Figure 6.
LlgA Activity Is Thermo-Regulated
(A) Intracellular growth analysis of WT Lm and mutants that are cured of the phage (Δφ) or deleted of lysis genes (Δ(lysis)φ) expressing llgA (pPL2-PactA-llgA) in BMDM cells. Data are representative of three independent experiments. Error bars represent the standard deviation of a triplicate.
(B and C) Growth analysis of WT Lm and bacteria expressing llgA-his from a TetR-dependent promoter (pPL2-PtetR-llgA-his), as well as a mutant deleted of the phage lysis genes (Δ(lysis)φ) harboring pPL2-PtetR-llgA-his with and without the addition of anhydrotetracycline (AT) at (B) 30°C and (C) 37°C. Data represent the mean of three independent experiments. Error bars represent the standard deviation.
(D) qRT-PCR analysis of llgA transcription levels at 30°C and 37°C in WT Lm and in Δ(lysis)φ and Δφ strains harboring pPL2-PtetR-llgA-his. Transcription levels are presented as a RQ, compared to the levels in WT bacteria grown at 30°C. mRNA levels were normalized to the levels of 16S rRNA. The data are representative of three independent experiments. Error bars indicate the 95% confidence interval.
(E) Western blot analysis of LlgA-6His protein obtained from WT Lm grown at 30°C and 37°C and from Δ(lysis)φ strain and a strain cured of the phage Δφ, all harboring pPL2-PtetR-llgA-his. Equal amounts of total protein were separated on a 15% SDS-PAGE that was blotted and probed with anti-6His antibody. The experiment was performed three times, and the figure shows a representative blot. Coomassie staining was used as a loading control (lower panel)
(F) qRT-PCR analysis of selected phage genes, representative of the early and late gene modules in WT Lm and in Δ(lysis)φ mutant harboring pPL2-PtetR-llgA-his grown in BHI at 30°C and 37°C. Transcription levels are presented as a RQ, compared to the levels in WT bacteria grown at 30°C. mRNA levels were normalized to the levels of 16S rRNA. The data are representative of three independent experiments. Error bars indicate the 95% confidence interval.
(G) qRT-PCR analysis of terS and llgA genes expressed from the pPL2 plasmid (pPL2-PtetR-llgA-terS) in Lm bacteria that are cured of the phage (Δφ) grown in BHI at 30°C and 37°C. Transcription levels are presented as a RQ, compared to the levels in bacteria grown at 30°C. mRNA levels were normalized to the levels of 16S rRNA. The data are representative of three independent experiments. Error bars indicate the 95% confidence interval.
(H) qRT-PCR analysis of terS and llgA genes expressed from amyE::Phs-llgA-terS in B. subtillis bacteria grown in LB at 30°C and 37°C with IPTG. Transcription levels are presented as a RQ, compared to the levels in bacteria grown at 30°C. mRNA levels were normalized to the levels of 16S rRNA. The data are representative of three independent experiments. Error bars indicate the 95% confidence interval.