ResearchPad - ribosomes https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Isolation of a novel species in the genus <i>Cupriavidus</i> from a patient with sepsis using whole genome sequencing]]> https://www.researchpad.co/article/elastic_article_14469 Whole genome sequencing (WGS) has become an accessible tool in clinical microbiology, and it allowed us to identify a novel Cupriavidus species. We isolated Gram-negative bacillus from the blood of an immunocompromised patient, and phenotypical and molecular identifications were performed. Phenotypic identification discrepancies were noted between the Vitek 2 (bioMérieux, Marcy-l’Étoile, France) and Vitek MS systems (bioMérieux). Using 16S rRNA gene sequencing, it was impossible to identify the pathogen to the species levels. WGS was performed using the Illumina MiSeq platform (Illumina, San Diego, CA), and genomic sequence database searching with a TrueBacTM ID-Genome system (ChunLab, Inc., Seoul, Republic of Korea) showed no strains with average nucleotide identity values higher than 95.0%, which is the cut-off for species-level identification. Phylogenetic analysis indicated that the bacteria was a new Cupriavidus species that formed a subcluster with Cupriavidus gilardii. WGS holds great promise for accurate molecular identification beyond 16S rRNA gene sequencing in clinical microbiology.

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<![CDATA[Substantial improvement of tetraene macrolide production in <i>Streptomyces diastatochromogenes</i> by cumulative drug resistance mutations]]> https://www.researchpad.co/article/elastic_article_7861 Tetraene macrolides remain one of the most reliable fungicidal agents as resistance of fungal pathogens to these antibiotics is relatively rare. The modes of action and biosynthesis of polyene macrolides had been the focus of research over the past few years. However, few studies have been carried out on the overproduction of polyene macrolides. In the present study, cumulative drug-resistance mutation was used to obtain a quintuple mutant G5-59 with huge tetraene macrolide overproduction from the starting strain Streptomyces diastatochromogenes 1628. Through DNA sequence analysis, the mutation points in the genes of rsmG, rpsL and rpoB were identified. Additionally, the growth characteristic and expression level of tetrRI gene (belonging to the large ATP binding regulator of LuxR family) involved in the biosynthesis of tetraene macrolides were analyzed. As examined with 5L fermentor, the quintuple mutant G5-59 grew very well and the maximum productivity of tetramycin A, tetramycin P and tetrin B was as high as 1735, 2811 and 1500 mg/L, which was 8.7-, 16- and 25-fold higher than that of the wild-type strain 1628, respectively. The quintuple mutant G5-59 could be useful for further improvement of tetraene macrolides production at industrial level.

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<![CDATA[Quantitative dynamics of Salmonella and E. coli in feces of feedlot cattle treated with ceftiofur and chlortetracycline]]> https://www.researchpad.co/article/Nd45d35d0-8623-4716-b387-5e4fac70c4ad

Antibiotic use in beef cattle is a risk factor for the expansion of antimicrobial-resistant Salmonella populations. However, actual changes in the quantity of Salmonella in cattle feces following antibiotic use have not been investigated. Previously, we observed an overall reduction in Salmonella prevalence in cattle feces associated with both ceftiofur crystalline-free acid (CCFA) and chlortetracycline (CTC) use; however, during the same time frame the prevalence of multidrug-resistant Salmonella increased. The purpose of this analysis was to quantify the dynamics of Salmonella using colony counting (via a spiral-plating method) and hydrolysis probe-based qPCR (TaqMan® qPCR). Additionally, we quantified antibiotic-resistant Salmonella by plating to agar containing antibiotics at Clinical & Laboratory Standards Institute breakpoint concentrations. Cattle were randomly assigned to 4 treatment groups across 16 pens in 2 replicates consisting of 88 cattle each. Fecal samples from Days 0, 4, 8, 14, 20, and 26 were subjected to quantification assays. Duplicate qPCR assays targeting the Salmonella invA gene were performed on total community DNA for 1,040 samples. Diluted fecal samples were spiral plated on plain Brilliant Green Agar (BGA) and BGA with ceftriaxone (4 μg/ml) or tetracycline (16 μg/ml). For comparison purposes, indicator non-type-specific (NTS) E. coli were also quantified by direct spiral plating. Quantity of NTS E. coli and Salmonella significantly decreased immediately following CCFA treatment. CTC treatment further decreased the quantity of Salmonella but not NTS E. coli. Effects of antibiotics on the imputed log10 quantity of Salmonella were analyzed via a multi-level mixed linear regression model. The invA gene copies decreased with CCFA treatment by approximately 2 log10 gene copies/g feces and remained low following additional CTC treatment. The quantities of tetracycline or ceftriaxone-resistant Salmonella were approximately 4 log10 CFU/g feces; however, most of the samples were under the quantification limit. The results of this study demonstrate that antibiotic use decreases the overall quantity of Salmonella in cattle feces in the short term; however, the overall quantities of antimicrobial-resistant NTS E. coli and Salmonella tend to remain at a constant level throughout.

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<![CDATA[Identification of soil bacteria capable of utilizing a corn ethanol fermentation byproduct]]> https://www.researchpad.co/article/5c8c1951d5eed0c484b4d3e6

A commercial corn ethanol production byproduct (syrup) was used as a bacterial growth medium with the long-term aim to repurpose the resulting microbial biomass as a protein supplement in aquaculture feeds. Anaerobic batch reactors were used to enrich for soil bacteria metabolizing the syrup as the sole nutrient source over an eight-day period with the goal of obtaining pure cultures of facultative organisms from the reactors. Amplification of the V4 variable region of the 16S rRNA gene was performed using barcoded primers to track the succession of microbes enriched for during growth on the syrup. The resulting PCR products were sequenced using Illumina MiSeq protocols, analyzed via the program QIIME, and the alpha-diversity was calculated. Seven bacterial families were the most prevalent in the bioreactor community after eight days of enrichment: Clostridiaceae, Alicyclobacillaceae, Ruminococcaceae, Burkholderiaceae, Bacillaceae, Veillonellaceae, and Enterobacteriaceae. Pure culture isolates obtained from the reactors, and additional laboratory stock strains, capable of facultative growth, were grown aerobically in microtiter plates with the syrup substrate to monitor growth yield. Reactor isolates of interest were identified at a species level using the full 16S rRNA gene and other biomarkers. Bacillus species, commonly used as probiotics in aquaculture, showed the highest biomass yield of the monocultures examined. Binary combinations of monocultures yielded no apparent synergism between organisms, suggesting competition for nutrients instead of cooperative metabolite conversion.

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<![CDATA[16S rRNA sequence embeddings: Meaningful numeric feature representations of nucleotide sequences that are convenient for downstream analyses]]> https://www.researchpad.co/article/5c7ee7c5d5eed0c4848f4d9c

Advances in high-throughput sequencing have increased the availability of microbiome sequencing data that can be exploited to characterize microbiome community structure in situ. We explore using word and sentence embedding approaches for nucleotide sequences since they may be a suitable numerical representation for downstream machine learning applications (especially deep learning). This work involves first encoding (“embedding”) each sequence into a dense, low-dimensional, numeric vector space. Here, we use Skip-Gram word2vec to embed k-mers, obtained from 16S rRNA amplicon surveys, and then leverage an existing sentence embedding technique to embed all sequences belonging to specific body sites or samples. We demonstrate that these representations are meaningful, and hence the embedding space can be exploited as a form of feature extraction for exploratory analysis. We show that sequence embeddings preserve relevant information about the sequencing data such as k-mer context, sequence taxonomy, and sample class. Specifically, the sequence embedding space resolved differences among phyla, as well as differences among genera within the same family. Distances between sequence embeddings had similar qualities to distances between alignment identities, and embedding multiple sequences can be thought of as generating a consensus sequence. In addition, embeddings are versatile features that can be used for many downstream tasks, such as taxonomic and sample classification. Using sample embeddings for body site classification resulted in negligible performance loss compared to using OTU abundance data, and clustering embeddings yielded high fidelity species clusters. Lastly, the k-mer embedding space captured distinct k-mer profiles that mapped to specific regions of the 16S rRNA gene and corresponded with particular body sites. Together, our results show that embedding sequences results in meaningful representations that can be used for exploratory analyses or for downstream machine learning applications that require numeric data. Moreover, because the embeddings are trained in an unsupervised manner, unlabeled data can be embedded and used to bolster supervised machine learning tasks.

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<![CDATA[Multiplexing polysome profiling experiments to study translation in Escherichia coli]]> https://www.researchpad.co/article/5c75ac71d5eed0c484d087b8

Polysome profiling is a widely used method to monitor the translation status of mRNAs. Although it is theoretically a simple technique, it is labor intensive. Repetitive polysome fractionation rapidly generates a large number of samples to be handled in the downstream processes of protein elimination, RNA extraction and quantification. Here, we propose a multiplex polysome profiling experiment in which distinct cellular extracts are pooled before loading on the sucrose gradient for fractionation. We used the multiplexing method to study translation in E. coli. Multiplexing polysome profiling experiments provided similar mRNA translation status to that obtained with the non-multiplex method with comparable distribution of mRNA copies between the polysome profiling fractions, similar ribosome occupancy and ribosome density. The multiplexing method was used for parallel characterization of gene translational responses to changing mRNA levels. When the mRNA level of two native genes, cysZ and lacZ was increased by transcription induction, their global translational response was similar, with a higher ribosome load leading to increased ribosome occupancy and ribosome densities. However the pattern and the magnitude of the translational response were gene specific. By reducing the number of polysome profiling experiments, the multiplexing method saved time and effort and reduced cost and technical bias. This method would be useful to study the translational effect of mRNA sequence-dependent parameters that often require testing multiple samples and conditions in parallel.

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<![CDATA[Nitrogen- and phosphorus-starved Triticum aestivum show distinct belowground microbiome profiles]]> https://www.researchpad.co/article/5c76fe27d5eed0c484e5b5dd

Many plants have natural partnerships with microbes that can boost their nitrogen (N) and/or phosphorus (P) acquisition. To assess whether wheat may have undiscovered associations of these types, we tested if N/P-starved Triticum aestivum show microbiome profiles that are simultaneously different from those of N/P-amended plants and those of their own bulk soils. The bacterial and fungal communities of root, rhizosphere, and bulk soil samples from the Historical Dryland Plots (Lethbridge, Canada), which hold T. aestivum that is grown both under N/P fertilization and in conditions of extreme N/P-starvation, were taxonomically described and compared (bacterial 16S rRNA genes and fungal Internal Transcribed Spacers—ITS). As the list may include novel N- and/or P-providing wheat partners, we then identified all the operational taxonomic units (OTUs) that were proportionally enriched in one or more of the nutrient starvation- and plant-specific communities. These analyses revealed: a) distinct N-starvation root and rhizosphere bacterial communities that were proportionally enriched, among others, in OTUs belonging to families Enterobacteriaceae, Chitinophagaceae, Comamonadaceae, Caulobacteraceae, Cytophagaceae, Streptomycetaceae, b) distinct N-starvation root fungal communities that were proportionally enriched in OTUs belonging to taxa Lulworthia, Sordariomycetes, Apodus, Conocybe, Ascomycota, Crocicreas, c) a distinct P-starvation rhizosphere bacterial community that was proportionally enriched in an OTU belonging to genus Agrobacterium, and d) a distinct P-starvation root fungal community that was proportionally enriched in OTUs belonging to genera Parastagonospora and Phaeosphaeriopsis. Our study might have exposed wheat-microbe connections that can form the basis of novel complementary yield-boosting tools.

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<![CDATA[Information about variations in multiple copies of bacterial 16S rRNA genes may aid in species identification]]> https://www.researchpad.co/article/5c706762d5eed0c4847c6f93

Variable region analysis of 16S rRNA gene sequences is the most common tool in bacterial taxonomic studies. Although used for distinguishing bacterial species, its use remains limited due to the presence of variable copy numbers with sequence variation in the genomes. In this study, 16S rRNA gene sequences, obtained from completely assembled whole genome and Sanger electrophoresis sequencing of cloned PCR products from Serratia fonticola GS2, were compared. Sanger sequencing produced a combination of sequences from multiple copies of 16S rRNA genes. To determine whether the variant copies of 16S rRNA genes affected Sanger sequencing, two ratios (5:5 and 8:2) with different concentrations of cloned 16S rRNA genes were used; it was observed that the greater the number of copies with similar sequences the higher its chance of amplification. Effect of multiple copies for taxonomic classification of 16S rRNA gene sequences was investigated using the strain GS2 as a model. 16S rRNA copies with the maximum variation had 99.42% minimum pairwise similarity and this did not have an effect on species identification. Thus, PCR products from genomes containing variable 16S rRNA gene copies can provide sufficient information for species identification except from species which have high similarity of sequences in their 16S rRNA gene copies like the case of Bacillus thuringiensis and Bacillus cereus. In silico analysis of 1,616 bacterial genomes from long-read sequencing was also done. The average minimum pairwise similarity for each phylum was reported with their average genome size and average “unique copies” of 16S rRNA genes and we found that the phyla Proteobacteria and Firmicutes showed the highest amount of variation in their copies of their 16S rRNA genes. Overall, our results shed light on how the variations in the multiple copies of the 16S rRNA genes of bacteria can aid in appropriate species identification.

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<![CDATA[Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation]]> https://www.researchpad.co/article/5c5df303d5eed0c484580b31

Hbs1 has been established as a central component of the cell’s translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.

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<![CDATA[Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs]]> https://www.researchpad.co/article/5c6b26afd5eed0c484289e7d

Current progress in the field of next-generation transcriptome sequencing have contributed significantly to the study of various malignancies including glioblastoma multiforme (GBM). Differential sequencing of transcriptomes of patients and non-tumor controls has a potential to reveal novel transcripts with significant role in GBM. One such candidate group of molecules are long non-coding RNAs (lncRNAs) which have been proved to be involved in processes such as carcinogenesis, epigenetic modifications and resistance to various therapeutic approaches. To maximize the value of transcriptome sequencing, a proper protocol for library preparation from tissue-derived RNA needs to be found which would produce high quality transcriptome sequencing data and increase the number of detected lncRNAs. It is important to mention that success of library preparation is determined by the quality of input RNA, which is in case of real-life tissue specimens very often altered in comparison to high quality RNA commonly used by manufacturers for development of library preparation chemistry. In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Libraries generated using SENSE kit were characterized by the most normal distribution of normalized average GC content, the least amount of over-represented sequences and the percentage of ribosomal RNA reads (0.3–1.5%) and highest numbers of uniquely mapped reads and reads aligning to coding regions. However, NEBNext kit performed better having relatively low duplication rates, even transcript coverage and the highest number of hits in Ensembl database for every biotype of our interest including lncRNAs. Our results indicate that out of three approaches the NEBNext library preparation kit was most suitable for the study of lncRNAs via transcriptome sequencing. This was further confirmed by highly consistent data reached in an independent validation on an expanded cohort.

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<![CDATA[SLC39A8 missense variant is associated with Crohn's disease but does not have a major impact on gut microbiome composition in healthy subjects]]> https://www.researchpad.co/article/5c5ca2e0d5eed0c48441ec3e

Background

Gene-microbiome interactions are important in aetiology and pathogenesis of inflammatory bowel disease, a chronic inflammatory disorder of the gastrointestinal tract consisting of Crohn’s disease and ulcerative colitis. Scarce studies on gene-microbiome interactions show very little overlap in their results. Therefore, it is of utmost importance that gene-microbiome studies are repeated. We aimed to replicate the association between the SLC39A8 [Thr]391 risk allele and gut microbiome composition in patients with inflammatory bowel disease and healthy controls.

Methods

We collected faecal samples, peripheral blood and extensive phenotype data from 291 patients with inflammatory bowel disease and 476 healthy controls. Carrier status information was obtained from whole exome sequencing data, generated using the Illumina HiSeq. The gut microbiome composition was determined by tag-sequencing the 16S rRNA gene. Associations between carrier status and disease were tested using the Wilcoxon-Mann-Whitney test. Associations between carriers and gut microbiome composition were determined using principal coordinate analyses, variance explained, alpha diversity and additive general linear models in inflammatory bowel disease, healthy controls and all groups combined.

Results

Crohn’s disease patients were more often carriers of the missense variant (21/171, 12.3%) than controls (30/476, 6.3%) (OR = 2.1, P = 0.01). We could not identify associations between carrier status and overall gut microbiome composition and microbial richness in all tested groups after correcting for potential confounding factors. We did identify 37 different operational taxonomical units to be associated with carrier status among the tested groups. Two of these 37 were identified before in the discovery study.

Conclusions

We could confirm the genetic association of the SLC39A8 [Thr]391 risk allele with Crohn’s disease but we could only limited replicate the association in gut microbiome composition. Independent replication of gene-microbiome studies is warranted to identify true biological mechanisms.

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<![CDATA[Comprehensive profiling of translation initiation in influenza virus infected cells]]> https://www.researchpad.co/article/5c521870d5eed0c4847983a1

Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactimidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. We do not detect globally higher levels of alternate translation initiation on host transcripts upon influenza infection or during the anti-viral response, but the subset of host transcripts induced by the anti-viral response is enriched for alternate initiation sites. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.

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<![CDATA[Ochrobactrum quorumnocens sp. nov., a quorum quenching bacterium from the potato rhizosphere, and comparative genome analysis with related type strains]]> https://www.researchpad.co/article/5c50c47fd5eed0c4845e8833

Ochrobactrum spp. are ubiquitous bacteria attracting growing attention as important members of microbiomes of plants and nematodes and as a source of enzymes for biotechnology. Strain Ochrobactrum sp. A44T was isolated from the rhizosphere of a field-grown potato in Gelderland, the Netherlands. The strain can interfere with quorum sensing (QS) of Gram-negative bacteria through inactivation of N-acyl homoserine lactones (AHLs) and protect plant tissue against soft rot pathogens, the virulence of which is governed by QS. Phylogenetic analysis based on 16S rRNA gene alone and concatenation of 16S rRNA gene and MLSA genes (groEL and gyrB) revealed that the closest relatives of A44T are O. grignonense OgA9aT, O. thiophenivorans DSM 7216T, O. pseudogrignonense CCUG 30717T, O. pituitosum CCUG 50899T, and O. rhizosphaerae PR17T. Genomes of all six type strains were sequenced, significantly expanding the possibility of genome-based analyses in Ochrobactrum spp. Average nucleotide identity (ANIb) and genome-to-genome distance (GGDC) values for A44T and the related strains were below the single species thresholds (95% and 70%, respectively), with the highest scores obtained for O. pituitosum CCUG 50899T (87.31%; 35.6%), O. rhizosphaerae PR17T (86.80%; 34.3%), and O. grignonense OgA9aT (86.30%; 33.6%). Distinction of A44T from the related type strains was supported by chemotaxonomic and biochemical analyses. Comparative genomics revealed that the core genome for the newly sequenced strains comprises 2731 genes, constituting 50–66% of each individual genome. Through phenotype-to-genotype study, we found that the non-motile strain O. thiophenivorans DSM 7216T lacks a cluster of genes related to flagella formation. Moreover, we explored the genetic background of distinct urease activity among the strains. Here, we propose to establish a novel species Ochrobactrum quorumnocens, with A44T as the type strain (= LMG 30544T = PCM 2957T).

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<![CDATA[Bacteria associated with moon jellyfish during bloom and post-bloom periods in the Gulf of Trieste (northern Adriatic)]]> https://www.researchpad.co/article/5c478c4ad5eed0c484bd154e

Jellyfish are a prominent component of the plankton community. They frequently form conspicuous blooms which may interfere with different human enterprises. Among the aspects that remain understudied are jellyfish associations with microorganisms having potentially important implications for organic matter cycling. To the best of our knowledge, this study is the first to investigate the bacterial community associated with live moon jellyfish (Aurelia solida, Scyohozoa) in the Adriatic Sea. Using 16S rRNA clone libraries and culture-based methods, we have analyzed the bacterial community composition of different body parts: the exumbrella surface, oral arms, and gastric cavity, and investigated possible differences in medusa-associated bacterial community structure at the time of the jellyfish population peak, and during the senescent phase at the end of bloom. Microbiota associated with moon jellyfish was different from ambient seawater bacterial assemblage and varied between different body parts. Betaproteobacteria (Burkholderia, Cupriavidus and Achromobacter) dominated community in the gastral cavity of medusa, while Alphaproteobacteria (Phaeobacter, Ruegeria) and Gammaproteobacteria (Stenotrophomonas, Alteromonas, Pseudoalteromonas and Vibrio) prevailed on ‘outer’ body parts. Bacterial community structure changed during senescent phase, at the end of the jellyfish bloom, showing an increased abundance of Gammaproteobacteria, exclusively Vibrio. The results of cultured bacterial isolates showed the dominance of Gammaproeteobacteria, especially Vibrio and Pseudoalteromonas in all body parts. Our results suggest that jellyfish associated bacterial community might have an important role for the host, and that anthropogenic pollution in the Gulf of Trieste might affect their community structure.

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<![CDATA[High throughput cultivation-based screening on porous aluminum oxide chips allows targeted isolation of antibiotic resistant human gut bacteria]]> https://www.researchpad.co/article/5c605a07d5eed0c4847cc7d2

The emergence of bacterial pathogens that are resistant to clinical antibiotics poses an increasing risk to human health. An important reservoir from which bacterial pathogens can acquire resistance is the human gut microbiota. However, thus far, a substantial fraction of the gut microbiota remains uncultivated and has been little-studied with respect to its resistance reservoir-function. Here, we aimed to isolate yet uncultivated resistant gut bacteria by a targeted approach. Therefore, faecal samples from 20 intensive care patients who had received the prophylactic antibiotic treatment selective digestive decontamination (SDD), i.e. tobramycin, polymyxin E, amphotericin B and cefotaxime, were inoculated anaerobically on porous aluminium oxide chips placed on top of poor and rich agar media, including media supplemented with the SDD antibiotics. Biomass growing on the chips was analysed by 16S rRNA gene amplicon sequencing, showing large inter-individual differences in bacterial cultivability, and enrichment of a range of taxonomically diverse operational taxonomic units (OTUs). Furthermore, growth of Ruminococcaceae (2 OTUs), Enterobacteriaceae (6 OTUs) and Lachnospiraceae (4 OTUs) was significantly inhibited by the SDD antibiotics. Strains belonging to 16 OTUs were candidates for cultivation to pure culture as they shared ≤95% sequence identity with the closest type strain and had a relative abundance of ≥2%. Six of these OTUs were detected on media containing SDD antibiotics, and as such were prime candidates to be studied regarding antibiotic resistance. One of these six OTUs was obtained in pure culture using targeted isolation. This novel strain was resistant to the antibiotics metrodinazole and imipenem. It was initially classified as member of the Ruminococcaceae, though later it was found to share 99% nucleotide identity with the recently published Sellimonas intestinalis BR72T. In conclusion, we show that high-throughput cultivation-based screening of microbial communities can guide targeted isolation of bacteria that serve as reservoirs of antibiotic resistance.

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<![CDATA[Impact of two neighbouring ribosomal protein clusters on biogenesis factor binding and assembly of yeast late small ribosomal subunit precursors]]> https://www.researchpad.co/article/5c605a0bd5eed0c4847cc84c

Many of the small ribosomal subunit proteins are required for the stabilisation of late small ribosomal subunit (SSU) precursors and for final SSU rRNA processing in S. cerevisiae. Among them are ribosomal proteins (r-proteins) which form a protein cluster around rpS0 (uS2) at the "neck" of the SSU (S0-cluster) and others forming a nearby protein cluster around rpS3 (uS3) at the SSU "beak". Here we applied semi-quantitative proteomics together with complementary biochemical approaches to study how incomplete assembly of these two r-protein clusters affects binding and release of SSU maturation factors and assembly of other r-proteins in late SSU precursors in S. cerevisiae. For each of the two clusters specific impairment of the local r-protein assembly state was observed in Rio2 associated SSU precursors. Besides, cluster-specific effects on the association of biogenesis factors were detected. These suggested a role of S0-cluster formation for the efficient release of the two nuclear export factors Rrp12 and Slx9 from SSU precursors and for the correct incorporation of the late acting biogenesis factor Rio2. Based on our and on previous results we propose the existence of at least two different r-protein assembly checkpoints during late SSU maturation in S. cerevisiae. We discuss in the light of recent SSU precursor structure models how r-protein assembly states might be sensed by biogenesis factors at the S0-cluster checkpoint.

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<![CDATA[Cophylogenetic analysis suggests cospeciation between the Scorpion Mycoplasma Clade symbionts and their hosts]]> https://www.researchpad.co/article/5c3fa5fed5eed0c484caaedb

Scorpions are predator arachnids of ancient origin and worldwide distribution. Two scorpion species, Vaejovis smithi and Centruroides limpidus, were found to harbor two different Mollicutes phylotypes: a Scorpion Mycoplasma Clade (SMC) and Scorpion Group 1 (SG1). Here we investigated, using a targeted gene sequencing strategy, whether these Mollicutes were present in 23 scorpion morphospecies belonging to the Vaejovidae, Carboctonidae, Euscorpiidae, Diplocentridae, and Buthidae families. Our results revealed that SMC is found in a species-specific association with Vaejovidae and Buthidae, whereas SG1 is uniquely found in Vaejovidae. SMC and SG1 co-occur only in Vaejovis smithi where 43% of the individuals host both phylotypes. A phylogenetic analysis of Mollicutes 16S rRNA showed that SMC and SG1 constitute well-delineated phylotypes. Additionally, we found that SMC and scorpion phylogenies are significantly congruent, supporting the observation that a cospeciation process may have occurred. This study highlights the phylogenetic diversity of the scorpion associated Mollicutes through different species revealing a possible cospeciation pattern.

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<![CDATA[A survey of argasid ticks and tick-associated pathogens in the Peripheral Oases around Tarim Basin and the first record of Argas japonicus in Xinjiang, China]]> https://www.researchpad.co/article/5c2d2eacd5eed0c484d9b0b4

Argasid ticks (Acari: Argasidae) carry and transmit a variety of pathogens of animals and humans, including viruses, bacteria and parasites. There are several studies reporting ixodid ticks (Acari: Ixodidae) and associated tick-borne pathogens in Xinjiang, China. However, little is known about the argasid ticks and argasid tick-associated pathogens in this area. In this study, a total of 3829 adult argasid ticks infesting livestock were collected at 12 sampling sites of 10 counties in the Peripheral Oases, which carry 90% of the livestock and humans population, around the Tarim Basin (southern Xinjiang) from 2013 to 2016. Tick specimens were identified to two species from different genera by morphology and sequences of mitochondrial 16S rRNA and 12S rRNA were derived to confirm the species designation. The results showed that the dominant argasid ticks infesting livestock in southern Xinjiang were Ornithodoros lahorensis (87.86%, 3364/3829). Ornithodoros lahorensis was distributed widely and were collected from 10 counties of southern Xinjiang. Argas japonicus was collected from Xinjiang for the first time. In addition, we screened these ticks for tick-associated pathogens and showed the presence of DNA sequences of Rickettsia spp. of Spotted fever group and Anaplasma spp. in the argasid ticks. This finding suggests the potential role for Argas japonicus as a vector of pathogens to livestock and humans.

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<![CDATA[Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo]]> https://www.researchpad.co/article/5c141f0dd5eed0c484d296ca

Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.

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<![CDATA[Evaluation of endogenous reference genes in Bactrocera cucurbitae by qPCR under different conditions]]> https://www.researchpad.co/article/5c21515ed5eed0c4843f9e08

Bactrocera cucurbitae (melon flies) are prominent invasive pests in southern China. To screen for a stable reference gene in melon flies suitable for comparing tissue samples subjected to different conditions in four categories (temperature, insect stage, days of age and gender), the expression of 12 candidate reference genes under different treatment conditions was analyzed by real-time fluorescent quantitative PCR. The results obtained from a comprehensive analysis with geNorm, NormFinder, BestKeeper and RefFinder software showed that the most stable reference gene was RPL60, and the least stable reference gene was actin-5. We used a heat shock protein gene (HSP-90) to verify the results, and the conclusion was consistent. When the reference gene RPL60 was used as the reference gene, the relative expression of HSP-90 was essentially constant with the prolongation of treatment time. When actin-5 was used, HSP-90 expression changed markedly with treatment time. The results of this study can be used for further research on gene expression inBactrocera cucurbitae.

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