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September 22, 2019  |  

Phosphagen kinase function in flagellated spores of the oomycete Phytophthora infestans integrates transcriptional regulation, metabolic dynamics and protein retargeting.

Flagellated spores play important roles in the infection of plants and animals by many eukaryotic microbes. The oomycete Phytophthora infestans, which causes potato blight, expresses two phosphagen kinases (PKs). These enzymes store energy in taurocyamine, and are hypothesized to resolve spatial and temporal imbalances between rates of ATP creation and use in zoospores. A dimeric PK is found at low levels in vegetative mycelia, but high levels in ungerminated sporangia and zoospores. In contrast, a monomeric PK protein is at similar levels in all tissues, although is transcribed primarily in mycelia. Subcellular localization studies indicate that the monomeric PK is mitochondrial. In contrast, the dimeric PK is cytoplasmic in mycelia and sporangia but is retargeted to flagellar axonemes during zoosporogenesis. This supports a model in which PKs shuttle energy from mitochondria to and through flagella. Metabolite analysis indicates that deployment of the flagellar PK is coordinated with a large increase in taurocyamine, synthesized by sporulation-induced enzymes that were lost during the evolution of zoospore-lacking oomycetes. Thus, PK function is enabled by coordination of the transcriptional, metabolic and protein targeting machinery during the life cycle. Since plants lack PKs, the enzymes may be useful targets for inhibitors of oomycete plant pathogens.© 2018 John Wiley & Sons Ltd.


September 22, 2019  |  

Antiviral adaptive immunity and tolerance in the mosquito Aedes aegyti

Mosquitoes spread pathogenic arboviruses while themselves tolerate infection. We here characterize an immunity pathway providing long-term antiviral protection and define how this pathway discriminates between self and non-self. Mosquitoes use viral RNAs to create viral derived cDNAs (vDNAs) central to the antiviral response. vDNA molecules are acquired through a process of reverse-transcription and recombination directed by endogenous retrotransposons. These vDNAs are thought to integrate in the host genome as endogenous viral elements (EVEs). Sequencing of pre-integrated vDNA revealed that the acquisition process exquisitely distinguishes viral from host RNA, providing one layer of self-nonself discrimination. Importantly, we show EVE-derived piRNAs have antiviral activity and are loaded onto Piwi4 to inhibit virus replication. In a second layer of self-non-self discrimination, Piwi4 preferentially loads EVE-derived piRNAs, discriminating against transposon-targeting piRNAs. Our findings define a fundamental virus-specific immunity pathway in mosquitoes that uses EVEs as a potent and specific antiviral transgenerational mechanism.


September 22, 2019  |  

Comparative genomics of Czech vaccine strains of Bordetella pertussis.

Bordetella pertussis is a strictly human pathogen causing the respiratory infectious disease called whooping cough or pertussis. B. pertussis adaptation to acellular pertussis vaccine pressure has been repeatedly highlighted, but recent data indicate that adaptation of circulating strains started already in the era of the whole cell pertussis vaccine (wP) use. We sequenced the genomes of five B. pertussis wP vaccine strains isolated in the former Czechoslovakia in the pre-wP (1954-1957) and early wP (1958-1965) eras, when only limited population travel into and out of the country was possible. Four isolates exhibit a similar genome organization and form a distinct phylogenetic cluster with a geographic signature. The fifth strain is rather distinct, both in genome organization and SNP-based phylogeny. Surprisingly, despite isolation of this strain before 1966, its closest sequenced relative appears to be a recent isolate from the US. On the genome content level, the five vaccine strains contained both new and already described regions of difference. One of the new regions contains duplicated genes potentially associated with transport across the membrane. The prevalence of this region in recent isolates indicates that its spread might be associated with selective advantage leading to increased strain fitness.


September 22, 2019  |  

Convergent evolution of complex genomic rearrangements in two fungal meiotic drive elements.

Meiotic drive is widespread in nature. The conflict it generates is expected to be an important motor for evolutionary change and innovation. In this study, we investigated the genomic consequences of two large multi-gene meiotic drive elements, Sk-2 and Sk-3, found in the filamentous ascomycete Neurospora intermedia. Using long-read sequencing, we generated the first complete and well-annotated genome assemblies of large, highly diverged, non-recombining regions associated with meiotic drive elements. Phylogenetic analysis shows that, even though Sk-2 and Sk-3 are located in the same chromosomal region, they do not form sister clades, suggesting independent origins or at least a long evolutionary separation. We conclude that they have in a convergent manner accumulated similar patterns of tandem inversions and dense repeat clusters, presumably in response to similar needs to create linkage between genes causing drive and resistance.


September 22, 2019  |  

An introduced crop plant is driving diversification of the virulent bacterial pathogen Erwinia tracheiphila.

Erwinia tracheiphila is the causal agent of bacterial wilt of cucurbits, an economically important phytopathogen affecting an economically important phytopathogen affecting few cultivated Cucurbitaceae few cultivated Cucurbitaceae host plant species in temperate eastern North America. However, essentially nothing is known about E. tracheiphila population structure or genetic diversity. To address this shortcoming, a representative collection of 88 E. tracheiphila isolates was gathered from throughout its geographic range, and their genomes were sequenced. Phylogenomic analysis revealed three genetic clusters with distinct hrpT3SS virulence gene repertoires, host plant association patterns, and geographic distributions. Low genetic heterogeneity within each cluster suggests a recent population bottleneck followed by population expansion. We showed that in the field and greenhouse, cucumber (Cucumis sativus), which was introduced to North America by early Spanish conquistadors, is the most susceptible host plant species and the only species susceptible to isolates from all three lineages. The establishment of large agricultural populations of highly susceptible C. sativus in temperate eastern North America may have facilitated the original emergence of E. tracheiphila into cucurbit agroecosystems, and this introduced plant species may now be acting as a highly susceptible reservoir host. Our findings have broad implications for agricultural sustainability by drawing attention to how worldwide crop plant movement, agricultural intensification, and locally unique environments may affect the emergence, evolution, and epidemic persistence of virulent microbial pathogens.IMPORTANCEErwinia tracheiphila is a virulent phytopathogen that infects two genera of cucurbit crop plants, Cucurbita spp. (pumpkin and squash) and Cucumis spp. (muskmelon and cucumber). One of the unusual ecological traits of this pathogen is that it is limited to temperate eastern North America. Here, we complete the first large-scale sequencing of an E. tracheiphila isolate collection. From phylogenomic, comparative genomic, and empirical analyses, we find that introduced Cucumis spp. crop plants are driving the diversification of E. tracheiphila into multiple lineages. Together, the results from this study show that locally unique biotic (plant population) and abiotic (climate) conditions can drive the evolutionary trajectories of locally endemic pathogens in unexpected ways. Copyright © 2018 Shapiro et al.


September 22, 2019  |  

Distribution of the pco gene cluster and associated genetic determinants among swine Escherichia coli from a controlled feeding trial.

Copper is used as an alternative to antibiotics for growth promotion and disease prevention. However, bacteria developed tolerance mechanisms for elevated copper concentrations, including those encoded by the pco operon in Gram-negative bacteria. Using cohorts of weaned piglets, this study showed that the supplementation of feed with copper concentrations as used in the field did not result in a significant short-term increase in the proportion of pco-positive fecal Escherichia coli. The pco and sil (silver resistance) operons were found concurrently in all screened isolates, and whole-genome sequencing showed that they were distributed among a diversity of unrelated E. coli strains. The presence of pco/sil in E. coli was not associated with elevated copper minimal inhibitory concentrations (MICs) under a variety of conditions. As found in previous studies, the pco/sil operons were part of a Tn7-like structure found both on the chromosome or on plasmids in the E. coli strains investigated. Transfer of a pco/sil IncHI2 plasmid from E. coli to Salmonellaenterica resulted in elevated copper MICs in the latter. Escherichia coli may represent a reservoir of pco/sil genes transferable to other organisms such as S. enterica, for which it may represent an advantage in the presence of copper. This, in turn, has the potential for co-selection of resistance to antibiotics.


September 22, 2019  |  

Endogenous rRNA sequence variation can regulate stress response gene expression and phenotype.

Prevailing dogma holds that ribosomes are uniform in composition and function. Here, we show that nutrient limitation-induced stress in E. coli changes the relative expression of rDNA operons to alter the rRNA composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes affect the expression of functionally coherent gene sets and alter the levels of the RpoS sigma factor, the master regulator of the general stress response. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility and are regulated by stress response proteins, RelA and RelE, as well as the metabolic enzyme and virulence-associated protein, AdhE. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.


September 22, 2019  |  

Comparative genome analysis and evaluation of probiotic characteristics of Lactobacillus plantarum strain JDFM LP11.

In the current study, the probiotic potential of approximately 250 strains of lactic acid bacteria (LAB) isolated from piglet fecal samples were investigated; among them Lactobacillus plantarum strain JDFM LP11, which possesses significant probiotic potential, with enhanced acid/bile tolerance, attachment to porcine intestinal epithelial cells (IPEC-J2), and antimicrobial activity. The genetic characteristics of strain JDFM LP11 were explored by performing whole genome sequencing (WGS) using a PacBio system. The circular draft genome have a total length of 3,206,883 bp and a total of 3,021 coding sequences were identified. Phylogenetically, three genes, possibly related to survival and metabolic activity in the porcine host, were identified. These genes encode p60, lichenan permease IIC component, and protein TsgA, which are a putative endopeptidase, a component of the phosphotransferase system (PTS), and a major facilitator in the gut environment, respectively. Our findings suggest that understanding the functional and genetic characteristics of L. plantarum strain JDFM LP11, with its candidate genes for gut health, could provide new opportunities and insights into applications in the animal food and feed additive industries.


September 22, 2019  |  

SKA: Split Kmer Analysis Toolkit for Bacterial Genomic Epidemiology

Genome sequencing is revolutionising infectious disease epidemiology, providing a huge step forward in sensitivity and specificity over more traditional molecular typing techniques. However, the complexity of genome data often means that its analysis and interpretation requires high-performance compute infrastructure and dedicated bioinformatics support. Furthermore, current methods have limitations that can differ between analyses and are often opaque to the user, and their reliance on multiple external dependencies makes reproducibility difficult. Here I introduce SKA, a toolkit for analysis of genome sequence data from closely-related, small, haploid genomes. SKA uses split kmers to rapidly identify variation between genome sequences, making it possible to analyse hundreds of genomes on a standard home computer. Tests on publicly available simulated and real-life data show that SKA is both faster and more efficient than the gold standard methods used today while retaining similar levels of accuracy for epidemiological purposes. SKA can take raw read data or genome assemblies as input and calculate pairwise distances, create single linkage clusters and align genomes to a reference genome or using a reference-free approach. SKA requires few decisions to be made by the user, which, along with its computational efficiency, allows genome analysis to become accessible to those with only basic bioinformatics training. The limitations of SKA are also far more transparent than for current approaches, and future improvements to mitigate these limitations are possible. Overall, SKA is a powerful addition to the armoury of the genomic epidemiologist. SKA source code is available from Github (https://github.com/simonrharris/SKA).


September 22, 2019  |  

Phylogenomics of colistin-susceptible and resistant XDR Acinetobacter baumannii.

Acinetobacter baumannii is a healthcare-associated pathogen with high rates of carbapenem resistance. Colistin is now routinely used for treatment of infections by this pathogen. However, colistin use has been associated with development of resistance to this agent.To elucidate the phylogenomics of colistin-susceptible and -resistant A. baumannii strain pairs from a cohort of hospitalized patients at a tertiary medical centre in the USA.WGS data from 21 pairs of colistin-susceptible and -resistant, XDR clinical strains were obtained and compared using phylogeny of aligned genome sequences, assessment of pairwise SNP differences and gene content.Fourteen patients had colistin-resistant strains that were highly genetically related to their own original susceptible strain with a median pairwise SNP distance of 5.5 (range 1-40 SNPs), while seven other strain pairs were divergent with =84 SNP differences. In addition, several strains from different patients formed distinct clusters on the phylogeny in keeping with closely linked transmission chains. The majority of colistin-resistant strains contained non-synonymous mutations within the pmrAB locus suggesting a central role for pmrAB mutations in colistin resistance. Excellent genotype-phenotype correlation was also observed for carbapenems, aminoglycosides and tetracyclines.The findings suggest that colistin resistance in the clinical setting arises through both in vivo evolution from colistin-susceptible strains and reinfection by unrelated colistin-resistant strains, the latter of which may involve patient-to-patient transmission.


September 22, 2019  |  

Molecular epidemiology of isolates with multiple mcr plasmids from a pig farm in Great Britain: the effects of colistin withdrawal in the short and long term.

The environment, including farms, might act as a reservoir for mobile colistin resistance (mcr) genes, which has led to calls for reduction of usage in livestock of colistin, an antibiotic of last resort for humans.To establish the molecular epidemiology of mcr Enterobacteriaceae from faeces of two cohorts of pigs, where one group had initially been treated with colistin and the other not, over a 5?month period following stoppage of colistin usage on a farm in Great Britain; faecal samples were also taken at ~20?months.mcr-1 Enterobacteriaceae were isolated from positive faeces and was WGS performed; conjugation was performed on selected Escherichia coli and colistin MICs were determined.E. coli of diverse ST harbouring mcr-1 and multiple resistance genes were isolated over 5?months from both cohorts. Two STs, from treated cohorts, contained both mcr-1 and mcr-3 plasmids, with some isolates also harbouring multiple copies of mcr-1 on different plasmids. The mcr-1 plasmids grouped into four Inc types (X4, pO111, I2 and HI2), with mcr-3 found in IncP. Multiple copies of mcr plasmids did not have a noticeable effect on colistin MIC, but they could be transferred simultaneously to a Salmonella host in vitro. Neither mcr-1 nor mcr-3 was detected in samples collected ~20?months after colistin cessation.We report for the first known time on the presence in Great Britain of mcr-3 from MDR Enterobacteriaceae, which might concurrently harbour multiple copies of mcr-1 on different plasmids. However, control measures, including stoppage of colistin, can successfully mitigate long-term on-farm persistence.


September 22, 2019  |  

Tracing back multidrug-resistant bacteria in fresh herb production: from chive to source through the irrigation water chain.

Environmental antibiotic-resistant bacteria (ARB) can be transferred to humans through foods. Fresh produce in particular is an ideal vector due to frequent raw consumption. A major contamination source of fresh produce is irrigation water. We hypothesized that water quality significantly affects loads of ARB and their diversity on fresh produce despite various other contamination sources present under agricultural practice conditions. Chive irrigated from an open-top reservoir or sterile-filtered water (control) was examined. Heterotrophic plate counts (HPC) and ARB were determined for water and chive with emphasis on Escherichia coli and Enterococcus spp. High HPC of freshly planted chive decreased over time and were significantly lower on control- vs. reservoir-irrigated chive at harvest (1.3 log (CFU/g) lower). Ciprofloxacin- and ceftazidime-resistant bacteria were significantly lower on control-irrigated chive at harvest and end of shelf life (up to 1.8 log (CFU/g) lower). Escherichia coli and Enterococcus spp. repeatedly isolated from water and chive proved resistant to up to six or four antibiotic classes (80% or 49% multidrug-resistant, respectively). Microbial source tracking identified E. coli-ST1056 along the irrigation chain and on chive. Whole-genome sequencing revealed that E. coli-ST1056 from both environments were clonal and carried the same transmissible multidrug-resistance plasmid, proving water as source of chive contamination. These findings emphasize the urgent need for guidelines concerning ARB in irrigation water and development of affordable water disinfection technologies to diminish ARB on irrigated produce.


September 22, 2019  |  

An IncX1 plasmid isolated from Salmonella enterica subsp. enterica serovar Pullorum carrying blaTEM-1B, sul2, arsenic resistant operons.

We have identified an IncX1 plasmid named pQJDSal1 from Salmonella enterica subsp. enterica serovar Pullorum (S. Pullorum). The plasmid is 67,685?bp in size and has 72 putative genes. pQJDSal1 harbors a conserved IncX1-type backbone with predicted regions for conjugation, replication and partitioning, as well as a toxin/antitoxin plasmid addiction system. Two regions (A and B) that have not been previously reported in IncX1 plasmids are inserted into the backbone. Region A (10.7?kb), inserted between parA and taxD, consists of a new Tn6168-like transposon containing an arsenic resistant operon arsB2CHR and sulfonamide resistance gene sul2. Region B contains another arsenic resistant operon arsADHR, resistance gene blaTEM-1B and three transposable elements. Conjugation experiments showed that pQJDSal1 could transfer from S. Pullorum to Escherichia coli (E. coli) J53. Statistical analysis of 70 sequenced IncX1 plasmids revealed that IncX1 plasmids harbored various antibiotic resistance genes. The results highlight the importance of IncX1 plasmids in disseminating antibiotic resistance genes.Copyright © 2018. Published by Elsevier Inc.


September 22, 2019  |  

Phenazines in plant-beneficial Pseudomonas spp.: biosynthesis, regulation, function and genomics.

Plant-beneficial phenazine-producing Pseudomonas spp. are proficient biocontrol agents of soil-dwelling plant pathogens. Phenazines are redox-active molecules that display broad-spectrum antibiotic activity toward many fungal, bacterial and oomycete plant pathogens. Phenazine compounds also play a role in the persistence and survival of Pseudomonas spp. in the rhizosphere. This mini-review focuses on plant-beneficial phenazine-producing Pseudomonas spp. from the P. fluorescens species complex, which includes numerous well-known phenazine-producing strains of biocontrol interest. In this review the current knowledge on phenazine biosynthesis and regulation, the role played by phenazines in biocontrol and rhizosphere colonization, as well as exciting new advances in the genomics of plant-beneficial phenazine-producing Pseudomonas spp. will be discussed.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.


September 22, 2019  |  

Genomic evidence for asymmetric introgression by sexual selection in the common wall lizard.

Strongly selected characters can be transferred from one lineage to another with limited genetic exchange, resulting in asymmetric introgression and a mosaic genome in the receiving population. However, systems are rarely sufficiently well studied to link the pattern of introgression to its underlying process. Male common wall lizards in western Italy exhibit exaggeration of a suite of sexually selected characters that make them outcompete males from a distantly related lineage that lack these characters. This results in asymmetric hybridization and adaptive introgression of the suite of characters following secondary contact. We developed genomewide markers to infer the demographic history of gene flow between different genetic lineages, identify the spread of the sexually selected syndrome, and test the prediction that introgression should be asymmetric and heterogeneous across the genome. Our results show that secondary contact was accompanied by gene flow in both directions across most of the genome, but with approximately 3% of the genome showing highly asymmetric introgression in the predicted direction. Demographic simulations reveal that this asymmetric gene flow is more recent than the initial secondary contact, and the data suggest that the exaggerated male sexual characters originated within the Italian lineage and subsequently spread throughout this lineage before eventually reaching the contact zone. These results demonstrate that sexual selection can cause a suite of characters to spread throughout both closely and distantly related lineages with limited gene flow across the genome at large.© 2018 John Wiley & Sons Ltd.


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