Closed genomes uncover a saltwater species ofCandidatusElectronema and shed new light on the boundary between marine and freshwater cable bacteria

Abstract

Cable bacteria of the Desulfobulbaceae family are centimeter-long filamentous bacteria, which are capable of conducting long-distance electron transfer. Currently, all cable bacteria are classified into two candidate genera: Candidatus Electronema, typically found in freshwater environments, and Candidatus Electrothrix, typically found in saltwater environments. This taxonomic framework is based on both 16S rRNA gene sequences and metagenome-assembled genome (MAG) phylogenies. However, most of the currently available MAGs are highly fragmented, incomplete, and thus likely miss key genes essential for deciphering the physiology of cable bacteria. To address this, we performed Nanopore long read (total 162.4 Gbp) and Illumina short read (total 148.3 Gbp) shotgun sequencing of selected environmental samples and a single-strain enrichment of Ca. Electronema aureum. We recovered multiple cable bacteria MAGs, including two circular and one single-contig. Phylogenomic analysis, also confirmed by 16S rRNA gene-based phylogeny, classified one circular MAG and the single-contig MAG as novel species of cable bacteria, which we propose to name Ca. Electronema halotolerans and Ca. Electrothrix laxa, respectively. The Ca. Electronema halotolerans, despite belonging to the previously recognized freshwater genus of cable bacteria, was retrieved from brackish-water sediment. Metabolic predictions showed several adaptations to a high salinity environment, similar to the ''saltwater'' Ca. Electrothrix species, indicating how Ca. Electronema halotolerans may be the evolutionary link between marine and freshwater cable bacteria lineages.

Dancing the Nanopore limbo – Nanopore metagenomics from small DNA quantities for bacterial genome reconstruction

“…In this study, we report how lower than recommended DNA input affects Nanopore long-read metagenomics by comparing different levels of metagenomic analysis from read QC to retrieval of MAGs. Many previously published Nanopore-based metagenomics studies lack information about the quantity of DNA used for library preparation [19,[24][25][26]32]. To the best of our knowledge, other published metagenomic studies quantified the amount of input DNA and met or exceeded the recommended amount of 1000 ng DNA for input material.…”

“…Examples for such regions are highly repetitive and/or conserved elements including multiple copies of the same transposable element in one genome [20], for which short read-based de novo assemblies are more likely to fail. Long-read metagenomics has successfully been used to for cMAG recovery from diverse samples, including activated sludge [21,22], water bodies [23], sediment [24] . CC-BY-NC-ND 4.0 International license perpetuity.…”

Extracellular electron transfer genes expressed by candidate flocking bacteria in cable bacteria sediment

“…16S rRNA amplicon sequences from all three time series experiments are available at NCBI in the Sequence Read Archive (SRA) and can be found under accession number PRJNA837365 and PRJNA1058976. Metagenomic data is available at the NCBI database under accession number PRJNA730231 (Bjerg et al, 2023) and from ENA under bio project number PRJEB52550 (Sereika et al, 2022). Metatranscriptomic data is available from the NCBI SRA-database under accession number PRJNA1057540.…”

Recovery of novel microbial genomes

“…Despite these efforts, the processed cable bacteria samples still contain other microorganisms and thus metagenomics is currently the only option for studying cable bacteria DNA. Even though MAGs for cable bacteria have been recovered from multiple environmental sources 161,166,167 , no closed or even singlecontig cable bacteria genomes have been published prior to Paper 2, likely due to reliance on short reads for assembling the metagenomes 168 .…”

“…The aim of Paper 2 was to apply long-read Nanopore sequencing for the recovery of cable bacteria MAGs, with an emphasis on recovering single-contig, circular MAGs 168 . Three different samples were sequenced, which were expected to contain members of both cable bacteria candidate genera: the freshwater-based Ca.…”