Daims, H. | bioRxiv Science

bioRxiv2017

Cultivation and genomic analysis of Candidatus Nitrosocaldus islandicus, a novel obligately thermophilic ammonia-oxidizing Thaumarchaeon

Ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaea are the only known aerobic ammonia oxidizers in geothermal environments. Although molecular data indicate the presence of phylogenetically diverse AOA from the Nitrosocaldus clade, group 1.1b and group 1.1a Thaumarchaea in terrestrial high-temperature habitats, only one enrichment culture of an AOA thriving above 50 {degrees}C has been reported and functionally analyzed. In this study, we physiologically and genomically characterized a novel Thaumarchaeon from the deep-branching Nitrosocaldaceae family of which we have obtained a high ([~]85 %) enrichment from biofilm of an Icelandic hot spring (73 {degrees}C). This AOA, which we provisionally refer to as \"Candidatus Nitrosocaldus islandicus\", is an obligately thermophilic, aerobic chemolithoautotrophic ammonia oxidizer, which stoichiometrically converts ammonia to nitrite at temperatures between 50 {degrees}C and 70 {degrees}C. Ca. N. islandicus encodes the expected repertoire of enzymes proposed to be required for archaeal ammonia oxidation, but unexpectedly lacks a nirK gene and also possesses no identifiable other enzyme for nitric oxide (NO) generation. Nevertheless, ammonia oxidation by this AOA appears to be NO-dependent as Ca. N. islandicus is, like all other tested AOA, inhibited by the addition of an NO scavenger. Furthermore, comparative genomics revealed that Ca. 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-oxidizing Thaumarchaea. Collectively, our findings suggest that metabolic versatility and DNA replication might differ substantially between obligately thermophilic and other AOA.

microbiology↗

bioRxiv2016

AmoA-targeted polymerase chain reaction primers for the specific detection and quantification of comammox Nitrospira in the environment

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be catalyzed by the concerted activity of ammonia and nitrite-oxidizing microorganisms. Only recently, complete ammonia oxidizers ( comammox), which oxidize ammonia to nitrate on their own, were identified in the bacterial genus Nitrospira, previously assumed to contain only canonical nitrite oxidizers. Nitrospira are widespread in nature, but for assessments of the distribution and functional importance of comammox Nitrospira in ecosystems, cultivation-independent tools to distinguish comammox from strictly nitrite-oxidizing Nitrospira are required. Here we developed new PCR primer sets that specifically target the amoA genes coding for subunit A of the distinct ammonia monooxygenase of comammox Nitrospira. While existing primers capture only a fraction of the known comammox amoA diversity, the new primer sets cover as much as 95% of the comammox amoA clade A and 92% of the clade B sequences in a reference database containing 326 comammox amoA genes with sequence information at the primer binding sites. Application of the primers to 13 samples from engineered systems (a groundwater well, drinking water treatment and wastewater treatment plants) and other habitats (rice paddy and forest soils, rice rhizosphere, brackish lake sediment and freshwater biofilm) detected comammox Nitrospira in all samples and revealed a considerable diversity of comammox in most habitats. Excellent primer specificity for comammox amoA was achieved by avoiding the use of highly degenerate primer preparations and by using equimolar mixtures of oligonucleotides that match existing comammox amoA genes. Quantitative PCR with these equimolar primer mixtures was highly sensitive and specific, and enabled the efficient quantification of clade A and clade B comammox amoA gene copy numbers in environmental samples. The measured relative abundances of comammox Nitrospira, compared to canonical ammonia oxidizers, were highly variable across environments. The new comammox amoA-targeted primers will enable more encompassing studies of nitrifying microorganisms in diverse ecosystems.

microbiology↗