Ammonia oxidation by the methane oxidising bacterium Methylococcus capsulatus strain bath

Soluble extracts of Methylococcus capsulatus (Bath) that readily oxidise methane to methanol will also oxidise ammonia to nitrite via hydroxylamine. The ammonia oxidising activity requires O₂, NADH and is readily inhibited by methane and specific inhibitors of methane mono-oxygenase activity. Hydroxylamine is oxidised to nitrite via an enzyme system that uses phenazine methosulphate (PMS) as an electron acceptor. The estimated K _mvalue for the ammonia hydroxylase activity was 87 mM but the kinetics of the oxidation were complex and may involve negative cooperativity.Abbreviations PMS Phenazine methosulphate - NADH nicotinamide adenine dinucleotide, reduced form - K _m Michaelis constant - NO ₂ ^- nitrite - NH₂OH hydroxylamine     

Target highlights in CASP13: Experimental target structures through the eyes of their authors

The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.     

Genetically encoded molecules for inducibly inactivating CaV channels

Voltage-gated Ca2+ (Ca(V)) channels are central to the biology of excitable cells, and therefore regulating their activity has widespread applications. We describe genetically encoded molecules for inducibly inhibiting Ca(V) channels (GEMIICCs). GEMIICCs are derivatives of Rem, a Ras-like GTPase that constitutively inhibits Ca2+ currents (I(Ca)). C terminus-truncated Rem(1-265) lost the ability to inhibit I(Ca) owing to loss of membrane targeting. Fusing the C1 domain of protein kinase Cgamma to yellow fluorescent protein (YFP)-Rem(1-265) generated a molecule that rapidly translocated from cytosol to plasma membrane with phorbol-12,13-dibutyrate in human embryonic kidney cells. Recombinant Ca(V)2.2 and Ca(V)1.2 channels were inhibited concomitantly with C1(PKCgamma)-YFP-Rem(1-265) membrane translocation. The generality of the approach was confirmed by creating a GEMIICC using rapamycin-dependent heterodimerization of YFP-FKBP-Rem(1-265) and a constitutively membrane-targeted rapamycin-binding domain. GEMIICCs reduced I(Ca) without diminishing gating charge, thereby ruling out decreased number of surface channels and voltage-sensor immobilization as mechanisms for inhibition. We introduce small-molecule-regulated GEMIICCs as potent tools for rapidly manipulating Ca2+ signals in excitable cells.     

Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells

Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to “ER stress” and activation of the “unfolded protein response” (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host.     

Divergence across diet,time and populations rules out parallel evolution in the gut microbiomes of Trinidadian guppies

Diverse microbial consortia profoundly influence animal biology, necessitating an understanding of microbiome variation in studies of animal adaptation. Yet, little is known about such variability among fish, in spite of their importance in aquatic ecosystems. The Trinidadian guppy, Poecilia reticulata, is an intriguing candidate to test microbiome-related hypotheses on the drivers and consequences of animal adaptation, given the recent parallel origins of a similar ecotype across streams. To assess the relationships between the microbiome and host adaptation, we used 16S rRNA amplicon sequencing to characterize gut bacteria of two guppy ecotypes with known divergence in diet, life history, physiology and morphology collected from low-predation (LP) and high-predation (HP) habitats in four Trinidadian streams. Guts were populated by several recurring, core bacteria that are related to other fish associates and rarely detected in the environment. Although gut communities of lab-reared guppies differed from those in the wild, microbiome divergence between ecotypes from the same stream was evident under identical rearing conditions, suggesting host genetic divergence can affect associations with gut bacteria. In the field, gut communities varied over time, across streams and between ecotypes in a stream-specific manner. This latter finding, along with PICRUSt predictions of metagenome function, argues against strong parallelism of the gut microbiome in association with LP ecotype evolution. Thus, bacteria cannot be invoked in facilitating the heightened reliance of LP guppies on lower-quality diets. We argue that the macroevolutionary microbiome convergence seen across animals with similar diets may be a signature of secondary microbial shifts arising some time after host-driven adaptation.