From gene to structure: Unraveling genomic dark matter inCa. Accumulibacter

Abstract Candidatus Accumulibacter is a unique and pivotal genus of polyphosphate-accumulating organisms (PAOs) prevalent in wastewater treatment plants, and plays mainstay roles in the global phosphorus cycle. Whereas, the efforts toward a complete understanding of their genetic and metabolic characteristics are largely hindered by major limitations in existing sequence-based annotation methods, leaving more than half of their protein-encoding genes unannotated. To address the challenge, we developed a comprehensive approach integrating pangenome analysis, gene-based protein structure and function prediction, and metatranscriptomic analysis, extending beyond the constraints of sequence-centric methodologies. The application to Ca. Accumulibacter allowed the establishment of the pan-Ca. Accumulibacter proteome structure database, providing references for >200,000 proteins. Benchmarking on 28 Ca. Accumulibacter genomes showed major increases in the average annotation coverage from 51% to 83%. Genetic and metabolic characteristics that had eluded exploration via conventional methods were unraveled. For instance, the identification of a previously unknown phosphofructokinase gene suggests that all Ca. Accumulibacter encoded a complete Embden-Meyerhof-Parnas pathway. A previously defined homolog of phosphate-specific transport system accessory protein (PhoU) was actually an inorganic phosphate transport (Pit) accessory protein, regulating Pit instead of the high-affinity phosphate transport (Pst), a key to the emergence of the polyphosphate-accumulating trait of Ca. Accumulibacter. Additional lineage members were found encoding complete denitrification pathways. This study offers a readily usable and transferable tool for the establishment of high-coverage annotation reference databases for diverse cultured and uncultured bacteria, facilitating the exploration and understanding of genomic dark matter in the bacterial domain. Synopsis A integrated and advanced approach unraveling key genomic dark matter in Ca. Accumulibacter and readily applicable to diverse bacteria for customerized high-coverage annotation reference database establishment Competing Interest Statement The authors have declared no competing interest.