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September 22, 2019

A hybrid-hierarchical genome assembly strategy to sequence the invasive golden mussel Limnoperna fortunei.

For more than 25 years, the golden mussel Limnoperna fortunei has aggressively invaded South American freshwaters, having travelled more than 5,000 km upstream across five countries. Along the way, the golden mussel has outcompeted native species and economically harmed aquaculture, hydroelectric powers, and ship transit. We have sequenced the complete genome of the golden mussel to understand the molecular basis of its invasiveness and search for ways to control it.We assembled the 1.6 Gb genome into 20548 scaffolds with an N50 length of 312 Kb using a hybrid and hierarchical assembly strategy from short and long DNA reads and transcriptomes. A total of 60717 coding genes were inferred from a customized transcriptome-trained AUGUSTUS run. We also compared predicted protein sets with those of complete molluscan genomes, revealing an exacerbation of protein-binding domains in L. fortunei. Conclusions: We built one of the best bivalve genome assemblies available using a cost-effective approach using Illumina pair-end, mate pair, and PacBio long reads. We expect that the continuous and careful annotation of L. fortunei’s genome will contribute to the investigation of bivalve genetics, evolution, and invasiveness, as well as to the development of biotechnological tools for aquatic pest control.© The Authors 2017. Published by Oxford University Press.

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September 22, 2019

Topical antibiotic use coselects for the carriage of mobile genetic elements conferring resistance to unrelated antimicrobials in Staphylococcus aureus.

Topical antibiotics, such as mupirocin and fusidic acid, are commonly used in the prevention and treatment of skin infections, particularly those caused by staphylococci. However, the widespread use of these agents is associated with increased resistance to these agents, potentially limiting their efficacy. Of particular concern is the observation that resistance to topical antibiotics is often associated with multidrug resistance, suggesting that topical antibiotics may play a role in the emergence of multidrug-resistant (MDR) strains. New Zealand (NZ) has some of the highest globally recorded rates of topical antibiotic usage and resistance. Using a combination of Pacific Biosciences single-molecule real-time (SMRT) whole-genome sequencing, Illumina short-read sequencing, and Bayesian phylogenomic modeling on 118 new multilocus sequence type 1 (ST1) communityStaphylococcus aureusisolates from New Zealand and 61 publically available international ST1 genome sequences, we demonstrate a strong correlation between the clinical introduction of topical antibiotics and the emergence of MDR ST1S. aureusWe also providein vitroexperimental evidence showing that exposure to topical antibiotics can lead to the rapid selection of MDRS. aureusisolates carrying plasmids that confer resistance to multiple unrelated antibiotics, from within a mixed population of competitor strains. These findings have important implications regarding the impact of the indiscriminate use of topical antibiotics. Copyright © 2018 Carter et al.

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September 22, 2019

By land, air, and sea: hemipteran diversity through the genomic lens

Thanks to a recent spate of sequencing projects, the Hemiptera are the first hemimetabolous insect order to achieve a critical mass of species with sequenced genomes, establishing the basis for comparative genomics of the bugs. However, as the most speciose hemimetabolous order, there is still a vast swathe of the hemipteran phylogeny that awaits genomic representation across subterranean, terrestrial, and aquatic habitats, and with lineage-specific and developmentally plastic cases of both wing polyphenisms and flightlessness. In this review, we highlight opportunities for taxonomic sampling beyond obvious pest species candidates, motivated by intriguing biological features of certain groups as well as the rich research tradition of ecological, physiological, developmental, and particularly cytogenetic investigation that spans the diversity of the Hemiptera.

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September 22, 2019

Genome sequences of Chlorella sorokiniana UTEX 1602 and Micractinium conductrix SAG 241.80: implications to maltose excretion by a green alga.

Green algae represent a key segment of the global species capable of photoautotrophic-driven biological carbon fixation. Algae partition fixed-carbon into chemical compounds required for biomass, while diverting excess carbon into internal storage compounds such as starch and lipids or, in certain cases, into targeted extracellular compounds. Two green algae were selected to probe for critical components associated with sugar production and release in a model alga. Chlorella sorokiniana UTEX 1602 – which does not release significant quantities of sugars to the extracellular space – was selected as a control to compare with the maltose-releasing Micractinium conductrix SAG 241.80 – which was originally isolated from an endosymbiotic association with the ciliate Paramecium bursaria. Both strains were subjected to three sequencing approaches to assemble their genomes and annotate their genes. This analysis was further complemented with transcriptional studies during maltose release by M. conductrix SAG 241.80 versus conditions where sugar release is minimal. The annotation revealed that both strains contain homologs for the key components of a putative pathway leading to cytosolic maltose accumulation, while transcriptional studies found few changes in mRNA levels for the genes associated with these established intracellular sugar pathways. A further analysis of potential sugar transporters found multiple homologs for SWEETs and tonoplast sugar transporters. The analysis of transcriptional differences revealed a lesser and more measured global response for M. conductrix SAG 241.80 versus C. sorokiniana UTEX 1602 during conditions resulting in sugar release, providing a catalog of genes that might play a role in extracellular sugar transport.© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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September 22, 2019

Sequence analysis of European maize inbred line F2 provides new insights into molecular and chromosomal characteristics of presence/absence variants.

Maize is well known for its exceptional structural diversity, including copy number variants (CNVs) and presence/absence variants (PAVs), and there is growing evidence for the role of structural variation in maize adaptation. While PAVs have been described in this important crop species, they have been only scarcely characterized at the sequence level and the extent of presence/absence variation and relative chromosomal landscape of inbred-specific regions remain to be elucidated.De novo genome sequencing of the French F2 maize inbred line revealed 10,044 novel genomic regions larger than 1 kb, making up 88 Mb of DNA, that are present in F2 but not in B73 (PAV). This set of maize PAV sequences allowed us to annotate PAV content and to analyze sequence breakpoints. Using PAV genotyping on a collection of 25 temperate lines, we also analyzed Linkage Disequilibrium in PAVs and flanking regions, and PAV frequencies within maize genetic groups.We highlight the possible role of MMEJ-type double strand break repair in maize PAV formation and discover 395 new genes with transcriptional support. Pattern of linkage disequilibrium within PAVs strikingly differs from this of flanking regions and is in accordance with the intuition that PAVs may recombine less than other genomic regions. We show that most PAVs are ancient, while some are found only in European Flint material, thus pinpointing structural features that may be at the origin of adaptive traits involved in the success of this material. Characterization of such PAVs will provide useful material for further association genetic studies in European and temperate maize.

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September 22, 2019

Biodegradation of decabromodiphenyl ether (BDE 209) by a newly isolated bacterium from an e-waste recycling area.

Polybrominated diphenyl ethers (PBDEs) have become widespread environmental pollutants all over the world. A newly isolated bacterium from an e-waste recycling area, Stenotrophomonas sp. strain WZN-1, can degrade decabromodiphenyl ether (BDE 209) effectively under aerobic conditions. Orthogonal test results showed that the optimum conditions for BDE 209 biodegradation were pH 5, 25 °C, 0.5% salinity, 150 mL minimal salt medium volume. Under the optimized condition, strain WZN-1 could degrade 55.15% of 65 µg/L BDE 209 under aerobic condition within 30 day incubation. Moreover, BDE 209 degradation kinetics was fitted to a first-order kinetics model. The biodegradation mechanism of BDE 209 by strain WZN-1 were supposed to be three possible metabolic pathways: debromination, hydroxylation, and ring opening processes. Four BDE 209 degradation genes, including one hydrolase, one dioxygenase and two dehalogenases, were identified based on the complete genome sequencing of strain WZN-1. The real-time qPCR demonstrated that the expression level of four identified genes were significantly induced by BDE 209, and they played an important role in the degradation process. This study is the first to demonstrate that the newly isolated Stenotrophomonas strain has an efficient BDE 209 degradation ability and would provide new insights for the microbial degradation of PBDEs.

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September 22, 2019

Bacterial artificial chromosome clones randomly selected for sequencing reveal genomic differences between soybean cultivars

This study pioneered the use of multiple technologies to combine the bacterial artificial chromosome (BAC) pooling strategy with high-throughput next- and third-generation sequencing technologies to analyse genomic difference. To understand the genetic background of the Chinese soybean cultivar N23601, we built a BAC library and sequenced 10 randomly selected clones followed by de novo assembly. Comparative analysis was conducted against the reference genome of Glycine max var. Williams 82 (2.0). Therefore, our result is an assessment of the reference genome. Our results revealed that 3517 single nucleotide polymorphisms (SNPs) and 662 insertion–deletions (InDels) occurred in ~1.2 Mb of the genomic region and that four of the 10 BAC clones contained 15 large structural variations (72?887?bp) compared with the reference genome. Gene annotation of the reference genome showed that Glyma.18g181000 was missing from the corresponding position of the 10 BAC clones. Additionally, there may be a problem with the assembly of some positions of the reference genome. Several gap regions in the reference genome could be supplemented by using the complete sequence of the 10 BAC clones. We believe that accurate and complete BAC sequence is a valuable resource that contributes to the completeness of the reference genome.

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September 22, 2019

Induced salt tolerance of perennial ryegrass by a novel bacterium strain from the rhizosphere of a desert shrub Haloxylon ammodendron.

Drought and soil salinity reduce agricultural output worldwide. Plant-growth-promoting rhizobacteria (PGPR) can enhance plant growth and augment plant tolerance to biotic and abiotic stresses.Haloxylon ammodendron, a C4 perennial succulent xerohalophyte shrub with excellent drought and salt tolerance, is naturally distributed in the desert area of northwest China. In our previous work, a bacterium strain numbered as M30-35 was isolated from the rhizosphere ofH. ammodendronin Tengger desert, Gansu province, northwest China. In current work, the effects of M30-35 inoculation on salt tolerance of perennial ryegrass were evaluated and its genome was sequenced to identify genes associated with plant growth promotion. Results showed that M30-35 significantly enhanced growth and salt tolerance of perennial ryegrass by increasing shoot fresh and dry weights, chlorophyll content, root volume, root activity, leaf catalase activity, soluble sugar and proline contents that contributed to reduced osmotic potential, tissue K? content and K?/Na? ratio, while decreasing malondialdehyde (MDA) content and relative electric conductivity (REC), especially under higher salinity. The genome of M30-35 contains 4421 protein encoding genes, 12 rRNA, 63 tRNA-encoding genes and four rRNA operons. M30-35 was initially classified as a new species inPseudomonasand named asPseudomonassp. M30-35. Thirty-four genes showing homology to genes associated with PGPR traits and abiotic stress tolerance were identified inPseudomonassp. M30-35 genome, including 12 related to insoluble phosphorus solubilization, four to auxin biosynthesis, four to other process of growth promotion, seven to oxidative stress alleviation, four to salt and drought tolerance and three to cold and heat tolerance. Further study is needed to clarify the correlation between these genes from M30-35 and the salt stress alleviation of inoculated plants under salt stress. Overall, our research indicated that desert shrubs appear rich in PGPRs that can help important crops tolerate abiotic stress.

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September 22, 2019

Basic characterization of natural transformation in a highly transformable Haemophilus parasuis strain SC1401.

Haemophilus parasuis causes Glässer’s disease and pneumonia, incurring serious economic losses in the porcine industry. In this study, natural competence was investigated in H. parasuis. We found competence genes in H. parasuis homologous to ones in Haemophilus influenzae and a high consensus battery of Sxy-dependent cyclic AMP (cAMP) receptor protein (CRP-S) regulons using bioinformatics. High rates of natural competence were found from the onset of stationary-phase growth condition to mid-stationary phase (OD600 from 0.29 to 1.735); this rapidly dropped off as cells reached mid-stationary phase (OD600 from 1.735 to 1.625). As a whole, bacteria cultured in liquid media were observed to have lower competence levels than those grown on solid media plates. We also revealed that natural transformation in this species is stable after 200 passages and is largely dependent on DNA concentration. Transformation competition experiments showed that heterogeneous DNA cannot outcompete intraspecific natural transformation, suggesting an endogenous uptake sequence or other molecular markers may be important in differentiating heterogeneous DNA. We performed qRT-PCR targeting multiple putative competence genes in an effort to compare bacteria pre-cultured in TSB++ vs. TSA++ and SC1401 vs. SH0165 to determine expression profiles of the homologs of competence-genes in H. influenzae. Taken together, this study is the first to investigate natural transformation in H. parasuis based on a highly naturally transformable strain SC1401.

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September 22, 2019

Pantoea ananatis genetic diversity analysis reveals limited genomic diversity as well as accessory genes correlated with onion pathogenicity.

Pantoea ananatis is a member of the family Enterobacteriaceae and an enigmatic plant pathogen with a broad host range. Although P. ananatis strains can be aggressive on onion causing foliar necrosis and onion center rot, previous genomic analysis has shown that P. ananatis lacks the primary virulence secretion systems associated with other plant pathogens. We assessed a collection of fifty P. ananatis strains collected from Georgia over three decades to determine genetic factors that correlated with onion pathogenic potential. Previous genetic analysis studies have compared strains isolated from different hosts with varying diseases potential and isolation sources. Strains varied greatly in their pathogenic potential and aggressiveness on different cultivated Allium species like onion, leek, shallot, and chive. Using multi-locus sequence analysis (MLSA) and repetitive extragenic palindrome repeat (rep)-PCR techniques, we did not observe any correlation between onion pathogenic potential and genetic diversity among strains. Whole genome sequencing and pan-genomic analysis of a sub-set of 10 strains aided in the identification of a novel series of genetic regions, likely plasmid borne, and correlating with onion pathogenicity observed on single contigs of the genetic assemblies. We named these loci Onion Virulence Regions (OVR) A-D. The OVR loci contain genes involved in redox regulation as well as pectate lyase and rhamnogalacturonase genes. Previous studies have not identified distinct genetic loci or plasmids correlating with onion foliar pathogenicity or pathogenicity on a single host pathosystem. The lack of focus on a single host system for this phytopathgenic disease necessitates the pan-genomic analysis performed in this study.

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September 22, 2019

Probiotic and anti-inflammatory potential of Lactobacillus rhamnosus 4B15 and Lactobacillus gasseri 4M13 isolated from infant feces.

A total of 22 Lactobacillus strains, which were isolated from infant feces were evaluated for their probiotic potential along with resistance to low pH and bile salts. Eight isolates (L. reuteri 3M02 and 3M03, L. gasseri 4M13, 4R22, 5R01, 5R02, and 5R13, and L. rhamnosus 4B15) with high tolerance to acid and bile salts, and ability to adhere to the intestine were screened from 22 strains. Further, functional properties of 8 Lactobacillus strains, such as anti-oxidation, inhibition of a-glucosidase activity, cholesterol-lowering, and anti-inflammation were evaluated. The properties were strain-specific. Particularly, two strains of L. rhamnosus, 4B15 (4B15) and L. gasseri 4M13 (4M13) showed considerably higher anti-oxidation, inhibition of a-glucosidase activity, and cholesterol-lowering, and greater inhibition of nitric oxide production than other strains. Moreover, the two selected strains substantially inhibited the release of inflammatory mediators such as TNF-a, IL-6, IL-1ß, and IL-10 stimulated the treatment of RAW 264.7 macrophages with LPS. In addition, whole genome sequencing and comparative genomic analysis of 4B15 and 4M13 indicated them as novel genomic strains. These results suggested that 4B15 and 4M13 showed the highest probiotic potential and have an impact on immune health by modulating pro-inflammatory cytokines.

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September 22, 2019

Characterizing the DNA methyltransferases of Haloferax volcanii via bioinformatics, gene deletion, and SMRT Sequencing.

DNA methyltransferases (MTases), which catalyze the methylation of adenine and cytosine bases in DNA, can occur in bacteria and archaea alongside cognate restriction endonucleases (REases) in restriction-modification (RM) systems or independently as orphan MTases. Although DNA methylation and MTases have been well-characterized in bacteria, research into archaeal MTases has been limited. A previous study examined the genomic DNA methylation patterns (methylome) of the halophilic archaeonHaloferax volcanii, a model archaeal system which can be easily manipulated in laboratory settings, via single-molecule real-time (SMRT) sequencing and deletion of a putative MTase gene (HVO_A0006). In this follow-up study, we deleted other putative MTase genes inH. volcaniiand sequenced the methylomes of the resulting deletion mutants via SMRT sequencing to characterize the genes responsible for DNA methylation. The results indicate that deletion of putative RM genesHVO_0794,HVO_A0006, andHVO_A0237in a single strain abolished methylation of the sole cytosine motif in the genome (Cm4TAG). Amino acid alignments demonstrated thatHVO_0794shares homology with characterized cytosine CTAG MTases in other organisms, indicating that this MTase is responsible for Cm4TAG methylation inH. volcanii. The CTAG motif has high density at only one of the origins of replication, and there is no relative increase in CTAG motif frequency in the genome ofH. volcanii, indicating that CTAG methylation might not have effectively taken over the role of regulating DNA replication and mismatch repair in the organism as previously predicted. Deletion of the putative Type I RM operonrmeRMS(HVO_2269-2271) resulted in abolished methylation of the adenine motif in the genome (GCAm6BN6VTGC). Alignments of the MTase (HVO_2270) and site specificity subunit (HVO_2271) demonstrate homology with other characterized Type I MTases and site specificity subunits, indicating that thermeRMSoperon is responsible for adenine methylation inH. volcanii. Together with HVO_0794, these genes appear to be responsible for all detected methylation inH. volcanii, even though other putative MTases (HVO_C0040,HVO_A0079) share homology with characterized MTases in other organisms. We also report the construction of a multi-RM deletion mutant (?RM), with multiple RM genes deleted and with no methylation detected via SMRT sequencing, which we anticipate will be useful for future studies on DNA methylation inH. volcanii.

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September 22, 2019

Assembly and analysis of a qingke reference genome demonstrate its close genetic relation to modern cultivated barley.

Qingke, the local name of hulless barley in the Tibetan Plateau, is a staple food for Tibetans. The availability of its reference genome sequences could be useful for studies on breeding and molecular evolution. Taking advantage of the third-generation sequencer (PacBio), we de novo assembled a 4.84-Gb genome sequence of qingke, cv. Zangqing320 and anchored a 4.59-Gb sequence to seven chromosomes. Of the 46,787 annotated ‘high-confidence’ genes, 31 564 were validated by RNA-sequencing data of 39 wild and cultivated barley genotypes with wide genetic diversity, and the results were also confirmed by nonredundant protein database from NCBI. As some gaps in the reference genome of Morex were covered in the reference genome of Zangqing320 by PacBio reads, we believe that the Zangqing320 genome provides the useful supplements for the Morex genome. Using the qingke genome as a reference, we conducted a genome comparison, revealing a close genetic relationship between a hulled barley (cv. Morex) and a hulless barley (cv. Zangqing320), which is strongly supported by the low-diversity regions in the two genomes. Considering the origin of Morex from its breeding pedigree, we then demonstrated a close genomic relationship between modern cultivated barley and qingke. Given this genomic relationship and the large genetic diversity between qingke and modern cultivated barley, we propose that qingke could provide elite genes for barley improvement.© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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September 22, 2019

A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering.

Despite the need for inducible promoters in strain development efforts, the majority of engineering in Saccharomyces cerevisiae continues to rely on a few constitutively active or inducible promoters. Building on advances that use the modular nature of both transcription factors and promoter regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein. Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain of the LexA protein, fused with the human estrogen binding domain and the viral activator domain, VP16. The resulting system with a bacterial DNA binding domain avoids the transcription of native S. cerevisiae genes, and the hybrid promoters can be induced using estradiol, a compound with no detectable impact on S. cerevisiae physiology. Using combinations of one, two or three operator sequence repeats and a set of native S. cerevisiae promoters, we obtained a series of hybrid promoters that can be induced to different levels, using the same synthetic TF and a given estradiol. This set of promoters, in combination with our synthetic TF, has the potential to regulate numerous genes or pathways simultaneously, to multiple desired levels, in a single strain.© 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

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September 22, 2019

Enhancing the adaptability of the deep-sea bacterium Shewanella piezotolerans WP3 to high pressure and low temperature by experimental evolution under H2O2 stress.

Oxidative stresses commonly exist in natural environments, and microbes have developed a variety of defensive systems to counteract such events. Although increasing evidence has shown that high hydrostatic pressure (HHP) and low temperature (LT) induce antioxidant defense responses in cells, there is no direct evidence to prove the connection between antioxidant defense mechanisms and the adaptation of bacteria to HHP and LT. In this study, using the wild-type (WT) strain of a deep-sea bacterium, Shewanella piezotolerans WP3, as an ancestor, we obtained a mutant, OE100, with an enhanced antioxidant defense capacity by experimental evolution under H2O2 stress. Notably, OE100 exhibited better tolerance not only to H2O2 stress but also to HHP and LT (20 MPa and 4°C, respectively). Whole-genome sequencing identified a deletion mutation in the oxyR gene, which encodes the transcription factor that controls the oxidative stress response. Comparative transcriptome analysis showed that the genes associated with oxidative stress defense, anaerobic respiration, DNA repair, and the synthesis of flagella and bacteriophage were differentially expressed in OE100 compared with the WT at 20 MPa and 4°C. Genetic analysis of oxyR and ccpA2 indicated that the OxyR-regulated cytochrome c peroxidase CcpA2 significantly contributed to the adaptation of WP3 to HHP and LT. Taken together, these results confirmed the inherent relationship between antioxidant defense mechanisms and the adaptation of a benthic microorganism to HHP and LT.IMPORTANCE Oxidative stress exists in various niches, including the deep-sea ecosystem, which is an extreme environment with conditions of HHP and predominantly LT. Although previous studies have shown that HHP and LT induce antioxidant defense responses in cells, direct evidence to prove the connection between antioxidant defense mechanisms and the adaptation of bacteria to HHP and LT is lacking. In this work, using the deep-sea bacterium Shewanella piezotolerans WP3 as a model, we proved that enhancement of the adaptability of WP3 to HHP and LT can benefit from its antioxidant defense mechanism, which provided useful insight into the ecological roles of antioxidant genes in a benthic microorganism and contributed to an improved understanding of microbial adaptation strategies in deep-sea environments.

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