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September 22, 2019

Sequencing of Panax notoginseng genome reveals genes involved in disease resistance and ginsenoside biosynthesis

Background: Panax notoginseng is a traditional Chinese herb with high medicinal and economic value. There has been considerable research on the pharmacological activities of ginsenosides contained in Panax spp.; however, very little is known about the ginsenoside biosynthetic pathway. Results: We reported the first de novo genome of 2.36 Gb of sequences from P. notoginseng with 35,451 protein-encoding genes. Compared to other plants, we found notable gene family contraction of disease-resistance genes in P. notoginseng, but notable expansion for several ATP-binding cassette (ABC) transporter subfamilies, such as the Gpdr subfamily, indicating that ABCs might be an additional mechanism for the plant to cope with biotic stress. Combining eight transcriptomes of roots and aerial parts, we identified several key genes, their transcription factor binding sites and all their family members involved in the synthesis pathway of ginsenosides in P. notoginseng, including dammarenediol synthase, CYP716 and UGT71. Conclusions: The complete genome analysis of P. notoginseng, the first in genus Panax, will serve as an important reference sequence for improving breeding and cultivation of this important nutraceutical and medicinal but vulnerable plant species.

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September 22, 2019

The integrative conjugative element clc (ICEclc) of Pseudomonas aeruginosa JB2.

Integrative conjugative elements (ICE) are a diverse group of chromosomally integrated, self-transmissible mobile genetic elements (MGE) that are active in shaping the functions of bacteria and bacterial communities. Each type of ICE carries a characteristic set of core genes encoding functions essential for maintenance and self-transmission, and cargo genes that endow on hosts phenotypes beneficial for niche adaptation. An important area to which ICE can contribute beneficial functions is the biodegradation of xenobiotic compounds. In the biodegradation realm, the best-characterized ICE is ICEclc, which carries cargo genes encoding for ortho-cleavage of chlorocatechols (clc genes) and aminophenol metabolism (amn genes). The element was originally identified in the 3-chlorobenzoate-degrader Pseudomonas knackmussii B13, and the closest relative is a nearly identical element in Burkholderia xenovorans LB400 (designated ICEclc-B13 and ICEclc-LB400, respectively). In the present report, genome sequencing of the o-chlorobenzoate degrader Pseudomonas aeruginosa JB2 was used to identify a new member of the ICEclc family, ICEclc-JB2. The cargo of ICEclc-JB2 differs from that of ICEclc-B13 and ICEclc-LB400 in consisting of a unique combination of genes that encode for the utilization of o-halobenzoates and o-hydroxybenzoate as growth substrates (ohb genes and hyb genes, respectively) and which are duplicated in a tandem repeat. Also, ICEclc-JB2 lacks an operon of regulatory genes (tciR-marR-mfsR) that is present in the other two ICEclc, and which controls excision from the host. Thus, the mechanisms regulating intracellular behavior of ICEclc-JB2 may differ from that of its close relatives. The entire tandem repeat in ICEclc-JB2 can excise independently from the element in a process apparently involving transposases/insertion sequence associated with the repeats. Excision of the repeats removes important niche adaptation genes from ICEclc-JB2, rendering it less beneficial to the host. However, the reduced version of ICEclc-JB2 could now acquire new genes that might be beneficial to a future host and, consequently, to the survival of ICEclc-JB2. Collectively, the present identification and characterization of ICEclc-JB2 provides insights into roles of MGE in bacterial niche adaptation and the evolution of catabolic pathways for biodegradation of xenobiotic compounds.

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September 22, 2019

Comparative genomics and genotype-phenotype associations in Bifidobacterium breve.

Bifidobacteria are common members of the gastro-intestinal microbiota of a broad range of animal hosts. Their successful adaptation to this particular niche is linked to their saccharolytic metabolism, which is supported by a wide range of glycosyl hydrolases. In the current study a large-scale gene-trait matching (GTM) effort was performed to explore glycan degradation capabilities in B. breve. By correlating the presence/absence of genes and associated genomic clusters with growth/no-growth patterns across a dataset of 20 Bifidobacterium breve strains and nearly 80 different potential growth substrates, we not only validated the approach for a number of previously characterized carbohydrate utilization clusters, but we were also able to discover novel genetic clusters linked to the metabolism of salicin and sucrose. Using GTM, genetic associations were also established for antibiotic resistance and exopolysaccharide production, thereby identifying (novel) bifidobacterial antibiotic resistance markers and showing that the GTM approach is applicable to a variety of phenotypes. Overall, the GTM findings clearly expand our knowledge on members of the B. breve species, in particular how their variable genetic features can be linked to specific phenotypes.

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September 22, 2019

The draft genomes of Elizabethkingia anophelis of equine origin are genetically similar to three isolates from human clinical specimens.

We report the isolation and characterization of two Elizabethkingia anophelis strains (OSUVM-1 and OSUVM-2) isolated from sources associated with horses in Oklahoma. Both strains appeared susceptible to fluoroquinolones and demonstrated high MICs to all cell wall active antimicrobials including vancomycin, along with aminoglycosides, fusidic acid, chloramphenicol, and tetracycline. Typical of the Elizabethkingia, both draft genomes contained multiple copies of ß-lactamase genes as well as genes predicted to function in antimicrobial efflux. Phylogenetic analysis of the draft genomes revealed that OSUVM-1 and OSUVM-2 differ by only 6 SNPs and are in a clade with 3 strains of Elizabethkingia anophelis that were responsible for human infections. These findings therefore raise the possibility that Elizabethkingia might have the potential to move between humans and animals in a manner similar to known zoonotic pathogens.

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September 22, 2019

Fusarium species complex causing Pokkah Boeng in China

Sugarcane is one of the most important crops for sugar production in sugarcane-growing areas. Many biotic and abiotic stresses affected the sugarcane production which leads to severe losses. Pokkah boeng is now playing a very important role due to its economic threats. Currently, the occurrence and rigorousness of pokkah boeng disease have been spread like wildfire from major sugarcane-growing countries. Pokkah boeng is a fungal disease that can cause serious yield losses in susceptible varieties. Infection of the disease is caused either by spores or ascospores. It may cause serious yield losses in commercial plantings. However, there have been many reported outbreaks of the disease which have looked spectacular but have caused trade and industry loss. Fusarium species complex is the major causal agent of this disease around the world, but some researchers have documented the increased importance of Fusarium. Three Fusarium species have been identified to cause the sugarcane pokkah boeng disease in China. Moreover, Fusarium may be accompanied of its mycotoxin production, genomic sequencing, and association with nitrogen application in China. Many studies on disease investigations, breeding of disease-resistant varieties, and strategy of disease control have also been carried out in China.

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September 22, 2019

Integrating long-range connectivity information into de Bruijn graphs.

The de Bruijn graph is a simple and efficient data structure that is used in many areas of sequence analysis including genome assembly, read error correction and variant calling. The data structure has a single parameter k, is straightforward to implement and is tractable for large genomes with high sequencing depth. It also enables representation of multiple samples simultaneously to facilitate comparison. However, unlike the string graph, a de Bruijn graph does not retain long range information that is inherent in the read data. For this reason, applications that rely on de Bruijn graphs can produce sub-optimal results given their input data.We present a novel assembly graph data structure: the Linked de Bruijn Graph (LdBG). Constructed by adding annotations on top of a de Bruijn graph, it stores long range connectivity information through the graph. We show that with error-free data it is possible to losslessly store and recover sequence from a Linked de Bruijn graph. With assembly simulations we demonstrate that the LdBG data structure outperforms both our de Bruijn graph and the String Graph Assembler (SGA). Finally we apply the LdBG to Klebsiella pneumoniae short read data to make large (12 kbp) variant calls, which we validate using PacBio sequencing data, and to characterize the genomic context of drug-resistance genes.Linked de Bruijn Graphs and associated algorithms are implemented as part of McCortex, which is available under the MIT license at https://github.com/mcveanlab/mccortex.Supplementary data are available at Bioinformatics online.

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September 22, 2019

Identification of the DNA methyltransferases establishing the methylome of the cyanobacterium Synechocystis sp. PCC 6803.

DNA methylation in bacteria is important for defense against foreign DNA, but is also involved in DNA repair, replication, chromosome partitioning, and regulatory processes. Thus, characterization of the underlying DNA methyltransferases in genetically tractable bacteria is of paramount importance. Here, we characterized the methylome and orphan methyltransferases in the model cyanobacterium Synechocystis sp. PCC 6803. Single molecule real-time (SMRT) sequencing revealed four DNA methylation recognition sequences in addition to the previously known motif m5CGATCG, which is recognized by M.Ssp6803I. For three of the new recognition sequences, we identified the responsible methyltransferases. M.Ssp6803II, encoded by the sll0729 gene, modifies GGm4CC, M.Ssp6803III, encoded by slr1803, represents the cyanobacterial dam-like methyltransferase modifying Gm6ATC, and M.Ssp6803V, encoded by slr6095 on plasmid pSYSX, transfers methyl groups to the bipartite motif GGm6AN7TTGG/CCAm6AN7TCC. The remaining methylation recognition sequence GAm6AGGC is probably recognized by methyltransferase M.Ssp6803IV encoded by slr6050. M.Ssp6803III and M.Ssp6803IV were essential for the viability of Synechocystis, while the strains lacking M.Ssp6803I and M.Ssp6803V showed growth similar to the wild type. In contrast, growth was strongly diminished of the ?sll0729 mutant lacking M.Ssp6803II. These data provide the basis for systematic studies on the molecular mechanisms impacted by these methyltransferases.

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September 22, 2019

Sequencing of pT5282-CTXM, p13190-KPC and p30860-NR, and comparative genomics analysis of IncX8 plasmids.

This study proposes a replicon-based scheme for typing IncX plasmids into nine separately clustering subgroups, including IncX1a, IncX1ß and IncX2-8. The complete nucleotide sequences of three IncX8 plasmids, namely pT5282-CTXM and p30860-NR from Enterobacter cloacae and p13190-KPC from Klebsiella pneumoniae, were determined and were compared with two other previously sequenced IncX8 plasmids (pCAV1043-58 and pCAV1741-16). These five plasmids possessed conserved IncX8 backbones with limited genetic variation with respect to gene content and organisation, and each of them carried one or three accessory modules that harboured resistance markers and metabolic gene clusters as well as transposons, insertion sequence (IS)-based transposition units and miniature inverted repeat transposable elements (MITEs), indicating that the relatively small IncX8 backbones were able to integrate various foreign genetic contents. The resistance genes blaCTX-M-3 and blaTEM-1 (ß-lactam resistance), blaKPC-2 (carbapenem resistance) and ?blaTEM-1, and tet(A) (tetracycline resistance) and mph(E) (macrolide resistance) were found in pT5282-CTXM, p13190-KPC and pCAV1741-16, respectively, whilst p30860-NR and pCAV1043-58 carried no resistance genes. The data presented here provide an insight into the diversification and evolution history of IncX8 plasmids. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

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September 22, 2019

Analysis of the draft genome of the red seaweed Gracilariopsis chorda provides insights into genome size evolution in Rhodophyta.

Red algae (Rhodophyta) underwent two phases of large-scale genome reduction during their early evolution. The red seaweeds did not attain genome sizes or gene inventories typical of other multicellular eukaryotes. We generated a high-quality 92.1 Mb draft genome assembly from the red seaweed Gracilariopsis chorda, including methylation and small (s)RNA data. We analyzed these and other Archaeplastida genomes to address three questions: 1) What is the role of repeats and transposable elements (TEs) in explaining Rhodophyta genome size variation, 2) what is the history of genome duplication and gene family expansion/reduction in these taxa, and 3) is there evidence for TE suppression in red algae? We find that the number of predicted genes in red algae is relatively small (4,803-13,125 genes), particularly when compared with land plants, with no evidence of polyploidization. Genome size variation is primarily explained by TE expansion with the red seaweeds having the largest genomes. Long terminal repeat elements and DNA repeats are the major contributors to genome size growth. About 8.3% of the G. chorda genome undergoes cytosine methylation among gene bodies, promoters, and TEs, and 71.5% of TEs contain methylated-DNA with 57% of these regions associated with sRNAs. These latter results suggest a role for TE-associated sRNAs in RNA-dependent DNA methylation to facilitate silencing. We postulate that the evolution of genome size in red algae is the result of the combined action of TE spread and the concomitant emergence of its epigenetic suppression, together with other important factors such as changes in population size.

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September 22, 2019

Genomes of ubiquitous marine and hypersaline Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira spp. encode a diversity of mechanisms to sustain chemolithoautotrophy in heterogeneous environments.

Chemolithoautotrophic bacteria from the genera Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira are common, sometimes dominant, isolates from sulfidic habitats including hydrothermal vents, soda and salt lakes and marine sediments. Their genome sequences confirm their membership in a deeply branching clade of the Gammaproteobacteria. Several adaptations to heterogeneous habitats are apparent. Their genomes include large numbers of genes for sensing and responding to their environment (EAL- and GGDEF-domain proteins and methyl-accepting chemotaxis proteins) despite their small sizes (2.1-3.1 Mbp). An array of sulfur-oxidizing complexes are encoded, likely to facilitate these organisms’ use of multiple forms of reduced sulfur as electron donors. Hydrogenase genes are present in some taxa, including group 1d and 2b hydrogenases in Hydrogenovibrio marinus and H. thermophilus MA2-6, acquired via horizontal gene transfer. In addition to high-affinity cbb3 cytochrome c oxidase, some also encode cytochrome bd-type quinol oxidase or ba3 -type cytochrome c oxidase, which could facilitate growth under different oxygen tensions, or maintain redox balance. Carboxysome operons are present in most, with genes downstream encoding transporters from four evolutionarily distinct families, which may act with the carboxysomes to form CO2 concentrating mechanisms. These adaptations to habitat variability likely contribute to the cosmopolitan distribution of these organisms.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

Genetic and biochemical characterization of 5-hydroxypicolinic acid metabolism in Alcaligenes faecalis JQ135.

5-Hydroxypicolinic acid (5HPA), a natural pyridine derivative, is microbially degraded in the environment. However, the physiological, biochemical, and genetic foundations of the 5HPA metabolism remain unknown. In this study, an operon (hpa), responsible for 5HPA degradation, was cloned from Alcaligenes faecalis JQ135. HpaM was a monocomponent FAD-dependent monooxygenase and shared low identity (only 28-31%) with reported monooxygenases. HpaM catalyzed the ortho decarboxylative hydroxylation of 5HPA, generating 2,5-dihydroxypyridine (2,5DHP). The monooxygenase activity of HpaM was FAD and NADH-dependent. The apparent Km values of HpaM for 5HPA and NADH were 45.4 µM and 37.8 µM, respectively. The genes hpaX, hpaD, and hpaF were found to encode 2,5DHP dioxygenase, N-formylmaleamic acid deformylase, and maleamate amidohydrolase, respectively; however, the three genes were not essential for 5HPA degradation in A. faecalis JQ135. Furthermore, the gene maiA, which encodes a maleic acid cis-trans isomerase, was essential for the metabolism of 5HPA, nicotinic acid, and picolinic acid in A. faecalis JQ135, indicating that it might be a key gene in the metabolism of pyridine derivatives. The genes and proteins identified in this study showed a novel degradation mechanism of pyridine derivatives.Importance Unlike the benzene ring, the uneven distribution of the electron density of pyridine ring influences the positional reactivity and the interaction with enzymes, e.g., the ortho and para oxidation are more difficult than the meta oxidations. Hydroxylation is an important oxidation process for the pyridine derivative metabolism. In previous reports, the ortho hydroxylation of pyridine derivatives were catalyzed by multicomponent molybdenum-containing monooxygenases, while the meta hydroxylations were catalyzed by monocomponent FAD-dependent monooxygenases. This study identified the new monocomponent FAD-dependent monooxygenase HpaM that catalyzed the ortho decarboxylative hydroxylation of 5HPA. In addition, we found that the maiA coding for maleic acid cis-trans isomerase was pivotal for the metabolism of 5HPA, nicotinic acid, and picolinic acid in A. faecalis JQ135. This study provides novel insights into the microbial metabolism of pyridine derivatives. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Identification and characterization of conjugative plasmids that encode ciprofloxacin resistance in Salmonella.

This study aimed to characterize novel conjugative plasmids that encode transferrable ciprofloxacin resistance in Salmonella In this study, 157 non-duplicated Salmonella isolates were recovered from food products, 55 out of which were found to be resistant to ciprofloxacin. Interestingly, 37 out of the 55 (67%) CipRSalmonella isolates did not harbor any mutations in the Quinolone resistance determine regions (QRDR). Interestingly, six Salmonella isolates were shown to carry two novel types of conjugative plasmids that could transfer ciprofloxacin resistance phenotype to E. coli J53 (AziR). The first type belonged to the ~110kb IncFIB type conjugative plasmid carrying qnrB-bearing and aac(6′)-Ib-cr-bearing mobile elements. Transfer of the plasmid between E. coli or Salmonella could confer CIP MIC to 1 to 2µg/ml. The second type of conjugative plasmid belonged to ~240kb IncH1/IncF plasmids carrying a single PMQR gene, qnrS Importantly, this type of conjugative ciprofloxacin resistance plasmids could be detected in clinical isolates of Salmonella Dissemination of these conjugative plasmids that confer ciprofloxaicn resistance poses serious public health impact and Salmonella infection control. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Analysis of the Gli-D2 locus identifies a genetic target for simultaneously improving the breadmaking and health-related traits of common wheat.

Gliadins are a major component of wheat seed proteins. However, the complex homoeologous Gli-2 loci (Gli-A2, -B2 and -D2) that encode the a-gliadins in commercial wheat are still poorly understood. Here we analyzed the Gli-D2 locus of Xiaoyan 81 (Xy81), a winter wheat cultivar. A total of 421.091 kb of the Gli-D2 sequence was assembled from sequencing multiple bacterial artificial clones, and 10 a-gliadin genes were annotated. Comparative genomic analysis showed that Xy81 carried only eight of the a-gliadin genes of the D genome donor Aegilops tauschii, with two of them each experiencing a tandem duplication. A mutant line lacking Gli-D2 (DLGliD2) consistently exhibited better breadmaking quality and dough functionalities than its progenitor Xy81, but without penalties in other agronomic traits. It also had an elevated lysine content in the grains. Transcriptome analysis verified the lack of Gli-D2 a-gliadin gene expression in DLGliD2. Furthermore, the transcript and protein levels of protein disulfide isomerase were both upregulated in DLGliD2 grains. Consistent with this finding, DLGliD2 had increased disulfide content in the flour. Our work sheds light on the structure and function of Gli-D2 in commercial wheat, and suggests that the removal of Gli-D2 and the gliadins specified by it is likely to be useful for simultaneously enhancing the end-use and health-related traits of common wheat. Because gliadins and homologous proteins are widely present in grass species, the strategy and information reported here may be broadly useful for improving the quality traits of diverse cereal crops.© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

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September 22, 2019

Characterization of LE3 and LE4, the only lytic phages known to infect the spirochete Leptospira.

Leptospira is a phylogenetically unique group of bacteria, and includes the causative agents of leptospirosis, the most globally prevalent zoonosis. Bacteriophages in Leptospira are largely unexplored. To date, a genomic sequence is available for only one temperate leptophage called LE1. Here, we sequenced and analysed the first genomes of the lytic phages LE3 and LE4 that can infect the saprophyte Leptospira biflexa using the lipopolysaccharide O-antigen as receptor. Bioinformatics analysis showed that the 48-kb LE3 and LE4 genomes are similar and contain 62% genes whose function cannot be predicted. Mass spectrometry led to the identification of 21 and 23 phage proteins in LE3 and LE4, respectively. However we did not identify significant similarities with other phage genomes. A search for prophages close to LE4 in the Leptospira genomes allowed for the identification of a related plasmid in L. interrogans and a prophage-like region in the draft genome of a clinical isolate of L. mayottensis. Long-read whole genome sequencing of the L. mayottensis revealed that the genome contained a LE4 phage-like circular plasmid. Further isolation and genomic comparison of leptophages should reveal their role in the genetic evolution of Leptospira.

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September 22, 2019

Citrobacter freundii fitness during bloodstream infection.

Sepsis resulting from microbial colonization of the bloodstream is a serious health concern associated with high mortality rates. The objective of this study was to define the physiologic requirements of Citrobacter freundii in the bloodstream as a model for bacteremia caused by opportunistic Gram-negative pathogens. A genetic screen in a murine host identified 177 genes that contributed significantly to fitness, the majority of which were broadly classified as having metabolic or cellular maintenance functions. Among the pathways examined, the Tat protein secretion system conferred the single largest fitness contribution during competition infections and a putative Tat-secreted protein, SufI, was also identified as a fitness factor. Additional work was focused on identifying relevant metabolic pathways for bacteria in the bloodstream environment. Mutations that eliminated the use of glucose or mannitol as carbon sources in vitro resulted in loss of fitness in the murine model and similar results were obtained upon disruption of the cysteine biosynthetic pathway. Finally, the conservation of identified fitness factors was compared within a cohort of Citrobacter bloodstream isolates and between Citrobacter and Serratia marcescens, the results of which suggest the presence of conserved strategies for bacterial survival and replication in the bloodstream environment.

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