Table 3 Phosphatases in cell extracts of impA, suhB mutants Substrate H37Rv ΔimpA ΔsuhB Fructose-1,6-bisP 26.04 ± 1.85 (5) 28.18 ± 0.92 (5) 32.70 ± 0.44 (5) Inositol-1-P 0.63 ± 0.13 (6) 0.79 ± 0.12 (5) 0.63 ± 0.25 (6) Inositol-2-P 1.20 ± 0.15 (4) 1.33 ± 0.22 (5) 1.03 ± 0.15 (6) Glycerol-2-P 0.08 ± 0.06 (12) -0.02 ± 0.03 (2) 0.39 ± 0.03 (2) Glycerol-3-P

-0.13 ± 0.12 (12) -0.08 ± 0.03 (2) 0 ± 0.21 (2) 5′ AMP 4.22 ± 0.36 (8) 4.13 ± 0.40 (2) 5.74 ± 0.04 (2) p-nitrophenyl-P 3.00 ± 0.35 (12) 3.55 ± 0.14 (2) 4.38 ± 0.36 (2) Values: nmol/min/mg protein, mean ± SEM (n). Differences Thiazovivin supplier between levels in mutants and the parent strain were not significant (P > 0.05; t-test). Table 4 Phosphatases in cell extracts of the cysQ mutants Substrate H37Rv ΔcysQ 203/12 ΔcysQ203/16 Fructose-1,6-bisP 18.94 ± 1.00 (6) 13.09 BAY 80-6946 manufacturer ± 1.24 (6) 12.41 ± 0.54 (7) Inositol-1-P 0.40 ± 0.09 (8) 0.49

± 0.17 (9) 0.57 ± 0.16 (9) Inositol-2-P 0.84 ± 0.12 (8) 0.90 ± 0.27 (10) 0.70 ± 0.23 (10) Glycerol-2-P 0.75 ± 0.32 (8) 1.02 ± 0.27 (10) 0.55 ± 0.15 (10) Glycerol-3-P -0.37 ± 0.28 (3) -0.35 ± 0.14 (3) 0.27 ± 0.45 (3) 5′ AMP 1.42 ± 0.31 (3) 1.69 ± 0.14 (3) 1.39 ± 0.03 (3) p-nitrophenyl-P 5.51 ± 0.36 (2) 3.64 ± 1.92 (2) 2.83 ± 0.25 (3) Values: nmol/min/mg protein, mean ± SEM (n). Level of FBPase in cysQ mutants relative to parent strain is significantly different (P < 0.05; t-test). Level of FBPase in H37Rv parent strain reported in table 4 is significantly different Tyrosine-protein kinase BLK (P < 0.05; t-test) to that reported in Table 3. PIM, LAM and mycothiol levels are normal in the impA, suhB and cysQ mutants Cell extracts DihydrotestosteroneDHT supplier of the mutant strains were prepared for the assay of inositol-containing molecules (cell envelope glycolipids and mycothiol). TLC analyses showed that PIMs were normal in the mutant strains (Figure

3A), whilst polyacrylamide gel electrophoresis (Figure 3B) and sugar compositional analysis (not shown) demonstrated normal levels of LAM and LM. Mycothiol levels were assayed by HPLC analysis; levels in the impA, suhB and cysQ mutants were similar to wild-type (see Figure 4). Figure 3 Analyses of cell wall major constituents of some representative mutants; the other strains exhibited profiles similar to those shown. (A) TLC analysis of extractable lipids. (B) SDS-PAGE of lipopolysaccharides. WT: M. tuberculosis H37Rv; ΔA: impA mutant; ΔB: suhB mutant; S: authentic standard of mycobacterial LAM and M. bovis BCG LM; TMM: trehalose monomycolate; PE: phosphatidylglycerol; PG: phosphatidylethanolamine; LAM: lipoarabinomannan; LM: lipomannan; PIM: phosphatidylinositol mannoside. Figure 4 HPLC analysis of mycothiol (marked with an arrow) in representative mutants; the other strains exhibited profiles similar to those shown. WT: M.