A diverse species library of the genus Mycobacterium.

a, Phylogenetic tree of mycobacterial species in our library calculated using the bcgTree pipeline v 1.1.0 [1–5]. b, Pathogenicity score. c, Doubling time. d, Genome size. e, Guanidine and cytosine percentage (GC%). f, Ribosomal copies (rrn operon). g, Gene ontology (GO) distribution. Colors from left to right represent the following GO categories: regulation of DNA-templated transcription (highlighted with a light orange arrow), transmembrane transport (highlighted with a dark orange arrow); amino acid, lipid, carbohydrate derivative, nucleobase-containing small molecule, and carbohydrate metabolic processes; generation of precursor metabolites and energy; sulfur compound, vitamin, and tRNA metabolic processes; DNA repair, protein modification process, signaling, cell wall organization or biogenesis, cellular modified amino acid metabolic process, DNA replication, DNA recombination, ribosome biogenesis, anatomical structure development, protein catabolic process, protein-containing complex assembly, protein maturation, nitrogen cycle metabolic process, intracellular protein transport, metal ion homeostasis, cell division, protein secretion, mRNA metabolic process, DNA integration, transport, organic substance transport, defense response to other organism, organic substance biosynthetic process, organic substance metabolic process, cellular process, nitrogen compound transport, regulation of gene expression, cellular biosynthetic process, other metabolic processes.

Antibiotic sensitivity mapping reveals complex patterns.

a, Heatmaps of overall MIC99 values. In the X axis, antibiotics are organized based on their mechanism of action; in the Y axis, mycobacterial species are organized phylogenetically in the left heatmap, and based on their response to the set of antibiotics tested (Manhattan clustering) in the right heatmap. Colors represent the standardized MIC99 (mean/SD and centered scaled). Lower MIC99 values are in brown/orange and higher MIC99 values in lilac/purple. The details of the data can be found in Supplementary Figure 2. b, Radar plots displaying the standardized MIC99 for all antibiotics tested. MIC99- values are normalized to be plotted in radar plots. All radar plots display the results for M. tuberculosis in orange. M. branderiis displayed in purple, M. conceptionensein dark blue and M. smegmatis in light blue. c, Violin plots showing the distribution of MIC99 values. In the X axis, the set of antibiotics tested; in the Y axis the MIC99 values in μg/mL. d, Relationship between mycobacterial doubling time (X axis) and MIC99 for the antibiotics BDQ, LZD and RIF (Y axis). Antibiotics targeting the cell wall are: isoniazid (INH), ethionamide (ETH), ethambutol (EMB), D-cycloserine (DCS), and meropenem (MEM); RNA/protein synthesis: rifampicin (RIF), streptomycin (STR), kanamycin (KAN), amikacin (AMK) capreomycin (CAP) and linezolid (LZD); DNA gyrase: moxifloxacin (MFX) and ofloxacin (OFX); folate metabolism: paraaminosalicylic acid (PAS); and ATP synthase: bedaquiline (BDQ).

Intra-bacterial antibiotic concentration does not correlate with potency.

a, Positive mode extracted ion chromatograms (EIC) of whole-cell extracts of mycobacteria treated with selected antibiotics. BDQ (m/z 555.1642), LZD (m/z 338.1511) and RIF (m/z 823.4124). b, Chemical structure of BDQ, LZD and RIF. c, relative intracellular antibiotic concentration, obtained by comparing the peak height of the samples with and injection of 10 μM of antibiotic, then normalized by the concentration of antibiotic used to treat the cells. d, Relative intracellular antibiotic concentrations in relation to the MIC99. Data in c and d come from independent experiments.

High-level rifampicin resistance is caused by rifamycin modification in selected mycobacteria.

a, RIF MIC99 values for the mycobacterial species in our library organized in decreasing MIC99 value order. b, Cultures of selected species on solid medium (7H10) containing RIF at different concentrations, starting at 1× M. tuberculosis MIC. c, Comparison of the amino acid residue sequence of the rifampicin resistance-determining region (RRDR) of RpoB in selected mycobacterial species; the only residue that differs is Ser 450 in M. branderi. d and e, Volcano plots showing the differential protein expression in whole-cells with and without RIF revealing inducible expression of RIF ADP-ribosyltransferase 1 (Arr-1) in M. smegmatis, M. conceptionenseand M. flavescens. f, Detection and quantification of ribosyl-RIF in whole-cell extracts by LC-MS.

Characterization of a novel rifabutin-ADP ribosyltransferase in mycobacteria.

a, Phylogenetic tree of mycobacterial RIF ADP-ribosyltransferases (RIF-ARTs) and related PFAM family PF12120 sequences. Many mycobacterial species encode the equivalent of M. smegmatis RIF-ART (MSMEG_1221; Arr-1/ms in purple), and some mycobacterial species encode a previously unidentified sister group we have named Arr-X (dark orange). See Supplementary Table 3 for detailed information of the sequences in the tree. b, Ribbon representation of the crystal structure of M. smegmatis RIF-ART (PDB code 2HW2; light blue) with RIF bound (red) overlaid with the AlphaFold2 models of M. conceptionense Arr-X (dark blue) and M. flavescens Arr-X (dark orange). c, Apparent velocity of reaction of each of the enzymes (X axis) with different rifamycins as substrate. d, M. conceptionensesingle and double knockdown (KD) arrstrains in the presence of rifabutin.

Detailed overall MIC99quantifications against the mycobacterial species in our library. X axes represent the species tested in phylogenetic order, and Y axes arethe MIC99 measurements in log10 scale. Dotted lines represent the mean MIC99 for each drug.

Log2(FC) MIC99 (Y axis) compared to the mean MIC99for each antibiotic. Species organized by phylogeny (X axis). Dotted line represents the mean MIC99 for each antibiotic.

Detailed view of the Mycobacterium holsaticum group MIC99 values

a, Phylogenetic distribution of RIF ADP-ribosyl transferases present in themycobacterial species in our library. b,Quantification of ribosyl-RIFin somemycobacterial species that encode Arr-1 and/or Arr-X.Not detected (ND).