Journal: bioRxiv

Article Title: Functional Characterization of 123 Genes from Mycobacteriophage LeBron Uncovers Cytotoxic Gene Families in Cluster L1

doi: 10.64898/2025.12.17.694915

Figure Lengend Snippet: a) Recombinant pExTra plasmids constructed in this study encode LeBron gene sequences downstream of the pTet promoter and upstream of mCherry . The two genes are arranged in an aTc-inducible operon, each with its own start and stop codons and ribosome binding site (RBS), ensuring independent translation of both protein products. b) Representative cytotoxicity assays illustrate the range of observed growth defects. Colonies of M. smegmatis mc2155 transformed with the indicated pExTra plasmids were resuspended, serially diluted, and spotted on 7H10 Kan media supplemented with 0, 10, or 100 ng/ml aTc. Triplicate colonies (A, B, C) were tested for each gene alongside a positive control strain (+, pExTra02 expressing wild-type Fruitloop gp52) and a negative control strain ( −, pExTra03 expressing Fruitloop gp52 I70S). Examples of each cytotoxicity score are shown: score 0 (LeBron gp30), score 1 (LeBron gp68), score 2 (LeBron gp73), and score 3 (LeBron gp47).

Article Snippet: Transformants were selected on 7H10 agar plates containing 10 μg/mL kanamycin (GoldBio) and incubated at 37°C for 5 days prior to phenotypic screening.

Techniques: Recombinant, Construct, Binding Assay, Transformation Assay, Positive Control, Expressing, Negative Control

Journal: iScience

Article Title: A potent phenylalkylamine disrupts mycobacterial membrane bioenergetics and augments bactericidal activity of bedaquiline

doi: 10.1016/j.isci.2025.112915

Figure Lengend Snippet: Bactericidal profiles of PhA2 and other phenylalkylamine actives in M. bovis BCG and Mab abscessus (A) Colony forming units (CFUs) of growing exponential phase M. bovis BCG cultures treated with actives (PhA1-3, PhA9, PhA11-13), VP and INH (control) at 1x, 2x, 4x MIC 90BCG . Mid-log phase cultures adjusted to OD 600 = 0.05 were treated with test compound (5 days, 37 o C, 110 rpm), then plated on 7H10 agar plates followed by enumeration of CFUs. Minimum bactericidal concentrations to reduce CFUs by 10-, 10 2 -, 10 3 -fold are given by MBC 90 , MBC 99 , MBC 99.9 , respectively, compared to drug-free control at time zero. CFUs were averaged from at least 3 independent determinations. MIC 90BCG of INH and VP are 3.4 μM (0.45 μg/mL) and 550 μM (250 μg/mL) respectively. MIC 90BCG of test compounds are PhA1 (25 μM, 13 μg/mL), PhA2 (18 μM, 9.4 μg/mL), PhA3 and PhA9 (22 μM, 12 μg/mL), PhA11 (25 μM, 13 μg/mL), PhA12 (29 μM, 16 μg/mL), PhA13 (23 μM, 13 μg/mL). (B) Time-kill curves of growing M. bovis BCG cultures treated with PhA1, PhA2, PhA3 at 2xMIC 90 BCG (26, 19, 24 μg/mL respectively). Also tested at 2xMIC 90 BCG were isoniazid (INH,0.9 μg/mL) and VP (500 μg/mL). At each time point, aliquots were withdrawn, plated on agar and incubated for 21 days to determine CFUs. Time-kill curves of growing M. bovis BCG treated with (C) PhA2 and (D) VP at concentrations of 0.25x to 2x MIC 90 , equivalent to 2.4–19 μg/mL PhA2 and 63–500 μg/mL VP. INH (control) was assessed at 2x MIC 90 (7 μM, 0.9 μg/mL). At each time point, aliquots were withdrawn, plated on agar and incubated for 21 days to determine CFUs. (E) Bactericidal activities of PhA2, VP and INH (control) under O 2 deprived conditions (Wayne model). PhA2 was tested at 2x MIC 90 (36 μM, 19 μg/mL), and 4x MIC 90 (72 μM, 38 μg/mL) VP was tested at 2x MIC 90 (1.1 mM, 500 μg/mL) and 4x MIC 90 (2.2 mM, 1 mg/mL). INH was tested at 4xMIC 90 (14 μM, 1.8 μg/mL). WCC 90 , WCC 99 , WCC 99.9 are concentrations required to reduce CFUs of nonreplicating M. bovis BCG by 10-, 10 2 -, 10 3 -fold respectively after 5 days treatment compared to drug-free cultures at time zero. CFUs were averaged from at least 2 independent determinations. Time-kill curves of Mab abscessus treated with (F) PhA2 and (G) VP at concentrations of 0.25x to 2x MIC 90 (PhA2: 11–90 μg/mL, VP: 275 μg/mL −2.2 mg/mL). At each time point, aliquots were withdrawn and incubated for 3 days to determine CFUs. MIC 90 abscessus of PhA2 and VP are 85 μM (45 μg/mL) and 2.4 mM (1.1 mg/mL). Except for (E), CFUs for other panels were determined from n = 3 separate runs and presented as mean ± SD (GraphPad Prism).

Article Snippet: Following the procedure set out in MIC determination against exponentially growing M. bovis BCG, aliquots of serially diluted samples at 1x, 2x, and 4x MIC 90 were plated on Middlebrook complete 7H10 agar plates, supplemented with 10% Middlebrook OADC enrichment and 0.5% glycerol, and incubated for at least three weeks at 37°C.

Techniques: Control, Incubation