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Asset Tag: Whole Genome Sequencing,

September 22, 2019

Genomic and genetic insights into a cosmopolitan fungus, Paecilomyces variotii (Eurotiales).

Species in the genus Paecilomyces, a member of the fungal order Eurotiales, are ubiquitous in nature and impact a variety of human endeavors. Here, the biology of one common species, Paecilomyces variotii, was explored using genomics and functional genetics. Sequencing the genome of two isolates revealed key genome and gene features in this species. A striking feature of the genome was the two-part nature, featuring large stretches of DNA with normal GC content separated by AT-rich regions, a hallmark of many plant-pathogenic fungal genomes. These AT-rich regions appeared to have been mutated by repeat-induced point (RIP) mutations. We developed methods for genetic transformation of P. variotii, including forward and reverse genetics as well as crossing techniques. Using transformation and crossing, RIP activity was identified, demonstrating for the first time that RIP is an active process within the order Eurotiales. A consequence of RIP is likely reflected by a reduction in numbers of genes within gene families, such as in cell wall degradation, and reflected by growth limitations on P. variotii on diverse carbon sources. Furthermore, using these transformation tools we characterized a conserved protein containing a domain of unknown function (DUF1212) and discovered it is involved in pigmentation.

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September 22, 2019

Genotypes and phenotypes of Enterococci isolated from broiler chickens

The objective of this study was to compare the resistance phenotypes to genotypes of enterococci from broiler and to evaluate the persistence and distribution of resistant genotypes in broiler fed bambermycin (BAM), penicillin (PEN), salinomycin (SAL), bacitracin (BAC) or a salinomycin/bacitracin combination (SALBAC) for 35 days. A total of 95 enterococci from cloacal (n=40), cecal (n=38) and litter collected on day 36 (n=17) samples were isolated weekly from day 7 to 36. All isolates were identified by API-20 Strep and their antimicrobial susceptibilities were evaluated using the Sensititre system with the commercially available NARMS’s plates of Gram positive bacteria. Whole genome sequencing (WGS) was used to assess their intra- and inter-genetic variability, with a focus on virulence and antibiotic resistance characteristics. All isolates were further characterized for hemolysin production (HEM), bile salt hydrolysis (BSH) and gelatinase (GEL) activities. Of the 95 isolates, E. faecium (n = 58) and E. faecalis (n = 24) were the most common Enterococcus species identified. Significant differences in the level of resistance for the E. faecium isolates to ciprofloxacin, macrolide, penicillin and tetracycline were observed among treatments. The bcrR, mefA and aac(6) genes were higher in BAM treatment than the other groups whereas bcrR, ermA, ermB, aphA(3) and tetL were more prevalent in PEN and BAC treatments. Overall, E. faecium isolates showed higher prevalence of antimicrobial resistance, but E. faecalis from litter also exhibited a significant level of resistance. A range of 4 to 15 different virulence genes was detected in E. faecalis. All isolates from litter but one (94.1%) showed BSH activities while 52.9% of them produced GEL. HEM activity was observed only in isolates collected on Day 7 (n= 9) and Day 14 (n= 1). This study confirmed that genetically diverse antimicrobial resistant enterococci harboring virulence factors can be promoted by the use of certain antimicrobials in feed and such enterococci could persist in broiler chickens and their litter, potentially contaminating the soil upon land application. This study underscores the need for ongoing monitoring the AMR enterococci.

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September 22, 2019

First draft genome for red sea bream of family Sparidae.

Reference genomes for all organisms on earth are now attainable owing to advances in genome sequencing technologies (Goodwin et al., 2016). Generally, species that contribute considerably to the economy or human welfare are sequenced and are considered more important than others. Furthermore, coastal indigenous people mainly depend on marine species for their food sources, which has resulted in the extinction of several marine species (Cisneros-Montemayor et al., 2016). Of these, an extinction risk assessment of marine fishes, mainly for sea breams (Family: Sparidae), has recently been conducted by way of a global extinction risk assessment from the dataset of the International Union for Conservation of Nature’s Red List Process, which mentions that around 25 species are threatened/near-threatened according to their body weight (Comeros-Raynal et al., 2016). Another report clearly showed the benefit of worldwide aquaculture production, which contributed to 47% of total seafood production, and also highlighted the over-fishing of sea breams (FAO, 2018). The Republic of Korea is the fourth largest seafood producer in the world, producing 3.3 million tons in 2015 and exporting seafood worth $1.6 billion in 2016; therefore, aquaculture- associated research is fundamental for Korea. In the present study, the red sea bream (Pagrus major), which belongs to the family Sparidae, which comprises 35 genera, 132 species, and 10 subspecies (de la Herran et al., 2001; NCBI, 2018), was assessed.

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September 22, 2019

A novel probiotic, Lactobacillus johnsonii 456, resists acid and can persist in the human gut beyond the initial ingestion period.

Probiotics are considered to have multiple beneficial effects on the human gastrointestinal tract, including immunomodulation, pathogen inhibition, and improved host nutrient metabolism. However, extensive characterization of these properties is needed to define suitable clinical applications for probiotic candidates. Lactobacillus johnsonii 456 (LBJ 456) was previously demonstrated to have anti-inflammatory and anti-genotoxic effects in a mouse model. Here, we characterize its resistance to gastric and bile acids as well as its ability to inhibit gut pathogens and adhere to host mucosa. While bile resistance and in vitro host attachment properties of LBJ 456 were comparable to other tested probiotics, LBJ 456 maintained higher viability at lower pH conditions compared to other tested strains. LBJ 456 also altered pathogen adhesion to LS 174T monolayers and demonstrated contact-dependent and independent inhibition of pathogen growth. Genome analyses further revealed possible genetic elements involved in host attachment and pathogen inhibition. Importantly, we show that ingestion of Lactobacillus johnsonii 456 over a one week yogurt course leads to persistent viable bacteria detectable even beyond the period of initial ingestion, unlike many other previously described probiotic species of lactic acid bacteria.

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September 22, 2019

Genome of Tenualosa ilisha from the river Padma, Bangladesh.

Hilsa shad (Tenualosa ilisha), is a popular fish of Bangladesh belonging to the Clupeidae family. An anadromous species, like the salmon and many other migratory fish, it is a unique species that lives in the sea and travels to freshwater rivers for spawning. During its entire life, Tenualosa ilisha migrates both from sea to freshwater and vice versa.The genome of Tenualosa ilisha collected from the river Padma of Rajshahi, Bangladesh has been sequenced and its de novo hybrid assembly and structural annotations are being reported here. Illumina and PacBio sequencing platforms were used for high depth sequencing and the draft genome assembly was found to be 816 MB with N50 size of 188 kb. MAKER gene annotation tool predicted 31,254 gene models. Benchmarking Universal Single-Copy Orthologs refer 95% completeness of the assembled genome.

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September 22, 2019

Development of New Tools to Detect Colistin-Resistance among Enterobacteriaceae Strains.

The recent discovery of the plasmid-mediated mcr-1 gene conferring resistance to colistin is of clinical concern. The worldwide screening of this resistance mechanism among samples of different origins has highlighted the urgent need to improve the detection of colistin-resistant isolates in clinical microbiology laboratories. Currently, phenotypic methods used to detect colistin resistance are not necessarily suitable as the main characteristic of the mcr genes is the low level of resistance that they confer, close to the clinical breakpoint recommended jointly by the CLSI and EUCAST expert systems (S?=?2?mg/L and R?>?2?mg/L). In this context, susceptibility testing recommendations for polymyxins have evolved and are becoming difficult to implement in routine laboratory work. The large number of mechanisms and genes involved in colistin resistance limits the access to rapid detection by molecular biology. It is therefore necessary to implement well-defined protocols using specific tools to detect all colistin-resistant bacteria. This review aims to summarize the current clinical microbiology diagnosis techniques and their ability to detect all colistin resistance mechanisms and describe new tools specifically developed to assess plasmid-mediated colistin resistance. Phenotyping, susceptibility testing, and genotyping methods are presented, including an update on recent studies related to the development of specific techniques.

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September 22, 2019

Description of Schaedlerella arabinophila gen. nov., sp. nov., a D-arabinose utilizing bacterium isolated from feces of C57BL/6J mice and a close relative of Clostridium sp. ASF 502

The use of gnotobiotics has gained large interest in recent years due to technological advances that have revealed the importance of host-associated microbiomes for host physiology and health. One of the oldest and most important gnotobiotics mouse model, the Altered Schaedler Flora (ASF) has been used for several decades. ASF comprises eight different bacterial species, which have been characterized to different extent, but only few are available through public strain collections. Here, the isolation of a close relative to one of the less studied ASF strains, Clostridium sp. ASF 502, is reported. Isolate TLL-A1, which shares 99.6% 16S rRNA gene sequence identity with Clostridium sp. ASF 502, was obtained from feces of C57BL/6J mice where is was detectable at a relative abundance of less than one percent. D-arabinose was used as sole carbon source in the anaerobic cultivation medium. Growth experiments with TLL-A1 on different carbon sources and analysis of its ~6.5 gigabase genome indicate that TLL-A1 harbors a large gene repertoire to utilize different carbohydrates for growth. Comparative genome analyses of TLL-A1 and Clostridium sp. ASF 502 reveal differences in genome content between the two strains, in particular with regards to carbohydrate activating enzymes. Based on physiology and genomic analysis it is proposed to name TLL-A1 to gen. nov. sp. nov Schaedlerella arabinophila TLL-A1 (DSMZ 106076T; KCTC 15657T). The closely related Clostridium sp. ASF 502 is proposed to be renamed to Schaedlerella arabinophila to reflect its taxonomic standing and to keep textquoterightASF 502textquoteright as strain designation.

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September 22, 2019

Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus.

Many cyanobacteria, which use light as an energy source via photosynthesis, have evolved the ability to guide their movement toward or away from a light source. This process, termed “phototaxis,” enables organisms to localize in optimal light environments for improved growth and fitness. Mechanisms of phototaxis have been studied in the coccoid cyanobacterium Synechocystis sp. strain PCC 6803, but the rod-shaped Synechococcus elongatus PCC 7942, studied for circadian rhythms and metabolic engineering, has no phototactic motility. In this study we report a recent environmental isolate of S. elongatus, the strain UTEX 3055, whose genome is 98.5% identical to that of PCC 7942 but which is motile and phototactic. A six-gene operon encoding chemotaxis-like proteins was confirmed to be involved in phototaxis. Environmental light signals are perceived by a cyanobacteriochrome, PixJSe (Synpcc7942_0858), which carries five GAF domains that are responsive to blue/green light and resemble those of PixJ from Synechocystis Plate-based phototaxis assays indicate that UTEX 3055 uses PixJSe to sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJSe controls both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis in Synechocystis.

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September 22, 2019

Sex chromosome evolution via two genes

The origin of sex chromosomes has been hypothesized to involve the linkage of factors with antagonistic effects on male and female function. Garden asparagus (Asparagus officinalis L.) is an ideal species to test this hypothesis, as the X and Y chromosomes are cytologically homomorphic and recently evolved from an ancestral autosome pair in association with a shift from hermaphroditism to dioecy. Mutagenesis screens paired with single-molecule fluorescence in situ hybridization (smFISH) directly implicate Y-specific genes that respectively suppress female organ development and are necessary for male gametophyte development. Comparison of contiguous X and Y chromosome shows that loss of recombination between the genes suppressing female function (SUPPRESSOR OF FEMALE FUNCTION, SOFF) and promoting male function (TAPETAL DEVELOPMENT AND FUNCTION 1, aspTDF1) is due to hemizygosity. We also experimentally demonstrate the function of aspTDF1. These finding provide direct evidence that sex chromosomes can evolve from autosomes via two sex determination genes: a dominant suppressor of femaleness and a promoter of maleness.

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September 22, 2019

De novo assembly of the Pasteuria penetrans genome reveals high plasticity, host dependency, and BclA-like collagens.

Pasteuria penetrans is a gram-positive endospore forming bacterial parasite of Meloidogyne spp. the most economically damaging genus of plant parasitic nematodes globally. The obligate antagonistic nature of P. penetrans makes it an attractive candidate biological control agent. However, deployment of P. penetrans for this purpose is inhibited by a lack of understanding of its metabolism and the molecular mechanics underpinning parasitism of the host, in particular the initial attachment of the endospore to the nematode cuticle. Several attempts to assemble the genomes of species within this genus have been unsuccessful. Primarily this is due to the obligate parasitic nature of the bacterium which makes obtaining genomic DNA of sufficient quantity and quality which is free from contamination challenging. Taking advantage of recent developments in whole genome amplification, long read sequencing platforms, and assembly algorithms, we have developed a protocol to generate large quantities of high molecular weight genomic DNA from a small number of purified endospores. We demonstrate this method via genomic assembly of P. penetrans. This assembly reveals a reduced genome of 2.64Mbp estimated to represent 86% of the complete sequence; its reduced metabolism reflects widespread reliance on the host and possibly associated organisms. Additionally, apparent expansion of transposases and prediction of partial competence pathways suggest a high degree of genomic plasticity. Phylogenetic analysis places our sequence within the Bacilli, and most closely related to Thermoactinomyces species. Seventeen predicted BclA-like proteins are identified which may be involved in the determination of attachment specificity. This resource may be used to develop in vitro culture methods and to investigate the genetic and molecular basis of attachment specificity.

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September 22, 2019

Integrative haplotype estimation with sub-linear complexity

The number of human genomes being genotyped or sequenced increases exponentially and efficient haplotype estimation methods able to handle this amount of data are now required. Here, we present a new method, SHAPEIT4, which substantially improves upon other methods to process large genotype and high coverage sequencing datasets. It notably exhibits sub-linear scaling with sample size, provides highly accurate haplotypes and allows integrating external phasing information such as large reference panels of haplotypes, collections of pre-phased variants and long sequencing reads. We provide SHAPET4 in an open source format on https://odelaneau.github.io/shapeit4/ and demonstrate its performance in terms of accuracy and running times on two gold standard datasets: the UK Biobank data and the Genome In A Bottle.

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September 22, 2019

CompStor Novos: a low cost yet fast assembly-based variant calling for personal genomes

Application of assembly methods for personal genome analysis from next generation sequencing data has been limited by the requirement for an expensive supercomputer hardware or long computation times when using ordinary resources. We describe CompStor Novos, achieving supercomputer-class performance in de novo assembly computation time on standard server hardware, based on a tiered-memory algorithm. Run on commercial off-the-shelf servers, Novos assembly is more precise and 10-20 times faster than that of existing assembly algorithms. Furthermore, we integrated Novos into a variant calling pipeline and demonstrate that both compute times and precision of calling point variants and indels compare well with standard alignment-based pipelines. Additionally, assembly eliminates bias in the estimation of allele frequency for indels and naturally enables discovery of breakpoints for structural variants with base pair resolution. Thus, Novos bridges the gap between alignment-based and assembly-based genome analyses. Extension and adaption of its underlying algorithm will help quickly and fully harvest information in sequencing reads for personal genome reconstruction.

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September 22, 2019

Comparative Analysis of the Genetic Basis of Branched Nonylphenol Degradation by Sphingobium amiense DSM 16289T and Sphingobium cloacae JCM 10874T.

Branched nonylphenol (BNP), a degradation product of nonylphenol polyethoxylates, exerts estrogenic effects on various organisms. The genes underlying BNP degradation by Sphingobium amiense DSM 16289T were analyzed by complete genome sequencing and compared with those of the versatile BNP-degrading Sphingobium cloacae JCM 10874T. An opdA homolog (opdADSM16289) encoding BNP degradation activity was identified in DSM 16289T, in contrast with JCM 10874T, possessing both the opdA homolog and nmoA. The degradation profile of different BNP isomers was examined by Escherichia coli transformants harboring opdADSM16289, opdAJCM10874, and nmoAJCM10874 to characterize and compare the expression activities of these genes.

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September 22, 2019

Mutators as drivers of adaptation in Streptococcus and a risk factor for host jumps and vaccine escape

Heritable hypermutable strains deficient in DNA repair genes (mutators) facilitate microbial adaptation as they may rapidly generate beneficial mutations. Mutators deficient in mismatch (MMR) and oxidised guanine (OG) repair are abundant in clinical samples and show increased adaptive potential in experimental infection models but their role in pathoadaptation is poorly understood. Here we investigate the role of mutators in epidemiology and evolution of the broad host pathogen, Streptococcus iniae, employing 80 strains isolated globally over 40 years. We determine phylogenetic relationship among S. iniae using 10,267 non-recombinant core genome single nucleotide polymorphisms (SNPs), estimate their mutation rate by fluctuation analysis, and detect variation in major MMR (mutS, mutL, dnaN, recD2, rnhC) and OG (mutY, mutM, mutX) genes. S. iniae mutation rate phenotype and genotype are strongly associated with phylogenetic diversification and variation in major streptococcal virulence determinants (capsular polysaccharide, hemolysin, cell chain length, resistance to oxidation, and biofilm formation). Furthermore, profound changes in virulence determinants observed in mammalian isolates (atypical host) and vaccine-escape isolates found in bone (atypical tissue) of vaccinated barramundi are linked to multiple MMR and OG variants and unique mutation rates. This implies that adaptation to new host taxa, new host tissue, and to immunity of a vaccinated host is promoted by mutator strains. Our findings support the importance of mutation rate dynamics in evolution of pathogenic bacteria, in particular adaptation to a drastically different immunological setting that occurs during host jump and vaccine escape events.Importance Host immune response is a powerful selective pressure that drives diversification of pathogenic microorganisms and, ultimately, evolution of new strains. Major adaptive events in pathogen evolution, such as transmission to a new host species or infection of vaccinated hosts, require adaptation to a drastically different immune landscape. Such adaptation may be favoured by hypermutable strains (or mutators) that are defective in normal DNA repair and consequently capable of generating multiple potentially beneficial and compensatory mutations. This permits rapid adjustment of virulence and antigenicity in a new immunological setting. Here we show that mutators, through mutations in DNA repair genes and corresponding shifts in mutation rate, are associated with major diversification events and virulence evolution in the broad host-range pathogen Streptococcus iniae. We show that mutators underpin infection of vaccinated hosts, transmission to new host species and the evolution of new strains.

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