DIA-Based Quantitative Proteomics Reveals Adaptive Responses and Potential Mechanisms of Se(IV) Resistance in Rhodococcus qingshengii PM1

Microbial reduction of soluble selenium oxyanions is a sustainable strategy for remediating selenium-contaminated environments, yet the molecular mechanisms underlying selenite tolerance in the genus Rhodococcus remain poorly understood. In this study, we investigated the proteomic adaptation of the highly tolerant strain Rhodococcus qingshengii PM1 under high-concentration selenite stress (50 mM Na₂SeO₃) using a data-independent acquisition (DIA)-based quantitative proteomics approach. A total of 3335 proteins were identified, and 3310 proteins were retained for downstream analysis. Comparative proteomics revealed 1411 differentially expressed proteins, including 972 upregulated and 439 downregulated proteins in the selenite-treated group. These changes indicate extensive systems-level proteomic reprogramming and support a growth–defense trade-off strategy. Strain PM1 strongly upregulated ferredoxin and multiple respiratory-chain- and oxidoreductase-associated proteins, suggesting a ferredoxin-associated electron-transfer network that may contribute to Se(IV) transformation and intracellular redox adjustment. In parallel, proteins involved in sulfur assimilation, cysteine/methionine and selenocompound metabolism, ergothioneine biosynthesis, GSH-associated metabolism, Trx/MSH thiol-redox systems, peroxidase/Ohr-Prx detoxification, metalloid/oxyanion resistance, urease-associated pH adaptation, DNA repair, and cell-envelope remodeling were induced, indicating activation of multilayered defense and homeostasis mechanisms. Conversely, proteins associated with central carbon metabolism, carbohydrate uptake, and ribosome-dependent translation were repressed, suggesting reduced growth investment and energy conservation under severe selenite pressure. Overall, this study provides a systems-level proteomic framework for understanding Se(IV) resistance in R. qingshengii PM1 and identifies candidate targets for future functional validation, strain engineering, and selenium/metal(loid) bioremediation.