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September 22, 2019

Investigating the central metabolism of Clostridium thermosuccinogenes.

Clostridium thermosuccinogenes is a thermophilic anaerobic bacterium able to convert various carbohydrates to succinate and acetate as main fermentation products. Genomes of the four publicly available strains have been sequenced, and the genome of the type strain has been closed. The annotated genomes were used to reconstruct the central metabolism, and enzyme assays were used to validate annotations and to determine cofactor specificity. The genes were identified for the pathways to all fermentation products, as well as for the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway. Notably, a candidate transaldolase was lacking, and transcriptomics during growth on glucose versus that on xylose did not provide any leads to potential transaldolase genes or alternative pathways connecting the C5 with the C3/C6 metabolism. Enzyme assays showed xylulokinase to prefer GTP over ATP, which could be of importance for engineering xylose utilization in related thermophilic species of industrial relevance. Furthermore, the gene responsible for malate dehydrogenase was identified via heterologous expression in Escherichia coli and subsequent assays with the cell extract, which has proven to be a simple and powerful method for the basal characterization of thermophilic enzymes.IMPORTANCE Running industrial fermentation processes at elevated temperatures has several advantages, including reduced cooling requirements, increased reaction rates and solubilities, and a possibility to perform simultaneous saccharification and fermentation of a pretreated biomass. Most studies with thermophiles so far have focused on bioethanol production. Clostridium thermosuccinogenes seems an attractive production organism for organic acids, succinic acid in particular, from lignocellulosic biomass-derived sugars. This study provides valuable insights into its central metabolism and GTP and PPi cofactor utilization. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Complete genome sequencing of exopolysaccharide-producing Lactobacillus plantarum K25 provides genetic evidence for the probiotic functionality and cold endurance capacity of the strain.

Lactobacillus plantarum (L. plantarum) K25 is a probiotic strain isolated from Tibetan kefir. Previous studies showed that this exopolysaccharide (EPS)-producing strain was antimicrobial active and cold tolerant. These functional traits were evidenced by complete genome sequencing of strain K25 with a circular 3,175,846-bp chromosome and six circular plasmids, encoding 3365 CDSs, 16 rRNA genes and 70 tRNA genes. Genomic analysis of L. plantarum K25 illustrates that this strain contains the previous reported mechanisms of probiotic functionality and cold tolerance, involving plantaricins, lysozyme, bile salt hydrolase, chaperone proteins, osmoprotectant, oxidoreductase, EPSs and terpenes. Interestingly, strain K25 harbors more genes that function in defense mechanisms, and lipid transport and metabolism, in comparison with other L. plantarum strains reported. The present study demonstrates the comprehensive analysis of genes related to probiotic functionalities of an EPS-producing L. plantarum strain based on whole genome sequencing.

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September 22, 2019

Tumor-specific mitochondrial DNA variants are rarely detected in cell-free DNA.

The use of blood-circulating cell-free DNA (cfDNA) as a “liquid biopsy” in oncology is being explored for its potential as a cancer biomarker. Mitochondria contain their own circular genomic entity (mitochondrial DNA, mtDNA), up to even thousands of copies per cell. The mutation rate of mtDNA is several orders of magnitude higher than that of the nuclear DNA. Tumor-specific variants have been identified in tumors along the entire mtDNA, and their number varies among and within tumors. The high mtDNA copy number per cell and the high mtDNA mutation rate make it worthwhile to explore the potential of tumor-specific cf-mtDNA variants as cancer marker in the blood of cancer patients. We used single-molecule real-time (SMRT) sequencing to profile the entire mtDNA of 19 tissue specimens (primary tumor and/or metastatic sites, and tumor-adjacent normal tissue) and 9 cfDNA samples, originating from 8 cancer patients (5 breast, 3 colon). For each patient, tumor-specific mtDNA variants were detected and traced in cfDNA by SMRT sequencing and/or digital PCR to explore their feasibility as cancer biomarker. As a reference, we measured other blood-circulating biomarkers for these patients, including driver mutations in nuclear-encoded cfDNA and cancer-antigen levels or circulating tumor cells. Four of the 24 (17%) tumor-specific mtDNA variants were detected in cfDNA, however at much lower allele frequencies compared to mutations in nuclear-encoded driver genes in the same samples. Also, extensive heterogeneity was observed among the heteroplasmic mtDNA variants present in an individual. We conclude that there is limited value in tracing tumor-specific mtDNA variants in blood-circulating cfDNA with the current methods available. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Biosynthesis of abscisic acid in fungi: identification of a sesquiterpene cyclase as the key enzyme in Botrytis cinerea.

While abscisic acid (ABA) is known as a hormone produced by plants through the carotenoid pathway, a small number of phytopathogenic fungi are also able to produce this sesquiterpene but they use a distinct pathway that starts with the cyclization of farnesyl diphosphate (FPP) into 2Z,4E-a-ionylideneethane which is then subjected to several oxidation steps. To identify the sesquiterpene cyclase (STC) responsible for the biosynthesis of ABA in fungi, we conducted a genomic approach in Botrytis cinerea. The genome of the ABA-overproducing strain ATCC58025 was fully sequenced and five STC-coding genes were identified. Among them, Bcstc5 exhibits an expression profile concomitant with ABA production. Gene inactivation, complementation and chemical analysis demonstrated that BcStc5/BcAba5 is the key enzyme responsible for the key step of ABA biosynthesis in fungi. Unlike what is observed for most of the fungal secondary metabolism genes, the key enzyme-coding gene Bcstc5/Bcaba5 is not clustered with the other biosynthetic genes, i.e., Bcaba1 to Bcaba4 that are responsible for the oxidative transformation of 2Z,4E-a-ionylideneethane. Finally, our study revealed that the presence of the Bcaba genes among Botrytis species is rare and that the majority of them do not possess the ability to produce ABA.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

Genome biology of a novel lineage of planctomycetes widespread in anoxic aquatic environments.

Anaerobic strains affiliated with a novel order-level lineage of the Phycisphaerae class were retrieved from the suboxic zone of a hypersaline cyanobacterial mat and anoxic sediments of solar salterns. Genome sequences of five isolates were obtained and compared with metagenome-assembled genomes representing related uncultured bacteria from various anoxic aquatic environments. Gene content surveys suggest a strictly fermentative saccharolytic metabolism for members of this lineage, which could be confirmed by the phenotypic characterization of isolates. Genetic analyses indicate that the retrieved isolates do not have a canonical origin of DNA replication, but initiate chromosome replication at alternative sites possibly leading to an accelerated evolution. Further potential factors driving evolution and speciation within this clade include genome reduction by metabolic specialization and rearrangements of the genome by mobile genetic elements, which have a high prevalence in strains from hypersaline sediments and mats. Based on genetic and phenotypic data a distinct group of strictly anaerobic heterotrophic planctomycetes within the Phycisphaerae class could be assigned to a novel order that is represented by the proposed genus Sedimentisphaera gen. nov. comprising two novel species, S. salicampi gen. nov., sp. nov. and S. cyanobacteriorum gen. nov., sp. nov.© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

High-quality assembly of the reference genome for scarlet sage, Salvia splendens, an economically important ornamental plant.

Salvia splendens Ker-Gawler, scarlet or tropical sage, is a tender herbaceous perennial widely introduced and seen in public gardens all over the world. With few molecular resources, breeding is still restricted to traditional phenotypic selection, and the genetic mechanisms underlying phenotypic variation remain unknown. Hence, a high-quality reference genome will be very valuable for marker-assisted breeding, genome editing, and molecular genetics.We generated 66 Gb and 37 Gb of raw DNA sequences, respectively, from whole-genome sequencing of a largely homozygous scarlet sage inbred line using Pacific Biosciences (PacBio) single-molecule real-time and Illumina HiSeq sequencing platforms. The PacBio de novo assembly yielded a final genome with a scaffold N50 size of 3.12 Mb and a total length of 808 Mb. The repetitive sequences identified accounted for 57.52% of the genome sequence, and ?54,008 protein-coding genes were predicted collectively with ab initio and homology-based gene prediction from the masked genome. The divergence time between S. splendens and Salvia miltiorrhiza was estimated at 28.21 million years ago (Mya). Moreover, 3,797 species-specific genes and 1,187 expanded gene families were identified for the scarlet sage genome.We provide the first genome sequence and gene annotation for the scarlet sage. The availability of these resources will be of great importance for further breeding strategies, genome editing, and comparative genomics among related species.

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September 22, 2019

N6-methyladenine DNA modification in the human genome.

DNA N6-methyladenine (6mA) modification is the most prevalent DNA modification in prokaryotes, but whether it exists in human cells and whether it plays a role in human diseases remain enigmatic. Here, we showed that 6mA is extensively present in the human genome, and we cataloged 881,240 6mA sites accounting for ~0.051% of the total adenines. [G/C]AGG[C/T] was the most significantly associated motif with 6mA modification. 6mA sites were enriched in the coding regions and mark actively transcribed genes in human cells. DNA 6mA and N6-demethyladenine modification in the human genome were mediated by methyltransferase N6AMT1 and demethylase ALKBH1, respectively. The abundance of 6mA was significantly lower in cancers, accompanied by decreased N6AMT1 and increased ALKBH1 levels, and downregulation of 6mA modification levels promoted tumorigenesis. Collectively, our results demonstrate that DNA 6mA modification is extensively present in human cells and the decrease of genomic DNA 6mA promotes human tumorigenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

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September 22, 2019

MIRU-profiler: a rapid tool for determination of 24-loci MIRU-VNTR profiles from assembled genomes of Mycobacterium tuberculosis.

Tuberculosis (TB) resulted in an estimated 1.7 million deaths in the year 2016. The disease is caused by the members of Mycobacterium tuberculosis complex, which includes Mycobacterium tuberculosis, Mycobacterium bovis and other closely related TB causing organisms. In order to understand the epidemiological dynamics of TB, national TB control programs often conduct standardized genotyping at 24 Mycobacterial-Interspersed-Repetitive-Units (MIRU)-Variable-Number-of-Tandem-Repeats (VNTR) loci. With the advent of next generation sequencing technology, whole-genome sequencing (WGS) has been widely used for studying TB transmission. However, an open-source software that can connect WGS and MIRU-VNTR typing is currently unavailable, which hinders interlaboratory communication. In this manuscript, we introduce the MIRU-profiler program which could be used for prediction of MIRU-VNTR profile from WGS of M. tuberculosis.The MIRU-profiler is implemented in shell scripting language and depends on EMBOSS software. The in-silico workflow of MIRU-profiler is similar to those described in the laboratory manuals for genotyping M. tuberculosis. Given an input genome sequence, the MIRU-profiler computes alleles at the standard 24-loci based on in-silico PCR amplicon lengths. The final output is a tab-delimited text file detailing the 24-loci MIRU-VNTR pattern of the input sequence.The MIRU-profiler was validated on four datasets: complete genomes from NCBI-GenBank (n = 11), complete genomes for locally isolated strains sequenced using PacBio (n = 4), complete genomes for BCG vaccine strains (n = 2) and draft genomes based on 250 bp paired-end Illumina reads (n = 106).The digital MIRU-VNTR results were identical to the experimental genotyping results for complete genomes of locally isolated strains, BCG vaccine strains and five out of 11 genomes from the NCBI-GenBank. For draft genomes based on short Illumina reads, 21 out of 24 loci were inferred with a high accuracy, while a number of inaccuracies were recorded for three specific loci (ETRA, QUB11b and QUB26). One of the unique features of the MIRU-profiler was its ability to process multiple genomes in a batch. This feature was tested on all complete M. tuberculosis genome (n = 157), for which results were successfully obtained in approximately 14 min.The MIRU-profiler is a rapid tool for inference of digital MIRU-VNTR profile from the assembled genome sequences. The tool can accurately infer repeat numbers at the standard 24 or 21/24 MIRU-VNTR loci from the complete or draft genomes respectively. Thus, the tool is expected to bridge the communication gap between the laboratories using WGS and those using the conventional MIRU-VNTR typing.

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September 22, 2019

Comparative analysis reveals unexpected genome features of newly isolated Thraustochytrids strains: on ecological function and PUFAs biosynthesis.

Thraustochytrids are unicellular fungal-like marine protists with ubiquitous existence in marine environments. They are well-known for their ability to produce high-valued omega-3 polyunsaturated fatty acids (?-3-PUFAs) (e.g., docosahexaenoic acid (DHA)) and hydrolytic enzymes. Thraustochytrid biomass has been estimated to surpass that of bacterioplankton in both coastal and oceanic waters indicating they have an important role in microbial food-web. Nevertheless, the molecular pathway and regulatory network for PUFAs production and the molecular mechanisms underlying ecological functions of thraustochytrids remain largely unknown.The genomes of two thraustochytrids strains (Mn4 and SW8) with ability to produce DHA were sequenced and assembled with a hybrid sequencing approach utilizing Illumina short paired-end reads and Pacific Biosciences long reads to generate a highly accurate genome assembly. Phylogenomic and comparative genomic analyses found that DHA-producing thraustochytrid strains were highly similar and possessed similar gene content. Analysis of the conventional fatty acid synthesis (FAS) and the polyketide synthase (PKS) systems for PUFAs production only detected incomplete and fragmentary pathways in the genome of these two strains. Surprisingly, secreted carbohydrate active enzymes (CAZymes) were found to be significantly depleted in the genomes of these 2 strains as compared to other sequenced relatives. Furthermore, these two strains possess an expanded gene repertoire for signal transduction and self-propelled movement, which could be important for their adaptations to dynamic marine environments.Our results demonstrate the possibility of a third PUFAs synthesis pathway besides previously described FAS and PKS pathways encoded in the genome of these two thraustochytrid strains. Moreover, lack of a complete set of hydrolytic enzymatic machinery for degrading plant-derived organic materials suggests that these two DHA-producing strains play an important role as a nutritional source rather than a nutrient-producer in marine microbial-food web. Results of this study suggest the existence of two types of saprobic thraustochytrids in the world’s ocean. The first group, which does not produce cellulosic enzymes and live as ‘left-over’ scavenger of bacterioplankton, serves as a dietary source for the plankton of higher trophic levels and the other possesses capacity to live on detrital organic matters in the marine ecosystems.

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September 22, 2019

Large-scale gene losses underlie the genome evolution of parasitic plant Cuscuta australis.

Dodders (Cuscuta spp., Convolvulaceae) are root- and leafless parasitic plants. The physiology, ecology, and evolution of these obligate parasites are poorly understood. A high-quality reference genome of Cuscuta australis was assembled. Our analyses reveal that Cuscuta experienced accelerated molecular evolution, and Cuscuta and the convolvulaceous morning glory (Ipomoea) shared a common whole-genome triplication event before their divergence. C. australis genome harbors 19,671 protein-coding genes, and importantly, 11.7% of the conserved orthologs in autotrophic plants are lost in C. australis. Many of these gene loss events likely result from its parasitic lifestyle and the massive changes of its body plan. Moreover, comparison of the gene expression patterns in Cuscuta prehaustoria/haustoria and various tissues of closely related autotrophic plants suggests that Cuscuta haustorium formation requires mostly genes normally involved in root development. The C. australis genome provides important resources for studying the evolution of parasitism, regressive evolution, and evo-devo in plant parasites.

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September 22, 2019

Emergence of a novel mobile colistin resistance gene, mcr-8, in NDM-producing Klebsiella pneumoniae.

The rapid increase in carbapenem resistance among gram-negative bacteria has renewed focus on the importance of polymyxin antibiotics (colistin or polymyxin E). However, the recent emergence of plasmid-mediated colistin resistance determinants (mcr-1, -2, -3, -4, -5, -6, and -7), especially mcr-1, in carbapenem-resistant Enterobacteriaceae is a serious threat to global health. Here, we characterized a novel mobile colistin resistance gene, mcr-8, located on a transferrable 95,983-bp IncFII-type plasmid in Klebsiella pneumoniae. The deduced amino-acid sequence of MCR-8 showed 31.08%, 30.26%, 39.96%, 37.85%, 33.51%, 30.43%, and 37.46% identity to MCR-1, MCR-2, MCR-3, MCR-4, MCR-5, MCR-6, and MCR-7, respectively. Functional cloning indicated that the acquisition of the single mcr-8 gene significantly increased resistance to colistin in both Escherichia coli and K. pneumoniae. Notably, the coexistence of mcr-8 and the carbapenemase-encoding gene blaNDM was confirmed in K. pneumoniae isolates of livestock origin. Moreover, BLASTn analysis of mcr-8 revealed that this gene was present in a colistin- and carbapenem-resistant K. pneumoniae strain isolated from the sputum of a patient with pneumonia syndrome in the respiratory intensive care unit of a Chinese hospital in 2016. These findings indicated that mcr-8 has existed for some time and has disseminated among K. pneumoniae of both animal and human origin, further increasing the public health burden of antimicrobial resistance.

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September 22, 2019

Molecular characterization of invasive meningococcal isolates in Burkina Faso as the relative importance of serogroups X and W increases, 2008-2012.

Neisseria meningitidis serogroup A disease in Burkina Faso has greatly decreased following introduction of a meningococcal A conjugate vaccine in 2010, yet other serogroups continue to pose a risk of life-threatening disease. Capsule switching among epidemic-associated serogroup A N. meningitidis strains could allow these lineages to persist despite vaccination. The introduction of new strains at the national or sub-national levels could affect the epidemiology of disease.Isolates collected from invasive meningococcal disease in Burkina Faso between 2008 and 2012 were characterized by serogrouping and molecular typing. Genome sequences from a subset of isolates were used to infer phylogenetic relationships.The ST-5 clonal complex (CC5) was identified only among serogroup A isolates, which were rare after 2010. CC181 and CC11 were the most common clonal complexes after 2010, having serogroup X and W isolates, respectively. Whole-genome phylogenetic analysis showed that the CC181 isolates collected during and after the epidemic of 2010 formed a single clade that was closely related to isolates collected in Niger during 2005 and Burkina Faso during 2007. Geographic population structure was identified among the CC181 isolates, where pairs of isolates collected from the same region of Burkina Faso within a single year had less phylogenetic diversity than the CC181 isolate collection as a whole. However, the reduction of phylogenetic diversity within a region did not extend across multiple years. Instead, CC181 isolates collected during the same year had lower than average diversity, even when collected from different regions, indicating geographic mixing of strains across years. The CC11 isolates were primarily collected during the epidemic of 2012, with sparse sampling during 2011. These isolates belong to a clade that includes previously described isolates collected in Burkina Faso, Mali, and Niger from 2011 to 2015. Similar to CC181, reduced phylogenetic diversity was observed among CC11 isolate pairs collected from the same regions during a single year.The population of disease-associated N. meningitidis strains within Burkina Faso was highly dynamic between 2008 and 2012, reflecting both vaccine-imposed selection against serogroup A strains and potentially complex clonal waves of serogroup X and serogroup W strains.

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September 22, 2019

Genome analysis of the ancient tracheophyte Selaginella tamariscina reveals evolutionary features relevant to the acquisition of desiccation tolerance.

Resurrection plants, which are the “gifts” of natural evolution, are ideal models for studying the genetic basis of plant desiccation tolerance. Here, we report a high-quality genome assembly of 301 Mb for the diploid spike moss Selaginella tamariscina, a primitive vascular resurrection plant. We predicated 27 761 protein-coding genes from the assembled S. tamariscina genome, 11.38% (2363) of which showed significant expression changes in response to desiccation. Approximately 60.58% of the S. tamariscina genome was annotated as repetitive DNA, which is an almost 2-fold increase of that in the genome of desiccation-sensitive Selaginella moellendorffii. Genomic and transcriptomic analyses highlight the unique evolution and complex regulations of the desiccation response in S. tamariscina, including species-specific expansion of the oleosin and pentatricopeptide repeat gene families, unique genes and pathways for reactive oxygen species generation and scavenging, and enhanced abscisic acid (ABA) biosynthesis and potentially distinct regulation of ABA signaling and response. Comparative analysis of chloroplast genomes of several Selaginella species revealed a unique structural rearrangement and the complete loss of chloroplast NAD(P)H dehydrogenase (NDH) genes in S. tamariscina, suggesting a link between the absence of the NDH complex and desiccation tolerance. Taken together, our comparative genomic and transcriptomic analyses reveal common and species-specific desiccation tolerance strategies in S. tamariscina, providing significant insights into the desiccation tolerance mechanism and the evolution of resurrection plants. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Heterogeneous and flexible transmission of mcr-1 in hospital-associated Escherichia coli.

The recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route of mcr-1 among Enterobacteriaceae species in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis of Escherichia coli isolates collected in a hospital in Hangzhou, China. We found that mcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread of mcr-1 The mcr-1 gene was found on either chromosomes or plasmids, but in most of the E. coli isolates, mcr-1 was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission of mcr-1 and the coexistence of mcr-1 with other genes encoding ß-lactamases and fluoroquinolone resistance in the E. coli isolates. These findings indicate that mcr-1 is heterogeneously disseminated in both commensal and pathogenic strains of E. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology of mcr-1 among hospital-associated E. coli strains. IMPORTANCE Colistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergent mcr-1 colistin resistance gene threatens the clinical utility of colistin and has gained global attention. How mcr-1 spreads in hospital settings remains unknown and was investigated by whole-genome sequencing of mcr-1-carrying Escherichia coli in this study. The findings revealed extraordinary flexibility of mcr-1 in its spread among genetically diverse E. coli hosts and plasmids, nosocomial transmission of mcr-1-carrying E. coli, and the continuous emergence of novel Inc types of plasmids carrying mcr-1 and new mcr-1 variants. Additionally, mcr-1 was found to be frequently associated with other genes encoding ß-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology of mcr-1 and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat. Copyright © 2018 Shen et al.

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September 22, 2019

Genomic variation among and within six Juglans species.

Genomic analysis in Juglans (walnuts) is expected to transform the breeding and agricultural production of both nuts and lumber. To that end, we report here the determination of reference sequences for six additional relatives of Juglans regia: Juglans sigillata (also from section Dioscaryon), Juglans nigra, Juglans microcarpa, Juglans hindsii (from section Rhysocaryon), Juglans cathayensis (from section Cardiocaryon), and the closely related Pterocarya stenoptera While these are ‘draft’ genomes, ranging in size between 640Mbp and 990Mbp, their contiguities and accuracies can support powerful annotations of genomic variation that are often the foundation of new avenues of research and breeding. We annotated nucleotide divergence and synteny by creating complete pairwise alignments of each reference genome to the remaining six. In addition, we have re-sequenced a sample of accessions from four Juglans species (including regia). The variation discovered in these surveys comprises a critical resource for experimentation and breeding, as well as a solid complementary annotation. To demonstrate the potential of these resources the structural and sequence variation in and around the polyphenol oxidase loci, PPO1 and PPO2 were investigated. As reported for other seed crops variation in this gene is implicated in the domestication of walnuts. The apparently Juglandaceae specific PPO1 duplicate shows accelerated divergence and an excess of amino acid replacement on the lineage leading to accessions of the domesticated nut crop species, Juglans regia and sigillata. Copyright © 2018 Stevens et al.

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