Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from Mycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of M. smegmatis in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.

替代核糖体亚基蛋白在不同细菌物种的基因组中普遍存在，但其功能意义尚存争议。研究微生物核糖体异质性的尝试主要依赖于将野生型菌株与缺失了亚基的突变体进行比较，但是这种方法不能直接比较从相同细胞环境中分离出来的其他核糖体同工型。在此，通过同时从耻垢分枝杆菌中纯化规范性和替代性RpsR核糖体，我们证明了替代核糖体与其规范对应物相比具有独特的翻译特征。替代核糖体和规范核糖体均积极参与蛋白质合成，尽管它们通过核糖体谱分析以不同的效率翻译基因的子集。我们还显示，替代核糖体在起始复合物形成中具有相对缺陷。此外，缺失了替代核糖体蛋白操纵子的耻垢分枝杆菌菌株在贫铁培养基中生长较差，从而揭示了替代核糖体在铁稳态中的作用。我们的工作证实了替代细菌核糖体在适应敌对环境方面的独特和非冗余贡献。