Figure 1.
The Mycobacterial cell wall and pathway for DPA biosynthesis.
A. The mycobacterial cell wall is a multilayered structure containing many components unique to these and closely related bacteria, including phosphatidylinositol mannosides (PIM), lipoarabinomannans (LAM), trehalose monomycolates (TMM), trehalose dimycolates (TDM) mycolic acids and arabinogalactan. The biosynthetic pathways for these unique components are a rich source of potential drug targets. Arabinose sugars are shown in orange. B. Decaprenylphosphoryl arabinose (DPA) is formed by the epimerization of decaprenylphosphoryl ribose (DPR) by DprE1 (Rv3790) and DprE2 (Rv3791). DPA serves as an arabinose donor in cell wall biosynthesis, contributing to arabinogalactan and LAM assembly (orange arrows).
Figure 2.
Homologs from M. leprae (ML0109), M. tuberculosis (Rv3790) and M. smegmatis (MSMEG_6382) were aligned using CLUSTALW [25]. Residues that are completely conserved are reverse shaded while similar residues are indicated in grey. The putative FAD-binding domain is shown by a solid line. The conserved cysteine residue that is altered in BTZ-resistant mycobacteria [11] is indicated by an asterix.
Figure 3.
Genetic strategy for conditional disruption of MSMEG_6382.
A. The recombination plasmid pPKC72 contained a cloned copy of MSMEG_6382 interrupted by a non-polar kanamycin resistance cassette (MSMEG_6382::aphA3), a gentamycin resistance marker (Gmr), a temperature-sensitive replication origin for M. smegmatis (oriMs (ts)), a replication origin for E. coli (oriEc) and a counterselectable marker encoding sucrose sensitivity (sacB). The construct was introduced into M. smegmatis at the permissive temperature (30°C). Integration of the plasmid by a single crossover at the position indicated was detected by growing the cells at the non-permissive temperature (42°C) in the presence of kanamycin. B. Genetic map of the single crossover, showing key restriction sites and fragments. C. Culturing the single crossover strain containing a rescue plasmid encoding MSMEG_6382 gave rise to a disrupted copy of MSMEG_6382 in the chromosome, producing the conditional knockout (6382CKO). Since the disruption of MSMEG_6382 coincided with the loss of the sacB gene, the conditional knockout strain could be selected on sucrose plates. Double lines indicate chromosomal DNA, single lines indicate plasmid DNA. Restriction fragments hybridising to the MSMEG_6382-specific probe are shown in kilobases (kb).
Figure 4.
Conditional disruption of MSMEG_6382.
Southern blot of genomic DNA digested with HindIII/NotI/XbaI. Lane 1, DNA molecular weight DNA markers of the sizes indicated (kilobases, kb); lane 2, wild-type M. smegmatis mc2155; lane 3, single crossover strain used to derive the conditional knockout; lane 4, conditional knockout of MSMEG_6382, designated 6382CKO.
Figure 5.
MSMEG_6382 is essential for growth of M. smegmatis in LB broth.
6382CKO was cultured at 30°C in LB containing Kn and Sm. At saturation, 5 ml was used to inoculate 200 ml of LB/Kn medium that had been prewarmed at the permissive (30°C •) or non-permissive (42°C ▪) temperature. Incubation was continued at the two temperatures and both cultures were sampled regularly with serial dilutions plated on LB plates containing Kn to determine colony forming units (CFUs) per ml. A wild-type M. smegmatis mc2155 strain containing the kanamycin resistance plasmid pMV261 was included as a control (30°C ▴, 42°C ▾). Unbroken lines represent the 6382CKO strain while broken lines represent the wild type (pMV261) control strain. These data represent means of triplicate samples ± standard deviation and are representative of three independent experiments.
Figure 6.
MSMEG_6382 is essential for growth of M. smegmatis on Middlebrook agar.
The conditional knockout strain 6382CKO was cultured at 30°C on Middlebrook 7H10 agar containing Kn and Sm, then subcultured onto Middlebrook 7H10 agar containing Kn at 30°C and 42°C and examined for growth. Wild-type M. smegmatis mc2155 control strain cultured at 30°C (A) and 42°C (B) on Middlebrook 7H10 agar without antibiotics; 6382CKO strain cultured at 30°C (C) and 42°C (D) on Middlebrook 7H10 agar containing Kn.