Expression of GlmM protein in wild type mc²155 strain and <i>M. smegmatis</i> AS strain.

<p>The expressions of <i>M. smegmatis</i> GlmM proteins in wild type mc²155 strain (A) and <i>M. smegmatis</i> AS strain (B) were detected by using anti-GlmM polyclonal antibody. The results showed that there were no obvious differences of GlmM protein expressions in wild type mc²155 strain with or without 20 ng/ml tetracycline. However, compared with uninduced <i>M. smegmatis</i> AS strain, the expression of GlmM protein was 70% decreased in <i>M. smegmatis</i> AS strain when induced with 20 ng/ml tetracycline. M. PageRuler prestained protein ladder; lane 1. GlmM proteins in uninduced cultures; lane 2. GlmM proteins in cultures induced with 20 ng/ml tetracycline.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Sequence alignment of Ec GlmM, Msm MSMEG_1556 and Mtb Rv3441c.

<p>Msm MSMEG_1556 protein was 38% identical to Ec GlmM and Mtb Rv3441c protein was 41% identical to Ec GlmM. Mtb Rv3441c protein and Msm MSMEG_1556 protein had 79% identities. * indicates the homology between all three organisms, # indicates the homology between <i>M. tuberculosis</i> and <i>M. smegmatis</i>, and ! indicates the homology between <i>E. coli</i> and <i>M. smegmatis</i> or <i>E. coli</i> and <i>M. tuberculosis</i>.</p

Specific phosphoglucosamine mutase activity of GlmM proteins.

<p>Specific phosphoglucosamine mutase activity of GlmM proteins.</p

Scanning electron micrographs of <i>M. smegmatis</i> LS2 strain after shifting temperature from 30°C to 42°C.

<p><i>M. smegmatis</i> LS2 grown at 30°C was switched to 42°C when OD₆₀₀ reached to 0.01, and <i>M. smegmatis</i> LS2 strain kept growing at 30°C was as control. The cells were harvested after temperature switched for 72 h, 120 h and 192 h, respectively. The micrographs showed that shape and size of <i>M. smegmatis</i> LS2 strain kept growing at 30°C (A, B, C) were similar to those of wild type strains, whereas the <i>M. smegmatis</i> LS2 strain at 42°C became longer shapes, “bulb” heads, rougher cell surface and even lysis (D, E, F). A, B, C. <i>M. smegmatis</i> LS2 grown at 30°C for 72, 120, 192 h (10000×); D, E, F. <i>M. smegmatis</i> LS2 grown at 42°C for 72, 120 and 192 h (10000×). H, I. <i>M. smegmatis</i> LS2 grown at 42°C for 120 and 192 h (20000×); G. Wild type <i>M. smegmatis</i> mc²155 (10000×).</p

Growth curves and CFU of <i>M. smegmatis</i> LS2 strain.

<p>A. Growth curves of <i>M. smegmatis</i> LS2 and <i>M. smegmatis</i> mc²155 carrying pCG76 plasmid at 30°C and 42°C. The cultures were grown in LB medium at 30°C and 42°C, and growth was monitored by measuring the absorbance of the cultures at 600 nm. <i>M. smegmatis</i> mc²155 strains carrying pCG76 plasmid had similar growth patterns at 30°C and 42°C. <i>M. smegmatis</i> LS2 was only able to grow at 30°C but not at 42°C. (⋄) <i>M. smegmatis</i> LS2 at 30°C; (⧫) <i>M. smegmatis</i> LS2 at 42°C; (○) <i>M. smegmatis</i> mc²155 carrying pCG76 at 30°C; (•) <i>M. smegmatis</i> mc²155 carrying pCG76 at 42°C. B. Growth curves of <i>M. smegmatis</i> LS2 after shifting temperature from 30°C to 42°C. <i>M. smegmatis</i> LS2 was grown at 30°C to 0.10 OD₆₀₀, and then transferred to a 42°C incubator. The culture grown at 30°C was as control. The OD₆₀₀ was determined at the interval of 24 hours. X-axis showed the time point after temperature transferred. (▪) <i>M. smegmatis</i> LS2 was grown at 30°C for 24 h, and then grown at 42°C; (□) <i>M. smegmatis</i> LS2 kept at 30°C was a control. C. CFU of <i>M. smegmatis</i> LS2 at 30°C and 42°C. CFU determinations were taken at the same time points as (B). Dilutions were spread on LB agar plates and the plates were incubated at 30°C before CFU were counted. Black bars were <i>M. smegmatis</i> LS2 at 30°C; White bars were <i>M. smegmatis</i> LS2 which grown at 30°C for 24 h and then grown at 42°C.</p

Biofilm formation in wild type mc²155 strain and <i>M. smegmatis</i> AS strain.

<p>Wild type mc²155 strain and <i>M. smegmatis</i> AS strain were incubated in modified M63 media with or without tetracycline in a polystyrene microtiter plate at 30°C for 5 days without disturbance. Crystal violet stained polystyrene wells (B) and the estimations of corresponding crystal violet uptake (A) were shown for the indicated strain. WT (−). Wild type mc²155 strain without tetracycline; WT (+). Wild type mc²155 strain induced with 20 ng/ml tetracycline; AS (−). <i>M. smegmatis</i> AS strain without tetracycline; AS (+). <i>M. smegmatis</i> AS strain induced with 20 ng/ml tetracycline. The experiment was carried out in triplicate and the graph indicates the mean value with standard deviation bars.</p

Bacterial growth of wild type mc²155 strain and <i>M. smegmatis</i> AS strain.

<p>The cultures were grown in LB broth containing 0.05% Tween 80 at 37°C with different concentration of tetracycline. The OD600 and CFU of the cultures were monitored at interval of 12 h. A. Growth curve of the wild type mc²155 strain; B. Growth curve of the <i>M. smegmatis</i> AS strain; C. CFU of the wild type mc²155 strain; D. CFU of the <i>M. smegmatis</i> AS strain. (▴) The cultures without tetracycline; (▪) The cultures induced with 10 ng/ml tetracycline; (○) The cultures induced with 20 ng/ml tetracycline; (•) The cultures induced with 30 ng/ml tetracycline; (□) The cultures induced with 50 ng/ml tetracycline. The values plotted are the mean value and standard deviation from triplicate experiments.</p

Identification of <em>M. tuberculosis</em> Rv3441c and <em>M. smegmatis</em> MSMEG_1556 and Essentiality of <em>M. smegmatis</em> MSMEG_1556

<div><p>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 <em>E. coli</em> BL21(DE3) and their phosphoglucosamine mutase activity were detected. In order to further investigate the essentiality of MSMEG_1556 for the growth of <em>M. smegmatis,</em> 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 <em>M. smegmatis</em>. This study provided evidence that GlmM enzyme could be as a potential target for developing anti-tuberculosis drugs.</p> </div

MICs of anti-tuberculosis drugs in wild type mc²155 strainand <i>M. smegmatis</i> AS strain.

<p>WT (−). Wild type mc²155 strain without tetracycline; WT (+). Wild type mc²155 strain induced with 20 ng/ml tetracycline; AS (−). <i>M. smegmatis</i> AS strain without tetracycline; AS (+). <i>M. smegmatis</i> AS strain induced with 20 ng/ml tetracycline. The experiment was carried out in triplicate, the mean value and standard deviation were indicated.</p