Figure 5.

Culture supernatant and high concentration of Mg²⁺ abolish the inhibitory effect caused by PknB-PASTA overexpression. (a) Culture supernatants abolish growth-inhibitory effect of PknB_TM-PASTA overexpression in a dose-dependent manner. Relative concentration of culture supernatant added is expressed as ‘fold concentration’. A value of onefold corresponds to undiluted culture supernatant. The apparent lag phase was calculated as a period of time when culture reached OD_{600 nm} 0.1. (b) Mg²⁺ (10 mM) relieves growth inhibition of TMP in a manner comparable with undiluted culture supernatant. (c) Effect of low Mg²⁺ supernatant on apparent lag phase of TMP strain. (d) Effect of synthetic muropeptides on growth of M. smegmatis. Mycobacteria from early stationary phase were washed in Sauton's medium twice and 5 × 10⁶ bacteria were inoculated in Sauton's medium, containing culture supernatant or synthetic muropeptides at final concentration of 10 µM. MPP, MurNAc-pentapeptide; DPP, GlcNAc-MurNAc-peptapeptide; ADPP, GlcNAc-1,6 anhydromuramyl-pentapeptide; TPP, tetra-saccharide-peptide; SN, 50% (v/v) culture supernatant. GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid; pentapeptide, l-Ala-γ-d-Glu-m-DAP-d-Ala-d-Ala. (b,d) Presented data are mean values of 5 replicates from two independent experiments. Standard deviations were 10% or less of average values and are not shown for clarity. SN, media were supplemented with culture supernatant.