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September 22, 2019

Assembling the genome of the African wild rice Oryza longistaminata by exploiting synteny in closely related Oryza species.

The African wild rice species Oryza longistaminata has several beneficial traits compared to cultivated rice species, such as resistance to biotic stresses, clonal propagation via rhizomes, and increased biomass production. To facilitate breeding efforts and functional genomics studies, we de-novo assembled a high-quality, haploid-phased genome. Here, we present our assembly, with a total length of 351?Mb, of which 92.2% was anchored onto 12 chromosomes. We detected 34,389 genes and 38.1% of the genome consisted of repetitive content. We validated our assembly by a comparative linkage analysis and by examining well-characterized gene families. This genome assembly will be a useful resource to exploit beneficial alleles found in O. longistaminata. Our results also show that it is possible to generate a high-quality, functionally complete rice genome assembly from moderate SMRT read coverage by exploiting synteny in a closely related Oryza species.

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September 22, 2019

Production of glycine-derived ammonia as a low-cost and long-distance antibiotic strategy by Streptomyces

Soil-inhabiting streptomycetes are Natures medicine makers, producing over half of all known antibiotics and many other bioactive natural products. However, these bacteria also produce many volatile compounds, and research into these molecules and their role in soil ecology is rapidly gaining momentum. Here we show that streptomycetes have the ability to kill bacteria over long distances via air-borne antibiosis. Our research shows that streptomycetes do so by producing surprisingly high amounts of the low-cost volatile antimicrobial ammonia, which travels over long distances and antagonises both Gram-positive and Gram-negative bacteria. Glycine is required as precursor to produce ammonia, and inactivation of the glycine cleavage system annihilated air-borne antibiosis. As a resistance strategy, E. coli cells acquired mutations resulting in reduced expression of the porin master regulator OmpR and its cognate kinase EnvZ, which was just enough to allow them to survive. We further show that ammonia enhances the activity of the more costly canonical antibiotics, suggesting that streptomycetes adopt a low-cost strategy to sensitize competitors for antibiosis over longer distances.

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September 22, 2019

Haematococcus lacustris: the makings of a giant-sized chloroplast genome.

Recent work on the chlamydomonadalean green alga Haematococcus lacustris uncovered the largest plastid genome on record: a whopping 1.35 Mb with >90 % non-coding DNA. A 500-word description of this genome was published in the journal Genome Announcements. But such a short report for such a large genome leaves many unanswered questions. For instance, the H. lacustris plastome was found to encode only 12 tRNAs, less than half that of a typical plastome, it appears to have a non-standard genetic code, and is one of only a few known plastid DNAs (ptDNAs), out of thousands of available sequences, not biased in adenine and thymine. Here, I take a closer look at the H. lacustris plastome, comparing its size, content and architecture to other large organelle DNAs, including those from close relatives in the Chlamydomonadales. I show that the H. lacustris plastid coding repertoire is not as unusual as initially thought, representing a standard set of rRNAs, tRNAs and protein-coding genes, where the canonical stop codon UGA appears to sometimes signify tryptophan. The intergenic spacers are dense with repeats, and it is within these regions where potential answers to the source of such extreme genomic expansion lie. By comparing ptDNA sequences of two closely related strains of H. lacustris, I argue that the mutation rate of the non-coding DNA is high and contributing to plastome inflation. Finally, by exploring publicly available RNA-sequencing data, I find that most of the intergenic ptDNA is transcriptionally active.

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September 22, 2019

Coculture of marine Streptomyces sp. with Bacillus sp. produces a newpiperazic acid-bearing cyclic peptide.

Microbial culture conditions in the laboratory, which conventionally involve the cultivation of one strain in one culture vessel, are vastly different from natural microbial environments. Even though perfectly mimicking natural microbial interactions is virtually impossible, the cocultivation of multiple microbial strains is a reasonable strategy to induce the production of secondary metabolites, which enables the discovery of new bioactive natural products. Our coculture of marine Streptomyces and Bacillus strains isolated together from an intertidal mudflat led to discover a new metabolite, dentigerumycin E (1). Dentigerumycin E was determined to be a new cyclic hexapeptide incorporating three piperazic acids, N-OH-Thr, N-OH-Gly, ß-OH-Leu, and a pyran-bearing polyketide acyl chain mainly by analysis of its NMR and MS spectroscopic data. The putative PKS-NRPS biosynthetic gene cluster for dentigerumycin E was found in the Streptomyces strain, providing clear evidence that this cyclic peptide is produced by the Streptomyces strain. The absolute configuration of dentigerumycin E was established based on the advanced Marfey’s method, ROESY NMR correlations, and analysis of the amino acid sequence of the ketoreductase domain in the biosynthetic gene cluster. In biological evaluation of dentigerumycin E (1) and its chemical derivatives [2-N,16-N-deoxydenteigerumycin E (2) and dentigerumycin methyl ester (3)], only dentigerumycin E exhibited antiproliferative and antimetastatic activities against human cancer cells, indicating that N-OH and carboxylic acid functional groups are essential for the biological activity.

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September 22, 2019

Insight into metabolic versatility of an aromatic compounds-degrading Arthrobacter sp. YC-RL1.

The genus Arthrobacter is ubiquitously distributed in different natural environments. Many xenobiotic-degrading Arthrobacter strains have been isolated and described; however, few have been systematically characterized with regard to multiple interrelated metabolic pathways and the genes that encode them. In this study, the biodegradability of seven aromatic compounds by Arthrobacter sp. YC-RL1 was investigated. Strain YC-RL1 could efficiently degrade p-xylene (PX), naphthalene, phenanthrene, biphenyl, p-nitrophenol (PNP), and bisphenol A (BPA) under both separated and mixed conditions. Based on the detected metabolic intermediates, metabolic pathways of naphthalene, biphenyl, PNP, and BPA were proposed, which indicated that strain YC-RL1 harbors systematic metabolic pathways toward aromatic compounds. Further, genomic analysis uncovered part of genes involved in the proposed pathways. Both intradiol and extradiol ring-cleavage dioxygenase genes were identified in the genome of strain YC-RL1. Meanwhile, gene clusters predicted to encode the degradation of biphenyl (bph), para-substituted phenols (npd) and protocatechuate (pca) were identified, and bphA1A2BCD was proposed to be a novel biphenyl-degrading gene cluster. The complete metabolic pathway of biphenyl was deduced via intermediates and functional gene analysis (bph and pca gene clusters). One of the these genes encoding ring-cleavage dioxygenase in bph gene cluster, a predicted 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) gene, was cloned and its activity was confirmed by heterologous expression. This work systematically illuminated the metabolic versatility of aromatic compounds in strain YC-RL1 via the combination of metabolites identification, genomics analysis and laboratory experiments. These results suggested that strain YC-RL1 might be a promising candidate for the bioremediation of aromatic compounds pollution sites.

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September 22, 2019

Conversion of methionine to cysteine in Lactobacillus paracasei depends on the highly mobile cysK-ctl-cysE gene cluster.

Milk and dairy products are rich in nutrients and are therefore habitats for various microbiomes. However, the composition of nutrients can be quite diverse, in particular among the sulfur containing amino acids. In milk, methionine is present in a 25-fold higher abundance than cysteine. Interestingly, a fraction of strains of the species L. paracasei – a flavor-enhancing adjunct culture species – can grow in medium with methionine as the sole sulfur source. In this study, we focus on genomic and evolutionary aspects of sulfur dependence in L. paracasei strains. From 24 selected L. paracasei strains, 16 strains can grow in medium with methionine as sole sulfur source. We sequenced these strains to perform gene-trait matching. We found that one gene cluster – consisting of a cysteine synthase, a cystathionine lyase, and a serine acetyltransferase – is present in all strains that grow in medium with methionine as sole sulfur source. In contrast, strains that depend on other sulfur sources do not have this gene cluster. We expanded the study and searched for this gene cluster in other species and detected it in the genomes of many bacteria species used in the food production. The comparison to these species showed that two different versions of the gene cluster exist in L. paracasei which were likely gained in two distinct events of horizontal gene transfer. Additionally, the comparison of 62 L. paracasei genomes and the two versions of the gene cluster revealed that this gene cluster is mobile within the species.

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September 22, 2019

Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications.

Modern genotyping techniques, such as SNP analysis and genotyping by sequencing (GBS), are hampered by poor DNA quality and purity, particularly in challenging plant species, rich in secondary metabolites. We therefore investigated the utility of a pre-wash step using a buffered sorbitol solution, prior to DNA extraction using a high salt CTAB extraction protocol, in a high throughput or miniprep setting. This pre-wash appears to remove interfering metabolites, such as polyphenols and polysaccharides, from tissue macerates. We also investigated the adaptability of the sorbitol pre-wash for RNA extraction using a lithium chloride-based protocol. The method was successfully applied to a variety of tissues, including leaf, cambium and fruit of diverse plant species including annual crops, forest and fruit trees, herbarium leaf material and lyophilized fungal mycelium. We consistently obtained good yields of high purity DNA or RNA in all species tested. The protocol has been validated for thousands of DNA samples by generating high data quality in dense SNP arrays. DNA extracted from Eucalyptus spp. leaf and cambium as well as mycelium from Trichoderma spp. was readily digested with restriction enzymes and performed consistently in AFLP assays. Scaled-up DNA extractions were also suitable for long read sequencing. Successful RNA quality control and good RNA-Seq data for Eucalyptus and cashew confirms the effectiveness of the sorbitol buffer pre-wash for high quality RNA extraction.

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September 22, 2019

Bacillus wiedmannii biovar thuringiensis: A specialized mosquitocidal pathogen with plasmids from diverse origins.

Bacillus cereus sensu lato also known as B. cereus group is composed of an ecologically diverse bacterial group with an increasing number of related species, some of which are medically or agriculturally important. Numerous e?orts have been undertaken to allow presumptive di?erentiation of B. cereus group species from one another. FCC41 is a Bacillus sp. strain toxic against mosquito species like Aedes aegypti, Aedes (Ochlerotatus) albifasciatus, Culex pipiens, Culex quinquefasciatus, and Culex apicinus, some of them responsible for the transmission of vector-borne diseases. Here, we report the complete genome sequence of FCC41 strain, which consists of one circular chromosome and eight circular plasmids ranging in size from 8 to 490?kb. This strain harbors six crystal protein genes, including cry24Ca, two cry4-like and two cry52-like, a cry41-like parasporin gene and multiple virulence factors. The phylogenetic analysis of the whole-genome sequence of this strain with molecular approaches places this strain into the Bacillus wiedmannii cluster. However, according with phenotypical characteristics such as the mosquitocidal activity due to the presence of Cry proteins found in the parasporal body and cry genes encoded in plasmids of different sizes, indicate that this strain could be renamed as B. wiedmannii biovar thuringiensis strain FCC41.

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September 22, 2019

Type II restriction modification system in Ureaplasma parvum OMC-P162 strain.

Ureaplasma parvum serovar 3 strain, OMC-P162, was isolated from the human placenta of a preterm delivery at 26 weeks’ gestation. In this study, we sequenced the complete genome of OMC-P162 and compared it with other serovar 3 strains isolated from patients with different clinical conditions. Ten unique genes in OMC-P162, five of which encoded for hypothetical proteins, were identified. Of these, genes UPV_229 and UPV_230 formed an operon whose open reading frames were predicted to code for a DNA methyltransferase and a hypothetical protein, respectively. DNA modification analysis of the OMC-P162 genome identified N4-methylcytosine (m4C) and N6-methyladenine (m6A), but not 5-methylocytosine (m5C). UPV230 recombinant protein displayed endonuclease activity and recognized the CATG sequence, resulting in a blunt cut between A and T. This restriction enzyme activity was identical to that of the cultivated OMC-P162 strain, suggesting that this restriction enzyme was naturally expressed in OMC-P162. We designated this enzyme as UpaP162. Treatment of pT7Blue plasmid with recombinant protein UPV229 completely blocked UpaP162 restriction enzyme activity. These results suggest that the UPV_229 and UPV_230 genes act as a type II restriction-modification system in Ureaplasma OMC-P162.

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September 22, 2019

Genome analysis of the yeast M14, an industrial brewing yeast strain widely used in China

The lager brewing yeast M14 is the most widely used yeast strain in the high gravity brewing process in China. To investigate the characteristics of this strain, the genome of the yeast M14 was sequenced and the genome annotation information is presented in this study. The current assembly contained 133 scaffolds and its total size was around 23?Mb with a GC content of 38.98%. The brewing yeast M14 is a hybrid Saccharomyces cerevisiae?×?Saccharomyces uvarum at the genomic level and its genome is comprised of one circular mitochondrial genome originating from S. uvarum. Furthermore, the functions of the 9,796 protein coding genes were annotated and their functions were analyzed using the Swiss-Prot database. Among them, the key genes responsible for typical lager brewing yeast characteristics, such as maltotriose uptake and sulfite production, were annotated and analyzed. Interestingly, nine specific genes present in the brewing yeast M14 were not found in the genome of either S. uvarum CBS 7001 or S. cerevisiae S288C, which are very close to strain M14 in the phylogenetic relationship. These nine genes encoding proteins were melibiase, DNA replication protein, fructose symporter, hypothetical protein, hypothetical protein M773_09155, LIF1, minor spike protein H, ribosomal protein S27, and mitochondrial chaperones, respectively. The genome sequence of the yeast strain M14 provides a new tool to better understand brewing yeast behavior in industrial beer production.

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September 22, 2019

Characterization of the lytic bacteriophage phiEaP-8 effective against both Erwinia amylovora and Erwinia pyrifoliae causing severe diseases in apple and pear.

Bacteriophages, bacteria-infecting viruses, have been recently reconsidered as a biological control tool for preventing bacterial pathogens. Erwinia amylovora and E. pyrifoliae cause fire blight and black shoot blight disease in apple and pear, respectively. In this study, the bacteriophage phiEaP-8 was isolated from apple orchard soil and could efficiently and specifically kill both E. amylovora and E. pyrifoliae. This bacteriophage belongs to the Podoviridae family. Whole genome analysis revealed that phiEaP-8 carries a 75,929 bp genomic DNA with 78 coding sequences and 5 tRNA genes. Genome comparison showed that phiEaP-8 has only 85% identity to known bacteriophages at the DNA level. PhiEaP-8 retained lytic activity up to 50°C, within a pH range from 5 to 10, and under 365 nm UV light. Based on these characteristics, the bacteriophage phiEaP-8 is novel and carries potential to control both E. amylovora and E. pyrifoliae in apple and pear.

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September 22, 2019

Endogenous rRNA sequence variation can regulate stress response gene expression and phenotype.

Prevailing dogma holds that ribosomes are uniform in composition and function. Here, we show that nutrient limitation-induced stress in E. coli changes the relative expression of rDNA operons to alter the rRNA composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes affect the expression of functionally coherent gene sets and alter the levels of the RpoS sigma factor, the master regulator of the general stress response. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility and are regulated by stress response proteins, RelA and RelE, as well as the metabolic enzyme and virulence-associated protein, AdhE. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Comparative genome analysis and evaluation of probiotic characteristics of Lactobacillus plantarum strain JDFM LP11.

In the current study, the probiotic potential of approximately 250 strains of lactic acid bacteria (LAB) isolated from piglet fecal samples were investigated; among them Lactobacillus plantarum strain JDFM LP11, which possesses significant probiotic potential, with enhanced acid/bile tolerance, attachment to porcine intestinal epithelial cells (IPEC-J2), and antimicrobial activity. The genetic characteristics of strain JDFM LP11 were explored by performing whole genome sequencing (WGS) using a PacBio system. The circular draft genome have a total length of 3,206,883 bp and a total of 3,021 coding sequences were identified. Phylogenetically, three genes, possibly related to survival and metabolic activity in the porcine host, were identified. These genes encode p60, lichenan permease IIC component, and protein TsgA, which are a putative endopeptidase, a component of the phosphotransferase system (PTS), and a major facilitator in the gut environment, respectively. Our findings suggest that understanding the functional and genetic characteristics of L. plantarum strain JDFM LP11, with its candidate genes for gut health, could provide new opportunities and insights into applications in the animal food and feed additive industries.

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September 22, 2019

Loss of bacitracin resistance due to a large genomic deletion among Bacillus anthracis strains.

Bacillus anthracis is a Gram-positive endospore-forming bacterial species that causes anthrax in both humans and animals. In Zambia, anthrax cases are frequently reported in both livestock and wildlife, with occasional transmission to humans, causing serious public health problems in the country. To understand the genetic diversity of B. anthracis strains in Zambia, we sequenced and compared the genomic DNA of B. anthracis strains isolated across the country. Single nucleotide polymorphisms clustered these strains into three groups. Genome sequence comparisons revealed a large deletion in strains belonging to one of the groups, possibly due to unequal crossing over between a pair of rRNA operons. The deleted genomic region included genes conferring resistance to bacitracin, and the strains with the deletion were confirmed with loss of bacitracin resistance. Similar deletions between rRNA operons were also observed in a few B. anthracis strains phylogenetically distant from Zambian strains. The structure of bacitracin resistance genes flanked by rRNA operons was conserved only in members of the Bacillus cereus group. The diversity and genomic characteristics of B. anthracis strains determined in this study would help in the development of genetic markers and treatment of anthrax in Zambia. IMPORTANCE Anthrax is caused by Bacillus anthracis, an endospore-forming soil bacterium. The genetic diversity of B. anthracis is known to be low compared with that of Bacillus species. In this study, we performed whole-genome sequencing of Zambian isolates of B. anthracis to understand the genetic diversity between closely related strains. Comparison of genomic sequences revealed that closely related strains were separated into three groups based on single nucleotide polymorphisms distributed throughout the genome. A large genomic deletion was detected in the region containing a bacitracin resistance gene cluster flanked by rRNA operons, resulting in the loss of bacitracin resistance. The structure of the deleted region, which was also conserved among species of the Bacillus cereus group, has the potential for both deletion and amplification and thus might be enabling the species to flexibly control the level of bacitracin resistance for adaptive evolution.

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September 22, 2019

Genome sequence and metabolic analysis of a fluoranthene-degrading strain Pseudomonas aeruginosa DN1.

Pseudomonas aeruginosa DN1, isolated from petroleum-contaminated soil, showed excellent degradation ability toward diverse polycyclic aromatic hydrocarbons (PAHs). Many studies have been done to improve its degradation ability. However, the molecular mechanisms of PAHs degradation in DN1 strain are unclear. In this study, the whole genome of DN1 strain was sequenced and analyzed. Its genome contains 6,641,902 bp and encodes 6,684 putative open reading frames (ORFs), which has the largest genome in almost all the comparative Pseudomonas strains. Results of gene annotation showed that this strain harbored over 100 candidate genes involved in PAHs degradation, including those encoding 25 dioxygenases, four ring-hydroxylating dioxygenases, five ring-cleaving dioxygenases, and various catabolic enzymes, transcriptional regulators, and transporters in the degradation pathways. In addition, gene knockout experiments revealed that the disruption of some key PAHs degradation genes in DN1 strain, such as catA, pcaG, pcaH, and rhdA, did not completely inhibit fluoranthene degradation, even though their degradative rate reduced to some extent. Three intermediate metabolites, including 9-hydroxyfluorene, 1-acenaphthenone, and 1, 8-naphthalic anhydride, were identified as the dominating intermediates in presence of 50 µg/mL fluoranthene as the sole carbon source according to gas chromatography mass spectrometry analysis. Taken together, the genomic and metabolic analysis indicated that the fluoranthene degradation by DN1 strain was initiated by dioxygenation at the C-1, 2-, C-2, 3-, and C-7, 8- positions. These results provide new insights into the genomic plasticity and environmental adaptation of DN1 strain.

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