Factors Related to the Oxygen Tolerance of Anaerobic Bacteria

The effect of atmospheric oxygen on the viability of 13 strains of anaerobic bacteria, two strains of facultative bacteria, and one aerobic organism was examined. There were great variations in oxygen tolerance among the bacteria. All facultative bacteria survived more than 72 h of exposure to atmospheric oxygen. The survival time for anaerobes ranged from less than 45 min for Peptostreptococcus anaerobius to more than 72 h for two Clostridium perfringens strains. An effort was made to relate the degree of oxygen tolerance to the activities of superoxide dismutase, catalase, and peroxidases in cell-free extracts of the bacteria. All facultative bacteria and a number of anaerobic bacteria possessed superoxide dismutase. There was a correlation between superoxide dismutase activity and oxygen tolerance, but there were notable exceptions. Polyacrylamide gel electropherograms stained for superoxide dismutase indicated that many of the anaerobic bacteria contained at least two electrophoretically distinct enzymes with superoxide dismutase activity. All facultative bacteria contained peroxidase, whereas none of the anaerobic bacteria possessed measurable amounts of this enzyme. Catalase activity was variable among the bacteria and showed no relationship to oxygen tolerance. The ability of the bacteria to reduce oxygen was also examined and related to enzyme content and oxygen tolerance. In general, organisms that survived for relatively long periods of time in the presence of oxygen but demonstrated little superoxide dismutase activity reduced little oxygen. The effects of medium composition and conditions of growth were examined for their influence on the level of the three enzymes. Bacteria grown on the surface of an enriched blood agar medium generally had more enzyme activity than bacteria grown in a liquid medium. The data indicate that superoxide dismutase activity and oxygen reduction rates are important determinants related to the tolerance of anaerobic bacteria to oxygen.     

Global Metabolic Response of Enterococcus faecalis to Oxygen

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

Metabolic Profiling of the Response to an Oral Glucose Tolerance Test Detects Subtle Metabolic Changes

Background

The prevalence of overweight is increasing globally and has become a serious health problem. Low-grade chronic inflammation in overweight subjects is thought to play an important role in disease development. Novel tools to understand these processes are needed. Metabolic profiling is one such tool that can provide novel insights into the impact of treatments on metabolism.

Methodology

To study the metabolic changes induced by a mild anti-inflammatory drug intervention, plasma metabolic profiling was applied in overweight human volunteers with elevated levels of the inflammatory plasma marker C-reactive protein. Liquid and gas chromatography mass spectrometric methods were used to detect high and low abundant plasma metabolites both in fasted conditions and during an oral glucose tolerance test. This is based on the concept that the resilience of the system can be assessed after perturbing a homeostatic situation.

Conclusions

Metabolic changes were subtle and were only detected using metabolic profiling in combination with an oral glucose tolerance test. The repeated measurements during the oral glucose tolerance test increased statistical power, but the metabolic perturbation also revealed metabolites that respond differentially to the oral glucose tolerance test. Specifically, multiple metabolic intermediates of the glutathione synthesis pathway showed time-dependent suppression in response to the glucose challenge test. The fact that this is an insulin sensitive pathway suggests that inflammatory modulation may alter insulin signaling in overweight men.     

Different Ionic Specificities of ATP Synthesis in Extremely Alkaliphilic Sulfate-Reducing and Acetogenic Bacteria

Ionic specificity of oxidative phosphorylation was studied in Natroniella acetigenaand Desulfonatronum lacustre, which are new alkaliphilic anaerobes that were isolated from soda lakes and have a pH growth optimum of 9.5–9.7. The ability of their cells to synthesize ATP in response to the imposition of artificial pH⁺and pNa⁺gradients was studied. As distinct from other marine and freshwater sulfate reducers and extremely alkaliphilic anaerobes, D. lacustreuses a Na⁺-translocating ATPase for ATP synthesis. The alkaliphilic acetogen N. acetigena, which develops at a much higher Na⁺concentration in the medium, generated primary pH⁺for ATP synthesis. Thus, the high Na⁺concentrations and alkaline pH values typical of soda lakes do not predetermine the type of bioenergetics of their inhabitants.     

Recovery and Analysis of Formyltetrahydrofolate Synthetase Gene Sequences from Natural Populations of Acetogenic Bacteria

Primers for PCR amplification of partial (1,102 of 1,680 bp) formyltetrahydrofolate synthetase (FTHFS) gene sequences were developed and tested. Partial FTHFS sequences were successfully amplified from DNA from pure cultures of known acetogens, from other FTHFS-producing organisms, from the roots of the smooth cordgrass, Spartina alterniflora, and from fresh horse manure. The amplimers recovered were cloned, their nucleotide sequences were determined, and their translated amino acid sequences were used to construct phylogenetic trees. We found that FTHFS sequences from homoacetogens formed a monophyletic cluster that did not contain sequences from nonhomoacetogens and that FTHFS sequences appear to be informative regarding major physiological features of FTHFS-producing organisms.     

Ethanol Tolerance in Bacteria

Abstract

One promising application of biotechnology is in making the production of energy and chemical products from plants (“biomass”) practical. Applications include new markets for crops and reduction of wastes by using field residues or “wastelands” as feedstocks in biomass energy production. In addition, engineered crops could synthesize complex compounds. However, conversion to widespread use of biomass could have undesirable impacts. For example, changes in cropping practices may reduce wildlife populations or disrupt food chains. Other negative impacts of an environmentally insensitive development might include increased release of CO₂ (worsening global warming), and production of wastes, either water, chemical or biological, that present disposal problems. Biotechnology applied to biomass and chemical production has the potential for aiding economic development while greatly improving environmental quality. Environmental concerns are expressed in this paper, in the belief that consideration of potential problems at the inception of a technology increases the likelihood that environmentally sound courses will be pursued.     

Effect of Yeast Extract on Growth and Metabolism of H2-Utilizing Acetogenic Bacteria from the Human Colon

The aim of this work was to determine the effect of yeast extract and of its vitamin contents on autotrophic and heterotrophic growth and metabolism of four acetogenic bacteria from the human colon. Yeast extract exerted a stimulatory effect on autotrophic growth of the colonic acetogens, but concentration of this compound above 1–2 g. L⁻¹ in the medium did not enhance utilization of H₂/CO₂. Vitamins provided by yeast extract were shown to be essential cofactors of the reductive pathway of acetate synthesis except for one Clostridium strain. Yeast extract was also necessary to maintain heterotrophic growth and acetate synthesis from glucose in acetogenic species, except in the Streptococcus strain. In the absence of yeast extract, vitamins could efficiently restore glucose fermentation via acetate. The reductive and oxidative pathways of acetate synthesis might, therefore, depend on vitamin cofactors supplied by yeast extract in most of the human acetogenic bacteria. Non-vitaminic factors appeared also to be involved in the metabolism of some of these acetogenic species. Received: 6 March 1998 / Accepted: 3 April 1998     

Diversity of H2/CO2-Utilizing Acetogenic Bacteria from Fecesof Non-Methane-Producing Humans

The purpose of this work was to study H₂/CO₂-utilizing acetogenic population in the colons of non-methane-producing individuals harboring low numbers of methanogenic archaea. Among the 50 H₂-consuming acetogenic strains isolated from four fecal samples and an in vitro semi-continuous culture enrichment, with H₂/CO₂ as sole energy source, 20 were chosen for further studies. All isolates were Gram-positive strict anaerobes. Different morphological types were identified, providing evidence of generic diversity. All acetogenic strains characterized used H₂/CO₂ to form acetate as the sole metabolite, following the stoichiometric equation of reductive acetogenesis. These bacteria were also able to use a variety of organic compounds for growth. The major end product of glucose fermentation was acetate, except for strains of cocci that mainly produced lactate. Yeast extract was not necessary, but was stimulatory for growth and acetogenesis from H₂/CO₂. Received: 28 December 1995 / Accepted: 30 January 1996     

Isolation of Acetogenic Bacteria That Induce Biocorrosion by Utilizing Metallic Iron as the Sole Electron Donor

Corrosion of iron occurring under anoxic conditions, which is termed microbiologically influenced corrosion (MIC) or biocorrosion, is mostly caused by microbial activities. Microbial activity that enhances corrosion via uptake of electrons from metallic iron [Fe(0)] has been regarded as one of the major causative factors. In addition to sulfate-reducing bacteria and methanogenic archaea in marine environments, acetogenic bacteria in freshwater environments have recently been suggested to cause MIC under anoxic conditions. However, no microorganisms that perform acetogenesis-dependent MIC have been isolated or had their MIC-inducing mechanisms characterized. Here, we enriched and isolated acetogenic bacteria that induce iron corrosion by utilizing Fe(0) as the sole electron donor under freshwater, sulfate-free, and anoxic conditions. The enriched communities produced significantly larger amounts of Fe(II) than the abiotic controls and produced acetate coupled with Fe(0) oxidation prior to CH₄ production. Microbial community analysis revealed that Sporomusa sp. and Desulfovibrio sp. dominated in the enrichments. Strain GT1, which is closely related to the acetogen Sporomusa sphaeroides, was eventually isolated from the enrichment. Strain GT1 grew acetogenetically with Fe(0) as the sole electron donor and enhanced iron corrosion, which is the first demonstration of MIC mediated by a pure culture of an acetogen. Other well-known acetogenic bacteria, including Sporomusa ovata and Acetobacterium spp., did not grow well on Fe(0). These results indicate that very few species of acetogens have specific mechanisms to efficiently utilize cathodic electrons derived from Fe(0) oxidation and induce iron corrosion.     

Metabolic Features of Methane- and Methanol-Utilizing Bacteria

Investigations of a wide range of methane- and methanol-utilizers showed a striking versatility of their metabolism dependent on the genotype and growth conditions. A correlation between pathways of carbon and nitrogen metabolism was found. It was most stringent in obligate methane-utilizers: the hexulosephosphate pathway bacteria assimilated NH₃ by the reductive amination of α-ketoglutarate or pyruvate whereas the serine pathway bacteria used the glutamate cycle (glutamine synthetase + glutamate-oxoglutarate aminotransferase). Multiple enzymic lesions were found in central metabolism of obligate methylotrophs, i.e. the absence of the enzymes of glycolytic and pentosephosphate pathways, gluconeogenesis, citric acid cycle and glyoxylate shunt. These metabolic blocks were not so profound and could be compensated in restricted and facultative methylotrophs during heterotrophic growth. The average levels of exogenous CO₂ fixation in methylotrophic bacteria with the hexulosephosphate, serine and ribulosebisphosphate pathways were found to be 10, 30 and 80% of their total cell carbon, respectively. These results served as a basis for biotechnological applications of metabolic potential of methylotrophs (production of biomass, polysaccharides and enzymes as well as for microbiological treatment of industrial waters containing toxic C₁- and C_n-compounds).     

Acetogenic and Sulfate-Reducing Bacteria Inhabiting the Rhizoplane and Deep Cortex Cells of the Sea Grass Halodule wrightii

Recent declines in sea grass distribution underscore the importance of understanding microbial community structure-function relationships in sea grass rhizospheres that might affect the viability of these plants. Phospholipid fatty acid analyses showed that sulfate-reducing bacteria and clostridia were enriched in sediments colonized by the sea grasses Halodule wrightii and Thalassia testudinum compared to an adjacent unvegetated sediment. Most-probable-number analyses found that in contrast to butyrate-producing clostridia, acetogens and acetate-utilizing sulfate reducers were enriched by an order of magnitude in rhizosphere sediments. Although sea grass roots are oxygenated in the daytime, colorimetric root incubation studies demonstrated that acetogenic O-demethylation and sulfidogenic iron precipitation activities were tightly associated with washed, sediment-free H. wrightii roots. This suggests that the associated anaerobes are able to tolerate exposure to oxygen. To localize and quantify the anaerobic microbial colonization, root thin sections were hybridized with newly developed ³³P-labeled probes that targeted (i) low-G+C-content gram-positive bacteria, (ii) cluster I species of clostridia, (iii) species of Acetobacterium, and (iv) species of Desulfovibrio. Microautoradiography revealed intercellular colonization of the roots by Acetobacterium and Desulfovibrio species. Acetogenic bacteria occurred mostly in the rhizoplane and outermost cortex cell layers, and high numbers of sulfate reducers were detected on all epidermal cells and inward, colonizing some 60% of the deepest cortex cells. Approximately 30% of epidermal cells were colonized by bacteria that hybridized with an archaeal probe, strongly suggesting the presence of methanogens. Obligate anaerobes within the roots might contribute to the vitality of sea grasses and other aquatic plants and to the biogeochemistry of the surrounding sediment.     

Metabolic Injury in Frozen Bacteria

S ummary : Aerobacter aerogenes populations, partly killed by freezing and thawing, usually showed more viable bacteria when plated on a rich medium than on a poor one. This 'metabolic injury' was not due to osmotic sensitivity or to toxic materials in the agar. Freezing and thawing was not significantly mutagenic and mutation to nutritional exigence did not account for the phenomenon. The survivors of freezing and thawing showed long lag phases in the recovery media, during which lag periods further mortality occurred.     

Quantitative Determination of H2-Utilizing Acetogenic and Sulfate-Reducing Bacteria and Methanogenic Archaea from Digestive Tract of Different Mammals

Total number of bacteria, cellulolytic bacteria, and H₂-utilizing microbial populations (methanogenic archaea, acetogenic and sulfate-reducing bacteria) were enumerated in fresh rumen samples from sheep, cattle, buffaloes, deer, llamas, and caecal samples from horses. Methanogens and sulfate reducers were found in all samples, whereas acetogens were not detected in some samples of each animal. Archaea methanogens were the largest H₂-utilizing populations in all animals, and a correlation was observed between the numbers of methanogens and those of cellulolytic microorganisms. Higher counts of acetogens were found in horses and llamas (1 × 10⁴ and 4 × 10⁴ cells ml⁻¹ respectively).     

Mechanisms of Oxygen Taxis in Bacteria

Since 1881 when Englemann reported aerotaxis in bacteria, an understanding of the molecular nature of the signal transduction remains a daring goal for microbiologists. This short review discusses known facts and recent advances in the field including the discovery of the flavoprotein receptor which drives Escherichia coli towards oxygen. Possible mechanisms of oxygen sensing in various bacterial species are considered in connection with the existing, often fragmental, data on phototaxis, redox taxis and taxis repellent effect of the reactive oxygen species (ROS).     

Oxygen Sensitivity of Various Anaerobic Bacteria

Anaerobes differ in their sensitivity to oxygen, as two patterns were recognizable in the organisms included in this study. Strict anaerobes were species incapable of agar surface growth at pO(2) levels greater than 0.5%. Species that were found to be strict anaerobes were Treponema macrodentium, Treponema denticola, Treponema oralis n. sp., Clostridium haemolyticum, Selenomonas ruminatium, Butyrivibrio fibrisolvens, Succinivibrio dextrinosolvens, and Lachnospira multiparus. Moderate anaerobes would include those species capable of growth in the presence of oxygen levels as high as 2 to 8%. The moderate anaerobes could be exposed to room atmosphere for 60 to 90 min without appreciable loss of viability. Species considered as moderate anaerobes were Bacteroides fragilis, B. melaninogenicus, B. oralis, Fusobacteria nucleatum, Clostridium novyi type A, and Peptostreptococcus elsdenii. The recognition of at least two general types of anaerobes would seem to have practical import in regard to the primary isolation of anaerobes from source material.     

Relations of Oligotrophic Bacteria to Oxygen

Polluted air and the derived photochemical smog are the sources of free radicals in the atmosphere. Organic peroxides present in the smog mediate formation of peroxide radical. Oxygen species are formed by purely physical mechanisms, for instance, energy consumption converts molecular oxygen to an excited singlet state. Six active oxygen species are known: ozone, atomic oxygen, perhydroxyl, superoxide, and singlet oxygen. Singlet oxygen is the most harmful oxygen product for living cells, while hydrogen peroxide is the least harmful. Molecular oxygen is hardly toxic for prokaryotes due to an efficient protection of microbial cells by specific enzymes. This work experimentally confirms the harmlessness of molecular oxygen.