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Systematic Proteomic Analysis of Protein Methylation in Prokaryotes and Eukaryotes Revealed Distinct Substrate Specificity.

Related Articles Systematic Proteomic Analysis of Protein Methylation in Prokaryotes and Eukaryotes Revealed Distinct Substrate Specificity. Proteomics. 2017 Nov 18;: Authors: Zhang M, Xu JY, Hu H, Ye BC, Tan M Abstract The studies of protein methylation mainly focus on lysine and arginine residues due to their diverse roles in essential cellular processes from gene expression to signal transduction. Nevertheless, atypical protein methylation occurring on amino acid residues, such as glutamine and glutamic acid, was largely neglected until recently. In addition, the systematic analysis for the distribution of methylation on different amino acids in various species is still lacking, which hinders our understanding of its functional roles. In this study, we deeply explored the methylated sites in three species E. coli, S. cerevisiae, and HeLa cells by employing mass spectrometry-based proteomic approach coupled with heavy methyl SILAC method. We identified a total of 234 methylated sites on 187 proteins with high localization confidence, including 94 unreported methylated sites on 9 different amino acid residues. KEGG and GO analysis showed the pathways enriched with methylated proteins were mainly involved in central metabolism for E. coli and S. cerevisiae, but related to spliceosome for HeLa cells. The analysis of methylation preference on different amino acids were conducted in three species. Protein N-terminal methylation was dominant in E. coli while lysine and arginine methylation were widely identified in S. cerevisiae and HeLa cells, respectively. To study whether some atypical protein methylation have biological relevance in the pathological process in mammalian cells, we focused on histone methylation in diet-induced obese (DIO) mouse. Two glutamate methylation sites showed statistical significance in DIO mice compared with chow-fed mice, suggesting their potential roles in diabetes and obesity. Together, these findings expanded the methylome database from microbes to mammals, which will benefit our further appreciation for the protein methylation as well as its possible functions on disease. This article is protected by copyright. All rights reserved. PMID: 29150981 [PubMed - as supplied by publisher]

Genotype and diet affect resistance, survival, and fecundity but not fecundity tolerance.

Related Articles Genotype and diet affect resistance, survival, and fecundity but not fecundity tolerance. J Evol Biol. 2017 Nov 18;: Authors: Kutzer MAM, Kurtz J, Armitage SAO Abstract Insects are exposed to a variety of potential pathogens in their environment, many of which can severely impact fitness and health. Consequently, hosts have evolved resistance and tolerance strategies to suppress or cope with infections. Hosts utilising resistance improve fitness by clearing or reducing pathogen loads and hosts utilising tolerance reduce harmful fitness effects per pathogen load. To understand variation in, and selective pressures on resistance and tolerance we asked to what degree they are shaped by host genetic background, whether plasticity in these responses depends upon dietary environment, and whether there are interactions between these two factors. Females from ten wild-type Drosophila melanogaster genotypes were kept on high or low protein (yeast) diets, and infected with one of two opportunistic bacterial pathogens, Lactococcus lactis or Pseudomonas entomophila. We measured host resistance as the inverse of bacterial load in the early infection phase. The slope linking fly fecundity and individual-level bacteria load provided our fecundity tolerance measure. Genotype and dietary yeast determined host fecundity and strongly affected survival after infection with pathogenic P. entomophila. There was considerable genetic variation in host resistance, a commonly found phenomenon resulting from e.g. varying resistance costs or frequency-dependent selection. Despite this variation and the reproductive cost of higher P. entomophila loads, the slopes linking bacteria load and fecundity did not vary across genotypes. Absence of genetic variation in tolerance may suggest that at this early infection stage fecundity tolerance is fixed or that any evolved tolerance mechanisms are not expressed under these infection conditions. This article is protected by copyright. All rights reserved. PMID: 29150962 [PubMed - as supplied by publisher]

Regulation of Calcification in Human Aortic Smooth Muscle Cells Infected with High-glucose-treated Porphyromonas Gingivalis.

Related Articles Regulation of Calcification in Human Aortic Smooth Muscle Cells Infected with High-glucose-treated Porphyromonas Gingivalis. J Cell Physiol. 2017 Nov 18;: Authors: Chen TC, Lin CT, Chien SJ, Chang SF, Chen CN Abstract Porphyromonas (P.) gingivalis infection leading to the periodontitis has been associated with the development of systemic diseases, including cardiovascular diseases and diabetes. However, the effect of a high concentration of glucose (HG) on the invasion efficiency of P. gingivalis and the consequent modulation of pathogenesis in vascular cells, especially in the vascular smooth muscle cells (VSMCs), remains unclear. Hence, the aim of this study was to investigate whether treating P. gingivalis with HG could change its invasion capability and result in VSMC calcification and the underlying mechanism. Human aortic SMCs (HASMCs) and P. gingivalis strain CCUG25226 were used in this study. We found that HGPg infection of HASMCs could initiate the HASMC calcification by stimulating the autocrine regulation of bone morphogenetic protein (BMP) 4 in HASMCs. The upregulation of BMP4 expression in HASMCs was mediated by toll-like receptor 4 and ERK1/2-p38 signaling after P. gingivalis infection. Moreover, the autocrine action of BMP4 in HGPg infection-initiated HASMC calcification upregulated BMP4-specific downstream smad1/5/8-runx2 signaling to increase the expressions of bone-related matrix proteins, i.e. osteopontin, osteocalcin, and alkaline phosphatase. This study elucidates the detailed mechanism of HGPg infection-initiated calcification of HASMCs and indicates a possible therapeutic role of BMP4 in P. gingivalis infection-associated vascular calcification. This article is protected by copyright. All rights reserved. PMID: 29150938 [PubMed - as supplied by publisher]

High fat diet-induced oxidative stress blocks hepatocyte nuclear factor 4α and leads to hepatic steatosis in mice.

Related Articles High fat diet-induced oxidative stress blocks hepatocyte nuclear factor 4α and leads to hepatic steatosis in mice. J Cell Physiol. 2017 Nov 18;: Authors: Yu D, Chen G, Pan M, Zhang J, He W, Liu Y, Nian X, Sheng L, Xu B Abstract Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease with manifestation of over-accumulation of fat in liver. Increasing evidences indicate that NAFLD may be in part caused by malfunction of very low density lipoprotein (VLDL) secretion. Hepatocyte nuclear factor 4α (HNF4α), a nuclear receptor protein, plays an important role in sustain hepatic lipid homeostasis via transcriptional regulation of genes involved in secretion of VLDL, such as apolipoprotein B (ApoB). However, the exact functional change of HNF4α in NAFLD remains to be elucidated. In the present study, we found that high fat diet (HFD) induced cytoplasmic retention of HNF4α in hepatocytes, which led to down-regulation of hepatic ApoB expression and its protein level in serum, as well as reduced secretion of VLDL. We further revealed that oxidative stress, elevated in fatty liver, was the key factor inducing the cytoplasmic retention of HNF4α in hepatocytes by activating protein kinase C (PKC)-mediated phosphorylation in HNF4α. Thus, our findings reveal a novel mechanism underlying HFD-induced fatty liver that oxidative stress impairs function of HNF4α on ApoB expression and VLDL secretion via PKC activation, eventually promoting fat accumulation in the liver. Therefore, oxidative stress/PKC/HNF4α pathway may be a novel target to treat diet-induced fatty liver. This article is protected by copyright. All rights reserved. PMID: 29150932 [PubMed - as supplied by publisher]

Sustaining an efficient and effective CHO cell line development platform by incorporation of 24-deep well plate screening and multivariate analysis.

Related Articles Sustaining an efficient and effective CHO cell line development platform by incorporation of 24-deep well plate screening and multivariate analysis. Biotechnol Prog. 2017 Nov 18;: Authors: Mora A, Zhang SS, Carson G, Nabiswa B, Hossler P, Yoon S Abstract Efficient and effective cell line screening is paramount towards a successful biomanufacturing program. Here we describe the implementation of 24-deep well plate screening of CHO lines as part of the cell line development platform at AbbVie. Incorporation of this approach accelerated the identification of the best candidate lines for process development. In an effort to quantify and predict process performance comparability, we compared cell culture performance in 24-deep well plates (24-DWP) and in shake flasks, for a panel of Chinese Hamster Ovary cell lines expressing a monoclonal antibody. The results in 24-DWP screening showed reduced growth profiles, but comparable viability profiles. Slow growers in 24-DWP achieved the highest productivity improvement upon scaling-up to shake flasks. Product quality of the protein purified from shake flasks and 24-DWP were also compared. The 24-DWP culture conditions were found to influence the levels of acidic species, reduce the G0 N-glycan species, and increase the high-mannose N-glycan species. Nevertheless, the identification of undesirable profiles is executed consistently with the scaled-up culture. We further employed multivariate data analysis to capture differences depending on the two scales and we could demonstrate that cell line profiles were adequately clustered, regardless of the vessel used for the development. In conclusion, the 24-DWP platform was reasonably predictive of the parameters crucial for upstream process development activities, and has been adapted as part of the AbbVie cell line development platform. This article is protected by copyright. All rights reserved. PMID: 29150912 [PubMed - as supplied by publisher]

CK4, CK6, cyclin D1, p16(INK4a) and EGFR expression in glioblastoma with a primitive neuronal component.

Related Articles CK4, CK6, cyclin D1, p16(INK4a) and EGFR expression in glioblastoma with a primitive neuronal component. J Neurooncol. 2017 Nov 17;: Authors: Xu G, Li JY Abstract Glioblastoma with primitive neuroectodermal tumor-like component (GBM-PNET) is a rare variant of glioblastoma, which was renamed as glioblastoma with a primitive neuronal component (GBM-PN) in new WHO classification of tumours of the central nervous system in 2016. There are few publications on the investigation of GBM-PN. In this study, PCR mRNA arrays on 6 cases of conventional GBM and 10 cases of GBM-PN showed high mRNA level of CDK4 in GBM-PN and low mRNA level of EGFR in GBM-PN. Immunohistochemical stains on tissue microarrays with 28 cases of conventional GBM and 13 cases of GBM-PN demonstrated that CDK4 was selectively expressed in the primitive neuronal component of all GBM-PN cases while EGFR was positive in conventional GBM and glial component of GBM-PN, but was negative in the primitive neuronal component of all GBM-PN cases. Immunohistochemical stains with antibodies against proteins that interact with CDK4 in cell cycle regulation, such as CDK6, cyclin D1 and p16(INK4a), were performed on these GBM-PN and GBM cases. CDK6 was patchily positive in rare cases of GBM-PN and cyclin D1 was negative in GBM-PN cases. p16(INK4a) is traditionally known as an inhibitor of CDK4 and CDK6. p16(INK4a) might not be the inhibitor of CDK4 in GBM-PN cases because seven GBM-PN cases were positive for both CDK4 and p16(INK4a). It indicates that CDK4 and p16(INK4a) might play a crucial role in GBM-PN pathogenesis. Since CDK4 and EGFR are highly expressed in the primitive neuronal component and in the glial component of GBM-PN respectively, the combination of CDK4/6 inhibitor and targeted therapy against EGFR might be potential effective therapeutic regimen for GBM-PN. CDK4 and EGFR immuohistochemical stain patterns make the diagnosis of GBM-PN much easier. PMID: 29150788 [PubMed - as supplied by publisher]

TSPO imaging using the novel PET ligand [(18)F]GE-180: quantification approaches in patients with multiple sclerosis.

Related Articles TSPO imaging using the novel PET ligand [(18)F]GE-180: quantification approaches in patients with multiple sclerosis. EJNMMI Res. 2017 Oct 26;7(1):89 Authors: Vomacka L, Albert NL, Lindner S, Unterrainer M, Mahler C, Brendel M, Ermoschkin L, Gosewisch A, Brunegraf A, Buckley C, Kümpfel T, Rupprecht R, Ziegler S, Kerschensteiner M, Bartenstein P, Böning G Abstract BACKGROUND: PET ligands targeting the translocator protein (TSPO) represent promising tools to visualise neuroinflammation. Here, we analysed parameters obtained in dynamic and static PET images using the novel TSPO ligand [(18)F]GE-180 in patients with relapsing remitting multiple sclerosis (RRMS) and an approach for semi-quantitative assessment of this disease in clinical routine. Seventeen dynamic [(18)F]GE-180 PET scans of RRMS patients were evaluated (90 min). A pseudo-reference region (PRR) was defined after identification of the least disease-affected brain area by voxel-based comparison with six healthy controls (HC) and upon exclusion of voxels suspected of being affected in static 60-90 min p.i. images. Standardised uptake value ratios (SUVR) obtained from static images normalised to PRR were correlated to the distribution volume ratios (DVR) derived from dynamic data with Logan reference tissue model. RESULTS: Group comparison with HC revealed white matter and thalamus as most affected regions. Fewest differences were found in grey matter, and normalisation to frontal cortex (FC) yielded the greatest reduction in variability of healthy grey and white matter. Hence, FC corrected for affected voxels was chosen as PRR, leading to time-activity curves of FC which were congruent to HC data (SUV60-90 0.37, U test P = 0.42). SUVR showed a very strong correlation with DVR (Pearson ρ > 0.9). Focal MS lesions exhibited a high SUVR (range, 1.3-3.2). CONCLUSIONS: This comparison with parameters from dynamic data suggests that SUVR normalised to corrected frontal cortex as PRR is suitable for the quantification of [(18)F]GE-180 uptake in lesions and different brain regions of RRMS patients. This efficient diagnostic protocol based on static [(18)F]GE-180 PET scans acquired 60-90 min p.i. allows the semi-quantitative assessment of neuroinflammation in RRMS patients in clinical routine. PMID: 29150726 [PubMed]

Impact of prolonged overfeeding on skeletal muscle mitochondria in healthy individuals.

Related Articles Impact of prolonged overfeeding on skeletal muscle mitochondria in healthy individuals. Diabetologia. 2017 Nov 17;: Authors: Toledo FGS, Johannsen DL, Covington JD, Bajpeyi S, Goodpaster B, Conley KE, Ravussin E Abstract AIMS/HYPOTHESES: Reduced mitochondrial capacity in skeletal muscle has been observed in obesity and type 2 diabetes. In humans, the aetiology of this abnormality is not well understood but the possibility that it is secondary to the stress of nutrient overload has been suggested. To test this hypothesis, we examined whether sustained overfeeding decreases skeletal muscle mitochondrial content or impairs function. METHODS: Twenty-six healthy volunteers (21 men, 5 women, age 25.3 ± 4.5 years, BMI 25.5 ± 2.4 kg/m(2)) underwent a supervised protocol consisting of 8 weeks of high-fat overfeeding (40% over baseline energy requirements). Before and after overfeeding, we measured systemic fuel oxidation by indirect calorimetry and performed skeletal muscle biopsies to measure mitochondrial gene expression, content and function in vitro. Mitochondrial function in vivo was measured by (31)P NMR spectroscopy. RESULTS: With overfeeding, volunteers gained 7.7 ± 1.8 kg (% change 9.8 ± 2.3). Overfeeding increased fasting NEFA, LDL-cholesterol and insulin concentrations. Indirect calorimetry showed a shift towards greater reliance on lipid oxidation. In skeletal muscle tissue, overfeeding increased ceramide content, lipid droplet content and perilipin-2 mRNA expression. Phosphorylation of AMP-activated protein kinase was decreased. Overfeeding increased mRNA expression of certain genes coding for mitochondrial proteins (CS, OGDH, CPT1B, UCP3, ANT1). Despite the stress of nutrient overload, mitochondrial content and mitochondrial respiration in muscle did not change after overfeeding. Similarly, overfeeding had no effect on either the emission of reactive oxygen species or on mitochondrial function in vivo. CONCLUSIONS/INTERPRETATION: Skeletal muscle mitochondria are significantly resilient to nutrient overload. The lower skeletal muscle mitochondrial oxidative capacity in human obesity is likely to be caused by reasons other than nutrient overload per se. TRIAL REGISTRATION: ClinicalTrials.gov NCT01672632. PMID: 29150696 [PubMed - as supplied by publisher]

Urothelial cancer proteomics provides both prognostic and functional information.

Related Articles Urothelial cancer proteomics provides both prognostic and functional information. Sci Rep. 2017 Nov 17;7(1):15819 Authors: de Velasco G, Trilla-Fuertes L, Gamez-Pozo A, Urbanowicz M, Ruiz-Ares G, Sepúlveda JM, Prado-Vazquez G, Arevalillo JM, Zapater-Moros A, Navarro H, Lopez-Vacas R, Manneh R, Otero I, Villacampa F, Paramio JM, Vara JAF, Castellano D Abstract Traditionally, bladder cancer has been classified based on histology features. Recently, some works have proposed a molecular classification of invasive bladder tumors. To determine whether proteomics can define molecular subtypes of  muscle invasive urothelial cancer (MIUC) and allow evaluating the status of biological processes and its clinical value. 58 MIUC patients who underwent curative surgical resection at our institution between 2006 and 2012 were included. Proteome was evaluated by high-throughput proteomics in routinely archive FFPE tumor tissue. New molecular subgroups were defined. Functional structure and individual proteins prognostic value were evaluated and correlated with clinicopathologic parameters. 1,453 proteins were quantified, leading to two MIUC molecular subgroups. A protein-based functional structure was defined, including several nodes with specific biological activity. The functional structure showed differences between subtypes in metabolism, focal adhesion, RNA and splicing nodes. Focal adhesion node has prognostic value in the whole population. A 6-protein prognostic signature, associated with higher risk of relapse (5 year DFS 70% versus 20%) was defined. Additionally, we identified two MIUC subtypes groups. Prognostic information provided by pathologic characteristics is not enough to understand MIUC behavior. Proteomics analysis may enhance our understanding of prognostic and classification. These findings can lead to improving diagnosis and treatment selection in these patients. PMID: 29150671 [PubMed - in process]

A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris.

Related Articles A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris. Sci Rep. 2017 Nov 17;7(1):15817 Authors: Royle KE, Polizzi K Abstract Although recent advances in E. coli self-assembly have greatly simplified cloning, these have not yet been harnessed for the high-throughput generation of expression strains in the early research and discovery phases of biopharmaceutical production. Here, we have refined the technique and incorporated it into a streamlined workflow for the generation of Pichia pastoris expression strains, reducing the timeline by a third and removing the reliance on DNA editing enzymes, which often require troubleshooting and increase costs. We have validated the workflow by cloning 24 human proteins of biopharmaceutical value, either as direct therapeutics or as research targets, which span a continuous range in size and GC content. This includes demonstrating the applicability of the workflow to three-part assemblies for a monoclonal antibody and its single-chain antibody fragments derivatives. This workflow should enable future research into recombinant protein production by P. pastoris and a synthetic biology approach to this industrial host. PMID: 29150665 [PubMed - in process]

Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium.

Related Articles Axonemal Lumen Dominates Cytosolic Protein Diffusion inside the Primary Cilium. Sci Rep. 2017 Nov 17;7(1):15793 Authors: Luo W, Ruba A, Takao D, Zweifel LP, Lim RYH, Verhey KJ, Yang W Abstract Transport of membrane and cytosolic proteins in primary cilia is thought to depend on intraflagellar transport (IFT) and diffusion. However, the relative contribution and spatial routes of each transport mechanism are largely unknown. Although challenging to decipher, the details of these routes are essential for our understanding of protein transport in primary cilia, a critically affected process in many genetic diseases. By using a high-speed virtual 3D super-resolution microscopy, we have mapped the 3D spatial locations of transport routes for various cytosolic proteins in the 250-nm-wide shaft of live primary cilia with a spatiotemporal resolution of 2 ms and <16 nm. Our data reveal two spatially distinguishable transport routes for cytosolic proteins: an IFT-dependent path along the axoneme, and a passive-diffusion route in the axonemal lumen that escaped previous studies. While all cytosolic proteins tested primarily utilize the IFT path in the anterograde direction, differences are observed in the retrograde direction where IFT20 only utilizes IFT, and approximately half of KIF17 and one third of α-tubulin utilizes diffusion besides IFT. PMID: 29150645 [PubMed - in process]

Ml proteins from Mesorhizobium loti and MucR from Brucella abortus: an AT-rich core DNA-target site and oligomerization ability.

Related Articles Ml proteins from Mesorhizobium loti and MucR from Brucella abortus: an AT-rich core DNA-target site and oligomerization ability. Sci Rep. 2017 Nov 17;7(1):15805 Authors: Baglivo I, Pirone L, Pedone EM, Pitzer JE, Muscariello L, Marino MM, Malgieri G, Freschi A, Chambery A, Roop Ii RM, Pedone PV Abstract Mesorhizobium loti contains ten genes coding for proteins sharing high amino acid sequence identity with members of the Ros/MucR transcription factor family. Five of these Ros/MucR family members from Mesorhizobium loti (Ml proteins) have been recently structurally and functionally characterized demonstrating that Ml proteins are DNA-binding proteins. However, the DNA-binding studies were performed using the Ros DNA-binding site with the Ml proteins. Currently, there is no evidence as to when the Ml proteins are expressed during the Mesorhizobium lo ti life cycle as well as no information concerning their natural DNA-binding site. In this study, we examine the ml genes expression profile in Mesorhizobium loti and show that ml1, ml2, ml3 and ml5 are expressed during planktonic growth and in biofilms. DNA-binding experiments show that the Ml proteins studied bind a conserved AT-rich site in the promoter region of the exoY gene from Mesorhizobium loti and that the proteins make important contacts with the minor groove of DNA. Moreover, we demonstrate that the Ml proteins studied form higher-order oligomers through their N-terminal region and that the same AT-rich site is recognized by MucR from Brucella abortus using a similar mechanism involving contacts with the minor groove of DNA and oligomerization. PMID: 29150637 [PubMed - in process]

A program for iron economy during deficiency targets specific Fe proteins.

Related Articles A program for iron economy during deficiency targets specific Fe proteins. Plant Physiol. 2017 Nov 17;: Authors: Hantzis LJ, Kroh GE, Jahn CE, Cantrell M, Peers G, Pilon M, Ravet K Abstract Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis thaliana adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue, during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon iron resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome-b6f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid iron-sulfur cofactor assembly pathway were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency. PMID: 29150559 [PubMed - as supplied by publisher]

Molecular characterization of an Endozoicomonas-like organism causing infection in king scallop Pecten maximus L.

Related Articles Molecular characterization of an Endozoicomonas-like organism causing infection in king scallop Pecten maximus L. Appl Environ Microbiol. 2017 Nov 17;: Authors: Cano I, van Aerle R, Ross S, Verner-Jeffreys DW, Paley RK, Rimmer G, Ryder D, Hooper P, Stone D, Feist SW Abstract One of the fastest growing fisheries in the UK is the king scallop Pecten maximus L., also currently rated as the second most valuable. Mass mortality events in scallops have been reported world-wide, often with the causative agent(s) remaining uncharacterized. In May 2013 and 2014, two mass mortality events affecting king scallops were recorded in the Lyme Bay Marine Protected Area (MPA), in South West England. Histopathological examination showed gill epithelial tissues infected with intracellular microcolonies of bacteria (IMC), resembling rickettsia-like organisms (RLOs), often with bacteria released in vascular spaces. Large colonies were associated with cellular and tissue disruption of the gills. Ultrastructural examination confirmed the intracellular location of these organisms in affected epithelial cells.16S rRNA gene sequences of the putative IMC obtained from infected king scallop gill samples, collected from both mortality events, were identical, and had a 99.4% identity to 16S rRNA gene sequences obtained from Candidatus endonucleobacter bathymodioli and 95% with Endozoicomonas spp.In situ hybridization assays using 16S rRNA gene probes confirmed the presence of the sequenced IMC gene in the gill tissues. Additional DNA sequences of the bacterium were obtained using high-throughput (Illumina) sequencing and bioinformatic analysis identified over 1000 genes with high similarity to protein sequences from Endozoicomonas spp. (ranging from 77-87% identity).Specific PCR assays were developed and applied to screen for the presence of IMC 16S rRNA gene sequences in king scallop gill tissues collected in the Lyme Bay MPA during 2015 and 2016. There was 100% prevalence of the IMC in these gill tissues and the 16S rRNA gene sequences identified were identical to the sequence found during the previous mortality event.ImportanceMolluscan mass mortalities associated with IMC have been reported worldwide for many years, however, apart from histological and ultrastructural characterization, characterization of the aetiological agents is limited. In the present work, we provide detailed molecular characterization of an Endozoicomonas-like organism (ELO) associated with an important commercial scallop species. PMID: 29150518 [PubMed - as supplied by publisher]

Multi-omics reveal the lifestyle of the acidophilic, mineral-oxidizing model species Leptospirillum ferriphilum(T).

Related Articles Multi-omics reveal the lifestyle of the acidophilic, mineral-oxidizing model species Leptospirillum ferriphilum(T). Appl Environ Microbiol. 2017 Nov 17;: Authors: Christel S, Herold M, Bellenberg S, El Hajjami M, Buetti-Dinh A, Pivkin IV, Sand W, Wilmes P, Poetsch A, Dopson M Abstract Leptospirillum ferriphilum plays a major role in acidic, metal rich environments where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of this model species' type strain is available, limiting the possibilities to investigate the strategies and adaptations Leptospirillum ferriphilum(T) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilum(T) DSM 14647 obtained by PacBio SMRT long read sequencing for use as a high quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as substrate and bioleaching cultures containing chalcopyrite (CuFeS2). Leptospirillum ferriphilum(T) adaptations to growth on chalcopyrite included a possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, expression and translation of genes responsible for chemotaxis and motility were enhanced.IMPORTANCELeptospirillum ferriphilum is one of the most important iron-oxidizers in the context of acidic and metal rich environments during moderately thermophilic biomining. A high-quality circular genome of Leptospirillum ferriphilum(T) coupled with functional omics data provides new insights into its metabolic properties, such as the novel identification of genes for atmospheric nitrogen fixation, and represents an essential step for further accurate proteomic and transcriptomic investigation of this acidophile model species in the future. Additionally, light is shed on Leptospirillum ferriphilum(T) adaptation strategies to growth on the copper mineral chalcopyrite. This data can be applied to deepen our understanding and optimization of bioleaching and biooxidation, techniques that present sustainable and environmentally friendly alternatives to many traditional methods for metal extraction. PMID: 29150517 [PubMed - as supplied by publisher]

Loop of Streptomyces feruloyl esterase plays an important role in its activity of releasing ferulic acid from biomass.

Related Articles Loop of Streptomyces feruloyl esterase plays an important role in its activity of releasing ferulic acid from biomass. Appl Environ Microbiol. 2017 Nov 17;: Authors: Uraji M, Tamura H, Mizohata E, Arima J, Wan K, Ogawa K, Inoue T, Hatanaka T Abstract Feruloyl esterases (FAEs) are key enzymes required for the production of ferulic acid from agricultural biomass. Previously, we identified and characterized R18, an FAE from Streptomyces cinnamoneus NBRC 12852, which showed no sequence similarity with the known FAEs. To determine the region involved in its catalytic activity, we constructed chimeric enzymes using R18 and its homolog (TH2-18) from S. cinnamoneus strain TH-2. Although R18 and TH2-18 showed 74% identity in their primary sequence, the recombinant proteins of these two FAEs (rR18 and rTH2-18) showed very different specific activities toward ethyl ferulate. By comparing the catalytic activities of the chimeras, a domain comprised of residues 140 to 154 was found to be crucial for the catalytic activity of R18. Further, we analyzed the crystal structure of rR18 at a resolution of 1.5 Å to elucidate the relationship between its activity and structure. rR18 possessed a typical catalytic triad, consisting of Ser-191, Asp-214, and His-268, which was characteristic of the serine esterase family. By structural analysis, the above domain was found to be present in a loop-like structure (the R18 loop), which possessed a disulfide bond conserved in genus Streptomyces Moreover, compared to its parental strain rTH2-18, the TH2-18 mutant, in which Pro and Gly residues were inserted into the domain responsible for forming the R18 loop, showed markedly higher kcat values using artificial substrates. We also showed that the FAE activity of TH2-18 toward corn bran, a natural substrate, was improved by the insertion of the Gly and Pro residues.IMPORTANCEStreptomyces species are widely distributed bacteria that are predominantly present in soil and function as decomposers in natural environments. They produce various enzymes, such as carbohydrate hydrolases, esterases, and peptidases, which decompose agricultural biomass. In this study, based on the genetic information of two Streptomyces cinnamoneus strains, we identified novel feruloyl esterases (FAEs) capable of producing ferulic acid from biomass. These two FAEs shared high similarity in their amino acid sequences but did not resemblance any known FAEs. By comparing chimeric proteins and performing crystal structure analysis, we confirmed that a flexible loop was important for the catalytic activity of Streptomyces FAEs. Furthermore, we determined that the catalytic activity of one FAE improved drastically by inserting only two amino acids into its loop-forming domain. Thus, differences in the amino acid sequence of the loop resulted in different catalytic activities. In conclusion, our findings provide a foundation for the development of novel enzymes for industrial use. PMID: 29150515 [PubMed - as supplied by publisher]

High prevalence and genetic diversity of large phiCD211/phiCDIF1296T-like prophages in Clostridioides difficile.

Related Articles High prevalence and genetic diversity of large phiCD211/phiCDIF1296T-like prophages in Clostridioides difficile. Appl Environ Microbiol. 2017 Nov 17;: Authors: Garneau JR, Sekulovic O, Dupuy B, Soutourina O, Monot M, Fortier LC Abstract Clostridioides difficile (formerly Clostridium difficile) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified in C. difficile so far, with a genome of 131-kbp. It shares morphological and genomic similarity with other large siphophages like phage 949 infecting Lactococcus lactis and phage c-st infecting Clostridium botulinum. A PhageTerm analysis indicated the presence of 378-bp direct terminal repeats at the phiCD211 genome termini. Among striking features of phiCD211, the presence of several transposase and integrase genes suggests past recombination events with other mobile genetic elements. Several gene products potentially influence the bacterial lifestyle and fitness, including a putative AcrB/AcrD/AcrF multidrug resistance protein, an EzrA septation ring formation regulator, and a spore protease. We also identified a CRISPR locus and a cas3 gene. We screened 2,584 C. difficile genomes available and detected 149 prophages sharing ≥80% nucleotide identity with phiCD211 (5% prevalence). Overall, phiCD211-like phages were detected in C. difficile strains corresponding to 21 different MLST groups, showing their high prevalence. Comparative genomic analyses revealed the existence of several clusters of highly similar phiCD211-like phages. Of note, large chromosome inversions were observed in some members, as well as multiple gene insertions and module exchanges. This highlights the great plasticity and gene coding potential of the phiCD211/phiCDIF1296T genome. Our analyses also suggest active evolution involving recombination with other mobile genetic elements.IMPORTANCEClostridioides difficile is a clinically important pathogen representing a serious threat to human health. Our hypothesis is that genetic differences between strains caused by the presence of integrated prophages could possibly explain the apparent differences observed in the virulence of different C. difficile strains. In this study, we provide a full characterization of phiCD211, also known as phiCDIF1296T, the largest phage known to infect C. difficile so far. Screening 2,584 C. difficile genomes revealed the presence of highly similar phiCD211-like phages in 5% of the strains analyzed, showing their high prevalence. Multiple genome comparisons suggest that evolution of the phiCD211-like phage community is dynamic, and some members have acquired genes that could possibly influence bacterial biology and fitness. Our study further supports the relevance of studying phages in C. difficile to better understand the epidemiology of this clinically important human pathogen. PMID: 29150513 [PubMed - as supplied by publisher]

Sulfolobus acidocaldarius uptakes pentoses via a cut2-type ABC transporter and metabolizes them through the aldolase-independent Weimberg pathway.

Related Articles Sulfolobus acidocaldarius uptakes pentoses via a cut2-type ABC transporter and metabolizes them through the aldolase-independent Weimberg pathway. Appl Environ Microbiol. 2017 Nov 17;: Authors: Wagner M, Shen L, Albersmeier A, van der Kolk N, Kim S, Cha J, Bräsen C, Kalinowski J, Siebers B, Albers SV Abstract Sulfolobus spp. possess a great metabolic versatility and grow heterotrophically on various carbon sources such as different sugars and peptides. Known sugar transporters in Archaea predominantly belong to ABC transport systems. Although several ABC transporters for sugar uptake have been characterized in the crenarchaeon Sulfolobus solfataricus, only one homologue of these transporters, the maltose/maltooligomer transporter, could be identified in the closely related Sulfolobus acidocaldarius Comparison of the transcriptome of S. acidocaldarius grown on peptides alone and peptides in presence of D-xylose allowed for the identification of the ABC transporter for D-xylose and L-arabinose transport and to gain deeper insights into pentose catabolism under the respective growth conditions. The D-xylose/L-arabinose substrate binding protein (SBP) (Saci_2122) of the ABC transporter is unique in Archaea and shares more similarity to bacterial SBPs of the Carbohydrate Uptake Transporter-2 (CUT2) family than to any characterized archaeal one. The identified pentose transporter is the first CUT2 family ABC transporter analyzed in the domain of Archaea. Single gene deletion mutants of the ABC transporter subunits exemplified the importance of the transport system for D-xylose and L-arabinose uptake. Next to the transporter operon, enzymes of the aldolase-independent pentose catabolism branch were found to be upregulated in N-Z-Amine and D-xylose medium. The α-ketoglutarate semialdehyde dehydrogenase (KGSADH; Saci_1938) seemed not to be essential for growth on pentoses. However, the deletion mutant of the 2-keto-3-deoxyarabinoate/xylonate dehydratase (KDXD/KDAD; Saci_1939) was no longer able to catabolize D-xylose or L-arabinose suggesting the absence of the aldolase-dependent branch in S. acidocaldariusImportance Thermoacidophilic microorganisms are emerging model organisms for biotechnological applications as their optimal growth conditions resemble conditions used in certain biotechnologies such as plant waste industrial degradation. Because of its high genome stability Sulfolobus acidocaldarius is especially suited as a platform organism for such applications. For the use in (ligno)cellulose degradation, it was important to understand the pentose uptake and metabolism in S. acidocaldarius This study revealed that only the aldolase-independent Weimberg pathway is required for growth of S. acidocaldarius on D-xylose and L-arabinose. Moreover, S. acidocaldarius employs a CUT2 ABC transporter for pentose uptake, which is more similar to bacterial than to archaeal ABC transporters. The identification of pentose inducible promoters will expedite the metabolic engineering of S. acidocaldarius for its development into a platform organism for (ligno)cellulose degradation. PMID: 29150511 [PubMed - as supplied by publisher]

The tape measure protein is involved in the heat stability of Lactococcus lactis phages.

Related Articles The tape measure protein is involved in the heat stability of Lactococcus lactis phages. Appl Environ Microbiol. 2017 Nov 17;: Authors: Geagea H, Labrie SJ, Subirade M, Moineau S Abstract Virulent lactococcal phages are still a major risk for milk fermentation processes as they may lead to slowdowns and low quality fermented dairy products, particularly cheeses. Some of the phage control strategies used by the industry rely on heat treatments. Recently, a few Lactococcus lactis phages were found to be highly thermo-resistant. To identify the genetic determinant(s) responsible for the thermal resistance of lactococcal phages, we used the virulent phage CB14 (sk1virus/936 group) to select for phage mutants with increased heat stability. By treating phage CB14 to successive low and high temperatures, we were able to select two CB14 derivatives with increased heat stability. Sequencing of their genome revealed the same nucleotide sequences as the wild-type phage CB14, except for a same-sized deletion (120 bp) in the gene coding for the tape measure protein (TMP) of each phage mutant, but at a different position. The TMP protein sequences of these mutant phages were compared with their homologues in other wild-type L. lactis phages with a wide diversity in heat stability. Comparative analysis showed that the same nucleotide deletion appears to have also occurred in the gene coding for the TMP of highly thermo-resistant lactococcal phages P1532 and P680. We propose that the TMP is, in part, responsible for the heat stability of the highly predominant lactococcal phages of the sk1virus group.Importance Virulent lactococcal phages still represent a major risk for milk fermentation as they may lead to slowdowns and low quality fermented dairy products. Heat treatment is one of the most commonly used methods to control these virulent phages in cheese byproducts. Recently, a few Lactococcus lactis phages, members of the sk1virus group, have emerged with high thermal stability. To our knowledge, the genetic determinant(s) responsible for this thermal resistance in lactococcal phages is unknown. A better understanding of the thermal stability of these emerging virulent lactococcal phages is needed to improve industrial control strategies. In this work, we report the identification of a phage structural protein that is involved in the heat stability of a virulent sk1virus phage. Identifying such genetic determinant for heat stability is a first step to understand the emergence of this group of thermostable phages. PMID: 29150509 [PubMed - as supplied by publisher]

Cry64Ba and Cry64Ca, two ETX/MTX2 Bacillus thuringiensis insecticidal proteins against hemipteran pests.

Related Articles Cry64Ba and Cry64Ca, two ETX/MTX2 Bacillus thuringiensis insecticidal proteins against hemipteran pests. Appl Environ Microbiol. 2017 Nov 17;: Authors: Liu Y, Wang Y, Shu C, Lin K, Song F, Bravo A, Soberón M, Zhang J Abstract Genetically modified crops that express insecticidal Bacillus thuringiensis (Bt) proteins have become a primary approach for control of lepidopteran (moth) and coleopteran (beetle) pests that feed by chewing the plants. However, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. In this study, we describe two Cry toxins (Cry64Ba and Cry64Ca) from a Bt 1012 strain showed toxicity against two important rice hemipteran pests, Laodelphax striatellus and Sogatella furcifera Both of these two proteins contain an ETX/MTX2 domain, and share common sequence features with the β-pore forming toxins. Co-expression of cry64Ba and cry64Ca genes in the acrystalliferous Bt strain HD73(-) resulted in high insecticidal activity against both hemipteran pests. No toxicity was observed on other pests such as Ostrinia furnacalis, Plutella xylostella or Colaphellus bowringi Also, no hemolytic activity or toxicity against cancer cells was detected. Binding assays showed specific binding of Cry64Ba/Cry64Ca toxin complex to brush border membrane vesicles (BBMV) isolated from L. striatellus Altogether, the Cry64Ba and Cry64Ca are Bt Cry toxins high effective against hemipteran pests and could provide a novel strategy for the environment-friendly biological control of rice planthoppers in transgenic plants.Importance: Rice is an important staple food in Asia whose production is threatened by rice planthoppers. To date, no effective Bacillus thuringiensis (Bt) protein has been identified against the rice planthoppers. We cloned two Bt toxin genes from Bt 1012 strain that showed toxicity against small brown planthopper (Laodelphax striatellus) and white-backed planthopper (Sogatella furcifera). The proteins encoded by cry64Ba and cry64Ca genes are the most efficient insecticidal Bt Cry proteins against hemipteran insects reported so far. Cry64Ba and Cry64Ca showed no toxicity against some Lepidopteran or Coleopteran pests. These two proteins should be able to be used for the integrated hemipteran pest management. PMID: 29150505 [PubMed - as supplied by publisher]
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