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

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. Also, a closed, circular genome of cable bacteria has not been published yet. To address this, we performed Nanopore long-read and Illumina short-read 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.

. Genome comparison metrics for cable bacteria MAGs. ANI using BLAST (ANIb) values and genome alignment fraction between all selected MAGs are presented. MAGs recovered in this study as well as relevant phylogenetic groups are highlighted.   Figure S4. Unique gene fractions for cable bacteria MAGs recovered in this study, compared to all cable bacteria reference MAGs (HQ and MQ), at different identity thresholds for gene clustering. "-excluded" refers to the unique gene fraction that was calculated by excluding a specified MAG or MAG group from gene clustering. Figure S5. Distribution of the NhaA Na+/H+ antiporter in cable bacteria genomes. A) Phylogenetic tree for NhaA protein sequences, recovered from high-quality cable bacteria MAGs, which were subsetted to species representatives. Different clades of NhaA sequences have been denoted in the tree. B) Phylogenetic tree for cable bacteria MAGs used in A alongside NhaA clade presence and absence status.       Habitat Intertidal zone (coastal habitat)

Miscellaneous, extraordinary features relevant for the description
Single-contig MAG achieved after read subsetting and re-assembly Sulfur metabolism and electron transport. All cable bacteria MAGs encode all key genes for the 2-step process, which includes an initial sulfide oxidation and subsequent sulfur disproportionation to sulfide and sulfate. These steps are mediated by a sulfide-quinone reductase (SQR), a polysulfide reductase (PSR) and the reversed dissimilatory sulfate reduction (DSR) pathway (Dataset S1). Homologs of a cytoplasmic rhodanese and the sulfur transferases TusA, likely involved in sulfur transport across the cytoplasmic membrane 1 , were also encoded by all MAGs (Dataset S1). The resulting sulfite is oxidized to sulfate by adenosine-5-phosphosulfate reductase (AprAB) and sulfate adenylyltransferase (Sat) mediate sulfite oxidation to sulfate, which is then transported out of the cell by the sulfate permase SulP (Dataset S1).
Electrons released in the process are hypothesized to be transferred to the quinone pool in the cytoplasmic membrane by the heterodisulfide reductase (DsrMK) and the quinonemodifying oxidoreductase (QmoABC) membrane complexes and all genes encoding these proteins were detected in all the MAGs (Dataset S1). According to the proposed model for LDET, electrons are transferred to the periplasm by either a cytochrome bc-like complexe, formed by a Rieske Fe-S domain protein with an adjacent membrane-bound cytochrome bdomain protein, and/or a homolog of CydA, subunit of the membrane-bound bd quinol oxidase. The potential for both these modules was encoded in the MAGs, while no complete terminal cytochrome bd-II oxidase was detected in the genomes (Dataset S1). Soluble periplasmic c-type cytochromes are then hypothesized to transfer the electrons to the conductive structure of the cable bacteria, also showed experimentally with Raman microspectroscopy 2 , and the genomes encode several c-type cytochromes, for instance homologs for the periplasmic cytochrome c peroxidase (Ccp), the ferrocytochrome c-552 (Cyt), or the diheme cytochrome MacA 1 (Dataset S1). The MAGs encode also for genes associated with type IV pili, including PilA, which could be part of the conductive fiber 1,3 (Dataset S1). The genome of Ca. Electrothrix laxa encodes all four subunits of the membranebound cytochrome c oxidase (Cco), with ~60% amino acid sequence identity to homologs in other Desulfobulbaceae species and most likely resulting of horizontal gene transfer, as previously observed for its close relative Ca. Electrothrix communis 1 . The other cable bacteria genomes did not encode membrane bound cytochrome oxidases, but homologs of the truncated hemoglobin proposed to participate in oxygen metabolism are present in all the MAGs 1 (Dataset S1).
Additionally, all the cable bacteria MAGs encode the NAD(P)H-quinone oxidoreductase complex (Nuo), excluding the NuoEFG subunits, and and F-type ATPase, to exploit the proton motive force (Dataset S1), as previously observed 1,3 .
Carbon metabolism and storage compounds. Cable bacteria are also known for the potential to fix CO2 and CO2 uptake has been previously experimentally confirmed 1 . All the MAGs encode the full Wood-Ljungdahl pathway and a periplasmic carbonic anhydrase (Cah), which converts bicarbonate into CO2 (Dataset S1). Additionally, the linked hdrABC genes encoding for the heterodisulfide reductase (HdrABC), potentially involved in CO2 fixation 1 , are encoded by Ca. Electrothrix laxa and Ca. Electronema halotolerans, and linked to a formate dehydrogenase (Dataset S1), and immediately followed by a homolog of mvhD encoding a methyl-viologen-reducing hydrogenase. Several pairs of HdrA-MvhD or HdrC-MvhD homologs are also observed in all the cable bacteria MAGs and are hypothesized to act as electron transfer modules in cytoplasmic redox reactions 1 . Additional carbon sources may be formate, which transport can be facilitated by a putative formate transporter (focA) and assimilated via the Wood-Ljungdahl pathway, or acetate, taken up by the symporter ActP and assimilated by the enzyme acetyl-CoA synthetase (Acs). The presence of polyphosphate (poly-P) granules in cable bacteria has been proven experimentally by microscopy or nanoSIMS and they are potentially involved as protection from oxidative stress and/or as a resource to support motility 1,4 . All the cable bacteria encode the genes for polyphosphate storage, including the phosphate transporters PstABC and Pit, and the polyphosphate kinases ppk1 and ppk2 (Dataset S1). Furthermore, all the MAGs encode the genetic potential for production and degradation of glycogen storages, which can be used as carbon and energy resource, as well as being involved in oxygen detoxification 1 .