The nonredundant roles of two 4'-phosphopantetheinyl transferases in vital processes of Mycobacteria

Abstract

Mycobacterium tuberculosis contains >20 enzymes that require activation by transfer of the 4'-phosphopantetheine moiety of CoA onto a conserved serine residue, a posttranslational modification catalyzed by 4'-phosphopantetheinyl transferases (PPTases). The modified proteins are involved in key metabolic processes such as cell envelope biogenesis and the production of virulence factors. We show that two PPTases conserved in all Mycobacterium spp. and in related genera activate two different subsets of proteins and are not functionally redundant. One enzyme, AcpS, activates the two fatty acid synthase systems of mycobacteria, whereas the other PPTase, PptT, acts on type-I polyketide synthases and nonribosomal peptide synthases, both of which are involved in the biosynthesis of virulence factors. We demonstrate that both PPTases are essential for Mycobacterium smegmatis viability and that PptT is required for the survival of Mycobacterium bovis bacillus Calmette-Guérin. These enzymes are thus central to the biology of mycobacteria and for mycobacterial pathogenesis and represent promising targets for new antituberculosis drugs.

Fig. 1.

Thermosensitivity of the M. smegmatis conditional mutants. M. smegmatis WT, PMM77 (ΔacpS:pC-acpSms), and PMM78 (ΔpptT:pC-pptTms) were grown in LB (with Km and streptomycin for the recombinant strains) at 30°C, streaked onto LB agar plates, and incubated for 2 days at 30°C (Left) or 42°C (Right).

Fig. 2.

Biochemical analysis of the cell envelope of C. glutamicum ΔacpS and ΔpptT recombinant strains. (A) Fatty acids and mycolic acids produced by C. glutamicum WT; the various mutants and the complemented strains were prepared from cells, derivatized into methyl esters, and separated by analytical TLC on Durasil 25TLC (Macherey & Nagel) with dichloromethane. Lipids were visualized by spraying the plates with 10% phosphomolybdic acid in ethanol and heating. (B) Trimethylsilyl derivatives of fatty acid methyl esters from C. glutamicum WT, ΔacpS, ΔpptT, and ΔpptT:pCGL-pptTms were analyzed by GC, as described in ref. . For each chromatogram, the portion between 36 and 44 min is magnified. Peaks corresponding to the C₁₆–C_18:1 fatty acids and the C₃₂, C_34:1, and C_36:2 corynomycolate methyl esters are indicated.

Fig. 3.

4′-Phosphopantetheinylation labeling assays using β-[β-¹⁴C]alanine. Various corynebacteria or recombinant E. coli strains were cultured in the presence of β-[β-¹⁴C]alanine and proteins from cell extracts of each strain were separated by SDS/PAGE. Polyacrylamide gels were stained with Coomassie blue (Lower) and placed against x-ray film for autoradiography (Upper). (A) Analysis of C. glutamicum WT, ΔpptT, Δpks13, and ΔacpS. Positions of Pks13 and Fas-I are indicated by arrows and arrowheads, respectively. M, ¹⁴C-methylated markers (220, 97.4, 66, 46, 30, 21.5, 14.3 kDa) (Sigma). (B–C) Analysis of strains of E. coli BL21ΔentD producing either Pks13 or Pks13* (S55A and S1266A mutated Pks13) from M. tuberculosis (B) or various type-I Pks from M. tuberculosis (C) with the WT PptT (+) from M. tuberculosis (strains transformed with pLSfp) or the mutated pptT-encoded PPTase (−) (strains transformed with pLSfpΔ). Positions of Pks13 and Pks13* (B) and Mas, PpsA, PpsB, PpsC, and PpsD (C) are indicated by arrows. M, ¹⁴C-methylated markers.

Fig. 4.

Effect of PptT depletion on growth of M. bovis bacillus Calmette–Guérin. M. bovis bacillus Calmette–Guérin WT and the PMM99 (ΔpptT:pC-pptTmb) mutant strain were grown in 7H9 containing ADC and ATc (50 ng/ml) (with Km and hygromycin for PMM99) at 37°C and streaked onto 7H11 plates supplemented with OADC with or without ATc (100 ng/ml). Plates were incubated for 20 days at 37°C.

Fig. 5.

Schematic diagram of the role played by AcpS and PptT in the fatty acid, mycolic acid, methyl-branched-containing lipid, and siderophore biosynthesis pathways in M. tuberculosis. Only proteins requiring 4′-phosphopantetheinylation in these pathways are indicated. Proteins activated by PptT are boxed in rectangles and proteins modified by AcpS are boxed in ovals. p-HBA, para-hydroxybenzoic acid; SL-1, sulfolipid 1; PAT, polyacyltrehaloses.