Figure 1.
Analysis of the domain structure of MarR and its binding with its upstream DNA sequence.
(A) Analysis of the domain structure of MarR and its genomic location. The Ms6508 gene encodes a typical MarR regulator containing an HTH-MARR domain. Ms6508 shares a 272-bp common upstream promoter region with the Ms6509–6510 gene cluster. (B) Bacterial one-hybrid assays for the interaction between MarR and the upstream sequence of the marRAB operon. A pair of pBXcmT/pTRG plasmids was co-transformed into the reporter strain and its growth was monitored together with that of self-activation controls on selective medium. Co-transformants containing the pBX-Rv2031/pTRG-Rv3133 plasmids (24) served as positive controls (CK+) and co-transformants containing the empty vectors pBX and pTRG served as negative controls (CK−). Only the MarR+Ms6508p co-transformant strains and a positive strain CK+ grew well on the screening medium, indicating that MarR specifically interacts with the upstream sequence of the marRAB operon, Ms6508p. (C) ChIP assays in wildtype and marR deletion mutant M. smegmatis strains. ChIP using preimmune (P) or immune sera (I) raised against MarR. The mycobacterial promoter Ms6141p was used as a negative control.
Figure 2.
Assays for the Ms6508–Ms6510 co-transcription by reverse transcription PCR.
(A) The operon structure of Ms6508–Ms6509–Ms6510. Primers were designed for assays and indicated by black arrows. (B) Reverse transcription PCR assays for Ms6508–Ms6510 co-transcription. mRNA (DNA-free) was used as negative controls. PCR procedure was as follows: reactions were degenerated at 95°C for 30 s, annealed at 60°C for 30 s, extended at 72°C for 30 s and under 35 cycles.
Figure 3.
Sequence alignment and domain analysis of Ms6508–Ms6510.
(A)The conserved amino acids residues of MarR are highlighted. ST1710, a MarR family regulator in Sulfolobus tokodaii; MTH313, a MarR family regulator in Methanobacterium thermoautotrophicum; b1530, a MarR family regulator in E. coli. (B) Domain assays for MarR and Ms6510. Ms6509 encodes a multidrug ABC transporter ATP-binding family protein and Ms6510 encodes a multidrug ABC transporter family protein.
Figure 4.
Identifying the DNA-binding motif of MarR.
(A) DNaseI footprinting assays were carried out on the coding and non-coding strands. Protection of the promoter DNA by MarR against DNaseI digestion was tested by increasing the amount of MarR (0–0.6 µM). The ladders are shown and the corresponding nucleotide sequence is listed (lanes 2–4). The protected regions on the coding and non-coding strands are indicated. (B) Sequence and structural characteristics of the promoter DNA region protected by MarR. The regions protected by MarR are underlined. The 21-bp sequences containing the inverted repeats (IR) separated by 1 bp are indicated by a pair of arrows. The translation start codon of MarR is indicated in bold. (C) EMSA assays for the DNA-binding activity of MarR on DNA substrates with wildtype IR sequence and IR-deleted mutant sequences. DNA substrates were co-incubated with 0.2–0.6 µM of the MarR protein. Cold DNA substrate containing IR motif (p4), but not unrelated substrate (p3) which does not contain IR motif, could competitively inhibit the binding of MarR to the labeled DNA substrate (p4*).
Figure 5.
Construction of the MarR knockout strain of M. smegmatis and southern blot assays.
(A) Schematic representation of the recombination strategy for removing marR from the genome of M. smegmatis. (B) A map of the recombinant vector pMindMs6508KO containing upstream and downstream sequences of marR, and the gene that confers resistance against hygromycin. (C) Schematic representation of the DNA fragments of the wildtype strain and the marR knockout strain treated with the restriction enzyme SalI. The probe is indicated with a black bar. (D) Southern blot assays. A 387 bp probe corresponding to the upstream sequences of marR in M. smegmatis was obtained by PCR and was labeled with digoxigenin dUTP (Boehringer Mannheim Inc., Germany).
Figure 6.
Expression assays for the effect of MarR on the target gene in wildtype and marR -deleted mutant strains.
(A) Determination of β-galactosidase activity (Miller units). The values presented represent averages of three independent experiments. For statistical analysis, two-way ANOVA with Bonferroni multiple comparison tests were performed. P≤0.05 was considered statistically significant. (B) Quantitative real-time PCR assays for differential expression of the target genes in wildtype, mutant and complementation strains (left panel), and the marR overexpression strain (right panel). Relative expression levels of the genes were normalized using the SigA gene as an invariant transcript, and an unrelated Ms6141 gene as a negative control. Data were analyzed using the 2ΔΔCt method [21]. Relative expression data were analyzed for statistical significance by the unpaired two-tailed Student’s T-test using GraphPad Prism (Version 5).
Figure 7.
Assays for the effects of MarR on RIF resistance in M. smegmatis.
Growth curves of the wild-ype, marR-overexpressed, deletion mutant and complementation strains were determined as described in Experimental Procedures. These mycobacterial strains were grown in 7H9 broth in the absence (A) and presence of 4 µg/ml RIF (B). Representative growth curves are shown.
Figure 8.
Effects of Ms6509–6510 genes on mycobacterial RIF resistance.
(A) Growth curves of the wildtype, Ms6509–6510 overexpression, deletion mutant and complementation strains in 7H9 broth in the absence of RIF. (B) Growth curves of these mycobacterial strains in 7H9 broth in the presence of 4 µg/ml RIF.
Figure 9.
Determination of RIF transport and absorption by M. smegmatis.
HPLC assays were performed as described in “Experimental Procedures”. The relationship of standard curve between RIF concentration and peak area was acquired through a series of concentration gradient standard RIF. A standard curve equation Y = 28403X−6721 (r = 0.9988) was obtained, in which Y represents peak area (µV×s), X represents RIF concentration (µg/ml) and r is the correlation coefficient. The samples peak area, from Msm/pMV261, Msm/pMV261-Ms6509–6510 and Msm Ms6509–6510: hyg/pMV261, was put into the standard curve equation and the RIF concentration of samples could be calculated. Each sample peak area was determined independently for three times. The quantity of the absorbed RIF by M. smegmatis was obtained by calculating the change of RIF concentration between two time points. Relative expression data were analyzed for statistical significance by the unpaired two-tailed Student’s T-test using GraphPad Prism (Version 5).