Nitrogen fixation and assimilation processes are vital to the functioning of any ecosystem. Nevertheless, studying these processes using 15N-based stable isotope probing was so far limited because of technical challenges related to the relative rarity of nitrogen in nucleic acids and proteins compared to carbon, and because of its absence in lipids. However, the recent adoption of high-throughput sequencing and statistical modelling methods to SIP studies increased the sensitivity of the method and enabled overcoming some of the challenges. This chapter describes in detail how to perform DNA- and RNA-SIP using 15N.

• MeSH
• bakteriální RNA chemie   genetika   izolace a purifikace   metabolismus   MeSH
• bakterie fixující dusík genetika   metabolismus   MeSH
• centrifugace - gradient hustoty MeSH
• DNA bakterií chemie   genetika   izolace a purifikace   metabolismus   MeSH
• fixace dusíku genetika   fyziologie   MeSH
• izotopové značení metody   MeSH
• izotopy dusíku metabolismus   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Niche specialization of nitrifying prokaryotes is usually studied with tools targeting molecules involved in the oxidation of ammonia and nitrite. The ecological significance of diverse CO2 fixation strategies used by nitrifiers is, however, mostly unexplored. By analyzing autotrophy-related genes in combination with amoA marker genes based on droplet digitial PCR and CARD-FISH counts targeting rRNA, we quantified the distribution of nitrifiers in eight stratified lakes. Ammonia oxidizing (AO) Thaumarchaeota using the 3-hydroxypropionate/4-hydroxybutyrate pathway dominated deep and oligotrophic lakes, whereas Nitrosomonas-related taxa employing the Calvin cycle were important AO bacteria in smaller lakes. The occurrence of nitrite oxidizing Nitrospira, assimilating CO2 with the reductive TCA cycle, was strongly correlated with the distribution of Thaumarchaeota. Recently discovered complete ammonia-oxidizing bacteria (comammox) belonging to Nitrospira accounted only for a very small fraction of ammonia oxidizers (AOs) present at the study sites. Altogether, this study gives a first insight on how physicochemical characteristics in lakes are associated to the distribution of nitrifying prokaryotes with different CO2 fixation strategies. Our investigations also evaluate the suitability of functional genes associated with individual CO2 assimilation pathways to study niche preferences of different guilds of nitrifying microorganisms based on an autotrophic perspective.

• MeSH
• amoniak metabolismus   MeSH
• Archaea klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• autotrofní procesy MeSH
• bakterie fixující dusík klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• dusitany metabolismus   MeSH
• jezera mikrobiologie   MeSH
• koloběh uhlíku * genetika   MeSH
• oxidace-redukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH