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September 22, 2019  |  

Genome-based evolutionary history of Pseudomonas spp.

Pseudomonas is a large and diverse genus of Gammaproteobacteria. To provide a framework for discovery of evolutionary and taxonomic relationships of these bacteria, we compared the genomes of type strains of 163 species and 3 additional subspecies of Pseudomonas, including 118 genomes sequenced herein. A maximum likelihood phylogeny of the 166 type strains based on protein sequences of 100 single-copy orthologous genes revealed thirteen groups of Pseudomonas, composed of two to sixty three species each. Pairwise average nucleotide identities and alignment fractions were calculated for the data set of the 166 type strains and 1224 genomes of Pseudomonas available in public databases. Results revealed that 394 of the 1224 genomes were distinct from any type strain, suggesting that the type strains represent only a fraction of the genomic diversity of the genus. The core genome of Pseudomonas was determined to contain 794 genes conferring primarily housekeeping functions. The results of this study provide a phylogenetic framework for future studies aiming to resolve the classification and phylogenetic relationships, identify new gene functions and phenotypes, and explore the ecological and metabolic potential of the Pseudomonas spp.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019  |  

Comparative genomics of Pseudomonas syringae reveals convergent gene gain and loss associated with specialization onto cherry (Prunus avium).

Genome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium), including pathovars P. syringae pv morsprunorum (Psm) races 1 and 2, P. syringae pv syringae (Pss) and P. syringae pv avii. Phylogenetic analyses revealed Psm races and P. syringae pv avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species. A maximum likelihood approach was used to predict effectors associated with pathogenicity on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters than Psm and P. syringae pv avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions. Cherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host.© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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September 22, 2019  |  

A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits.

Rose is the world’s most important ornamental plant, with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Roses are outbred and can have various ploidy levels. Our objectives were to develop a high-quality reference genome sequence for the genus Rosa by sequencing a doubled haploid, combining long and short reads, and anchoring to a high-density genetic map, and to study the genome structure and genetic basis of major ornamental traits. We produced a doubled haploid rose line (‘HapOB’) from Rosa chinensis ‘Old Blush’ and generated a rose genome assembly anchored to seven pseudo-chromosomes (512?Mb with N50 of 3.4?Mb and 564 contigs). The length of 512?Mb represents 90.1-96.1% of the estimated haploid genome size of rose. Of the assembly, 95% is contained in only 196 contigs. The anchoring was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features, including the pericentromeric regions, through annotation of transposable element families and positioned centromeric repeats using fluorescent in situ hybridization. The rose genome displays extensive synteny with the Fragaria vesca genome, and we delineated only two major rearrangements. Genetic diversity was analysed using resequencing data of seven diploid and one tetraploid Rosa species selected from various sections of the genus. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and the number of flower petals. A rose APETALA2/TOE homologue is proposed to be the major regulator of petal number in rose. This reference sequence is an important resource for studying polyploidization, meiosis and developmental processes, as we demonstrated for flower and prickle development. It will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny across Rosaceae.

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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  |  

Variant O89 O-antigen of E. coli is associated with group 1 capsule loci and multidrug resistance.

Bacterial surface polysaccharides play significant roles in fitness and virulence. In Gram-negative bacteria such as Escherichia coli, major surface polysaccharides are lipopolysaccharide (LPS) and capsule, representing O- and K-antigens, respectively. There are multiple combinations of O:K types, many of which are well-characterized and can be related to ecotype or pathotype. In this investigation, we have identified a novel O:K permutation resulting through a process of major genome reorganization in a clade of E. coli. A multidrug-resistant, extended-spectrum ß-lactamase (ESBL)-producing strain – E. coli 26561 – represented a prototype of strains combining a locus variant of O89 and group 1 capsular polysaccharide. Specifically, the variant O89 locus in this strain was truncated at gnd, flanked by insertion sequences and located between nfsB and ybdK and we apply the term O89m for this variant. The prototype lacked colanic acid and O-antigen loci between yegH and hisI with this tandem polysaccharide locus being replaced with a group 1 capsule (G1C) which, rather than being a recognized E. coli capsule type, this locus matched to Klebsiella K10 capsule type. A genomic survey identified more than 200 E. coli strains which possessed the O89m locus variant with one of a variety of G1C types. Isolates from our collection with the combination of O89m and G1C all displayed a mucoid phenotype and E. coli 26561 was unusual in exhibiting a mucoviscous phenotype more recognized as a characteristic among Klebsiella strains. Despite the locus truncation and novel location, all O89m:G1C strains examined showed a ladder pattern typifying smooth LPS and also showed high molecular weight, alcian blue-staining polysaccharide in cellular and/or extra-cellular fractions. Expression of both O-antigen and capsule biosynthesis loci were confirmed in prototype strain 26561 through quantitative proteome analysis. Further in silico exploration of more than 200 E. coli strains possessing the O89m:G1C combination identified a very high prevalence of multidrug resistance (MDR) – 85% possessed resistance to three or more antibiotic classes and a high proportion (58%) of these carried ESBL and/or carbapenemase. The increasing isolation of O89m:G1C isolates from extra-intestinal infection sites suggests that these represents an emergent clade of invasive, MDR E. coli.

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September 22, 2019  |  

The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen.

Chytridiomycota species (chytrids) belong to a basal lineage in the fungal kingdom. Inhabiting terrestrial and aquatic environments, most are free-living saprophytes but several species cause important diseases: e.g. Batrachochytrium dendrobatidis, responsible for worldwide amphibian decline; and Synchytrium endobioticum, causing potato wart disease. S. endobioticum has an obligate biotrophic lifestyle and isolates can be further characterized as pathotypes based on their virulence on a differential set of potato cultivars. Quarantine measures have been implemented globally to control the disease and prevent its spread. We used a comparative approach using chytrid mitogenomes to determine taxonomical relationships and to gain insights into the evolution and recent history of introductions of this plant pathogen.We assembled and annotated the complete mitochondrial genome of 30 S. endobioticum isolates and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum is linear with terminal inverted repeats which was validated by tailing and PCR amplifying the telomeric ends. Surprisingly, no conservation in organisation and orientation of mitochondrial genes was observed among the Chytridiomycota except for S. endobioticum and its sister species Synchytrium microbalum. However, the mitochondrial genome of S. microbalum is circular and comprises only a third of the 72.9 Kbp found for S. endobioticum suggesting recent linearization and expansion. Four mitochondrial lineages were identified in the S. endobioticum mitochondrial genomes. Several pathotypes occur in different lineages, suggesting that these have emerged independently. In addition, variations for polymorphic sites in the mitochondrial genome of individual isolates were observed demonstrating that S. endobioticum isolates represent a community of different genotypes. Such communities were shown to be complex and stable over time, but we also demonstrate that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype associated with increased virulence.Mitochondrial genomic variation shows that S. endobioticum has been introduced into Europe multiple times, that several pathotypes emerged multiple times, and that isolates represent communities of different genotypes. Our study represents the most comprehensive dataset of chytrid mitogenomes, which provides new insights into the extraordinary dynamics and evolution of mitochondrial genomes involving linearization, expansion and reshuffling.

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September 22, 2019  |  

Ma orthologous genes in Prunus spp. shed light on a noteworthy NBS-LRR cluster conferring differential resistance to root-knot nematodes.

Root-knot nematodes (RKNs) are considerable polyphagous pests that severely challenge plants worldwide and especially perennials. The specific genetic resistance of plants mainly relies on the NBS-LRR genes that are pivotal factors for pathogens control. In Prunus spp., the Ma plum and RMja almond genes possess different spectra for resistance to RKNs. While previous works based on the Ma gene allowed to clone it and to decipher its peculiar TIR-NBS-LRR (TNL) structure, we only knew that the RMja gene mapped on the same chromosome as Ma. We carried out a high-resolution mapping using an almond segregating F2 progeny of 1448 seedlings from resistant (R) and susceptible (S) parental accessions, to locate precisely RMja on the peach genome, the reference sequence for Prunus species. We showed that the RMja gene maps in the Ma resistance cluster and that the Ma ortholog is the best candidate for RMja. This co-localization is a crucial step that opens the way to unravel the molecular determinants involved in the resistance to RKNs. Then we sequenced both almond parental NGS genomes and aligned them onto the RKN susceptible reference peach genome. We produced a BAC library of the R parental accession and, from two overlapping BAC clones, we obtained a 336-kb sequence encompassing the RMja candidate region. Thus, we could benefit from three Ma orthologous regions to investigate their sequence polymorphism, respectively, within plum (complete R spectrum), almond (incomplete R spectrum) and peach (null R spectrum). We showed that the Ma TNL cluster has evolved orthologs with a unique conserved structure comprised of five repeated post-LRR (PL) domains, which contain most polymorphism. In addition to support the Ma and RMja orthologous relationship, our results suggest that the polymorphism contained in the PL sequences might underlie differential resistance interactions with RKNs and an original immune mechanism in woody perennials. Besides, our study illustrates how PL exon duplications and losses shape TNL structure and give rise to atypical PL domain repeats of yet unknown role.

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September 22, 2019  |  

Extraordinary genome instability and widespread chromosome rearrangements during vegetative growth

The haploid genome of the pathogenic fungus Zymoseptoria tritici is contained on “core” and “accessory” chromosomes. While 13 core chromosomes are found in all strains, as many as eight accessory chromosomes show presence/absence variation and rearrangements among field isolates. The factors influencing these presence/absence polymorphisms are so far unknown. We investigated chromosome stability using experimental evolution, karyotyping, and genome sequencing. We report extremely high and variable rates of accessory chromosome loss during mitotic propagation in vitro and in planta Spontaneous chromosome loss was observed in 2 to >50% of cells during 4 weeks of incubation. Similar rates of chromosome loss in the closely related Zymoseptoria ardabiliae suggest that this extreme chromosome dynamic is a conserved phenomenon in the genus. Elevating the incubation temperature greatly increases instability of accessory and even core chromosomes, causing severe rearrangements involving telomere fusion and chromosome breakage. Chromosome losses do not affect the fitness of Zymoseptoria tritici in vitro, but some lead to increased virulence, suggesting an adaptive role of this extraordinary chromosome instability. Copyright © 2018 by the Genetics Society of America.

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September 22, 2019  |  

Dissemination and persistence of extended-spectrum cephalosporin-resistance encoding IncI1-blaCTXM-1 plasmid among Escherichia coli in pigs.

This study investigated the ecology, epidemiology and plasmid characteristics of extended-spectrum cephalosporin (ESC)-resistant E. coli in healthy pigs over a period of 4 years (2013-2016) following the withdrawal of ESCs. High carriage rates of ESC-resistant E. coli were demonstrated in 2013 (86.6%) and 2014 (83.3%), compared to 2015 (22%) and 2016 (8.5%). ESC resistance identified among E. coli isolates was attributed to the carriage of an IncI1 ST-3 plasmid (pCTXM1-MU2) encoding blaCTXM-1. Genomic characterisation of selected E. coli isolates (n?=?61) identified plasmid movement into multiple commensal E. coli (n?=?22 STs). Major STs included ST10, ST5440, ST453, ST2514 and ST23. A subset of the isolates belong to the atypical enteropathogenic E. coli (aEPEC) pathotype that harboured multiple LEE pathogenic islands. pCTXM1-MU2 was similar (99% nt identity) to IncI1-ST3 plasmids reported from Europe, encoded resistance to aminoglycosides, sulphonamides and trimethoprim, and carried colicin Ib. pCTXM1-MU2 appears to be highly stable and readily transferable. This study demonstrates that ESC resistance may persist for a protracted period following removal of direct selection pressure, resulting in the emergence of ESC-resistance in both commensal E. coli and aEPEC isolates of potential significance to human and animal health.

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September 22, 2019  |  

Genomic analysis of the Phalaenopsis pathogen Dickeya sp. PA1, representing the emerging species Dickeya fangzhongdai.

Dickeya sp. strain PA1 is the causal agent of bacterial soft rot in Phalaenopsis, an important indoor orchid in China. PA1 and a few other strains were grouped into a novel species, Dickeya fangzhongdai, and only the orchid-associated strains have been shown to cause soft rot symptoms.We constructed the complete PA1 genome sequence and used comparative genomics to explore the differences in genomic features between D. fangzhongdai and other Dickeya species.PA1 has a 4,979,223-bp circular genome with 4269 predicted protein-coding genes. D. fangzhongdai was phylogenetically similar to Dickeya solani and Dickeya dadantii. The type I to type VI secretion systems (T1SS-T6SS), except for the stt-type T2SS, were identified in D. fangzhongdai. The three phylogenetically similar species varied significantly in terms of their T5SSs and T6SSs, as did the different D. fangzhongdai strains. Genomic island (GI) prediction and synteny analysis (compared to D. fangzhongdai strains) of PA1 also indicated the presence of T5SSs and T6SSs in strain-specific regions. Two typical CRISPR arrays were identified in D. fangzhongdai and in most other Dickeya species, except for D. solani. CRISPR-1 was present in all of these Dickeya species, while the presence of CRISPR-2 varied due to species differentiation. A large polyketide/nonribosomal peptide (PK/NRP) cluster, similar to the zeamine biosynthetic gene cluster in Dickeya zeae rice strains, was discovered in D. fangzhongdai and D. solani. The D. fangzhongdai and D. solani strains might recently have acquired this gene cluster by horizontal gene transfer (HGT).Orchid-associated strains are the typical members of D. fangzhongdai. Genomic analysis of PA1 suggested that this strain presents the genomic characteristics of this novel species. Considering the absence of the stt-type T2SS, the presence of CRISPR loci and the zeamine biosynthetic gene cluster, D. fangzhongdai is likely a transitional form between D. dadantii and D. solani. This is supported by the later acquisition of the zeamine cluster and the loss of CRISPR arrays by D. solani. Comparisons of phylogenetic positions and virulence determinants could be helpful for the effective quarantine and control of this emerging species.

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September 22, 2019  |  

Comparative genomic analysis of Pseudomonas amygdali pv. lachrymans NM002: Insights into its potential virulence genes and putative invasion determinants.

Pseudomonas amygdali pv. lachrymans is currently of important plant pathogenic bacteria that causes cucumber angular leaf spot worldwide. The pathogen has been studied for its roles in pathogenicity and plant inheritance resistance. To further delineate traits critical to virulence, invasion and survival in the phyllosphere, we reported the first complete genome of P. amygdali pv. lachrymans NM002. Analysis of the whole genome in comparison with three closely-related representative pathovars of P. syringae identified the conservation of virulence genes, including flagella and chemotaxis, quorum-sensing systems, two-component systems, and lipopolysaccharide and antiphagocytosis. It also revealed differences of invasion determinants, such as type III effectors, phytotoxin (coronatine, syringomycin and phaseolotoxin) and cell wall-degrading enzyme, which may contribute to infectivity. The aim of this study was to derive genomic information that would reveal the probable molecular mechanisms underlying the virulence, infectivity and provide a better understanding of the pathogenesis of the P. syringae pathovars. Copyright © 2018. Published by Elsevier Inc.

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September 22, 2019  |  

A strain of an emerging Indian Xanthomonas oryzae pv. oryzae pathotype defeats the rice bacterial blight resistance gene xa13 without inducing a clade III SWEET gene and is nearly identical to a recent Thai isolate.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) injects transcription activator-like effectors (TALEs) that bind and activate host “susceptibility” (S) genes important for disease. Clade III SWEET genes are major S genes for bacterial blight. The resistance genes xa5, which reduces TALE activity generally, and xa13, a SWEET11 allele not recognized by the cognate TALE, have been effectively deployed. However, strains that defeat both resistance genes individually were recently reported in India and Thailand. To gain insight into the mechanism(s), we completely sequenced the genome of one such strain from each country and examined the encoded TALEs. Strikingly, the two strains are clones, sharing nearly identical TALE repertoires, including a TALE known to activate SWEET11 strongly enough to be effective even when diminished by xa5. We next investigated SWEET gene induction by the Indian strain. The Indian strain induced no clade III SWEET in plants harboring xa13, indicating a pathogen adaptation that relieves dependence on these genes for susceptibility. The findings open a door to mechanistic understanding of the role SWEET genes play in susceptibility and illustrate the importance of complete genome sequence-based monitoring of Xoo populations in developing varieties with effective disease resistance.

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September 22, 2019  |  

Genome sequence of the potato pathogenic fungus Alternaria solani HWC-168 reveals clues for its conidiation and virulence.

Alternaria solani is a known air-born deuteromycete fungus with a polycyclic life cycle and is the causal agent of early blight that causes significant yield losses of potato worldwide. However, the molecular mechanisms underlying the conidiation and pathogenicity remain largely unknown.We produced a high-quality genome assembly of A. solani HWC-168 that was isolated from a major potato-producing region of Northern China, which facilitated a comprehensive gene annotation, the accurate prediction of genes encoding secreted proteins and identification of conidiation-related genes. The assembled genome of A. solani HWC-168 has a genome size 32.8 Mb and encodes 10,358 predicted genes that are highly similar with related Alternaria species including Alternaria arborescens and Alternaria brassicicola. We identified conidiation-related genes in the genome of A. solani HWC-168 by searching for sporulation-related homologues identified from Aspergillus nidulans. A total of 975 secreted protein-encoding genes, which might act as virulence factors, were identified in the genome of A. solani HWC-168. The predicted secretome of A. solani HWC-168 possesses 261 carbohydrate-active enzymes (CAZy), 119 proteins containing RxLx[EDQ] motif and 27 secreted proteins unique to A. solani.Our findings will facilitate the identification of conidiation- and virulence-related genes in the genome of A. solani. This will permit new insights into understanding the molecular mechanisms underlying the A. solani-potato pathosystem and will add value to the global fungal genome database.

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September 22, 2019  |  

Genome wide characterization of enterotoxigenic Escherichia coli serogroup O6 isolates from multiple outbreaks and sporadic infections from 1975-2016.

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea globally, particularly among children under the age of five in developing countries. ETEC O6 is the most common ETEC serogroup, yet the genome wide population structure of isolates of this serogroup is yet to be determined. In this study, we have characterized 40 ETEC O6 isolates collected between 1975-2016 by whole genome sequencing (WGS) and by phenotypic antimicrobial susceptibility testing. To determine the relatedness of isolates, we evaluated two methods-whole genome high-quality single nucleotide polymorphism (whole genome-hqSNP) and core genome SNP analyses using Lyve-SET and Parsnp respectively. All isolates were tested for antimicrobial susceptibility using a panel of 14 antibiotics. ResFinder 2.1 and a custom quinolone resistance determinants workflow were used for resistance determinant detection. VirulenceFinder 1.5 was used for prediction of the virulence genes. Thirty-seven isolates clustered into three major clades (I, II, III) by whole genome-hqSNP and core genome SNP analyses, while three isolates included in the whole genome-hqSNP analysis only did not cluster with clades I-III by both analyses and formed a distantly related outgroup, designated clade IV. Median number of pairwise whole genome-hqSNPs in clonal ETEC O6 outbreaks ranged from 0 to 5. Of the 40 isolates tested for antimicrobial susceptibility, 18 isolates were pansusceptible. Twenty-two isolates were resistant to at least one antibiotic, nine of which were multidrug resistant. Phenotypic antimicrobial resistance (AR) correlated with AR determinants in 22 isolates. Thirty-two isolates harbored both enterotoxin virulence genes while the remaining 8 isolates had only one of the two virulence genes. In summary, whole genome-hqSNP and core genome SNP analyses from this study revealed similar evolutionary relationships and an overall diversity of ETEC O6 isolates independent of time of isolation. Less than 5 pairwise hqSNPs between ETEC O6 isolates is circumstantially indicative of an outbreak cluster. Findings from this study will be a basis for quicker outbreak detection and control by efficient subtyping by WGS.

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September 22, 2019  |  

Emerging multidrug-resistant hybrid pathotype shiga toxin-producing Escherichia coli O80 and related strains of clonal complex 165, Europe.

Enterohemorrhagic Escherichia coli serogroup O80, involved in hemolytic uremic syndrome associated with extraintestinal infections, has emerged in France. We obtained circularized sequences of the O80 strain RDEx444, responsible for hemolytic uremic syndrome with bacteremia, and noncircularized sequences of 35 O80 E. coli isolated from humans and animals in Europe with or without Shiga toxin genes. RDEx444 harbored a mosaic plasmid, pR444_A, combining extraintestinal virulence determinants and a multidrug resistance-encoding island. All strains belonged to clonal complex 165, which is distantly related to other major enterohemorrhagic E. coli lineages. All stx-positive strains contained eae-?, ehxA, and genes characteristic of pR444_A. Among stx-negative strains, 1 produced extended-spectrum ß-lactamase, 1 harbored the colistin-resistance gene mcr1, and 2 possessed genes characteristic of enteropathogenic and pyelonephritis E. coli. Because O80-clonal complex 165 strains can integrate intestinal and extraintestinal virulence factors in combination with diverse drug-resistance genes, they constitute dangerous and versatile multidrug-resistant pathogens.

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