Building the phage-host-nutrient interaction data to scale from genes-to-ecosystems: Towards predictive modeling of wild microbial and viral communities dynamics
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Duhaime, Melissa↗
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Predicting promoters in phage genomes using PhagePromoter
Abstract Summary The growing interest in phages as antibacterial agents has led to an increase in the number of sequenced phage genomes, increasing the need for intuitive bioinformatics tools for performing genome annotation. The identification of phage promoters is indeed the most difficult step of this process. Due to the lack of online tools for phage promoter prediction, we developed PhagePromoter, a tool for locating promoters in phage genomes, using machine learning methods. This is the first online tool for predicting promoters that uses phage promoter data and the first to identify both host and phage promoters with different motifs. Availability and implementation This tool was integrated in the Galaxy framework and it is available online at: https://bit.ly/2Dfebfv. Supplementary information Supplementary data are available at Bioinformatics online.
Sampaio, Marta↗
Microbial Dark Matter & Beyond
Eddy Rubin, DOE JGI Director, at the 9th Annual Genomics of Energy & Environment Meeting on March 19, 2014 in Walnut Creek, CA. The talk is related to a study published in the journal Science.
59 BASIC BIOLOGICAL SCIENCES↗
Large-scale maps of variable infection efficiencies in aquatic Bacteroidetes phage-host model systems: Variable phage-host infection interactions
Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. In this paper, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding and modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions.
59 BASIC BIOLOGICAL SCIENCES↗
Geochemical constraints on bacteriophage infectivity in terrestrial environments
Abstract Lytic phages can be potent and selective inhibitors of microbial growth and can have profound impacts on microbiome composition and function. However, there is uncertainty about the biogeochemical conditions under which phage predation modulates microbial ecosystem function, particularly in terrestrial systems. Ionic strength is critical for infection of bacteria by many phages, but quantitative data is limited on the ion thresholds for phage infection that can be compared with environmental ion concentrations. Similarly, while carbon composition varies in the environment, we do not know how this variability influences the impact of phage predation on microbiome function. Here, we measured the half-maximal effective concentrations (EC50) of 80 different inorganic ions for the infection of E. coli with two canonical dsDNA and ssRNA phages, T4 and MS2, respectively. Many alkaline earth metals and alkali metals enabled lytic infection but the ionic strength thresholds varied for different ions between phages. Additionally, using a freshwater nitrate-reducing microbiome, we found that the ability of lytic phages to influence nitrate reduction end-products depended upon the carbon source as well as ionic strength. For all phage:host pairs, the ion EC50s for phage infection exceeded the ion concentrations found in many terrestrial freshwater systems. Thus, our findings support a model where phages most influence terrestrial microbial functional ecology in hot spots and hot moments such as metazoan guts, drought influenced soils, or biofilms where ion concentration is locally or transiently elevated and nutrients are available to support the growth of specific phage hosts.
Carlson, Hans K. (ORCID:0000000215835313)↗
The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins
Abstract RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron–electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.
Biochemistry & Molecular Biology↗
Experimental validation that human microbiome phages use alternative genetic coding
Previous bioinformatic analyses of metagenomic data have indicated that bacteriophages can use genetic codes different from those of their host bacteria. In particular, reassignment of stop codon TAG to glutamine (a variation known as ‘genetic code 15’) has been predicted. Here, we use LC-MS/MS-based metaproteomics of human fecal samples to provide experimental evidence of the use of genetic code 15 in two crAss-like phages. Furthermore, the proteomic data from several phage structural proteins supports the reassignment of the TAG stop codon to glutamine late in the phage infection cycle. Thus, our work experimentally validates the expression of genetic code 15 in human microbiome phages.
59 BASIC BIOLOGICAL SCIENCES↗
Breaking the Ice: A Review of Phages in Polar Ecosystems
Bacteriophages, or phages, are viruses that infect and replicate within bacterial hosts, playing a significant role in regulating microbial populations and ecosystem dynamics. However, phages from extreme environments such as polar regions remain relatively understudied due to challenges such as restricted ecosystem access and low biomass. In this study, understanding the diversity, structure, and functions of polar phages is crucial for advancing our knowledge of the microbial ecology and biogeochemistry of these environments. In this review, we will explore the current state of knowledge on phages from the Arctic and Antarctic, focusing on insights gained from -omic studies, phage isolation, and virus-like particle abundance data. Metagenomic studies of polar environments have revealed a high diversity of phages with unique genetic characteristics, providing insights into their evolutionary and ecological roles. Phage isolation studies have identified novel phage–host interactions and contributed to the discovery of new phage species. Virus-like particle abundance and lysis rate data, on the other hand, have highlighted the importance of phages in regulating bacterial populations and nutrient cycling in polar environments. Overall, this review aims to provide a comprehensive overview of the current state of knowledge about polar phages, and by synthesizing these different sources of information, we can better understand the diversity, dynamics, and functions of polar phages in the context of ongoing climate change, which will help to predict how polar ecosystems and residing phages may respond to future environmental perturbations.
09 BIOMASS FUELS↗
MVP: a modular viromics pipeline to identify, filter, cluster, annotate, and bin viruses from metagenomes
While numerous computational frameworks and workflows are available for recovering prokaryote and eukaryote genomes from metagenome data, only a limited number of pipelines are designed specifically for viromics analysis. With many viromics tools developed in the last few years alone, it can be challenging for scientists with limited bioinformatics experience to easily recover, evaluate quality, annotate genes, dereplicate, assign taxonomy, and calculate relative abundance and coverage of viral genomes using state-of-the-art methods and standards. Here, we describe Modular Viromics Pipeline (MVP) v.1.0, a user-friendly pipeline written in Python and providing a simple framework to perform standard viromics analyses. MVP combines multiple tools to enable viral genome identification, characterization of genome quality, filtering, clustering, taxonomic and functional annotation, genome binning, and comprehensive summaries of results that can be used for downstream ecological analyses. Overall, MVP provides a standardized and reproducible pipeline for both extensive and robust characterization of viruses from large-scale sequencing data including metagenomes, metatranscriptomes, viromes, and isolate genomes. As a typical use case, we show how the entire MVP pipeline can be applied to a set of 20 metagenomes from wetland sediments using only 10 modules executed via command lines, leading to the identification of 11,656 viral contigs and 8,145 viral operational taxonomic units (vOTUs) displaying a clear beta-diversity pattern. Further, acting as a dynamic wrapper, MVP is designed to continuously incorporate updates and integrate new tools, ensuring its ongoing relevance in the rapidly evolving field of viromics. MVP is available at https://gitlab.com/ccoclet/mvp and as versioned packages in PyPi and Conda.
59 BASIC BIOLOGICAL SCIENCES↗
The development of Nanosota-1 as anti-SARS-CoV-2 nanobody drug candidates
Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking viral receptor angiotensin-converting enzyme 2 (ACE2). The lead drug candidate possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD ~3000 times more tightly than ACE2 did and inhibited SARS-CoV-2 pseudovirus ~160 times more efficiently than ACE2 did. Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy against live SARS-CoV-2 infection in both hamster and mouse models. Unlike conventional antibodies, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented an excellent in vivo stability and a high tissue bioavailability. As effective and inexpensive drug candidates, Nanosota-1 may contribute to the battle against COVID-19.
60 APPLIED LIFE SCIENCES↗
A Phage Foundry Framework to Systematically Develop Viral Countermeasures to Combat Antibiotic-Resistant Bacterial Pathogens
At its current rate, the rise of antimicrobial-resistant (AMR) infections is predicted to paralyze our industries and healthcare facilities while becoming the leading global cause of loss of human life. With limited new antibiotics on the horizon, we need to invest in alternative solutions. Bacteriophages (phages)–viruses targeting bacteria–offer a powerful alternative approach to tackle bacterial infections. Despite recent advances in using phages to treat recalcitrant AMR infections, the field lacks systematic development of phage therapies scalable to different applications. We propose a Phage Foundry framework to establish metrics for phage characterization and to fill the knowledge and technological gaps in phage therapeutics. Coordinated investment in AMR surveillance, sampling, characterization, and data sharing procedures will enable rational exploitation of phages for treatments. A fully realized Phage Foundry will enhance the sharing of knowledge, technology, and viral reagents in an equitable manner and will accelerate the biobased economy.
59 BASIC BIOLOGICAL SCIENCES↗
$\mathrm{COBRA}$ improves the completeness and contiguity of viral genomes assembled from metagenomes
Viruses are often studied using metagenome-assembled sequences, but genome incompleteness hampers comprehensive and accurate analyses. Contig Overlap Based Re-Assembly (COBRA) resolves assembly breakpoints based on the de Bruijn graph and joins contigs. Here we benchmarked COBRA using ocean and soil viral datasets. COBRA accurately joined the assembled sequences and achieved notably higher genome accuracy than binning tools. From 231 published freshwater metagenomes, we obtained 7,334 bacteriophage clusters, ~83% of which represent new phage species. Notably, ~70% of these were circular, compared with 34% before COBRA analyses. We expanded sampling of huge phages (≥200 kbp), the largest of which was curated to completion (717 kbp). Improved phage genomes from Rotsee Lake provided context for metatranscriptomic data and indicated the in situ activity of huge phages, whiB-encoding phages and cysC- and cysH-encoding phages. COBRA improves viral genome assembly contiguity and completeness, thus the accuracy and reliability of analyses of gene content, diversity and evolution.
54 ENVIRONMENTAL SCIENCES↗
MetaboDirect: an analytical pipeline for the processing of FT-ICR MS-based metabolomic data
Background: Microbiomes are now recognized as the main drivers of ecosystem function ranging from the oceans and soils to humans and bioreactors. However, a grand challenge in microbiome science is to characterize and quantify the chemical currencies of organic matter (i.e., metabolites) that microbes respond to and alter. Critical to this has been the development of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), which has drastically increased molecular characterization of complex organic matter samples, but challenges users with hundreds of millions of data points where readily available, user-friendly, and customizable software tools are lacking. Results: Here, we build on years of analytical experience with diverse sample types to develop MetaboDirect, an open-source, command-line-based pipeline for the analysis (e.g., chemodiversity analysis, multivariate statistics), visualization (e.g., Van Krevelen diagrams, elemental and molecular class composition plots), and presentation of direct injection high-resolution FT-ICR MS data sets after molecular formula assignment has been performed. When compared to other available FT-ICR MS software, MetaboDirect is superior in that it requires a single line of code to launch a fully automated framework for the generation and visualization of a wide range of plots, with minimal coding experience required. Among the tools evaluated, MetaboDirect is also uniquely able to automatically generate biochemical transformation networks (ab initio) based on mass differences (mass difference network-based approach) that provide an experimental assessment of metabolite connections within a given sample or a complex metabolic system, thereby providing important information about the nature of the samples and the set of microbial reactions or pathways that gave rise to them. Finally, for more experienced users, MetaboDirect allows users to customize plots, outputs, and analyses. Conclusion: Application of MetaboDirect to FT-ICR MS-based metabolomic data sets from a marine phage-bacterial infection experiment and a Sphagnum leachate microbiome incubation experiment showcase the exploration capabilities of the pipeline that will enable the research community to evaluate and interpret their data in greater depth and in less time. It will further advance our knowledge of how microbial communities influence and are influenced by the chemical makeup of the surrounding system. The source code and User’s guide of MetaboDirect are freely available through (https://github.com/Coayala/MetaboDirect) and (https://metabodirect.readthedocs.io/en/latest/), respectively.
54 ENVIRONMENTAL SCIENCES↗
Large freshwater phages with the potential to augment aerobic methane oxidation
There is growing evidence that phages with unusually large genomes are common across various microbiomes, but little is known about their genetic inventories or potential ecosystem impacts. In the present study, we reconstructed large phage genomes from freshwater lakes known to contain bacteria that oxidize methane. Of manually curated genomes, 22 (18 are complete), ranging from 159 kilobase (kb) to 527 kb in length, were found to encode the pmoC gene, an enzymatically critical subunit of the particulate methane monooxygenase, the predominant methane oxidation catalyst in nature. The phage-associated PmoC sequences show high similarity to (>90%), and affiliate phylogenetically with, those of coexisting bacterial methanotrophs, including members of Methyloparacoccus, Methylocystis and Methylobacter spp. In addition, pmoC-phage abundance patterns correlate with those of the coexisting bacterial methanotrophs, supporting host–phage relationships. Future work is needed to determine whether phage-associated PmoC has similar functions to additional copies of PmoC encoded in bacterial genomes, thus contributing to growth on methane. Transcriptomics data from Lake Rotsee (Switzerland) showed that some phage-associated pmoC genes were highly expressed in situ and, of interest, that the most rapidly growing methanotroph was infected by three pmoC-phages. Thus, augmentation of bacterial methane oxidation by pmoC-phages during infection could modulate the efflux of this potent greenhouse gas into the environment.
59 BASIC BIOLOGICAL SCIENCES↗
A phage nucleus-associated RNA-binding protein is required for jumbo phage infection
Abstract Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.
59 BASIC BIOLOGICAL SCIENCES↗
Regulation of infection efficiency in a globally abundant marine Bacteriodetes virus
Microbes impact human health and disease, industrial processes and natural ecosystems, but do so under the influence of viruses. Problematically, knowledge of viral infection efficiencies and outcomes (e.g. lysis, lysogeny) derives from few model systems that over-represent efficient, lytic infections and under-represent virus-host natural diversity. Here we sought to understand how infection efficiency is regulated in an environmental Bacteroidetes virus that represents a globally abundant viral group and has drastically different infection efficiencies when infecting two nearly identical bacterial strains. To this end, we quantified bacterial virus (phage) and host DNA, transcripts and phage particles throughout the infection of both bacterial hosts. While the phage transcriptome was similar during both infections, host transcriptional differences appeared to have altered infection efficiency. Specifically, host transcriptomes suggested that the phage failed to repress early host expression in the inefficient nfection, thereby allowing the host to respond against infection by delaying phage DNA replication and protein translation. Further measurements showed that phage DNA and particle production were delayed (by >30 minutes) and reduced (by >50%) in the inefficient versus efficient infection as the host over-expressed DNA degradation genes and under-expressed translation genes, respectively. Together these results suggest that multiple levels of regulation can impact infection efficiencies as failure to repress host transcription allowed the host to defend against both phage DNA and protein production. Given that this phage type is ubiquitous and abundant in the global oceans and that variably efficient viral infections are likely common in any ecosystem with varying phage-host abundances and physiological states, these data provide a critically needed foundation for understanding and modeling viral infection efficiency in nature.
60 APPLIED LIFE SCIENCES↗
Systematic and scalable genome-wide essentiality mapping to identify nonessential genes in phages
Phages are one of the key ecological drivers of microbial community dynamics, function, and evolution. Despite their importance in bacterial ecology and evolutionary processes, phage genes are poorly characterized, hampering their usage in a variety of biotechnological applications. Methods to characterize such genes, even those critical to the phage life cycle, are labor intensive and are generally phage specific. Here, we develop a systematic gene essentiality mapping method scalable to new phage–host combinations that facilitate the identification of nonessential genes. As a proof of concept, we use an arrayed genome-wide CRISPR interference (CRISPRi) assay to map gene essentiality landscape in the canonical coliphages λ and P1. Results from a single panel of CRISPRi probes largely recapitulate the essential gene roster determined from decades of genetic analysis for lambda and provide new insights into essential and nonessential loci in P1. We present evidence of how CRISPRi polarity can lead to false positive gene essentiality assignments and recommend caution towards interpreting CRISPRi data on gene essentiality when applied to less studied phages. Finally, we show that we can engineer phages by inserting DNA barcodes into newly identified inessential regions, which will empower processes of identification, quantification, and tracking of phages in diverse applications.
59 BASIC BIOLOGICAL SCIENCES↗
Identification and Characterization of ten Escherichia coli Strains Encoding Novel Shiga Toxin 2 Subtypes, Stx2n as Well as Stx2j, Stx2m, and Stx2o, in the United States
The sharing of genome sequences in online data repositories allows for large scale analyses of specific genes or gene families. This can result in the detection of novel gene subtypes as well as the development of improved detection methods. Here, we used publicly available WGS data to detect a novel Stx subtype, Stx2n in two clinical E. coli strains isolated in the USA. During this process, additional Stx2 subtypes were detected; six Stx2j, one Stx2m strain, and one Stx2o, were all analyzed for variability from the originally described subtypes. Complete genome sequences were assembled from short- or long-read sequencing and analyzed for serotype, and ST types. The WGS data from Stx2n- and Stx2o-producing STEC strains were further analyzed for virulence genes pro-phage analysis and phage insertion sites. Nucleotide and amino acid maximum parsimony trees showed expected clustering of the previously described subtypes and a clear separation of the novel Stx2n subtype. WGS data were used to design OMNI PCR primers for the detection of all known stx1 (283 bp amplicon), stx2 (400 bp amplicon), intimin encoded by eae (221 bp amplicon), and stx2f (438 bp amplicon) subtypes. These primers were tested in three different laboratories, using standard reference strains. An analysis of the complete genome sequence showed variability in serogroup, virulence genes, and ST type, and Stx2 pro-phages showed variability in size, gene composition, and phage insertion sites. The strains with Stx2j, Stx2m, Stx2n, and Stx2o showed toxicity to Vero cells. Stx2j carrying strain, 2012C-4221, was induced when grown with sub-inhibitory concentrations of ciprofloxacin, and toxicity was detected. Taken together, these data highlight the need to reinforce genomic surveillance to identify the emergence of potential new Stx2 or Stx1 variants. The importance of this surveillance has a paramount impact on public health. Per our description in this study, we suggest that 2017C-4317 be designated as the Stx2n type-strain.
59 BASIC BIOLOGICAL SCIENCES↗