AbstractMost autotrophs use the Calvin–Benson–Bassham (CBB) cycle for carbon fixation. In contrast, all currently described autotrophs from the Campylobacterota (previously Epsilonproteobacteria) use the reductive tricarboxylic acid cycle (rTCA) instead. We discovered campylobacterotal epibionts (“CandidatusThiobarba”) of deep-sea mussels that have acquired a complete CBB cycle and may have lost most key genes of the rTCA cycle. Intriguingly, the phylogenies of campylobacterotal CBB cycle genes suggest they were acquired in multiple transfers from Gammaproteobacteria closely related to sulfur-oxidizing endosymbionts associated with the mussels, as well as from Betaproteobacteria. We hypothesize that “Ca. Thiobarba” switched from the rTCA cycle to a fully functional CBB cycle during its evolution, by acquiring genes from multiple sources, including co-occurring symbionts. We also found key CBB cycle genes in free-living Campylobacterota, suggesting that the CBB cycle may be more widespread in this phylum than previously known. Metatranscriptomics and metaproteomics confirmed high expression of CBB cycle genes in mussel-associated “Ca. Thiobarba”. Direct stable isotope fingerprinting showed that “Ca. Thiobarba” has typical CBB signatures, suggesting that it uses this cycle for carbon fixation. Our discovery calls into question current assumptions about the distribution of carbon fixation pathways in microbial lineages, and the interpretation of stable isotope measurements in the environment.
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.