Context. A member of the candidate phylum radiation (CPR) of bacteria was recently shown to lyse cells of Gordonia amarae which is known to cause foaming in wastewater treatment plants(Batinovic et al. 2021). The ability to kill G. amarae suggests that CPR bacteria could potentially be used as a biocontrol agent to destabilize wastewater foams. However, the diversity, distribution and host range of CPR bacteria in wastewater treatment plants is not well understood, in part due to the poor performance of common primers used for amplicon-based monitoring. CPR bacteria escape general primer-based detection due to the high divergence of their 16S rRNA gene sequences. Here, we designed and tested primers that extended the diversity of CPR bacteria detectable by amplicon surveys.
Aim. The development of improved primers that show a broad coverage of CPR bacteria to monitor their community composition in wastewater treatment plants.
Methods. Universal primers 341F/806R were modified for CPR bacteria. Amplicon coverage was assessed in-silico with metagenome-assembled genomes (MAGs) constructed from combined Illumina and Nanopore sequencing of an activated sludge sample collected from a wastewater treatment plant in Klosterneuburg, Austria. Amplicon sequencing with the V3-V4 region was performed on the same activated sludge sample using both original and modified primers.
Findings. In silico analysis predicted that 75% of the CPR MAGs in the activated sludge were covered by general V3-V4 primers and 92.5% were covered by the modified primers. On activated sludge samples, newly designed primers amplified a broader range of CPR bacteria than the general V3-V4 primers, in particular Microgenomatia and Parcubacteria. (Figure).
Utilization. The newly designed primers will allow efficient identification, characterization, and surveillance of populations of CPR bacteria in wastewater treatment plants, providing a robust basis to further explore the various functions of these conspicuous microbes in wastewater treatment.
