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Abstract Terrestrial organic matter (tOM) plays a critical role in aquatic ecosystems, influencing carbon processes and greenhouse gas emissions. Here, we investigate the impact of tOM on methane production in littoral and pelagic sediments from the Mississippi River headwaters using a microcosm approach. Contrary to our expectations, tOM addition universally increased methane production across lentic sediments, with no significant difference between littoral and pelagic zones. Methane production was influenced by select sediment microorganisms, primarily methanogens and lignocellulose degrading bacteria, which responded similarly across different sediment habitats. The study highlights the role of cytochrome-containing methanogens and their syntrophic relationships with fermentative bacteria, emphasizing the significance of microbial community structure in sediment methane dynamics. Our findings suggest that increasing tOM loads to freshwater systems could have broader implications for methane emissions, driven by specific microbial interactions. Author Contribution StatementHMS and TLH conceived the study and obtained the funds. HMS led fieldwork and microcosm set-up. HMS and LAD analyzed gas samples and HMS performed the data analysis and graphical representation of the results. HMS wrote the first draft of the manuscript, and all authors contributed significantly to the preparation of the final draft. Scientific Significance StatementAs human activities and climate change increase the amount of organic material entering lakes and rivers, understanding the effects this has on greenhouse gas emissions is crucial. Our study reveals that adding terrestrial organic matter to freshwater sediments universally boosts methane production, a potent greenhouse gas. Through the exploration of microbial communities responsible for this process, our research highlights how changes in terrestrial organic matter export to aquatic systems could increase methane emissions from sediments. Data Availability StatementAdditional Supporting Information can be found in the online version of this article, including an extended version of methods and supplementary tables. Sequencing data associated with this paper is available on NCBI, BioProject PRJNA1164797. 

ABSTRACT Transformation, the uptake of DNA directly from the environment, is a major driver of gene flow in microbial populations. In bacteria, DNA uptake requires a nuclease that processes dsDNA to ssDNA, which is subsequently transferred into the cell and incorporated into the genome. However, the process of DNA uptake in archaea is still unknown. Previously, we cataloged genes essential to natural transformation inMethanococcus maripaludis, but few homologs of bacterial transformation‐associated genes were identified. Here, we characterize one gene, MMJJ_16440 (named here asecnA), to be an extracellular nuclease. We show that EcnA is Ca²⁺‐activated, present on the cell surface, and essential for transformation. While EcnA can degrade several forms of DNA, the highest activity was observed with ssDNA as a substrate. Activity was also observed with circular dsDNA, suggesting that EcnA is an endonuclease. This is the first biochemical characterization of a transformation‐associated protein in a member of the archaeal domain and suggests that both archaeal and bacterial transformation initiate in an analogous fashion. 

Abstract Natural transformation, the process whereby a cell acquires DNA directly from the environment, is an important driver of evolution in microbial populations, yet the mechanism of DNA uptake is only characterized in bacteria. To expand our understanding of natural transformation in archaea, we undertook a genetic approach to identify a catalog of genes necessary for transformation inMethanococcus maripaludis. Using an optimized method to generate random transposon mutants, we screened 6144 mutant strains for defects in natural transformation and identified 25 transformation-associated candidate genes. Among these are genes encoding components of the type IV-like pilus, transcription/translation associated genes, genes encoding putative membrane bound transport proteins, and genes of unknown function. Interestingly, similar genes were identified regardless of whether replicating or integrating plasmids were provided as a substrate for transformation. Using allelic replacement mutagenesis, we confirmed that several genes identified in these screens are essential for transformation. Finally, we identified a homolog of a membrane bound substrate transporter inMethanoculleus thermophilusand verified its importance for transformation using allelic replacement mutagenesis, suggesting a conserved mechanism for DNA transfer in multiple archaea. These data represent an initial characterization of the genes important for transformation which will inform efforts to understand gene flow in natural populations. Additionally, knowledge of the genes necessary for natural transformation may assist in identifying signatures of transformation machinery in archaeal genomes and aid the establishment of new model genetic systems for studying archaea.