AbstractCable 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.
AbstractCable bacteria of theDesulfobulbaceaefamily are centimeter-long filamentous bacteria, which are capable of conducting long-distance electron transfer. Currently, all cable bacteria are classified into two candidate genera:CandidatusElectronema, typically found in freshwater environments, andCandidatusElectrothrix, 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 ofCa. 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 nameCa. Electronema halotolerans andCa. Electrothrix laxa, respectively. TheCa. 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 howCa. Electronema halotolerans may be the evolutionary link between marine and freshwater cable bacteria lineages.
ABSTRACT
Nitrification is a key process of the biogeochemical nitrogen cycle and of biological wastewater treatment. The second step, nitrite oxidation to nitrate, is catalyzed by phylogenetically diverse, chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Uncultured NOB from the genus “
Candidatus
Nitrotoga” are widespread in natural and engineered ecosystems. Knowledge about their biology is sparse, because no genomic information and no pure “
Ca
. Nitrotoga” culture was available. Here we obtained the first “
Ca
. Nitrotoga” isolate from activated sludge. This organism, “
Candidatus
Nitrotoga fabula,” prefers higher temperatures (>20°C; optimum, 24 to 28°C) than previous “
Ca
. Nitrotoga” enrichments, which were described as cold-adapted NOB. “
Ca
. Nitrotoga fabula” also showed an unusually high tolerance to nitrite (activity at 30 mM NO
2
−
) and nitrate (up to 25 mM NO
3
−
). Nitrite oxidation followed Michaelis-Menten kinetics, with an apparent
K
m
(
K
m
(app)
) of ~89 µM nitrite and a
V
max
of ~28 µmol of nitrite per mg of protein per h. Key metabolic pathways of “
Ca
. Nitrotoga fabula” were reconstructed from the closed genome. “
Ca
. Nitrotoga fabula” possesses a new type of periplasmic nitrite oxidoreductase belonging to a lineage of mostly uncharacterized proteins. This novel enzyme indicates (i) separate evolution of nitrite oxidation in “
Ca
. Nitrotoga” and other NOB, (ii) the possible existence of phylogenetically diverse, unrecognized NOB, and (iii) together with new metagenomic data, the potential existence of nitrite-oxidizing archaea. For carbon fixation, “
Ca
. Nitrotoga fabula” uses the Calvin-Benson-Bassham cycle. It also carries genes encoding complete pathways for hydrogen and sulfite oxidation, suggesting that alternative energy metabolisms enable “
Ca
. Nitrotoga fabula” to survive nitrite depletion and colonize new niches.
IMPORTANCE
Nitrite-oxidizing bacteria (NOB) are major players in the biogeochemical nitrogen cycle and critical for wastewater treatment. However, most NOB remain uncultured, and their biology is poorly understood. Here, we obtained the first isolate from the environmentally widespread NOB genus “
Candidatus
Nitrotoga” and performed a detailed physiological and genomic characterization of this organism (“
Candidatus
Nitrotoga fabula”). Differences between key phenotypic properties of “
Ca
. Nitrotoga fabula” and those of previously enriched “
Ca
. Nitrotoga” members reveal an unexpectedly broad range of physiological adaptations in this genus. Moreover, genes encoding components of energy metabolisms outside nitrification suggest that “
Ca
. Nitrotoga” are ecologically more flexible than previously anticipated. The identification of a novel nitrite-oxidizing enzyme in “
Ca
. Nitrotoga fabula” expands our picture of the evolutionary history of nitrification and might lead to discoveries of novel nitrite oxidizers. Altogether, this study provides urgently needed insights into the biology of understudied but environmentally and biotechnologically important microorganisms.