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September 22, 2019

Xanthomonas citri jumbo phage XacN1 exhibits a wide host range and high complement of tRNA genes.

Xanthomonas virus (phage) XacN1 is a novel jumbo myovirus infecting Xanthomonas citri, the causative agent of Asian citrus canker. Its linear 384,670?bp double-stranded DNA genome encodes 592 proteins and presents the longest (66?kbp) direct terminal repeats (DTRs) among sequenced viral genomes. The DTRs harbor 56 tRNA genes, which correspond to all 20 amino acids and represent the largest number of tRNA genes reported in a viral genome. Codon usage analysis revealed a propensity for the phage encoded tRNAs to target codons that are highly used by the phage but less frequently by its host. The existence of these tRNA genes and seven additional translation-related genes as well as a chaperonin gene found in the XacN1 genome suggests a relative independence of phage replication on host molecular machinery, leading to a prediction of a wide host range for this jumbo phage. We confirmed the prediction by showing a wider host range of XacN1 than other X. citri phages in an infection test against a panel of host strains. Phylogenetic analyses revealed a clade of phages composed of XacN1 and ten other jumbo phages, indicating an evolutionary stable large genome size for this group of phages.

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September 22, 2019

Assimilation of cyanide and cyano-derivatives by Pseudomonas pseudoalcaligenes CECT5344: from omic approaches to biotechnological applications.

Mining, jewellery and metal-processing industries use cyanide for extracting gold and other valuable metals, generating large amounts of highly toxic wastewater. Biological treatments may be a clean alternative under the environmental point of view to the conventional physical or chemical processes used to remove cyanide and related compounds from these industrial effluents. Pseudomonas pseudoalcaligenes CECT5344 can grow under alkaline conditions using cyanide, cyanate or different nitriles as the sole nitrogen source, and is able to remove up to 12 mM total cyanide from a jewellery industry wastewater that contains cyanide free and complexed to metals. Complete genome sequencing of this bacterium has allowed the application of transcriptomic and proteomic techniques, providing a holistic view of the cyanide biodegradation process. The complex response to cyanide by the cyanotrophic bacterium P. pseudoalcaligenes CECT5344 and the potential biotechnological applications of this model organism in the bioremediation of cyanide-containing industrial residues are reviewed.

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September 22, 2019

The hardy rubber tree genome provides insights into the evolution of polyisoprene biosynthesis.

Eucommia ulmoides, also called hardy rubber tree, is an economically important tree; however, the lack of its genome sequence restricts the fundamental biological research and applied studies of this plant species. Here, we present a high-quality assembly of its ~1.2-Gb genome (scaffold N50 = 1.88 Mb) with at least 26 723 predicted genes for E. ulmoides, the first sequenced genome of the order Garryales, which was obtained using an integrated strategy combining Illumina sequencing, PacBio sequencing, and BioNano mapping. As a sister taxon to lamiids and campanulids, E. ulmoides underwent an ancient genome triplication shared by core eudicots but no further whole-genome duplication in the last ~125 million years. E. ulmoides exhibits high expression levels and/or gene number expansion for multiple genes involved in stress responses and the biosynthesis of secondary metabolites, which may account for its considerable environmental adaptability. In contrast to the rubber tree (Hevea brasiliensis), which produces cis-polyisoprene, E. ulmoides has evolved to synthesize long-chain trans-polyisoprene via farnesyl diphosphate synthases (FPSs). Moreover, FPS and rubber elongation factor/small rubber particle protein gene families were expanded independently from the H. brasiliensis lineage. These results provide new insights into the biology of E. ulmoides and the origin of polyisoprene biosynthesis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Genotype assembly, biological activity and adaptation of spatially separated isolates of Spodoptera litura nucleopolyhedrovirus.

The cotton leafworm Spodoptera litura is a polyphagous insect. It has recently made a comeback as a primary insect pest of cotton in Pakistan due to reductions in pesticide use on the advent of genetically modified cotton, resistant to Helicoverpa armigera. Spodoptera litura nucleopolyhedrovirus (SpltNPV) infects S. litura and is recognized as a potential candidate to control this insect. Twenty-two NPV isolates were collected from S. litura from different agro-ecological zones (with collection sites up to 600?km apart) and cropping systems in Pakistan to see whether there is spatial dispersal and adaptation of the virus and/or adaptation to crops. Therefore, the genetic make-up and biological activity of these isolates was measured. Among the SpltNPV isolates tested for speed of kill in 3rd instar larvae of S. litura, TAX1, SFD1, SFD2 and GRW1 were significantly faster killing isolates than other Pakistani isolates. Restriction fragment length analysis of the DNA showed that the Pakistan SpltNPV isolates are all variants of a single SpltNPV biotype. The isolates could be grouped into three genogroups (A-C). The speed of kill of genogroup A viruses was higher than in group C according to a Cox’ proportional hazards analysis. Sequence analysis showed that the Pakistan SpltNPV isolates are more closely related to each other than to the SpltNPV type species G2 (Pang et al., 2001). This suggests a single introduction of SpltNPV into Pakistan. The SpltNPV-PAK isolates are distinct from Spodoptera littoralis nucleopolyhedrovirus. There was a strong correlation between geographic spread and the genetic variation of SpltNPV, and a marginally significant correlation between the latter and the cropping system. The faster killing isolates may be good candidates for biological control of S. litura in Pakistan. Copyright © 2018 Elsevier Inc. All rights reserved.

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September 22, 2019

Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation.

The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome.The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads.The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease.

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September 22, 2019

Genomic insights into the Acidobacteria reveal strategies for their success in terrestrial environments.

Members of the phylum Acidobacteria are abundant and ubiquitous across soils. We performed a large-scale comparative genome analysis spanning subdivisions 1, 3, 4, 6, 8 and 23 (n?=?24) with the goal to identify features to help explain their prevalence in soils and understand their ecophysiology. Our analysis revealed that bacteriophage integration events along with transposable and mobile elements influenced the structure and plasticity of these genomes. Low- and high-affinity respiratory oxygen reductases were detected in multiple genomes, suggesting the capacity for growing across different oxygen gradients. Among many genomes, the capacity to use a diverse collection of carbohydrates, as well as inorganic and organic nitrogen sources (such as via extracellular peptidases), was detected – both advantageous traits in environments with fluctuating nutrient environments. We also identified multiple soil acidobacteria with the potential to scavenge atmospheric concentrations of H2 , now encompassing mesophilic soil strains within the subdivision 1 and 3, in addition to a previously identified thermophilic strain in subdivision 4. This large-scale acidobacteria genome analysis reveal traits that provide genomic, physiological and metabolic versatility, presumably allowing flexibility and versatility in the challenging and fluctuating soil environment.© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

Predominant gut Lactobacillus murinus strain mediates anti-inflammaging effects in calorie-restricted mice.

Calorie restriction (CR), which has a potent anti-inflammaging effect, has been demonstrated to induce dramatic changes in the gut microbiota. Whether the modulated gut microbiota contributes to the attenuation of inflammation during CR is unknown, as are the members of the microbial community that may be key mediators of this process.Here, we report that a unique Lactobacillus-predominated microbial community was rapidly attained in mice within 2 weeks of CR, which decreased the levels of circulating microbial antigens and systemic inflammatory markers such as tumour necrosis factor alpha (TNF-a). Lactobacillus murinus CR147, an isolate in the most abundant operational taxonomic unit (OTU) enriched by CR, downregulated interleukin-8 production in TNF-a-stimulated Caco-2 cells and significantly increased the lifespan and the brood size of the nematode Caenorhabditis elegans. In gnotobiotic mice colonized with the gut microbiota from old mice, this strain decreased their intestinal permeability and serum endotoxin load, consequently attenuating the inflammation induced by the old microbiota.Our study demonstrated that a strain of Lactobacillus murinus was promoted in CR mice and causatively contributed to the attenuation of ageing-associated inflammation.

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September 22, 2019

DNA strand-exchange patterns associated with double-strand break-induced and spontaneous mitotic crossovers in Saccharomyces cerevisiae.

Mitotic recombination can result in loss of heterozygosity and chromosomal rearrangements that shape genome structure and initiate human disease. Engineered double-strand breaks (DSBs) are a potent initiator of recombination, but whether spontaneous events initiate with the breakage of one or both DNA strands remains unclear. In the current study, a crossover (CO)-specific assay was used to compare heteroduplex DNA (hetDNA) profiles, which reflect strand exchange intermediates, associated with DSB-induced versus spontaneous events in yeast. Most DSB-induced CO products had the two-sided hetDNA predicted by the canonical DSB repair model, with a switch in hetDNA position from one product to the other at the position of the break. Approximately 40% of COs, however, had hetDNA on only one side of the initiating break. This anomaly can be explained by a modified model in which there is frequent processing of an early invasion (D-loop) intermediate prior to extension of the invading end. Finally, hetDNA tracts exhibited complexities consistent with frequent expansion of the DSB into a gap, migration of strand-exchange junctions, and template switching during gap-filling reactions. hetDNA patterns in spontaneous COs isolated in either a wild-type background or in a background with elevated levels of reactive oxygen species (tsa1? mutant) were similar to those associated with the DSB-induced events, suggesting that DSBs are the major instigator of spontaneous mitotic recombination in yeast.

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September 22, 2019

Insights into the evolution of host association through the isolation and characterization of a novel human periodontal pathobiont, Desulfobulbus oralis.

The human oral microbiota encompasses representatives of many bacterial lineages that have not yet been cultured. Here we describe the isolation and characterization of previously uncultured Desulfobulbus oralis, the first human-associated representative of its genus. As mammalian-associated microbes rarely have free-living close relatives, D. oralis provides opportunities to study how bacteria adapt and evolve within a host. This sulfate-reducing deltaproteobacterium has adapted to the human oral subgingival niche by curtailing its physiological repertoire, losing some biosynthetic abilities and metabolic independence, and by dramatically reducing environmental sensing and signaling capabilities. The genes that enable free-living Desulfobulbus to synthesize the potent neurotoxin methylmercury were also lost by D. oralis, a notably positive outcome of host association. However, horizontal gene acquisitions from other members of the microbiota provided novel mechanisms of interaction with the human host, including toxins like leukotoxin and hemolysins. Proteomic and transcriptomic analysis revealed that most of those factors are actively expressed, including in the subgingival environment, and some are secreted. Similar to other known oral pathobionts, D. oralis can trigger a proinflammatory response in oral epithelial cells, suggesting a direct role in the development of periodontal disease.IMPORTANCE Animal-associated microbiota likely assembled as a result of numerous independent colonization events by free-living microbes followed by coevolution with their host and other microbes. Through specific adaptation to various body sites and physiological niches, microbes have a wide range of contributions, from beneficial to disease causing. Desulfobulbus oralis provides insights into genomic and physiological transformations associated with transition from an open environment to a host-dependent lifestyle and the emergence of pathogenicity. Through a multifaceted mechanism triggering a proinflammatory response, D. oralis is a novel periodontal pathobiont. Even though culture-independent approaches can provide insights into the potential role of the human microbiome “dark matter,” cultivation and experimental characterization remain important to studying the roles of individual organisms in health and disease.

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September 22, 2019

Synchronous termination of replication of the two chromosomes is an evolutionary selected feature in Vibrionaceae.

Vibrio cholerae, the causative agent of the cholera disease, is commonly used as a model organism for the study of bacteria with multipartite genomes. Its two chromosomes of different sizes initiate their DNA replication at distinct time points in the cell cycle and terminate in synchrony. In this study, the time-delayed start of Chr2 was verified in a synchronized cell population. This replication pattern suggests two possible regulation mechanisms for other Vibrio species with different sized secondary chromosomes: Either all Chr2 start DNA replication with a fixed delay after Chr1 initiation, or the timepoint at which Chr2 initiates varies such that termination of chromosomal replication occurs in synchrony. We investigated these two models and revealed that the two chromosomes of various Vibrionaceae species terminate in synchrony while Chr2-initiation timing relative to Chr1 is variable. Moreover, the sequence and function of the Chr2-triggering crtS site recently discovered in V. cholerae were found to be conserved, explaining the observed timing mechanism. Our results suggest that it is beneficial for bacterial cells with multiple chromosomes to synchronize their replication termination, potentially to optimize chromosome related processes as dimer resolution or segregation.

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September 22, 2019

Unique genetic cassettes in a Thermoanaerobacterium contribute to simultaneous conversion of cellulose and monosugars into butanol.

The demand for cellulosic biofuels is on the rise because of the anticipation for sustainable energy and less greenhouse gas emissions in the future. However, production of cellulosic biofuels, especially cellulosic butanol, has been hampered by the lack of potent microbes that are capable of converting cellulosic biomass into biofuels. We report a wild-type Thermoanaerobacterium thermosaccharolyticum strain TG57, which is capable of using microcrystalline cellulose directly to produce butanol (1.93 g/liter) as the only final product (without any acetone or ethanol produced), comparable to that of engineered microbes thus far. Strain TG57 exhibits significant advances including unique genes responsible for a new butyrate synthesis pathway, no carbon catabolite repression, and the absence of genes responsible for acetone synthesis (which is observed as the main by-product in most Clostridium strains known today). Furthermore, the use of glucose analog 2-deoxyglucose posed a selection pressure to facilitate isolation of strain TG57 with deletion/silencing of carbon catabolite repressor genes-the ccr and xylR genes-and thus is able to simultaneously ferment glucose, xylose, and arabinose to produce butanol (7.33 g/liter) as the sole solvent. Combined analysis of genomic and transcriptomic data revealed unusual aspects of genome organization, numerous determinants for unique bioconversions, regulation of central metabolic pathways, and distinct transcriptomic profiles. This study provides a genome-level understanding of how cellulose is metabolized by T. thermosaccharolyticum and sheds light on the potential of competitive and sustainable biofuel production.

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September 22, 2019

Molecular characterization of IMP-1-producing Enterobacter cloacae complex isolates in Tokyo.

Although KPC enzymes are most common among carbapenemases produced by Enterobacter cloacae complex globally, the epidemiology varies from one country to another. While previous studies have suggested that IMP enzymes are most common in Japan, detailed analysis has been scarce thus far. Here, we carried out a molecular epidemiological study and plasmid analysis of IMP-1-producing E. cloacae complex isolates collected from three hospitals in central Tokyo using whole-genome sequencing. Seventy-one isolates were classified into several sequence types (STs), and 49 isolates were identified as Enterobacter hormaechei ST78. Isolates of ST78 were divided into three clades by core-genome single nucleotide polymorphism (SNP)-based phylogenetic analysis. Whereas isolates of clade 3 were isolated from only one hospital, isolates of clade 1 and 2 were identified from multiple hospitals. Ten of 12 clade 1 isolates and 1 of 4 clade 2 isolates carried blaIMP-1 on IncHI2 plasmids, with high similarity of genetic structures. In addition, these plasmids shared backbone structures with IncHI2 plasmids carrying blaIMP reported from other countries of the Asia-Pacific region. All isolates of clade 3 except one carried blaIMP-1 in In1426 on IncW plasmids. An isolate of clade 3, which lacked IncW plasmids, carried blaIMP-1 in In1426 on an IncFIB plasmid. These observations suggest that IMP-producing E. cloacae complex isolates with a diversity of host genomic backgrounds have spread in central Tokyo, and they indicate the possible contribution of IncHI2 plasmids toward this phenomenon. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Occurrence, evolution, and functions of DNA phosphorothioate epigenetics in bacteria.

The chemical diversity of physiological DNA modifications has expanded with the identification of phosphorothioate (PT) modification in which the nonbridging oxygen in the sugar-phosphate backbone of DNA is replaced by sulfur. Together with DndFGH as cognate restriction enzymes, DNA PT modification, which is catalyzed by the DndABCDE proteins, functions as a bacterial restriction-modification (R-M) system that protects cells against invading foreign DNA. However, the occurrence of dnd systems across a large number of bacterial genomes and their functions other than R-M are poorly understood. Here, a genomic survey revealed the prevalence of bacterial dnd systems: 1,349 bacterial dnd systems were observed to occur sporadically across diverse phylogenetic groups, and nearly half of these occur in the form of a solitary dndBCDE gene cluster that lacks the dndFGH restriction counterparts. A phylogenetic analysis of 734 complete PT R-M pairs revealed the coevolution of M and R components, despite the observation that several PT R-M pairs appeared to be assembled from M and R parts acquired from distantly related organisms. Concurrent epigenomic analysis, transcriptome analysis, and metabolome characterization showed that a solitary PT modification contributed to the overall cellular redox state, the loss of which perturbed the cellular redox balance and induced Pseudomonas fluorescens to reconfigure its metabolism to fend off oxidative stress. An in vitro transcriptional assay revealed altered transcriptional efficiency in the presence of PT DNA modification, implicating its function in epigenetic regulation. These data suggest the versatility of PT in addition to its involvement in R-M protection.

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September 22, 2019

Ploidy variation in Kluyveromyces marxianus separates dairy and non-dairy isolates.

Kluyveromyces marxianus is traditionally associated with fermented dairy products, but can also be isolated from diverse non-dairy environments. Because of thermotolerance, rapid growth and other traits, many different strains are being developed for food and industrial applications but there is, as yet, little understanding of the genetic diversity or population genetics of this species. K. marxianus shows a high level of phenotypic variation but the only phenotype that has been clearly linked to a genetic polymorphism is lactose utilisation, which is controlled by variation in the LAC12 gene. The genomes of several strains have been sequenced in recent years and, in this study, we sequenced a further nine strains from different origins. Analysis of the Single Nucleotide Polymorphisms (SNPs) in 14 strains was carried out to examine genome structure and genetic diversity. SNP diversity in K. marxianus is relatively high, with up to 3% DNA sequence divergence between alleles. It was found that the isolates include haploid, diploid, and triploid strains, as shown by both SNP analysis and flow cytometry. Diploids and triploids contain long genomic tracts showing loss of heterozygosity (LOH). All six isolates from dairy environments were diploid or triploid, whereas 6 out 7 isolates from non-dairy environment were haploid. This also correlated with the presence of functional LAC12 alleles only in dairy haplotypes. The diploids were hybrids between a non-dairy and a dairy haplotype, whereas triploids included three copies of a dairy haplotype.

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September 22, 2019

Cross-species comparison of the gut: Differential gene expression sheds light on biological differences in closely related tenebrionids.

The gut is one of the primary interfaces between an insect and its environment. Understanding gene expression profiles in the insect gut can provide insight into interactions with the environment as well as identify potential control methods for pests. We compared the expression profiles of transcripts from the gut of larval stages of two coleopteran insects, Tenebrio molitor and Tribolium castaneum. These tenebrionids have different life cycles, varying in the duration and number of larval instars. T. castaneum has a sequenced genome and has been a model for coleopterans, and we recently obtained a draft genome for T. molitor. We assembled gut transcriptome reads from each insect to their respective genomes and filtered mapped reads to RPKM>1, yielding 11,521 and 17,871 genes in the T. castaneum and T. molitor datasets, respectively. There were identical GO terms in each dataset, and enrichment analyses also identified shared GO terms. From these datasets, we compiled an ortholog list of 6907 genes; 45% of the total assembled reads from T. castaneum were found in the top 25 orthologs, but only 27% of assembled reads were found in the top 25 T. molitor orthologs. There were 2281 genes unique to T. castaneum, and 2088 predicted genes unique to T. molitor, although improvements to the T. molitor genome will likely reduce these numbers as more orthologs are identified. We highlight a few unique genes in T. castaneum or T. molitor that may relate to distinct biological functions. A large number of putative genes expressed in the larval gut with uncharacterized functions (36 and 68% from T. castaneum and T. molitor, respectively) support the need for further research. These data are the first step in building a comprehensive understanding of the physiology of the gut in tenebrionid insects, illustrating commonalities and differences that may be related to speciation and environmental adaptation. Published by Elsevier Ltd.

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