Editor: Roger Buxton
An rmlA gene encoding d-glucose-1-phosphate thymidylyltransferase is essential for mycobacterial growth
Article first published online: 3 SEP 2007
FEMS Microbiology Letters
Volume 275, Issue 2, pages 237–243, October 2007
How to Cite
Qu, H., Xin, Y., Dong, X. and Ma, Y. (2007), An rmlA gene encoding d-glucose-1-phosphate thymidylyltransferase is essential for mycobacterial growth. FEMS Microbiology Letters, 275: 237–243. doi: 10.1111/j.1574-6968.2007.00890.x
- Issue published online: 3 SEP 2007
- Article first published online: 3 SEP 2007
- Received 11 April 2007; revised 8 July 2007; accepted 16 July 2007.First published online September 2007.
- Mycobacterium tuberculosis;
- Mycobacterium smegmatis;
- mycobacterial cell wall;
- d-glucose-1-phosphate thymidylyltransferase
The rhamnose-GlcNAc disaccharide is a critical linker which connects arabinogalactan to peptidoglycan via a phosphodiester linkage. The biosynthesis of dTDP-rhamnose is catalysed by four enzymes, and the first reaction is catalysed by an rmlA gene encoding d-glucose-1-phosphate thymidylyltransferase (RmlA). We generated a Mycobacterium smegmatis mc2155 mutant lacking the rmlA gene via a homologous recombination method. We tested the requirement for the rmlA gene and the effect of a lack of RmlA on bacterial cell morphology. The results demonstrate that the rmlA gene is essential for mycobacterial growth and that lack of RmlA activity has profound negative effects on bacterial cell morphology. RmlA is thus a potential target for the development of new antituberculosis drugs.
The mycobacterial cell wall is a complex structure composed of peptidoglycan, arabinogalactan and mycolic acids. The d-N-acetylglucosamine–l-rhamnose disaccharide connects the galactan region of arabinogalactan to the peptidoglycan via a phosphodiester linkage (Brennan & Nikaido, 1995; Crick et al., 2004). Therefore, the disaccharide is a critical linker to the structural integrity of the cell wall and is thus required for mycobacterial viability. The l-rhamnose of the disaccharide linker is from a precursor, dTDP-rhamnose. dTDP-rhamnose is synthesized from d-glucose-1-phosphate and dTTP via a biosynthetic pathway that consists of four distinct enzymes (Stevenson et al., 1994; Ma et al., 1997; Tsukioka et al., 1997a, b): d-glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-d-glucose-4, 6-dehydratase (RmlB), dTDP-4-keto-6-deoxyglucose-3, 5-epimerase (RmlC) and dTDP-6-deoxy-l-lyxo-4-hexulose reductase (RmlD). RmlA–D enzymes are encoded by the genes rmlA–D, previously named rfbA–D (Reeves et al., 1996). Briefly, RmlA catalyses the reaction of d-glucose-1-phosphate and dTTP to produce dTDP-d-glucose and PPi (Pyrophosphate). RmlB oxidizes dTDP-d-glucose to form dTDP-6-deoxy-d-xylo-4-hexulose. RmlC converts dTDP-6-deoxy-d-xylo-4-hexulose to dTDP-6-deoxy-l-lyxo-4-hexulose, and RmlD catalyzes the reaction of dTDP-6-deoxy-l-lyxo-4-hexulose and NADPH to generate dTDP-rhamnose and NADP. The rhamnosyl transferase encoded by the wbbL gene transfers the rhamnosyl residue of dTDP-rhamnose into d-N-acetylglucosaminosyl-1-phosphate to form a d-N-acetylglucosamine-l-rhamnose disaccharide linker. Mycobacterium tuberculosis rmlA–D genes are not located in one locus in the genome (Cole et al., 1998). The rmlA (Rv0334) gene is isolated from any other rhamnosyl formation enzymes, the rmlB (Rv3464) and rmlC (Rv3465) genes are together in one operon, and the rmlD (Rv3266c) gene is found in an operon with wbbL (Rv3265c) and manB (Rv3264c) (Cole et al., 1998).
Mycobacterium tuberculosis is a remarkably successful pathogen that has latently infected one-third of the World's population. One in every ten of these individuals will develop tuberculosis at some point in their lifetime and 2 million people die of tuberculosis each year (Warner & Mizrahi, 2004; Zhang et al., 2006). There have been no new drugs to combat tuberculosis in nearly 40 years; the identification of more drug targets for the development of antituberculosis drugs is therefore urgently required. It is obvious that the disaccharide linker is an excellent drug target given that inhibition of the disaccharide biosynthesis could affect the integrity of the mycobacterial cell wall, which is required for the survival and growth of mycobacteria in the host. In previous studies, we have generated Mycobacterium smegmatis mc2155 mutants with the rmlB–C and rmlD genes knocked out, respectively, and performed tests of the essential requirement for the rmlB, rmlC and rmlD genes for mycobacterial growth. The results provided the direct evidence that M. tuberculosis rmlB, rmlC and rmlD genes are valid targets (Ma et al., 2002; Li et al., 2006). We also established M. tuberculosis RmlB–D enzyme assays to screen inhibitors for developing new tuberculosis therapeutics (Ma et al., 2001).
In the present study, we generated an M. smegmatis mc2155 rmlA gene knock-out strain via a homologous recombination strategy and tested the essential requirement for the rmlA gene for mycobacterial growth. We also observed the morphology of the mc2155 rmlA gene knock-out cells by scanning electron microscopy (SEM) to determine the effects of RmlA activity on the morphological phenotype of M. smegmatis.
Materials and methods
Bacterial strains and plasmids
The characteristics of all bacterial strains and plasmids used in this study are detailed in Table 1. Escherichia coli NovaBlue cells were routinely grown in Luria-Bertani (LB) broth or on LB agar plates at 37°C. Mycobacterium smegmatis mc2155 cells were routinely grown in LB broth containing 0.05% Tween 80 or on LB agar plates at 37°C. The rmlA knock-out strain mc2155 was grown at 30 and 42°C. The final concentrations of antibiotics used were as follows: ampicillin (Ap), 100 μg mL−1 for NovaBlue; kanamycin (Km), 50 μg mL−1 for NovaBlue and 25 μg mL−1 for mc2155; gentamicin (Gm), 5 μg mL−1 for NovaBlue and mc2155; and streptomycin (Sm), 25 μg mL−1 for NovaBlue and 12.5 μg mL−1 for mc2155.
|E. coli NovaBlue||For constructing plasmids||Novagen|
|M. tuberculosis H37Rv||Pathogenic; for amplifying M. tuberculosis rmlA gene||ATCC|
|M. smegmatis mc2155||Nonpathogenic; for amplifying M. smegmatis rmlA gene and achieving homologous recombination at rmlA locus||ATCC|
|mc2155 mutant-1||mc2155 with pPR27-rmlA::KmR integrated into rmlA locus||This work|
|mc2155 mutant-2||mc2155 with knocked rmlA gene in presence of pCG76-Mtb rmlA||This work|
|pMD18-T||For cloning PCR product with A′ at 3′ ends||Takara|
|pUC4 K||For disrupting M. smegmatis rmlA by kmR cassette||GE Healthcare|
|pPR27-xylE||Carries sacB and xylE genes; carries replication origins for E. coli and mycobacteria||Guilhot et al. (1994)|
|pET23b-Phsp60||Carries M. bovis BCG hsp60 promoter||Guilhot et al. (1994)|
|pCG76||Carries replication origins for E. coli and mycobacteria||Li et al. (2006)|
|pMD-rmlA||M. smegmatis rmlA gene with its upstream sequence was cloned to EcoRV site of pMD18-T||This work|
|pMD-rmlA::KmR||The KmR cassette was inserted to StuI site of pMD-rmlA||This work|
|pPR27-rmlA::KmR||M. smegmatis rmlA::KmR was cloned to NotI and SpeI sites of pPR27-xylE||This work|
|pMD-Mtb rmlA||M. tuberculosis rmlA gene was cloned to EcoRV site of pMD18-T||This work|
|pET23b-Phsp60-Mtb rmlA||M. tuberculosis rmlA gene was cloned to NdeI and XhoI sites of pET23b-Phsp60||This work|
|pCG76-Mtb rmlA||Phsp60-Mtb rmlA was cloned to XbaI and XhoI sites of pCG76||This work|
Preparation of M. smegmatis mc2155 genomic DNA and Southern blot analysis
mc2155 cells from 5 mL of culture were harvested for genomic DNA preparation as described (Li et al., 2006). mc2155 genomic DNA was dissolved in 15 μL TE buffer and stored at 4°C for further use.
The genomic DNA was digested by SmaI and the resulting DNA fragments was separated by running a 0.8% agarose gel. The DNA was transferred to Nytran membrane (Schleicher & Schuell) as described (Li et al., 2006). Southern hybridization was performed using a DIG High Prime Labeling and Detection Starter Kit I (Roche). The membrane was prehybridized at 42°C for 1 h in DIG Easy Hyb and hybridized via a digoxigenin-labeled rmlA probe overnight at 42°C. After the membrane was washed with 2 × SSC containing 0.1% SDS and 0.5 × SSC containing 0.1% SDS, the hybridized DNA bands were detected by colorimetric solution.
Construction of conditional replication plasmid and rescue plasmid
Mycobacterium tuberculosis H37Rv RmlA (Rv0334) protein sequence was acquired from the TubercuList (http://genolist.pasteur.fr/TubercuList/). Mycobacterium tuberculosis RmlA protein sequence was used as a query in BLASTP to identify the most homologous gene in the M. smegmatis mc2155 genome. The M. smegmatis rmlA gene (867 bp) with its upstream sequence (506 bp) was amplified from mc2155 genomic DNA by using the M. smegmatis rmlA-1 primer (5′AACTAGTGGCGACCCCCCTTTACCCGGATG 3′, underlined sequence is the SpeI site) and M. smegmatis rmlA-2 primer (5′TGCGGCCGCCTACTCTCGATCCAGAAGTTG 3′, underlined sequence is the NotI site). The PCR product of 1373 bp was purified and ligated to pMD18-T to generate pMD-rmlA (Table 1). The KmR cassette from pUC4 K was inserted to the StuI site of the rmlA gene, yielding pMD-rmlA::KmR (Table 1). The rmlA::kmR fragment (2.63 kb) was ligated into NotI and SpeI sites of pPR27-xylE (Li et al., 2006), resulting in a conditional replication plasmid pPR27-rmlA::KmR (Table 1, Fig. 1a), which was used to achieve the first single crossover at the rmlA locus of the M. smegmatis mc2155 genome.
The M. tuberculosis rmlA gene was amplified from M. tuberculosis H37Rv genomic DNA (supplied by Colorado State University via an NIH contract) by using M. tuberculosis rmlA-1 primer (5′CATATG ATGCGCGGGATCATCTTGGC 3′, underlined sequence is the NdeI site) and M. tuberculosis rmlA-2 primer (5′CTCGAGTCAGTTGCGCTCCAACAACTC 3′ underlined sequence is the XhoI site). Mycobacterium tuberculosis rmlA was cloned into pMD18-T vector to generate a pMD-Mtb rmlA (Table 1). The M. tuberculosis rmlA gene was ligated into NdeI and XhoI sites of pET23b-Phsp60 (Li et al., 2006) to generate pET23b-Phsp60-Mtb rmlA (Table 1). The Phsp60-Mtb rmlA fragment was ligated to pCG76 (Guilhot et al., 1994), resulting in a rescue plasmid pCG76-Mtb rmlA (Table 1).
Selection of mc2155 mutant-1 strains with integrated rmlA::KmR in the genome
Electrocompetent mc2155 cells were prepared as described (Guilhot et al., 1994), and pPR27-rmlA::KmR was electroporated to mc2155 cells. Transformants were grown on LB agar plates containing Km and Gm at 30°C. One colony was propagated in LB broth containing 0.05% Tween 80, Km and Gm at 30°C and the cells were spread on LB agar plates containing Km and Gm at 42°C. The mc2155 mutant-1 strains (Table 1) with the first single crossover event were selected using Southern blot.
Selection of mc2155 mutant-2 (rmlA gene knock-out) strains
The rescue plasmid pCG76-Mtb rmlA was electroporated into the mc2155 mutant-1 strain. Transformants were grown on LB agar plates containing Km and Sm at 30°C. One colony was inoculated into LB broth containing Km and Sm, and incubated at 30°C. The cells were spread on LB agar plates containing 10% sucrose, Km and Sm. Five mc2155 mutant-2 (rmlA knock-out) strains (nos. 1–5) (Table 1) with the second single crossover event were selected via Southern blot.
Growth of the mc2155 rmlA knock-out strain
Five mc2155 rmlA knock-out strains (nos. 1–5) were inoculated in LB broth containing 0.05% Tween 80 and appropriate antibiotics, and incubated at both 30 and 42°C. The wild-type mc2155 carrying pCG76 was used as a control. Absorbance at 600 nm (A600nm) was detected at intervals of 24 h and the growth curves at both 30 and 42°C were obtained.
Morphology of the mc2155 rmlA knock-out strain after shifting from 30 to 42°C
The mc2155 rmlA knock-out strain (no. 4) was grown in LB broth containing 0.05% Tween 80 and Km at 30°C for 20 h (A600nm was 0.026), and the cells were transferred to a 42°C incubator. A600nm was detected at intervals of 24 h (see Fig. 3a), and the cells grown at 42°C for 72 and 120 h were harvested for SEM observation. The cells were fixed with 2.5% glutaraldehyde and 1% OsO4. After dehydration through a graded series of ethanol (20, 40, 60, 70, 80, 90, 100%), the cells were applied to a silicon wafer slide. The cells were examined with a JSM-6360 scanning electron microscope (JEOL) at an accelerating voltage of 28 kV.
Construction of conditional replication plasmid and rescue plasmid
Conditional replication plasmid pPR27-rmlA::KmR (Table 1) was constructed to select mc2155 mutant-1 strains, which have undergone the first homologous recombination at the rmlA locus of the genome. In plasmid pPR27-rmlA::KmR, the KmR cassette was introduced inside Sm rmlA, so it directly led to the disruption of Sm rmlA. Sm rmlA::KmR would be integrated to the mc2155 genome after the first single crossover event occurred. The parent plasmid pPR27 (Pelicic et al., 1997) is a shuttle vector containing the replication origins for both E. coli and mycobacteria. The replication origin for mycobacteria has mutations sensitive to temperature; thus, it can replicate at 30°C (permissive temperature) but is efficiently lost at 42°C (nonpermissive temperature).
Rescue plasmid pCG76-Mtb rmlA (Table 1) was constructed for complementation of rmlA::KmR in the mc2155 mutant-2 (rmlA gene knock-out) genome with the second single crossover event. The M. tuberculosis rmlA gene was transcribed by the promoter of heat shock protein 60 from Mycobacterium bovis BCG. The parent plasmid pCG76 has the same temperature-sensitive mycobacterial replication origin as pPR27 and thus can replicate at 30°C but not at 42°C.
Selection of mc2155 mutant-1 strains with integrated rmlA::KmR in the genome
The mc2155 transformants with pPR27-rmlA::KmR were selected on LB agar plates containing Km and Gm at 30°C and all colonies became yellow-pigmented when catechol was sprayed on the plates owing to expression of the xylE gene (Curcic et al., 1994) in the pPR27-rmlA::KmR plasmid. The yellow colony was propagated in LB broth containing Km and Gm at 30°C, and spread on LB agar plates containing Km and Gm at 42°C. The Km-resistant colonies on the plates have necessarily integrated rmlA::KmR into the mc2155 genome at 42°C. Southern hybridization analysis of 17 yellow colonies revealed that six showed integration of rmlA::KmR upstream of the rmlA locus (Fig. 1a) and one colony showed integration of rmlA::KmR downstream of the rmlA locus. Figure 1(b) shows two colonies with integration of rmlA::KmR upstream of the rmlA locus.
Selection of mc2155 mutant-2 (rmlA gene knock-out) strains
To attempt the second single crossover event, rescue plasmid pCG76-Mtb rmlA was electroporated to mc2155 mutant-1 (with the pathway 1) cells and spread on LB agar plates containing sucrose, Km and Sm and incubated at 30°C. Under selection of sucrose and expression of M. tuberculosis rmlA in the mc2155 mutant-1, the GmR, sacB (Pelicic et al., 1996), xylE and M. smegmatis rmlA genes will be deleted from the genome of the mc2155 mutant-1 when the second single crossover event occurs, resulting in generation of mc2155 mutant-2 (rmlA gene knock-out) strains (Fig. 1a). Thus, only the white colonies grown on LB agar plates containing sucrose, Km and Sm were candidates for rmlA gene knock-out. Genomic DNA from five white colonies was digested by SmaI and hybridized using the M. smegmatis rmlA probe. All five colonies showed bands at 3.24 and 8.14 kb as expected (Fig. 1c) for the second single crossover event. The 6.5-kb band was from the pCG76-Mtb rmlA plasmid.
Essentialness of the rmlA gene for mycobacterial growth
To confirm whether the rmlA gene is essential for mycobacterial growth, the growth curves of five mc2155 rmlA knock-out strains (nos. 1–5) at both 30 and 42°C were determined; similar patterns were observed for all, and the growth curve for no. 4 is shown in Fig. 2. The results clearly showed that rmlA knock-out strain mc2155 grew only at 30°C but not at 42°C at which pCG76-Mtb rmlA was unable to replicate. In contrast, wild-type mc2155 containing pCG76 grew at both 30 and 42°C, confirming that the M. tuberculosis rmlA gene was essential for mycobacterial growth.
Morphological change of mc2155 rmlA knock-out strains after shifting from 30 to 42°C
To determine whether decreasing RmlA activity has effects on the morphology of mc2155 rmlA knock-out cells a temperature shift experiment was performed to acquire a certain amount of mc2155 rmlA knock-out cells. The mc2155 rmlA knock-out strain (no. 4) with pCG76-Mtb rmlA was grown at 30°C for 20 h to produce M. tuberculosis RmlA enzyme, and then the cells were grown at 42°C. A600nm over time was obtained as shown in Fig. 3(a). The expressed M. tuberculosis RmlA protein from pCG76-Mtb rmlA in mc2155 rmlA knock-out cells at 30°C allowed the cells to grow at 42°C for a certain period and even multiplied for the first 24 h after the temperature shift to 42°C. The morphological phenotypes of mc2155 rmlA knock-out cells and wild type mc2155 cells were examined via SEM (Fig. 3b–f). The mc2155 rmlA knock-out cells grown at 30°C for 72 h (Fig. 3b) and 120 h (Fig. 3d) exhibited the normal rod-like shape of wild-type mc2155 (Fig. 3f), whereas the mc2155 rmlA knock-out cells grown at 42°C for 72 h appeared significantly longer (Fig. 3c). Some of mc2155 rmlA knock-out cells grown at 42°C for 120 h had irregular surface wrinkles and even lysed (Fig. 3e). These SEM results indicate that lack of RmlA activity will cause dramatic morphological changes in the bacteria prior to cell lysis.
l-Rhamnose is also present in both Gram-negative and Gram-positive bacteria. l-Rhamnose is a common component of the O-antigen of lipopolysaccharides (LPS) of Gram-negative bacteria such as E. coli (Stevenson et al., 1994), Salmonella enterica (Jiang et al., 1991) and Shigella flexneri (Macpherson et al., 1994). In Gram-positive bacteria such as Lactococcus lactis (Boels et al., 2004), Streptococcus mutans (Tsukioka et al., 1997a, b), l-rhamnose is a component of cell-wall polysaccharides on their cell surfaces. dTDP-rhamnose is a precursor of l-rhamnose and the biosynthetic pathway of dTDP-rhamnose is ubiquitous and highly conserved in both Gram-negative and Gram-positive bacteria, but the essential requirement for rml genes for both Gram-negative and Gram-positive bacterial growth has not yet been investigated.
The rhamnose-GlcNAc disaccharide is a critical linker to the structural integrity of the mycobacterial cell wall. Neither l-rhamnose nor the genes encoding RmlA–D and rhamnosyl transferase have been identified in humans so far (Giraud & Naismith, 2000). Thus, inhibitors of RmlA–D enzymes and rhamnosyl transferase (WbbL) are unlikely to interfere with metabolic pathways in humans. Our previous genetic approaches have provided the direct evidence that M. tuberculosis rmlB, rmlC, rmlD and wbbL genes are valid targets (Ma et al., 2002; Mills et al., 2004; Li et al., 2006). Here we have investigated the essential requirement for the rmlA gene in M. smegmatis and the effect of a lack of RmlA on cellular morphology.
We used M. smegmatis mc2155 as a model organism to test the essential requirement for rmlA for bacterial growth, as M. tuberculosis and M. smegmatis have a basic cell-wall structure (Daffe et al., 1993). BLAST analysis also showed that the organization of rmlA–D genes in the M. smegmatis mc2155 genome is the same as that in the M. tuberculosis H37Rv genome. The results show that RmlA enzyme clearly is essential for mycobacterial growth, because an mc2155 rmlA gene knock-out strain carrying rescue plasmid pCG76-Mtb rmlA can grow only at 30°C but not at 42°C when the rescue plasmid does not replicate. This result is consistent with the report that M. tuberculosis rmlA is an essential gene using an insertional mutagenesis technology (Sassetti et al., 2003).
KasA (β-ketoacyl-ACP synthase) is a key enzyme of mycolic acid biosynthesis in mycobacteria, and scanning electron micrographs of kasA mutants have revealed that KasA depletion results in the cell surface having a crumpled appearance prior to lysis (Bhatt et al., 2005). Arabinosyltransferases (EmbA, EmbB and EmbC) are involved in the biosynthesis of arabinan in the mycobacterial cell wall (Crick et al., 2004). Escuyer et al. (2001) generated M. smegmatis embA, embB and embC mutants and observed morphological alterations of emb mutants. The embB mutant showed drastically altered morphology with size shortening, swelling and distortion. The embA mutant was also altered in its morphology with size shortening, slight distortion and swelling but to a lesser extent than with the embB mutant; the embC mutant exhibited even greater size shortening. Their results point to the probability that the emb mutants had an altered cell wall. We examined morphological changes of mc2155 rmlA knock-out cells as the biosynthesis of the RmlA enzyme decreased in the temperature shift experiment. The SEM data indicate that morphological alterations (enlongation and lysis over time) of mc2155 rmlA knock-out cells correlated with lack of RmlA enzyme. Thus, the present results demonstrate that RmlA, d-glucose-1-phosphate thymidylyltransferase, can be used as a target to develop new antituberculosis drugs.
This work was supported by funds provided through the National Basic Research Program of China (2006CB504400) and the National Natural Science Foundation of China (30270320).
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