Daims, Holger

Nitrobacterstrain NHB1 is a nitrite-oxidising bacterium previously co-enriched with the neutrophilic ammonia-oxidising bacteriumNitrosospiraAHB1, a consortium that nitrifies in acidic conditions in co-culture. Here we characterise the growth of the isolateNitrobacterstrain NHB1 as a function of pH and nitrite (NO2-) concentration, and its influence on the activity of acidophilic soil ammonia-oxidising archaea (AOA). NHB1 is acidotolerant and grows optimally at pH 6.0 (range 5.0 - 7.5) at initial NO2-concentrations of 500 μM. However, the optimum decreases to pH 5.0 at lower initial NO2-concentrations typically found in soil, with detectable growth down to pH 3.5. NHB1 has a comparatively high affinity for NO2-with an apparent-half-saturation constant (54 μM) one order of magnitude lower than its closest relative, the neutrophilic strainNitrobacter hamburgensisX14. In co-culture, NHB1 enhances the growth of acidophilic AOA. Specifically,Nitrosotalea devaniterraeNd1 andNitrosotalea sinensisNd2 are sensitive to NO2--derived compounds and only oxidise ~200-300 μM ammonia (NH3) in batch cultures. However, in co-culture with NHB1, pH ranges were lowered by ~0.5 pH units and both strains could oxidise up to 3 mM NH3, only limited by buffering capacity. NHB1 possesses a cyanase facilitating reciprocal cross-feeding via generating cyanate-derived NH3and utilising AOA-derived NO2-. Removal of NO2-is likely crucial for the growth of nitrifiers in acidic soils and this study highlights the importance of considering substrate and metabolic product concentrations when characterising physiology. Genome analysis reveals NHB1 is distinct from validated species and the name "Nitrobacter laanbroekii" is proposed.

Abstract Nitrospirales, including the genus Nitrospira, are environmentally widespread chemolithoautotrophic nitrite-oxidizing bacteria. These mostly uncultured microorganisms gain energy through nitrite oxidation, fix CO2, and thus play vital roles in nitrogen and carbon cycling. Over the last decade, our understanding of their physiology has advanced through several new discoveries, such as alternative energy metabolisms and complete ammonia oxidizers (comammox Nitrospira). These findings mainly resulted from studies of terrestrial species, whereas less attention has been given to marine Nitrospirales. In this study, we cultured three new marine Nitrospirales enrichments and one isolate. Three of these four NOB represent new Nitrospira species while the fourth represents a novel genus. This fourth organism, tentatively named “Ca. Nitronereus thalassa”, represents the first cultured member of a Nitrospirales lineage that encompasses both free-living and sponge-associated nitrite oxidizers, is highly abundant in the environment, and shows distinct habitat distribution patterns compared to the marine Nitrospira species. Partially explaining this, “Ca. Nitronereus thalassa” harbors a unique combination of genes involved in carbon fixation and respiration, suggesting differential adaptations to fluctuating oxygen concentrations. Furthermore, “Ca. Nitronereus thalassa” appears to have a more narrow substrate range compared to many other marine nitrite oxidizers, as it lacks the genomic potential to utilize formate, cyanate, and urea. Lastly, we show that the presumed marine Nitrospirales lineages are not restricted to oceanic and saline environments, as previously assumed.

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.

AbstractAmmonia-oxidizing archaea (AOA) within the phylumThaumarchaeaare the only known aerobic ammonia oxidizers in geothermal environments. Although molecular data indicate the presence of phylogenetically diverse AOA from theNitrosocaldusclade, group 1.1b and group 1.1aThaumarchaeain terrestrial high-temperature habitats, only one enrichment culture of an AOA thriving above 50 °C has been reported and functionally analyzed. In this study, we physiologically and genomically characterized a novelThaumarchaeonfrom the deep-branchingNitrosocaldaceaefamily of which we have obtained a high (∼85 %) enrichment from biofilm of an Icelandic hot spring (73 °C). This AOA, which we provisionally refer to as “CandidatusNitrosocaldus islandicus”, is an obligately thermophilic, aerobic chemolithoautotrophic ammonia oxidizer, which stoichiometrically converts ammonia to nitrite at temperatures between 50 °C and 70 °C.Ca.N. islandicus encodes the expected repertoire of enzymes proposed to be required for archaeal ammonia oxidation, but unexpectedly lacks anirKgene and also possesses no identifiable other enzyme for nitric oxide (NO) generation. Nevertheless, ammonia oxidation by this AOA appears to be NO-dependent asCa.N. islandicus is, like all other tested AOA, inhibited by the addition of an NO scavenger. Furthermore, comparative genomics revealed thatCa.N. islandicus has the potential for aromatic amino acid fermentation as its genome encodes an indolepyruvate oxidoreductase(iorAB)as well as a type 3b hydrogenase, which are not present in any other sequenced AOA. A further surprising genomic feature of this thermophilic ammonia oxidizer is the absence of DNA polymerase D genes - one of the predominant replicative DNA polymerases in all other ammonia-oxidizingThaumarchaea.Collectively, our findings suggest that metabolic versatility and DNA replication might differ substantially between obligately thermophilic and other AOA.