Correspondence: Yufang Ma, Department of Biochemistry and Molecular Biology, Dalian Medical University, 9 W. Lushun South Road, Dalian 116044, China. Tel.: +86 411 8611 0338; fax: +86 411 8611 0378; e-mail: email@example.com
The disaccharide d-N-acetylglucosamine-l-rhamnose plays an important role in the mycobacterial cell wall as a linker connecting arabinogalactan and peptidoglycan via a phosphodiester linkage. The first step of the disaccharide linker is the formation of decaprenyl phosphate-GlcNAc, which is catalyzed by GlcNAc-1-phosphate transferase. In Gram-negative bacteria, the wecA gene specifies the UDP-GlcNAc: undecaprenyl phosphate GlcNAc-1-phosphate transferase (WecA), which catalyzes the first step in the biosynthesis of lipopolysaccharide O-antigen. Mycobacterium tuberculosis Rv1302 and Mycobacterium smegmatis MSMEG_4947 show homology to Escherichia coli WecA protein. We cloned Rv1302 and MSMEG_4947 and introduced plasmids pYJ-1 (carrying Rv1302) and pYJ-2 (carrying MSMEG_4947) into a wecA-defective strain of E. coli MV501, respectively. Lipopolysaccharide analysis demonstrated that lipopolysaccharide synthesis in MV501 (pYJ-1) and MV501 (pYJ-2) was restored upon complementation with Rv1302 and MSMEG_4947, respectively. This provides the first evidence that Rv1302 and MSMEG_4947 have the same function as E. coli WecA. We also generated an M. smegmatis MSMEG_4947 knockout mutant using a homologous recombination strategy. The disruption of MSMEG_4947 in the M. smegmatis genome resulted in the loss of viability at a nonpermissive temperature. Scanning electron microscopy and transmission electron microscopy results showed that the lack of the MSMEG_4947 protein causes drastic morphological changes in M. smegmatis.
The cell wall core of mycobacteria consists of mycolic acids, arabinogalactan and peptidoglycan. The esterified arabinogalactan with the mycolic acids is attached to the peptidoglycan via a disaccharide linker, d-N-acetylglucosamine-l-rhamnose (d-N-GlcNAc-l-Rha) (Brennan, 2003; Crick et al., 2004) (Fig. 1a). The disaccharide linker is biosynthesized on a lipid carrier, decaprenyl phosphate (C50-P) (Barry et al., 2007; Berg et al., 2007). GlcNAc-1-phosphate transferase transfers GlcNAc-1-phosphate from undecaprenyl phosphate (UDP)-GlcNAc to the carrier, yielding C50-P-P-GlcNAc. The rhamnosyl transferase (WbbL) (Mills et al., 2004; Grzegorzewicz et al., 2008) encoded by Rv3265c attaches the rhamnosyl residue (Rha) to C50-P-P-GlcNAc to produce C50-P-P-GlcNAc-Rha (Fig. 1b), which is then further elongated with galactan and arabinan and finally mycolylated arabinogalactan attached to the peptidoglycan. However, GlcNAc-1-phosphate transferase has not yet been identified in mycobacteria.
Lipopolysaccharides found in the outer membrane of Gram-negative bacteria are made up of a hydrophobic lipid (lipid A), a hydrophilic core polysaccharide chain and a hydrophilic O-antigenic polysaccharide side chain (O-antigen). In most cases, O-specific chains are formed by repeating units of oligosaccharides that exhibit a strain-specific structural diversity (Reeves et al., 1996). The biosynthesis of an O repeating unit starts on the cytosolic face of the plasma membrane with the formation of a sugar–phosphodiester linkage with a lipid carrier. After the initiation reaction, additional sugars are incorporated to complete the O unit in reactions catalyzed by specific glycosyltransferases, which are either soluble cytosolic enzymes or peripheral membrane proteins associated with the plasma membrane by ionic interactions (Feldman et al., 1999; Samuel & Reeves, 2003). The GlcNAc is the first sugar of the O unit and the wecA gene (formerly called rfe) specifies the UDP-GlcNAc: undecaprenyl phosphate (Und-P) GlcNAc-1-phosphate transferase (WecA) that catalyzes the first step in the biosynthesis of O unit (Alexander & Valvano, 1994; Raetz & Whitfield, 2002; Schäffer et al., 2002). That is, WecA from Gram-negative bacteria transfers GlcNAc-1-phosphate from UDP-GlcNAc to Und-P (C55-P), forming C55-P-P-GlcNAc. This reaction is similar to the formation of C50-P-P-GlcNAc in mycobacteria, although decaprenyl phosphate, rather than the usual Und-P, plays the central role as the carrier lipid in all known cell wall biosynthetic processes in mycobacteria (Scherman et al., 1996; Mahapatra et al., 2005; Mikušováet al., 2005).
Mycobacterium tuberculosis Rv1302 shows high homology to Escherichia coli WecA protein (Amer & Valvano, 2001). Rv1302 and E. coli WecA have 28% identity (85/305) and 44% (137/305) positivity. A Mycobacterium smegmatis MSMEG_4947 ortholog was found by a blastp search using M. tuberculosis Rv1302 protein as a query; Rv1302 and MSMEG_4947 have 79% identity (301/380) and 83% positivity (316/380); and MSMEG_4947 and E. coli WecA have 29% (92/313) and 44% (138/313), respectively. Escherichia coli WecA has been well characterized as UDP-GlcNAc: Und-P-GlcNAc-1-phosphate transferase, in which the two aspartic acids of a DGID motif have been reported to bind polyprenyl phosphate (Amer & Valvano, 2002), and arginine as well as the HIHH motif in cytosolic domain V have been proposed to recognize UDP-GlcNAc (Amer & Valvano, 2001). The DGLD motif and HLHH motif are found in both Rv1302 and MSMEG_4947.
In this study, to ascertain the function of M. tuberculosis Rv1302 and M. smegmatis MSMEG_4947, we cloned Rv1302 and MSMEG_4947 to investigate the complementation of Rv1302 and MSMEG_4947 on the wecA-defective strain E. coli MV501 (Alexander & Valvano, 1994). To test the viability of MSMEG_4947 for mycobacteria, we constructed M. smegmatis MSMEG_4947 knockout mutant using a homologous recombination strategy and observed the morphology changes in the MSMEG_4947 knockout mutant using scanning electron microscopy (SEM) and transmission electron microscopy (TEM).
Materials and methods
Bacterial strains and plasmids
The characteristics of all the bacterial strains and plasmids used in this study are detailed in Table 1. Escherichia coli NovaBlue, E. coli K-12 and E. coli MV501 were grown routinely in Luria–Bertani (LB) broth or on LB agar plates at 37 °C. Mycobacterium smegmatis mc2155 was grown routinely in LB broth containing 0.05% Tween 80 or on LB agar plates at 37 °C. Mycobacterium smegmatis MSMEG_4947 knockout mutant was grown at 30 or 42 °C. Antibiotics were added in the following concentrations: ampicillin, 100 μg mL−1; tetracycline, 20 μg mL−1; kanamycin, 50 μg mL−1 for NovaBlue and 25 μg mL−1 for mc2155; gentamicin, 5 μg mL−1; and streptomycin, 25 μg mL−1 for NovaBlue and 12.5 μg mL−1 for mc2155.
Table 1. Bacterial strains and plasmids used in this study
Escherichia coli NovaBlue
Used for cloning and propagation of plasmids
E. coli K-12
Used as a DNA template to amplify the wecA gene
Myobacterium tuberculosis H37Rv
Used as a DNA template to amplify Rv1302
Myobacterium smegmatis mc2155
Used as a DNA template for amplification of MSMEG_4947 with its upstream sequence
The E. coli wecA gene was cloned to the EcoRV site of pMD18-T
Rv1302 was cloned to the EcoRV site of pMD18-T
MSMEG_4947 with its upstream sequence was cloned to the EcoRV site of pMD18-T
The kanR cassette was inserted into the BglII site of MSMEG_4947 in pYJ-2
Conditional replication plasmid, MSMEG_4947∷kanR was cloned to the NotI and SpeI sites of pPR27-xylE
Rv1302 was cloned to the NdeI and BamHI sites of pET23b-Phsp60
Rescue plasmid, Phsp60-Rv1302 was cloned to the XbaI and BamHI sites of pCG76
Cloning E. coli wecA, M. tuberculosis Rv1302 and M. smegmatis MSMEG___4947
The genomic DNA of E. coli K-12 was prepared as described previously (Chen & Kuo, 1993), with modification. Escherichia coli wecA gene was amplified from E. coli K-12 genomic DNA using the forward primer 5′-GCCATATGAATTTACTGACAGTGAG-3′ and the reverse primer 5′-TTCTCGAGTTATTTGGTTAAATTGGGGC-3′ and was cloned into pMD18-T, yielding pYJ (Table 1). Rv1302 was amplified from M. tuberculosis H37Rv genomic DNA (supplied by Colorado State University via an NIH contract) using the forward primer 5′-GGCGCATATGCAGTACGGTCTCGAGGTG-3′ and the reverse primer 5′-TAATGGATCCCTAGTCCAGGTCCGGGTCGTAG-3′, and was cloned into pMD18-T to yield pYJ-1 (Table 1). The genomic DNA of M. smegmatis mc2155 was prepared as described previously (Jackson et al., 2000). MSMEG_4947 with its upstream sequence (550 bp) was amplified from mc2155 genomic DNA using the forward primer 5′-ATGACTAGTGCGACATGCCCGTCGGCGTG-3′ and the reverse primer 5′-ATGCGGCCGCTCACGGCTCCTGCGCACCGTC-3′ and cloned into pMD18-T to generate pYJ-2 (Table 1). The nucleotide sequences of the E. coli wecA gene, Rv1302 and MSMEG_4947 were confirmed by DNA sequencing.
Preparation and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of lipopolysaccharides
pYJ, pYJ-1 and pYJ-2 were transformed, respectively, to E. coli MV501 strain, in which the wecA gene is defective, generating MV501 (pYJ), MV501 (pYJ-1) and MV501 (pYJ-2) (Table 1). The pUC18 plasmid was transformed to MV501, yielding MV501 (pUC18) as a control. The lipopolysaccharides from MV501 (pYJ), MV501 (pYJ-1), MV501 (pYJ-2) and MV501 (pUC18) cell envelopes were extracted with hot phenol and analyzed by SDS-PAGE, followed by silver staining as described previously (Tsai & Frasch, 1982; Hitchcock & Brown, 1983; Valvano & Crosa, 1989), with a slight modification. The total cell envelopes obtained from 50 mL cultures were suspended in 200 μL of water, and treated with an equal volume of 90% phenol at 65 °C for 15 min, followed by centrifugation at 16 000 g. The aqueous phase was extracted once with ethyl ether and mixed in a 1 : 1 ratio with a tracking dye solution (125 mM Tris-HCl, pH 6.8, 2% SDS, 20% glycerol, 0.002% bromophenol blue and 10% mercaptoethanol), and then boiled for 5 min. The lipopolysaccharides were loaded onto a 15% polyacrylamide gel containing 4 M urea and the gel was stained by silver staining solution.
Construction of the MSMEG___4947 gene knockout mutant
The kanR from pUC4K was inserted into the BglII site of pYJ-2 to disrupt MSMEG_4947, yielding pYJ-3 (Table 1). pYJ-3 was digested by SpeI and NotI and the DNA fragment of MSMEG_4947∷kanR was ligated to the SpeI and NotI sites of pPR27-xylE to yield a conditional replication plasmid pYJ-4 (Table 1). pYJ-1 was digested by NdeI and BamHI and Rv1302 was ligated to the NdeI and BamHI sites of pET23b-Phsp60 to generate pYJ-5 (Table 1). pYJ-5 was digested by XbaI and BamHI and the Phsp60-Rv1302 was ligated to the XbaI and BamHI sites of pCG76 to yield a rescue plasmid pYJ-6 (Table 1).
Mycobacterium smegmatis mc2155 electrocompetent cells were prepared as described (Pelicic et al., 1996). The pYJ-4 was electroporated to the competent cells with Electroporator 2510 (Eppendorf). Transformants were grown on LB agar plates containing kanamycin and gentamicin at 30 °C. One colony was propagated in LB broth containing 0.05% Tween 80, kanamycin and gentamicin at 30 °C and the cells were spread on LB agar plates containing kanamycin and gentamicin at 42 °C. The mc2155 mutant strains with the first single crossover event were selected using a Southern blot, as described (Li et al., 2006).
The rescue plasmid pYJ-6 was electroporated into the mc2155 mutant. Transformants were grown on LB agar plates containing kanamycin and streptomycin at 30 °C. One colony was inoculated into LB broth containing kanamycin and streptomycin, and incubated at 30 °C. The cells were spread on an LB agar plate containing 10% sucrose, kanamycin and streptomycin. The MSMEG_4947 knockout mutant strains (Table 1) with the second single crossover event were selected via a Southern blot.
Growth of the MSMEG___4947 knockout mutant
Five MSMEG_4947 knockout mutants (nos 1–5) were inoculated into 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. A600 nm was detected at intervals of 24 h and the growth curves at both 30 and 42 °C were obtained.
Morphology of the MSMEG___4947 knockout mutant after shifting from 30 to 42 °C
The MSMEG_4947 knockout mutant (no. 2) was grown in LB broth containing 0.05% Tween 80 and kanamycin at 30 °C for 24 h (A600 nm was 0.064), and then transferred to a 42 °C incubator for continuous growth. The cells grown at 42 °C for 72 and 144 h were harvested and fixed in ice-cold 2.5% glutaraldehyde. The cells were washed with phosphate-buffered saline and postfixed with 1% osmium tetroxide and then dehydrated in a graded ethanol series (20–100%). The cells for SEM observation were critical-point dried and applied to a silicon wafer slide. The cells were then examined using a JSM-6360 scanning electron microscope (JEOL) (Qu et al., 2008). The cells (grown at 42 °C for 144 h) for TEM observation were embedded in the Epon 812 embedding kit and cut into ultrathin sections. The sections were double-stained with uranyl acetate and lead nitrate and then examined using a JEM-2000EX TEM (JEOL).
Complementation of the E. coli MV501 strain with the E. coli wecA gene, Rv1302 and MSMEG___4947
The lipopolysaccharides prepared from MV501 (pYJ), MV501 (pYJ-1), MV501 (pYJ-2) and MV501 (pUC18) cells were analyzed by SDS-PAGE, followed by silver staining (Fig. 2). The lipopolysaccharides from MV501 (pYJ), MV501 (pYJ-1) and MV501 (pYJ-2) cells showed a ladder-like banding pattern characteristic of O side-chain material. The results suggested that Rv1302 and MSMEG_4947 have the same function as E. coli WecA and both Rv1302 and MSMEG_4947 utilize C55-P and UDP-GlcNAc as substrates to initiate the synthesis of the O7 polysaccharide that is covalently linked to the lipid A-core oligosaccharide of E. coli O7:K1 strain VW187 (Valvano & Crosa, 1989).
Generation of the M. smegmatis MSMEG___4947 knockout mutant
The MSMEG_4947 in conditional replication plasmid pYJ-4 was disrupted by inserting a kanR cassette. A two-step homologous recombination procedure (Li et al., 2006) was used to achieve the allelic replacement of the MSMEG_4947 gene by MSMEG_4947∷kanR. MSMEG_4947 (406 amino acids) shares 79% identity with Rv1302 (404 amino acids); therefore, the rescue plasmid pYJ-6 carrying Rv1302 was constructed for complementation studies. The MSMEG_4947 knockout mutant was confirmed by a Southern blot using MSMEG_4947 as a probe (Fig. 3).
Requirement of MSMEG___4947 for M. smegmatis growth
The growth of five MSMEG_4947 knockout mutants (nos 1–5) was investigated at both 30 and 42 °C. All five MSMEG_4947 knockout mutants had similar growth patterns and the growth curve of no. 2 mutant is shown in Fig. 4a. The results clearly showed that the MSMEG_4947 knockout mutant grew only at 30 °C and not at 42 °C. The rescue plasmid pYJ-6 was unable to replicate at 42 °C and, therefore, no more Rv1302 protein was generated. In contrast, wild-type mc2155 containing pCG76 grew at both 30 and 42 °C, confirming that MSMEG_4947 was essential for the growth of M. smegmatis.
Morphological alterations of the MSMEG___4947 knockout mutant
To investigate whether a decrease in WecA has effects on the morphology of the MSMEG_4947 knockout mutant, a certain amount of cells was acquired by performing a temperature shift experiment. The MSMEG_4947 knockout mutant (no. 2) was grown at 30 °C for 24 h to produce Rv1302 protein, and then grown at 42 °C. A600 nm was measured at 24-h intervals (Fig. 4b) and the cells were harvested for observation of the morphological phenotype (Fig. 5). MSMEG_4947 knockout cells grown at 30 °C for 72 (Fig. 5a) and 144 h (Fig. 5c) had a smooth cell surface and exhibited the normal rod-like shape seen in the wild-type mc2155 cells (Qu et al., 2008). In contrast, the MSMEG_4947 knockout cells grown at 42 °C for 72 (Fig. 5b) and 144 h (Fig. 5d) had a clearly different shape and some cells grown for 144 h had an irregular surface with a crumpled appearance and were even lysed (Fig. 5d). TEM analysis showed that MSMEG_4947 knockout cells grown at 42 °C for 144 h (Fig. 5f) grew larger (in diameter) and were pear-shaped, in contrast to MSMEG_4947 knockout cells grown at 30 °C (Fig. 5e). Vacuoles were also observed in MSMEG_4947 knockout cells grown at 42 °C for 144 h (Fig. 5f). These SEM and TEM results indicate that the lack of WecA will cause drastic morphological alterations before lysis.
The disaccharide linker d-N-GlcNAc-l-Rha is a critical structure for the integration of mycolylated arabinogalactan and peptidoglycan of the mycobacterial cell wall. The biosynthesis of the disaccharide linker is initiated by a transfer of GlcNAc-1-phosphate from UDP-GlcNAc to the acceptor C50-P, yielding C50-P-P-GlcNAc, which is similar to the first step of the O-antigen biosynthesis in Gram-negative bacteria (e.g. E. coli). Escherichia coli WecA has been well characterized as UDP-GlcNAc: Und-P-GlcNAc-1-phosphate transferase to catalyze the first step in the synthesis of E. coli WecA of O-antigen (Amer & Valvano, 2002); M. tuberculosis Rv1302 and M. smegmatis MSMEG_4947 have significant homology to E. coli WecA.
In our study, we cloned Rv1302 and MSMEG_4947 to construct pYJ-1 and pYJ-2 plasmids, respectively. MV501 (pYJ-1) and MV501 (pYJ-2) were generated by transforming pYJ-1 and pYJ-2 to an E. coli wecA-defective strain MV501 (Alexander & Valvano, 1994), respectively. MV501 (pYJ) control was also generated by transforming pYJ carrying the E. coli wecA gene to MV501. The E. coli wecA mutation carried by the MV501 strain abolishes the expression of the O7-specific polysaccharide, but does not affect the synthesis of the lipid A-core. The lipopolysaccharides from MV501 (pYJ-1) and MV501 (pYJ-2) was restored upon complementation with Rv1302 and MSMEG_4947, respectively, and the pattern of O-antigen from MV501 (pYJ-1) and MV501 (pYJ-2) was the same as that from MV501 (pYJ). This suggests that Rv1302 and MSMEG_4947 have a WecA transferase function that catalyzes a transfer of GlcNAc-1-phosphate to the lipid carrier C55-P that is involved in the formation of the O7 repeating unit. However, mycobacteria use C50-P as a lipid carrier in all known cell wall biosynthetic pathways (Scherman et al., 1996; Mahapatra et al., 2005; Mikušováet al., 2005). We speculate that Rv1302 and MSMEG_4947 could utilize either C50-P or C55-P as a substrate. Al-Dabbagh et al. (2008) tested the Thermotoga maritima WecA activity using polyisoprenyl phosphate of different sizes, from C15-P to C75-P; their data showed that a minimal length of 35 carbons was required for the lipid substrate. Therefore, it is necessary to clarify the substrate specificity using purified Rv1302 and MSMEG_4947 proteins. Rv1302 and MSMEG_4947 proteins are predicted to be membrane proteins with 11 transmembrane domains and therefore it is difficult to produce high yields of these proteins in E. coli. Overexpression of Rv1302 and MSMEG_4947 proteins in certain E. coli expression strains is currently underway in our laboratory for further characterization.
It is obvious that the disaccharide linker plays an important role by joining mycolylated arabinogalactan and peptidoglycan. The growth curves of the M. smegmatis MSMEG_4947 knockout mutant at 30 and 42 °C show that MSMEG_4947 is essential for the growth of M. smegmatis. The SEM and TEM examinations of the MSMEG_4947 knockout mutant demonstrate that the disruption of MSMEG_4947 affected cellular appearance and structure. Therefore, a lack of WecA protein results in the destruction of cell wall structure, eventually leading to cell death.
We would like to thank Prof. M.A. Valvano for providing the E. coli MV501 strain. This work was supported by the National Basic Research Program of China (2006CB504400) and the Key Project of Major Infectious Diseases (2008ZX10003-006).