Substrate specificity of branched chain amino acid aminotransferases: The substitution of glycine to serine in the active site determines the substrate specificity for α-ketoglutarate

A branched chain aminotransferase from Thermoproteus tenax has been identi fi ed, cloned, over-expressed and biochemically characterised. A molecular modelling approach has been used to predict the 3D structure allowing its comparison with other related enzymes. This enzyme has high similarity to a previously characterised aminotransferase from Thermoproteus uzoniensis however its substrate speci fi city shows key differences towards the substrate α -ketoglutarate. Examination of the active sites of the two related enzymes reveals a single amino acid substitution of a glycine residue to a serine residue which could be responsible for this difference. When Gly104 in T. tenax was mutated to a serine residue and the resultant enzyme characterised, this single amino acid change resulted in a dramatic reduction in activity towards α - ketoglutarate with an 18-fold reduction in Vmax and a 20-fold Km increase, resulting in a 370-fold lower catalytic ef fi ciency. Structural comparisons between the two related Thermoproteus enzymes and another branched chain aminotransferase from Geoglobus acetivorans has revealed that the serine residue affects the fl exibility of a key loop involved in catalysis. This subtle difference has provided further insight into our understanding of the substrate speci fi city of these industrially important enzymes

C/EBP-induced transdifferentiation reveals granulocyte-macrophage precursor-like plasticity of B cells

The lymphoid-myeloid transdifferentiation potentials of members of the C/EBP family (C/EBP{alpha}, {beta}, {delta}, and {epsilon}) were compared in v-Abl-immortalized primary B cells. Conversion of B cells to macrophages was readily induced by the ectopic expression of any C/EBP, and enhanced by endogenous C/EBP{alpha} and {beta} activation. High transgene expression of C/EBP{beta} or C/EBP{epsilon}, but not of C/EBP{alpha} or C/EBP{delta}, also induced the formation of granulocytes. Granulocytes and macrophages emerged in a mutually exclusive manner. C/EBP{beta}-expressing B cells produced granulocyte-macrophage progenitor (GMP)-like progenitors when subjected to selective pressure to eliminate lymphoid cells. The GMP-like progenitors remained self-renewing and cytokine-independent, and continuously produced macrophages and granulocytes. In addition to their suitability to study myelomonocytic lineage bifurcation, lineage-switched GMP-like progenitors could reflect the features of the lympho-myeloid lineage switch observed in leukemic progression

Genomic characterization of murine monocytes reveals C/EBPβ transcription factor dependence of Ly6C(-) cells

Monocytes are circulating, short-lived mononuclear phagocytes, which in mice and man comprise two main subpopulations. Murine Ly6C(+) monocytes display developmental plasticity and are recruited to complement tissue-resident macrophages and dendritic cells on demand. Murine vascular Ly6C(-) monocytes patrol the endothelium, act as scavengers, and support vessel wall repair. Here we characterized population and single cell transcriptomes, as well as enhancer and promoter landscapes of the murine monocyte compartment. Single cell RNA-seq and transplantation experiments confirmed homeostatic default differentiation of Ly6C(+) into Ly6C(-) monocytes. The main two subsets were homogeneous, but linked by a more heterogeneous differentiation intermediate. We show that monocyte differentiation occurred through de novo enhancer establishment and activation of pre-established (poised) enhancers. Generation of Ly6C(-) monocytes involved induction of the transcription factor C/EBP{beta} and C/EBP{beta}-deficient mice lacked Ly6C(-) monocytes. Mechanistically, C/EBP{beta} bound the Nr4a1 promoter and controlled expression of this established monocyte survival factor

Activation of Methanogenesis in Arid Biological Soil Crusts Despite the Presence of Oxygen

Methanogenesis is traditionally thought to occur only in highly reduced, anoxic environments. Wetland and rice field soils are well known sources for atmospheric methane, while aerated soils are considered sinks. Although methanogens have been detected in low numbers in some aerated, and even in desert soils, it remains unclear whether they are active under natural oxic conditions, such as in biological soil crusts (BSCs) of arid regions. To answer this question we carried out a factorial experiment using microcosms under simulated natural conditions. The BSC on top of an arid soil was incubated under moist conditions in all possible combinations of flooding and drainage, light and dark, air and nitrogen headspace. In the light, oxygen was produced by photosynthesis. Methane production was detected in all microcosms, but rates were much lower when oxygen was present. In addition, the δ13C of the methane differed between the oxic/oxygenic and anoxic microcosms. While under anoxic conditions methane was mainly produced from acetate, it was almost entirely produced from H2/CO2 under oxic/oxygenic conditions. Only two genera of methanogens were identified in the BSC-Methanosarcina and Methanocella; their abundance and activity in transcribing the mcrA gene (coding for methyl-CoM reductase) was higher under anoxic than oxic/oxygenic conditions, respectively. Both methanogens also actively transcribed the oxygen detoxifying gene catalase. Since methanotrophs were not detectable in the BSC, all the methane produced was released into the atmosphere. Our findings point to a formerly unknown participation of desert soils in the global methane cycle

Deltaproteobacteria (Pelobacter) and Methanococcoides are responsible for choline-dependent methanogenesis in a coastal saltmarsh sediment

Coastal saltmarsh sediments represent an important source of natural methane emissions, much of which originates from quaternary and methylated amines, such as choline and trimethylamine. In this study, we combine DNA stable isotope probing with high throughput sequencing of 16S rRNA genes and 13C2-choline enriched metagenomes, followed by metagenome data assembly, to identify the key microbes responsible for methanogenesis from choline. Microcosm incubation with 13C2-choline leads to the formation of trimethylamine and subsequent methane production, suggesting that choline-dependent methanogenesis is a two-step process involving trimethylamine as the key intermediate. Amplicon sequencing analysis identifies Deltaproteobacteria of the genera Pelobacter as the major choline utilizers. Methanogenic Archaea of the genera Methanococcoides become enriched in choline-amended microcosms, indicating their role in methane formation from trimethylamine. The binning of metagenomic DNA results in the identification of bins classified as Pelobacter and Methanococcoides. Analyses of these bins reveal that Pelobacter have the genetic potential to degrade choline to trimethylamine using the choline-trimethylamine lyase pathway, whereas Methanococcoides are capable of methanogenesis using the pyrrolysine-containing trimethylamine methyltransferase pathway. Together, our data provide a new insight on the diversity of choline utilizing organisms in coastal sediments and support a syntrophic relationship between Bacteria and Archaea as the dominant route for methanogenesis from choline in this environment

The rumen microbial metagenome associated with high methane production in cattle

Acknowledgements The Rowett Institute of Nutrition and Health and SRUC are funded by the Rural and Environment Science and Analytical Services Division (RESAS) of the Scottish Government. The project was supported by Defra and the DA funded Agricultural Greenhouse Gas Inventory Research Platform, the Technology Strategy Board (Project No: TP 5903–40240) and the Biotechnology and Biological Sciences Research Council (BBSRC; BB/J004243/1, BB/J004235/1). Our thanks are due to the excellent support staff at the SRUC Beef and Sheep Research Centre, Edinburgh, and to Silvia Ramos Garcia for help in interrogating the data. MW and RR contributed equally to the paper and should be considered as joint last authors.Peer reviewedPublisher PD