Hierarchical evolution of the proteobacterial essential chaperone SecB reveals non-neutral synonymous evolution under extreme purifying selection

SecB is an essential cytosolic chaperone that mediates post-translational protein export through the bacterial Sec pathway. Although the secB gene evolves under strong purifying selection, its synonymous and higher-order sequence evolution have not been examined in a lineage-resolved, integrative framework across the Proteobacteria phylum. A comprehensive comparative analysis of secB codon usage and selection patterns was conducted using broad proteobacterial sampling. Compositional metrics (GC1/GC2/GC3), neutrality plots, effective number of codons–GC3 relationships, relative synonymous codon usage, parity rule 2 bias, dinucleotide observed/expected ratios, codon-pair organization, codon adaptation index (CAI), and codon-based maximum-likelihood models were integrated within a unified analytical framework. Across all classes, ω values were uniformly < 1, confirming pervasive purifying selection at the protein level. Lineage-dependent modulation of selection intensity, with significant relaxation in Alphaproteobacteria and intensification in Gammaproteobacteria was also detected. GC3 exhibited substantial variability, whereas GC1 and GC2 remained tightly constrained, indicating buffering of amino-acid–encoding positions. CAI was significantly associated with the effective number of codons and GC3, supporting lineage-structured synonymous organization consistent with mutation–selection balance. Dinucleotide biases were conserved but varied in magnitude among the proteobacterial classes. These findings demonstrate that secB evolution is characterized by stable protein-level constraints combined with lineage-modulated synonymous and contextual organization. This integrative case analysis refines understanding of essential-gene evolution and generates experimentally testable predictions regarding translational robustness and synonymous constraint across proteobacterial lineages.