Expression of divergent methyl/alkyl coenzyme M reductases from uncultured archaea.

Methanogens and anaerobic methane-oxidizing archaea (ANME) are important players in the global carbon cycle. Methyl-coenzyme M reductase (MCR) is a key enzyme in methane metabolism, catalyzing the last step in methanogenesis and the first step in anaerobic methane oxidation. Divergent mcr and mcr-like genes have recently been identified in uncultured archaeal lineages. However, the assembly and biochemistry of MCRs from uncultured archaea remain largely unknown. Here we present an approach to study MCRs from uncultured archaea by heterologous expression in a methanogen, Methanococcus maripaludis. Promoter, operon structure, and temperature were important determinants for MCR production. Both recombinant methanococcal and ANME-2 MCR assembled with the host MCR forming hybrid complexes, whereas tested ANME-1 MCR and ethyl-coenzyme M reductase only formed homogenous complexes. Together with structural modeling, this suggests that ANME-2 and methanogen MCRs are structurally similar and their reaction directions are likely regulated by thermodynamics rather than intrinsic structural differences.

Tuning Gene Expression by Phosphate in the Methanogenic Archaeon Methanococcus maripaludis.

Methanococcus maripaludis is a rapidly growing, hydrogenotrophic, and genetically tractable methanogen with unique capabilities to convert formate and CO2 to CH4. The existence of genome-scale metabolic models and an established, robust system for both large-scale and continuous cultivation make it amenable for industrial applications. However, the lack of molecular tools for differential gene expression has hindered its application as a microbial cell factory to produce biocatalysts and biochemicals. In this study, a library of differentially regulated promoters was designed and characterized based on the pst promoter, which responds to the inorganic phosphate concentration in the growth medium. Gene expression increases by 4- to 6-fold when the medium phosphate drops to growth-limiting concentrations. Hence, this regulated system decouples growth from heterologous gene expression without the need for adding an inducer. The minimal pst promoter is identified and contains a conserved AT-rich region, a factor B recognition element, and a TATA box for phosphate-dependent regulation. Rational changes to the factor B recognition element and start codon had no significant impact on expression; however, changes to the transcription start site and the 5' untranslated region resulted in the differential protein production with regulation remaining intact. Compared to a previous expression system based upon the histone promoter, this regulated expression system resulted in significant improvements in the expression of a key methanogenic enzyme complex, methyl-coenzyme M reductase, and the potentially toxic arginine methyltransferase MmpX.

Study of Fe-S Cluster Proteins in Methanococcus maripaludis , a Model Archaeal Organism.

Iron-sulfur (Fe-S) clusters are among the oldest and most versatile cofactors present in all domains of life. Many bacterial and eukaryotic Fe-S proteins have been well-characterized, whereas the archaeal ones are less studied. Fe-S proteins are particularly abundant and play essential roles in methanogenic archaea. Methanococcus maripaludis is a model methanogen with available genetic tools. Here, we describe the techniques for anaerobic cultivation of M. maripaludis with formate, liposome-mediated transformation, expression and anoxic affinity purification of Fe-S proteins, Fe-S cluster reconstitution, and analysis of Fe-S proteins by UV-visible absorption spectroscopy.

Identification of the fungal endophyte of Ammophila breviligulata (American beachgrass) as Epichloë amarillans.

The grass Ammophila breviligulata (American beachgrass) is known to host an endophyte of the genus Epichloë. Based on morphological characteristics it was originally identified as Acremonium typhinum var. ammophilae and is currently designated as Epichloë typhina var. ammophilae. However, the Epichloë species has not previously been identified based on DNA sequence data. Based on phylogenetic placement of beta-tubulin and translation elongation factor 1-alpha DNA sequences the endophyte is identified as a member of E. amarillans rather than E. typhina.

Pickering-Emulsion-Templated Encapsulation of a Hydrophilic Amine and Its Enhanced Stability Using Poly(allyl amine).

Efficient encapsulation of tetraethylenepentamine (TEPA), as an example aliphatic amine, was achieved by an emulsion-templated, in situ polymerization. Hydrophobically modified clay nanoplatelets were employed as emulsifiers to obtain water-in-oil (W/O) dispersions followed by interfacial polymerization between a portion of the TEPA cargo and polymethylene polyphenylene isocyanate (PMPPI). The resultant capsules exhibit spherical shape, desirable thermal stability, modest barrier properties, and shear-induced release in an epoxide monomer mixture. Most importantly, a significant gain in capsule barrier properties was realized by introducing poly(allyl amine) (pAAm) as an interface-selective reactive additive in the Pickering emulsions. In addition to the fundamental interest of pAAm localization and interface-selective reactivity, this microencapsulation system for aliphatic amines has technological potential in coating, self-healing, and drug-delivery applications.

Wide variety of bioserotypes of enteropathogenic Yersinia in tonsils of English pigs at slaughter.

The tonsils of 630 pigs from 45 English farms using three different rearing methods (Assured British Pigs, Open Management and Organic) were examined between 2003 and 2005 in order to investigate if the low incidence of human yersiniosis could be attributed to a low prevalence of enteropathogenic Yersinia among English pigs. In addition, different isolation methods were compared, possible differences in prevalence among pigs were studied, as well as the prevalence of different bioserotypes of enteropathogenic Yersinia. A high prevalence and a wide diversity of bioserotypes of enteropathogenic Yersinia compared to other European countries were observed. The prevalence of pathogenic Yersinia enterocolitica was 44% and of Yersinia pseudotuberculosis 18%. Overall, 60% of pigs carried enteropathogenic Yersinia. Y. pseudotuberculosis was detected on 78% of farms and Y. enterocolitica on 69%. The most common bioserotypes of Y. enterocolitica were 2/O:9 (33%) and 2/O:5 (26%), and of Y. pseudotuberculosis 2/O:3 (34%), 1/O:1 (26%) and 1/O:4 (24%). Cold enrichment gave the highest isolation rate for both species. Y. enterocolitica was more prevalent (P<0.001) and Y. pseudotuberculosis less prevalent (P<0.05) in winter than in summer in Eastern England. Y. enterocolitica was more common in Eastern England and in assured British pigs, whereas Y. pseudotuberculosis was more common in Western England and in organic pigs. Y. pseudotuberculosis 1/O:1 was predominant (P<0.05) in Western England. Types 1/O:4 (P<0.05) and 2/O:3 (P<0.001) predominated in Eastern England. The high prevalence of Y. enterocolitica bioserotypes 2/O:9 and 2/O:5 found in this study suggests that English pigs are an important reservoir of these bioserotypes whereas in other European countries bioserotype 4/O:3 predominates.

Modification of a French pressure cell to improve microbial cell disruption.

A procedure for modification of the valve stem of a 40 K French pressure cell is described. The modification should be done by a machinist and requires a metalworking lathe. After modification of the valve stem, a torlon 4203 plastic ball is used between the valve stem and valve seat to control the pressure within the cell. The torlon plastic ball is a key component needed to obtain the high pressures required for efficient disruption of microbial cells.

The local environment of Cu+ in Cu-Y zeolite and its relationship to the synthesis of dimethyl carbonate.

Cu-exchanged Y zeolite was investigated in order to determine the location of the copper cations relative to the zeolite framework and to determine which Cu cations are active for the oxidative carbonylation of methanol to dimethyl carbonate (DMC). Cu-Y zeolite was prepared by vapor-phase exchange of H-Y with CuCl. The oxidation state, local coordination, and bond distances of Al and Cu were determined using Al K-edge and Cu K-edge X-ray absorption spectroscopy (XAS). Complimentary information was obtained by H2 temperature-programmed reduction and by in-situ infrared spectroscopy. Cu-Y has a Cu/Al ratio of unity and very little occluded CuCl. The average Al-O and Al-Cu bond distances are 1.67 angstroms and 2.79 angstroms, respectively, and the average Cu-O and Cu-Si(Al) bond distances are 1.99 angstroms and 3.13 angstroms, respectively. All of the Cu exchanged is present as Cu+ in sites I', II, and III'. Cu-Y is active for the oxidative carbonylation of methanol, and at low reactant contact time produces DMC as the primary product. With increasing reactant contact time, DMC formation decreases in preference to the formation of dimethoxy methane (DMM) and methylformate (MF). The formation of DMM and MF is attributed to the hydrogenation of DMC and the hydrogenolysis of DMM, respectively. Observation of the catalyst under reaction conditions reveals that most of the copper cations remain as Cu+, but some oxidation of Cu+ to Cu2+ does occur. It is also concluded that only those copper cations present in site II and III' positions are accessible to the reactants, and hence are catalytically active. The dominant adsorbed species on the surface are methoxy groups, and adsorbed CO is present as a minority species. The relationship of these observations to the kinetics of DMC synthesis is discussed.

An in situ Al K-edge XAS investigation of the local environment of H+- and Cu+-exchanged USY and ZSM-5 zeolites.

Aluminum coordination in the framework of USY and ZSM-5 zeolites containing charge-compensating cations (NH4+, H+, or Cu+) was investigated by Al K-edge EXAFS and XANES. This work was performed using a newly developed in-situ cell designed especially for acquiring soft X-ray absorption data. Both tetrahedrally and octahedrally coordinated Al were observed for hydrated H-USY and H-ZSM-5, in good agreement with 27Al NMR analyses. Upon dehydration, water desorbed from the zeolite, and octahedrally coordinated Al was converted progressively to tetrahedrally coordinated Al. These observations confirmed the hypothesis that the interaction of water with Brønsted acid protons can lead to octahedral coordination of Al without loss of Al from the zeolite lattice. When H+ is replaced with NH4+ or Cu+, charge compensating species that absorb less water, less octahedrally coordinated Al was observed. Analysis of Al K-edge EXAFS data indicates that the Al-O bond distance for tetrahedrally coordinated Al in dehydrated USY and ZSM-5 is 1.67 angstroms. Simulation of k3chi(k) for Cu+ exchanged ZSM-5 leads to an estimated distance between Cu+ and framework Al atoms of 2.79 angstroms.

Liquid-phase oxidation of alkylaromatics by a H-atom transfer mechanism with a new heterogeneous CoSBA-15 catalyst.

A new pseudo-tetrahedral Co(II) complex has been grafted onto the surface of SBA-15 and successfully utilized for the catalytic oxidization of alkylaromatic substrates with tert-butyl hydroperoxide (TBHP)via an H-atom transfer mechanism.

Atomic level control over surface species via a molecular precursor approach: isolated Cu(I) sites and Cu nanoparticles supported on mesoporous silica.

A nonaqueous molecular precursor grafting approach was employed for the generation of well-defined surface structures featuring Cu on a mesoporous silica support. X-ray absorption measurements (XANES and EXAFS) were used to determine that [CuOSi(OtBu)3]4 provided 100% isolated Cu(I) sites upon grafting (without thermal treatment), whereas [CuOtBu]4 gave isolated species with most of the original Cu-O-Cu linkages intact, but in a more relaxed straight chain form. Upon heating under inert conditions, the vast majority of Cu in the materials from [CuOSi(OtBu)3]4 remained as isolated Cu(I) sites (up to 88% isolation), with significant stabilization provided from the -OSi(OtBu)3 ligands. In stark contrast, approximately 100% of the Cu in the materials generated from [CuOtBu]4 was readily reduced upon heating, forming isolated Cu metal particles with an average diameter of 0.7 nm.

A small bowel volvulus caused by a mesenteric lipoma: report of a case.

A 31-year-old man underwent a laparotomy for acute intestinal obstruction symptoms, which he had intermittently experienced for 14 years. The cause of the obstruction was due to a volvulus of the small bowel caused by a mesenteric lipoma. This is a rare finding, which is ideally diagnosed by computed tomography, with surgery the best and most highly recommended treatment. This particular presentation, to the best of our knowledge, has not yet been previously reported in the English language.

Antioxidant vitamin supplements do not reduce reactive oxygen species activity in Helicobacter pylori gastritis in the short term.

Reactive oxygen species have been implicated in Helicobacter pylori-mediated gastric carcinogenesis, whereas diets high in antioxidant vitamins C and E are protective. We have examined the effect of vitamin C and E supplements in combination with H. pylori eradication on reactive oxygen species activity in H. pylori gastritis. H. pylori-positive patients were randomized into four groups: triple therapy alone (Bismuth chelate, tetracycline, and metronidazole for 2 weeks), vitamins alone (200mg vitamin C and 50mg vitamin E, both twice per day for 4 weeks), both treatments or neither. Plasma and mucosal ascorbic acid, malondialdehyde and reactive oxygen species were determined before and after treatment. Compared with normal controls (n 61), H. pylori-positive patients (n 117) had higher mucosal reactive oxygen species and malondialdehyde levels and lower plasma ascorbic acid. Plasma ascorbic acid doubled in both groups of patients receiving vitamins and mucosal levels also increased. Malondialdehyde and reactive oxygen species fell in patients in whom H. pylori was eradicated but vitamin supplements were not effective either alone or in combination with H. pylori eradication. Supplements of vitamins C and E do not significantly reduce mucosal reactive oxygen species damage in H. pylori gastritis.