Biochar alters the soil microbiome and soil function: results of next generation amplicon sequencing across Europe

Jenkins JR, Viger M, Arnold EC, Harris ZM, Ventura M, Miglietta F, Girardin C, Edwards RJ, Rumpel C, Fornasier F, Zavalloni C, Tonon G, Alberti G & Taylor G (2016): Biochar alters the soil microbiome and soil function: results of next generation amplicon sequencing across Europe. GCB Bioenergy Adv. Access DOI: 10.1111/gcbb.12371

Abstract

Wide scale application of biochar to soil has been suggested as a mechanism to offset increases in CO2 emissions through the long-term sequestration of a carbon rich and inert substance to the soil, but the implications of this for soil diversity and function remain to be determined. Biochar is capable of inducing changes in soil bacterial communities, but the exact impacts of its application are poorly understood. Using three European sites (UK SRC, short rotation coppice, French grassland (FR) and Italian SRF, short rotation forestry (IT)) treated with identical biochar applications; we undertook 16S and ITS amplicon DNA sequencing. In addition, we carried out assessments of community change over time and N and P mobilisation in the UK.

Significant changes in bacterial and community structure occurred due to treatment, although the nature of the changes varied by site. STAMP differential abundance analysis showed enrichment of Gemmatimonadete and Acidobacteria in UK biochar plots one year after application, whilst control plots exhibited enriched Gemmataceae, Isosphaeraceae and Koribacteraceae. Increased mobility of ammonium and phosphates were also detected after one year, coupled with a shift from acid to alkaline phophomonoesterase activity, which may suggest an ecological and functional shift towards a more copiotrophic ecology. Italy also exhibited enrichments, in both the Proteobacteria (driven by an increase in the order Rhizobiales) and the Gemmatimonadetes. No significant change in the abundance of individual taxa were noted in FR, although a small significant change in unweighted UNIFRAC occurred, indicating variation in the identities of taxa present due to treatment. Fungal β diversity was affected by treatment in IT and FR, but was unaffected in UK samples. The effects of time and site were greater than that of biochar application in UK samples. Overall, this report gives a tantalising view of the soil microbiome at several sites across Europe, and suggests that although application of biochar has significant effects on microbial communities, these may be small compared with the highly variable soil microbiome that is found in different soils and changes with time.

Parallel evolution in Streptococcus pneumoniae biofilms

Churton NWV, Misra RV, Howlin RP, Allan RN, Jefferies J, Faust SN, Gharbia SE, Edwards RJ, Clarke SC & Webb JS (2016): Parallel evolution in Streptococcus pneumoniae biofilms. Genome Biology and Evolution Adv. Access doi: 10.1093/gbe/evw072

Abstract

Streptococcus pneumoniae is a commensal human pathogen and the causative agent of various invasive and non-invasive diseases. Carriage of the pneumococcus in the nasopharynx is thought to be mediated by biofilm formation, an environment where isogenic populations frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. We employed metabolic characterization and whole genome sequencing of biofilm-derived S. pneumoniae serotype 22F pneumococcal SCVs to investigate diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles, and increased biofilm formation compared to the ancestral strain. Whole genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51-264 bp, one insertion resulting in a coding frameshift, and seven nonsense single nucleotide substitutions that result in a truncated gene product. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae, and therefore may have important implications for colonization, carriage and persistence of the organism. Furthermore, recurrent mutation of the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development.

HSP-4 endoplasmic reticulum (ER) stress pathway is not activated in a C. elegans model of ethanol intoxication and withdrawal

Ient B, Edwards RJ, Mould R, Hannah M, Holden-Dye LM & O’Connor V (2012): HSP-4 endoplasmic reticulum (ER) stress pathway is not activated in a C. elegans model of ethanol intoxication and withdrawal. Invertebrate Neuroscience 12(2): 93-102.

Abstract

Acute and chronic exposure of Caenorhabditis elegans to concentrations of ethanol in the range 250-350 mM elicits distinct behaviours. Previous genetic analysis highlights specific neurobiological substrates for these effects. However, ethanol may also elicit cellular stress responses which may contribute to the repertoire of ethanol-induced behaviours. Here, we have studied the effect of ethanol on an important arm of the cellular stress pathways, which emanates from the endoplasmic reticulum (ER) in response to several conditions including heat shock and chemical or genetic perturbations that lead to protein misfolding. HSP-4 is a heat shock protein and homologue of mammalian BiP. It is a pivotal upstream component of the ER stress response. Therefore, we used a C. elegans heat shock protein mutant, hsp-4, and a strain carrying a transcriptional reporter, Phsp-4::gfp, to test the role of the ER following chronic ethanol conditioning. We found no evidence for an overt ER response during acute or prolonged exposure to concentrations of ethanol that lead to defined ethanol-induced behaviours. Furthermore, whilst hsp-4 was strongly induced by tunicamycin, pre-exposure of C. elegans to low doses of tunicamycin followed by ethanol was not sufficient to induce an additive ER stress response. Behavioural analysis of an hsp-4 mutant indicated no difference compared to wild type in susceptibility to ethanol intoxication and withdrawal. There is a clear precedent for a significance of ER stress pathways particularly in clinical conditions associated with toxic or pathological effects of high doses of alcohol consumption. The concentrations of ethanol used in this C. elegans study equate to the highest blood alcohol levels measured in patients with chronic alcohol dependency. Taken together, these observations imply that the classic ER stress pathway in C. elegans is relatively refractory to induction by ethanol.

PMID: 22661239

Shotgun proteomic analysis of Emiliania huxleyi, a marine phytoplankton species of major biogeochemical importance

Jones BM*, Edwards RJ*, Skipp PJ, O’Connor CD & Iglesias-Rodriguez MD (2011): Shotgun Proteomic Analysis of Emiliania huxleyi, a Marine Phytoplankton Species of Major Biogeochemical Importance. Marine Biotechnology 13(3): 496-504. *Joint first authors

Abstract

Emiliania huxleyi is a unicellular marine phytoplankton species known to play a significant role in global biogeochemistry. Through the dual roles of photosynthesis and production of calcium carbonate (calcification), carbon is transferred from the atmosphere to ocean sediments. Almost nothing is known about the molecular mechanisms that control calcification, a process that is tightly regulated within the cell. To initiate proteomic studies on this important and phylogenetically remote organism, we have devised efficient protein extraction protocols and developed a bioinformatics pipeline that allows the statistically robust assignment of proteins from MS/MS data using preexisting EST sequences. The bioinformatics tool, termed BUDAPEST (Bioinformatics Utility for Data Analysis of Proteomics using ESTs), is fully automated and was used to search against data generated from three strains. BUDAPEST increased the number of identifications over standard protein database searches from 37 to 99 proteins when data were amalgamated. Proteins involved in diverse cellular processes were uncovered. For example, experimental evidence was obtained for a novel type I polyketide synthase and for various photosystem components. The proteomic and bioinformatic approaches developed in this study are of wider applicability, particularly to the oceanographic community where genomic sequence data for species of interest are currently scarce.

PMID: 20924652

Selective toxicity of the anthelmintic emodepside revealed by heterologous expression of human KCNMA1 in Caenorhabditis elegans

Crisford A, Murray C, O’Connor V, Edwards RJ, Kruger N, Welz C, von Samson-Himmelstjerna G, Harder A, Walker RJ & Holden-Dye L. (2011): Selective Toxicity of the Anthelmintic Emodepside Revealed by Heterologous Expression of Human KCNMA1 in Caenorhabditis elegans. Molecular Pharmacology 79 (6): 1031-43.

Abstract

Emodepside is a resistance-breaking anthelmintic of a new chemical class, the cyclooctadepsipeptides. A major determinant of its anthelmintic effect is the calcium-activated potassium channel SLO-1. SLO-1 belongs to a family of channels that are highly conserved across the animal phyla and regulate neurosecretion, hormone release, muscle contraction, and neuronal network excitability. To investigate the selective toxicity of emodepside, we performed transgenic experiments in which the nematode SLO-1 channel was swapped for a mammalian ortholog, human KCNMA1. Expression of either the human channel or Caenorhabditis elegans slo-1 from the native slo-1 promoter in a C. elegans slo-1 functional null mutant rescued behavioral deficits that otherwise resulted from loss of slo-1 signaling. However, worms expressing the human channel were 10- to 100-fold less sensitive to emodepside than those expressing the nematode channel. Strains expressing the human KCNMA1 channel were preferentially sensitive to the mammalian channel agonists NS1619 and rottlerin. In the C. elegans pharyngeal nervous system, slo-1 is expressed in neurons, not muscle, and cell-specific rescue experiments have previously shown that emodepside inhibits serotonin-stimulated feeding by interfering with SLO-1 signaling in the nervous system. Here we show that ectopic overexpression of slo-1 in pharyngeal muscle confers sensitivity of the muscle to emodepside, consistent with a direct interaction of emodepside with the channel. Taken together, these data predict an emodepside-selective pharmacophore harbored by SLO-1. This has implications for the development of this drug/target interface for the treatment of helminth infections.

PMID: 21415309

Protein interactions with the platelet integrin alpha(IIb) regulatory motif

Raab M, Daxecker H, Edwards RJ, Treumann A, Murphy D & Moran N (2010): Protein interactions with the platelet integrin alpha(IIb) regulatory motif. Proteomics 10: 2790-2800.

Abstract

Integrins are transmembrane proteins regulating cellular shape, mobility and the cell cycle. A highly conserved signature motif in the cytoplasmic tail of the integrin alpha-subunit, KXGFFKR, plays a critical role in regulating integrin function. To date, six proteins have been identified that target this motif of the platelet-specific integrin alpha(IIb)beta(3). We employ peptide-affinity chromatography followed-up with LC-MS/MS analysis as well as protein chips to identify new potential regulators of integrin function in platelets and put them into their biological context using information from protein:protein interaction (PPI) databases. Totally, 44 platelet proteins bind with high affinity to an immobilized LAMWKVGFFKR-peptide. Of these, seven have been reported in the PPI literature as interactors with integrin alpha-subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin-tagged alpha-integrin cytoplasmic peptides. Two of these proteins are also identified in the peptide-affinity experiments, one is also found in the PPI databases and a further one is present in the data to all three approaches. Finally, novel short linear interaction motifs are common to a number of proteins identified.

PMID: 20486118

Bioinformatic discovery of novel bioactive peptides

Edwards RJ*, Moran N*, Devocelle M, Kiernan A, Meade G, Signac W, Foy M, Park SDE, Dunne E, Kenny D & Shields DC (2007): Bioinformatic discovery of novel bioactive peptides. Nature Chem. Biol. 3(2):108-112. *Joint first authors

Abstract

Short synthetic oligopeptides based on regions of human proteins that encompass functional motifs are versatile reagents for understanding protein signaling and interactions. They can either mimic or inhibit the parent protein’s activity and have been used in drug development. Peptide studies typically either derive peptides from a single identified protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. Our objective was to determine whether rational bioinformatic design of oligopeptides specifically targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function. High-throughput in vitro platelet function assays of palmitylated cell-permeable oligopeptides corresponding to these regions identified many agonists and antagonists of platelet function. Many bioactive peptides were from adhesion molecules, including a specific CD226-derived inhibitor of inside-out platelet signaling. Systematic screens of this nature are highly efficient tools for discovering short signaling motifs in molecular signaling pathways.

Comment in

A shortcut to peptides to modulate platelets. Nat Chem Biol. 2007.

PMID: 17220901

Monitoring Modulators of Platelet Aggregation in a Microtitre Plate Assay

Moran N, Kiernan A, Dunne E, Edwards RJ, Shields DC & Kenny D (2006): Monitoring Modulators of Platelet Aggregation in a Microtitre Plate Assay. Anal. Biochem. 357(1):77-84.

Abstract

Platelets play a central role in maintaining biological hemostasis. Inappropriate platelet activation is responsible for thrombotic diseases such as myocardial infarction and stroke. Therefore, novel agents that can inhibit platelet activation are necessary. However, assays that monitor platelet aggregation are generally time-consuming and require high volumes of blood and specialized equipment. Therefore, a medium- to high-throughput assay that can monitor platelet aggregation would be considered useful. Such an assay should be sensitive, comparable to the “gold standard” assay of platelet aggregometry, and able to monitor multiple samples simultaneously but with low assay volumes. We have developed such a microtiter assay. It can assay an average of 60 independent treatments per 60 ml blood donation and demonstrates greater sensitivity than the current gold standard assay, namely platelet aggregation in stirring conditions in a platelet aggregometer. The microtiter plate (MTP) assay can detect known inhibitors of platelet function such as indomethacin, aspirin, and ReoPro. It is highly reproducible when using standard doses of agonists such as thrombin receptor-activating peptide (20 microM) and collagen (0.19 mg/ml). Finally, the MTP assay is rapid and sensitive and can detect unknown platelet-modulating agents from a library of compounds.

PMID: 16920064

A simple method for genome-wide screening for advantageous insertions of mobile DNAs in Escherichia coli

Edwards RJ, Sockett RE & Brookfield JFY (2002): A simple method for genome-wide screening for advantageous insertions of mobile DNAs in Escherichia coli. Curr Biol. 12(10):863-867.

Abstract

Laboratory evolution in Escherichia coli has revealed that fitness typically increases in experimental populations. These changes are sometimes associated with changes in insertion sequence positions, some of which may themselves cause advantageous phenotypes. We have a novel and general method for identifying genes in Escherichia coli, whose knockout by mobile DNA insertions is beneficial in experimental evolution. Insertion sites in favored clones can be identified by reference to genomic information. We have implemented the method using modified Tn10 transposons bearing kanamycin and chloramphenicol resistance cassettes. Results are consistent across replicated experiments, demonstrating that the insertions are themselves creating selective advantages, rather than hitch-hiking with favorable base substitutions. The successful clones have subsequently been confirmed to have a fitness advantage relative to the progenitor strain. In experiments in shaking culture, we find that advantageous insertions usually fall in operons required in the pathways creating flagella. The method allows a rapid genome-wide screening for advantageous insertions in arbitrary environmental conditions. It allows investigation of the extent to which transient mutations generating environment-dependent selective advantages may help to explain the persistence of mobile DNAs in primarily clonal organisms, such as E. coli.

PubMed: 12015126