Biology subjects

Ettwiller, L.

Publications and source records attributed to Ettwiller, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Microbial Community Multi-omic Analysis of Marsh Sediment post Crustacean Shell Compost Enrichment: pathogen emergence and community response

Changes in nutrient availability can rapidly alter microbial processes in natural environments, with implications in biogeochemical cycling and pathogen emergence. Short-term, functional responses of microbial communities to nutrient amendment in coastal communities remain poorly understood, particularly in temperate environments. A 48-hour microcosm pulse experiment was completed in which paired metagenomic and metatranscriptomic sequencing were employed to examine how the decomposition of chitin rich substrates, namely crab and lobster shell compost, alters salt marsh microbiome structure and function. Within 48 hours of amendment, pronounced shifts in community metabolism were observed, including increased chitin degradation and utilization, stress-response, and sporulation. These responses coincided with marked decreases in genes associated with key biogeochemical processes, including carbon fixation, sulfur oxidation and reduction, and other metabolic pathways. Shell compost addition also enriched putative pathogens and virulence-associated genes, accompanied by modest transcriptional activation, notably aerolysin A (aerA), which encodes the pore-forming exotoxin aerolysin. These results demonstrate temperate salt marsh sediment microbiomes can undergo shifts in community composition and function that is associated with chitin-rich nutrient perturbation. The sensitivity of temperate coastal systems to organic matter input and the potential for ecological and public-health relevant outcomes are underscored, notably given that chitin is among the most abundant and readily available bionutrients in aquatic ecosystems globally.

microbiology↗

bioRxiv2025

Association Studies of Phage Metagenomes from Aquatic Environment Identify Known and Potentially New Key Players in the 2,6-Diaminopurine Biosynthesis Pathway

Advances in next-generation sequencing (NGS) and bioinformatics have expanded the potential of metagenomic studies, particularly for unculturable organisms such as phages with heavily modified genomes. In the evolutionary arms race between phages and their hosts, some phages develop a strategy to fully modify their DNAs to evade host-encoded antiphage defenses. Here, we developed a restriction-enzyme based method to selectively enrich all phages containing 2-aminoadenine (diaminopurine or dZ) directly from purified metagenomic DNA from environmental samples. Applying our previously established metagenomic genome-phenome association (metaGPA) pipeline, we identified 116 protein domains significantly associated with dZ phages. The top candidate domains fully recapitulated the phage-encoded dZ biosynthetic pathway. Study of individual enzymes, including PurZ and YfbR-like dATPase at residue resolution revealed key amino acids for the distinction between dZ and dA substrate. Additionally, we identified and validated two novel DNA polymerases with enhanced dZ incorporation activity and uncovered nucleoside 2-deoxyribosyltransferase (NDT) as a potential new component of the dZ biosynthetic pathway. Collectively, these findings refine current models of the dZ biosynthesis pathway, potentially reveal previously unrecognized enzymatic components, and expand the understanding of phage biology with implications for synthetic biology and DNA engineering.

genomics↗

bioRxiv2025

A Bait-and-Switch strategy links phenotypes to genes coding for Polymer-Degrading Enzymes in Intact Microbiomes

Advances in next generation sequencing have made it possible to explore microbial community dynamics and regulation of functionally important genes through metagenomics and metatranscriptomics. However, the use of meta-omics to link enzyme function directly with complex, community-level phenotypes remain largely unexplored. To overcome this gap, we developed a novel framework that integrates ecological concepts by microbial community perturbation with association analysis to a targeted phenotype. Specifically, we introduce a hypothesis-free "bait and switch" strategy demonstrated through salt marsh soil microcosm pulse experiments to detect and characterize novel enzymes responsible for chitin degradation. Soil microbial communities were "baited" with shell compost, a chitin-rich substrate, to trigger community succession toward chitin degraders and gene upregulation of chitinases. A "switch" was then employed, by addition of glucose, inducing rapid downregulation of genes putatively responsible for chitin degradation. Results demonstrate the feasibility of this approach to identify functionally important enzymes, in this example, 48 hours after chitin addition. The bait and switch community perturbation provides a framework for discovery of polymer degrading enzymes present in complex microbial communities and serves as a proof of concept applicable for linking enzyme function with emergent community level phenotypes.

microbiology↗

bioRxiv2025

A novel NGS-compatible Enzymatic Strategy Enables Carryover Contamination Removal and Enhances Sequencing Performance.

Carryover contamination during DNA amplification can often lead to false positives. Traditional mitigation strategies often include physical separation and/or enzymatic decontamination. However, these methods have limitations, including logistical constraints and polymerase compatibility issues especially for Next Generation Sequencing (NGS). Here, we introduce a novel approach using 7-deaza-dGTP and Fpg for carryover amplicon degradation. When incorporated into library preparation, Fpg degrades 7-deaza-deoxyguanosine amplicons providing carryover protection comparable to the established dUTP/UDG strategy with over 95% carryover contamination removal. Unlike currently available carryover enzyme/dNTP solutions, 7-deaza-deoxyguanosine is compatible with many polymerases and does not impact substitution frequencies during sequencing. Additionally, due to its chemical properties, incorporating 7-deaza-dGTP during amplification improves GC bias during sequencing. In turn, this method is compatible with NGS, supports broader polymerase compatibility, and improves sequencing performance, particularly in AT- and GC-rich regions.

genomics↗

bioRxiv2024

Proxi-RIMS-seq2 applied to native microbiomes uncovers hundreds of known and novel m5C methyltransferase specificities.

Methylation patterns in bacteria can be used to study Restriction-Modification (RM) or other defense systems with novel properties. While m4C and m6A methylation is well characterized mainly through PacBio sequencing, the landscape of m5C methylation is under-characterized. To bridge this gap, we performed RIMS-seq2 on microbiomes composed of resolved assemblies of distinct genomes through proximity ligation. This high-throughput approach enables the identification of m5C methylated motifs and links them to cognate methyltransferases directly on native microbiomes without the need to isolate bacterial strains. Methylation patterns can also be identified on viral DNA and compared to host DNA, strengthening evidence for virus-host interaction. Applied to three different microbiomes, the method unveils over 1900 motifs that were deposited in REBASE. The motifs include a novel 8-base recognition site (CATm5CGATG) that was experimentally validated by characterizing its cognate methyltransferase. Our findings suggest that microbiomes harbor arrays of untapped m5C methyltransferase specificities, providing insights to bacterial biology and biotechnological applications.

genomics↗

bioRxiv2023

Type II restriction of 2-aminoadenine (dZ) modified DNA and production of dZ-modified plasmid in E. coli

The modified DNA base 2,6 aminopurine (2-aminoadenine, (d)Z base) was originally found in phages to counteract host encoded restriction systems. However, only a limited number of restriction endonucleases (REases) have been tested on dZ-modified DNA. Herein we report the results of 147 REases activity on dZ-modified PCR DNA. Among the enzymes tested, 53.1% are resistant or partially resistant, and 46.9% are sensitive when the restriction sites contain 1 to 6 modified bases. Sites with 4-6 dZ substitutions are most likely resistant to Type II restriction. Our results support the notion that dZ-modified phage genomes are evolved to combat host- encoded restriction systems. dZ-modified DNA can also "slow down" phage T5 exonuclease degradation, but it has no effect on RecBCD digestion. When two genes for dZ biosynthesis and one gene for dATP hydrolysis from Salmonella phage PMBT28 (purZ (adenylosuccinate synthetase), datZ (dATP triphosphohydrolase), and mazZ ((d)GTP-specific diphosphohydrolase) were cloned into E. coli plasmid, dZ incorporation level reached 19-20% dZ/(dZ+dA). dZ level can be further increased to 28.9-44.3% with co-expression of a DNA polymerase gene from the same phage. High level of dZ incorporation in recombinant plasmid is possible by co-expression of purZ, mazZ, datZ and phage DNA helicase, dpoZ (DNA polymerase) and ssb (single-stranded DNA binding protein SSB). This work has a general interest for molecular biologists working on dZ DNA modification and restriction systems. It provides a foundation for future research on screening dZ-dependent Type IV restriction systems. The results presented herein may have implication in gene therapy utilizing dZ-modified DNA, provided that human RNA polymerase variants can efficiently perform transcription from a dZ-modified template.

microbiology↗

bioRxiv2023

Simultaneous assessment of human genome and methylome data in a single experiment using limited deamination of methylated cytosine.

Multi-omics requires concerted recording of independent information, ideally from a single experiment. In this study, we introduce RIMS-seq2, a high-throughput technique to simultaneously sequence genomes and overlay methylation information while requiring only a small modification of the experimental protocol for high throughput DNA sequencing to include a controlled deamination step. Importantly, the rate of deamination of 5mC is negligible and thus, do not interfere with standard DNA sequencing and data processing. Thus, RIMS-seq2 libraries from whole or targeted genome sequencing show the same germline variation calling accuracy and sensitivity as compared to standard DNA-seq. Additionally, regional methylation levels provide an accurate map of the human methylome.

genomics↗

bioRxiv2023

Prokaryotic winged helix domains as dsDNA adenine methylation sensors

Winged helix (wH) domains, also termed winged helix-turn-helix (wHTH) domains, are widespread in all kingdoms of life, and have diverse roles. In the context of DNA binding and DNA modification sensing, some eukaryotic wH domains are known as sensors of non-methylated CpG. In contrast, the prokaryotic wH domains in DpnI and phi.HhiV4I act as sensors of adenine methylation in 6mApT (6mA = N6mA) context. DNA binding modes and interactions with the probed dinucleotide are vastly different in the two cases. Here, we show that the role of the wH domain as a sensor of adenine methylation is widespread in prokaryotes. We present previously uncharacterized examples of PD-(D/E)XK--wH (FcyTI, Psp4BI), PUA--wH--HNH (HtuIII, Hsa13891I), wH--GIY-YIG (Ahi29725I, Apa233I) and PLD--wH (Aba4572I, CbaI) fusion endonucleases that sense adenine methylation in the Dam G6mATC, and possibly other, slightly more relaxed contexts. Representatives of the wH domain endonuclease fusion families with the exception of the PLD--wH family could be purified, and an in vitro preference for adenine methylation in the Dam context could be demonstrated. Like most other MDREs, the new fusion endonucleases except those in the PD-(D/E)XK--wH family cleave close to, but outside the recognition sequence. Taken together, our data illustrate the widespread combinatorial use of prokaryotic wH domains as adenine methylation sensors.

molecular biology↗

bioRxiv2023

FFPE DNA shows two major error profiles derived from deamination of cytosine and methylcytosine that can be mitigated using distinct repair strategies.

Avoiding damage-induced sequencing errors is a critical step for the accurate identification of medium to rare frequency mutations in DNA samples. In the case of FFPE samples, deamination of cytosine moieties represents a major damage resulting in the loss of DNA material and sequencing errors. In this study, we demonstrated that, while damage from deamination of both cytosine and methylated cytosine moieties results in elevated C to T transition, the error profiles and mediation strategies are different and easily distinguishable. While damage-induced sequencing errors from cytosine deamination is driven by the end-repair step commonly used in NGS workflow, DNA damage resulting from deamination of methylated cytosine is another major contributor to sequencing errors at CpG sites. Uracil DNA glycosylase and human thymine DNA glycosylase can respectively eliminate and mitigate both damages in FFPE DNA samples, therefore increasing sequencing accuracy notably for the identification of moderate allelic frequency variants.

genomics↗

bioRxiv2023

Genomes of the human filarial parasites Mansonella perstans and Mansonella ozzardi

The filarial parasites Mansonella ozzardi and Mansonella perstans, causative agents of mansonellosis, infect hundreds of millions of people worldwide, yet remain among the most understudied of the human filarial pathogens. M. ozzardi is highly prevalent in Latin American countries and Caribbean Islands, while M. perstans is predominantly found in sub-Saharan Africa as well as in a few areas in South America. In addition to the differences in their geographical distribution, the two parasites are transmitted by different insect vectors, as well as exhibit differences in their responses to commonly used anthelminthic drugs. The lack of genome information has hindered investigations into the biology and evolution of Mansonella parasites and understanding the molecular basis of the clinical differences between species. In the current study, high quality genomes of two independent clinical isolates of M. perstans from Cameroon and two M. ozzardi isolates one from Brazil and one from Venezuela are reported. The genomes are approximately 76 Mb in size, encode about 10,000 genes each, and are largely complete based on BUSCO scores of about 90%, similar to other completed filarial genomes. These sequences represent the first genomes from Mansonella parasites and enabled a comparative genomic analysis of the similarities and differences between Mansonella and other filarial parasites. Horizontal DNA transfers (HDT) from mitochondria (nuMTs) as well as transfers from genomes of endosymbiotic Wolbachia bacteria (nuWTs) to the host nuclear genome were identified and analyzed. Sequence comparisons and phylogenetic analysis of known targets of anti-filarial drugs diethylcarbamazine (DEC), ivermectin and mebendazole revealed that all known target genes were present in both species, except for the DEC target encoded by gon-2 gene, which is fragmented in genome assemblies from both M. ozzardi isolates. These new reference genome sequences will provide a valuable resource for further studies on biology, symbiosis, evolution and drug discovery.

genomics↗

bioRxiv2022

Methyl-SNP-seq reveals dual readouts of methylome and variome at molecule resolution.

Covalent modifications of genomic DNA are crucial for most organisms to survive. Amplicon-based high throughput sequencing technologies erase all DNA modifications to retain only sequence information for the four canonical nucleobases, necessitating specialized technologies for ascertaining epigenetic information. To also capture base modification information, we developed Methyl-SNP-seq, a technology that takes advantage of the complementarity of the double helix to extract the methylation and original sequence information from a single DNA molecule. More specifically, Methyl-SNP-seq uses bisulfite conversion of one of the strands to identify cytosine methylation while retaining the sequence of the other strand. As both strands are locked together to link the dual readouts on a single paired-end read, Methyl-SNP-seq allows detecting methylation status of any DNA even without a reference genome. Because one of the strands retains the original 4 nucleotide composition, Methyl-SNP-seq can also be used in conjunction with standard sequence-specific probes for targeted enrichment and amplification. We demonstrate the usefulness of this technology in a broad spectrum of applications ranging from allele-specific methylation analysis in humans to identification of methyltransferase specificity in complex bacterial communities.

genomics↗

bioRxiv2022

Modification-Dependent Restriction Endonuclease-based sequencing method (EcoWI-seq) maps the genome-wide landscape of phosphorothioate modification at base resolution.

Phosphorothioation (PT), in which a non-bridging oxygen is replaced by a sulfur, is one of the rare modifications discovered in bacteria and archaea that occurs on the sugar-phosphate backbone as opposed to the nucleobase moiety of DNA. While PT modification is widespread in the prokaryotic kingdom, how PT modifications are distributed in the genomes and their exact roles in the cell remain to be defined. In this study, we developed a simple and convenient technique called EcoWI-seq based on a modification-dependent restriction endonuclease to identify genomic positions of PT modifications. EcoWI-seq shows similar performance than other PT modification detection techniques and additionally, is easily scalable while requiring little starting material. As a proof of principle, we applied EcoWI-seq to map at base resolution the PT modifications in the genomes of both the Salmonella enterica cerro 87 and E. coli expressing the dnd+ gene cluster. Specifically, we address whether the partial establishment of modified PT positions is a stochastic or deterministic process. EcoWI-seq reveals a systematic usage of the same subset of target sites in clones for which the PT modification has been independently established.

microbiology↗

bioRxiv2021

Nanopore ReCappable Sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.

genomics↗

bioRxiv2021

Reassembling a cannon in the DNA defense arsenal: genetics of StySA, a BREX phage exclusion system in Salmonella lab strains

Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA. Recent investigation has uncovered BREX systems, RM-like activities that employ host protection by DNA modification but replication arrest without evident nuclease action on unmodified phage DNA. We show that the historical stySA RM locus of Salmonella enterica sv Typhimurium is a BREX homolog. The stySA29 allele of the hybrid strain LB5000 carries a mutated version of the ancestral LT2 BREX system. Surprisingly, both a restriction and a methylation defect are observed for this lineage despite lack of mutations in brxX, the modification gene homolog. Instead, flanking genes pglZ and brxC each carry multiple mutations () in C-terminal domains. To avoid plasmid artifacts and potential stoichiometric interference, we chose to investigate this system in situ, replacing the mutated pglZ and brxC genes with wild type (WT). PglZ-WT supports methylation in the presence of either BrxC or BrxC-WT but not in the presence of a deletion/insertion allele, {Delta}brxC::cat. Restriction of phage L requires both BrxC-WT and PglZ-WT, implicating the BrxC C-terminus specifically in restriction activity. Disruption of four other CDS with cat cassettes still permitted modification, suggesting that BrxC, PglZ and BrxX are principal components of the modification activity. BrxL is required for restriction only. A partial disruption of brxL disrupts transcription globally.

genetics↗

bioRxiv2021

Genomes of Wolbachia endosymbionts from the human filarial parasites Mansonella perstans and Mansonella ozzardi

The intracellular endosymbiotic proteobacteria Wolbachia have evolved across the phyla nematoda and arthropoda. In Wolbachia phylogeny, supergroup F is the only clade known so far with members from both arthropod and filarial nematode hosts and therefore can provide unique insights into their evolution and biology. In this study, 4 new supergroup F Wolbachia genomes have been assembled using a metagenomic assembly and binning approach, wMoz and wMpe from the human filarial parasites Mansonella ozzardi and Mansonella perstans, and wOcae and wMoviF from the blue mason bee Osmia caerulescens and the sheep ked Melophagus ovinus respectively. A comprehensive phylogenomic analysis revealed two independent origins of filarial Wolbachia in supergroup F, most likely from ancestral arthropod hosts. The analysis also reveals that the switch from arthropod to filarial host is accompanied by a convergent pseudogenization and loss of the bacterioferritin gene, a phenomenon found to be shared by all filarial Wolbachia, even those outside supergroup F. These observations indicate that differences in heme metabolism might be a key feature distinguishing filarial and arthropod Wolbachia. The new genomes provide a valuable resource for further studies on symbiosis, evolution, and the discovery of new antibiotics to treat mansonellosis. Significance statementWolbachia are bacterial endosymbionts of medically important parasitic filarial nematodes and arthropods. The evolutionary history and biological roles of Wolbachia in these different hosts are not well understood. The supergroup F in Wolbachia phylogeny harbors members from both filarial and arthropod hosts, providing an unparalleled opportunity for genomic comparisons to uncover distinguishing characteristics. This study provides new genomes from filarial and arthropod Wolbachia from this unique supergroup. Their phylogenomic analysis reveals multiple, independent transfers of Wolbachia from arthropod to filariae. Remarkably, these transfers were associated with a convergent loss of the bacterioferritin gene, a key regulator of heme metabolism. Heme supplementation is considered a critical component of Wolbachia - filaria symbiosis. We demonstrate bacterioferritin loss is a novel feature exclusive to filarial Wolbachia.

genomics↗

bioRxiv2021

A Genome-Phenome Association study in native microbiomes identifies a mechanism for cytosine modification in DNA and RNA

Shotgun metagenomic sequencing is a powerful approach to study microbiomes in an unbiased manner and of increasing relevance for identifying novel enzymatic functions. However, the potential of metagenomics to relate from microbiome composition to function has thus far been underutilized. Here, we introduce the Metagenomics Genome-Phenome Association (MetaGPA) study framework, which allows to link genetic information in metagenomes with a dedicated functional phenotype. We applied MetaGPA to identify enzymes associated with cytosine modifications in environmental samples. From the 2365 genes that met our significance criteria, we confirm known pathways for cytosine modifications and proposed novel cytosine-modifying mechanisms. Specifically, we characterized and identified a novel nucleic acid modifying enzyme, 5-hydroxymethylcytosine carbamoyltransferase, that catalyzes the formation of a previously unknown cytosine modification, 5-carbamoyloxymethylcytosine, in DNA and RNA. Our work introduces MetaGPA as a novel and versatile tool for advancing functional metagenomics.

genomics↗

bioRxiv2021

Rapid Identification of Methylase Specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and Single Molecule, Real-Time (SMRT) Sequencing usually misses m5C. Here, we present a new method called RIMS-seq (Rapid Identification of Methylase Specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.

genomics↗

bioRxiv2020

Identification of high confidence human poly(A) RNA isoform scaffolds using nanopore sequencing

Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional methods the 5' and 3' ends of poly(A) RNA cannot be identified unambiguously. This is due in part to the architecture of the nanopore/enzyme-motor complex, and in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoform scaffolds among [~]4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5' m7G caps, we exchanged the biological cap for a modified cap attached to a 45-nucleotide oligomer. This oligomer adaptation method improved 5' end sequencing and ensured correct identification of the 5' m7G capped ends. Second, among these 5'-capped nanopore reads, we screened for ionic current signatures consistent with a 3' polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds, most of which (257,721) aligned to protein-coding genes. Of these, 4,876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data confirmed the validity of these high-confidence RNA scaffolds.

genomics↗