Nitrosopumilus adriaticus sp. nov. and Nitrosopumilus piranensis sp. nov., two ammonia-oxidizing archaea from the Adriatic Sea and members of the class Nitrososphaeria.

Two mesophilic, neutrophilic and aerobic marine ammonia-oxidizing archaea, designated strains NF5T and D3CT, were isolated from coastal surface water of the Northern Adriatic Sea. Cells were straight small rods 0.20-0.25 µm wide and 0.49-2.00 µm long. Strain NF5T possessed archaella as cell appendages. Glycerol dibiphytanyl glycerol tetraethers with zero to four cyclopentane moieties (GDGT-0 to GDGT-4) and crenarchaeol were the major core lipids. Menaquinone MK6 : 0 was the major respiratory quinone. Both isolates gained energy by oxidizing ammonia (NH3) to nitrite (NO2-) and used bicarbonate as a carbon source. Strain D3CT was able use urea as a source of ammonia for energy production and growth. Addition of hydrogen peroxide (H2O2) scavengers (catalase or α-keto acids) was required to sustain growth. Optimal growth occurred between 30 and 32 °C, pH 7.1 and 7.3 and between 34 and 37‰ salinity. The cellular metal abundance ranking of both strains was Fe>Zn>Cu>Mn>Co. The genomes of strains NF5T and D3CT have a DNA G+C content of 33.4 and 33.8 mol%, respectively. Phylogenetic analyses of 16S rRNA gene sequences revealed that both strains are affiliated with the class Nitrososphaeria, sharing ~85 % 16S rRNA gene sequence identity with Nitrososphaera viennensis EN76T. The two isolates are separated by phenotypic and genotypic characteristics and are assigned to distinct species within the genus Nitrosopumilus gen. nov. according to average nucleotide identity thresholds of their closed genomes. Isolates NF5T (=JCM 32270T =NCIMB 15114T) and D3CT (=JCM 32271T =DSM 106147T =NCIMB 15115T) are type strains of the species Nitrosopumilusadriaticus sp. nov. and Nitrosopumiluspiranensis sp. nov., respectively.

Evaluation of the reliability of maize reference assays for GMO quantification.

A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize.

Physiological and genomic characterization of two novel marine thaumarchaeal strains indicates niche differentiation.

Ammonia-oxidizing Archaea (AOA) are ubiquitous throughout the oceanic water column; however, our knowledge on their physiological and ecological diversity in different oceanic regions is rather limited. Here, we report the cultivation and characterization of two novel Nitrosopumilus strains, originating from coastal surface waters of the Northern Adriatic Sea. The combined physiological and genomic information revealed that each strain exhibits different metabolic and functional traits, potentially reflecting contrasting life modes. Strain NF5 contains many chemotaxis-related genes and is able to express archaella, suggesting that it can sense and actively seek favorable microenvironments such as nutrient-rich particles. In contrast, strain D3C is non-motile and shows higher versatility in substrate utilization, being able to use urea as an alternative substrate in addition to ammonia. Furthermore, it encodes a divergent, second copy of the AmoB subunit of the key enzyme ammonia monooxygenase, which might have an additional catalytic function and suggests further metabolic versatility. However, the role of this gene requires further investigation. Our results provide evidence for functional diversity and metabolic versatility among phylogenetically closely related thaumarchaeal strains, and point toward adaptations to free-living versus particle-associated life styles and possible niche differentiation among AOA in marine ecosystems.