Table 1.
Sensitivity of C. glutamicum strains to alkylating agents tested by disk diffusion assay.
Table 2.
Sensitivity of C. glutamicum strains to oxidizing and reducing agents tested by disk diffusion assay.
Table 3.
Sensitivity of C. glutamicum strains to various classes of antibiotics tested by disk diffusion assay.
Table 4.
The minimum inhibitory concentrations (MICs) of various antibiotics for C. glutamicum strains.
Figure 1.
Sensitivity assays of C. glutamicum strains to heavy metals.
A–D The growth (A600) of the C. glutamicum WT(pXMJ19), Δmca(pXMJ19) and Δmca(pXMJ19-mca) strains after 24 h at 30°C in LB medium containing increasing concentrations of Cd2+(A), Ni2+ (B), Cr2+(C) and Cu2+(D) was recorded. Mean values with standard deviations (error bars) from at least three repeats are shown. *: P≤0.05.
Figure 2.
The amidase activity of C. glutamicum Mca.
The levels of reactant (MSmB) and product (AcCysmB) of the amidase activity following reaction of mBBr and MSH in different C. glutamicum strains were determined. Cells grown in LB (A600 = 1.6) were treated with monobromobimane (mBBr), to form a fluorescent bimane derivative of mycothiol (MSmB). Mca cleaved MSmB to AcCysmB (N-acetylcystein S-conjugate of bimane) that was released into the medium. The amounts of MSmB and AcCysmB in the cytoplasm and the medium were measured by HPLC and were presented as µmol g−1 dried cell weight. Mean values with standard deviations (error bars) from at least three repeats were shown. **: P≤0.01. *: P≤0.05.
Figure 3.
Catalytic activity of C. glutamicum Mca.
A. GlcN standard curve. Solutions of GlcN (0–100 µM) in buffer (50 mM HEPES, 50 mM NaCl, and 1 mM TCEP, pH 7.5) were diluted with borate (0.75 M, pH 9.0) and mixed with FSA (2.3 mM), followed by measurement of the resulting fluorescence. The observed increase in fluorescence was drawn against glucosamine concentration to generate the standard curve. A linear equation was fitted to data. B. Mca-catalyzed reaction. GlcNAc (2 mM) was pre-incubated at 30°C in assay buffer (50 mM HEPES, 50 mM NaCl, and 1 mM TCEP, pH 7.5) and the reaction was started by the addition of Mca (6.4 µM). At different time points, aliquots of reaction mixture were terminated by 5% trichloroacetic acid, diluted with borate (0.75 M, pH 9.0) and mixed with FSA (2.3 mM), followed by measurement of the resulting fluorescence. The glucosamine standard curve (A) was used to transform the observed rate of the reaction into µ min−1.
Table 5.
Michaelis-Menten parameters of Mca for N-deacetylation of GlcNAc and for amidase activity of MSmB.
Figure 4.
Effects of divalent metal cations and pH on C. glutamicum Mca activity.
A–E. Catalytic activity of Mca in the presence of Co2+(A), Mn2+(B), Ni2+(C), Zn2+(D) and Fe2+(E), respectively, was analyzed with GlcNAc or MSmB as substrates. Apo-Mca was incubated with stoichiometric amounts of metal ions. After 30 min, the enzyme was diluted into assay buffer containing the substrate GlcNAc (5 mM) or MSmB (1 mM). The amidase activity (Left Y axis) and deacetylase activity (Right Y axis) were measured as described in “Materials and Methods”. F. Deacetylation of GlcNAc and amidase activity of MSmB by Zn2+-Mca at different pH levels. The V/K values were measured with 5 mM GlcNAc as substrate for deacetylase activity (Left Y axis) or 1 mM MSmB as substrate for amidase activity (Right Y axis) under six different pH values. pKa values of 6.5 and 9.5 were determined by fitting Equation 1 to the data (bars represent standard error of the mean).
Table 6.
Steady-state kinetic parameters of C. glutamicum Mca mutants for amidase activity of mycothiol bimane (MSmB).
Table 7.
Steady-state kinetic parameters of C. glutamicum Mca mutants for N-deacetylation of N-acetyl-D-glucosamine (GlcNAc).
Figure 5.
Positive regulation of C. glutamicum mca expression by SigH.
A. β-Galactosidase analysis of the mca promoter activity was performed using the transcriptional Pmca::lacZ chromosomal fusion reporter expressed in the wild type, ΔsigH mutant, and the complementary strain ΔsigH(pXMJ19-sigH). 100 µl of exponentially growing C. glutamicum cells induced with different toxic agents at indicated concentrations for 30 min was added to the enzyme reaction system. β-Galactosidase activity was assayed as described in “Materials and Methods”. Mean values with standard deviations (error bar) from at least three repeats are shown. **, P≤0.01. B. qRT-PCR assay revealed that expression of mca was under strict positive regulation of SigH. Exponentially growing C. glutamicum cells were exposed to different toxic agents at indicated concentrations for 30 min. The levels of mca expression were determined by quantitative RT-PCR. The mRNA levels were presented relative to the value obtained from wild type cells without treatment. The values represent the mean results from three independent cultivations, with standard errors. **: P≤0.01. C. Interactions between SigH and the mca promoter analyzed by EMSA. The increasing amounts of SigH used were 0, 0.5, 1.5, and 3.0 µg (lane 1, 2, 3, and 4, respectively). As a negative control, a 400 bp fragment from the mca coding region amplified with primers Control-F and Control-R instead of the 400 bp mca promoter was incubated with 3.0 µg His6-SigH in the binding assay (lane 5). (*) Free DNA, and (**) major DNA-protein complex.