Active sludge microcosms with and without BrdU addition

Most microorganisms cannot be cultured in isolation, necessitating sophisticatedmethods for studying their (eco)physiology. While numerous approaches can probethe activity of given microbes in enrichment cultures, no single technique can rendersimultaneous data on both metabolic capacities and mobile genetic elements. Here,we apply long-read sequencing to monitor the incorporation of non-canonical basesin genome-resolved metagenomic datasets and elucidate the replication patterns ofboth bacteria and phages. This technology enables the simultaneous reconstructionof both prokaryotic and viral genomes (alongside genomics downstream analyseslike metabolic predictions), in addition to providing information regarding theirreplication in enrichment cultures. By spiking the base analog5-bromo-2'-deoxyuridine (BrdU) into activated sludge microcosms, we determinedthat 114 of the 118 high-quality genomes recovered were actively replicating inenrichment cultures from activated sludge and identified both slow (low BrdUincorporation and change in abundance) and rapidly replicating organisms (highBrdU incorporation and change in abundance). Some of the genomes detectedexhibited regions rich in BrdU that were predicted to represent prophages in theirlytic cycle. Ultimately, this novel means of monitoring the replication responses ofmicrobes, and deciphering their genomes and active mobile genetic elements willadvance and empower strategies aimed at isolating previously uncultivatedmicrobes in pure culture.