Biology subjects

Temperton, B.

Publications and source records attributed to Temperton, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Biocontainment of phages inhibits bacterial clearance in micro niches

Phage therapy offers a promising solution to the antimicrobial resistance crisis. However, a major concern preventing the adoption of phage therapy is the potential for unintended consequences of phage release; both in regard to preventing the spread of phage resistance, and the proliferation of a non-endemic virus into the microbial ecosystem. Conditional replication (biocontainment) of phages through bioengineering may address these concerns, but the impact on bactericidal efficacy is unknown. Here, we created a biocontained T7 phage (T7{Delta}capsid) lacking the major structural capsid gene, gp10AB, that can only replicate on Escherichia coli strains expressing gp10AB in trans, and assessed its bactericidal efficacy compared with wild-type T7. Congruent with model predictions, T7{Delta}capsid was only able to clear a well-mixed culture of E. coli at a multiplicity of infection (MOI) of 10 or higher, whereas wild-type T7 prohibited growth at an MOI of 0.1. The reduction in efficacy was more evident in a complex structured environment within a microfluidic device, where phage success depends on its ability to penetrate a microbial niche via propagation. In this environment, T7{Delta}capsid was unable to propagate into the bacterial population and unlike wild-type T7, had no impact on the populations growth. This study shows that whilst biocontainment of phages may improve the biosafety of phage therapy, it comes at the cost of its propagation efficacy and niche penetration in relevant environments.

microbiology↗

bioRxiv2026

Lipopolysaccharide truncation and restoration drives a trade-off in resistance to two phages in Pseudomonas aeruginosa

Phage therapy is a promising treatment for multidrug resistant bacterial infections, and for patients no longer able to tolerate antibiotic treatments. A major challenge for phage therapy is emergent phage resistance, which target bacteria acquire by structurally modifying or masking phage receptors to prevent adsorption. Functionally diverse phage cocktails that target a broad range of receptors are less prone to resistance as there is a higher fitness cost associated with modifying multiple receptors. Expanding phage libraries with well-characterised phages that target a broad range of receptors would aid in timely and strategic design of functionally diverse phage cocktails. Here, we aimed to isolate phages targeting novel receptors by enriching wastewater samples on a Pseudomonas aeruginosa PAO1 {Delta}pilA {Delta}galU unmarked deletion mutant lacking O-antigen, outer core lipopolysaccharide (LPS) and type IV pili (T4P) - the three most common Pseudomonas phage receptors. This led to the isolation of a novel phage, named Vale. Vale was predicted to bind the LPS inner core as it could only infect strains with truncated LPS, suggesting that the outer core LPS blocks Vale from accessing the inner core. We identified a trade-oM in resistance to Vale and another phage, Tor, that targets the LPS outer core, mediated by host-derived LPS modifications. The PAO1 host evolved resistance to Tor by 100-200kb genomic deletions, which resulted in LPS truncation and sensitivity to Vale. Complete LPS restoration in the {Delta}pilA {Delta}galU mutant conferred resistance to Vale and sensitivity to Tor in two out of three replicates. Combined treatment with Tor and Vale delayed the emergence of resistance in PAO1 for at least three times longer than individual phage treatments. This study provides an example of how using phage receptors to strategically design phage cocktails can minimise the likelihood of emergent phage resistance. Graphical abstractA summary of the LPS modifications, genomic mutations and phage susceptibilities of Tor and Vale resistant mutants. "Parent strain" refers to PAO1 {Delta}hsdR. {Delta}pilA {Delta}galU refers to PAO1 {Delta}hsdR {Delta}pilA {Delta}galU. The parent strain gains resistance to Tor via LPS truncation associated with 100-200 kb genomic deletions, resulting in sensitivity to Vale. {Delta}pilA {Delta}galU gains resistance to Vale by restoring its LPS, conferring sensitivity to Tor in two out of three repeats. As resistance to one phage sensitises bacteria to the other, combined treatment with both phages suppresses phage resistance for longer than individual treatments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/700494v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1efee0forg.highwire.dtl.DTLVardef@f64bfeorg.highwire.dtl.DTLVardef@1f71130org.highwire.dtl.DTLVardef@1897bd6_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Arbitrium phages can manipulate each other's lysis - lysogeny decisions

Many viruses can switch between lytic replication and dormancy (or lysogeny). It was recently discovered that some viruses that infect bacteria (known as bacteriophage, or phage) employ peptide-based ("arbitrium") communication systems to optimise their lysis/lysogeny switch: high peptide concentrations signal a lack of susceptible hosts and trigger lysogeny, while low peptide concentrations signal an abundance of uninfected hosts and prompt lysis. Here we demonstrate that Arbitrium-phages belonging to different species and genera can influence each others infection dynamics by secreting similar communication peptides, leading to early lysogenisation of the signal-receiving phage, and elevated fitness of the signal-emitting phage. Antagonistic coevolution between signal emitting and signal receiving phages to manipulate each others infection behaviours may explain the rapid diversification of arbitrium systems and their frequent horizontal exchange to escape the noise of cross talk.

ecology↗

bioRxiv2025

Eliminating the type I restriction endonuclease from Pseudomonas aeruginosa PAO1 for optimised phage isolation

Phage therapy is a promising treatment for multidrug-resistant bacterial infections. Due to their high host specificity, phages must be matched to the target clinical strains. Efficiently identifying appropriate phages and producing sufficient titres for clinical use requires comprehensive phage libraries and multiple propagation hosts. An idealised system would use a highly promiscuous bacterial host to isolate a broader range of phages and streamline optimised phage production. Anti-phage defences constrain bacterial host promiscuity, such as restriction-modification systems that recognise and cleave foreign DNA. Here, the type I restriction endonuclease, HsdR, was deleted from Pseudomonas aeruginosa PAO1 to make a more promiscuous phage isolation and propagation host. Removal of this endonuclease more than doubled the efficiency of phage propagation and yielded seven times more phages from freshwater samples than wildtype PAO1 - an important step in producing an optimised P. aeruginosa strain for isolating and propagating phages for clinical phage therapy. Graphical abstract(Created in BioRender.com) [A] The type I R-M system comprises three subunits HsdS, HsdM and HsdR. One HsdS and two HsdM subunits form the methyltransferase, and the addition of two HsdR subunits to this complex forms the restriction endonuclease. [B] In the wildtype PAO1, the type I R-M system destroys phage DNA while protecting the host. The HsdS subunit of the restriction endonuclease binds to unmethylated recognition sequences. Then, HsdR translocates the DNA, pulling it together in both directions until it collides with another restriction endonuclease, cleaving the DNA and preventing phage proliferation. The methyltransferase protects the host DNA through the methylation of recognition sequences, preventing the restriction endonuclease from binding. [C] In {Delta}hsdR, the restriction endonuclease cannot form, preventing phage DNA cleavage and increasing phage proliferation. The methyltransferase is still active, so both host and phage DNA are methylated, providing phage progeny with protection from R-M systems in future hosts. O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/656992v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@9418f8org.highwire.dtl.DTLVardef@f3703dorg.highwire.dtl.DTLVardef@4dcde6org.highwire.dtl.DTLVardef@8d6fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Viral chronotypes and their role in shaping seasonal viral dynamics in the Western English Channel

Marine viruses are key players of ocean biogeochemistry, profoundly influencing microbial community ecology and evolution. Despite their importance, few studies have explored the temporal dynamics of viral genome abundances in marine environments. Viral dynamics are complex, influenced by multiple factors such as host population dynamics and environmental conditions. To disentangle the complexity of viral communities, we developed an unsupervised machine learning framework to classify viral genomes into "chronotypes" based on temporal abundance patterns. Analysing an inter-seasonal monthly time-series of surface viral metagenomes from the Western English Channel, we identified chronotypes and compared their functional and evolutionary profiles. Results revealed a consistent annual cycle with steep compositional changes from winter to summer and steadier transitions from summer to winter. Seasonal chronotypes were enriched in potential auxiliary metabolic genes like ferrochelatases and 2OG-Fe(II) oxygenases compared to non-seasonal types. Chronotypes clustered into four groups based on their correlation profiles with environmental parameters, primarily driven by temperature and nutrients. Viral genomes exhibited a rapid turnover of polymorphisms, akin to Red Queen dynamics. However, within seasonal chronotypes, some sequences exhibited annual polymorphism recurrence, which declined over a 16-month period, suggesting that a fraction of the seasonal viral populations evolve more slowly. Classification into chronotypes revealed viral genomic signatures linked to temporal patterns, likely reflecting metabolic adaptations to environmental fluctuations and host dynamics. This novel framework enables the identification of long-term trends in viral composition, environmental influences on genomic structure, and potential viral interactions.

microbiology↗

bioRxiv2024

Seasonal exometabolites are regulated by essential microbial metabolisms in the oligotrophic ocean

Predictions of how the biogeochemical reservoir of marine dissolved organic matter (DOM) will respond to future ocean changes require an improved understanding of the thousands of individual microbe-molecule interactions which regulate the transformation and fate of DOM. Bulk characterizations of organic matter can mask this complex network of interactions comprised of rich chemical and taxonomic diversity. Here, we present a three-year, depth-resolved time-series of the seasonal dynamics of the exometabolome and the bacterioplankton community at the Bermuda Atlantic Time-series Study (BATS) site. We find both time-series to be highly structured and compositionally distinct across sampling depths. Putative exometabolite identifications (gonyol, glucose 6-sulfate, succinate, and trehalose) indicate that at least a portion of the exometabolome contains rapidly remineralized, labile molecules. We hypothesize that apparent seasonal accumulation of these labile molecules could result from environmental conditions that alter community composition on a seasonal timescale and thus shift the relative proportions of microbial functions that produce and consume the substrates. Critically, we found the composition of seasonal DOM features was more stable interannually than the microbial community structure. By estimating redundancy of metabolic functions responsible for cycling these molecules in BATS metagenomes, we propose a paradigm whereby core microbial metabolisms, either those utilized by all or by a subset of marine microbes, are better predictors of DOM composition than microbial taxonomies. The molecular-level characterization of DOM achieved herein highlights the metabolic imprint of microbial activity in DOM composition and greatly enhances our understanding of the dynamics regulating Earths largest reservoir of organic carbon. Significance statementMarine dissolved organic matter (DOM) is a major carbon reservoir that acts as a critical control on Earths climate. DOM dynamics are largely regulated by a complex web of microbial interactions, but the mechanisms underpinning these processes are not well understood. In a three-year time-series, we found thousands of DOM molecules and microbial taxa exhibited seasonal patterns. Critically, the identity of the microbes was more variable between years than the composition of the DOM molecules. We suggest that shared metabolisms encoded by genes that conduct core microbial functions are responsible for the more stable composition of DOM. This work links DOM molecules with microbial biodiversity, and presents testable predictors of DOM composition in our changing oceans.

microbiology↗

bioRxiv2024

Heterotrophic prokaryotes internal carbon recycling compensates mismatches between phytoplankton production and heterotrophic prokaryotic consumption

Molecular observational tools are great for characterizing the composition and genetic endowment of microbial communities, but cannot measure fluxes, which are critical for the understanding of ecosystems. To overcome these limitations, we use a mechanistic inference approach to estimate dissolved organic carbon (DOC) production and consumption by phytoplankton operational taxonomic units (OTUs) and heterotrophic prokaryotic amplicon sequences variants (ASVs), and infer carbon fluxes between members of this microbial community from Western English Channel (WEC) time-series data. Our analyses focus on phytoplankton spring and summer blooms, as well as bacteria summer blooms. In spring blooms, phytoplankton DOC production exceeds heterotrophic prokaryotic consumption, but in bacterial summer blooms heterotrophic prokaryotes consume almost 3 times more DOC than produced by the phytoplankton. This mismatch is compensated by heterotrophic prokaryotic DOC release by death, presumably viral lysis. In both types of summer blooms, large amounts of the DOC liberated by heterotrophic prokaryotes are reused, i.e. internally recycled, and fluxes between different heterotrophic prokaryotes are at the same level as fluxes between phytoplankton and heterotrophic prokaryotes. Contextualized, internal recycling accounts for approximately 75% and 30% of the estimated net primary production (0.16 vs 0.22 and 0.08 vs 0.29 {micro}mol l-1 d-1) in bacteria and phytoplankton summer blooms, respectively, and thus represents a major component of the WEC carbon cycle. We conclude that internal recycling compensates mismatches between phytoplankton DOC production and heterotrophic prokaryotic consumption, and encourage future analyses on aquatic carbon cycles to consider fluxes between heterotrophic prokaryotes, i.e. internal recycling.

microbiology↗

bioRxiv2023

In field use of water samples for genomic surveillance of ISKNV infecting tilapia fish in Lake Volta, Ghana

Viral outbreaks are a constant threat to aquaculture, limiting production for better global food security. A lack of diagnostic testing and monitoring in resource-limited areas hinders the capacity to respond rapidly to disease outbreaks and to prevent viral pathogens becoming endemic in fisheries productive waters. Recent developments in diagnostic testing for emerging viruses, however, offers a solution for rapid in situ monitoring of viral outbreaks. Genomic epidemiology has furthermore proven highly effective in detecting viral mutations involved in pathogenesis and assisting in resolving chains of transmission. Here, we demonstrate the application of an in-field epidemiological tool kit to track viral outbreaks in aquaculture on farms with reduced access to diagnostic labs, and with non-destructive sampling. Inspired by the "lab in a suitcase" approach used for genomic surveillance of human viral pathogens and wastewater monitoring of COVID19, we evaluated the feasibility of real-time genome sequencing surveillance of the fish pathogen, Infectious spleen and kidney necrosis virus (ISKNV) in Lake Volta. Viral fractions from water samples collected from cages holding Nile tilapia (Oreochromis niloticus) with suspected ongoing ISKNV infections were concentrated and used as a template for whole genome sequencing, using a previously developed tiled PCR method for ISKNV. Mutations in ISKNV in samples collected from the water surrounding the cages matched those collected from infected caged fish, illustrating that water samples can be used for detecting predominant ISKNV variants in an ongoing outbreak. This approach allows for the detection of ISKNV and tracking of the dynamics of variant frequencies, and may thus assist in guiding control measures for the rapid isolation and quarantine of infected farms and facilities.

genomics↗

bioRxiv2022

Long-read powered viral metagenomics in the oligotrophic Sargasso Sea

In the summer months, the waters of the Sargasso Sea are nutrient-limited and strongly stratified, serving as a model system for the predicted warmer and nutrient-limited oceans of the Anthropocene. The dominant microorganisms of surface waters are key drivers of the global carbon cycle. However, the viruses of the Sargasso Sea that shape these host communities and influence host biogeochemical function are not well understood. Here, we apply a hybrid sequencing approach that combines short- and long reads to survey Sargasso Sea phage communities via metagenomics at the viral maximum (80m) and mesopelagic (200m) depths. Taxonomically, we identified 2,301 Sargasso Sea phage populations (~species-level taxonomy) across 186 genera. Over half of the phage populations lacked representation in other global ocean viral metagenomes, whilst 177 phage genera lacked representation in phage isolate databases. Viral fraction and cell-associated viral communities captured in short-read data were distinct and decoupled at both depths, possibly indicating low active lytic viral replication in the Sargasso Sea, with viral turnover occurring across periods longer than the sampling period of three days. Inclusion of long read data was critical for (1) the identification of 79 ecologically important and common viral genomes; (2) capturing the extent of viral genome microdiversity; and (3) enabling the recovery of hypervariable regions in viral genomes predicted to encode proteins involved in host recognition, DNA synthesis and DNA packaging. Host prediction was only possible for ~4% of viral populations. Genomes of phages known to infect Prochlorococcus and Pelagibacter were poorly represented in our data, supporting recent evidence of low infection levels in the dominant bacterial taxa of oligotrophic regions. SubjectsBioinformatics, Genomics, Marine Biology, Microbiology, Virology Sequence data accession numbersPRJNA767318

microbiology↗

bioRxiv2022

The Diversity and Ubiquity of Antibiotic Resistant Genes in Finfish Culture Ponds in Bangladesh

In Bangladesh, fish provide over 60% of animal-source food with 56.2% of this coming from aquaculture produced predominantly in rural freshwater ponds. Increasing demand for fish products is driving intensification and resulting in higher disease prevalence, posing a risk to food security. Biosecurity is often absent in rural aquaculture practices in Bangladesh and antibiotics are commonly used to treat and prevent disease outbreaks. Antibiotics are often administered incorrectly - a key factor associated with the development of antimicrobial resistance (AMR). AMR can be disseminated rapidly within microbial ecosystems via mobile genetic elements, posing a risk for humans and animals infected with AMR pathogens as treatments with antibiotics become ineffective. Early AMR detection and understanding of the spread of antimicrobial resistant genes (ARGs) in rural aquaculture practices is critical for both food security and human health protection. Here, we apply a metagenomic approach to assess the ARG composition in pond water from six finfish (tilapia and pangasius) farms in the Mymensingh division of North-central Bangladesh. We found microbial communities within the ponds had similar alpha and beta diversities, with multiple ARGs predicted to confer resistance to eighteen different classes of antimicrobials. The most common ARGs conferred resistance to aminoglycosides and sulphonamides and were present in taxa associated with both fish and human pathogens. This ARG diversity potentially confers resistance to a wide variety of antibiotic classes and questions the effectiveness of current and future treatment of diseases with antibiotics in earthen aquaculture ponds. The microbial and ARG compositions between fish ponds within each farm were similar, which may relate to parallels in farming practices creating similar microbial selection pressures and thus comparable microbial populations. Without a more controlled approach towards antibiotic usage, will inevitably further exacerbate the challenges in treating and preventing disease outbreaks as aquaculture production intensifies in Bangladesh. HighlightsO_LIARGs in Bangladesh rural fishponds indicate resistance to 18 different antibiotics C_LIO_LIThe most common AMR were to aminoglycosides and sulphonamides C_LIO_LIARGs were present in plasmids and taxa-associated pathogens C_LIO_LIFarming practices strongly influence microbial and ARG compositions C_LIO_LIIdentified ARGs question antibiotic treatment of disease in rural aquaculture C_LI

microbiology↗

bioRxiv2022

Pelagibacter phage Skadi - An abundant polar specialist that exemplifies ecotypic niche specificity among the most abundant viruses on Earth

Bacteria in the SAR11 clade are the most abundant members of surface marine bacterioplankton and are a critical component of global biogeochemical cycles. Similarly, pelagiphages that predate SAR11 are both ubiquitous and highly abundant in the oceans. These viruses are predicted to shape SAR11 community structures and increase carbon turnover throughout the oceans. Yet, ecological drivers of host and niche specificity of pelagiphage populations are poorly understood. Here we report the global distribution of a novel pelagiphage called Skadi isolated from the Western English Channel using a cold-water ecotype of SAR11 (HTCC1062) as bait. Skadi is closely related to the globally dominant pelagiphage HTVC010P. We show that, along with other HTVC010P-type viruses, Skadi belongs to a distinct viral family within the order Caudovirales for which we propose the name Ubiqueviridae. Metagenomic read recruitment identifies Skadi as one of the most abundant pelagiphages on Earth. Skadi is a polar specialist, replacing HTVC010P at high latitudes. Experimental evaluation of Skadi host-range against cold- and warm-water SAR11 ecotypes supported cold-water specialism. Read mapping from the Global Ocean Viromes project (GOV2) showed that relative abundance of Skadi correlated negatively with temperature, and positively with nutrients, available oxygen and chlorophyll concentrations. In contrast, relative abundance of HTVC010P correlated negatively with oxygen and positively with salinity, with no significant correlation to temperature. The majority of other pelagiphages were scarce in most marine provinces, with a few representatives constrained to discrete ecological niches. Our results suggest that pelagiphage populations persist within a global viral seed-bank, with environmental parameters and host availability selecting for a few ecotypes that dominate ocean viromes.

microbiology↗

bioRxiv2022

Influence of short and long term processes on SAR11 communities in open ocean and coastal systems

SAR11 bacteria dominate the surface ocean and are major players in converting fixed carbon back to atmospheric carbon dioxide. The SAR11 clade is comprised of niche-specialized ecotypes that display distinctive spatiotemporal transitions. We analysed SAR11 ecotype seasonality in two long-term 16S rRNA amplicon time series representing different North Atlantic regimes: the Sargasso Sea (subtropical ocean-gyre; BATS) and the temperate coastal Western English Channel (WEC). Using phylogenetically resolved amplicon sequence variants (ASVs), we evaluated seasonal environmental constraints on SAR11 ecotype periodicity. Despite large differences in temperature and nutrient availability between the two sites, at both SAR11 succession was defined by summer and winter clusters of ASVs. Summer cluster was dominated by ecotype Ia.3 in both sites. Winter clusters were dominated by ecotypes Ib and IIa.A at BATS and Ia.1 and IIa.B at WEC. A two-year weekly analysis within the WEC time series showed that the response of SAR11 communities to short-term environmental fluctuations was variable. In 2016, community shifts were abrupt and synchronised to environmental shifts. However, in 2015, changes were gradual and decoupled from environmental fluctuations, likely due to increased mixing from strong winds. We demonstrate that interannual weather variability disturb the pace of SAR11 seasonal progression.

microbiology↗

bioRxiv2021

Relationships between pond water and tilapia skin microbiomes in aquaculture ponds in Malawi

Intensification of fish farming practices is being driven by the demand for increased food production to support a rapidly growing global human population, particularly in lower-middle income countries. Intensification of production, however, increases the risk of disease outbreaks and thus the likelihood for crop losses. The microbial communities that colonise the skin mucosal surface of fish are poorly understood, but are important in maintaining fish health and resistance against disease. This skin microbial community is susceptible to disruption through stressors associated with transport, handling and the environment of intensive practices, and this risks the propagation of disease-causing pathogens. In this study, we characterised the microbial assemblages found on tilapia skin -- the most widely farmed finfish globally -- and in the surrounding water of seven earthen aquaculture ponds from two pond systems in distinct geographic regions in Malawi. Metabarcoding approaches were used to sequence the prokaryotic and microeukaryotic communities. We found 92% of prokaryotic amplicon sequence variants were common to both skin and water samples. Differentially enriched and core taxa, however, differed between the skin and water samples. In tilapia skin, Cetobacterium, Paucibacter, Pseudomonas and Comamonadaceae were enriched, whereas, the cyanobacteria Cyanobium, Microcystis and/or Synechocystis, and the diatom Cyclotella, were most prevalent in pond water. Ponds that clustered together according to their water prokaryotic communities also had similar microeukaryotic communities indicating strong environmental influences on prokaryotic and microeukaryotic community structures. While strong site-specific clustering was observed in pond water, the grouping of tilapia skin prokaryotes by pond site was less distinct, suggesting fish microbiota have a greater buffering capacity against environmental influences. The characterised diversity, structure and variance of microbial communities associated with tilapia culture in Malawi provide the baseline for studies on how future intensification practices may lead to microbial dysbiosis and disease onset. HighlightsO_LIFish skin and pond water communities differ structurally, but share common taxa C_LIO_LIPond locations have a stronger influence on water versus fish skin microbiome community structure C_LIO_LISelected skin-associated taxa could be used to monitor dysbiotic events in aquaculture C_LIO_LITaxa with opportunistic pathogen potential were identified at low abundance C_LI

microbiology↗

bioRxiv2021

Genomic evidence for inter-class host transition between abundant streamlined heterotrophs by a novel and ubiquitous marine Methylophage

The methylotrophic OM43 clade are Gammaproteobacteria that comprise some of the smallest free-living cells known and have highly streamlined genomes. OM43 represents an important microbial link 0between marine primary production and remineralisation of carbon back to the atmosphere. Bacteriophages shape microbial communities and are major drivers of microbial mortality and global marine biogeochemistry. Recent cultivation efforts have brought the first viruses infecting members of the OM43 clade into culture. Here we characterize a novel myophage infecting OM43 called Melnitz. Melnitz was isolated independently on three separate occasions (with isolates sharing >99.95% average nucleotide identity) from water samples from a subtropical ocean gyre (Sargasso Sea) and temperate coastal (Western English Channel) systems. Metagenomic recruitment from global ocean viromes confirmed that Melnitz is globally ubiquitous, congruent with patterns of host abundance. Bacteria with streamlined genomes such as OM43 and the globally dominant SAR11 clade use riboswitches as an efficient method to regulate metabolism. Melnitz encodes a two-piece tmRNA (ssrA), controlled by a glutamine riboswitch, providing evidence that riboswitch use also occurs for regulation during phage infection of streamlined heterotrophs. Virally encoded tRNAs and ssrA found in Melnitz were phylogenetically more closely related to those found within the alphaproteobacterial SAR11 clade and their associated myophages than those within their gammaproteobacterial hosts. This suggests the possibility of an ancestral inter-class host transition event between SAR11 and OM43. Melnitz and a related myophage that infects SAR11 were unable to infect hosts of the SAR11 and OM43, respectively, suggesting host transition rather than a broadening of host range. IMPORTANCEIsolation and cultivation of viruses is the foundation on which the mechanistic understanding of virus-host interactions and ground-truthing is based. This study isolated and characterised the first myophage known to infect the OM43 clade, expanding our knowledge of this understudied group of microbes. The near-identical genomes of four strains of Melnitz isolated from different marine provinces and global abundance estimations from metagenomic data suggest that this viral population is globally ubiquitous. Genome analysis revealed several unusual features in Melnitz and related genomes recovered from viromes, such as a curli operon and virally encoded tmRNA controlled by a glutamine riboswitch, neither of which are found in the host. Further phylogenetic analysis of shared genes indicates that this group of viruses infecting the gammaproteobacterial OM43 shares a recent common ancestor with viruses infecting the abundant alphaproteobacterial SAR11 clade. Host ranges are affected by compatible cell surface receptors, successful circumvention of superinfection exclusion systems and the presence of required accessory proteins, which typically limits phages to singular narrow groups of closely related bacterial hosts. This study provides intriguing evidence that for streamlined heterotrophic bacteria, virus-host transitioning is not necessarily restricted to phylogenetically related hosts, but is a function of shared physical and biochemical properties of the cell.

microbiology↗

bioRxiv2020

MetaPop: A pipeline for macro- and micro-diversity analyses and visualization of microbial and viral metagenome-derived populations

BackgroundMicrobes and their viruses are hidden engines driving Earths ecosystems from the oceans and soils to humans and bioreactors. Though gene marker approaches can now be complemented by genome-resolved studies of inter- (macrodiversity) and intra- (microdiversity) population variation, analytical tools to do so remain scattered or under-developed. ResultsHere we introduce MetaPop, an open-source bioinformatic pipeline that provides a single interface to analyze and visualize microbial and viral community metagenomes at both the macro- and micro-diversity levels. Macrodiversity estimates include population abundances and - and {beta}-diversity. Microdiversity calculations include identification of single nucleotide polymorphisms, novel codon-constrained linkage of SNPs, nucleotide diversity ({pi} and {theta}) and selective pressures (pN/pS and Tajimas D) within and fixation indices (FST) between populations. MetaPop will also identify genes with distinct codon usage. Following rigorous validation, we applied MetaPop to the gut viromes of autistic children that underwent fecal microbiota transfers and their neurotypical peers. The macrodiversity results confirmed our prior findings for viral populations (microbial shotgun metagenomes were not available), that diversity did not significantly differ between autistic and neurotypical children. However, by also quantifying microdiversity, MetaPop revealed lower average viral nucleotide diversity ({pi}) in autistic children. Analysis of the percentage of genomes detected under positive selection was also lower among autistic children, suggesting that higher viral {pi} in neurotypical children may be beneficial because it allows populations to better bet hedge in changing environments. Further, comparisons of microdiversity pre- and post-FMT in the autistic children revealed that the delivery FMT method (oral versus rectal) may influence viral activity and engraftment of microdiverse viral populations, with children who received their FMT rectally having higher microdiversity post-FMT. Overall, these results show that analyses at the macro-level alone can miss important biological differences. ConclusionsThese findings suggest that standardized population and genetic variation analyses will be invaluable for maximizing biological inference, and MetaPop provides a convenient tools package to explore the dual impact of macro- and micro-diversity across microbial communities.

bioinformatics↗

bioRxiv2020

VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature

Microbes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequencing has greatly improved our virus ecology toolkit-- and revealed many critical ecosystem roles for viruses -- microdiverse populations and fine-scale genomic traits are missed. Some of these microdiverse populations are abundant and the missed regions may be of interest for identifying selection pressures that underpin evolutionary constraints associated with hosts and environments. Though long-read sequencing promises complete virus genomes on single reads, it currently suffers from high DNA requirements and sequencing errors that limit accurate gene prediction. Here we introduce VirION2, an integrated short- and long-read metagenomic wet-lab and informatics pipeline that updates our previous method (VirION) to further enhance the utility of long-read viral metagenomics. Using a viral mock community, we first optimized laboratory protocols (polymerase choice, DNA shearing size, PCR cycling) to enable 76% longer reads (now median length of 6,965 bp) from 100-fold less input DNA (now 1 nanogram). Using a virome from a natural seawater sample, we compared viromes generated with VirION2 against other library preparation options (unamplified, original VirION, and short-read), and optimized downstream informatics for improved long-read error correction and assembly. VirION2 assemblies combined with short-read based data ( enhancedviromes), provided significant improvements over VirION libraries in the recovery of longer and more complete viral genomes, and our optimized error-correction strategy using long- and short-read data achieved 99.97% accuracy. In the seawater virome, VirION2 assemblies captured 5,161 viral populations (including all of the virus populations observed in the other assemblies), 30% of which were uniquely assembled through inclusion of long-reads, and 22% of the top 10% most abundant virus populations derived from assembly of long-reads. Viral populations unique to VirION2 assemblies had significantly higher microdiversity, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow (updated at protocols.io) can help researchers better investigate the virosphere, even from challenging low-biomass samples. Our new protocols are available to the research community on protocols.io as a living document to facilitate dissemination of updates to keep pace with the rapid evolution of long read sequencing technology. Taken together, the addition of long-reads uniquely enabled the recovery of 22% of the most abundant viruses--that would have been missed in short-read only assemblies.

microbiology↗

bioRxiv2020

Efficient Dilution-to-Extinction isolation of novel virus-host model systems for fastidious heterotrophic bacteria

Microbes and their associated viruses are key drivers of biogeochemical processes in marine and soil biomes. While viruses of phototrophic cyanobacteria are well-represented in model systems, challenges of isolating marine microbial heterotrophs and their viruses have hampered experimental approaches to quantify the importance of viruses in nutrient recycling. A resurgence in cultivation efforts has improved the availability of fastidious bacteria for hypothesis testing, but this has not been matched by similar efforts to cultivate their associated bacteriophages. Here, we describe a high-throughput method for isolating important virus-host systems for fastidious heterotrophic bacteria that couples advances in culturing of hosts with sequential enrichment and isolation of associated phages. Applied to six monthly samples from the Western English Channel, we first isolated one new member of the globally dominant bacterial SAR11 clade and three new members of the methylotrophic bacterial clade OM43. We used these as bait to isolate 117 new phages including the first known siphophage infecting SAR11, and the first isolated phage for OM43. Genomic analyses of 13 novel viruses revealed representatives of three new viral genera, and infection assays showed that the viruses infecting SAR11 have ecotype-specific host-ranges. Similar to the abundant human-associated phage {Phi}CrAss001, infection dynamics within the majority of isolates suggested either prevalent lysogeny or chronic infection, despite a lack of associated genes; or host phenotypic bistability with lysis putatively maintained within a susceptible subpopulation. Broader representation of important virus-host systems in culture collections and genomic databases will improve both our understanding of virus-host interactions, and accuracy of computational approaches to evaluate ecological patterns from metagenomic data.

microbiology↗

bioRxiv2020

Expanding the diversity of bacterioplankton isolates and modeling isolation efficacy with large scale dilution-to-extinction cultivation

Cultivated bacterioplankton representatives from diverse lineages and locations are essential for microbiology, but the large majority of taxa either remain uncultivated or lack isolates from diverse geographic locales. We paired large scale dilution-to-extinction (DTE) cultivation with microbial community analysis and modeling to expand the phylogenetic and geographic diversity of cultivated bacterioplankton and to evaluate DTE cultivation success. Here, we report results from 17 DTE experiments totaling 7,820 individual incubations over three years, yielding 328 repeatably transferable isolates. Comparison of isolates to microbial community data of source waters indicated that we successfully isolated 5% of the observed bacterioplankton community throughout the study. 43% and 26% of our isolates matched operational taxonomic units and amplicon single nucleotide variants, respectively, within the top 50 most abundant taxa. Isolates included those from previously uncultivated clades such as SAR11 LD12 and Actinobacteria acIV, as well as geographically novel members from other ecologically important groups like SAR11 subclade IIIa, SAR116, and others; providing the first isolates in eight putatively new genera and seven putatively new species. Using a newly developed DTE cultivation model, we evaluated taxon viability by comparing relative abundance with cultivation success. The model i) revealed the minimum attempts required for successful isolation of taxa amenable to growth on our media, and ii) identified possible subpopulation viability variation in abundant taxa such as SAR11 that likely impacts cultivation success. By incorporating viability in experimental design, we can now statistically constrain the effort necessary for successful cultivation of specific taxa on a defined medium. ImportanceEven before the coining of the term "great plate count anomaly" in the 1980s, scientists had noted the discrepancy between the number of microorganisms observed under the microscope and the number of colonies that grew on traditional agar media. New cultivation approaches have reduced this disparity, resulting in the isolation of some of the "most wanted" bacterial lineages. Nevertheless, the vast majority of microorganisms remain uncultured, hampering progress towards answering fundamental biological questions about many important microorganisms. Furthermore, few studies have evaluated the underlying factors influencing cultivation success, limiting our ability to improve cultivation efficacy. Our work details the use of dilution-to-extinction (DTE) cultivation to expand the phylogenetic and geographic diversity of available axenic cultures. We also provide a new model of the DTE approach that uses cultivation results and natural abundance information to predict taxon-specific viability and iteratively constrain DTE experimental design to improve cultivation success.

microbiology↗