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September 22, 2019

Characterization of the SN35N strain-specific exopolysaccharide encoded in the whole circular genome of a plant-derived Lactobacillus plantarum.

Lactobacillus plantarum SN35N, which has been previously isolated from pear, secretes exopolysaccharide (EPS). The aim of the present study is to characterize the EPS chemically and to find the EPS-biosynthesizing gene cluster. The present study demonstrates that the strain produces an acidic EPS carrying phosphate residue, which is composed of glucose, galactose, and mannose at a molecular ratio of 15.0?:?5.7?:?1.0. We also show that acidic EPS strongly inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35), promoting an inflammatory reaction. In the present study, we also determined the complete genome sequence of the SN35N strain, demonstrating that the genome is a circular DNA with 3267626?bp, and the number of predicted coding genes is 3146, with a GC content of 44.51%. In addition, the strain harbors four plasmids, designated pSN35N-1, -2, -3, and -4. Although four EPS-biosynthesizing genes, designated lpe1, lpe2, lpe3, and lpe4, are present in the SN35N chromosomal DNA, another EPS gene cluster, lpe5, is located in the pSN35N-3 plasmid, composed of 35425?bp. EPS low-producing mutants, which were obtained by treating SN35N cells with novobiocin, lost the lpe5 gene cluster in the plasmid-curing experiment, suggesting that the gene cluster for the biosynthesis of acidic EPS is present in the plasmid. The present study shows the chemical characterization of the acidic EPS and its inhibitory effect to the hyaluronidase.

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September 22, 2019

Translating genomics into practice for real-time surveillance and response to carbapenemase-producing Enterobacteriaceae: evidence from a complex multi-institutional KPC outbreak.

Until recently, Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks.All KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each isolate. This was complemented with PacBio long-read sequencing on selected isolates to establish high-quality reference sequences and interrogate characteristics of KPC-encoding plasmids.Initial investigations indicated that the outbreak was widespread, with 86 KPC-producing Enterobacteriaceae isolates (K. pneumoniae 92%) identified from 35 different locations across metropolitan and rural Victoria between 2012 and 2015. Initial combined analyses of the epidemiological and genomic data resolved the outbreak into distinct nosocomial transmission networks, and identified healthcare facilities at the epicentre of KPC transmission. New cases were assigned to transmission networks in real-time, allowing focussed infection control efforts. PacBio sequencing confirmed a secondary transmission network arising from inter-species plasmid transmission. Insights from Bayesian transmission inference and analyses of within-host diversity informed the development of state-wide public health and infection control guidelines, including interventions such as an intensive approach to screening contacts following new case detection to minimise unrecognised colonisation.A real-time combined epidemiological and genomic investigation proved critical to identifying and defining multiple transmission networks of KPC Enterobacteriaceae, while data from either investigation alone were inconclusive. The investigation was fundamental to informing infection control measures in real-time and the development of state-wide public health guidelines on carbapenemase-producing Enterobacteriaceae surveillance and management.

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September 22, 2019

Complete genome sequence of Lactobacillus pentosus SLC13, isolated from mustard pickles, a potential probiotic strain with antimicrobial activity against foodborne pathogenic microorganisms.

Lactobacillus pentosus SLC13 is a high exopolysaccharide (EPS)-producing strain with broad-spectrum antimicrobial activity and the ability to grow in simulated gastrointestinal conditions. SLC13 was isolated from mustard pickles in Taiwan for potential probiotic applications. To better understand the molecular base for its antimicrobial activity and high EPS production, entire genome of SLC13 was determined by PacBio SMRT sequencing.L. pentosus SLC13 contains a genome with a 3,520,510-bp chromosome and a 62,498-bp plasmid. GC content of the complete genome was 46.5% and that of plasmid pSLC13 was 41.3%. Sequences were annotated at the RAST prokaryotic genome annotation server, and the results showed that the genome contained 3172 coding sequences and 82 RNA genes. Seventy-six protein-coding sequences were identified on the plasmid pSLC13. A plantaricin gene cluster, which is responsible for bacteriosins biosynthesis and could be associated with its broad-spectrum antimicrobial activity, was identified based on comparative genomic analysis. Two gene clusters involved in EPS production were also identified.This genomic sequence might contribute to a future application of this strain as probiotic in productive livestock potentially inhibiting competing and pathogenic organisms.

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September 22, 2019

The genomes of Crithidia bombi and C. expoeki, common parasites of bumblebees.

Trypanosomatids (Trypanosomatidae, Kinetoplastida) are flagellated protozoa containing many parasites of medical or agricultural importance. Among those, Crithidia bombi and C. expoeki, are common parasites in bumble bees around the world, and phylogenetically close to Leishmania and Leptomonas. They have a simple and direct life cycle with one host, and partially castrate the founding queens greatly reducing their fitness. Here, we report the nuclear genome sequences of one clone of each species, extracted from a field-collected infection. Using a combination of Roche 454 FLX Titanium, Pacific Biosciences PacBio RS, and Illumina GA2 instruments for C. bombi, and PacBio for C. expoeki, we could produce high-quality and well resolved sequences. We find that these genomes are around 32 and 34 MB, with 7,808 and 7,851 annotated genes for C. bombi and C. expoeki, respectively-which is somewhat less than reported from other trypanosomatids, with few introns, and organized in polycistronic units. A large fraction of genes received plausible functional support in comparison primarily with Leishmania and Trypanosoma. Comparing the annotated genes of the two species with those of six other trypanosomatids (C. fasciculata, L. pyrrhocoris, L. seymouri, B. ayalai, L. major, and T. brucei) shows similar gene repertoires and many orthologs. Similar to other trypanosomatids, we also find signs of concerted evolution in genes putatively involved in the interaction with the host, a high degree of synteny between C. bombi and C. expoeki, and considerable overlap with several other species in the set. A total of 86 orthologous gene groups show signatures of positive selection in the branch leading to the two Crithidia under study, mostly of unknown function. As an example, we examined the initiating glycosylation pathway of surface components in C. bombi, finding it deviates from most other eukaryotes and also from other kinetoplastids, which may indicate rapid evolution in the extracellular matrix that is involved in interactions with the host. Bumble bees are important pollinators and Crithidia-infections are suspected to cause substantial selection pressure on their host populations. These newly sequenced genomes provide tools that should help better understand host-parasite interactions in these pollinator pathogens.

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September 22, 2019

An ancient integration in a plant NLR is maintained as a trans-species polymorphism

Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens. Nucleotide-binding leucine-rich repeat (NLR) proteins are one class of cytoplasmic immune receptors whose genes commonly show signatures of adaptive evolution. While it is known that balancing selection contributes to maintaining high intraspecific allelic diversity, the evolutionary mechanism that influences the transmission of alleles during speciation remains unclear. The barley Mla locus has over 30 described alleles conferring isolate-specific resistance to barley powdery mildew and contains three NLR families (RGH1, RGH2, and RGH3). We discovered (using sequence capture and RNAseq) the presence of a novel integrated Exo70 domain in RGH2 in the Mla3 haplotype. Allelic variation across barley accessions includes presence/absence of the integrated domain in RGH2. Expanding our search to several Poaceae species, we found shared interspecific conservation in the RGH2-Exo70 integration. We hypothesise that balancing selection has maintained allelic variation at Mla as a trans-species polymorphism over 24 My, thus contributing to and preserving interspecific allelic diversity during speciation.

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September 22, 2019

Pangenome analyses of the wheat pathogen Zymoseptoria tritici reveal the structural basis of a highly plastic eukaryotic genome.

Structural variation contributes substantially to polymorphism within species. Chromosomal rearrangements that impact genes can lead to functional variation among individuals and influence the expression of phenotypic traits. Genomes of fungal pathogens show substantial chromosomal polymorphism that can drive virulence evolution on host plants. Assessing the adaptive significance of structural variation is challenging, because most studies rely on inferences based on a single reference genome sequence.We constructed and analyzed the pangenome of Zymoseptoria tritici, a major pathogen of wheat that evolved host specialization by chromosomal rearrangements and gene deletions. We used single-molecule real-time sequencing and high-density genetic maps to assemble multiple genomes. We annotated the gene space based on transcriptomics data that covered the infection life cycle of each strain. Based on a total of five telomere-to-telomere genomes, we constructed a pangenome for the species and identified a core set of 9149 genes. However, an additional 6600 genes were exclusive to a subset of the isolates. The substantial accessory genome encoded on average fewer expressed genes but a larger fraction of the candidate effector genes that may interact with the host during infection. We expanded our analyses of the pangenome to a worldwide collection of 123 isolates of the same species. We confirmed that accessory genes were indeed more likely to show deletion polymorphisms and loss-of-function mutations compared to core genes.The pangenome construction of a highly polymorphic eukaryotic pathogen showed that a single reference genome significantly underestimates the gene space of a species. The substantial accessory genome provides a cradle for adaptive evolution.

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September 22, 2019

Nuclear and mitochondrial genomes of the hybrid fungal plant pathogen Verticillium longisporum display a mosaic structure

Allopolyploidization, genome duplication through interspecific hybridization, is an important evolutionary mechanism that can enable organisms to adapt to environmental changes or stresses. This increased adaptive potential of allopolyploids can be particularly relevant for plant pathogens in their quest for host immune response evasion. Allodiploidization likely caused the shift in host range of the fungal pathogen plant Verticillium longisporum, as V. longisporum mainly infects Brassicaceae plants in contrast to haploid Verticillium spp. In this study, we investigated the allodiploid genome structure of V. longisporum and its evolution in the hybridization aftermath. The nuclear genome of V. longisporum displays a mosaic structure, as numerous contigs consists of sections of both parental origins. V. longisporum encountered extensive genome rearrangements, whereas the contribution of gene conversion is negligible. Thus, the mosaic genome structure mainly resulted from genomic rearrangements between parental chromosome sets. Furthermore, a mosaic structure was also found in the mitochondrial genome, demonstrating its bi-parental inheritance. In conclusion, the nuclear and mitochondrial genomes of V. longisporum parents interacted dynamically in the hybridization aftermath. Conceivably, novel combinations of DNA sequence of different parental origin facilitated genome stability after hybridization and consecutive niche adaptation of V. longisporum.

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September 22, 2019

The novel phages phiCD5763 and phiCD2955 represent two groups of big plasmidial Siphoviridae phages of Clostridium difficile.

Until recently, Clostridium difficile phages were limited to Myoviruses and Siphoviruses of medium genome length (32–57 kb). Here we report the finding of phiCD5763, a Siphovirus with a large extrachromosomal circular genome (132.5 kb, 172 ORFs) and a large capsid (205.6 ± 25.6 nm in diameter) infecting MLST Clade 1 strains of C. difficile. Two subgroups of big phage genomes similar to phiCD5763 were identified in 32 NAPCR1/RT012/ST-54 C. difficile isolates from Costa Rica and in whole genome sequences (WGS) of 41 C. difficile isolates of Clades 1, 2, 3, and 4 from Canada, USA, UK, Belgium, Iraq, and China. Through comparative genomics we discovered another putative big phage genome in a non-NAPCR1 isolate from Costa Rica, phiCD2955, which represents other big phage genomes found in 130 WGS of MLST Clade 1 and 2 isolates from Canada, USA, Hungary, France, Austria, and UK. phiCD2955 (131.6 kb, 172 ORFs) is related to a previously reported C. difficile phage genome, phiCD211/phiCDIF1296T. Detailed genome analyses of phiCD5763, phiCD2955, phiCD211/phiCDIF1296T, and seven other putative C. difficile big phage genome sequences of 131–136 kb reconstructed from publicly available WGS revealed a modular gene organization and high levels of sequence heterogeneity at several hotspots, suggesting that these genomes correspond to biological entities undergoing recombination. Compared to other C. difficile phages, these big phages have unique predicted terminase, capsid, portal, neck and tail proteins, receptor binding proteins (RBPs), recombinases, resolvases, primases, helicases, ligases, and hypothetical proteins. Moreover, their predicted gene load suggests a complex regulation of both phage and host functions. Overall, our results indicate that the prevalence of C. difficile big bacteriophages is more widespread than realized and open new avenues of research aiming to decipher how these viral elements influence the biology of this emerging pathogen.

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September 22, 2019

Screening and genomic characterization of filamentous hemagglutinin-deficient Bordetella pertussis.

Despite high vaccine coverage, pertussis cases in the United States have increased over the last decade. Growing evidence suggests that disease resurgence results, in part, from genetic divergence of circulating strain populations away from vaccine references. The United States employs acellular vaccines exclusively, and current Bordetella pertussis isolates are predominantly deficient in at least one immunogen, pertactin (Prn). First detected in the United States retrospectively in a 1994 isolate, the rapid spread of Prn deficiency is likely vaccine driven, raising concerns about whether other acellular vaccine immunogens experience similar pressures, as further antigenic changes could potentially threaten vaccine efficacy. We developed an electrochemiluminescent antibody capture assay to monitor the production of the acellular vaccine immunogen filamentous hemagglutinin (Fha). Screening 722 U.S. surveillance isolates collected from 2010 to 2016 identified two that were both Prn and Fha deficient. Three additional Fha-deficient laboratory strains were also identified from a historic collection of 65 isolates dating back to 1935. Whole-genome sequencing of deficient isolates revealed putative, underlying genetic changes. Only four isolates harbored mutations to known genes involved in Fha production, highlighting the complexity of its regulation. The chromosomes of two Fha-deficient isolates included unexpected structural variation that did not appear to influence Fha production. Furthermore, insertion sequence disruption of fhaB was also detected in a previously identified pertussis toxin-deficient isolate that still produced normal levels of Fha. These results demonstrate the genetic potential for additional vaccine immunogen deficiency and underscore the importance of continued surveillance of circulating B. pertussis evolution in response to vaccine pressure. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Characterization of plasmids harboring blaCTX-M and blaCMY genes in E. coli from French broilers.

Resistance to extended-spectrum cephalosporins (ESC) is a global health issue. The aim of this study was to analyze and compare plasmids coding for resistance to ESC isolated from 16 avian commensal and 17 avian pathogenic Escherichia coli (APEC) strains obtained respectively at slaughterhouse or from diseased broilers in 2010-2012. Plasmid DNA was used to transform E. coli DH5alpha, and the resistances of the transformants were determined. The sequences of the ESC-resistance plasmids prepared from transformants were obtained by Illumina (33 plasmids) or PacBio (1 plasmid). Results showed that 29 of these plasmids contained the blaCTX-M-1 gene and belonged to the IncI1/ST3 type, with 27 and 20 of them carrying the sul2 or tet(A) genes respectively. Despite their diverse origins, several plasmids showed very high percentages of identity. None of the blaCTX-M-1-containing plasmid contained APEC virulence genes, although some of them were detected in the parental strains. Three plasmids had the blaCMY-2 gene, but no other resistance gene. They belonged to IncB/O/K/Z-like or IncFIA/FIB replicon types. The blaCMY-2 IncFIA/FIB plasmid was obtained from a strain isolated from a diseased broiler and also containing a blaCTX-M-1 IncI1/ST3 plasmid. Importantly APEC virulence genes (sitA-D, iucA-D, iutA, hlyF, ompT, etsA-C, iss, iroB-E, iroN, cvaA-C and cvi) were detected on the blaCMY-2 plasmid. In conclusion, our results show the dominance and high similarity of blaCTX-M-1 IncI1/ST3 plasmids, and the worrying presence of APEC virulence genes on a blaCMY-2 plasmid.

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September 22, 2019

Convergent evolution driven by rifampin exacerbates the global burden of drug-resistant Staphylococcus aureus.

Mutations in the beta-subunit of bacterial RNA polymerase (RpoB) cause resistance to rifampin (Rifr), a critical antibiotic for treatment of multidrug-resistantStaphylococcus aureus.In vitrostudies have shown that RpoB mutations confer decreased susceptibility to other antibiotics, but the clinical relevance is unknown. Here, by analyzing 7,099S. aureusgenomes, we demonstrate that the most prevalent RpoB mutations promote clinically relevant phenotypic plasticity resulting in the emergence of stableS. aureuslineages, associated with increased risk of therapeutic failure through generation of small-colony variants (SCVs) and coresistance to last-line antimicrobial agents. We found eight RpoB mutations that accounted for 93% (469/505) of the total number of Rifrmutations. The most frequently selected amino acid substitutions affecting residue 481 (H481N/Y) were associated with worldwide expansions of Rifrclones spanning decades. Recreating the H481N/Y mutations confirmed no impact onS. aureusgrowth, but the H481N mutation promoted the emergence of a subpopulation of stable RifrSCVs with reduced susceptibility to vancomycin and daptomycin. Recreating the other frequent RpoB mutations showed similar impacts on resistance to these last-line agents. We found that 86% of all Rifrisolates in our global sample carried the mutations promoting cross-resistance to vancomycin and 52% to both vancomycin and daptomycin. As four of the most frequent RpoB mutations confer only low-level Rifr, equal to or below some international breakpoints, we recommend decreasing these breakpoints and reconsidering the appropriate use of rifampin to reduce the fixation and spread of these clinically deleterious mutations. IMPORTANCE Increasing antibiotic resistance in the major human pathogenStaphylococcus aureusis threatening the ability to treat patients with these infections. Recent laboratory studies suggest that mutations in the gene commonly associated with rifampin resistance may also impact susceptibility to other last-line antibiotics inS. aureus; however, the overall frequency and clinical impact of these mutations are unknown. By mining a global collection of clinicalS. aureusgenomes and by mutagenesis experiments, this work reveals that common rifampin-inducedrpoBmutations promote phenotypic plasticity that has led to the global emergence of stable, multidrug-resistantS. aureuslineages that are associated with increased risk of therapeutic failure through coresistance to other last-line antimicrobials. We recommend decreasing susceptibility breakpoints for rifampin to allow phenotypic detection of criticalrpoBmutations conferring low resistance to rifampin and reconsidering the appropriate use of rifampin to reduce the fixation and spread of these deleterious mutations globally.

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September 22, 2019

Candidatus Nitrosocaldus cavascurensis, an ammonia oxidizing, extremely thermophilic archaeon with a highly mobile genome.

Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first enrichment of a thermophilic representative with a sequenced genome, which facilitates the search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota.CandidatusNitrosocaldus cavascurensis has been enriched from a hot spring in Ischia, Italy. It grows optimally at 68°C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 23 h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome ofCa.N. cavascurensis harbors anamoAXCB gene cluster encoding ammonia monooxygenase and genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although abona fidegene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the potential importance of this compound for AOA metabolism.Ca.N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits.

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September 22, 2019

The genome of the Hi5 germ cell line from Trichoplusia ni, an agricultural pest and novel model for small RNA biology.

We report a draft assembly of the genome of Hi5 cells from the lepidopteran insect pest,Trichoplusia ni, assigning 90.6% of bases to one of 28 chromosomes and predicting 14,037 protein-coding genes. Chemoreception and detoxification gene families revealT. ni-specific gene expansions that may explain its widespread distribution and rapid adaptation to insecticides. Transcriptome and small RNA data from thorax, ovary, testis, and the germline-derived Hi5 cell line show distinct expression profiles for 295 microRNA- and >393 piRNA-producing loci, as well as 39 genes encoding small RNA pathway proteins. Nearly all of the W chromosome is devoted to piRNA production, andT. nisiRNAs are not 2´-O-methylated. To enable use of Hi5 cells as a model system, we have established genome editing and single-cell cloning protocols. TheT. nigenome provides insights into pest control and allows Hi5 cells to become a new tool for studying small RNAs ex vivo.© 2018, Fu et al.

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September 22, 2019

The repeat structure of two paralogous genes, Yersinia ruckeri invasin (yrInv) and a “Y. ruckeri invasin-like molecule”, (yrIlm) sheds light on the evolution of adhesive capacities of a fish pathogen.

Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_3758, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812bp) and has a molecular mass of 256.4kDa. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced. Y. ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y. ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen. Copyright © 2017 Elsevier Inc. All rights reserved.

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September 22, 2019

A hybrid-hierarchical genome assembly strategy to sequence the invasive golden mussel Limnoperna fortunei.

For more than 25 years, the golden mussel Limnoperna fortunei has aggressively invaded South American freshwaters, having travelled more than 5,000 km upstream across five countries. Along the way, the golden mussel has outcompeted native species and economically harmed aquaculture, hydroelectric powers, and ship transit. We have sequenced the complete genome of the golden mussel to understand the molecular basis of its invasiveness and search for ways to control it.We assembled the 1.6 Gb genome into 20548 scaffolds with an N50 length of 312 Kb using a hybrid and hierarchical assembly strategy from short and long DNA reads and transcriptomes. A total of 60717 coding genes were inferred from a customized transcriptome-trained AUGUSTUS run. We also compared predicted protein sets with those of complete molluscan genomes, revealing an exacerbation of protein-binding domains in L. fortunei. Conclusions: We built one of the best bivalve genome assemblies available using a cost-effective approach using Illumina pair-end, mate pair, and PacBio long reads. We expect that the continuous and careful annotation of L. fortunei’s genome will contribute to the investigation of bivalve genetics, evolution, and invasiveness, as well as to the development of biotechnological tools for aquatic pest control.© The Authors 2017. Published by Oxford University Press.

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