Pseudomonas chloritidismutans sp. nov., a non-denitrifying chlorate-reducing bacterium

A Gram-negative, facultatively anaerobic, rod-shaped, dissimilatory chlorate-reducing bacterium, strain AW-1(T), was isolated from biomass of an anaerobic chlorate-reducing bioreactor. Phylogenetic analysis of the 16S rDNA sequence showed 100␜equence similarity to Pseudomonas stutzeri DSM 50227 and 98.6␜equence similarity to the type strain of P. stutzeri (DSM 5190(T)). The species P. stutzeri possesses a high degree of genotypic and phenotypic heterogeneity. Therefore, eight genomic groups, termed genomovars, have been proposed based upon DeltaT(m) values, which were used to evaluate the quality of the pairing within heteroduplexes formed by DNA--DNA hybridization. In this study, DNA--DNA hybridization between strain AW-1(T) and P. stutzeri strains DSM 50227 and DSM 5190(T) revealed respectively 80.5 and 56.5␜imilarity. DNA--DNA hybridization between P. stutzeri strains DSM 50227 and DSM 5190(T) revealed 48.4␜imilarity. DNA--DNA hybridization indicated that strain AW-1(T) is not related at the species level to the type strain of P. stutzeri. However, strain AW-1(T) and P. stutzeri DSM 50227 are related at the species level. The physiological and biochemical properties of strain AW-1(T) and the two P. stutzeri strains were compared. A common characteristic of P. stutzeri strains is the ability to denitrify. However, in growth experiments, strain AW-1(T) could use only chlorate or oxygen as an electron acceptor and not nitrate, perchlorate or bromate. Strain AW-1(T) is the first chlorate-reducing bacterium described that does not possess another oxyanion-reduction pathway. Cell extracts of strain AW-1(T) showed chlorate and bromate reductase activities but not nitrate reductase activity. P. stutzeri strains DSM 50227 and DSM 5190(T) could use nitrate or oxygen as an electron acceptor, but not chlorate. Chlorate reductase activity, in addition to nitrate reductase activity, was detected in cell extracts of both P. stutzeri strains. Chlorite dismutase activity was absent in extracts of both P. stutzeri strains but was present in extracts of strain AW-1(T). Based on the hybridization experiments and the physiological and biochemical data, it is proposed that strain AW-1(T) be classified as a novel species of Pseudomonas, Pseudomonas chloritidismutans sp. nov. The type strain is strain AW-1(T) (=DSM 13592(T)=ATCC BAA-443(T))

Extremofiele micro-organismen: some like it hot

Een duik nemen in de Noordzee op nieuwjaarsdag vinden we behoorlijk stoer. De Russen doen er een schepje bovenop door het begin van de winter te vieren met een duik in ijswater van nul graden. Dat valt best wel mee, zeker wanneer je dit afwisselt met een bezoek aan de sauna met een luchttemperatuur van 90 graden. Hiermee hebben we de extremen die we als mens kunnen trotseren echter wel gehad. Wat te denken van een duik in kokend water, zwemmen in een zwembad gevuld met azijn of ammonia, of kokend zwavelzuur? Wat voor ons onmogelijk is, is voor veel micro-organismen de normaalste zaak van de werel

Substrate and product inhibition of hydrogen production by the extreme thermophile, Caldicellulosiruptor saccharolyticus.

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H2 in the gas phase ( partial hydrogen pressure (pH2) of (1-2) · 104 Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H2 ( pH2 of 5.6 · 104 Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70°C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 255-262, 2003

Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain pnitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold 2 enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.This work was supported by the Hotzyme project (grant agreement no. 265933) financed by the European Union 7th Framework Programme FP7/2007-2013. WF is funded by a BBSRC PhD studentship. MI would like to thank the BBSRC funded ERA-IB grant BB/L002035/1 and the University of Exeter for support. The authors would like to thank the Diamond Synchrotron Light Source for access to beamline I03 (proposals No. MX8889 and No. MX11945) and the beamline scientists for assistance. The work of ML was funded by the Graduate School VLAG Wageningen, the Netherlan

The role of ethanol oxidation during carboxydotrophic growth of clostridium autoethanogenum

The WoodLjungdahl pathway is an ancient metabolic route used by acetogenic carboxydotrophs to convert CO into acetate, and some cases ethanol. When produced, ethanol is generally seen as an end product of acetogenic metabolism, but here we show that it acts as an important intermediate and co-substrate during carboxydotrophic growth of Clostridium autoethanogenum. Depending on CO availability, C. autoethanogenum is able to rapidly switch between ethanol production and utilization, hereby optimizing its carboxydotrophic growth. The importance of the aldehyde ferredoxin:oxidoreductase (AOR) route for ethanol production in carboxydotrophic acetogens is known; however, the role of the bifunctional alcohol dehydrogenase AdhE (AldAdh) route in ethanol metabolism remains largely unclear. We show that the mutant strain C. autoethanogenum adhE1a, lacking the Ald subunit of the main bifunctional aldehyde/alcohol dehydrogenase (AdhE, CAETHG\_3747), has poor ethanol oxidation capabilities, with a negative impact on biomass yield. This indicates that the AdhAld route plays a major role in ethanol oxidation during carboxydotrophic growth, enabling subsequent energy conservation via substrate-level phosphorylation using acetate kinase. Subsequent chemostat experiments with C. autoethanogenum show that the wild type, in contrast to adhE1a, is more resilient to sudden changes in CO supply and utilizes ethanol as a temporary storage for reduction equivalents and energy during CO-abundant conditions, reserving these stored assets for more CO-limited conditions. This shows that the direction of the ethanol metabolism is very dynamic during carboxydotrophic acetogenesis and opens new insights in the central metabolism of C. autoethanogenum and similar acetogens.info:eu-repo/semantics/publishedVersio

Biohydrogen Production from Glycerol using Thermotoga spp

Given the highly reduced state of carbon in glycerol and its availability as a substantial byproduct of biodiesel production, glycerol is of special interest for sustainable biofuel production. Glycerol was used as a substrate for biohydrogen production using the hyperthermophilic bacterium, Thermotoga maritima and Thermotoga neapolitana. Both species metabolized glycerol to mainly acetate and hydrogen. At glycerol concentrations of 2.5 g/L, hydrogen was produced with a yield of 2.75 and 2.65 mol H2/mol glycerol consumed by T. maritima and T. neapolitana respectively. Additionally, the effect of initial pH (ranging between pH 5.0-8.5) and yeast extract concentrations (0.5, 1, 2, 4 g/L) on glycerol fermentation by T. neapolitana was investigated in batch systems. An initial pH value of around 7 was optimal for hydrogen production by T. neapolitana. Lower concentration of yeast extract resulted in a lower H2 production, however increasing the concentration from 2 to 4 g/L did not affect H2 productio

Characterization of the chlorate reductase from Pseudomonas chloritidismutans

A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate. Differences were also found in N-terminal sequences, molecular weight, and subunit composition. Metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are also found in other dissimilatory oxyanion reductase