Effects of alternative dietary substrates on competition between human colonic bacteria in an anaerobic fermentor system

Duplicate anaerobic fermentor systems were used to examine changes in a community of human fecal bacteria supplied with different carbohydrate energy sources. A panel of group-specific fluorescent in situ hybridization probes targeting 16S rRNA sequences revealed that the fermentors supported growth of a greater proportion of Bacteroides and a lower proportion of gram-positive anaerobes related to Faecalibacterium prausnitzii, Ruminococcus flavefaciens-Ruminococcus bromii, Eubacterium rectale-Clostridium coccoides, and Eubacterium cylindroides than the proportions in the starting fecal inoculum. Nevertheless, certain substrates, such as dahlia inulin, caused a pronounced increase in the number of bacteria related to R. flavefaciens-R. bromii and E. cylindroides. The ability of three strictly anaerobic, gram-positive bacteria to compete with the complete human fecal flora was tested in the same experiment by using selective plating to enumerate the introduced strains. The Roseburia-related strain A2-183(F) was able to grow on all substrates despite the fact that it was unable to utilize complex carbohydrates in pure culture, and it was assumed that this organism survived by cross-feeding. In contrast, Roseburia intestinalis L1-82(R) and Eubacterium sp. strain A2-194(R) survived less well despite the fact that they were able to utilize polysaccharides in pure culture, except that A2-194(R) was stimulated 100-fold by inulin. These results suggest that many low-G+C-content gram-positive obligate anaerobes may be selected against during in vitro incubation, although several groups were stimulated by inulin. Thus, considerable caution is necessary when workers attempt to predict the in vivo effects of probiotics and prebiotics from their effects in vitro.     

Citric-acid cycle key enzyme activities during in vitro growth and metacyclogenesis of Leishmania infantum promastigotes.

The activities of 5 key regulatory enzymes in most energetic systems, namely citrate synthase (EC 4.1.3.7, CS), NADP-specific isocitrate dehydrogenase (EC 1.1.1.42, ICDH), succinate dehydrogenase (EC 1.3.99.1, SDH), L-malate dehydrogenase (EC 1.1.1.37, MDH), and decarboxylating malic enzyme (EC 1.1.1.40, ME), were measured during the growth and metacyclogenesis of a cutaneous (CL) and a visceral (VL) strain of Leishmania infantum. As occurs with other Leishmania species, infective promastigotes were present along all phases of growth, but their percentages were higher at the early stationary phase for VL and the end of the same phase for CL. High CS and SDH activities were detected in both strains, as compared with other trypanosomatids, bringing more evidence for an actively functional citric-acid cycle in L. infantum. Both strains showed higher levels of CS, ICDH, and MDH and lower SDH and ME activities when more metacyclic promastigotes were present, but in VL these changes paralleled an increase in glucose consumption, whereas in CL these changes coincided with an NH3 hyperproduction. This suggests that the energy metabolism during L. infantum growth and metacyclogenesis is affected by regulated enzymes that probably respond to changes in the culture medium in the levels of glucose and amino acids.     

Acetate oxidation to CO2 in anaerobic bacteria via a novel pathway not involving reactions of the citric acid cycle

In several sulfate-reducing bacteria capable of complete oxidation of acetate (or acetyl CoA), the citric acid cycle is not operative. No 2-oxoglutarate dehydrogenase activity was found in these organisms, and the labelling pattern of oxaloacetate excludes its synthesis via 2-oxo-glutarate. These sulfate-reducers contained, however, high activities of the enzymes carbon monoxide dehydrogenase and formate dehydrogenase and catalyzed an isotope exchange between CO₂ and the carboxyl group of acetate (or acetyl CoA), showing a direct C-C-cleavage of activated acetic acid. These findings suggest that in the investigated sulfate-reducers acetate is oxidized to CO₂ via C₁ intermediates. The proposed pathway provides a possible explanation for the reported different fluoroacetate sensitivity of acetate oxidation by anaerobic bacteria, for mini-methane formation, as well as for the postulated anaerobic methane oxidation by special sulfate-reducers.     

Growth characteristic of anaerobic ammonium-oxidizing bacteria in an anaerobic biological filtrated reactor

The doubling time of anaerobic ammonium-oxidizing (anammox) bacteria in an anaerobic biological filtrated (ABF) reactor was determined. Fluorescence in situ hybridization analysis was used to detect and count anammox bacteria cells in anammox sludge. As a result, the populations of anammox bacteria at 14th and 21st days were 1.1×10⁶ and 1.7×10⁷ cells/ml reactor, respectively. From these results, the doubling time of anammox bacteria was calculated as 1.8 days, and the specific growth rate (μ) was 0.39 day⁻¹. This result indicated that the anammox bacteria have higher growth rate than the reported value (doubling time, 11 days). Furthermore, it was clearly demonstrated that nitrogen conversion rate was proportional to the population of anammox bacteria. Maintaining the ideal environment for the growth of anammox bacteria in the ABF reactor might lead to faster growth. This is the first report of the growth rate of anammox bacteria based on the direct counting of anammox bacteria.     

Endosymbiotic purple non-sulphur bacteria in an anaerobic ciliated protozoon

Abstract The marine ciliate Strombidium purpureum Kahl harbours endosymbiotic purple non-sulphur bacteria. The bacteria contain bacteriochlorophyll a and the carotenoid spirilloxanthin, and they have photosynthetic membranes and cell walls. The ciliates require light for survival and growth under anaerobic conditions; in the dark the cells prefer microaerobic conditions. The ciliates show a photosensory behaviour, and they accumulate in light at wave lenghts corresponding to the absorption spectrum of the symbionts. The findings are discussed in terms of theories on the endosymbiotic origin of mitochondria.     

Studies on anaerobic proteolytic bacteria of Leh,India

Five anaerobic proteolytic bacteria were isolated from water bodies of Leh, India, where the ambient temperature varies from –25 to 25 °C. Isolates showed growth at all temperatures ranging from 5 to 37 °C except SPL-4 and SPL-5 which showed no growth at 5 °C. The cultures could grow and produce proteases on various protein substrates and the yield varied with the substrates. Two of the cultures showed the presence of spores. Acetate was the dominant VFA during hydrolysis of protein substrates.     

The parasexual cycle in Candida albicans provides an alternative pathway to meiosis for the formation of recombinant strains

Candida albicans has an elaborate, yet efficient, mating system that promotes conjugation between diploid a and α strains. The product of mating is a tetraploid a/α cell that must undergo a reductional division to return to the diploid state. Despite the presence of several “meiosis-specific” genes in the C. albicans genome, a meiotic program has not been observed. Instead, tetraploid products of mating can be induced to undergo efficient, random chromosome loss, often producing strains that are diploid, or close to diploid, in ploidy. Using SNP and comparative genome hybridization arrays we have now analyzed the genotypes of products from the C. albicans parasexual cycle. We show that the parasexual cycle generates progeny strains with shuffled combinations of the eight C. albicans chromosomes. In addition, several isolates had undergone extensive genetic recombination between homologous chromosomes, including multiple gene conversion events. Progeny strains exhibited altered colony morphologies on laboratory media, demonstrating that the parasexual cycle generates phenotypic variants of C. albicans. In several fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, the conserved Spo11 protein is integral to meiotic recombination, where it is required for the formation of DNA double-strand breaks. We show that deletion of SPO11 prevented genetic recombination between homologous chromosomes during the C. albicans parasexual cycle. These findings suggest that at least one meiosis-specific gene has been re-programmed to mediate genetic recombination during the alternative parasexual life cycle of C. albicans. We discuss, in light of the long association of C. albicans with warm-blooded animals, the potential advantages of a parasexual cycle over a conventional sexual cycle.     

Glutathione amide and its perthiol in anaerobic sulfur bacteria.

Chromatium species produced the novel biological thiol glutathione amide, gamma-L-glutamyl-L-cysteinylglycine amide (GASH), when grown photoheterotrophically. GASH was largely converted to the corresponding perthiol during photoautotrophic growth on sulfide, suggesting that GASH may have a function in anaerobic sulfide metabolism. This unprecedented form of glutathione metabolism was probably present in anaerobic ancestors of modern cyanobacteria and purple bacteria.     

Application of a tetrazolium dye as an indicator of viability in anaerobic bacteria.

The use of the redox dye 5-cyano-2,3,-ditolyl tetrazolium chloride (CTC) for evaluating the metabolic activity of aerobic bacteria has gained wide application in recent years. In this study, we examined the utility of CTC in capturing the metabolic activity of anaerobic bacteria. In addition, the factors contributing to abiotic reduction of CTC were also examined. CTC was used in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF), that targets bacterial cell wall proteins, to quantitate the active fraction of total bacterial numbers. Facultative anaerobic bacteria, including Escherichia coli grown fermentatively, and Pseudomonas chlorophis, P. fluorescens, P. stutzeri, and P. pseudoalcalegenes subsp. pseudoalcalegenes grown under nitrate-reducing conditions, actively reduced CTC during all phases of growth. Greater than 95% of these cells accumulated intracellular CTC-formazan crystals during the exponential phase. Obligate anaerobic bacteria, including Syntrophus aciditrophicus grown fermentatively, Geobacter sulfurreducens grown with fumarate as the electron acceptor, Desulfovibrio desulfuricans subsp. desulfuricans and D. halophilus grown under sulfate-reducing conditions, Methanobacterium formicicum grown on formate, H2 and CO2, and Methanobacterium thermoautotrophicum grown autotrophically on H2 and CO2 all reduced CTC to intracellular CTC-formazan crystals. The optimal CTC concentration for all organisms examined was 5 mM. Anaerobic CTC incubations were not required for quantification of anaerobically grown cells. CTC-formazan production by all cultures examined was proportional to biomass production, and CTC reduction was observed even in the absence of added nutrients. CTC was reduced by culture fluids containing ferric citrate as electron acceptor following growth of either G. metallireducens or G. sulfurreducens. Abiotic reduction of CTC was observed in the presence of ascorbic acid, cysteine hydrochloride, dithiothreitol, NADH, NADPH, Fe(II)Cl2, sodium thioglycolic acid and sodium sulfide. These results suggest that while CTC can be used to capture the metabolic activity of anaerobic bacteria, care must be taken to avoid abiotic reduction of CTC.     

Phylogenetic and functional diversity of propionate-oxidizing bacteria in an anaerobic digester sludge

The phylogenetic and functional diversity of syntrophic propionate-oxidizing bacteria (POB) present in an anaerobic digester was investigated by microautoradiography combined with fluorescent in situ hybridization (MAR–FISH) that can directly link 16S rRNA phylogeny with in situ metabolic function. The syntrophic POB community in the anaerobic digester sludge consisted of at least four phylogenetic groups: Syntrophobacter, uncultured short rod Smithella (Smithella sp. SR), uncultured long rod Smithella (Smithella sp. LR), and an unidentified group. The activities of these POB groups were dependent on the propionate concentrations. The uncultured Smithella sp. SR accounted for 52–62% of the total active POB under all the propionate concentrations tested (0.5–15 mM). In contrast, uncultured Smithella sp. LR was active only at lower propionate concentrations and became a dominant active POB at 0.5 mM of propionate. Syntrophobacter accounted for 16–31% of the total active POB above 2.5 mM propionate, whereas the active Syntrophobacter population became low (ca. 6%) at 0.5 mM of propionate. The anaerobic digester was operated in a fill and draw mode, resulting in periodical changes in propionate concentration ranging from 0 to 10 mM. These phylogenetically and functionally diverse, to some extent functionally redundant, active POB communities were dynamically responding to the periodical changes in propionate concentration.     

Signal sequence-independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle

Protein targeting by the signal recognition particle (SRP) and the bacterial SRP receptor FtsY requires a series of closely coordinated steps that monitor the presence of a substrate, the membrane, and a vacant translocon. Although the influence of substrate binding on FtsY-SRP complex formation is well documented, the contribution of the membrane is largely unknown. In the current study, we found that negatively charged phospholipids stimulate FtsY-SRP complex formation. Phospholipids act on a conserved positively charged amphipathic helix in FtsY and induce a conformational change that strongly enhances the FtsY-lipid interaction. This membrane-bound, signal sequence-independent FtsY-SRP complex is able to recruit RNCs to the membrane and to transfer them to the Sec translocon. Significantly, the same results were also observed with an artificial FtsY-SRP fusion protein, which was tethered to the membrane via a transmembrane domain. This indicates that substrate recognition by a soluble SRP is not essential for cotranslational targeting in Escherichia coli. Our findings reveal a remarkable flexibility of SRP-dependent protein targeting, as they indicate that substrate recognition can occur either in the cytosol via ribosome-bound SRP or at the membrane via a preassembled FtsY-SRP complex.     

Simple and convenient method for culturing anaerobic bacteria.

A simple and convenient method for culturing anaerobic bacteria is described. Cultures can be grown in commercially available flasks normally used for preparation of sterile external solutions. A special disposable rubber flask closure maintains anaerobic conditions in the flask after autoclaving. Growth of a variety of anaerobic oral bacteria was comparable to that obtained after anaerobic incubation of broth cultures in Brewer Anaerobic Jars.     

Metabolism of alkylbenzenes, alkanes, and other hydrocarbons in anaerobic bacteria

Aromatic and aliphatic hydrocarbons are the main constituents of petroleum and its refined products. Whereas degradation of hydrocarbons by oxygen-respiring microorganisms has been known for about a century, utilization of hydrocarbons under anoxic conditions has been investigated only during the past decade. Diverse strains of anaerobic bacteria have been isolated that degrade toluene anaerobically, using nitrate, iron(III), or sulfate as electron acceptors. Also, other alkylbenzenes such as m-xylene or ethylbenzene are utilized by a number of strains. The capacity for anaerobic utilization of alkylbenzenes has been observed in members of the -, -, - and -subclasses of the Proteobacteria. Furthermore, denitrifying bacteria and sulfate-reducing bacteria with the capacity for anaerobic alkane degradation have been isolated, which are members of the - and -subclass, respectively. The mechanism of the activation of hydrocarbons as apolar molecules in the absence of oxygen is of particular interest.The biochemistry of anaerobic toluene degradation has been studied in detail. Toluene is activated by addition to fumarate to yield benzylsuccinate, which is then further metabolized via benzoyl-CoA. The toluene-activating enzyme presents a novel type of glycine radical protein. Another principle of anaerobic alkylbenzene activation has been observed in the anaerobic degradation of ethylbenzene. Ethylbenzene in denitrifying bacteria is dehydrogenated to 1-phenylethanol and further to acetophenone; the latter is also metabolized to benzoyl-CoA. Naphthalene is presumably activated under anoxic conditions by a carboxylation reaction. Investigations into the pathway of anaerobic alkane degradation are only at the beginning. The saturated hydrocarbons are mostlikely activated by addition of a carbon compound rather than by desaturation and hydration, as speculated about in some early studies. An anaerobic oxidation of methane with sulfate as electron acceptor has been documented in aquatic sediments. The process is assumed to involve a reversal of methanogenesis catalyzed by Archaea, and scavenge of an electron-carrying metabolite by sulfate-reducing bacteria. Among unsaturated non-aromatic hydrocarbons, anaerobic bacterial degradation has been demonstrated and investigated with n-alkenes, alkenoic terpenes and the alkyne, acetylene.     

Interactions between amino-acid-degrading bacteria and methanogenic bacteria in anaerobic digestion

The degradation of amino acids in anaerobic digestion was examined in terms of the interactions between amino-acid-degrading bacteria and methanogenic bacteria. Certain amino acids were degraded oxidatively by dehydrogenation, with methanogenic bacteria acting as H(2) acceptors. The inhibition of methanogenesis by chloroform also inhibited the degradation of these amino acids and/or caused variations in the composition of volatile acids produced from them. The presence of glycine reduced the inhibitory effect caused by chloroform, probably because glycine acted as an H(2) acceptor in place of methanogenic bacteria. This fact suggested that the coupled oxidation-reduction reactions between two amino acids-one acting as the H(2) donor and the other acting as the H(2) acceptor-may occur in the anaerobic digestion of proteins or amino-acid mixtures. The conversion of some proteins to volatile acids was not affected when methanogenesis was inhibited by chloroform. This suggested that the component amino acids of proteins may be degraded by the coupled oxidation-reduction reactions and that the degradation of proteins may not be dependent on the activity of methanogenic bacteria as H(2) acceptors.     

Application, eco-physiology and biodiversity of anaerobic ammonium-oxidizing bacteria

The demand for new and sustainable systems for nitrogen removal has increased dramatically in the last decade. It is clear that the conventional systems cannot deal with the increasing nitrogen loads in a cost effective way. As an alternative, the implementation of the anammox (anaerobic ammonium oxidation) process in the treatment of wastewater with high ammonium concentrations has been started. The compact anammox reactors can sustain high nitrogen loads without any problems. The highest observed anammox capacity is 8.9 kg N removed m^-3 reactor day^-1. The first 75 m³ anammox reactor is operating in Rotterdam, the Netherlands, combined with the partial nitrification process Single reaction system for High Ammonium Removal Over Nitrite (SHARON). Partial nitrification and anammox can also be combined in one reactor systems like Completely Autotrophic Nitrogen removal Over Nitrite (CANON) or Oxygen Limited Ammonium removal via Nitrification Denitrification (OLAND) where aerobic ammonium-oxidizing bacteria (AOB) and anammox bacteria cooperate under oxygen-limitation. These systems remove about 1.5 kg N m^-3 reactor day^-1. In addition to ammonium, urea can also be converted in the CANON system after a two-week adaptation period. The ecophysiological properties of the anammox bacteria make them very well suited to convert ammonium and nitrite. The K_s values for ammonium and nitrite are below 5 M. However, nitrite above 10 mM is detrimental for the anammox process, and oxygen reversibly inhibits the process at concentrations as low as 1 M. Acetate and propionate can be used by the anammox bacteria to convert nitrite and nitrate, whereas methanol and ethanol severely inhibit the anammox reaction. The enzyme hydroxylamine/hydrazine oxidoreductase (HAO), one of the key enzymes, is located in the anammoxosome, which is a membrane bound organelle. The membranes of the anammox bacteria contain unique ladderane lipids and hopanoids. The bacteria responsible for the anammox reaction are related to the Planctomycetes. The first anammox bacteria were isolated via Percoll centrifugation and characterized as Candidatus Brocadia anammoxidans. Survey of different wastewater treatment plants using anammox specific 16S rRNA gene primers and anammox specific oligonucleotide probes has revealed the presence of at least three other anammox bacteria, which have been tentatively named Candidatus Kuenenia stuttgartiensis, Candidatus Scalindua wagneri and Candidatus Scalindua brodae. A close relative of the latter, Candidatus Scalindua sorokinii was found to be responsible for about 50% of the nitrogen conversion in the anoxic zone of the Black Sea, making the anammox bacteria an important player in the oceanic nitrogen cycle.     

Regulation of the tricarboxylic acid cycle in gram-positive, facultatively anaerobic bacilli.

The facultative anaerobes Bacillus polymyxa Hino G, B. polymyxa Hino J, and B.macerans were observed to have imcomplete tricarboxylic acid cycles. They were devoid of malate dehydrogenase and all had very low levels of alpha-ketoglutarate dehydrogenase. B. polymyxa Hino J was devoid of alpha-ketoglutarate dehydrogenase when grown aerobically and anerobically. Citrate synthase from B. polymyxa was inhibited by adenosine triphosphate but not reduced nicotinamide adenine dinucleotide and resembled enzymes from other gram-positive bacteria in this respect. Like the citrate synthases from gram-negative, facultative anaerobes and chemolithotrophs, the enzyme from B. polymyxa was inhibited by alpha-ketoglutarate. Inhibition by adenosine triphosphate was shown to be competitive with acetyl-coenzyme A and alpha-ketoglutarate inhibition was competitive with oxaloacetate.