Exploring the upper pH limits of nitrite oxidation: diversity, ecophysiology, and adaptive traits of haloalkalitolerantNitrospira

Abstract

Nitrite-oxidizing bacteria of the genus Nitrospira are key players of the biogeochemical nitrogen cycle. However, little is known about their occurrence and survival strategies in extreme pH environments. Here, we report on the discovery of physiologically versatile, haloalkalitolerant Nitrospira that drive nitrite oxidation at exceptionally high pH. Nitrospira distribution, diversity, and ecophysiology were studied in hypo-and subsaline, highly alkaline (pH 8.9-10.3) lakes by amplicon sequencing, metagenomics, and cultivation-based approaches. Surprisingly, diverse Nitrospira populations from lineages I, II and IV were found. Furthermore, the ability of Nitrospira enrichment cultures to oxidize nitrite at neutral to highly alkaline pH of 10.5 was demonstrated. Metagenomic analysis of a newly enriched Nitrospira lineage IV species, "Candidatus Nitrospira alkalitolerans", revealed numerous adaptive features of this organism to its extreme environment. Among them were a sodiumdependent N-type ATPase and NADH:quinone oxidoreductase next to the proton-driven forms usually found in Nitrospira. Other functions aid in pH and cation homeostasis and osmotic stress defense. "Ca. Nitrospira alkalitolerans" also possesses group 2a and 3b [NiFe] hydrogenases, suggesting it can use hydrogen as alternative energy source. These results reveal how Nitrospira cope with strongly fluctuating pH and salinity conditions and expand our knowledge of nitrogen cycling in extreme habitats. 2 25 30 35 40 45 50 55 60 enrichments.

Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira marina and its proteomic response to oxygen-limited conditions

“…This feature is shared with the closely related Ca. N. alkalitolerans [9] and with the more distantly related marine NOB Nitrospina gracilis [55], but absent in all other thus far sequenced Nitrospira species. In addition to the canonical H + -translocating F 1 F O -ATPase (complex V), N. marina also encodes a putative alternative Na + -translocating N-ATPase (Table S5), which potentially contributes to the maintenance of the membrane potential and the generation of a sodium motive force (SMF) as suggested for Ca.…”

“…In addition to the canonical H + -translocating F 1 F O -ATPase (complex V), N. marina also encodes a putative alternative Na + -translocating N-ATPase (Table S5), which potentially contributes to the maintenance of the membrane potential and the generation of a sodium motive force (SMF) as suggested for Ca. N. alkalitolerans [9]. Furthermore, a H + -translocating pyrophosphatase (H + -PPase) with homology to Leptospira/protozoan/plant-type enzymes [56] was identified.…”

“…Nitrospina gracilis 3/211 and Nitrococcus mobilis Nb-231 [55,59], Ca. N. alkalitolerans [9] and in comammox Nitrospira [14,28] (Table S4). In addition to catalyzing the reversible, NAD + -dependent oxidation of hydrogen, these so-called sulfhydrogenases are able to reduce elemental sulfur (S o ) or polysulfides to hydrogen sulfide (H S) [60].…”

“…Two of these putative BicA-like transporters are co-localized with genes encoding Na + /H + antiporters (NhaB family), which could drive the uptake of HCO 3 via Na + extrusion under alkaline conditions as suggested for the cyanobacterium Aphanothece halophytica [62] and Ca. N. alkalitolerans [9]. N. marina also encodes one putative SulP-related bicarbonate transporter fused to a carbonic anhydrase and four genes encoding putative alpha, beta and gamma carbonic anhydrases (Table S5), which can convert the imported HCO 3 to CO 2 for inorganic carbon fixation via the rTCA cycle.…”

“…The genus Nitrospira consists of at least six phylogenetic sublineages and is the most diverse NOB genus [3]. Nitrospira are ubiquitously present in natural and engineered ecosystems, including oceans [4,5], freshwater habitats [6], soils [7,8], saline-alkaline lakes [9], hot springs [10], wastewater treatment plants [11][12][13], and aquaculture biofilters [14,15]. In human-made ecosystems, Nitrospira is generally considered to be adapted to low NO 2 concentrations [16].…”