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bioRxiv2020

A mysterious 80 nm amoeba virus with a near-complete "ORFan genome" challenges the classification of DNA viruses

Here we report the discovery of Yaravirus, a new lineage of amoebal virus with a puzzling origin and phylogeny. Yaravirus presents 80 nm-sized particles and a 44,924 bp dsDNA genome encoding for 74 predicted proteins. More than 90% (68) of Yaravirus predicted genes have never been described before, representing ORFans. Only six genes had distant homologs in public databases: an exonuclease/recombinase, a packaging-ATPase, a bifunctional DNA primase/polymerase and three hypothetical proteins. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially representing a novel capsid protein with no significant homology with NCLDV major capsid proteins but with a predicted double-jelly roll domain. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists. Significance statementMost of the known viruses of amoeba have been seen to share many features that eventually prompted authors to classify them into common evolutionary groups. Here we describe Yaravirus, an entity that could represent either the first isolated virus of Acanthamoeba spp. out of the group of NCLDVs or, in alternative evolutive scenario, it is a distant and extremely reduced virus of this group. Contrary to what is observed in other isolated viruses of amoeba, Yaravirus is not represented by a large/giant particle and a complex genome, but at the same time carries an important number of previously undescribed genes, including one encoding a novel major capsid protein. Metagenomic approaches also testified for the rarity of Yaravirus in the environment.

microbiology↗

bioRxiv2020

Molecular characterization of a novel cytorhabdovirus with a unique genomic organization infecting yerba mate (Ilex paraguariensis) in Argentina

The genome of a novel rhabdovirus was detected in yerba mate (Ilex paraguariensis St. Hil.). The newly identified virus, tentatively named yerba mate virus A (YmVA), has a genome of 14,961 nucleotides. Notably, eight open reading frames were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, including two novel accessory genes, in the order 3-N-P-3-4-M-G-L-8-5. Sequence identity of the encoded proteins as well as phylogenetic analysis suggest that YmVA is a new member of the genus Cytorhabdovirus, family Rhabdoviridae. YmVA unique genomic organization and phylogenetic relationships indicate that this virus likely represents a distinct evolutionary lineage within the cytorhabdoviruses.

microbiology↗

bioRxiv2020

Horizontal gene transfer in the human and skin commensal Malassezia: a bacterially-derived flavohemoglobin is required for NO resistance and host interaction

The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions. Malassezia are the most abundant fungal skin inhabitant of warm-blooded animals, and have been implicated in skin diseases and systemic disorders, including Crohns disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within the Malassezia genus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in M. sympodialis, we demonstrated that bacterially-derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNAseq analysis revealed that endogenous accumulation of nitric oxide resulted in upregulation of genes involved in stress response, and downregulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure of Malassezia flavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent of Malassezia flavohemoglobin. Lastly, we identified additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin. Significance statementMalassezia species are the main fungal components of the mammalian skin microbiome and are associated with a number of skin disorders. Recently, Malassezia has also been found in association with Crohns Disease and with pancreatic cancer. The elucidation of the molecular bases of skin adaptation by Malassezia is critical to understand its role as commensal and pathogen. In this study we employed evolutionary, molecular, biochemical, and structural analyses to demonstrate that the bacterially-derived flavohemoglobins acquired by Malassezia through horizontal gene transfer resulted in a gain of function critical for nitric oxide detoxification and resistance to nitrosative stress. Our study underscores horizontal gene transfer as an important force modulating Malassezia evolution and niche adaptation.

microbiology↗

bioRxiv2020

Comparative genomics reveals different population structures associated with host and geographic origin in antimicrobial-resistant Salmonella enterica

Genetic variation in a pathogen, including the causative agent of salmonellosis, Salmonella enterica, can occur as a result of eco-evolutionary forces triggered by dissimilarities of ecological niches. Here, we applied comparative genomics to study 90 antimicrobial resistant (AMR) S. enterica isolates from bovine and human hosts in New York state and Washington state to understand host- and geographic-associated population structure. Results revealed distinct presence/absence profiles of functional genes and pseudogenes (e.g., virulence genes) associated with bovine and human isolates. Notably, bovine isolates contained significantly more transposase genes but fewer transposase pseudogenes than human isolates, suggesting the occurrence of large-scale transposition in genomes of bovine and human isolates at different times. The high correlation between transposase genes and AMR genes, as well as plasmid replicons, highlights the potential role of horizontally transferred transposons in promoting adaptation to antibiotics. By contrast, a number of potentially geographic-associated single-nucleotide polymorphisms (SNPs), rather than geographic-associated genes, were identified. Interestingly, 38% of these SNPs were in genes annotated as cell surface protein-encoding genes, including some essential for antibiotic resistance and host colonization. Overall, different evolutionary forces and limited recent inter-population transmission appear to shape AMR S. enterica population structure in different hosts and geographic origins. Originality/Significance StatementSalmonella enterica, which is the causative agent of salmonellosis, poses a growing public health concern due to the emergence and spread of antimicrobial resistant (AMR) strains. The mechanisms underlying the population structure associated with different hosts and geographic origins of AMR S. enterica are underexplored due to limited genome-wide studies assessing the impact of ecological niches on genetic variations. By employing comparative genomics, our study provided insights into the genomic profiles of AMR S. enterica associated with two distinct hosts and two distant geographic locations, improving the mechanistic understanding of how bacterial population structure is shaped by different ecological niches. Our findings have broad implications for elucidating the impact of ecological and evolutionary forces on the adaptation, antimicrobial resistance, and pathogenicity of bacteria. Also, specific genetic markers we identified may help predict host or geographic origin of AMR Salmonella isolates, which could benefit the source tracking (e.g., host and geographic origins) of human disease cases and contamination events caused by AMR S. enterica.

microbiology↗

bioRxiv2020

Genetic regulation of the bacterial omega-3 polyunsaturated fatty acid biosynthesis pathway

A characteristic among many marine Gammaproteobacteria is the biosynthesis and incorporation of omega-3 polyunsaturated fatty acids into membrane phospholipids. Biosynthesis of eicosapentaenoic (EPA) and/or docosahexaenoic (DHA) acids is accomplished using a polyketide/fatty acid synthase mechanism encoded by a set of five genes pfaABCDE. This unique fatty acid synthesis (FAS) pathway co-exists with the canonical Type II dissociated fatty acid synthesis pathway, which is responsible for the biosynthesis of saturated, monounsaturated, and hydroxylated fatty acids used in phospholipid and lipid A biosynthesis. In this work, a genetic approach was undertaken to elucidate genetic regulation of the pfa genes in the model marine bacterium Photobacterium profundum SS9. Using a reporter gene fusion, we showed that expression of the pfa operon is down regulated in response to exogenous fatty acids, particularly long chain monounsaturated fatty acids. This regulation occurs independently of the canonical fatty acid regulators, FabR and FadR, present in P. profundum SS9. Transposon mutagenesis and screening of a library of mutants identified a novel transcriptional regulator, which we have designated pfaF, to be responsible for the observed regulation of the pfa operon in P. profundum SS9. Gel mobility shift and DNase I footprinting assays confirmed that PfaF binds the pfaA promoter and identified the PfaF binding site. ImportanceThe production of polyunsaturated fatty acids (PUFA) by marine Gammaproteobacteria, particularly those from deep-sea environments, has been known for decades. These unique fatty acids are produced by a polyketide-type mechanism and subsequently incorporated into the phospholipid membrane. While much research has focused on the biosynthesis genes, their products and the phylogenetic distribution of these gene clusters, no prior studies have detailed the genetic regulation of this pathway. This study describes how this pathway is regulated under various culture conditions and has identified and characterized a fatty acid responsive transcriptional regulator specific to the PUFA biosynthesis pathway.

microbiology↗

bioRxiv2020

Deep Sequencing Reveals Compartmentalized HIV-1 in the Semen of Men with and without STI-associated Urethritis

Concurrent sexually transmitted infections (STI) can increase the probability of HIV-1 transmission primarily by increasing the viral load present in semen. In this study, we explored the relationship of HIV-1 in blood and seminal plasma in the presence and absence of urethritis and after treatment of the concurrent STI. Primer ID deep sequencing of the V1/V3 region of the HIV-1 env gene was done for paired blood and semen samples from ART-naive men living in Malawi with (n = 19) and without (n = 5) STI-associated urethritis; for a subset of samples full length env genes were generated for sequence analysis and to test entry phenotype. Cytokine concentrations in the blood and semen were also measured, and a reduction in the levels of pro-inflammatory cytokines was observed following STI treatment. We observed no difference in the prevalence of diverse compartmentalized semen-derived lineages in men with or without STI-associated urethritis, and these viral populations were largely stable during STI treatment. Clonal amplification of one or a few viral sequences accounted for nearly 50% of the viral population indicating a recent bottleneck followed by limited viral replication. We documented a case of superinfection where the new strain was restricted to the genital tract. We conclude that the male genital tract is a site where virus can be brought in from the blood, where localized sustained replication can occur, where a superinfecting strain can persist, and where specific genotypes can be amplified perhaps initially by cellular proliferation but further by limited viral replication. ImportanceHIV-1 is a sexually transmitted infection that co-exists with other STIs. Here we examine the impact of a concurrent STI resulting in urethritis on the HIV-1 population within the male genital tract. We found that viral populations remain largely stable even with treatment of the STI. These results show that viral populations within the male genital tract are defined by factors beyond transient inflammation associated with a concurrent STI.

microbiology↗

bioRxiv2020

Delivery mode impacts newborn gut colonization efficiency

Delivery mode is the variable with the greatest influence on the infant gut microbiome composition in the first few months of life. Children born by Cesarean section (C-section) lack species from the Bacteroides genus in their gut microbial community, and this difference can be detectable until 6-18 months of age. One hypothesis is that these differences stem from lack of exposure to the maternal vaginal microbiome, as children born by C-section do not pass through the birth canal; however, Bacteroides species are not common members of the vaginal microbiome, thus this explanation seems inadequate. Here, we set out to re-evaluate this hypothesis by collecting rectal and vaginal samples before delivery from 73 mothers with paired stool from their infants in the first two weeks of life. We compared microbial profiles of infants born by planned, pre-labor C-section to those born by emergent, post-labor surgery (where the child was in the birth canal, but eventually delivered through an abdominal incision), and found no significant differences in the microbiome between these two groups. Both groups showed the characteristic signature lack of Bacteroides species, despite their difference in exposure to the birth canal. Surprisingly, this signature was only evident in samples from week two of life, but not in the first week. Children born by C-section often had high abundance of Bacteroides in their first few days of life, but these were not stable colonizers of the infant gut, as they were not detectable by week two. Finally, we used metagenomic sequencing to compare microbial strains in maternal vaginal and rectal samples and samples from their infants; we found evidence for mother-to-child transmission of rectal rather than vaginal strains. These results challenge birth canal exposure as the dominant factor in infant gut microbiome establishment and implicate colonization efficiency rather than exposure as a dictating factor of the newborn gut microbiome composition.

microbiology↗

bioRxiv2020

Biofilms mechanically damage epithelia by buckling.

During chronic infections and in microbiota, bacteria predominantly colonize their hosts as multicellular structures called biofilms. Despite their ubiquity in vivo, we still lack a basic understanding of how they interact with biological tissues, and ultimately how they influence host physiology. A common assumption is that biofilms interact with their hosts biochemically. However, the contributions of mechanics, while being central to the process of biofilm formation, have been vastly overlooked as a factor influencing host physiology. Specifically, how biofilms form on soft, tissue-like materials remains unknown. Here we show that biofilms can deform soft substrates by transmission of internally-generated mechanical stresses. We found that biofilms from both Vibrio cholerae and Pseudomonas aeruginosa can induce large deformations of soft synthetic hydrogels. Using a combination of mechanical measurements and mutants in matrix components, we found that biofilms deform their substrates by simultaneous buckling and adhesion. Specifically, mechanical constraints opposing growth causes biofilm buckling, while matrix components maintaining surface adhesion transmit buckling forces to the substrate. Finally, we demonstrate that biofilms can generate sufficient mechanical stress to deform and disrupt soft epithelial cell monolayers, suggesting that these forces can damage a host independently of typical virulence factors. Altogether, our results illustrate that forces generated by bacterial communities play an important role not only in biofilm morphogenesis but also in host physiology, suggesting a mechanical mode of infection.

microbiology↗

bioRxiv2020

Transposon mobilization during infection promotes drug resistance in the human fungal pathogen Cryptococcus deneoformans

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeast-like fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37{degrees} compared to 30{degrees} in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNAi pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30{degrees} to the level observed at 37{degrees}. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host. SIGNIFICANCE STATEMENTThe incidence of infections due to fungal pathogens has dramatically increased in the past few decades with similar increases in human populations with weakened or suppressed immune systems. Understanding the mechanisms by which organisms rapidly adapt during human infection to enhance virulence and evolve drug resistance is important for developing effective treatments. We find that transposon mobilization in the human pathogen Cryptococcus causes genomic mutations in a murine model of infection and promotes resistance to antifungal drugs in vitro. Thermotolerance is a key virulence determinant for pathogenic fungi during the environment-to-host transition, and we demonstrate that a temperature increase is sufficient to trigger transposon mobilization in vitro. The link between temperature stress and transposon-associated mutations may significantly impact adaptation to the host during infection, including the acquisition of drug resistance.

microbiology↗

bioRxiv2020

Characterization of Escherichia coli ExbD protein modification in vivo

The TonB system of Escherichia coli couples the protonmotive force of the cytoplasmic membrane to active transport of nutrients across the outer membrane. In the cytoplasmic membrane, this system consists of three known proteins, TonB, ExbB, and ExbD. ExbB and ExbD appear to harvest the protonmotive force and transmit it to TonB, which then makes direct physical contact with TonB-dependent active transport proteins in the outer membrane. Using two-dimensional gel electrophoresis, we found that ExbD exists as two different species with the same apparent molecular mass but with different pIs. The more basic ExbD species was consistently present, while the more acidic species arose when cells were starved for iron by the addition of iron chelators. The cause of the modification was, however, more complex than simple iron starvation. Absence of either TonB or ExbB protein also gave rise to modified ExbD under iron-replete conditions where the wild-type strain exhibited no ExbD modification. The effect of the tonB or exbB mutations were not entirely due to iron limitation since an equally iron-limited aroB mutation did not replicate the ExbD modification. This constitutes the first report of in vivo modification for any of the TonB system proteins.

microbiology↗

bioRxiv2020

Insights into Cross-species Evolution of Novel Human Coronavirus 2019-nCoV and Defining Immune Determinants for Vaccine Development

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak in the city of Wuhan, China during December 2019, has now spread to various countries across the globe triggering a heightened containment effort. This human pathogen is a member of betacoronavirus genus carrying 30 kilobase of single positive-sense RNA genome. Understanding the evolution, zoonotic transmission, and source of this novel virus would help accelerating containment and prevention efforts. The present study reported detailed analysis of SARS-CoV-2 genome evolution and potential candidate peptides for vaccine development. This new coronavirus genotype might have been evolved from a bat-coronavirus by accumulating non-synonymous mutations, indels, and recombination events. Structural proteins Spike (S), and Membrane (M) had extensive mutational changes, whereas Envelope (E) and Nucleocapsid (N) proteins were very conserved suggesting differential selection pressures exerted on SARS-CoV-2 during evolution. Interestingly, SARS-CoV-2 Spike protein contains a 39 nucleotide sequence insertion relative to SARS-like bat-SL-CoVZC45/2017. Furthermore, we identified eight high binding affinity (HBA) CD4 T-cell epitopes in the S, E, M and N proteins, which can be commonly recognized by HLA-DR alleles of Asia and Asia-Pacific Region population. These immunodominant epitopes can be incorporated in universal subunit SARS-CoV-2 vaccine. Diverse HLA types and variations in the epitope binding affinity may contribute to the wide range of immunopathological outcomes of circulating virus in humans. Our findings emphasize the requirement for continuous surveillance of SARS-CoV-2 strains in live animal markets to better understand the viral adaptation to human host and to develop practical solutions to prevent the emergence of novel pathogenic SARS-CoV-2 strains.

microbiology↗

bioRxiv2020

Analysis of metagenome-assembled genomes from the mouse gut microbiota reveals distinctive strain-level characteristics

The laboratorial mouse harbors a unique gut microbiota with potential value for human microbiota-associated studies. Mouse gut microbiota has been explored at the genus and species levels, but features rarely been showed at the strain level. The identification of 833,051 and 658,438 nonredundant genes of faeces and gut content samples from the laboratorial C57/BL mice showed over half of these genes were newly found compared to the previous mouse gut microbial gene catalogue. Metagenome-assembled genomes (MAGs) was used to reconstruct 46 nonredundant MAGs belonging to uncultured specieses. These MAGs included members across all phyla in mouse gut (i.e. Firmicutes, Bacteroidetes, Proteobacteria, Deferribacteres, Verrucomicrobia, and Tenericutes) and allowed a strain-level delineating of the mouse gut microbiota. Comparison of MAGs with human gut colonies revealed distinctive genomic and functional characteristics of mouses Bacteroidetes and Firmicutes strains. Genomic characteristics of rare phyla in mouse gut microbiota were demonstrated by MAG approach, including strains of Mucispirillum schaedleri, Parasutterella excrementihominis, Helicobacter typhlonius, and Akkermansia muciniphila. ImportanceThe identification of nonredundant genes suggested the existence of unknown microbes in the mouse gut samples. The metagenome-assembled genomes (MAGs) instantiated the specificity of mouse gut species and revealed an intestinal microbial correlation between mouse and human. The cultivation of faeces and gut contents sample validated the existence of MAGs and estimate their accuracy. Full-length 16S ribosomal RNA gene sequencing enabled taxonomic characterization. This study highlighted a unique ecosystem in the gut of laboratorial mice that obviously differed with the human gut flora at the strain level. The outcomes may be beneficial to researches based on laboratorial mouse models.

microbiology↗

bioRxiv2020

The immune response and microbiota profiles during co-infection with P. vivax and soil-transmitted helminths

Co-infection with soil-transmitted helminths (STH) and Plasmodium spp. parasites is a common occurrence in tropical low-income countries, but the consequences of this interaction remain poorly understood. Here, we performed a multi-omic analysis on peripheral blood and fecal samples from 130 individuals in Tierralta, Cordoba, Colombia who were infected with P. vivax alone (n = 33), co-infected with P. vivax and STH (n = 27), infected with STH alone (n = 39) or were infected with neither P. vivax nor STH (n = 31). In addition to Complete Blood Count (CBC) with differential, transcriptional profiling of peripheral blood samples was performed by RNA-Seq, fecal microbial communities were determined by 16S ribosomal RNA gene sequencing and circulating cytokine levels were measured by bead-based immunoassays. Differences in blood cell counts were driven primarily by P. vivax infection, including an increased percentage of neutrophils that was associated with a transcriptional signature of neutrophil activation in the blood. P. vivax infection was also associated with increased levels of IL-6, IL-8 and IL-10, and these cytokine levels were not affected by STH co-infection. Surprisingly, P. vivax infection was more strongly associated with changes in the microbiome than STH infection. Children infected with P. vivax exhibited elevated Bacteroides and reduced Prevotella and Clostridiaceae, but these differences were not observed in individuals co-infected with STH. We also observed that P. vivax parasitemia was higher in the STH-infected population. When we used machine learning to identify the most important predictors of P. vivax parasite burden from all measured variables, bacterial taxa were the strongest predictors of parasitemia levels. In contrast, circulating TGF-{beta} was the strongest predictor of T. trichiura egg burden. This study provides unexpected evidence that the gut microbiota may have a stronger link with P. vivax than with STH infection.

microbiology↗

bioRxiv2020

A Prokaryotic Membrane Sculpting BAR Domain Protein

Bin/Amphiphysin/RVS (BAR) domain proteins belong to a superfamily of coiled-coil proteins influencing membrane curvature in eukaryotes and are associated with vesicle biogenesis, vesicle-mediated protein trafficking, and intracellular signaling. Here we report the first prokaryotic BAR domain protein, BdpA, from Shewanella oneidensis MR-1, known to produce redox-active membrane vesicles and micrometer-scale outer membrane extensions (OMEs). BdpA is required for uniform size distribution of membrane vesicles and scaffolding OMEs into a consistent diameter and curvature. Cryogenic transmission electron microscopy reveals a strain lacking BdpA produces lobed, disordered OMEs rather than membrane tubes produced by the wild type strain. Overexpression of BdpA promotes OME formation during conditions where they are less common. Heterologous expression results in OME production in Marinobacter atlanticus and Escherichia coli. Based on the ability of BdpA to alter membrane curvature in vivo, we propose that BdpA and its homologs comprise a newly identified class of prokaryotic BAR (P-BAR) domains.

microbiology↗

bioRxiv2020

Evolution and variation of 2019-novel coronavirus

BackgroundThe current outbreak caused by novel coronavirus (2019-nCoV) in China has become a worldwide concern. As of 28 January 2020, there were 4631 confirmed cases and 106 deaths, and 11 countries or regions were affected. MethodsWe downloaded the genomes of 2019-nCoVs and similar isolates from the Global Initiative on Sharing Avian Influenza Database (GISAID and nucleotide database of the National Center for Biotechnology Information (NCBI). Lasergene 7.0 and MEGA 6.0 softwares were used to calculate genetic distances of the sequences, to construct phylogenetic trees, and to align amino acid sequences. Bayesian coalescent phylogenetic analysis, implemented in the BEAST software package, was used to calculate the molecular clock related characteristics such as the nucleotide substitution rate and the most recent common ancestor (tMRCA) of 2019-nCoVs. ResultsAn isolate numbered EPI_ISL_403928 showed different phylogenetic trees and genetic distances of the whole length genome, the coding sequences (CDS) of ployprotein (P), spike protein (S), and nucleoprotein (N) from other 2019-nCoVs. There are 22, 4, 2 variations in P, S, and N at the level of amino acid residues. The nucleotide substitution rates from high to low are 1{middle dot}05 x 10-2 (nucleotide substitutions/site/year, with 95% HPD interval being 6.27 x 10-4 to 2.72 x 10-2) for N, 5.34 x 10-3 (5.10 x 10-4, 1.28 x 10-2) for S, 1.69 x 10-3 (3.94 x 10-4, 3.60 x 10-3) for P, 1.65 x 10-3 (4.47 x 10-4, 3.24 x 10-3) for the whole genome, respectively. At this nucleotide substitution rate, the most recent common ancestor (tMRCA) of 2019-nCoVs appeared about 0.253-0.594 year before the epidemic. ConclusionOur analysis suggests that at least two different viral strains of 2019-nCoV are involved in this outbreak that might occur a few months earlier before it was officially reported.

microbiology↗

bioRxiv2020

Niche preference of Escherichia coli in a peri-urban pond ecosystem

Escherichia coli comprises of diverse strains with a large accessory genome, indicating functional diversity and the ability to adapt to a range of niches. Specific strains would display greatest fitness in niches matching their combination of phenotypic traits. Given this hypothesis, we sought to determine whether E. coli in a peri-urban pond and associated cattle pasture display niche preference. Samples were collected from water, sediment, aquatic plants, water snails associated with the pond as well as bovine feces from cattle in an adjacent pasture. Isolates (120) were obtained after plating on Membrane Lactose Glucuronide Agar (MLGA). We used the uidA and mutS sequences for all isolates to determine phylogeny by maximum likelihood, and population structure through gene flow analysis. PCR was used to allocate isolates to phylogroups and to determine the presence of pathogenicity / virulence genes (stxI, stxII, eaeA, hlyA, ST and LT). Antimicrobial resistance was determined using a disk diffusion assay for Tetracycline, Gentamicin, Ciprofloxacin, Meropenem, Ceftriaxone, and Azitrhomycin. Our results showed that isolates from water, sediment and water plants were similar by phylogroup distribution, virulence gene distribution and antibiotic resistance while both snail and feces populations were significantly different. Few of the feces isolates were significantly similar to aquatic ones, and most of the snail isolates were also different. Population structure analysis indicated three genetic backgrounds associated with bovine, snail and aquatic environments. Collectively these data support niche preference of E. coli isolates occurring in this ecosystem.

microbiology↗

bioRxiv2020

A plant pathogen utilizes effector proteins for microbiome manipulation

During colonization of their hosts, pathogens secrete effector proteins to promote disease development through various mechanisms. Increasing evidence shows that the host microbiome plays a crucial role in health, and that hosts actively shape their microbiomes to suppress disease. We hypothesized that pathogens evolved to manipulate host microbiomes to their advantage in turn. Here, we show that the fungal plant pathogen Verticillium dahliae utilizes effector proteins for niche colonization through selective manipulation of host microbiomes by suppressing microbes with antagonistic activities. Moreover, we show that effector proteins are similarly exploited for microbiome manipulation in the soil environment, where the fungus resides in absence of a host. In conclusion, we demonstrate that pathogens utilize effector proteins to modulate microbiome compositions and propose that their effector catalogs represent an untapped resource for novel antibiotics.

microbiology↗

bioRxiv2020

Expression and purification of p72 trimers as subunit vaccine candidate

The authors have withdrawn this manuscript because the result is invalid. We intend to revise it on our technical approach and interpretation of the results. The authors do not wish for this manuscript to be cited as a reference for this project. Please contact the corresponding author if you have any questions.

microbiology↗