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. 2017 Jun 28;12(6):e0179513. doi: 10.1371/journal.pone.0179513

Fig 1. Confirmation of disruption of the bioA gene in M. tuberculosis.

Fig 1

(a) Diagrammatic representation of the bioA locus in M. tuberculosis and MtbΔbioA. The bioA gene is the first gene of an operon consisting of three downstream genes bioF1, bioD and Rv1571. The figure depicts the disruption of the bioA gene in MtbΔbioA by homologous recombination. Arrows show the location of the bioA gene specific primers F-bioA and R-bioA employed for the confirmation of disruption of the bioA gene by PCR. (b) PCR based confirmation of disruption of bioA gene in MtbΔbioA. A 1.3 kb PCR amplification product was obtained with the M. tuberculosis genomic DNA as template (lane 1) and 2.4 kb PCR amplification product was obtained with MtbΔbioA genomic DNA as template (lane 2). λHindIII DNA molecular mass marker and 100 bp ladder were loaded in lanes 3 and 4, respectively. (c) Confirmation of disruption of bioA in MtbΔbioA by immunoblot analysis. 10 μg of cell lysate of M. tuberculosis (lane 1), MtbΔbioA (lane 2) and MtbΔbioA.comp (lane 3) strains were separated on a 12.5% polyacrylamide gel and immunoblot analysis was carried out with anti-BioA polyclonal antiserum. BioA (~48 kDa band) was detected in the cell lysate of the M. tuberculosis and MtbΔbioA.comp. The disruption of bioA in MtbΔbioA was confirmed by the absence of the ~48 kDa band in lane 2. Protein molecular weight marker and 150 ng of purified BioA were loaded in lanes 4 and 5, respectively. (d, e, f) Influence of disruption of the bioA gene on the growth of M. tuberculosis in culture media. M. tuberculosis, MtbΔbioA and MtbΔbioA.comp were cultured separately in (d) MB7H9 medium, (e) modified 7H9 medium (prepared without biotin) and (f) modified 7H9 medium supplemented with 1 mg/L of biotin. Growth at 37°C/200 rpm was monitored for 14 days by measuring the absorbance at 600 nm. MtbΔbioA failed to grow in modified 7H9 medium and the growth was restored to wild type levels when modified 7H9 was supplemented with 1 mg/L or 0.5 mg/L of biotin. Two independent growth analysis experiments were carried out in duplicates and the values of absorbance are represented as the mean (±SE).