Figure 1.
Biotin synthesis pathway and construction of Mtb ΔbioA.
(A) Shown are the reactions catalyzed by KAPA synthase (BioF), DAPA synthase (BioA), DTB synthase (BioD) and biotin synthase (BioB), which convert pimeloyl-CoA to biotin, and biotin ligase, which attaches the cofactor to biotin-dependent enzymes. (B) The upper panel displays the genetic organization of the bioA region in wt Mtb; the lower panel displays that of ΔbioA. Gray boxes represent open reading frames of the genes specified above or below, the striped boxes mark the putative bioA promoter, and the black boxes represent hygR. The localization of recognition sites for the restriction endonuclease DraIII are labeled “D”. (C) Southern blot of DraIII-digested genomic DNA from wt Mtb and ΔbioA. The expected sizes for the DraIII fragments hybridizing to the bioF1-derived probe that was used in this blot are 3068 bp for wt and 4021 bp for ΔbioA.
Figure 2.
In vitro characterization of Mtb ΔbioA.
(A) Growth of ΔbioA in Sauton's medium (left) or Sauton's medium supplemented with KAPA, DTB or biotin. Data are from individual cultures and representative of at least two independent experiments. (B)Growth of ΔbioA in Sauton's medium with varying concentrations of biotin. Data are from individual cultures and representative of at least two independent experiments. (C) Immunoblots performed with both an anti-biotin antiserum and rabbit serum recognizing Mtb's dihydrolipoamide acyltransferase (DlaT). The lower panel shows the signal from the anti-biotin antibody; the upper panel shows the DlaT signal, which served as the loading control. Protein extracts were prepared after the indicated days of cultivation in biotin-free Sauton's medium. Data are from individual cultures and representative of at least two independent experiments. (D) Survival of wt and ΔbioA in Sauton's media with and without biotin. Data are averages from triplicate cultures and representative of at least two independent experiments. Error bars indicate standard deviations.
Figure 3.
Growth of wt Mtb, ΔbioA, and bioA TetON-1 in liquid media and mice.
(A) Growth of wt, ΔbioA, and bioA TetON-1 in Sauton's media, Sauton's media with atc or Sauton's media with biotin. Squares, circles, and triangles represent data for wt, ΔbioA, and bioA TetON-1, respectively. Data are from individual cultures and representative of several independent experiments. (B)Growth of ΔbioA and bioA TetON-1 in mouse lungs. Circles represent data for ΔbioA, triangles represent data for bioA TetON-1. Doxy was either given from day 1 of the infection or not at all. Data displayed by open symbols and dotted lines are from doxy-free mice, closed symbols represent data from doxy-fed mice. Data are averages from four mice per group; error bars represent the standard error of the mean.
Figure 4.
BioA protein levels of wt Mtb, ΔbioA, and bioA TetON mutants.
(A) Immunoblots performed with anti-BioA antiserum using total protein extracts prepared from cultures grown with biotin. The plus and minus signs indicate presence or absence of atc in the culture medium. The first lane on the left contained purified BioA. (B) The putative Shine-Dalgarno sequence of bioA TetON-1 is shown in bold. The mutations that were introduced and distinguish the different TetON mutants are shown below. “ATG” indicates the start of the bioA.
Figure 5.
Growth of Mtb bioA TetON-5 in biotin free liquid media and of all mutants in mice without doxy.
(A) Growth of wt and bioA TetON-5 in biotin-free Sauton's medium with and without atc. Squares represent data for wt, triangles represent data for bioA TetON-5. Open symbols indicate data from medium without atc, closed symbols represent data from medium containing atc. Data are from individual cultures and representative of at least two independent experiments. (B) Growth of wt, ΔbioA and the different bioA TetON mutants in mouse lungs. Closed symbols represent CFUs from day 1 post infection, open symbols represent data from day 21. The dotted line indicates the limit of detection (which was lower for wt and ΔbioA than for the TetON mutants due to the different amounts of lung homogenates that were plated). Each symbol represents CFUs obtained from one mouse.
Figure 6.
Growth and survival of Mtb bioA TetON-5 in mouse lungs and spleens.
(A) Growth and persistence in lungs. Squares represent data for wt, circles represent data for bioA TetON-5. Mtb bioA TetON-5 was analyzed in mice that did not receive doxy (red circles), received doxy from day 1 to day 10 (blue circles), from day 1 to day 28 (purple circles), from day 1 to 56 (brown circles) or received doxy throughout the infection (black circles). Data are averages from at least four mice and two independent infections; error bars represent the standard error of the mean. The limit of detection was 4 CFUs per lung. Dotted lines end in data points for which most of the lungs contained 4 or fewer CFUs. The red asterisk indicates that no CFUs were recovered from any of the no-doxy mice 112 days post infection. (B) Persistence in spleens. The blue asterisk indicates that on day 224 no CFUs were recovered from any of the mice that received doxy up to day 10. Otherwise as described for (A).
Figure 7.
Quantitative BioA immunoblotting of bioA TetON-1 and bioA TetON-5.
Immunoblots were performed as described in the main text and in the material and methods.
Table 1.
Relative BioA protein levels and activities.