Unraveling the rate-limiting step in microorganisms' mediation of denitrification and phosphorus absorption/transport processes in a highly regulated river-lake system

River–lake ecosystems are indispensable hubs for water transfers and flow regulation engineering, which have frequent and complex artificial hydrological regulation processes, and the water quality is often unstable. Microorganisms usually affect these systems by driving the nutrient cycling process. Thus, understanding the key biochemical rate-limiting steps under highly regulated conditions was critical for the water quality stability of river–lake ecosystems. This study investigated how the key microorganisms and genes involving nitrogen and phosphorus cycling contributed to the stability of water by combining 16S rRNA and metagenomic sequencing using the Dongping river–lake system as the case study. The results showed that nitrogen and phosphorus concentrations were significantly lower in lake zones than in river inflow and outflow zones (p < 0.05). Pseudomonas, Acinetobacter, and Microbacterium were the key microorganisms associated with nitrate and phosphate removal. These microorganisms contributed to key genes that promote denitrification (nirB/narG/narH/nasA) and phosphorus absorption and transport (pstA/pstB/pstC/pstS). Partial least squares path modeling (PLS-PM) revealed that environmental factors (especially flow velocity and COD concentration) have a significant negative effect on the key microbial abundance (p < 0.001). Our study provides theoretical support for the effective management and protection of water transfer and the regulation function of the river–lake system.