Figure 6. plcA-egfp fusion gene expression of M. tuberculosis H37RvΔPLC::Pr_plcA-egfp during growth in phosphate limiting conditions.

(A) The curve shows the phosphate concentration in samples over time of in vitro growth. Histogram represents increase of the culture fluorescence intensity due to GFP expression over time. Results shown are representative of 2 independent experiments. Note that towards the end of the experiment the phosphate concentration slightly increased, which is plausibly due to lysis of some of the older bacterial cells. (B) Measurement of fluorescence divided into GFP and red fluorescence in a culture of H37RvΔPLC::Pr_plcA-egfp complemented with a DsRed expressing plasmid. DS-red is expressed via a constitutive promoter while GFP expression is dependent on plcA promoter activity. (C) Promoter activity of M. tuberculosis H37RvΔPLC::Pr_plcA-egfp in presence of decreasing phosphate concentration due to in vitro growth of culture during 7 days at 37 °C under shaking conditions. Measures show the ratio between fluorescence and absorbance, the first reflecting GFP expression levels and the latter reflecting cell density. (D) Survival of M. tuberculosis H37Rv WT and mutant strains in broth that provides phosphatidylcholine as the sole phosphate source. Results shown represent two different experiments. For each experiment the different strains tested were plated and counted in triplicate.