Enhanced heterologous expression of the cytochrome c from uncultured anaerobic methanotrophic archaea / 生物工程学报

Cytochrome c is a type of heme proteins that are widely distributed in living organisms. It consists of heme and apocytochrome c, and has potential applications in bioelectronics, biomedicine and pollutant degradation. However, heterologous overexpression of cytochrome c is still challenging. To date, expression of the cytochrome c from uncultured anaerobic methanotrophic archaea has not been reported, and nothing is known about the function of this cytochrome c. A his tagged cytochrome c was successfully expressed in E. coli by introducing a thrombin at the N-terminus of CytC4 and co-expressing CcmABCDEFGH, which is responsible for the maturation of cytochrome c. Shewanella oneidensis, which naturally has enzymes for cytochrome c maturation, was then used as a host to further increase the expression of CytC4. Indeed, a significantly higher expression of CytC4 was achieved in S. oneidensis when compared with in E. coli. The successful heterologous overexpression of CytC4 will facilitate the exploitation of its physiological functions and biotechnological applications.

Expression and characterization of a bifunctional thermal β-glucosidase IuBgl3 from thermophilic archaeon Infirmifilum uzonense / 生物工程学报

β-glucosidase has important applications in food, medicine, biomass conversion and other fields. Therefore, exploring β-glucosidase with strong stability and excellent properties is a research hotspot. In this study, a GH3 family β-glucosidase gene named Iubgl3 was successfully cloned from Infirmifilum uzonense. Sequence analysis showed that the full length of Iubgl3 was 2 106 bp, encoding 702 amino acids, with a theoretical molecular weight of 77.0 kDa. The gene was cloned and expressed in E. coli and the enzymatic properties of purified IuBgl3 were studied. The results showed that the optimal pH and temperature for pNPG hydrolysis were 5.0 and 85 ℃, respectively. The enzyme has good thermal stability, and more than 85% of enzyme activity can be retained after being treated at 80 ℃ for2 h. This enzyme has good pH stability and more than 85% of its activity can be retained after being treated at pH 4.0-11.0 for 1 h. It was found that the enzyme had high hydrolysis ability to p-nitrophenyl β-d-glucoside (pNPG) and p-nitrophenyl β-d-xylopyranoside (pNPX). When pNPG was used as the substrate, the kinetic parameters Km and Vmax were 0.38 mmol and 248.55 μmol/(mg·min), respectively, and the catalytic efficiency kcat/Km was 6 149.20 s-1mmol-1. Most metal ions had no significant effect on the enzyme activity of IuBgl3. SDS completely inactivated the enzyme, while EDTA increased the enzyme activity by 30%. This study expanded the β-glucosidase gene diversity of the thermophilic archaea GH3 family and obtained a thermostable acid bifunctional enzyme with good industrial application potential.

Advances of structure, function, and catalytic mechanism of methyl-coenzyme M reductase / 生物工程学报

Methanogens are unique microorganisms for methane production and the main contributor of the biogenic methane in atmosphere. Methyl-coenzyme M reductase (Mcr) catalyzes the last step of methane production in methanogenesis and the first step of methane activation in anaerobic oxidation of methane. The genes encoding this enzyme are highly conserved and are widely used as a marker in the identification and phylogenetic study of archaea. There has been a longstanding interest in its unique cofactor F430 and the underpinning mechanisms of enzymatic cleavage of alkane C-H bond. The recent breakthroughs of high-resolution protein and catalytic-transition-state structures further advanced the structure-function study of Mcr. In particular, the recent discovery of methyl-coenzyme M reductase-like (Mcr-like) enzymes that activates the anaerobic degradation of non-methane alkanes has attracted much interest in the molecular mechanisms of C-H activation without oxygen. This review summarized the advances on function-structure-mechanism study of Mcr/Mcr-like enzymes. Additionally, future directions in anaerobic oxidation of alkanes and greenhouse-gas control using Mcr/Mcr-like enzymes were proposed.

The effect of introduction of chicken manure on the biodiversity and performance of an anaerobic digester

Background: Ammonium stress is a prime limiting phenomenon that occurs during methane formation from poultry manure. It is caused by elevated ammonium nitrogen concentrations that result from substrate decomposition. The amounts of methane formed depend on the activity of methanogenic microbes. Results: During the research reported in this paper, the response of a mesophilic consortium inhabiting a biogas reactor to rising load of poultry manure was investigated. The taxonomic composition of bacterial population was mostly typical, however syntrophic bacteria were not detected. This absence resulted in limitation of succession of some methanogenic microorganisms, especially obligate hydrogenotrophs. The methanogenic activity of the consortium was totally dependent on the activity of Methanosaeta. Inhibition of methanoganesis was noticed at ammonium nitrogen concentration of 3.68 g/L, total cessation occurred at 5.45 g/L. Significant amounts of acetic acid in the fermentation pulp accompanied the inhibition. Conclusions: The effectiveness of the consortium was totally dependent on the metabolic activity of the acetoclastic Methanoseata genus and lack of SAOB did not allow hydrogenotrophic methanogens to propagate and lead to cessation of biogas production at an elevated ammonium concentration at which acetoclastic methanogens were inhibited.

Characterization of a hyperthermophilic sulphur-oxidizing biofilm produced by archaea isolated from a hot spring

Background: Sulphur-oxidizing microorganisms are widely used in the biofiltration of total reduced sulphur compounds (odorous and neurotoxic) produced by industries such as the cellulose and petrochemical industries, which include high-temperature process steps. Some hyperthermophilic microorganisms have the capability to oxidize these compounds at high temperatures (N60°C), and archaea of this group, for example, Sulfolobus metallicus, are commonly used in biofiltration technology. Results: In this study, a hyperthermophilic sulphur-oxidizing strain of archaea was isolated from a hot spring (Chillán, Chile) and designated as M1. It was identified as archaea of the genus Sulfolobus (99% homology with S. solfataricus 16S rDNA). Biofilms of this culture grown on polyethylene rings showed an elemental sulphur oxidation rate of 95.15 ± 15.39 mg S l-1 d-1, higher than the rate exhibited by the biofilm of the sulphur-oxidizing archaea S. metallicus (56.8 ± 10.91 mg l-1 d-1). Conclusions: The results suggest that the culture M1 is useful for the biofiltration of total reduced sulphur gases at high temperatures and for other biotechnological applications.

Anaerobic microbiota: spatial-temporal changes in the sediment of a tropical coastal lagoon with ephemeral inlet in the Gulf of Mexico / Microbiota anaerobia: cambios espacio-temporales en el sedimento de una laguna costera tropical con boca efímera en el Golfo de México

Abstract:Sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) share common niches in coastal sediments during the terminal phases of the anaerobic mineralization of organic matter. The purpose of this study was to analyze the spatial - temporal variation of SRB and MA in the sediments of a tropical coastal lagoon with ephemeral inlet (La Mancha, Veracruz, Gulf of Mexico) and its relationship with environmental changes. A total of 24 sediment samples were collected during the dry (April, May), rainy (July, September) and Northern (November, February) seasons in the period 2013-2014. Microbiological analyses included the quantification of the viable SRB and MA with different substrates, as well as mineralization experiments to determine the effect of sulfate on acetate oxidation. The analyzed environmental variables in the sediments included: temperature, pH, Eh, salinity, sulfates, H2S, volatile solids, carbohydrates, and granulometric characteristics. Major changes occurred between the dry and rainy seasons. During the dry season, sulfate-reducing abundance was significantly greater with lactate (8.3x105 - 1.2x107 cells / g) and propionate (1.8x105 - 6.6x106 cells / g) as substrates, while the MA that use methanol were dominant (4.2x105 - 9.1x106 cells / g). In contrast, during the rainy season, hydrogenophylic (2.6x105 - 8.3x106 cells/g) and acetoclastic (5.4x105-6.4x106 cells / g) MA increased significantly and SRB decreased in the analyzed substrates. An apparent competition for acetate was observed, with a greater oxidation in the media with sulfates in the dry season (0.06 mM acetate / g sediment / day), and a greater oxidation in the media without sulfates in the rainy season (0.02 mM acetate / g sediment / day). SRB and MA were present throughout the sediment column, however SRB dominated in the first centimeters of the sediment while MA were abundant in deeper layers. In conclusion, SRB and MA together played a role in the mineralization of organic matter in the sediments of La Mancha lagoon, with sulfate-reduction dominating in the dry season (closed inlet) and methanogenesis during the rainy season (open inlet). Changes in rainfall and river input in this lagoon significantly affect salinity and sulfate content, the main factors that regulate the dynamics of SRB and MA in the sediments. Rev. Biol. Trop. 64 (4): 1759-1770. Epub 2016 December 01.

Resumen:Las bacterias sulfatorreductoras (BSR) y las arqueas metanogénicas (AM) comparten nichos comunes en los sedimentos costeros durante las fases terminales de la mineralización anaerobia de la materia orgánica. El objetivo del presente estudio fue analizar la variación espacio-temporal de las BSR y AM en los sedimentos de una laguna costera tropical con una boca efímera (La Mancha, Veracruz, Golfo de México) y su relación con los cambios ambientales. Un total de 24 muestras de sedimentos fueron recolectadas en las temporadas de secas (abril, mayo), lluvias (julio, septiembre) y nortes (noviembre, febrero) en el periodo 2013 - 2014. Los análisis microbiológicos incluyeron la cuantificación de las BSR y AM viables con diferentes sustratos, así como experimentos de mineralización para determinar el efecto de los sulfatos en la oxidación del acetato. Las variables ambientales analizadas en los sedimentos incluyeron la temperatura, pH, Eh, salinidad, sulfatos, H2S, sólidos volátiles, carbohidratos y las características granulométricas. Los principales cambios se presentaron entre las estaciones de secas y lluvias. En la temporada de secas la abundancia sulfatorreductora fue significativamente superior con lactato (8.3x105 - 1.2x107 células / g) y propionato (1.8x105 - 6.6x106 células / g) como sustratos, mientras que las AM que emplean metanol dominaron (4.2x105 - 9.1x106 células / g). Por el contrario, en la época de lluvias aumentaron significativamente las AM hidrogenofílicas (2.6x105-8.3x106 células / g) y acetoclásticas (5.4x105-6.4x106 células / g), disminuyendo las BSR con los sustratos analizados. Se determinó una competencia aparente por el acetato. Su oxidación fue mayor en los medios con presencia de sulfatos en las temporadas de secas (0.06 mM acetato / g sedimento / día), mientras que aumentó en los medios sin sulfatos en la época de lluvias (0.02 mM acetato / g sedimento / día). Las BSR y las AM estuvieron presentes a lo largo de la columna sedimentaria; sin embargo, las BSR dominaron en los primeros centímetros del sedimento y las AM abundaron a mayor profundidad. En conclusión las BSR y AM participaron de manera conjunta en la mineralización de la materia orgánica en los sedimentos de la laguna de La Mancha, dominando la sulfatorreducción en la temporada de secas (boca cerrada) y la metanogénesis en la época de lluvias (boca abierta). Los cambios en el aporte fluvial y la precipitación en esta laguna afectaron significativamente la salinidad y el contenido de sulfatos, siendo los principales factores que regularon la dinámica de las BSR y AM en los sedimentos.

Protein thermostability in Archaea and Eubacteria

In order to survive at high temperatures, thermophilic prokaryotes (Archaea and Eubacteria) adopt different strategies. Among several important contributing factors for stability of proteins are CG-rich codons, the ratio of charged amino acids compared to uncharged amino acids, ionic interactions, amino acid preferences and their distribution, post-translational modifications, and solute accumulation. However, these factors may differ from taxon to taxon, both within and between species depending upon the composition of proteins found in these organisms. This is exemplified in the case of differences in strategies adopted by soluble proteins and membrane proteins. Therefore, it appears that no single factor or combination of factors together can be universally attributed to the provision of thermal stability in proteins.

Extremely thermophilic microorganisms and their polymer-hydrolytic enzymes

Thermophilic and hyperthermophilic microorganisms are found as normal inhabitants of continental and submarine volcanic areas, geothermally heated sea-sediments and hydrothermal vents and thus are considered extremophiles. Several present or potential applications of extremophilic enzymes are reviewed, especially polymer-hydrolysing enzymes, such as amylolytic and hemicellulolytic enzymes. The purpose of this review is to present the range of morphological and metabolic features among those microorganisms growing from 70ºC to 100ºC and to indicate potential opportunities for useful applications derived from these features

Products of non-reductive CO2 assimilation in the halophilic archaebacterium Haloferax mediterranei.

The products of CO2 fixation by heterotrophically grown Haloferax mediterranei were analysed. The main 14C-labelled alpha-ketoacid detected following incubation with NaH14CO3 and pyruvate or propionate was pyruvate. In presence of these organic acids and NH4+, 14CO2 was incorporated into glutamic and aspartic acids and alanine.