Biosynthesis of selenium-enriched proteins with distinct selenium incorporation patterns in Bifidobacterium longum by reshaping protein quality control and sulfur metabolic pathways.

The UGA-independent substitution of methionine (Met) and cysteine (Cys) with their selenium (Se) analogues, selenomethionine (SeMet) and selenocysteine (Sec), represents a non-canonical but widespread pathway for the biosynthesis of selenium-enriched proteins. Although well-documented across prokaryotes and eukaryotes, the associated cellular adaptive strategies and phenotypes remain poorly understood. Here, we investigated these substitution patterns and their functional consequences in Bifidobacterium longum (B. longum), a probiotic bacterium that adapts efficiently to high Se stress. Using high-resolution mass spectrometry, we systematically identified and compared SeMet and Sec incorporation sites within the B. longum proteome under Se-enriched conditions. SeMet incorporation proved extensive, substituting over 90% of Met residues, with limited cellular damage. Ribosomal proteins exhibited the highest SeMet incorporation, which did not significantly alter the translational rate. In contrast, Sec incorporation was markedly restricted, characterized by significantly fewer substitution sites and lower substitution proportions. This restriction, accompanied by severely delayed bacterial growth, indicates a profound state of cellular stress, which was further corroborated by the upregulation of protein quality control machinery and the remodeling of sulfur metabolism pathways. However, a subset of proteins with a high probability of Sec incorporation remained, primarily found in catalytic enzymes, yet not localized within their active sites. Notably, both SeMet and Sec incorporations occurred preferentially in high-abundance proteins, without distinct sequence preferences. This work provides the first systematic comparison of SeMet and Sec incorporation patterns in a bacterial proteome, establishing a framework to analyze noncanonical Se incorporation and the specific adaptation strategies bacteria employ against environmental Se challenges.