Isolating and characterizing translationally active fraction of anammox microbiota using bioorthogonal non-canonical amino acid tagging

Deciphering active and inactive populations is crucial to the understanding of how microorganisms function in their native environment. In this study, we propose a rapid, sensitive and cost-effective approach to isolating and characterizing translationally active and inactive fractions of anammox consortia using a combination of bioorthogonal non-canonical amino acid tagging (BONCAT), fluorescence activated cell sorting (FACS) and 16S rRNA gene sequencing methods. The approach can detect translationally active anammox bacteria as early as after 8 h incubation in the presence of 50 µM homopropargylglycine (HPG). Overall, the relative abundance of translationally active taxa obtained by BONCAT displayed good correlation with measurement from RNA-based method, while the active community structure of the two methods was distinct from each other. Integration of the two approaches will be particularly useful for providing insight into the active members of microbial communities. Based on BONCAT-method, we firstly report that about 70% cells of anammox biomass were translationally active, and the community structure of the active fraction is significantly distinct from that of the inactive fraction. The fold-change differences between translationally active and inactive fractions demonstrated population-wide and taxon-specific translational heterogeneity within anammox consortia. Surprisingly, more than a quarter of anammox bacteria were translationally inactive even in suitable habitat, which may be attributed to the fiercely intraspecies competition when density-dependent resources become limited. Together, our BONCAT application offers new insight into the ecophysiology of anammox consortia and lays the foundation for expanding the approach into other biological wastewater treatment systems.