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Assessing horizontal gene transfer in the rhizosphere of Brachypodium distachyon using fabricated ecosystems (EcoFABs)

ABSTRACT Horizontal gene transfer (HGT) is a major process by which genes are transferred between microbes in the rhizosphere. However, examining HGT remains challenging due to the complexity of mimicking conditions within the rhizosphere. Fabricated ecosystems (EcoFABs) have been used to investigate several complex processes in plant-associated environments. Here we show that EcoFABs are efficient tools to examine and measure HGT frequency in the rhizosphere. We provide the first demonstration of gene transfer via a triparental conjugation system in the Brachypodium distachyon rhizosphere in an EcoFAB using Pseudomonas putida KT2440 as both donor and recipient bacterial strain with the donor containing a mobilizable and non-self-transmissible plasmid. We observed that the frequency of plasmid transfer in the rhizosphere is potentially dependent on the plant developmental stage and the composition and amount of root exudates. The frequency of plasmid transfer also increased with higher numbers of donor cells. We demonstrate the transfer of plasmid from P. putida to another B. distachyon root colonizer, Burkholderia sp. OAS925, showing HGT within a rhizosphere microbial community. Environmental stresses also influenced the rate and efficiency of HGT in the rhizosphere between different species and genera. This study provides a robust workflow to evaluate transfer of engineered plasmids in the rhizosphere when such plasmids are potentially introduced in a field or other plant-associated environments. IMPORTANCE We report the use of EcoFABs to investigate the HGT process in a rhizosphere environment. It highlights the potential of EcoFABs in recapitulating the dynamic rhizosphere conditions as well as their versatility in studying plant-microbe interactions. This study also emphasizes the importance of studying the parameters impacting the HGT frequency. Several factors such as plant developmental stages, nutrient conditions, number of donor cells, and environmental stresses influence gene transfer within the rhizosphere microbial community. This study paves the way for future investigations into understanding the fate and movement of engineered plasmids in a field environment.

Priya, Shweta↗

Diverse fates of ancient horizontal gene transfers in extremophilic red algae

Abstract Horizontal genetic transfer (HGT) is a common phenomenon in eukaryotic genomes. However, the mechanisms by which HGT‐derived genes persist and integrate into other pathways remain unclear. This topic is of significant interest because, over time, the stressors that initially favoured the fixation of HGT may diminish or disappear. Despite this, the foreign genes may continue to exist if they become part of a broader stress response or other pathways. The conventional model suggests that the acquisition of HGT equates to adaptation. However, this model may evolve into more complex interactions between gene products, a concept we refer to as the ‘Integrated HGT Model’ (IHM). To explore this concept further, we studied specialized HGT‐derived genes that encode heavy metal detoxification functions. The recruitment of these genes into other pathways could provide clear examples of IHM. In our study, we exposed two anciently diverged species of polyextremophilic red algae from the Galdieria genus to arsenic and mercury stress in laboratory cultures. We then analysed the transcriptome data using differential and coexpression analysis. Our findings revealed that mercury detoxification follows a ‘one gene‐one function’ model, resulting in an indivisible response. In contrast, the ars H gene in the arsenite response pathway demonstrated a complex pattern of duplication, divergence and potential neofunctionalization, consistent with the IHM. Our research sheds light on the fate and integration of ancient HGTs, providing a novel perspective on the ecology of extremophiles.

59 BASIC BIOLOGICAL SCIENCES↗

Catabolic pathway acquisition by rhizosphere bacteria readily enables growth with a root exudate component but does not affect root colonization

Horizontal gene transfer (HGT) is a fundamental evolutionary process that plays a key role in bacterial evolution. The likelihood of a successful transfer event is expected to depend on the precise balance of costs and benefits resulting from pathway acquisition. Most experimental analyses of HGT have focused on phenotypes that have large fitness benefits under appropriate selective conditions, such as antibiotic resistance. However, many examples of HGT involve phenotypes that are predicted to provide smaller benefits, such as the ability to catabolize additional carbon sources. We have experimentally simulated the consequences of one such HGT event in the laboratory, studying the effects of transferring a pathway for catabolism of the plant-derived aromatic compound salicyl alcohol between rhizosphere isolates from the Pseudomonas genus. We find that pathway acquisition enables rapid catabolism of salicyl alcohol with only minor disruptions to the existing metabolic and regulatory networks of the new host. However, this new catabolic potential does not confer a measurable fitness advantage during competitive growth in the rhizosphere. We conclude that the phenotype of salicyl alcohol catabolism is readily transferable but is selectively neutral under environmentally relevant conditions. We propose that this condition is common and that HGT of many pathways will be self-limiting because the selective benefits are small.

59 BASIC BIOLOGICAL SCIENCES↗

Toxin-Antitoxin Systems Reflect Community Interactions Through Horizontal Gene Transfer

Bacterial evolution through horizontal gene transfer (HGT) reflects their community interactions. In this way, HGT networks do well at mapping community interactions, but offer little toward controlling them—an important step in the translation of synthetic strains into natural contexts. Toxin–antitoxin (TA) systems serve as ubiquitous and diverse agents of selection; however, their utility is limited by their erratic distribution in hosts. Here we examine the heterogeneous distribution of TAs as a consequence of their mobility. By systematically mapping TA systems across a 10,000 plasmid network, we find HGT communities have unique and predictable TA signatures. We propose these TA signatures arise from plasmid competition and have further potential to signal the degree to which plasmids, hosts, and phage interact. To emphasize these relationships, we construct an HGT network based solely on TA similarity, framing specific selection markers in the broader context of bacterial communities. This work both clarifies the evolution of TA systems and unlocks a common framework for manipulating community interactions through TA compatibility.

59 BASIC BIOLOGICAL SCIENCES↗

Genetic Transfer in Action: Uncovering DNA Flow in an Extremophilic Microbial Community

ABSTRACT Horizontal genetic transfer (HGT) is a significant driver of genomic novelty in all domains of life. HGT has been investigated in many studies however, the focus has been on conspicuous protein‐coding DNA transfers that often prove to be adaptive in recipient organisms and are therefore fixed longer‐term in lineages. These results comprise a subclass of HGTs and do not represent exhaustive (coding and non‐coding) DNA transfer and its impact on ecology. Uncovering exhaustive HGT can provide key insights into the connectivity of genomes in communities and how these transfers may occur. In this study, we use the term frequency‐inverse document frequency (TF‐IDF) technique, that has been used successfully to mine DNA transfers within real and simulated high‐quality prokaryote genomes, to search for exhaustive HGTs within an extremophilic microbial community. We establish a pipeline for validating transfers identified using this approach. We find that most DNA transfers are within‐domain and involve non‐coding DNA. A relatively high proportion of the predicted protein‐coding HGTs appear to encode transposase activity, restriction‐modification system components, and biofilm formation functions. Our study demonstrates the utility of the TF‐IDF approach for HGT detection and provides insights into the mechanisms of recent DNA transfer.

Microbiology↗

Genomics discovery of giant fungal viruses from subsurface oceanic crustal fluids

Abstract The oceanic igneous crust is a vast reservoir for microbial life, dominated by diverse and active bacteria, archaea, and fungi. Archaeal and bacterial viruses were previously detected in oceanic crustal fluids at the Juan de Fuca Ridge (JdFR). Here we report the discovery of two eukaryotic Nucleocytoviricota genomes from the same crustal fluids by sorting and sequencing single virions. Both genomes have a tRNATyr gene with an intron (20 bps) at the canonical position between nucleotide 37 and 38, a common feature in eukaryotic and archaeal tRNA genes with short introns (<100 bps), and fungal genes acquired through horizontal gene transfer (HGT) events. The dominance of Ascomycota fungi as the main eukaryotes in crustal fluids and the evidence for HGT point to these fungi as the putative hosts, making these the first putative fungi-Nucleocytoviricota specific association. Our study suggests active host-viral dynamics for the only eukaryotic group found in the subsurface oceanic crust and raises important questions about the impact of viral infection on the productivity and biogeochemical cycling in this ecosystem.

Bhattacharjee, Ananda S.↗

Ancient Rapid Radiation Explains Most Conflicts Among Gene Trees and Well-Supported Phylogenomic Trees of Nostocalean Cyanobacteria

Prokaryotic genomes are often considered to be mosaics of genes that do not necessarily share the same evolutionary history due to widespread horizontal gene transfers (HGTs). Consequently, representing evolutionary relationships of prokaryotes as bifurcating trees has long been controversial. However, studies reporting conflicts among gene trees derived from phylogenomic data sets have shown that these conflicts can be the result of artifacts or evolutionary processes other than HGT, such as incomplete lineage sorting, low phylogenetic signal, and systematic errors due to substitution model misspecification. Here, we present the results of an extensive exploration of phylogenetic conflicts in the cyanobacterial order Nostocales, for which previous studies have inferred strongly supported conflicting relationships when using different concatenated phylogenomic data sets. We found that most of these conflicts are concentrated in deep clusters of short internodes of the Nostocales phylogeny, where the great majority of individual genes have low resolving power. We then inferred phylogenetic networks to detect HGT events while also accounting for incomplete lineage sorting. Our results indicate that most conflicts among gene trees are likely due to incomplete lineage sorting linked to an ancient rapid radiation, rather than to HGTs. Moreover, the short internodes of this radiation fit the expectations of the anomaly zone, i.e., a region of the tree parameter space where a species tree is discordant with its most likely gene tree. In this work, we demonstrated that concatenation of different sets of loci can recover up to 17 distinct and well-supported relationships within the putative anomaly zone of Nostocales, corresponding to the observed conflicts among well-supported trees based on concatenated data sets from previous studies. Our findings highlight the important role of rapid radiations as a potential cause of strongly conflicting phylogenetic relationships when using phylogenomic data sets of bacteria. We propose that polytomies may be the most appropriate phylogenetic representation of these rapid radiations that are part of anomaly zones, especially when all possible genomic markers have been considered to infer these phylogenies.

59 BASIC BIOLOGICAL SCIENCES↗

Reference-free structural variant detection in microbiomes via long-read co-assembly graphs

Motivation: The study of bacterial genome dynamics is vital for understanding the mechanisms underlying microbial adaptation, growth, and their impact on host phenotype. Structural variants (SVs), genomic alterations of 50 base pairs or more, play a pivotal role in driving evolutionary processes and maintaining genomic heterogeneity within bacterial populations. While SV detection in isolate genomes is relatively straightforward, metagenomes present broader challenges due to the absence of clear reference genomes and the presence of mixed strains. In response, our proposed method rhea, forgoes reference genomes and metagenome-assembled genomes (MAGs) by encompassing all metagenomic samples in a series (time or other metric) into a single co-assembly graph. The log fold change in graph coverage between successive samples is then calculated to call SVs that are thriving or declining. Results: We show rhea to outperform existing methods for SV and horizontal gene transfer (HGT) detection in two simulated mock metagenomes, particularly as the simulated reads diverge from reference genomes and an increase in strain diversity is incorporated. We additionally demonstrate use cases for rhea on series metagenomic data of environmental and fermented food microbiomes to detect specific sequence alterations between successive time and temperature samples, suggesting host advantage. Our approach leverages previous work in assembly graph structural and coverage patterns to provide versatility in studying SVs across diverse and poorly characterized microbial communities for more comprehensive insights into microbial gene flux.

59 BASIC BIOLOGICAL SCIENCES↗

Horizontal Gene Transfer and CRISPR Targeting Drive Phage-Bacterial Host Interactions and Coevolution in “Pink Berry” Marine Microbial Aggregates

Phages, which are viruses that infect bacteria, are important components of all microbial systems, in which they drive the turnover of organic matter by lysing host cells, facilitate horizontal gene transfer (HGT), and coevolve with their bacterial hosts. Bacteria resist phage infection, which is often costly or lethal, through a diversity of mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗

Convergent evolution and horizontal gene transfer in Arctic Ocean microalgae

Microbial communities in the world ocean are affected strongly by oceanic circulation, creating characteristic marine biomes. The high connectivity of most of the ocean makes it difficult to disentangle selective retention of colonizing genotypes (with traits suited to biome specific conditions) from evolutionary selection, which would act on founder genotypes over time. The Arctic Ocean is exceptional with limited exchange with other oceans and ice covered since the last ice age. To test whether Arctic microalgal lineages evolved apart from algae in the global ocean, we sequenced four lineages of microalgae isolated from Arctic waters and sea ice. Here we show convergent evolution and highlight geographically limited HGT as an ecological adaptive force in the form of PFAM complements and horizontal acquisition of key adaptive genes. Notably, ice-binding proteins were acquired and horizontally transferred among Arctic strains. A comparison with Tara Oceans metagenomes and metatranscriptomes confirmed mostly Arctic distributions of these IBPs. The phylogeny of Arctic-specific genes indicated that these events were independent of bacterial-sourced HGTs in Antarctic Southern Ocean microalgae.

59 BASIC BIOLOGICAL SCIENCES↗

Plasmid-Encoded Traits Vary across Environments

Plasmids are well known for their role in the transmission of antibiotic resistance-conferring genes. Beyond human and clinical settings, however, they disseminate many other types of genes, including those that contribute to microbially driven ecosystem processes.

59 BASIC BIOLOGICAL SCIENCES↗