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April 21, 2020  |  

Complete Genome Sequence Analysis and Characterization of Selected Iron Regulation Genes of Pasteurella Multocida Serotype A Strain PMTB2.1.

Although more than 100 genome sequences of Pasteurella multocida are available, comprehensive and complete genome sequence analysis is limited. This study describes the analysis of complete genome sequence and pathogenomics of P. multocida strain PMTB2.1. The genome of PMTB2.1 has 2176 genes with more than 40 coding sequences associated with iron regulation and 140 virulence genes including the complete tad locus. The tad locus includes several previously uncharacterized genes such as flp2, rcpC and tadV genes. A transposable phage resembling to Mu phages was identified in P. multocida that has not been identified in any other serotype yet. The multi-locus sequence typing analysis assigned the PMTB2.1 genome sequence as type ST101, while the comparative genome analysis showed that PMTB2.1 is closely related to other P. multocida strains with the genomic distance of less than 0.13. The expression profiling of iron regulating-genes of PMTB2.1 was characterized under iron-limited environment. Results showed significant changes in the expression profiles of iron-regulating genes (p < 0.05) whereas the highest expression of fecE gene (281 fold) at 30 min suggests utilization of the outer-membrane proteins system in iron acquisition at an early stage of growth. This study showed the phylogenomic relatedness of P. multocida and improved annotation of important genes and functional characterization of iron-regulating genes of importance to the bacterial growth.


April 21, 2020  |  

Genome assembly and annotation of the Trichoplusia ni Tni-FNL insect cell line enabled by long-read technologies.

Trichoplusiani derived cell lines are commonly used to enable recombinant protein expression via baculovirus infection to generate materials approved for clinical use and in clinical trials. In order to develop systems biology and genome engineering tools to improve protein expression in this host, we performed de novo genome assembly of the Trichoplusiani-derived cell line Tni-FNL.By integration of PacBio single-molecule sequencing, Bionano optical mapping, and 10X Genomics linked-reads data, we have produced a draft genome assembly of Tni-FNL.Our assembly contains 280 scaffolds, with a N50 scaffold size of 2.3 Mb and a total length of 359 Mb. Annotation of the Tni-FNL genome resulted in 14,101 predicted genes and 93.2% of the predicted proteome contained recognizable protein domains. Ortholog searches within the superorder Holometabola provided further evidence of high accuracy and completeness of the Tni-FNL genome assembly.This first draft Tni-FNL genome assembly was enabled by complementary long-read technologies and represents a high-quality, well-annotated genome that provides novel insight into the complexity of this insect cell line and can serve as a reference for future large-scale genome engineering work in this and other similar recombinant protein production hosts.


April 21, 2020  |  

Diverse Vectors and Mechanisms Spread New Delhi Metallo-ß-Lactamases among Carbapenem-Resistant Enterobacteriaceae in the Greater Boston Area.

New Delhi metallo-beta-lactamases (NDMs) are an uncommon but emerging cause of carbapenem resistance in the United States. Genomic factors promoting their domestic spread remain poorly characterized. A prospective genomic surveillance program among Boston-area hospitals identified multiple new occurrences of NDM-carrying strains of Escherichia coli and Enterobacter cloacae complex in inpatient and outpatient settings, representing the first occurrences of NDM-mediated resistance since initiating genomic surveillance in 2011. Cases included domestic patients with no international exposures. PacBio sequencing of isolates identified strain characteristics, resistance genes, and the complement of mobile vectors mediating spread. Analyses revealed a common 3,114-bp region containing the blaNDM gene, with carriage of this conserved region among unique strains by diverse transposon and plasmid backbones. Functional studies revealed a broad capacity for blaNDM transmission by conjugation, transposition, and complex interplasmid recombination events. NDMs represent a rapidly spreading form of drug resistance that can occur in inpatient and outpatient settings and in patients without international exposures. In contrast to Tn4401-based spread of Klebsiella pneumoniae carbapenemases (KPCs), diverse transposable elements mobilize NDM enzymes, commonly with other resistance genes, enabling naive strains to acquire multi- and extensively drug-resistant profiles with single transposition or plasmid conjugation events. Genomic surveillance provides effective means to rapidly identify these gene-level drivers of resistance and mobilization in order to inform clinical decisions to prevent further spread.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Into the Thermus Mobilome: Presence, Diversity and Recent Activities of Insertion Sequences Across Thermus spp.

A high level of transposon-mediated genome rearrangement is a common trait among microorganisms isolated from thermal environments, probably contributing to the extraordinary genomic plasticity and horizontal gene transfer (HGT) observed in these habitats. In this work, active and inactive insertion sequences (ISs) spanning the sequenced members of the genus Thermus were characterized, with special emphasis on three T. thermophilus strains: HB27, HB8, and NAR1. A large number of full ISs and fragments derived from different IS families were found, concentrating within megaplasmids present in most isolates. Potentially active ISs were identified through analysis of transposase integrity, and domestication-related transposition events of ISTth7 were identified in laboratory-adapted HB27 derivatives. Many partial copies of ISs appeared throughout the genome, which may serve as specific targets for homologous recombination contributing to genome rearrangement. Moreover, recruitment of IS1000 32 bp segments as spacers for CRISPR sequence was identified, pointing to the adaptability of these elements in the biology of these thermophiles. Further knowledge about the activity and functional diversity of ISs in this genus may contribute to the generation of engineered transposons as new genetic tools, and enrich our understanding of the outstanding plasticity shown by these thermophiles.


April 21, 2020  |  

Complete genome sequence and comparative analysis of Synechococcus sp. CS-601 (SynAce01), a cold-adapted cyanobacterium from an olligotrophic Antarctic habitat.

Marine picocyanobacteria belonging to Synechococcus are major contributors to the global carbon cycle, however the genomic information of its cold-adapted members has been lacking to date. To fill this void the genome of a cold-adapted planktonic cyanobacterium Synechococcus sp. CS-601 (SynAce01) has been sequenced. The genome of the strain contains a single chromosome of approximately 2.75 MBp and GC content of 63.92%. Gene prediction yielded 2984 protein coding sequences and 44 tRNA genes. The genome contained evidence of horizontal gene transfer events during its evolution. CS-601 appears as a transport generalist with some specific adaptation to an oligotrophic marine environment. It has a broad repertoire of transporters of both inorganic and organic nutrients to survive in inhospitable environments. The cold adaptation of the strain exhibited characteristics of a psychrotroph rather than psychrophile. Its salt adaptation strategy is likely to rely on the uptake and synthesis of osmolytes, like glycerol or glycine betaine. Overall, the genome reveals two distinct patterns of adaptation to the inhospitable environment of Antarctica. Adaptation to an oligotrophic marine environment is likely due to an abundance of genes, probably acquired horizontally, that are associated with increased transport of nutrients, osmolytes, and light harvesting. On the other hand, adaptations to low temperatures are likely due to prolonged evolutionary changes.


April 21, 2020  |  

LR_Gapcloser: a tiling path-based gap closer that uses long reads to complete genome assembly.

Completing a genome is an important goal of genome assembly. However, many assemblies, including reference assemblies, are unfinished and have a number of gaps. Long reads obtained from third-generation sequencing (TGS) platforms can help close these gaps and improve assembly contiguity. However, current gap-closure approaches using long reads require extensive runtime and high memory usage. Thus, a fast and memory-efficient approach using long reads is needed to obtain complete genomes.We developed LR_Gapcloser to rapidly and efficiently close the gaps in genome assembly. This tool utilizes long reads generated from TGS sequencing platforms. Tested on de novo assembled gaps, repeat-derived gaps, and real gaps, LR_Gapcloser closed a higher number of gaps faster and with a lower error rate and a much lower memory usage than two existing, state-of-the art tools. This tool utilized raw reads to fill more gaps than when using error-corrected reads. It is applicable to gaps in the assemblies by different approaches and from large and complex genomes. After performing gap-closure using this tool, the contig N50 size of the human CHM1 genome was improved from 143 kb to 19 Mb, a 132-fold increase. We also closed the gaps in the Triticum urartu genome, a large genome rich in repeats; the contig N50 size was increased by 40%. Further, we evaluated the contiguity and correctness of six hybrid assembly strategies by combining the optimal TGS-based and next-generation sequencing-based assemblers with LR_Gapcloser. A proposed and optimal hybrid strategy generated a new human CHM1 genome assembly with marked contiguity. The contig N50 value was greater than 28 Mb, which is larger than previous non-reference assemblies of the diploid human genome.LR_Gapcloser is a fast and efficient tool that can be used to close gaps and improve the contiguity of genome assemblies. A proposed hybrid assembly including this tool promises reference-grade assemblies. The software is available at http://www.fishbrowser.org/software/LR_Gapcloser/.


April 21, 2020  |  

Complete Sequence of a Novel Multidrug-Resistant Pseudomonas putida Strain Carrying Two Copies of qnrVC6.

This study aimed at identification and characterization of a novel multidrug-resistant Pseudomonas putida strain Guangzhou-Ppu420 carrying two copies of qnrVC6 isolated from a hospital in Guangzhou, China, in 2012. Antimicrobial susceptibility was tested by Vitek2™ Automated Susceptibility System and Etest™ strips, and whole-genome sequencing facilitated analysis of its multidrug resistance. The genome has a length of 6,031,212?bp and an average G?+?C content of 62.01%. A total of 5,421 open reading frames were identified, including eight 5S rRNA, seven 16S rRNA, and seven 23S rRNA, and 76 tRNA genes. Importantly, two copies of qnrVC6 gene with three ISCR1 around, a blaVIM-2 carrying integron In528, a novel gcu173 carrying integron In1348, and six antibiotic resistance genes were identified. This is the first identification of two copies of the qnrVC6 gene in a single P. putida isolate and a class 1 integron In1348.


April 21, 2020  |  

Adaptive archaic introgression of copy number variants and the discovery of previously unknown human genes

As they migrated out of Africa and into Europe and Asia, anatomically modern humans interbred with archaic hominins, such as Neanderthals and Denisovans. The result of this genetic introgression on the recipient populations has been of considerable interest, especially in cases of selection for specific archaic genetic variants. Hsieh et al. characterized adaptive structural variants and copy number variants that are likely targets of positive selection in Melanesians. Focusing on population-specific regions of the genome that carry duplicated genes and show an excess of amino acid replacements provides evidence for one of the mechanisms by which genetic novelty can arise and result in differentiation between human genomes.Science, this issue p. eaax2083INTRODUCTIONCharacterizing genetic variants underlying local adaptations in human populations is one of the central goals of evolutionary research. Most studies have focused on adaptive single-nucleotide variants that either arose as new beneficial mutations or were introduced after interbreeding with our now-extinct relatives, including Neanderthals and Denisovans. The adaptive role of copy number variants (CNVs), another well-known form of genomic variation generated through deletions or duplications that affect more base pairs in the genome, is less well understood, despite evidence that such mutations are subject to stronger selective pressures.RATIONALEThis study focuses on the discovery of introgressed and adaptive CNVs that have become enriched in specific human populations. We combine whole-genome CNV calling and population genetic inference methods to discover CNVs and then assess signals of selection after controlling for demographic history. We examine 266 publicly available modern human genomes from the Simons Genome Diversity Project and genomes of three ancient homininstextemdasha Denisovan, a Neanderthal from the Altai Mountains in Siberia, and a Neanderthal from Croatia. We apply long-read sequencing methods to sequence-resolve complex CNVs of interest specifically in the Melanesianstextemdashan Oceanian population distributed from Papua New Guinea to as far east as the islands of Fiji and known to harbor some of the greatest amounts of Neanderthal and Denisovan ancestry.RESULTSConsistent with the hypothesis of archaic introgression outside Africa, we find a significant excess of CNV sharing between modern non-African populations and archaic hominins (P = 0.039). Among Melanesians, we observe an enrichment of CNVs with potential signals of positive selection (n = 37 CNVs), of which 19 CNVs likely introgressed from archaic hominins. We show that Melanesian-stratified CNVs are significantly associated with signals of positive selection (P = 0.0323). Many map near or within genes associated with metabolism (e.g., ACOT1 and ACOT2), development and cell cycle or signaling (e.g., TNFRSF10D and CDK11A and CDK11B), or immune response (e.g., IFNLR1). We characterize two of the largest and most complex CNVs on chromosomes 16p11.2 and 8p21.3 that introgressed from Denisovans and Neanderthals, respectively, and are absent from most other human populations. At chromosome 16p11.2, we sequence-resolve a large duplication of >383 thousand base pairs (kbp) that originated from Denisovans and introgressed into the ancestral Melanesian population 60,000 to 170,000 years ago. This large duplication occurs at high frequency (>79%) in diverse Melanesian groups, shows signatures of positive selection, and maps adjacent to Homo sapienstextendashspecific duplications that predispose to rearrangements associated with autism. On chromosome 8p21.3, we identify a Melanesian haplotype that carries two CNVs, a ~6-kbp deletion, and a ~38-kbp duplication, with a Neanderthal origin and that introgressed into non-Africans 40,000 to 120,000 years ago. This CNV haplotype occurs at high frequency (44%) and shows signals consistent with a partial selective sweep in Melanesians. Using long-read sequencing genomic and transcriptomic data, we reconstruct the structure and complex evolutionary history for these two CNVs and discover previously undescribed duplicated genes (TNFRSF10D1, TNFRSF10D2, and NPIPB16) that show an excess of amino acid replacements consistent with the action of positive selection.CONCLUSIONOur results suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation that is absent from current reference genomes.Large adaptive-introgressed CNVs at chromosomes 8p21.3 and 16p11.2 in Melanesians.The magnifying glasses highlight structural differences between the archaic (top) and reference (bottom) genomes. Neanderthal (red) and Denisovan (blue) haplotypes encompassing large CNVs occur at high frequencies in Melanesians (44 and 79%, respectively) but are absent (black) in all non-Melanesians. These CNVs create positively selected genes (TNFRSF10D1, TNFRSF10D2, and NPIPB16) that are absent from the reference genome.Copy number variants (CNVs) are subject to stronger selective pressure than single-nucleotide variants, but their roles in archaic introgression and adaptation have not been systematically investigated. We show that stratified CNVs are significantly associated with signatures of positive selection in Melanesians and provide evidence for adaptive introgression of large CNVs at chromosomes 16p11.2 and 8p21.3 from Denisovans and Neanderthals, respectively. Using long-read sequence data, we reconstruct the structure and complex evolutionary history of these polymorphisms and show that both encode positively selected genes absent from most human populations. Our results collectively suggest that large CNVs originating in archaic hominins and introgressed into modern humans have played an important role in local population adaptation and represent an insufficiently studied source of large-scale genetic variation.


April 21, 2020  |  

Sensitivity to the two peptide bacteriocin plantaricin EF is dependent on CorC, a membrane-bound, magnesium/cobalt efflux protein.

Lactic acid bacteria produce a variety of antimicrobial peptides known as bacteriocins. Most bacteriocins are understood to kill sensitive bacteria through receptor-mediated disruptions. Here, we report on the identification of the Lactobacillus plantarum plantaricin EF (PlnEF) receptor. Spontaneous PlnEF-resistant mutants of the PlnEF-indicator strain L. plantarum NCIMB 700965 (LP965) were isolated and confirmed to maintain cellular ATP levels in the presence of PlnEF. Genome comparisons resulted in the identification of a single mutated gene annotated as the membrane-bound, magnesium/cobalt efflux protein CorC. All isolates contained a valine (V) at position 334 instead of a glycine (G) in a cysteine-ß-synthase domain at the C-terminal region of CorC. In silico template-based modeling of this domain indicated that the mutation resides in a loop between two ß-strands. The relationship between PlnEF, CorC, and metal homeostasis was supported by the finding that PlnEF-resistance was lost when PlnEF was applied together with high concentrations of Mg2+ , Co2+ , Zn2+ , or Cu2+ . Lastly, PlnEF sensitivity was increased upon heterologous expression of LP965 corC but not the G334V CorC mutant in the PlnEF-resistant strain Lactobacillus casei BL23. These results show that PlnEF kills sensitive bacteria by targeting CorC. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


April 21, 2020  |  

Aquella oligotrophica gen. nov. sp. nov.: A new member of the family Neisseriaceae isolated from laboratory tap water.

A bacterial strain designated as P08T was isolated from laboratory tap water during a water quality assessment in University of Malaya, Malaysia. The strain was a Gram-negative, rod-shaped, nonmotile, and aerobic bacterium. Complete genome of P08T comprised of a 2,820,660 bp chromosome with a G + C content of 36.43%. Both 16S rRNA phylogeny and phylogenetic tree inferred from the core gene matrix demonstrated that P08T formed a hitherto unknown subline within the family Neisseriaceae. Ortho average nucleotide identity (OrthoANI) values and the percentage of conserved proteins (POCP) calculated from complete genome sequence indicated low relatedness between P08T and its phylogenetic neighbors. Respiratory quinone analysis revealed Q-8 as the only detectable quinone. The predominant cellular fatty acids were identified as C14:0 , iso-C15:0 , and summed feature 3 (C16:1 ?7c/C16:1 ?6c). The polar lipids consisted of uncharacterized aminolipid, phosphatidylglycerol, and phosphatidylethanolamine. All aspects of phenotypic and phylogenetic data suggested that strain P08T represents a novel genus within family Neisseriaceae, for which the name Aquella gen. nov. is proposed. The type species of the genus is Aquella oligotrophica sp. nov., and the type strain is P08T (=LMG 29629T =DSM 100970T ). © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


April 21, 2020  |  

Rapid antigen diversification through mitotic recombination in the human malaria parasite Plasmodium falciparum.

Malaria parasites possess the remarkable ability to maintain chronic infections that fail to elicit a protective immune response, characteristics that have stymied vaccine development and cause people living in endemic regions to remain at risk of malaria despite previous exposure to the disease. These traits stem from the tremendous antigenic diversity displayed by parasites circulating in the field. For Plasmodium falciparum, the most virulent of the human malaria parasites, this diversity is exemplified by the variant gene family called var, which encodes the major surface antigen displayed on infected red blood cells (RBCs). This gene family exhibits virtually limitless diversity when var gene repertoires from different parasite isolates are compared. Previous studies indicated that this remarkable genome plasticity results from extensive ectopic recombination between var genes during mitotic replication; however, the molecular mechanisms that direct this process to antigen-encoding loci while the rest of the genome remains relatively stable were not determined. Using targeted DNA double-strand breaks (DSBs) and long-read whole-genome sequencing, we show that a single break within an antigen-encoding region of the genome can result in a cascade of recombination events leading to the generation of multiple chimeric var genes, a process that can greatly accelerate the generation of diversity within this family. We also found that recombinations did not occur randomly, but rather high-probability, specific recombination products were observed repeatedly. These results provide a molecular basis for previously described structured rearrangements that drive diversification of this highly polymorphic gene family.


April 21, 2020  |  

Complete genome sequence of Helicobacter pylori B128 7.13 and a single-step method for the generation of unmarked mutations.

Helicobacter pylori represents an interesting model of bacterial pathogenesis given that most infections are asymptomatic, while a minority of infections cause severe gastric disease. H pylori strain B128 7.13 is used extensively to understand H pylori pathophysiology. Due to extensive restriction-modification systems, the fact that only some H pylori strains are naturally transformable, the inability of common plasmid and transposon vectors to replicate in this bacterium, as well as the limited number of antibiotic cassettes that are functional in H pylori, there are relatively few genetic tools for the mutagenesis of this bacterium.Here, we use PacBio and Illumina sequencing to reveal the complete genome sequence of H pylori B128 7.13. Furthermore, we describe a system to generate markerless and scarless mutations on the H pylori chromosome using the counter-selection marker, galactokinase from Escherichia coli.We show that this mutagenesis strategy can be used to generate in-frame insertions, gene deletions, and multiple independent mutations in B128 7.13. Using the closed genome as a reference, we also report the absence of second site chromosomal mutations and/or rearrangements in our mutagenized strains. We compare the genome sequence of H pylori B128 7.13 with a closely related strain, H pylori B8, and reveal one notable region of difference, which is a 1430 bp insertion encoding a H pylori-specific DUF874 family protein of unknown function.This article reports the closed genome of the important H pylori B128 7.13 strain and a mutagenesis method that can be adopted by researchers as an alternative strategy to generate isogenic mutants of H pylori in order to further our understanding of this bacterium. © 2019. The Authors. Helicobacter Published by John Wiley & Sons Ltd.


April 21, 2020  |  

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.


April 21, 2020  |  

Effector gene reshuffling involves dispensable mini-chromosomes in the wheat blast fungus.

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome.


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