescens

Veillonella alcalescens, a strict anaerobe, was found to possess a nitrate reductase system which has characteristics of both assimilatory and respiratory nitrate reduction. The nitrate reductase has been identified tentatively as a particulate enzyme which utilizes a variety of electron donors for the reduction of nitrate. By use of ¹⁵N-labeled nitrate, it was shown that under appropriate conditions nitrate nitrogen is incorporated into cell material. V. alcalescens grown on pyruvate and nitrate has a greater growth rate than cells grown on pyruvate alone. Growth can occur in a medium with hydrogen and nitrate as the sole energy source. Ammonium chloride decreases the rate of nitrate reduction but does not completely inhibit reduction or incorporation. The results suggest that nitrate assimilation and respiration are not as distinct as in some other organisms.     

微生物介导硝酸盐还原耦合亚铁氧化过程的动力学及其影响因素

【目的】探究不同菌浓度和亚铁浓度条件下,Acidovorax sp. strain BoFeN1介导的厌氧亚铁氧化耦合硝酸盐还原过程的动力学和次生矿物。【方法】构建包含菌BoFeN1、硝酸盐、亚铁的厌氧培养体系,测试硝酸根、亚硝酸根、乙酸根、亚铁等浓度,并收集次生矿物,采用XRD、SEM进行矿物种类和形貌表征。【结果】在微生物介导硝酸盐还原耦合亚铁氧化的体系中,高菌浓度促进硝酸盐还原,对亚铁氧化也有一定促进作用;高浓度亚铁在低菌浓度下氧化反应速率和程度降低,但是在高菌浓度下无明显影响;亚铁浓度越高次生矿物结晶度越高,但对硝酸盐还原具有一定抑制作用。在微生物介导亚硝酸盐还原耦合亚铁氧化的体系中,高的菌浓度和亚铁浓度都会促进亚硝酸盐还原,但亚铁氧化的次生矿物会对亚硝酸盐的微生物还原产生较强的抑制作用,次生矿物的种类和结晶度主要受亚铁浓度影响。【结论】硝酸盐还原主要是生物反硝化作用,亚硝酸盐还原包含生物反硝化和化学反硝化两部分,在硝酸盐体系中亚铁氧化与次生矿物生成是受生物和化学反硝化作用的共同影响,但亚硝酸盐体系中亚铁氧化与次生矿物生成主要是受化学反硝化作用影响。该研究可为深入理解厌氧微生物介导铁氮耦合反应机制提供基础数据和理论支撑。     

Tetrapyrrole utilization by protoheme-synthesizing anaerobes

Protoheme-synthesizing anaerobic bacteria were grown in the presence of metal-free deuteroporphyrin IX or selected metal chelates of deuteroporphyrin IX to determine whether anaerobes that synthesize protoheme-containing cytochrome b de novo would use preformed tetrapyrroles. Characteristic reduced versus oxidized difference spectra of whole cells and pyridine hemochromogen spectra revealed thatSelenomonas ruminantium subsp.lactilytica andBacteroides succinogenes synthesized protoheme-containing b-type cytochromes de novo during growth in the presence of the manganese or magnesium chelates of deuteroporphyrin IX, but thatBacteroides ruminicola subsp.brevis synthesized deuteroheme-containing cytochrome b under the same conditions. During growth of the latter organism in the presence of the vanadium, molybdenum, cobalt, or nickel chelates of deuteroporphyrin, protoheme cytochrome b was formed.Bacteroides ruminicola subsp.brevis had a unique and metal-specific ability to use preformed tetrapyrroles for cytochrome synthesis; that ability was absent in other cytochrome-synthesizing rumen bacteria.