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bioRxiv2020

Principles of dengue virus evolvability derived from genotype-fitness maps in human and mosquito cells

Dengue virus (DENV), an arbovirus infecting over 100 million annually, cycles between human and mosquito hosts1. Examining how DENV adapts to such different host environments could uncover principles of arbovirus transmission and emergence. Here we combine sequential passaging and ultra-deep sequencing to examine the sequence dynamics and fitness changes of DENV populations adapting to human and mosquito cells, identifying the contributions of beneficial and deleterious mutations in shaping the fitness landscape driving host-specific paths of viral adaptation. We find DENV phenotypic adaptation is best described by the collective fitness contributions of all the alleles present in the population. Accordingly, while increased fitness during adaptation to each host is driven by host-specific beneficial mutations, it is reduced by the consistently replenished genetic load of deleterious mutations. Of note, host-specific beneficial mutations are in discrete regions across the genome, revealing molecular mechanisms of adaptation. Some of these clusters comprise phenotypically redundant mutations that may provide evolutionary robustness to transmission bottlenecks. Our results also suggest DENV adaptation is facilitated through variation in intrinsically disordered protein regions while transmembrane and structured domains evolve under stronger biophysical constraints. Importantly, the adaptation strategies uncovered in our simple system mirror macro-evolutionary changes observed across DENV serotypes and Zika virus and may suggest general principles of evolvability in arbovirus evolution.

microbiology↗

bioRxiv2020

A hierarchical transcriptional network controls appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae

Rice blast is a pervasive and devastating disease that threatens rice production across the world. In spite of its importance to global food security, however, the underlying biology of plant infection by the blast fungus Magnaporthe oryzae remains poorly understood. In particular, it is unclear how the fungus elaborates a specialised infection cell, the appressorium, in response to surface signals from the rice leaf. Here, we report the identification of a network of temporally co-regulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We have functionally characterised this network of transcription factors and demonstrated the operation of a hierarchical transcriptional control system. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which represses hyphal-associated gene expression and simultaneously induces major physiological changes required for appressorium development, including cell cycle arrest, autophagic cell death, turgor generation and melanin biosynthesis. Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organisation, polarised exocytosis and effector gene expression necessary for plant tissue invasion.

microbiology↗

bioRxiv2020

Growth phase-dependent responses of an in vitro gut microbiome to metformin

In vitro gut microbiota models are often used to study drug-microbiome interaction. Similar to culturing individual microbial strains, the biomass accumulation of in vitro gut microbiota follows a logistic growth curve. Current studies on in vitro gut microbiome responses introduce drug stimulation during different growth stages, e.g. lag phase or stationary phase. However, in vitro gut microbiota in different growth phases may respond differently to same stimuli. Therefore, in this study, we used a 96-deep well plate-based culturing model (MiPro) to culture the human gut microbiota. Metformin, as the stimulus, was added at the lag, log and stationary phases of growth. Microbiome samples were collected at different time points for optical density and metaproteomic functional analysis. Results show that in vitro gut microbiota responded differently to metformin added during different growth phases, in terms of the growth curve, alterations of taxonomic and functional compositions. The addition of drugs at log phase leads to the greatest decline of bacterial growth. Metaproteomic analysis suggested that the strength of the metformin effect on the gut microbiome functional profile was ranked as lag phase > log phase > stationary phase. Our results showed that metformin added at lag phase resulted in a significantly reduced abundance of the Clostridiales order as well as an increased abundance of the Bacteroides genus, which was different from stimulation during the rest of the growth phase. Metformin also resulted in alterations of several pathways, including energy production and conversion, lipid transport and metabolism, translation, ribosomal structure and biogenesis. Our results indicate that the timing for drug stimulation should be considered when studying drug-microbiome interactions in vitro.

microbiology↗

bioRxiv2020

Activator protein-1 transactivation of the major immediate early locus is a determinant of cytomegalovirus reactivation from latency

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continue to evolve. We previously showed the HCMV-encoded G-protein coupled receptor US28 is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to wild type. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Further, AP-1 is critical for de-repression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1-independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency. Significance StatementHuman cytomegalovirus (HCMV) is a common pathogen that infects the majority of the population for life. This infection poses little threat in immunologically healthy individuals, but can be fatal in people with compromised immune systems. Our understanding of the mechanisms underlying latency and reactivation remains incomplete. Here, we show the cellular transcription factor, AP-1, is a key to regulating HCMV reactivation. Our findings reveal AP-1 binding to the major immediate early enhancer/promoter is critical for switching this locus from one that is repressed during latency to one that is highly active following reactivation. Our work provides a novel mechanism HCMV exploits to reactivate, highlighting AP-1 as a potential target to prevent HCMV reactivation.

microbiology↗

bioRxiv2020

Engineering isoprenoid quinone production in yeast

Isoprenoid quinones are bioactive molecules that include an isoprenoid chain and a quinone head. They are traditionally found to be involved in primary metabolism, where they act as electron transporters, but specialized isoprenoid quinones are also produced by all domains of life. Here, we report the engineering of a bakers yeast strain, Saccharomyces cerevisiae EPYFA3, for the production of isoprenoid quinones. Our yeast strain was developed through overexpression of the shikimate pathway in a well-established recipient strain (S. cerevisiae EPY300) where the mevalonate pathway is overexpressed. As a proof of concept, our new host strain was used to overproduce the endogenous isoprenoid quinone coenzyme Q6, resulting in a final four-fold production increase. EPYFA3 represents a valuable platform for the heterologous production of high value isoprenoid quinones. EPYFA3 will also facilitate the elucidation of isoprenoid quinone biosynthetic pathways. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/932020v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@dcb86borg.highwire.dtl.DTLVardef@cf9a11org.highwire.dtl.DTLVardef@18c00aaorg.highwire.dtl.DTLVardef@7fd429_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

Biotic and abiotic factors predict the biogeography of soil microbes in the Serengeti

Field-based observational research is the first step in understanding the factors that predict the biogeography and community structure of soil microbes. The Serengeti National Park in Tanzania is an ideal location for this type of research because active volcanoes generate strong environmental gradients due to ash deposition and a rain shadow. Also, as one of the last remaining naturally grazed ecosystems on Earth, the Serengeti provides insights about the influence of herbivory on microbial communities. We used 16S rRNA amplicons to characterize bacterial and archaeal communities in soils from a 13-year herbivore removal experiment to study the influence of environmental factors and grazing on the natural distribution of soil microbes. We collected soil samples from seven sites, each with three naturally grazed plots and three plots that were fenced to prevent grazing by large mammalian herbivores. Soil fertility (phosphorus, nitrogen, iron, calcium, organic matter), texture, and pH were measured at each plot. Beta diversity of bacterial and archaeal communities was most strongly correlated with soil texture (R2 = 32.4%). The abundance of many operational taxonomic units (OTUs) were correlated with soil texture, and the evenness of taxa within samples increased with fine-textured soil. Removal of grazing shifted community structure, with 31 OTUs that were significant indicator taxa of the ungrazed treatment and three OTUs that were significant indicators of the grazed treatment. ImportanceOur results show that in this regional scale study, soil texture was the best environmental predictor, and grazing by large mammals also structures bacterial and archaeal communities. When large mammals are removed, as humans have been doing for millenia, there are cascading effects into the microbial world that can influence ecosystem functions like carbon and nitrogen cycles. These empirical findings from a natural tropical savannah can help inform models of the global distribution and function of soil microbes.

microbiology↗

bioRxiv2020

Enterococcus faecalis manganese exporter MntE alleviates manganese toxicity and is required for mouse gastrointestinal colonization

Bacterial pathogens encounter a variety of nutritional environments in the human host, including nutrient metal restriction and overload. Uptake of manganese (Mn) is essential for Enterococcus faecalis growth and virulence; however, it is not known how this organism prevents Mn toxicity. In this study, we examine the role of the highly conserved MntE transporter in E. faecalis Mn homeostasis and virulence. We show that inactivation of mntE results in growth restriction in the presence of excess Mn, but not other metals, demonstrating its specific role in Mn detoxification. Upon growth in the presence of excess Mn, an mntE mutant accumulates intracellular Mn, iron (Fe), and magnesium (Mg), supporting a role for MntE in Mn and Fe export, and a role for Mg in offsetting Mn toxicity. Growth of the mntE mutant in excess Fe also results in increased levels of intracellular Fe, but not Mn or Mg, providing further support for MntE in Fe efflux. Inactivation of mntE in the presence of excess iron also results in the upregulation of glycerol catabolic genes and enhanced biofilm growth, and addition of glycerol is sufficient to augment biofilm growth for both the mntE mutant and its wild type parental strain, demonstrating that glycerol availability significantly enhances biofilm formation. Finally, we show that mntE contributes to infection of the antibiotic-treated mouse gastrointestinal (GI) tract, suggesting that E. faecalis encounters excess Mn in this niche. Collectively, these findings demonstrate that the manganese exporter MntE plays a crucial role in E. faecalis metal homeostasis and virulence.

microbiology↗

bioRxiv2020

Lateral gene transfer drives metabolic flexibility in the anaerobic methane oxidising archaeal family Methanoperedenaceae

Anaerobic oxidation of methane (AOM) is an important biological process responsible for controlling the flux of methane into the atmosphere. Members of the archaeal family Methanoperedenaceae (formerly ANME-2d) have been demonstrated to couple AOM to the reduction of nitrate, iron, and manganese. Here, comparative genomic analysis of 16 Methanoperedenaceace metagenome-assembled genomes (MAGs), recovered from diverse environments, revealed novel respiratory strategies acquired through lateral gene transfer (LGT) events from diverse archaea and bacteria. Comprehensive phylogenetic analyses suggests that LGT has allowed members of the Methanoperedenaceae to acquire genes for the oxidation of hydrogen and formate, and the reduction of arsenate, selenate and elemental sulfur. Numerous membrane-bound multi-heme c type cytochrome complexes also appear to have been laterally acquired, which may be involved in the direct transfer of electrons to metal oxides, humics and syntrophic partners. ImportanceAOM by microorganisms limits the atmospheric release of the potent greenhouse gas methane and has consequent importance to the global carbon cycle and climate change modelling. While the oxidation of methane coupled to sulphate by consortia of anaerobic methanotrophic (ANME) archaea and bacteria is well documented, several other potential electron acceptors have also been reported to support AOM. In this study we identify a number of novel respiratory strategies that appear to have been laterally acquired by members of the Methanoperedenaceae as they are absent in related archaea and other ANME lineages. Expanding the known metabolic potential for members of the Methanoperedenaceae provides important insight into their ecology and suggests their role in linking methane oxidation to several global biogeochemical cycles.

microbiology↗

bioRxiv2020

Brucella species circulating in wildlife in Serengeti ecosystem, Tanzania

BackgroundBrucellosis is a bacterial zoonosis of public health and economic importance world-wide. It affects a number of domestic animals, wildlife and humans. This study was carried out to determine circulating Brucella species in wildlife in Serengeti ecosystem using molecular techniques. MethodologyA total of 189 samples including EDTA blood, serum and amniotic fluid from buffalos, lions, wildebeest, impala, zebra and hyena that were collected in relation to different cross-sectional studies conducted in the Serengeti ecosystem in Tanzania were used. Multiplex polymerase chain reaction AMOS-PCR and quantitative Real-Time PCR (qPCR) targeting the genus specific surface protein bcsp31 gene and the insertion sequence IS711 element downstream of the alkB gene for B. abortus and BMEI1162 gene for B. melitensis were employed on the samples. ResultsResults indicated that out of 189 samples examined, 12 (6.4%) and 22 (11.6%) contained Brucella DNA as detected by AMOS-PCR and qPCR, respectively. Most of the positive samples were from lions (52.6%) and buffaloes (19.6%). Other animals that were positive included wildebeest, impala, zebra and hyena. Out of 22 positive samples, 16 (66.7%) were identified as B. abortus and the rest were B. melitensis. ConclusionDetection of zoonotic Brucella species in wildlife suggests that livestock and humans at the interface areas where there is high interaction are at risk of acquiring the infection. Therefore, public education to interrupt risky transmission practices is needed. The findings also shed light on the transmission dynamics around interface areas and the role of wildlife in transmission and maintenance of Brucella infection in the region.

microbiology↗

bioRxiv2020

Duplications drive diversity in Bordetella pertussis on an underestimated scale.

Bacterial genetic diversity is often described using solely base pair changes despite a wide variety of other mutation types likely being major contributors. Tandem duplications of genomic loci are thought to be widespread among bacteria but due to their often intractable size and instability, comprehensive studies of the range and genome dynamics of these mutations are rare. We define a methodology to investigate duplications in bacterial genomes based on read depth of genome sequence data as a proxy for copy number. We demonstrate the approach with Bordetella pertussis, whose insertion sequence element-rich genome provides extensive scope for duplications to occur. Analysis of genome sequence data for 2430 B. pertussis isolates identified 272 putative duplications, of which 94% were located at 11 hotspot loci. We demonstrate limited phylogenetic connection for the occurrence of duplications, suggesting unstable and sporadic characteristics. Genome instability was further described in-vitro using long read sequencing via the Nanopore platform. Clonally derived laboratory cultures produced heterogenous populations containing multiple structural variants. Short read data was used to predict 272 duplications, whilst long reads generated on the Nanopore platform enabled the in-depth study of the genome dynamics of tandem duplications in B. pertussis. Our work reveals the unrecognised and dynamic genetic diversity of B. pertussis and, as the complexity of the B. pertussis genome is not unique, highlights the need for a holistic and fundamental understanding of bacterial genetics.

microbiology↗

bioRxiv2020

TaxisPy: A Python-based Software for the Quantitative Analysis of Bacterial Chemotaxis

Several species of bacteria are able to modify their swimming behavior in response to chemical attractants or repellents. Methods for the quantitative analysis of bacterial chemotaxis such as quantitative capillary assays are tedious and time-consuming. Computer-based video analysis of swimming bacteria represents a valuable method to directly assess their chemotactic response. Even though multiple studies have used this approach to elucidate various aspects of the bacterial chemotaxis, to date, no computer software for such analyses is freely available. Here, we introduce TaxisPy, a Python-based software for the quantitative analysis of bacterial chemotaxis. The software comes with an intuitive graphical user interface and can be easily accessed through Docker on any operating system. Using a video of freely swimming cells as input, TaxisPy estimates the cultures average tumbling frequency over time. We demonstrate the utility of the software by assessing the effect of different concentrations of the attractant shikimate on the swimming behavior of Pseudomonas putida F1 and by capturing the adaptation process that Escherichia coli undergoes after being exposed to L-aspartate.

microbiology↗

bioRxiv2020

Bacteriophage SP01 Gene Product 56 (gp56) Inhibits Bacillus subtilis Cell Division by Interacting with DivIC/FtsL to Prevent Pbp2B/FtsW Recruitment

Previous work identified gp56, encoded by the lytic bacteriophage SP01, as responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here we show that expression of the predicted 9.3-kDa gene product 56 (gp56) of SP01 inhibits latter stages of B. subtilis cell division without altering FtsZ ring assembly. GFP-tagged gp56 localizes to the membrane at the site of division. While its localization permits recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analysis suggest that gp56 localization and activity depends on its interaction with mid-recruited proteins DivIC and/or FtsL. Together these data support a model where gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis. IMPORTANCEResearch over the past decades has uncovered bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. Phage factors that cause cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanism of several identified phage factors that inhibit cytokinesis remain unexplored, including gp56 of bacteriophage SP01 of Bacillus subtilis. Here, we show that unlike related published examples of phage inhibition of cyotkinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and block recruitment of proteins needed for the septal cell wall synthesis.

microbiology↗

bioRxiv2020

Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS and transplanted patients, and in congenitally infected newborns. Despite the availability of several antiviral drugs, their utility is limited by poor bioavailability, toxicity, and resistant strains emergence. Therefore, it is crucial to identify new targets of therapeutic intervention. The dimerization of HCMV DNA polymerase processivity factor UL44 plays an essential role in the viral life cycle being required for oriLyt-dependent DNA replication. We validated the existence of UL44 homodimers both in vitro and in living cells by a variety of approaches, including GST pulldown, thermal shift, FRET and BRET assays. Dimerization occurred with an affinity comparable to that of the UL54/UL44 interaction, and was impaired by amino acid substitutions at the dimerization interface. Subsequently, we performed an in-silico screening to select 18 small molecules (SMs) potentially interfering with UL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the 18 selected SMs led to the identification of four active SMs. The most active one also inhibited AD169 in plaque reduction assays, and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, treatment of infected cells specifically reduced viral gene expression starting from 48 h post infection, consistent with activity on viral DNA synthesis. Therefore, SMs inhibitors of UL44 dimerization could represent a new class of HCMV inhibitors, alternative to those targeting the DNA polymerase catalytic subunit or the viral terminase complex. IMPORTANCEHCMV is a ubiquitous infectious agent causing life-lasting infections in humans. HCMV primary infections and reactivation in non-immunocompetent individuals often result in life-threatening conditions. Antiviral therapy mainly targets the DNA polymerase catalytic subunit UL54 and is often limited by toxicity and selection of drug-resistant viral strains, making the identification of new targets of therapeutic intervention crucial for a successful management of HCMV infections. The significance of our work is in identifying the dimerization of the DNA polymerase processivity factor UL44 as an alternative antiviral target. We could show that full length UL44 dimerizes in a cellular context with high affinity and that such interaction could be targeted by small molecules, thus inhibiting the replication of several HCMV strains, including a drug-resistant mutant. Thus, our work could pave the way to the development of a new class of anti-HCMV compounds that act by targeting UL44 dimerization.

microbiology↗

bioRxiv2020

Severe acute respiratory syndrome-related coronavirus - The species and its viruses, a statement of the Coronavirus Study Group

The present outbreak of lower respiratory tract infections, including respiratory distress syndrome, is the third spillover, in only two decades, of an animal coronavirus to humans resulting in a major epidemic. Here, the Coronavirus Study Group (CSG) of the International Committee on Taxonomy of Viruses, which is responsible for developing the official classification of viruses and taxa naming (taxonomy) of the Coronaviridae family, assessed the novelty of the human pathogen tentatively named 2019-nCoV. Based on phylogeny, taxonomy and established practice, the CSG formally recognizes this virus as a sister to severe acute respiratory syndrome coronaviruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus and designates it as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To facilitate communication, the CSG further proposes to use the following naming convention for individual isolates: SARS-CoV-2/Isolate/Host/Date/Location. The spectrum of clinical manifestations associated with SARS-CoV-2 infections in humans remains to be determined. The independent zoonotic transmission of SARS-CoV and SARS-CoV-2 highlights the need for studying the entire (virus) species to complement research focused on individual pathogenic viruses of immediate significance. This research will improve our understanding of virus-host interactions in an ever-changing environment and enhance our preparedness for future outbreaks.

microbiology↗

bioRxiv2020

A novel factor essential for unconventional secretion of chitinase Cts1

Subcellular targeting of proteins is essential to orchestrate cytokinesis in eukaryotic cells. During cell division of Ustilago maydis, for example, chitinases must be specifically targeted to the fragmentation zone at the site of cell division to degrade remnant chitin and thus separate mother and daughter cells. Chitinase Cts1 is exported to this location via an unconventional secretion pathway putatively operating in a lock-type manner. The underlying mechanism is largely unexplored. Here, we applied a forward genetic screen based on UV mutagenesis to identify components essential for Cts1 export. The screen revealed a novel factor termed Jps1 lacking known protein domains. Deletion of the corresponding gene confirmed its essential role for Cts1 secretion. Localization studies demonstrated that Jps1 colocalizes with Cts1 in the fragmentation zone of dividing yeast cells. While loss of Jps1 leads to exclusion of Cts1 from the fragmentation zone and strongly reduced unconventional secretion, deletion of the chitinase does not disturb Jps1 localization. Yeast-two hybrid experiments suggest that the two proteins interact. In essence, we identified a novel component of unconventional secretion that functions in the fragmentation zone to enable export of Cts1. We hypothesize that Jps1 acts as an anchoring factor, supporting the proposed novel lock-type mechanism of unconventional secretion.

microbiology↗

bioRxiv2020

Canopy position has a stronger effect than tree species identity on phyllosphere bacterial diversity in a floodplain hardwood forest

The phyllosphere is a challenging microbial habitat in which microorganisms can flourish on organic carbon released by plant leaves but are also exposed to harsh environmental conditions. Here, we assessed the relative importance of canopy position - top, mid, and bottom at a height between 31 m and 20 m - and tree species identity for shaping the phyllosphere microbiome in a floodplain hardwood forest. Leaf material was sampled from three tree species - maple (Acer pseudoplatanus L.), oak (Quercus robur L.), and lime (Tilia cordata MILL.) - at the Leipzig canopy crane facility (Germany). Estimated bacterial species richness (Chao1) and bacterial abundances approximated by quantitative PCR of 16S rRNA genes exhibited clear vertical trends with a strong increase from the top to the mid and bottom position of the canopy. 30 Operational Taxonomic Units (OTUs) formed the core microbiome, which accounted for 77% of all sequence reads. These core OTUs showed contrasting trends in their vertical distribution within the canopy, pointing to different ecological preferences and tolerance to presumably more extreme conditions at the top position of the canopy. Co-occurrence analysis revealed distinct tree species-specific OTU networks, and 55-57% of the OTUs were unique to each tree species. Overall, the phyllosphere microbiome harbored surprisingly high fractions of Actinobacteria of up to 46%. Our results clearly demonstrate strong effects of the position in the canopy on phyllosphere bacterial communities in a floodplain hardwood forest and - in contrast to other temperate or tropical forests - a strong predominance of Actinobacteria.

microbiology↗

bioRxiv2020

Evidence of recombination in coronaviruses implicating pangolin origins of nCoV-2019

A novel coronavirus (nCoV-2019) was the cause of an outbreak of respiratory illness detected in Wuhan, Hubei Province, China in December of 2019. Genomic analyses of nCoV-2019 determined a 96% resemblance with a coronavirus isolated from a bat in 2013 (RaTG13); however, the receptor binding motif (RBM) of these two genomes share low sequence similarity. This divergence suggests a possible alternative source for the RBM coding sequence in nCoV-2019. We identified high sequence similarity in the RBM between nCoV-2019 and a coronavirus genome reconstructed from a viral metagenomic dataset from pangolins possibly indicating a more complex origin for nCoV-2019.

microbiology↗

bioRxiv2020

High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic Nanopore sequencing

The rise of antimicrobial resistant Neisseria gonorrhoeae is a significant public health concern. Against this background, rapid culture-independent diagnostics may allow targeted treatment and prevent onward transmission. We have previously shown metagenomic sequencing of urine samples from men with urethral gonorrhoea can recover near-complete N. gonorrhoeae genomes. However, disentangling the N. gonorrhoeae genome from metagenomic samples and robustly identifying antimicrobial resistance determinants from error-prone Nanopore sequencing is a substantial bioinformatics challenge. Here we demonstrate an N. gonorrhoeae diagnostic workflow for analysis of metagenomic sequencing data obtained from clinical samples using R9.4.1 Nanopore sequencing. We compared results from simulated and clinical infections with data from known reference strains and Illumina sequencing of isolates cultured from the same patients. We evaluated three Nanopore variant callers and developed a random forest classifier to filter called SNPs. Clair was the most suitable variant caller after SNP filtering. A minimum depth of 20x reads was required to confidently identify resistant determinants over the entire genome. Our findings show that metagenomic Nanopore sequencing can provide reliable diagnostic information in N. gonorrhoeae infection.

microbiology↗