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.

Two mesophilic, neutrophilic and aerobic marine ammonia-oxidizing archaea, designated strains NF5 T and D3C T , 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 NF5 T 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 MK 6 : 0 was the major respiratory quinone. Both isolates gained energy by oxidizing ammonia (NH 3) to nitrite (NO 2-) and used bicarbonate as a carbon source. Strain D3C T was able use urea as a source of ammonia for energy production and growth. Addition of hydrogen peroxide (H 2 O 2) scavengers (catalase or a-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 NF5 T and D3C T 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 EN76 T. 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 NF5 T (=JCM 32270 T =NCIMB 15114 T) and D3C T (=JCM 32271 T =DSM 106147 T =NCIMB 15115 T) are type strains of the species Nitrosopumilus adriaticus sp. nov. and Nitrosopumilus piranensis sp. nov., respectively.

The potential link between the microbial dynamics and the environmental parameters was investigated in a semi-enclosed and highly dynamic coastal system (Gulf of Trieste, northern Adriatic Sea, NE Mediterranean Sea). Our comprehensive 2-year time-series study showed that despite the shallowness of this area, there was a significant difference between the surface and the bottom bacterial community structure. The bottom bacterial community was more diverse than the surface one and influenced by sediment re-suspension. The surface seawater temperature had a profound effect on bacterial productivity, while the bacterial community structure was more affected by freshwater-borne nutrients and phytoplankton blooms. Phytoplankton blooms caused an increase of Gammaproteobacteria (Alteromonadaceae, SAR86 and Vibrionaceae) and shift in dominance from SAR11 to Rhodobacteraceae taxon at the surface. Our results propose the importance of the water mass movements as drivers of freshwater-borne nutrients and of allochthonous microbial taxa. This study emphasizes the prediction power based on association networks analyses that are fed with long-term measurements of microbial and environmental parameters. These interaction maps offer valuable insights into the response of marine ecosystem to climate- and anthropogenic-driven stressors.

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