New insights into functional divergence and adaptive evolution of uncultured bacteria in anammox community by complete genome-centric analysis.

Anaerobic ammonium-oxidation (anammox) bacteria play a crucial role in global nitrogen cycling and wastewater nitrogen removal, but they share symbiotic relationships with various other microorganisms. Functional divergence and adaptive evolution of uncultured bacteria in anammox community remain underexplored. Although shotgun metagenomics based on short reads has been widely used in anammox research, metagenome-assembled genomes (MAGs) are often discontinuous and highly contaminated, which limits in-depth analyses of anammox communities. Here, for the first time, we performed Pacific Biosciences high-fidelity (HiFi) long-read sequencing on the anammox granule sludge sample from a lab-scale bioreactor, and obtained 30 accurate and complete metagenome-assembled genomes (cMAGs). These cMAGs were obtained by selecting high-quality circular contigs from initial assemblies of long reads generated by HiFi sequencing, eliminating the need for Illumina short reads, binning, and reassembly. One new anammox species affiliated with Candidatus Jettenia and three species affiliated with novel families were found in this anammox community. cMAG-centric analysis revealed functional divergence in general and nitrogen metabolism among the anammox community members, and they might adopt a cross-feeding strategy in organic matter, cofactors, and vitamins. Furthermore, we identified 63 mobile genetic elements (MGEs) and 50 putative horizontal gene transfer (HGT) events within these cMAGs. The results suggest that HGT events and MGEs related to phage and integration or excision, particularly transposons containing tnpA in anammox bacteria, might play important roles in the adaptive evolution of this anammox community. The cMAGs generated in the present study could be used to establish of a comprehensive database for anammox bacteria and associated microorganisms. These findings highlight the advantages of HiFi sequencing for the studies of complex mixed cultures and advance the understanding of anammox communities.