Cell wall core galactofuran synthesis is essential for growth of mycobacteria

Abstract

The mycobacterial cell wall core consists of an outer lipid (mycolic acid) layer attached to peptidoglycan via a galactofuranosyl-containing polysaccharide, arabinogalactan. This structural arrangement strongly suggests that galactofuranosyl residues are essential for the growth and viability of mycobacteria. Galactofuranosyl residues are formed in nature by a ring contraction of UDP-galactopyranose to UDP-galactofuranose catalyzed by the enzyme UDP-galactopyranose mutase (Glf). In Mycobacterium tuberculosis the glf gene overlaps, by 1 nucleotide, a gene, Rv3808c, that has been shown to encode a galactofuranosyl transferase. We demonstrate here that glf can be knocked out in Mycobacterium smegmatis by allelic replacement only in the presence of two rescue plasmids carrying functional copies of glf and Rv3808c. The glf rescue plasmid was designed with a temperature-sensitive origin of replication and the M. smegmatis glf knockout mutant is unable to grow at the higher temperature at which the glf-containing rescue plasmid is lost. In a separate experiment, the Rv3808c rescue plasmid was designed with a temperature-sensitive origin of replication and the glf-bearing plasmid was designed with a normal original of replication; this strain was also unable to grow at the nonpermissive temperature. Thus, both glf and Rv3808c are essential for growth. These findings and the fact that galactofuranosyl residues are not found in humans supports the development of UDP-galactopyranose mutase and galactofuranosyl transferase as important targets for the development of new antituberculosis drugs.

FIG. 1

Formation of UDP-Galf (A) and galactofuran (B). The role of galactofuranosyl residues of linking peptidoglycan to mycolic acids via arabinan is also shown. From the galactofuran structure, it is estimated that four galactofuranosyl transferase activities (Gal Tran A to D) are needed to form the galactofuran (with the remaining residues being assembled by the activities Gal Tran C and D); it is possible that some of these activities are combined into one polypeptide. Rv3808c presumably encodes one or more of these galactofuranosyl transferase activities (15). Galp, galactopyranose; Rhap, rhamnosylpyranose.

FIG. 2

Key plasmids (pFP101, pCG76:TBglf, and pMVHG1:Rv3808c) used in this study. The plasmids pCG76:Rv3808c and pMVHG1:TBglf are strictly analogous to CG76:TBglf and pMVHG1:Rv3808c. TS oriM, temperature-sensitive oriM; Amp^R, ampicillin resistance cassette; Gm^R; gentamicin resistance cassette; Str/sp, streptomycin/spectinomycin resistance cassette; Hyg^R, hygromycin resistance cassette.

FIG. 3

Two possible pathways for homologous recombination between pFP101 and the M. smegmatis chromosome. Crossover upstream of kan yields a functional glf gene (with its promoter) along with functional Rv3808c and any transcriptionally linked genes further downstream. Crossover downstream from kan yields no functional glf gene, and the interrupted glf gene upstream from Rv3808c is likely to inhibit transcription of Rv3808c and any transcriptionally linked genes downstream from it. If glf, Rv3808c, or any other downstream ORF expressed from the glf promoter is essential, only single-crossover events of type 1 should occur. Also illustrated are the NruI fragments used to distinguish which single-crossover event occurred.

FIG. 4

Southern blot analysis of M. smegmatis of the single homologous-recombination event at the glf locus of M. smegmatis. Lane 1 is wild-type M. smegmatis; lanes 2 to 18 are 17 clones selected from the LB broth-KAN plates at 42°C. The DNA was cleaved with NruI, and the 1,595-bp glf-containing fragment was used as the probe template. The DNAs in lanes 3, 5, 9, 11, 13, 14, and 16 resulted from type 1 homologous recombination (Fig. 3), as bands at 1.2 and 15.6 kb are evident, whereas the DNAs in lanes 2, 4, 6, 7, 8, 10, 12, 15, 17, and 18 come from clones with illegitimate recombination, as wild-type glf at 2.4 kb is evident, as are the expected two bands of various sizes.

FIG. 5

Southern blot analysis of M. smegmatis FP102 (the glf knockout strain) containing plasmids pMVHG1:Rv3808c and pCG76:TBglf. The DNA was cleaved with NruI, and the 1,595-bp glf-containing M. smegmatis fragment was used as the probe template. Lanes 1 to 3 are controls; lanes 4 to 6 are positive for the second crossover event. Lane 1, plasmid pCG76:TBglf only; lane 2, M. smegmatis mc²155 wild type; lane 3, M. smegmatis FP101 (first single-crossover bacterium [see also Fig. 4]) with plasmids pMVHG1:Rv3808c and pCG76:TBglf before selection on sucrose for the second crossover event; lanes 4 to 6, three colonies of M. smegmatis FP102 (Table 1) containing plasmids pMVHG1:Rv3808c and pCG76:TBglf. The origins of the bands at 1.2 and 2.4 kb in M. smegmatis FP102 are illustrated. Both the wild type (lane 2) and M. smegmatis FP102 yield a band near 2.4 kb (2.39 kb for the wild type and 2.41 kb for M. smegmatis FP102). However, the band near 2.4 kb also hybridizes with a probe made from the KAN resistance cassette in the case of M. smegmatis FP102 (lanes 4 to 6) but does not hybridize in the case of wild-type M. smegmatis (lane 2) (data not presented). The bands in lanes 3 to 6 (M. smegmatis FP102) at ≈11 and 0.5 kb come from plasmid pCG76:TBglf (see lane 1) being present in M. smegmatis FP102.

FIG. 6

Growth curves of M. smegmatis strains at 30 and 40°C. Shown are results with M. smegmatis mc²155 containing plasmids pMVHG1:Rv3808c and pCG76:TBglf at 30°C (▴) or at 40°C (▵), M. smegmatis FP102 (Table 1) at 30°C (●) or at 40°C (○), and M. smegmatis FP103 (Table 1) at 30°C (▪) or at 40°C (□). The medium was LB broth in all cases, and antibiotics were present as detailed in Materials and Methods. M. smegmatis mc²155 containing plasmids pCG76:Rv3808c and pMVHG1:TBglf, a second control construct, grew at both 30 and 40°C (data not presented). The slight lag in growth seen for the two knockout strains of M. smegmatis at 30°C is likely due to a weaker inoculum, as evidenced by the optical density (OD) at 600 nm at time zero.