Most in silico evolutionary studies commonly assumed that core genes are essential for cellular function, while accessory genes are dispensable, particularly in nutrient-rich environments. However, this assumption is seldom tested genetically within the pangenome context. Here we conducted a robust pangenomic Tn-seq analysis of fitness genes in a nutrient-rich medium for Sinorhizobium strains with a canonical open pangenome. The Hidden Markov Model-based method is the most robust to variations among mutant libraries per strain and non-sensitive to data size. Fitness genes, categorized as essential (ES), advantage (GA) and disadvantage (GD) for growth, are enriched in core genes, while nonessential genes (NE) are over-represented in accessory genes. Connectivity degrees in predicted protein-protein interaction networks decline in the order of ES, GA, GD and NE, and those in the co-fitness network decreases from ES to GA/GD to NE. Notably, 1599 out of 3284 core genes display differential essentiality across test strains. Within pangenome core, both shared quasi-essential (ES and GA) and strain-dependent fitness genes are enriched in similar functional categories. Our analysis demonstrates a considerable fuzzy essential zone determined by co-fitness connectivity degrees in Sinorhizobium pangenome, and highlights the power of co-fitness network in understanding genetic basis of ever-increasing prokaryotic pangenome data.

The main findings of this study were that we clarified that the roles and essentiality of nodD1 and nolA are host dependent. Importantly, for the first time, NolA was found to positively regulate T3SS effector gene nopP to mediate incompatibility on mung bean.