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September 22, 2019

Genomic characterization and probiotic potency of Bacillus sp. DU-106, a highly effective producer of L-lactic acid isolated from fermented yogurt.

Bacillus sp. DU-106, a newly isolated member of Bacillus cereus group, exhibits the predominant ability to produce L-lactic acid. The probiotic potency of test strain revealed its survivability at acidic pH, bile salts and viability in simulated gastric juice in vitro. The acute oral toxicity test indicated its no toxicity to laboratory mice in vivo. We further determined the complete genome of strain DU-106 to understand genetic basis as a potential probiotic. It has a circular chromosome and three plasmids for a total genome 5,758,208 bp in size with a G + C content of 35.10%. Genes associated with lactate synthesis were found in the DU-106 genome. We also annotated various stress-related, bile salt resistance, and adhesion-related domains in this strain, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival under gastrointestinal tract. Moreover, strain DU-106 genome lacks the virulence genes encodes cereulide synthetase, enterotoxin FM, and cytotoxin K. These phenotypic and genomic probiotic potencies facilitate its potential candidate as probiotic starter in food industry.

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September 22, 2019

Methylation of the reelin gene promoter in peripheral blood and its relationship with the cognitive function of schizophrenia patients.

There is a decrease in the expression of the reelin gene (RELN) in the brain of schizophrenia patients, which can underlie observed cognitive abnormalities. It is suggested that this decrease is caused by the hypermethylation of the RELN promoter. The aim of the study was to investigate methylation of the RELN promoter in the peripheral blood of schizophrenia patients and its association with their cognitive deficits. A modified SMRT-BS (single-molecule real-time bisulfite sequencing) was used. We determined the methylation rate of 170 CpG sites within a 1465 bp DNA region containing the entire CpG island in the RELN promoter in 51 schizophrenia patients and 52 healthy controls. All subjects completed a battery of neuropsychological tests. There were no DNA methylation changes associated with schizophrenia. Most CpGs sites were unmethylated in both groups. At the same time, there was a variability in the methylation level of different regions within the promoter. The methylation level in the area from -258 to -151 bp relative to RELN transcription start site was a significant predictor of the index of patients’ cognitive functioning if sex, age, smoking, education, and polymorphism rsl858815 had been considered. The positive correlation between the methylation rate in this region and cognitive index suggests that the hypomethylation of the RELN promoter could contribute to the development of cognitive deficits in schizophrenia.

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September 22, 2019

De novo assembly, delivery and expression of a 101 kb human gene in mouse cells

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger, mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a pipeline for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kb. The DNA assembly step is supported by an integrated robotic workcell. We assemble the 101 kb human HPRT1 gene in yeast, deliver it to mouse cells, and show expression of the human protein from its full-length gene. This pipeline provides a framework for producing systematic, designer variants of any mammalian gene locus for functional evaluation in cells.

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September 22, 2019

Comparative genomics of degradative Novosphingobium strains with special reference to the microcystin-degrading Novosphingobium sp. THN1

Bacteria in genus Novosphingobium associated with biodegradation of substrates are prevalent in environments such as lakes, soil, sea, wood and sediments. To better understand the characteristics linked to their wide distribution and metabolic versatility, we report the whole genome sequence of Novosphingobium sp. THN1, a microcystin-degrading strain previously isolated by Jiang et al. (2011) from cyanobacteria-blooming water samples from Lake Taihu, China. We performed a genomic comparison analysis of Novosphingobium sp. THN1 with 21 other degradative Novosphingobium strains downloaded from GenBank. Phylogenetic trees were constructed using 16S rRNA genes, core genes, protein-coding sequences, and average nucleotide identity of whole genomes. Orthologous protein analysis showed that the 22 genomes contained 674 core genes and each strain contained a high proportion of distributed genes that are shared by a subset of strains. Inspection of their genomic plasticity revealed a high number of insertion sequence elements and genomic islands that were distributed on both chromosomes and plasmids. We also compared the predicted functional profiles of the Novosphingobium protein-coding genes. The flexible genes and all protein-coding genes produced the same heatmap clusters. The COG annotations were used to generate a dendrogram correlated with the compounds degraded. Furthermore, the metabolic profiles predicted from KEGG pathways showed that the majority of genes involved in central carbon metabolism, nitrogen, phosphate, sulfate metabolism, energy metabolism and cell mobility (above 62.5%) are located on chromosomes. Whereas, a great many of genes involved in degradation pathways (21–50%) are located on plasmids. The abundance and distribution of aromatics-degradative mono- and dioxygenases varied among 22 Novosphingoibum strains. Comparative analysis of the microcystin-degrading mlr gene cluster provided evidence for horizontal acquisition of this cluster. The Novosphingobium sp. THN1 genome sequence contained all the functional genes crucial for microcystin degradation and the mlr gene cluster shared high sequence similarity (=85%) with the sequences of other microcystin-degrading genera isolated from cyanobacteria-blooming water. Our results indicate that Novosphingobium species have high genomic and functional plasticity, rearranging their genomes according to environment variations and shaping their metabolic profiles by the substrates they are exposed to, to better adapt to their environments.

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September 22, 2019

A comparison of genotypic and phenotypic methods for analyzing the susceptibility to sulfamethoxazole and trimethoprim in Edwardsiella piscicida.

In a study of 39 isolates of Edwardsiella piscicida made from Korean aquaculture sites, sul genes were detected in 16 isolates and dfr genes in 19. Ten isolates were shown to contain both sul and dfr genes. MIC and disc diffusion zones assays were performed to measure the phenotypic susceptibilities of the 39 isolates. Normalized resistance interpretation was applied to these data to categorize isolates as either fully susceptible or as manifesting reduced susceptibility. The standard CLSI protocols specify the use of a mixture of sulfamethoxazole/trimethoprim (20:1) in both MIC and disc diffusion tests. Using the CLSI MIC protocol, 100% of the isolates containing dfr genes, but only 75% of the isolates containing sul genes, were categorized as manifesting reduced susceptibility. Using the CLSI disc diffusion protocol, only 58% of the isolates containing dfr genes and 69% of those containing sul genes were categorized as manifesting reduced susceptibility. When the single agent trimethoprim was substituted for the combined mixture in both the MIC and disc diffusion protocols, 100% of the dfr-positive isolates were categorized as NWT. When the single-agent sulfamethoxazole was substituted, the analysis of the MIC characterized 100% and the disc zone data 94% of the sul-positive isolates as manifesting reduced susceptibility. It is argued that the use of trimethoprim and sulfamethoxazole as single agents in phenotypic susceptibility tests would provide more meaningful data than the currently recommended use of these two agents combined.

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September 22, 2019

4.5 years within-patient evolution of a colistin resistant KPC-producing Klebsiella pneumoniae ST258.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) has emerged globally over the last decade as a major nosocomial pathogen that threatens patient care. These highly resistant bacteria are mostly associated with a single Kp clonal group, CG258, but the reasons for its host and hospital adaptation remain largely unknown.We analyzed the in vivo evolution of a colistin-resistant KPC-Kp CG258 strain that contaminated a patient following an endoscopy and was responsible for a fatal bacteremia 4.5 years later. Whole-genome sequencing was performed on 17 KPC-Kp isolates from this patient; single-nucleotide polymorphisms were analyzed and their implication in antimicrobial resistance and bacterial host adaptation investigated.The patient KPC-Kp strain diversified over 4.5 years at a rate of 7.5 substitutions per genome per year, resulting in broad phenotypic modifications. After 2 years of carriage, all isolates restored susceptibility to colistin. Higher expression of the fimbriae conferred the ability to produce more biofilm, and the isolate responsible for a bacteremia grew in human serum. The convergent mutations occurring in specific pathways, such as the respiratory chain and the cell envelope, revealed a complex long-term adaptation of KPC-Kp.Broad genomic and phenotypic diversification and the parallel selection of pathoadaptive mutations might contribute to long-term carriage and virulence of KPC-Kp CG258 strains and to the dissemination of this clone.

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September 22, 2019

Dissemination and persistence of extended-spectrum cephalosporin-resistance encoding IncI1-blaCTXM-1 plasmid among Escherichia coli in pigs.

This study investigated the ecology, epidemiology and plasmid characteristics of extended-spectrum cephalosporin (ESC)-resistant E. coli in healthy pigs over a period of 4 years (2013-2016) following the withdrawal of ESCs. High carriage rates of ESC-resistant E. coli were demonstrated in 2013 (86.6%) and 2014 (83.3%), compared to 2015 (22%) and 2016 (8.5%). ESC resistance identified among E. coli isolates was attributed to the carriage of an IncI1 ST-3 plasmid (pCTXM1-MU2) encoding blaCTXM-1. Genomic characterisation of selected E. coli isolates (n?=?61) identified plasmid movement into multiple commensal E. coli (n?=?22 STs). Major STs included ST10, ST5440, ST453, ST2514 and ST23. A subset of the isolates belong to the atypical enteropathogenic E. coli (aEPEC) pathotype that harboured multiple LEE pathogenic islands. pCTXM1-MU2 was similar (99% nt identity) to IncI1-ST3 plasmids reported from Europe, encoded resistance to aminoglycosides, sulphonamides and trimethoprim, and carried colicin Ib. pCTXM1-MU2 appears to be highly stable and readily transferable. This study demonstrates that ESC resistance may persist for a protracted period following removal of direct selection pressure, resulting in the emergence of ESC-resistance in both commensal E. coli and aEPEC isolates of potential significance to human and animal health.

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September 22, 2019

Development and validation of 58K SNP-array and high-density linkage map in Nile tilapia (O. niloticus).

Despite being the second most important aquaculture species in the world accounting for 7.4% of global production in 2015, tilapia aquaculture has lacked genomic tools like SNP-arrays and high-density linkage maps to improve selection accuracy and accelerate genetic progress. In this paper, we describe the development of a genotyping array containing more than 58,000 SNPs for Nile tilapia (Oreochromis niloticus). SNPs were identified from whole genome resequencing of 32 individuals from the commercial population of the Genomar strain, and were selected for the SNP-array based on polymorphic information content and physical distribution across the genome using the Orenil1.1 genome assembly as reference sequence. SNP-performance was evaluated by genotyping 4991 individuals, including 689 offspring belonging to 41 full-sib families, which revealed high-quality genotype data for 43,588 SNPs. A preliminary genetic linkage map was constructed using Lepmap2 which in turn was integrated with information from the O_niloticus_UMD1 genome assembly to produce an integrated physical and genetic linkage map comprising 40,186 SNPs distributed across 22 linkage groups (LGs). Around one-third of the LGs showed a different recombination rate between sexes, with the female being greater than the male map by a factor of 1.2 (1632.9 to 1359.6 cM, respectively), with most LGs displaying a sigmoid recombination profile. Finally, the sex-determining locus was mapped to position 40.53 cM on LG23, in the vicinity of the anti-Müllerian hormone (amh) gene. These new resources has the potential to greatly influence and improve the genetic gain when applying genomic selection and surpass the difficulties of efficient selection for invasively measured traits in Nile tilapia.

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September 22, 2019

Amycomicin is a potent and specific antibiotic discovered with a targeted interaction screen.

The rapid emergence of antibiotic-resistant pathogenic bacteria has accelerated the search for new antibiotics. Many clinically used antibacterials were discovered through culturing a single microbial species under nutrient-rich conditions, but in the environment, bacteria constantly encounter poor nutrient conditions and interact with neighboring microbial species. In an effort to recapitulate this environment, we generated a nine-strain actinomycete community and used 16S rDNA sequencing to deconvolute the stochastic production of antimicrobial activity that was not observed from any of the axenic cultures. We subsequently simplified the community to just two strains and identified Amycolatopsis sp. AA4 as the producing strain and Streptomyces coelicolor M145 as an inducing strain. Bioassay-guided isolation identified amycomicin (AMY), a highly modified fatty acid containing an epoxide isonitrile warhead as a potent and specific inhibitor of Staphylococcus aureus Amycomicin targets an essential enzyme (FabH) in fatty acid biosynthesis and reduces S. aureus infection in a mouse skin-infection model. The discovery of AMY demonstrates the utility of screening complex communities against specific targets to discover small-molecule antibiotics.

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September 22, 2019

Genome plasticity of agr-defective Staphylococcus aureus during clinical infection.

Therapy for bacteremia caused by Staphylococcus aureus is often ineffective, even when treatment conditions are optimal according to experimental protocols. Adapted subclones, such as those bearing mutations that attenuate agr-mediated virulence activation, are associated with persistent infection and patient mortality. To identify additional alterations in agr-defective mutants, we sequenced and assembled the complete genomes of clone pairs from colonizing and infected sites of several patients in whom S. aureus demonstrated a within-host loss of agr function. We report that events associated with agr inactivation result in agr-defective blood and nares strain pairs that are enriched in mutations compared to pairs from wild-type controls. The random distribution of mutations between colonizing and infecting strains from the same patient, and between strains from different patients, suggests that much of the genetic complexity of agr-defective strains results from prolonged infection or therapy-induced stress. However, in one of the agr-defective infecting strains, multiple genetic changes resulted in increased virulence in a murine model of bloodstream infection, bypassing the mutation of agr and raising the possibility that some changes were selected. Expression profiling correlated the elevated virulence of this agr-defective mutant to restored expression of the agr-regulated ESAT6-like type VII secretion system, a known virulence factor. Thus, additional mutations outside the agr locus can contribute to diversification and adaptation during infection by S. aureus agr mutants associated with poor patient outcomes. Copyright © 2018 Altman et al.

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September 22, 2019

Genomic assemblies of newly sequenced Trypanosoma cruzi strains reveal new genomic expansion and greater complexity.

Chagas disease is a complex illness caused by the protozoan Trypanosoma cruzi displaying highly diverse clinical outcomes. In this sense, the genome sequence elucidation and comparison between strains may lead to disease understanding. Here, two new T. cruzi strains, have been sequenced, Y using Illumina and Bug2148 using PacBio, assembled, analyzed and compared with the T. cruzi annotated genomes available to date. The assembly stats from the new sequences show effective improvement of T. cruzi genome over the actual ones. Such as, the largest contig assembled (1.3?Mb in Bug2148) in de novo attempts and the highest mean assembly coverage (71X for Y). Our analysis reveals a new genomic expansion and greater complexity for those multi-copy gene families related to infection process and disease development, such as Trans-sialidases, Mucins and Mucin Associated Surface Proteins, among others. On one side, we demonstrate that multi-copy gene families are located near telomeric regions of the “chromosome-like” 1.3?Mb contig assembled of Bug2148, where they likely suffer high evolutive pressure. On the other hand, we identified several strain-specific single copy genes that might help to understand the differences in infectivity and physiology among strains. In summary, our results indicate that T. cruzi has a complex genomic architecture that may have promoted its evolution.

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September 22, 2019

Multisite de novo mutations in human offspring after paternal exposure to ionizing radiation.

A genome-wide evaluation of the effects of ionizing radiation on mutation induction in the mouse germline has identified multisite de novo mutations (MSDNs) as marker for previous exposure. Here we present the results of a small pilot study of whole genome sequencing in offspring of soldiers who served in radar units on weapon systems that were emitting high-frequency radiation. We found cases of exceptionally high MSDN rates as well as an increased mean in our cohort: While a MSDN mutation is detected in average in 1 out of 5 offspring of unexposed controls, we observed 12 MSDNs in altogether 18 offspring, including a family with 6 MSDNs in 3 offspring. Moreover, we found two translocations, also resulting from neighboring mutations. Our findings indicate that MSDNs might be suited in principle for the assessment of DNA damage from ionizing radiation also in humans. However, as exact person-related dose values in risk groups are usually not available, the interpretation of MSDNs in single families would benefit from larger molecular epidemiologic studies on this new biomarker.

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September 22, 2019

A multiplex homology-directed DNA repair assay reveals the impact of more than 1,000 BRCA1 missense substitution variants on protein function.

Loss-of-function pathogenic variants in BRCA1 confer a predisposition to breast and ovarian cancer. Genetic testing for sequence changes in BRCA1 frequently reveals a missense variant for which the impact on cancer risk and on the molecular function of BRCA1 is unknown. Functional BRCA1 is required for the homology-directed repair (HDR) of double-strand DNA breaks, a critical activity for maintaining genome integrity and tumor suppression. Here, we describe a multiplex HDR reporter assay for concurrently measuring the effects of hundreds of variants of BRCA1 for their role in DNA repair. Using this assay, we characterized the effects of 1,056 amino acid substitutions in the first 192 residues of BRCA1. Benchmarking these results against variants with known effects on DNA repair function or on cancer predisposition, we demonstrate accurate discrimination of loss-of-function versus benign missense variants. We anticipate that this assay can be used to functionally characterize BRCA1 missense variants at scale, even before the variants are observed in results from genetic testing. Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Antiviral adaptive immunity and tolerance in the mosquito Aedes aegyti

Mosquitoes spread pathogenic arboviruses while themselves tolerate infection. We here characterize an immunity pathway providing long-term antiviral protection and define how this pathway discriminates between self and non-self. Mosquitoes use viral RNAs to create viral derived cDNAs (vDNAs) central to the antiviral response. vDNA molecules are acquired through a process of reverse-transcription and recombination directed by endogenous retrotransposons. These vDNAs are thought to integrate in the host genome as endogenous viral elements (EVEs). Sequencing of pre-integrated vDNA revealed that the acquisition process exquisitely distinguishes viral from host RNA, providing one layer of self-nonself discrimination. Importantly, we show EVE-derived piRNAs have antiviral activity and are loaded onto Piwi4 to inhibit virus replication. In a second layer of self-non-self discrimination, Piwi4 preferentially loads EVE-derived piRNAs, discriminating against transposon-targeting piRNAs. Our findings define a fundamental virus-specific immunity pathway in mosquitoes that uses EVEs as a potent and specific antiviral transgenerational mechanism.

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September 22, 2019

Multi-population genomic analysis of malaria parasites indicates local selection and differentiation at the gdv1 locus regulating sexual development.

Parasites infect hosts in widely varying environments, encountering diverse challenges for adaptation. To identify malaria parasite genes under locally divergent selection across a large endemic region with a wide spectrum of transmission intensity, genome sequences were obtained from 284 clinical Plasmodium falciparum infections from four newly sampled locations in Senegal, The Gambia, Mali and Guinea. Combining these with previous data from seven other sites in West Africa enabled a multi-population analysis to identify discrete loci under varying local selection. A genome-wide scan showed the most exceptional geographical divergence to be at the early gametocyte gene locus gdv1 which is essential for parasite sexual development and transmission. We identified a major structural dimorphism with alternative 1.5?kb and 1.0?kb sequence deletions at different positions of the 3′-intergenic region, in tight linkage disequilibrium with the most highly differentiated single nucleotide polymorphism, one of the alleles being very frequent in Senegal and The Gambia but rare in the other locations. Long non-coding RNA transcripts were previously shown to include the entire antisense of the gdv1 coding sequence and the portion of the intergenic region with allelic deletions, suggesting adaptive regulation of parasite sexual development and transmission in response to local conditions.

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