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bioRxiv2017

The ecological genetics of Pseudomonas syringae residing on the kiwifruit leaf surface

Interactions between commensal microbes and invading pathogens are understudied, despite their likely effects on pathogen population structure and infection processes. We describe the population structure and genetic diversity of a broad range of co-occurring Pseudomonas syringae isolated from infected and uninfected kiwifruit during an outbreak of bleeding canker disease caused by P. syringae pv. actinidiae (Psa) in New Zealand. Overall population structure was clonal and affected by ecological factors including infection status and cultivar. Most isolates are members of a new clade in phylogroup 3 (PG3a), also present on kiwifruit leaves in China and Japan. Stability of the polymorphism between pathogenic Psa and commensal P. syringae PG3a isolated from the same leaf was tested using reciprocal invasion from rare assays in vitro and in planta. P. syringae G33C (PG3a) inhibited Psa NZ54, while the presence of Psa NZ54 enhanced the growth of P. syringae G33C. This effect could not be attributed to virulence activity encoded by the Type 3 secretion system of Psa. Together our data contribute toward the development of an ecological perspective on the genetic structure of pathogen populations.\n\nORIGINALITY-SIGNIFICANT STATEMENTBacterial pathogen populations are often studied with little consideration of co-occurring microbes and yet interactions between pathogens and commensals can affect both population structure and disease progression. A fine-scale sampling of commensals present on kiwifruit leaves during an outbreak of bleeding canker disease caused by P. syringae pv. actinidiae reveals a clonal population structure. A new clade of non-pathogenic P. syringae (PG3a) appears to be associated with kiwifruit on a global scale. The presence of PG3a on kiwifruit has significant effects on the outcome of infection by P. syringae pv. actinidiae. This emphasises the value of studying the effect of co-occurring bacteria on pathogen-plant interactions.

microbiology↗

bioRxiv2017

Fitness traits of deoxynivalenol and nivalenol-producing Fusarium graminearum species complex strains from wheat

Fusarium graminearum of the 15-acetyl(A)deoxynivalenol(D0N) chemotype is the main cause of Fusarium head blight (FHB) of wheat in south of Brazil. However, 3-ADON and nivalenol(NIV) chemotypes have been found in other members of the species complex causing FHB in wheat. To improve our understanding of the pathogen ecology, we assessed a range of fitness-related traits in a sample of 30 strains representatives of 15-ADON (F. graminearum), 3-ADON (F. cortaderiae and F. austroamericanum) and NIV (F. meridionale and F. cortaderiae). These included: perithecia formation on three cereal-based substrates, mycelial growth at two suboptimal temperatures, sporulation and germination, pathogenicity towards a susceptible and a moderately resistant cultivar and sensitivity to tebuconazole. The most important trait favoring F. graminearum was its 2x higher sexual fertility (> 40% PPI = perithecia production index) than the other species (< 30% PPI); PPI varied among substrates (maize > rice > wheat). In addition, sensitivity to tebuconazole appeared lower in F. graminearum which had the only strain with EC50 > 1 ppm. In the pathogenicity assays, the DON-producers were generally more aggressive (1.5 to 2x higher final severity) towards the two cultivars, with 3-ADON or 15-ADON leading to higher area under the severity curve than the NIV strains in the susceptible and moderately resistant cv., respectively. There was significant variation among strains of a same species with regards asexual fertility (mycelial growth, macroconidia production and germination), which suggest a strain-rather than a species-specific differences. These results contribute new knowledge to improve our understanding of the pathogen-related traits that may explain the dominance of certain members of the species complex in specific wheat agroecosystems.

microbiology↗

bioRxiv2017

Distribution of Sigma factors delineates segregation of virulent and avirulent Mycobacterium

Sigma factors, in combination with RNA polymerase and several transcription factors play specific role in expression of housekeeping as well as various stress responsive genes in mycobacterial species. The genus Mycobacterium includes a wide range of species under major pathogens, opportunists and non-pathogens. The number and combination of sigma factors is extremely diversified among Mycobacterium. We have performed comparative genome analysis among 40 different species of Mycobacterium whose whole genome sequence is available, in order to identify the distribution of sigma factors. The study illustrate that SigC, SigD, SigG, SigH, SigK and SigI are dominant among the true pathogens. Moreover, 16S rDNA based phylogenetic analyses distinctly differentiate the slow growing Mycobacterium from the fast growers, and clusters the true pathogens from the opportunists and non-pathogens. While evaluating the similarity coefficient upon the allotment of sigma factors of different Mycobacterium species through UPGMA dendrogram analysis, it is apparent that the true pathogens are grouped separately following the similar trend observed from evolutionary approach. Sigma factors playing dominant role in pathogenicity are found stable in nature with high aliphatic index thereby remain flexible at a wide range of temperature. The comparative distribution of six well known virulence factors of Mycobacterium - PhoP, PcaA, FbpA, Mce1B, KatG and PE_PGRS and various sigma factors justify the allotment pattern of mycobacterial sigma factors among pathogenic species. The pathogenicity responsible sigma factors elicit close resemblance with few notable characters of the known virulence factors. Thus the analysis renders that the distribution of sigma factors of different species of Mycobacterium can be a potential tool to predict the pathogenicity index of this genus.

microbiology↗

bioRxiv2017

Host adaptation mediated by intergenic evolution in a bacterial pathogen

Bacterial pathogens evolve during the course of infection as they adapt to the selective pressures that confront them inside the host. Identification of adaptive mutations and their contributions to pathogen fitness remain a central challenge. Although mutations can either target intergenic or coding regions in the pathogen genome, studies of host adaptation have focused predominantly on molecular evolution within coding regions whereas the role of intergenic mutations remains unclear. Here, we address this issue and investigate the extent to which intergenic mutations contribute to the evolutionary response of pathogens to host environments, and if intergenic mutations have distinct roles in host adaptation. We characterize intergenic evolution in 44 lineages of a clinically important bacterial pathogen, Pseudomonas aeruginosa, as they adapt to their hosts. We identify 88 intergenic regions in which parallel evolution occur. At the genetic level, we find that mutations in these regions under selection are located primarily within regulatory elements upstream of transcriptional start sites. At the functional level, we show that these mutations both create or destroy regulatory interactions in connection to transcriptional processes, and are directly responsible for evolution of important pathogenic phenotypes including antibiotic sensitivity. Importantly, we find that intergenic mutations are more likely to be selected than coding region mutations, and that intergenic mutations enable essential genes to become targets of evolution. In summary, our results highlight the evolutionary significance of intergenic mutations in creating host-adapted variants, and that intergenic and coding regions have different qualitative and quantitative contributions to this process.\n\nSignificancePathogens adapt to their host during infection, but the contribution and function of non-coding intergenic sequences to adaptation is poorly understood. Here, genome-wide identification of adaptive mutations within intergenic regions demonstrates that these sequences constitute an important part of the genetic basis for host adaptation. We find that intergenic mutations are abundant relative to adaptive mutations within coding sequences, and can contribute directly to evolution of pathogen relevant traits. Importantly, we find that intergenic mutations modify expression of essential genes and thus make contributions that are functionally distinct from coding mutations. These results improve our understanding of the evolutionary processes in vivo, and can potentially assist in refining predictions of pathogen evolution, disease outcome, and antibiotic resistance development.

microbiology↗

bioRxiv2017

Characterisation of two Toxoplasma PROPPINs homologous to Atg18/WIPI suggests they have evolved distinct specialised functions

Toxoplasma gondii is a parasitic protist possessing a limited set of proteins involved in the autophagy pathway, a self-degradative machinery for protein and organelle recycling. This distant eukaryote has even repurposed part of this machinery, centered on protein ATG8, for a non-degradative function related to the maintenance of the apicoplast, a parasite-specific organelle. However, some evidence also suggest Toxoplasma is able to generate autophagic vesicles upon stress, and that some autophagy-related proteins, such as ATG9, might be involved solely in the canonical autophagy function. Here, we have characterised two Toxoplasma proteins containing WD-40 repeat that can bind lipids for their recruitment to vesicular structures upon stress. They belong to the PROPPIN family and are homologues to ATG18/WIPI, which are known to be important for the autophagic process. We conducted a functional analysis of these two Toxoplasma PROPPINs. One of them is dispensable for normal in vitro growth, although it may play a role for parasite survival in specific stress conditions or for parasite fitness in the host, through a canonical autophagy-related function. The other, however, seems important for parasite viability in normal growth conditions and could be primarily involved in a non-canonical function. These divergent roles for two proteins from the same family illustrate the functional versatility of the autophagy-related machinery in Toxoplasma.

microbiology↗

bioRxiv2017

Human Herpes Virus 6 (HHV-6) - Pathogen or Passenger? A pilot study of clinical laboratory data and next generation sequencing

ABSTRACT\n\nBackgroundHuman herpes virus 6 (HHV-6) is a ubiquitous organism that can cause a variety of clinical syndromes ranging from short-lived rash and fever through to life-threatening encephalitis.\n\nObjectivesWe set out to generate observational data regarding the epidemiology of HHV-6 infection in clinical samples from a UK teaching hospital and to compare different diagnostic approaches.\n\nStudy designFirst, we scrutinized HHV-6 detection in samples submitted to our hospital laboratory through routine diagnostic pathways. Second, we undertook a pilot study using Illumina next generation sequencing (NGS) to determine the frequency of HHV-6 in CSF and respiratory samples that were initially submitted to the laboratory for other diagnostic tests.\n\nResultsOf 72 samples tested for HHV-6 by PCR at the request of a clinician, 24 (33%) were positive for HHV-6. The majority of these patients were under the care of the haematology team (30/41, 73%), and there was a borderline association between HHV-6 detection and both Graft versus Host Disease (GvHD) and Central nervous system (CNS) disease (p=0.05 in each case). We confirmed detection of HHV-6 DNA using NGS in 4/20 (20%) CSF and respiratory samples.\n\nConclusionsHHV-6 is common in clinical samples submitted from a high-risk haematology population, and enhanced screening of this group should be considered. NGS can be used to identify HHV-6 from a complex microbiomee, but further controls are required to define the sensitivity and specificity, and to correlate these results with clinical disease. Our results underpin ongoing efforts to develop NGS technology for viral diagnostics.

microbiology↗

bioRxiv2017

CLIQ-BID: A method to quantify bacteria-induced damage to eukaryotic cells by automated live-imaging of bright nuclei

Pathogenic bacteria induce eukaryotic cell damage which range from discrete modifications of signalling pathways, to morphological alterations and even to cell death. Accurate quantitative detection of these events is necessary for studying host-pathogen interactions and for developing strategies to protect host organisms from bacterial infections. Investigation of morphological changes is cumbersome and not adapted to high-throughput and kinetics measurements. Here, we describe a simple and cost-effective method based on automated analysis of live cells with stained nuclei, which allows real-time quantification of bacteria-induced eukaryotic cell damage at single-cell resolution. We demonstrate that this automated high-throughput microscopy approach permits screening of libraries composed of interference-RNA, bacterial strains, antibodies and chemical compounds in ex vivo infection settings. The use of fluorescently-labelled bacteria enables the concomitant detection of changes in bacterial growth. Using this method named CLIQ-BID (Cell Live Imaging Quantification of Bacteria Induced Damage), we were able to distinguish the virulence profiles of different pathogenic bacterial species and clinical strains.

microbiology↗

bioRxiv2017

Antenatal microbial colonisation of mammalian gut

The widely accepted dogma of intrauterine sterility and initial colonisation of the newborn during birth has been blurred by recent observations of microbial presence in meconium, placenta and amniotic fluid. Given the importance of a maternal-derived in utero infant seeding, it is crucial to exclude potential environmental or procedural contaminations, and to assess foetal colonisation before parturition. To ascertain antenatal microbial colonisation in mammals, we analysed sterilely collected intestinal tissues from rodent foetuses in parallel with experimental controls, and tissues from autoptic human foetuses. Next generation sequencing (NGS) showed the presence of pioneer microbes in both rat and human intestines, as well as in rodent placentas and amniotic fluids. Live microbes were isolated from culture-dependent analyses from homogenized rat foetal intestines.\n\nMicrobial communities showed foetus- and dam-dependent clustering, confirming the high interindividual variability of microbiota even in the antenatal period. Fluorescent in situ hybridisation analysis confirmed the microbes existence in the lumen of the developing gut.\n\nThese findings have vast implications for an emerging field of enhancing the management of healthy pregnancies, and for understanding how the infant microbiome starts and it is thus shaped.

microbiology↗

bioRxiv2017

The structure of the influenza A virus genome

Influenza A viruses (IAVs) are segmented single-stranded negative sense RNA viruses that constitute a major threat to human health. The IAV genome consists of eight RNA segments contained in separate viral ribonucleoprotein complexes (vRNPs) that are packaged together into a single virus particle1,2. While IAVs are generally considered to have an unstructured single-stranded genome, it has also been suggested that secondary RNA structures are required for selective packaging of the eight vRNPs into each virus particle3,4. Here, we employ high-throughput sequencing approaches to map both the intra and intersegment RNA interactions inside influenza virions. Our data demonstrate that a redundant network of RNA-RNA interactions is required for vRNP packaging and virus growth. Furthermore, the data demonstrate that IAVs have a much more structured genome than previously thought and the redundancy of RNA interactions between the different vRNPs explains how IAVs maintain the potential for reassortment between different strains, while also retaining packaging selectivity. Our study establishes a framework towards further work into IAV RNA structure and vRNP packaging, which will lead to better models for predicting the emergence of new pandemic influenza strains and will facilitate the development of antivirals specifically targeting genome assembly.

microbiology↗

bioRxiv2018

Persistent Chikungunya Virus Replication in Human Cells is Associated with Presence of Stable Cytoplasmic Granules Containing Non-structural Protein 3

Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months or even years in patients. The non-structural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used sub-diffraction multi-color microscopy for a spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate viral RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and G3BP Stress Granule Assembly factor; (ii) were next to double-stranded RNA foci, and nsP1-positive structures; and (iii) made contact with markers of the cytoskeleton and cellular structures, such as early endosomes and nucleopores. Analysis of protein turnover and mobility by live-cell microscopy revealed that granules could persist for hours to days, can accumulate newly synthesized protein, and move at differently through the cytoplasm. Granules also had a static internal architecture and were stable in cell lysates. Whereas cells with active replication and stable nsP3-granules did not respond to oxidative stress, refractory cells that had cleared the non-cytotoxic replicon could. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein-complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus).\n\nImportanceChikungunya virus (CHIKV) is a re-emerging alphavirus transmitted by mosquitos and causes widespread transient sickness but also chronic disease affecting muscles and joints. Although no approved vaccines or antivirals are available, a better understanding of the viral life cycle and the role of individual viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and persistent CHIKV model systems now allow researchers to study viral proteins within controlled laboratory environments. Here we established human cells that stably replicate viral RNA and express a tagged version of non-structural protein 3. The ability to track this viral protein within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms.

microbiology↗

bioRxiv2017

Missing pstS locus associated with an insertion in tetM in vancomycin resistant Enterococcus faecium

Multi-locus sequencing typing (MLST) is widely used to monitor the phylogeny of microbial outbreaks. However, several strains of vancomycin-resistant Enterococcus faecium (VREfm) with a missing MLST locus (pstS) have recently emerged in Australia, with a few cases also reported in England. Here, we identified similarly distinct strains circulating in two closely located hospitals in Scotland. Whole genome sequencing of five VREfm strains isolated from these hospitals identified four pstS-null strains across both hospitals, while the fifth was of a multi-locus sequence type (ST) 262, which is the first documented in the UK. All five Scottish isolates had an insertion in the tetM gene, which is associated with increased susceptibility to tetracyclines, providing no other tetracycline-resistant gene is present. Such an insertion, which encompasses a dfrG gene and two currently uncharacterised genes, was additionally identified in all tested VanA-type pstS-null VREfm strains (5 English and 18 Australian). Phylogenetic comparison with other VREfm genomes indicates that the four pstS-null Scottish isolates sequenced in this study are more closely related to pstS-null strains from Australia rather than the English pstS-null isolates. Given how rapidly such pstS-null strains have expanded in Australia, the emergence of this clone in Scotland raises concerns for a potential outbreak.

microbiology↗

bioRxiv2017

The primary transcriptome, small RNAs, and regulation of antimicrobial resistance in Acinetobacter baumannii

AO_SCPCAPBSTRACTC_SCPCAPWe present the first high-resolution determination of transcriptome architecture in the priority pathogen Acinetobacter baumannii. Pooled RNA from 16 laboratory conditions was used for differential RNA-seq (dRNA-seq) to identify 3731 transcriptional start sites (TSS) and 110 small RNAs, including the first identification in A. baumannii of sRNAs encoded at the 3 end of coding genes. Most sRNAs were conserved among sequenced A. baumannii genomes, but were only weakly conserved or absent in other Acinetobacter species. Single nucleotide mapping of TSS enabled prediction of -10 and -35 RNA polymerase binding sites and revealed an unprecedented base preference at position +2 that hints at an unrecognized transcriptional regulatory mechanism. To apply functional genomics to the problem of antimicrobial resistance, we dissected the transcriptional regulation of the drug efflux pump responsible for chloramphenicol resistance, craA. The two craA promoters were both down-regulated >1000-fold when cells were shifted to nutrient limited medium. This conditional down-regulation of craA expression renders cells sensitive to chloramphenicol, a highly effective antibiotic for the treatment of multidrug resistant infections. An online interface that facilitates open data access and visualization is provided as \"AcinetoCom\" (http://bioinf.gen.tcd.ie/acinetocom/).

microbiology↗

bioRxiv2017

Discovery and biosynthesis of the antibiotic bicyclomycin in distant bacterial classes

Bicyclomycin (BCM) is a clinically promising antibiotic that is biosynthesised by Streptomyces cinnamoneus DSM 41675. BCM is structurally characterized by a core cyclo(L-Ile-L-Leu) 2,5-diketopiperazine (DKP) that is extensively oxidized. Here, we identify the BCM biosynthetic gene cluster, which shows that the core of BCM is biosynthesised by a cyclodipeptide synthase and the oxidative modifications are introduced by five 2-oxoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase. The discovery of the gene cluster enabled the identification of BCM pathways encoded in the genomes of hundreds of Pseudomonas aeruginosa isolates distributed globally, and heterologous expression of the pathway from P. aeruginosa SCV20265 demonstrated that the product is chemically identical to BCM produced by S. cinnamoneus. Overall, putative BCM gene clusters have been found in at least seven genera spanning Actinobacteria and Proteobacteria (Alpha-, Beta- and Gamma-). This represents a rare example of horizontal gene transfer of an intact biosynthetic gene cluster across such distantly related bacteria, and we show that these gene clusters are almost always associated with mobile genetic elements.\n\nIMPORTANCEBicyclomycin is the only natural product antibiotic that selectively inhibits the transcription termination factor Rho. This mechanism of action, combined with its proven biological safety and its activity against clinically relevant Gram-negative bacterial pathogens, makes it a very promising antibiotic candidate. Here, we report the identification of the bicyclomycin biosynthetic gene cluster in the known producing organism Streptomyces cinnamoneus, which will enable the engineered production of new bicyclomycin derivatives. The identification of this gene cluster also led to the discovery of hundreds of bicyclomycin pathways encoded in highly diverse bacteria, including the opportunistic pathogen Pseudomonas aeruginosa. This wide distribution of a complex biosynthetic pathway is very unusual, and provides an insight into how a pathway for an antibiotic can be transferred between diverse bacteria.

microbiology↗

bioRxiv2017

Prevalence of dual clarithromycin and metronidazole resistance in Helicobacter pylori estimated by molecular methods in Santiago, Chile

BackgroundCurrent available treatments for Helicobacter pylori eradication are chosen according to local clarithromycin and metronidazole resistance prevalence. The aim of this study was to estimate, by means of molecular methods, both clarithromycin and metronidazole resistance in gastric mucosa from patients infected with H.pylori.\n\nMethodsA total of 191 DNA samples were analyzed. DNA was purified from gastric mucosa obtained from patients who underwent an upper gastrointestinal endoscopy at an university hospital from Santiago, Chile, between 2011 and 2014. H.pylori was detected by real-time PCR. A 5exonuclease assay was developed to detect A2142G and A2143G mutations among Hpylori-positive samples. rdxA gene was sequenced in samples harboring A2142G and A2143G mutations in order to detect mutations that potentially confer dual clarithromycin and metronidazole resistance.\n\nResultsNinety-three (93) out of 191 DNA samples obtained from gastric mucosa were H. pylori-positive (48.7%). Clarithromycin-resistance was detected in 29 samples (31.2% [95%CI 22.0%-41.6%]). The sequencing of rdxA gene revealed that two samples harbored truncating mutations in rdxA, one sample had an in-frame deletion, and 11 had amino acid changes that likely cause metronidazole resistance.\n\nConclusionsWe estimated a prevalence of clarithomycin-resistance of 31.8% in Santiago, Chile. The proportion of dual clarithromycin and metronidazole resistance could be, at least, 15.0%. Our results require further confirmation. Nevertheless, they are significant as an initial approximation in re-evaluating the guidelines for H.pylori eradication currently used in Chile.

microbiology↗

bioRxiv2017

Division of labor during biofilm matrix production

Organisms as simple as bacteria can engage in complex collective actions, such as group motility and fruiting body formation. Some of these actions involve a division of labor, where phenotypically specialized clonal subpopulations, or genetically distinct lineages cooperate with each other by performing complementary tasks. Here, we combine experimental and computational approaches to investigate potential benefits arising from division of labor during biofilm matrix production. We show that both phenotypic and genetic strategies for a division of labor can promote collective biofilm formation in the soil bacterium Bacillus subtilis. In this species, biofilm matrix consists of two major components; EPS and TasA. We observed that clonal groups of B. subtilis phenotypically segregate into three subpopulations composed of matrix non-producers, EPS-producers, and generalists, which produce both EPS and TasA. This incomplete phenotypic specialization was outperformed by a genetic division of labor, where two mutants, engineered as specialists, complemented each other by exchanging EPS and TasA. The relative fitness of the two mutants displayed a negative frequency dependence both in vitro and on plant roots, with strain frequency reaching a stable equilibrium at 30% TasA-producers, corresponding exactly to the population composition where group productivity is maximized. Using individual-based modelling, we show that asymmetries in strain ratio can arise due to differences in the relative benefits that matrix compounds generate for the collective; and that genetic division of labor can be favored when it breaks metabolic constraints associated with the simultaneous production of two matrix components.\n\nHighlights- matrix components EPS and TasA are costly public goods in B. subtilis biofilms\n\n- genetic division of labor using {Delta}eps and {Delta}tasA fosters maximal biofilm productivity\n\n- {Delta}eps and {Delta}tasA cooperation is evolutionary stable in laboratory and ecological systems\n\n- costly metabolic coupling of public goods favors genetic division of labor

microbiology↗

bioRxiv2017

Comprehensive analysis of Verticillium nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion

BackgroundThe vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop.\n\nResultsThe secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed.\n\nConclusionsSeveral putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.

microbiology↗

bioRxiv2017

Characterizing co-expression networks underpinning maize stalk rot virulence in Fusarium verticillioides through computational subnetwork module analyses

Fusarium verticillioides is recognized as an important stalk rot pathogen of maize worldwide, but our knowledge of genetic mechanisms underpinning this pathosystem is limited. Previously, we identified a striatin-like protein Fsr1 that plays an important role in stalk rot. To further characterize transcriptome networks downstream of Fsr1, we performed next-generation sequencing (NGS) to investigate relative read abundance and also to infer co-expression networks utilizing the preprocessed expression data through partial correlation. We used a probabilistic pathway activity inference strategy to identify functional subnetwork modules likely involved in virulence. Each subnetwork modules consisted of multiple correlated genes with coordinated expression patterns, but the collective activation levels were significantly different in F. verticillioides wild type versus the mutant. We also identified putative hub genes from predicted subnetworks for functional validation and network robustness studies through mutagenesis, virulence and qPCR studies. Our results suggest that these genes are important virulence genes that regulate the expression of closely correlated genes, demonstrating that these are important hubs of their respective subnetworks. Lastly, we used key F. verticillioides virulence genes to computationally predict a subnetwork of maize genes that potentially respond to fungal genes by applying cointegration-correlation-expression strategy.

microbiology↗

bioRxiv2017

The essential genome of Escherichia coli K-12

Transposon-Directed Insertion-site Sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries and therefore it remains unclear whether the two methodologies are comparable. To address this, a high density transposon library was constructed in Escherichia coli K-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false positive identification of essential gene candidates, statistical data analysis included corrections for both gene length and genome length. Through this analysis new essential genes and genes previously incorrectly designated as essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects and fine resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis datasets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.\n\nIMPORTANCEIncentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes in E. coli, we constructed a very high density transposon mutant library. Initial automated analysis of the resulting data revealed many discrepancies when compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high density TraDIS sequencing data for each putative essential gene for the model laboratory organism, Escherichia coli. This paper is important because it provides a better understanding of the essential genes of E. coli, reveals the limitations of relying on automated analysis alone and a provides new standard for the analysis of TraDIS data.

microbiology↗