Project Summary Evolutionarily distant bacterial species respond to zinc starvation by reprogramming their ribosome assembly, in which the constitutive ribosomal proteins with zinc-binding motifs CXXC (C+) are substituted with alternative zinc-free counterparts (C-) through a transcriptional de- repression mechanism involving the zinc uptake regulator, ZurB. The alternative ribosomes assembled with C- ribosomal proteins reduce the zinc requirement for cellular growth under zinc-limiting conditions. Mycobacterium tuberculosis (Mtb), the etiological agent for tuberculosis (TB), has four C+/C- ribosomal protein pairs. All four genes encoding C- proteins of the pair are organized in an operon and are co-expressed through a ZurB-repressible promoter. Co- expression implies simultaneous substitution of all four C+ proteins by C- paralogs, raising questions about the influence of the substitutions on ribosomal response to antibiotics. We have found that the alternative C- ribosomes in both Mtb and Mycobacterium smegmatis not only reduce the zinc requirement for cellular growth, but also confer tolerance to clinically relevant anti-TB ribosomal antibiotics. We further observed that Mtb express more alternative ribosomes during chronic infection than in early acute phase. We thus hypothesize that expression of alternative ribosomes are the primary reasons underlying the inefficacies of the clinically established ribosomal antibiotics against TB. To facilitate improved targeting of mycobacterial ribosomes we propose to: a) elucidate the mechanistic basis of differential responses of constitutive (C+) and alternative (C-) ribosomes to antibiotics (aim 1), b) identify strategies to target alternative (C-) ribosomes (aim 2) and c) determine the role of alternative ribosomes in pathogenesis of Mtb (aim 3). These studies will ultimately lead to new strategies to target both constitutive and alternative ribosomes in mycobacteria, and thus allow effective clearance of mycobacterial infections.