Site-2 protease-like protein 2 mediates phenol tolerance in Rhodococcus ruber through transcriptomic and functional...

Organic compounds such as phenol are highly toxic to most microorganisms, as they disrupt membrane stabilization and cellular functions. In the tolerant Rhodococcus ruber SD3 (R. ruber), a previously identified site-2 protease like protein 1 (S2PLP1) was associated with phenol tolerance, although its mechanism remained unclear. Here, through bioinformatic and membrane localization analysis, we identified putative site-2 protease like protein 2 (S2PLP2) as a hydrophobic transmembrane protein that harbored conserved HExxH (where H represents histidine, E represents glutamic acid, and x denotes any amino acid) and LDG motifs, two peptidase_M50 domains (PFAM02163), and two CBS domains. Heterologous expression and purification of S2PLP2 enabled the generation of specific antibodies. Western blot analysis demonstrated a significant upregulation of S2PLP2 expression under phenol stress. Overexpression of s2plp2 enhanced bacterial growth rates at 0.02% and 0.04% phenol concentrations. Transcriptomic profiling of the overexpression strain identified 682 upregulated and 944 downregulated genes. Key upregulated functional categories included efflux pumps, membrane-stabilizing proteins, oxidative stress defense systems, proteostasis maintenance factors, and energy metabolism pathways. To elucidate protein-protein interactions, we employed GST pull-down combined with mass spectrometry and identified 316 potential binding partners, among which 22 were single-pass transmembrane proteins. Notably, the SPFH/Band7/PHB domain-containing protein (SBP, UniProt ID: A0A098BIA4) showed the strongest binding affinity to S2PLP2(H68C), with a K_d value of 625 ± 4.42 nM as determined by microscale thermophoresis assays. These findings provide new insights into the biological role of S2PLP2 in Rhodococcus ruber.