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PubMed1999

Effect of cyclin E overexpression on lovastatin-induced G1 arrest and RhoA inactivation in NIH3T3 cells.

The HMG-CoA reductase inhibitor, lovastatin, blocks targeting of the Rho and Ras families of small GTPases to their active sites by inhibiting protein prenylation. Control NIH3T3 cells, and those overexpressing human cyclin E protein were treated with lovastatin for 24 h to determine the effects of cyclin E overexpression on lovastatin-induced growth arrest and cell rounding. Lovastatin treatment (10 microM) of control 3T3 cells resulted in growth arrest at G1 accompanied by actin stress fiber disassembly, cell rounding, and decreased active RhoA from the membranous protein fraction. By contrast, in NIH3T3 cells overexpressing cyclin E, lovastatin did not cause loss of RhoA from the membrane (active) protein fraction, actin stress fiber disassembly, cell rounding or growth arrest within 24 h. Analysis of cell cycle proteins showed that 24 h of lovastatin treatment in the control cells caused an elevation in the levels of the cyclin-dependent kinase inhibitor p27(kip1), inhibition of both cyclin E- and cyclin A-dependent kinase activity, and decreased levels of hyperphosphorylated retinoblastoma protein (pRb). By contrast, lovastatin treatment of the cyclin E overexpressors did not suppress either cyclin E- or cyclin A-dependent kinase activity, nor did it alter the level of maximally phosphorylated pRb, despite increased levels of p27(kip1). However, by 72 h, the cyclin E overexpressors rounded up but remained attached to the substratum, indicating a delayed response to lovastatin. In contrast with lovastatin, inactivation of membrane-bound Rho proteins (i.e., GTP-bound RhoA, RhoB, RhoC) with botulinum C3 transferase caused cell rounding and G1 growth arrest in both cell types but did not inhibit cyclin E-dependent histone kinase activity in the cyclin E overexpressors. In addition, 24 h of cycloheximide treatment caused depletion of RhoA from the membrane (active) fraction in neo cells, but in the cells overexpressing cyclin E, RhoA remained in the active (membrane-associated) fraction. Our observations suggest that (1) RhoA activation occurs downstream of cyclin E-dependent kinase activation, and (2) overexpression of cyclin E decreased the turnover rate of active RhoA.

3T3 Cells↗
PubMed1995

Viral dynamics in human immunodeficiency virus type 1 infection.

The dynamics of HIV-1 replication in vivo are largely unknown yet they are critical to our understanding of disease pathogenesis. Experimental drugs that are potent inhibitors of viral replication can be used to show that the composite lifespan of plasma virus and virus-producing cells is remarkably short (half-life approximately 2 days). Almost complete replacement of wild-type virus in plasma by drug-resistant variants occurs after fourteen days, indicating that HIV-1 viraemia is sustained primarily by a dynamic process involving continuous rounds of de novo virus infection and replication and rapid cell turnover.

Antiviral Agents↗
PubMed1980

Neutral proteases of human polymorphonuclear granulocytes: putative mediators of pulmonary damage.

Tissue proteolytic enzymes are currently believed to be critical to the pathogenesis of panacinar emphysema. Polymorphonuclear leukocytes (Polys) have several enzymes including elastase and cathepsin G in their azurophil granules. They have collagenase in their specific granules. We have found that this collagenase is doubly latent. It has the lysosomal type of latency that depends on the impermeability of the unit membrane that surrounds each specific granule. In addition it has a latency that is converted to activity by proteolytic enzymes. The cathepsin G of the azurophil granule is a potent activator of this latent collagenase once the collagenase is released from its membrane dependent latency. Thus latency of enzymes, the nature of the latency and accessibility of the latent enzymes to activating mechanisms must all be taken into account in any analysis of their contribution to pathogenesis of local lung disease. Equally important is that fact that polys are not a prominent cellular component of normal lung. Polys must be attracted to the lung by chemotactic peptides. These peptides must be released by the interaction of inflammatory stimuli, such as smoke particles, with complement components or they must be provided by other sources. The hypothesis that lung damage in panacinar emphysema is mediated by polys and their proteases is attractive and suggestive evidence supporting this is available. However, more evidence that takes into full account the cell biology of the proteases any poly turnover in the lung are needed to extend the hypothesis and to form a rational basis for therapeutic and prophylactic measures.

Cathepsin G↗
PubMed1993

Concentrations and fluxes of organic carbon substrates in the aquatic environment.

Data concerning concentrations and fluxes of dissolved organic compounds (DOC) from marine and lacustrine environments are reviewed and discussed. Dissolved free amino acids and carbohydrates comprised the main fraction in the labile organic carbon pool. Dissolved free amino acids in marine waters varied between 3-1400 nM and those of fresh waters between 2.6-4124 nM. Dissolved free carbohydrates varied between 0.4-5000 nM in marine systems and between 14-111 nM in fresh waters, The turnover times of both substrate pools varied in marine waters between 1.4 hours and 948 days and in fresh waters between 2 hours and 51 days. Measurements of stable 12/13C-ratio and 14C-isotope dating in ocean deep water samples revealed DOC turnover times between 2000-6000 years. Studies on carbon flows within the aquatic food webs revealed that about 50% of photosynthetically fixed carbon was channelled via DOC to the bacterioplankton. Excreted organic carbon varied between 1-70% of photosynthetically fixed carbon in marine waters and between 1-99% in fresh waters. The labile organic carbon pool represented only 10-30% of the DOC. The majority (70-90%) of the DOC was recalcitrant to microbial assimilation. Only 10-20% of the DOC could be easily chemically identified. Most of the large bulk material represented dissolved humic matter and neither the chemical structure nor the ecological function of the DOC is as yet clearly understood.

Amino Acids↗
PubMed1997

Role of 2,6-dideoxy-2,6-diaminoglucose in activation of a eukaryotic phospholipase C by aminoglycoside antibiotics.

Recent emergence of microbial resistance to aminoglycoside antibiotics, and the documented cytotoxicity associated with their use, calls for sustained efforts at understanding the effects of the compounds on eukaryotic cells. Using a glycosyl phosphatidylinositol (GPI)-phospholipase C (GPI-PLC) from the protozoan parasite Trypanosoma brucei, we demonstrate that a eukaryotic PLC can be activated 6-fold by aminoglycosides. Neomycin B protected GPI-PLC from a reduction in activity at pH 6.5, and increased the turnover number (kcat) of the enzyme. In structure-activity studies with the neomycin group, 2-deoxy-streptamine was mildly stimulatory; the concentration required to activate GPI-PLC 2-fold (SC200) was 310 microM. Neamine was 150-fold more active (SC200 = 2 microM) than 2-deoxy-streptamine, indicating that a 2,6-dideoxy-2, 6-diaminoglucose substituent at the 4-position of 2-deoxystreptamine plays an important role in activation of GPI-PLC. Ribostamycin and neomycin B also had SC200's of 2 microM, implying that the ribose group in ribostamycin is not involved in activation of GPI-PLC. These conclusions were affirmed in studies with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. A 2, 6-dideoxy-2,6-diaminoglucose substitution at the 4-OH of 2-deoxystreptamine activates the enzyme 17-fold, while a second 2, 6-dideoxy-2,6-diaminoglucose moiety on the ribose ring of ribostamycin provides an additional 3.5-fold stimulation. Possible implications of these observations for the effects of aminoglycosides on eukaryote cells are discussed.

Animals↗
PubMed1977

The mechanism of action of nitro-heterocyclic antimicrobial drugs. Primary target of 1-methyl-2-nitro-5-vinylimidazole is DNA.

The antimicrobial drug 1-methyl-2-nitro-5-vinylimidazole (MEV) preferentially blocked DNA synthesis, was mutagenic and induced coliphage lambda in Escherichia coli. The antibacterial effects of MEV are the consequences of repairable damage to DNA, as shown by hypersensitivity of recA and uvr strains to MEV and related drugs, stimulation by MEV of DNA turnover which was dependent on the product of the uvrA gene, and the presence of cross-links in DNA from MEV-treated bacteria.

Anti-Bacterial Agents↗
PubMed2003

Trimethylbenzoic acids as metabolite signatures in the biogeochemical evolution of an aquifer contaminated with jet fuel hydrocarbons.

Evolution of trimethylbenzoic acids in the KC-135 aquifer at the former Wurtsmith Air Force Base (WAFB), Oscoda, MI was examined to determine the functionality of trimethylbenzoic acids as key metabolite signatures in the biogeochemical evolution of an aquifer contaminated with JP-4 fuel hydrocarbons. Changes in the composition of trimethylbenzoic acids and the distribution and concentration profiles exhibited by 2,4,6- and 2,3,5-trimethylbenzoic acids temporally and between multilevel wells reflect processes indicative of an actively evolving contaminant plume. The concentration levels of trimethylbenzoic acids were 3-10 orders higher than their tetramethylbenzene precursors, a condition attributed to slow metabolite turnover under sulfidogenic conditions. The observed degradation of tetramethylbenzenes into trimethylbenzoic acids obviates the use of these alkylbenzenes as non-labile tracers for other degradable aromatic hydrocarbons, but provides rare field evidence on the range of high molecular weight alkylbenzenes and isomeric assemblages amenable to anaerobic degradation in situ. The coupling of actual tetramethylbenzene loss with trimethylbenzoic acid production and the general decline in the concentrations of these compounds demonstrate the role of microbially mediated processes in the natural attenuation of hydrocarbons and may be a key indicator in the overall rate of hydrocarbon degradation and the biogeochemical evolution of the KC-135 aquifer.

Benzoates↗
PubMed1991

Influence of rumen undegradable protein levels on feed intake and milk production of dairy cows.

Twenty-seven dairy cows in midlactation were utilized in two experiments using 15 and 12 cows to determine effects of varying the delivery of ruminally undegraded protein on feed intake, milk production, and some rumen and plasma characteristics. In Experiment 1, cows consumed alfalfa silage ad libitum and one of three barley-based concentrates with either soybean meal (a rapidly rumen degraded protein source), corn gluten meal (a slowly degraded protein source), or an equal mixture of the two, fed at the rate of .36 kg/kg of milk produced. In Experiment 2, cows were fed total mixed diets based upon alfalfa silage, barley, and either soybean meal, corn gluten meal, or a mixture of soybean meal and whey powder (a protein source very rapidly degraded in the rumen). In sacco incubation procedures were used to estimate degradability of protein in all diets. All diets exceeded Agricultural Research Council recommendations for rumen degraded and undegraded protein as well as NRC recommendations for degraded protein. However, one to three of the six total diets, depending upon assumed ruminal turnover rates, did not meet NRC recommendations for undegraded protein. Production parameters, include DMI as well as milk yield and composition, were not influenced by diet in either experiment. Results do not support NRC recommendations for ruminally undegraded protein for midlactation dairy cows producing about 30 kg/d of milk and broadly support the lower recommendations of the Agricultural Research Council. Results also appear to question use of dietary energy intake to predict net rumen microbial protein yield.

Animal Feed↗
PubMed1982

Microbial dynamics of an epilithic mat community in a high alpine stream.

Studies were conducted to examine interrelationships between the heterotrophic and phototrophic populations within an epilithic community in the outlet stream of a high alpine lake. Levels of nitrates, phosphates, and total organic compounds in the lake were consistently near the lower limits of detectability. Microscopic examination of the community by phase-contrast light microscopy and scanning electron microscopy revealed diatoms, filamentous algae, and bacteria embedded within a dense gelatinous matrix. Chlorophyll a and primary productivity measurements had peak values in early August, with subsequent declines. Bacterial heterotrophic activity, as measured by V(max), turnover rate, and relative activity, increased significantly as the phototrophic community declined. This trend in heterotrophic activity was not accompanied by an increase in total bacterial numbers as determined by epi-illuminated fluorescence microscopy. These results suggest that the phototrophic community responded to changes in, or interactions among, various chemical and physical factors throughout the study period. The catabolic activity of the sessile bacteria appeared to be positively influenced by changes in the mat environment resulting from the decline of the phototrophic populations.

Journal Article↗
PubMed2004

Acetoclastic and hydrogenotrophic methane production and methanogenic populations in an acidic West-Siberian peat bog.

Sites in the West Siberian peat bog 'Bakchar' were acidic (pH 4.2-4.8), low in nutrients, and emitted CH4 at rates of 0.2-1.5 mmol m(-2) h(-1). The vertical profile of delta13CH4 and delta13CO2 dissolved in the porewater indicated increasing isotope fractionation and thus increasing contribution of H2/CO2-dependent methanogenesis with depth. The anaerobic microbial community at 30-50 cm below the water table produced CH4 with optimum activity at 20-25 degrees C and pH 5.0-5.5 respectively. Inhibition of methanogenesis with 2-bromo-ethane sulphonate showed that acetate, phenyl acetate, phenyl propionate and caproate were important intermediates in the degradation pathway of organic matter to CH4. Further degradation of these intermediates indicated that 62-72% of the CH4 was ultimately derived from acetate, the remainder from H2/CO2. Turnover times of [2-14C]acetate were on the order of 2 days (15, 25 degrees C) and accounted for 60-65% of total CH4 production. Conversion of 14CO2 to 14CH4 accounted for 35-43% of total CH4 production. These results showed that acetoclastic and hydrogenotrophic methanogenesis operated closely at a ratio of approximately 2 : 1 irrespective of the incubation temperature (4, 15 and 25 degrees C). The composition of the archaeal community was determined in the peat samples by terminal restriction fragment length polymorphism (T-RFLP) analysis and sequencing of amplified SSU rRNA gene fragments, and showed that members of Methanomicrobiaceae, Methanosarcinaceae and Rice cluster II (RC-II) were present. Other, presumably non-methanogenic archaeal clusters (group III, RC-IV, RC-V, RC-VI) were also detected. Fluorescent in situ hybridization (FISH) showed that the number of Bacteria decreased (from 24 x 10(7) to 4 x 10(7) cells per gram peat) with depth (from 5 to 55 cm below the water table), whereas the numbers of Archaea slightly increased (from 1 x 10(7) to 2 x 10(7) cells per gram peat). Methanosarcina spp. accounted for about half of the archaeal cells. Our results show that both hydrogenotrophic and acetoclastic methanogenesis are an integral part of the CH4-producing pathway in acidic peat and were represented by appropriate methanogenic populations.

Acetic Acid↗
PubMed1994

Role of bile in non-specific defence mechanisms of the gut.

The effect of depriving the intestine of bile for 48 hours was studied to determine any influence on various parameters of innate immunity in the gastrointestinal tract. Groups of rats were prepared by bile duct cannulation (with or without fluid replacement) or bile duct ligation. Normal and sham operated animals were used for comparing the thickness of the mucus layer and the cells contained therein, enumeration of goblet cells, and measurement of villus size. Histological examination indicated that the intestinal tissues of treated and control rats were similar. Though villus size and numbers of goblet cells were unaffected, a significant reduction occurred in the thickness of the mucus blanket in the duodenal regions of rats deprived of bile, and there were significantly lower numbers of mucus associated enterocytes and lymphocytes, suggesting a lower turnover rate of the epithelium. The balance of the bacterial populations in the caecum and intestine was altered by bile deprivation-increased numbers of coliform organisms were found in both regions. The range of factors, including antibodies and other known constituents, present in bile may contribute to the maintenance of tissue integrity and influence the balance in indigenous bacterial populations in the intestine. Disturbance of the host's biliary system and concomitant effects on the microbial flora may weaken the overall processes of defence in the intestine.

Animals↗
PubMed1996

Characterization of a Cys115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-GlcNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin.

The antibiotic fosfomycin inhibits bacterial cell wall biosynthesis by inactivation of UDP-GlcNAc enolpyruvyl tranferase (MurA). Prior work has established that Cys115 of Escherichia coli and Enterobacter cloacae MurA is the active site nucleophile alkylated by fosfomycin and implicated this residue in the formation of a covalent phospholactyl-enzyme adduct derived from substrate, phosphoenolpyruvate (PEP). On the basis of sequencing information from putative MurA homolog from Mycobacterium tuberculosis, we generated a C115D mutant of E. coli MurA that was highly active but fully resistant to time-dependent inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition vs PEP. In contrast to the broad pH-independent behavior of wild-type (WT) MurA, C115D mutant activity titrated across the pH range examined (pH 5.5-9) with an apparent pKa approximately 6, with kcatC115D ranging from approximately 10kcatWT at pH 5.5 to <0.1kcatWT at pH9.0. Km(PEP)115D was relatively constant in the pH range examined and increased approximately 100-fold relative to Km(PEP)WT. A fosfomycin-resistant C115E mutant with -1% activity of the C115D mutant was found to follow a pH dependence similar to that observed for C115D MurA. The contrasting pH dependences of WT and C115D MurA was also observed in the reaction with the pseudosubstrate, (Z)-3-fluorophosphoenolpyruvate, strongly suggesting a role for Cys/Asp115 as the general acid in the protonation of C-3 of PEP during MurA-catalyzed enol ether transfer. The difference in nucleophilicity between the carboxylate side chains of Asp115 and Glu115 and the thiolate group of Cys115 suggests that covalent enzyme adduct formation is not required for catalytic turnover and, furthermore, provides a chemical rationale for the resistance of the C115D and C115E mutants to fosfomycin inactivation.

Alkyl and Aryl Transferases↗
PubMedDate not supplied

Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase.

Transglutaminase-mediated site-specific and covalent immobilization of an enzyme to chemically modified agarose was explored. Using Escherichia coli alkaline phosphatase (AP) as a model, two designed specific peptide tags containing a reactive lysine (Lys) residue with different length Gly-Ser linkers for microbial transglutaminase (MTG) were genetically attached to N- or C-termini. For solid support, agarose gel beads were chemically modified with beta-casein to display reactive glutamine (Gln) residues on the support surface. Recombinant APs were enzymatically and covalently immobilized to casein-grafted agarose beads. Immobilization by MTG markedly depended on either the position or the length of the peptide tags incorporated to AP, suggesting steric constraint upon enzymatic immobilization. Enzymatically immobilized AP showed comparable catalytic turnover (k(cat)) to the soluble counterpart and comparable operational stability with chemically immobilized AP. These results indicate that attachment of a suitable specific peptide tag to the right position of a target protein is crucial for MTG-mediated formulation of highly active immobilized proteins.

Alkaline Phosphatase↗
PubMed1982

Estimation of the proportion of non-ammonia-nitrogen reaching the lower gut of the ruminant derived from bacterial and protozoal nitrogen.

1. A method for estimating the proportions of bacterial- and protozoal-N in the total non-ammonia-N reaching the lower gut of the ruminant under steady-state conditions was evaluated. Three trials using two different diets were conducted with a Holstein steer equipped with a rumen cannula and duodenal re-entrant cannulas. 2. An intraruminal primed infusion of (15NH4)2SO4 was administered for 68 h during each trial. Bacteria and protozoa samples were isolated from rumen fluid at approximately 6 h intervals during each infusion period. Total non-ammonia-N was isolated from duodenal digesta samples taken at approximately the same times. All of these samples were analysed for 15N enrichment. A computer program was used to fit equations to the 15N-enrichment curves of bacterial- and protozoal-N. Models of both bacterial- and protozoal-N kinetics consisted of a small pool which equilibrated rapidly with rumen NH3 and a large pool with a fractional turnover rate of 0.045-0.070/h for bacterial-N and 0.056-0.069/h for protozoal-N. 3. Abomasal fluid turnover was estimated by a single injection of polyethylene glycol (molecular weight 4000) into the rumen followed by sampling of rumen fluid and duodenal digesta. 4. Estimates of abomasal fluid turnover, bacterial-N turnover, and protozoal-N turnover were entered into an equation which was adjusted by computer iteration to fit the 15N-enrichment curve of duodenal digesta non-NH3-N generated from each (15NH4)2SO4 infusion period. The computer fit of this equation to the observed results gave estimates of 0:39-0.45 and 0.22-0.41 for the proportion of duodenal non-NH3-N derived from bacterial-N and protozoal-N respectively. 5. This method is potentially useful in estimating microbial protein passage to the lower gut in ruminants. Sampling digesta from the omasum rather than the duodenum would simplify the method and possibly increase the reliability of the estimates.

Abomasum↗
PubMed2003

The fibrinolytic system in dissemination and matrix protein deposition during a mycobacterium infection.

The fibrinolytic system is known to play an important role in the inflammatory response to bacterial infections. In the present study, relationships between protein components of the fibrinolytic system and infectivity by Mycobacterium avium were analyzed. Infections were initiated through noninvasive intratracheal administration of M. avium 724 in mice individually deficient for plasminogen, tissue-type plasminogen activator, urokinase-type plasminogen activator, and urokinase-type plasminogen activator receptor, along with wild-type control mice. There were no differences in lung colony counts among all mouse genotypes throughout a 10-week infection. However, in tissue-type plasminogen activator and plasminogen-deficient mice an earlier dissemination of M. avium to other organs was observed. Nevertheless, the M. avium growth rates in the liver, spleen, and lung did not differ between the various mouse populations throughout a 10-week infection. Histochemical and immunohistochemical analyses at 5 and 10 weeks after infection demonstrated that plasminogen-deficient mice, compared to wild-type mice, had enhanced fibrin and fibronectin deposition, as well as increased neutrophil infiltration within liver granulomas. These results suggest that plasmin(ogen) plays a role in the turnover of extracellular matrix proteins within granulomas and has a limited effect in the early dissemination of M. avium from lungs. Thus, plasmin(ogen) functions in limiting progressive fibrosis in the granuloma during a chronic mycobacterial infection.

Animals↗
PubMed1994

Simulation of the dynamics of protozoa in the rumen.

A modified mathematical model is described that simulates the dynamics of rumen micro-organisms, with specific emphasis on the rumen protozoa. The model is driven by continuous inputs of nutrients and consists of nineteen state variables, which represent the N, carbohydrate, fatty acid and microbial pools in the rumen. Several protozoal characteristics were represented in the model, including preference for utilization of starch and sugars compared with fibre, and of insoluble compared with soluble protein; engulfment and storage of starch; no utilization of NH3 to synthesize amino acids; engulfment and digestion of bacteria and protozoa; selective retention within the rumen; death and lysis related to nutrient availability. Comparisons between model predictions and experimental observations showed reasonable agreement for protozoal biomass in the rumen, but protozoal turnover time was not predicted well. Sensitivity analyses highlighted the need for more reliable estimates of bacterial engulfment rate, protozoal maintenance requirement, and death rate. Simulated protozoal biomass was increased rapidly in response to increases in dietary starch content, but further increases in starch content of a high-concentrate diet caused protozoal mass to decline. Increasing the sugar content of a concentrate diet, decreased protozoa, while moderate elevations of the sugar content on a roughage diet increased protozoal biomass. Simulated protozoal biomass did not change in response to variations in dietary neutral-detergent fibre (NDF) content. Reductions in dietary N resulted in an increased protozoal biomass. Depending on the basal intake level and dietary composition, protozoal concentration in the rumen was either increased or decreased by changes in feed intake level. Such changes in relative amounts of protozoal and bacterial biomass markedly affected the supply of nutrients available for absorption. The integration of protozoal, bacterial and dietary characteristics through mathematical representation provided an improved understanding of mechanisms of protozoal responses to changes in dietary inputs.

Animals↗
PubMed2006

Estimating high-affinity methanotrophic bacterial biomass, growth, and turnover in soil by phospholipid fatty acid 13C labeling.

A time series phospholipid fatty acid (PLFA) 13C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH4 concentrations using synthetic air containing 2 parts per million of volume of 13CH4. Flowthrough chambers maintained a stable CH4 concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of 13C label into the microbial biomass. Incorporation of the 13C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The delta13C values of individual PLFAs showed that 13C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of 13C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant 13C-labeled PLFA was 18:1omega7c, with 16:1omega5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 x 10(6) cells g(-1) of soil (dry weight). While recycling of 13C label from the methanotrophic biomass must occur, it is a slower process than initial 13CH4 incorporation, with only about 5 to 10% of 13C-labeled PLFAs reflecting this process. Thus, 13C-labeled PLFA distributions determined at any time point during 13CH4 incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum 13C labeling for methanotrophic biomass determinations.

Alphaproteobacteria↗
PubMed1988

Control of Interspecies Electron Flow during Anaerobic Digestion: Significance of Formate Transfer versus Hydrogen Transfer during Syntrophic Methanogenesis in Flocs.

Microbial formate production and consumption during syntrophic conversion of ethanol or lactate to methane was examined in purified flocs and digestor contents obtained from a whey-processing digestor. Formate production by digestor contents or purified digestor flocs was dependent on CO(2) and either ethanol or lactate but not H(2) gas as an electron donor. During syntrophic methanogenesis, flocs were the primary site for formate production via ethanol-dependent CO(2) reduction, with a formate production rate and methanogenic turnover constant of 660 muM/h and 0.044/min, respectively. Floc preparations accumulated fourfold-higher levels of formate (40 muM) than digestor contents, and the free flora was the primary site for formate cleavage to CO(2) and H(2) (90 muM formate per h). Inhibition of methanogenesis by CHCl(3) resulted in formate accumulation and suppression of syntrophic ethanol oxidation. H(2) gas was an insignificant intermediary metabolite of syntrophic ethanol conversion by flocs, and its exogenous addition neither stimulated methanogenesis nor inhibited the initial rate of ethanol oxidation. These results demonstrated that >90% of the syntrophic ethanol conversion to methane by mixed cultures containing primarily Desulfovibrio vulgaris and Methanobacterium formicicum was mediated via interspecies formate transfer and that <10% was mediated via interspecies H(2) transfer. The results are discussed in relation to biochemical thermodynamics. A model is presented which describes the dynamics of a bicarbonate-formate electron shuttle mechanism for control of carbon and electron flow during syntrophic methanogenesis and provides a novel mechanism for energy conservation by syntrophic acetogens.

Journal Article↗