Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG_1556 and Essentiality of M. smegmatis MSMEG_1556

The normal growth of mycobacteria attributes to the integrity of cell wall core which consists of peptidoglycan (PG), arabinogalactan (AG) and mycolic acids. N-acetyl glucosamine (GlcNAc) is an essential component in both PG and AG of mycobacterial cell wall. The biosynthetic pathway for UDP-N-acetylglucosamine (UDP-GlcNAc), as a sugar donor of GlcNAc, is different in prokaryotes and eukaryotes. The conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, which is catalyzed by phosphoglucosamine mutase (GlmM), is unique to prokaryotes. Bioinformatic analysis showed that Msm MSMEG_1556 and Mtb Rv3441c are homologous to Ec GlmM. In this study, soluble Msm MSMEG_1556 protein and Mtb Rv3441c protein were expressed in E. coli BL21(DE3) and their phosphoglucosamine mutase activity were detected. In order to further investigate the essentiality of MSMEG_1556 for the growth of M. smegmatis, we generated a conditional MSMEG_1556 knockout mutant, which harbored thermo-sensitive rescue plasmid carrying Mtb Rv3441c. As the rescue plasmid was unable to complement MSMEG_1556 deficiency at 42°C, MSMEG_1556 knockout mutant did not grow. The dramatic morphological changes of MSMEG_1556 knockout mutant after temperature shift from 30°C to 42°C have been observed by scanning electron microscope. These results demonstrated that MSMEG_1556 is essential for growth of M. smegmatis. This study provided evidence that GlmM enzyme could be as a potential target for developing anti-tuberculosis drugs.


Introduction
Nowadays, two millions deaths each year (2% increased incidence) caused by Mycobacterium tuberculosis has been considered to be a major public health threat [1]. However, vaccine failed to provide protective immunity and any efforts to control tuberculosis were compromised as they evolved into stronger, more drugresistant forms [2,3,4,5]. As we know, the cell wall of all Mycobacterium species is very waxy, hydrophobic, and thicker than other bacteria, the low permeability and resistance of cell wall substantially contributes to the defense of adverse factors [6,7]. Thereby, the enzymes involved in the metabolic pathways of the cell wall are potential excellent targets for new anti-tuberculosis drugs [8,9].
The mycobacterial cell wall consists of the mycolate and peptidoglycan (PG) layer held together by arabinogalactan (AG) layer [10,11]. AG is attached to the muramic acid residue of the PG through a disaccharide linker (a-L-rhamnosyl-a-D-N-acetylglucosaminosyl-1-phosphate), and the glycan of PG is a disaccharide repeat unit (N-acetylmuramic acid-N-acetyl glucosamine). UDP-N-acetylglucosamine (UDP-GlcNAc) is an important pre-cursor for the synthesis of PG layer, and also a direct glycosyl donor for disaccharide linker, therefore, it perhaps plays a vital role in mycobacterial growth [12,13,14]. Three enzymes glutamine fructose-6-phosphate transferase (GlmS), phosphoglucosamine mutase (GlmM), glucosamine-1-phosphate acetyl transferase/N-Acetylglucosamine-1-phosphate urididyl transferase (GlmU) involve in the metabolic pathway of UDP-GlcNAc in E. coli [13,15,16,17,18]. It is noteworthy that reactions catalyzed by GlmM and GlmU are unique to prokaryotes (Fig. 1). The function of Msm GlmU and Mtb GlmU had been identified and the GlmU had been confirmed being essential for growth of M. smegmatis and M. tuberculosis [19,20]. Therefore, GlmM may also be essential for mycobacteria and could be used as a potential target of antituberculosis drug.
The bioinformatics analysis data showed that Msm MSMEG_1556 and Mtb Rv3441c are homologous to Ec GlmM. We proposed Msm MSMEG_1556 protein and Mtb Rv3441c protein catalyzing the same reaction in biosynthetic pathway of UDP-GlcNAc as in E. coli. In this study, we identified that both Msm MSMEG_1556 and Mtb Rv3441c protein had the phosphoglucosamine mutase activity. The essentiality of MSMEG_1556 for mycobacteria was determined by observing growth curve, colony forming unit (CFU) and morphology of MSMEG_1556 knockout mutant. In addition, dinitrothiocyanobenzene (DNTB) chromogenic method [20,21] in microtiter plate for the measurement of phosphoglucosamine mutases activity was described.

Bacterial strains, plasmids, and culture conditions
The bacterial strains and plasmids used in this study were listed in Table S1. E.coli DH5a, NovaBlue and BL21(DE3) cells were grown in Luria-Bertani (LB) medium at 37uC routinely. M. smegmatis mc 2 155 strain was grown in LB broth containing 0.05% Tween 80 or on LB agar at 37uC routinely. M. smegmatis mc 2 155 was used for cloning MSMEG_1556 gene with its upstream region and constructing a conditional M. smegmatis MSMEG_1556 gene knockout strain by allelic exchange recombinant experiments. Sucrose was added to the LB agar at final concentration of 10% when required. Antibiotics were added at the following final concentrations: ampicillin (Amp), 100 mg/ml; gentamicin (Gen), 5 mg/ml for E. coli; kanamycin (Kan), 50 mg/ml for E. coli and 25 mg/ml for M. smegmatis; streptomycin (Str), 25 mg/ml for E. coli and 12.5 mg/ml for M. smegmatis.
The purified Ec GlmM protein, Msm MSMEG_1556 protein and Mtb Rv3441c protein were run on 12% SDS-PAGE and transferred to a nitrocellulose membrane (Pall Corp) in blotting buffer (20 mM Tris-base, 150 mM glycine and 20% methanol). The membrane was blocked with 1% BSA in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) and incubated with (anti)-polyhistidine clone His-1 antibody (Sigma) at 1:5000 dilution followed by washing with TBST. The membrane was incubated with antimouse-IgG-AP conjugate and the protein band was visualized in BCIP/NBT solution.
The Mtb GlmU protein was purified as described previously [19].

Assaying phosphoglucosamine mutase of Ec GlmM protein, Msm MSMEG_1556 protein and Mtb Rv3441c protein
The phosphoglucosamine mutase activity of Ec GlmM protein, Msm MSMEG_1556 protein and Mtb Rv3441c protein were determined by coupled assay, in which the GlcNH 2 -1-P converted from GlcNH 2 -6-P by the mutase was quantitatively converted into UDP-GlcNAc in the presence of purified GlmU enzyme [24]. The by-product CoA-SH from the reaction catalyzed by GlmU acetyltransferase activity was then measured by a colorimetric assay coupled with 5, 59-dithio-bis-(2-nitrobenzoic acid) (DTNB) (Fig. 2). The reaction mixture of 50 ml contained 50 mM Tris-HCl, pH8.0, 2.5 mM MgCl 2 , 1 mM GlcNH 2 -6-P, 0.6 mM acetyl CoA, 0.7 mM Glc-1, 6-diP, purified Mtb GlmU protein (1 mg) and purified Ec GlmM protein (0.625 mg), Msm MSMEG_1556 protein (0.625 mg) or Mtb Rv3441c protein (0.625 mg) in a 96well microtiter plate. A control without GlmM protein was used to correct the trial errors from the reaction of GlmU protein with GlcNH 2 -6-P. The reaction was incubated at 37uC for 30 min and terminated by adding 50 ml of stop solution containing 50 mM Tris-HCl, pH 7.5, 6 M guanidine hydrochloride and then incubated for 10 min by the addition of 50 ml of Ellman's reagent solution containing 0.2 mM DTNB in buffer with 50 mM Tris-HCl, pH 7.5, and 1 mM EDTA. TNB, the product generated from the reaction of CoA-SH and DTNB, was monitored at 405 nm by Benchmark Plus plate reader (Thermo). To confirm product CoA-SH the reaction mixture was applied on a Nova-Pak C18 (3.96150 mm, 4 mm) and CoA-SH was separated at a flow rate of 1 ml/min under phosphate buffer (pH 6.5)-methanol (95:5) and monitored at 259 nm. One unit of specific enzyme activity was defined as one nmol CoA-SH produced by one mg protein per minute under the specific condition.
Construction of conditional M. smegmatis MSMEG_1556 gene knockout strain Msm MSMEG_1556 gene (1362 bp) with its upstream sequence (500 bp) was amplified from mc 2 155 genomic DNA by using Msm glmM3 and Msm glmM4 primers ( Table 1). The PCR product (1862 bp) was purified and cloned into pMD18-T vector to yield pLS3 (pMD18-Msm MSMEG_1556) plasmid (Table S1). After confirmation by DNA sequencing, the kan R cassette from pUC4K was inserted to the XhoI site of pLS3, resulting in pLS4 (pMD18-Msm MSMEG_1556::kan R ) plasmid (Table S1). pLS4 was digested by NotI and SpeI and the Msm MSMEG_1556::kan R fragment was cloned to the NotI and SpeI sites of pPR27-xylE to generate a conditional replication plasmid pLS5 (pPR27-Msm MSMEG_1556::kan R ) (Table S1). pLS5 also harbors the counterselectable marker sacB from Bacillus subtilis for use in selecting the double crossover event in the presence of sucrose.
Since both Mtb Rv3441c gene and Msm MSMEG_1556 gene were identified as glmM gene to encode phosphoglucosamine mutase, Mtb Rv3441c gene was used for construction of the rescue plasmid. pLS2 was digested by NdeI and BamHI and the Mtb Rv3441c gene was ligated to the NdeI and BamHI sites of pET23b-Phsp60, yielding pLS6 (pET23b-Phsp60-Mtb Rv3441c) plasmid. pLS6 was digested by XbaI and BamHI and Phsp60-Mtb Rv3441c fragment was cloned to the XbaI and BamHI sites of pCG76 to generate a rescue plasmid pLS7 (pCG76-Phsp60-Mtb Rv3441c) [25].
M. smegmatis mc 2 155 electro-competent cells were prepared as described [26]. The conditional replication plasmid pLS5 was electroporated into mc 2 155 competent cells by Electroporator 2510 (Eppendorf). The transformants were then grown on LB agar plates containing Kan and Gen at 30uC. One colony was incubated in LB broth containing 0.05% Tween 80, Kan and Gen at 30uC and the culture was then plated out onto an LB agar plate containing Kan and Gen followed by incubation at 42uC for 6 days. Since plasmid pLS5 was not able to replicate at 42uC, the MSMEG_1556::kan R in pLS5 made single crossover at upstream or downstream of the MSMEG_1556 locus in M. smegmatis genome of colonies grown at 42uC. M. smegmatis LS1 mutants (M. smegmatis mc 2 155 with pLS5 plasmid integrated into the MSMEG_1556 gene locus) (Table S1) through the first homologous recombination were selected by Southern blot [11].
The rescue plasmid pLS7 was electroporated into M. smegmatis LS1 electro-competent cells and the transformants were grown on an LB agar plate containing Kan, Gen and Str and grown at 30uC. One colony was incubated in LB broth containing 0.05% Tween 80, Kan, Gen and Str and grown at 30uC. The culture was then plated out onto LB agar plate containing Kan, Str and 10% sucrose. M. smegmatis LS2 (mcD1556::pLS7), a MSMEG_1556 Figure 2. Assay of phosphoglucosamine mutase. Phosphoglucosamine mutase catalyzes the conversion of GlcNH 2 -6-P to GlcNH 2 -1-P. GlcNH 2 -1-P and acetyl CoA were catalyzed by glucosamine-1-phosphate acetyltransferase activity of GlmU to produce GlcNAc-1-P and CoA-SH which was detected by DTNB. TNB, the product generated from the reaction of CoA-SH and DTNB, could be monitored at 405 nm. doi:10.1371/journal.pone.0042769.g002  (Table S1) where Msm MSMEG_1556 gene was knocked out through the second homologues recombination was selected by using Southern blot. To confirm M. smegmatis LS2, the DNA fragment containing Msm MSMEG_1556::kan R and its flanking sequences at upstream and downstream of Msm MSMEG_1556 was amplified from M. smegmatis LS2 genome by using Msm glmM5 and Msm glmM6 primers ( Table 1). The amplified PCR products of Msm MSMEG_1556::kan R from M. smegmatis LS2 and Msm MSMEG_1556 from wild type M. smegmatis mc 2 155 were distinguished by 1% agrose gel.

Southern blot analysis
The MSMEG_1556 DNA probe was prepared by using DIG High Prime Labeling and Detection Starter Kit I (Roche). The pLS3 was digested by SpeI and NotI and Msm MSMEG_1556 was purified. One microliter of the purified Msm MSMEG_1556 was used as a template to generate probe. The genomic DNA isolated from different colonies was digested overnight by SmaI enzyme. The resulting DNA fragments were separated by a 0.8% agarose gel. The DNA was then transferred to Hybond-N + membrane (GE Healthcare). DNA hybridization and detection of the probe were performed as recommended by Roche.

Growth of M. smegmatis LS2 strain
The growth of M. smegmatis LS2 was determined by observing growth curve and CFU, respectively. To measure growth curve, M. smegmatis LS2 was inoculated into 5 ml of LB broth containing 0.05% Tween 80 and appropriate antibiotics, and the cells were incubated at 30uC and 42uC respectively. M. smegmatis mc 2 155 carrying the rescue plasmid pLS7 and wild type M. smegmatis mc 2 155 were used as controls. The OD 600 of the cultures was monitored at interval of 24 h and the growth curves at both 30uC and 42uC were obtained.
To measure CFU, M. smegmatis LS2 was grown in LB medium containing 0.05% Tween 80 and Kan at 30uC until the OD 600 reached 0.10, and the culture was transferred to a 42uC incubator. The culture grown at 30uC was as control. The OD 600 was determined at the interval of 24 hours. For CFU determinations (taken at the same time points), dilutions were spread on LB agar plates containing Str and Kan. The plates were incubated at 30uC before CFU were counted.

Expression of MSMEG_1556 protein in M. smegmatis LS2 strain
Western blot was used to analyze the expression levels of GlmM protein in M. smegmatis LS2. M. smegmatis LS2 was grown in 500 ml LB broth at 30uC, and then switched to 42uC when OD 600 of the culture was 0.01. M. smegmatis LS2 kept growing at 30uC was as control. The culture after temperature shifting for 72 h was harvested and the cells were resuspended in 4 ml lysis buffer with 1 mM PMSF followed by sonication. After centrifugation at 20,0006g for 30 minutes, the resulting supernatant was collected for Western blot analyses. The protocol was followed as previously described but (anti)-MSMEG_1556 antibody (prepared in our lab, unpublished data) was used to instead of the (anti)-polyhistidine clone His-1 antibody.
Morphology of M. smegmatis LS2 strain after shifting temperature from 30uC to 42uC M. smegmatis LS2 was grown in 20 ml LB broth at 30uC, and then switched to 42uC when OD 600 of the culture was 0.01. M. smegmatis LS2 kept growing at 30uC was as control. The cells were harvested after shifting temperature for 72 h, 120 h and 192 h, respectively. Scanning electron microscopy (SEM) samples were prepared as previously described [19]. Briefly, the cell pellet was washed three times in the 0.1 M Phosphate buffer (pH 7.4) and fixed with 2.5% glutaraldehyde followed by fixation with 1% OsO4. The cells were then dehydrated in a graded series of ethanol (20, 40, 60, 70, 80, 90, and 100%). After critical point dry the cells were applied to a silicon wafer slide and coated by gold.  Table 2. Specific phosphoglucosamine mutase activity of GlmM proteins.

Proteins
Specific enzyme activity of (nmol?min 21  The cells were then observed by using a JSM-6360 at 15 kV of accelerating voltage.

Msm MSMEG_1556 and Mtb Rv3441c were homologous to Ec GlmM
The bioinformatics analysis data showed that Msm MSMEG_1556 protein was 38% identical to Ec GlmM and Mtb Rv3441c protein was 41% identical to Ec GlmM. Msm MSMEG_1556 protein and Mtb Rv3441c protein had 79% identities (Fig. 3). The results demostrated that Rv3441c is the ortholog of GlmM in M. tuberculosis and MSMEG_1556 is the ortholog of GlmM in M. smegmatis.

Soluble Ec GlmM protein, Msm MSMEG_1556 protein and Mtb Rv3441c protein were overexpressed
Soluble Ec GlmM protein, Msm MSMEG_1556 protein and Mtb Rv3441c protein were overexpressed in BL21(DE3) respectively and purified by Ni-NTA affinity chromatography. The purified proteins were analyzed by SDS-PAGE and Western blot. The results showed that purified Ec GlmM protein, Msm MSMEG_1556 protein and Mtb Rv3441c protein had the expected molecular weight of 48.99 kD, 47.99 kD, and 47.31 kD, respectively (Fig. 4).

Msm MSMEG_1556 protein and Mtb Rv3441c protein had phosphoglucosamine mutase activity
The specific enzyme activity of Ec GlmM, which could be as a positive control, was 156 nmol?min 21 ?mg 21 . The specific enzyme activity of Msm MSMEG_1556 protein and Mtb Rv3441c protein were 145 and 151 nmol?min 21 ?mg 21 , respectively. The results demonstrated that both Msm MSMEG_1556 protein and Mtb Rv3441c protein had phosphoglucosamine mutase activity same as Ec GlmM protein (Table 2). In the reaction catalyzed by Mtb Rv3441c protein, the product CoA-SH was also detected by Nova-Pak C18 and its retention time was 9.1 min, which is consistent with CoA-SH standard (data not shown).

Conditional M. smegmatis MSMEG_1556 gene knockout strain LS2 was constructed
Once conditional replication plasmid pLS5 was electroporated to M. smegmatis mc 2 155, the MSMEG_1556::kan R in pLS5 made single crossover at upstream or downstream of the MSMEG_1556 locus in M. smegmatis genome of colonies grown at 42uC, resulting in M. smegmatis LS1 mutants which integrated the MSMEG_1556::kan R at the MSMEG_1556 locus through the first homologous recombination. M. smegmatis LS1 mutants were selected by Southern blot (data not shown). The results suggested that MSMEG_1556 was an essential gene. When rescue plasmid pLS7 was electroporated to M. smegmatis LS1 mutant, the colonies grown on LB agar plate containing Kan, Str and Sucrose at 30uC had undergone the second homologous recombination, resulting in MSMEG_1556 gene knockout. Five M. smegmatis LS2 (MSMEG_1556 gene knockout) strains were selected by Southern blot. All five M. smegmatis LS2 strains showed the expected DNA fragments of 2.12 kb and 2.55 kb (Fig. 5A, 5C), whereas wild type M. smegmatis mc 2 155 showed an expected DNA fragment of 3.38 kb (Fig. 5B, 5C). The PCR result indicated that all five M. smegmatis LS2 strains had the expected PCR product of 3.15 kb, and wild type M. smegmatis mc 2 155 showed the expected PCR product of 1.88 kb (Fig. 5D).

MSMEG_1556 gene was essential for mycobacterial growth
Growth curves of M. smegmatis LS2 and M. smegmatis mc 2 155 carrying pCG76 at 30uC and 42uC were obtained to determine the essentiality of MSMEG_1556 for the growth of mycobacteria. The results showed that M. smegmatis LS2 was only able to grow at 30uC but not at 42uC, whereas M. smegmatis mc 2 155 carrying pCG76 was able to grow at both 30uC and 42uC (Fig. 6A), which was consistent to the growth curve of wild type M. smegmatis mc 2 155 (data not shown). To measure CFU, M. smegmatis LS2 was grown at 30uC to 0.10 OD 600 , and then the culture was switched to an incubator at 42uC. The OD 600 and CFU over time were obtained as shown (Fig. 6B, 6C). Since Rv3441c protein was produced in M. smegmatis LS2 cells at 30uC, the cells multiplied for the first 24 hours after the temperature shift. Then dramatically, with increased incubation time at 42uC, the CFUs dropped as the amount of Rv3441c protein decreased. These results demonstrated that MSMEG_1556 gene is essential in M. smegmatis.

Rv3441c protein was not detected in M. smegmatis LS2 strain
Western blot was used to analyze the expression levels of Rv3441c protein. (Anti)-MSMEG_1556 antibody performed a cross reaction with Rv3441c protein (Fig. 7A), demonstrated that (anti)-MSMEG_1556 antibody could be used for detect the Rv3441c protein in M. smegmatis LS2 strain. Western blot results showed that when M. smegmatis LS2 was grown at 42uC, the expression of Rv3441c protein was not detected compared to the Rv3441c protein in M. smegmatis LS2 grown at 30uC (Fig. 7B). The MSMEG_1556 in wild type M. smegmatis was also detected. The data indicated that lacking Rv3441c protein in M. smegmatis LS2 had effect on bacterial growth at 42uC.

M. smegmatis LS2 grown at 42uC had a morphological change
The morphology of M. smegmatis mc 2 155, M. smegmatis LS2 grown at 30uC and M. smegmatis LS2 grown at 42uC were observed by using SEM. The results showed that the cellular shape and size of M. smegmatis LS2 grown at 42uC (switched from 30uC) changed compared to those kept growing at 30uC and wild type M. smegmatis mc 2 155. As the incubation time extended, M. smegmatis LS2 strain at 42uC became longer shapes, ''bulb'' heads, rougher cell surface and even lysis (Fig. 8D, 8E, 8F). Whereas, the shape and size of M. smegmatis LS2 strain kept growing at 30uC (Fig. 8A, 8B, 8C) were similar to those of wild type M. smegmatis mc 2 155 (Fig. 8G).

Discussion
UDP-GlcNAc is an essential common precursor of bacterial cell wall PG and outer membrane lipopolysaccharide, as well as important for the synthesis of the enterobacterial common antigen [13,14]. The pathway for UDP-GlcNAc synthesis in prokaryotes is somewhat different from that in eukaryotes [17]. Three enzymes, glutamine fructose-6-phosphate transferase (GlmS), phosphoglucosamine mutase (GlmM) and glucosamine-1-phosphate acetyl transferase/N-Acetylglucosamine-1-phosphate urididyl transferase (GlmU), catalyzed the formation of UDP-GlcNAc in E. coli have been identified [13,15,16,17,18]. GlmM catalyzes the conversion of GlcNH 2 -6-P to GlcNH 2 -1-P. The homologs of GlmM were also found in other Gram-negative bacteria e.g. H. influenzae, H. pylori, P. aeruginosa [27,28,29] and Gram-positive bacteria e.g. S. gordonii and S. aureus [30,31]. The glmM gene was essential for cell growth in Gram-negative bacteria (e.g. E. coli). The inactivation of glmM gene in E. coli was followed by various alterations of cell shape and finally cells were lysed [13]. This was most probably due to the progressive depletion of precursors for PG and lipopolysaccharide synthesis. However, the mutation of glmM gene only reduced the growth rate and increase cell autolysis in Gram-postive bacteria [30,32]. Furthermore, the glmM mutation also reduced the carrying pCG76 at 42uC. B. Growth curves of M. smegmatis LS2 after shifting temperature from 30uC to 42uC. M. smegmatis LS2 was grown at 30uC to 0.10 OD 600 , and then transferred to a 42uC incubator. The culture grown at 30uC was as control. The OD 600 was determined at the interval of 24 hours. X-axis showed the time point after temperature transferred. (&) M. smegmatis LS2 was grown at 30uC for 24 h, and then grown at 42uC; (%) M. smegmatis LS2 kept at 30uC was a control. C. CFU of M. smegmatis LS2 at 30uC and 42uC. CFU determinations were taken at the same time points as (B). Dilutions were spread on LB agar plates and the plates were incubated at 30uC before CFU were counted. Black bars were M. smegmatis LS2 at 30uC; White bars were M. smegmatis LS2 which grown at 30uC for 24 h and then grown at 42uC. doi:10.1371/journal.pone.0042769.g006 formation of bacterial biofilm and increased sensitivity to penicillins [30,32].
In mycobacteria, UDP-GlcNAc is an important sugar donor for both formation of dissarchride linker and biosynthesis of peptidoglycan in mycobacterial cell wall, therefore, lacking UDP-GlcNAc could have effect on the structural integration of mycobacterial cell wall and further on their cell morphology. Ec GlmM has been well characterized as phosphoglucosamine mutase to catalyze the second step in the synthesis of E. coli UDP-GlcNAc [13], and Msm MSMEG_1556 protein and Mtb Rv3441c protein have significant homology to Ec GlmM.
To detect the phosphoglucosamine mutase activity of Msm MSMEG_1556 protein and Mtb Rv3441c protein, it is required to acquire soluble protein. Mtb Rv3441c protein was produced by using pET16b vector, unfortunately, Mtb Rv3441c protein was insoluble (data not shown). To avoid the formation of inclusion bodies, a cold-shock expression vector pCold II with the Rv3441c gene was constructed. This vector was designed to perform efficient protein expression utilizing promoter derived from cspA gene, which was one of the cold-shock gene. When the incubation temperature of E. coli host cells was reduced sufficiently, the growth is temporarily halted and almost of protein expression decrease, while expression of a group of proteins called ''coldshock proteins'' was specifically induced. A significant overproduction of soluble Mtb Rv3441c protein in E. coli BL21(DE3) was observed. The Msm MSMEG_1556 protein was also produced by using the same protocol.
In our study, we also set up an enzyme assay for detection of phosphoglucosamine mutase activity. DNTB chromogenic method was more convenient and time saving, which facilitates highthroughput inhibitor screening, compared with previous methods by autoradiography and HPLC [13,17].
The activity of both Msm MSMEG_1556 protein and Mtb Rv3441c protein was dependent on the presence of Mg 2+ . Glc-1,6-diP was also required for activity. The purified proteins exhibited little activity without Glc-1,6-diP, however, phosphoglucosamine mutase activity was remarkably enhanced in the presence of this compound. The phosphorylated phosphoglucosamine mutase was assumed to be active [13]. It is unclear that Glc-1,6-diP is a  phosphorylating agent or activator. We attempt to co-crystallize Glc-1,6-diP and Mtb Rv3441c protein so as to reveal this mechanism in follow-up research.
Mtb Rv3441c gene (annotated as mrsA in TubercuList Server) has been proved to be essential for the growth of cells by using Himar1-based transposon site hybridization (TraSH) methodology [33]. To assess the effect of mutated glmM gene on cell growth, morphology, cell wall structure, etc., we used a model mycobacterial strain, M. smegmatis, to construct conditional MSMEG_1556 gene knockout strain LS2 by inserting kan R cassette. M. smegmatis LS2 was unable to grow at 42uC (non-permissive temperature) since the rescue plasmid carrying Mtb Rv3441c gene could not replicate. It demonstrated that the MSMEG_1556 gene is essential for the growth of M. smegmatis. Furthermore, we found that GlmM protein was not expressed in M. smegmatis LS2 strain. To observe morphological change of LS2 strain, the LS2 cells were harvested after shifting temperature from 30uC (permissive temperature) to 42uC. The morphological analysis of LS2 by SEM revealed that the LS2 cells were longer and had rougher surface compared to the wild type cells. With the increased incubation time at 42uC, many cells fused and lysed eventually. These results suggested that lacking GlmM could block the PG synthesis and make the structure of cell wall changed, resulting in cell death. Therefore, GlmM is a potential target for development of anti-tuberculosis drugs.

Supporting Information
Table S1 Bacterial strains and plasmids used in this study. (DOC)