Codon usage optimization contributes to evolutionary dynamics of TetM following its acquisition via interspecies and intergeneric recombination events in recipient bacteria

Abstract Background Tetracycline resistance in bacteria is predominantly mediated by the tetM gene, which exhibits an exceptionally broad distribution across Gram-positive and Gram-negative bacteria. Although the molecular mechanism underlying tetM-mediated resistance is well characterized, the evolutionary forces shaping the tetM gene—particularly the relative roles of purifying selection, episodic positive selection, interspecies and intergeneric recombination, and post-transfer codon usage adaptation—remain incompletely understood or uncertain. To address these gaps, we performed a comprehensive evolutionary analysis of tetM across large natural bacterial populations. Results We analyzed 2,838 GenBank-deposited tetM sequences, representing 409 distinct allelic types spanning a wide range of bacterial species and genera. Neutrality and diversity analyses revealed moderate polymorphism (ps = 0.2526), a mildly negative Tajima’s D (–0.268), and low d N /d S ratios (~ 0.15), collectively indicating strong pervasive purifying selection. Codon-based likelihood tests (PAML and HyPhy) detected no evidence of widespread positive selection across the full tetM dataset; however, when analyses were restricted to phylogenetically coherent subsets, episodic diversifying selection affecting a small fraction of codons (~ 4.1%) was detected, indicating lineage-specific adaptation. Conserved-region mapping revealed pronounced conservation of functionally critical GTPase-associated motifs, including GTP/Mg²⁺ binding and G4 elements. The Switch I and Switch II regions exhibited greater sequence tolerance, consistent with preserved structural flexibility. Linkage disequilibrium patterns, allelic network structure, and phylogenetic analyses collectively provided strong evidence for extensive interspecies and intergeneric recombination involving both internal tetM loci and the entire gene. Identical tetM alleles were shared across phylogenetically distant taxa, including a large spectrum of human and animal pathogens, as well as commensal and environmental bacteria, with mammalian gut-associated species serving as key reservoirs. Codon usage analyses further demonstrated that post-transfer adaptation of tetM is not uniform: significant synonymous convergence toward host-preferred codons at fourfold-degenerate sites was observed in multiple recipient lineages (P ≤ 0.043), indicating codon optimization across this gene. Conclusions The evolution of tetM is governed by strong functional constraint, episodic lineage-specific diversification, and frequent recombination-mediated dissemination, including whole-gene transfer. Host-specific codon usage adaptation is suggested to contribute to functional integration and long-term persistence of tetM, facilitating the widespread maintenance of tetracycline resistance across diverse bacterial populations.