High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism (original) (raw)
Figure 3
Genes required for growth on cholesterol.
(A) Mce4 mutants are specifically underrepresented in the cholesterol-grown pool. Transposon libraries grown for 12 generations in media with either glycerol or cholesterol as a primary carbon source were compared by deep sequencing. Normalized sequence reads for individual insertions sites throughout the Mce4 operon are shown following growth in glycerol (blue) and cholesterol (red). The average underrepresentation of Mce4 mutants in the cholesterol-grown pool corresponds to a predicted 31% growth disadvantage per generation. (B) The number of sequence reads per TA provides an accurate estimate of relative growth rates. The experimentally determined growth curves of a Mce4 deletion mutant in the indicated media are compared to the growth rate of Mce4 mutants predicted in panel A. Log phase growth is plotted as percentage of initial bacterial number. (C) Mutants predicted to be required for cholesterol utilization display the predicted phenotypes. Transposon mutants were grown in minimal media with the indicated primary carbon sources and growth was monitored by optical density. This experiment was repeated three times with similar results. (D) Identification of genes that are differentially required for growth. For each gene, the ratio of normalized sequence reads per insertion site (cholesterol pool/glycerol pool) is plotted on the x-axis (“fold change”). Y-axis represents the significance of each of these changes in representation (p value). A hyperbolic function was used to define genes that were differentially represented. The asymptotes of these curves are 0 for the log2 fold change and −0.07 for the log10 p value. Genes containing fewer than two TA sites were excluded from the analysis. Genes in blue represent those within the predicted cholesterol region.