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Characterization of the interaction and cross-regulation of three Mycobacterium tuberculosis RelBE modules.

Yang M, Gao C, Wang Y, Zhang H, He ZG - PLoS ONE (2010)

Bottom Line: RelBE modules appear to be autoregulated in an atypical manner compared to other TA systems; however, the molecular mechanisms and potential interactions between different RelBE modules remain to be elucidated.The RelBE-like modules exerted complex cross-regulation effects on mycobacterial growth.Conversely, relF enhanced the toxicity of the relE toxin gene, while relB increased the toxicity of relK.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.

ABSTRACT
RelBE represents a typical bacterial toxin-antitoxin (TA) system. Mycobacterium tuberculosis H37Rv, the pathogen responsible for human tuberculosis, contains three RelBE-like modules, RelBE, RelFG, and RelJK, which are at least partly expressed in human macrophages during infection. RelBE modules appear to be autoregulated in an atypical manner compared to other TA systems; however, the molecular mechanisms and potential interactions between different RelBE modules remain to be elucidated. In the present study, we characterized the interaction and cross-regulation of these Rel toxin-antitoxin modules from this unique pathogen. The physical interactions between the three pairs of RelBE proteins were confirmed and the DNA-binding domain recognized by three RelBE-like pairs and domain structure characteristics were described. The three RelE-like proteins physically interacted with the same RelB-like protein, and could conditionally regulate its binding with promoter DNA. The RelBE-like modules exerted complex cross-regulation effects on mycobacterial growth. The relB antitoxin gene could replace relF in cross-neutralizing the relG toxin gene. Conversely, relF enhanced the toxicity of the relE toxin gene, while relB increased the toxicity of relK. This is the first report of interactions between different pairs of RelBE modules of M. tuberculosis.

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Self-interactions of three M. tuberculosis relBE-like modules.Electrophoretic mobility shift assays (EMSA) were used to detect the binding of RelBE proteins with their operon promoters. A fixed amount of 32P-labeled DNA substrate was incubated with various amounts of proteins in a total volume of 15 µL of an EMSA buffer. Electrophoresis was performed and gels were exposed to a storage-phosphor screen overnight as described in the “Materials and Methods”. The images were acquired by Typhoon Scanner (GE Healthcare). Both DNA substrate and protein/DNA complexes are indicated by arrows on the left of the figure. (A) Different amount of each RelB-like protein (3.75 µM, 7.5 µM, and 15 µM ) interacts with their promoter DNA. (B) The concentration of Rv1247c remains constant at 7.5 µM. The interaction between various ratio of RelE/RelB (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (C) The concentration of RelF remains constant at 7.5 µM. The interaction between different ratio of RelF/RelG (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (D) The concentration of RelJ remains constant at 3.75 µM. The interaction between different ratio of RelK/RelJ (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (E) Competitive assays. Three non-labeled promoter DNA substrates (5-fold, 10-fold or 50-fold) were used to compete with their corresponding labeled DNA substrates. The species of promoter DNA was indicated on top of each panel in the figure.
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pone-0010672-g002: Self-interactions of three M. tuberculosis relBE-like modules.Electrophoretic mobility shift assays (EMSA) were used to detect the binding of RelBE proteins with their operon promoters. A fixed amount of 32P-labeled DNA substrate was incubated with various amounts of proteins in a total volume of 15 µL of an EMSA buffer. Electrophoresis was performed and gels were exposed to a storage-phosphor screen overnight as described in the “Materials and Methods”. The images were acquired by Typhoon Scanner (GE Healthcare). Both DNA substrate and protein/DNA complexes are indicated by arrows on the left of the figure. (A) Different amount of each RelB-like protein (3.75 µM, 7.5 µM, and 15 µM ) interacts with their promoter DNA. (B) The concentration of Rv1247c remains constant at 7.5 µM. The interaction between various ratio of RelE/RelB (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (C) The concentration of RelF remains constant at 7.5 µM. The interaction between different ratio of RelF/RelG (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (D) The concentration of RelJ remains constant at 3.75 µM. The interaction between different ratio of RelK/RelJ (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (E) Competitive assays. Three non-labeled promoter DNA substrates (5-fold, 10-fold or 50-fold) were used to compete with their corresponding labeled DNA substrates. The species of promoter DNA was indicated on top of each panel in the figure.

Mentions: Promoter DNA (described as 1247p, 2865p, and 3357p below) was used as substrate to further investigate the in vitro association of RelBE with promoters. As shown in Fig. 2A, of the three RelB-like proteins (from 3.75 to 15 µM), only RelJ associated strongly with its promoter as a singular protein and produced a substantial shifted protein/DNA complex band (Fig. 2A). In contrast, no complex was observed for either the single RelB or RelF antitoxin proteins under similar experimental conditions.


Characterization of the interaction and cross-regulation of three Mycobacterium tuberculosis RelBE modules.

Yang M, Gao C, Wang Y, Zhang H, He ZG - PLoS ONE (2010)

Self-interactions of three M. tuberculosis relBE-like modules.Electrophoretic mobility shift assays (EMSA) were used to detect the binding of RelBE proteins with their operon promoters. A fixed amount of 32P-labeled DNA substrate was incubated with various amounts of proteins in a total volume of 15 µL of an EMSA buffer. Electrophoresis was performed and gels were exposed to a storage-phosphor screen overnight as described in the “Materials and Methods”. The images were acquired by Typhoon Scanner (GE Healthcare). Both DNA substrate and protein/DNA complexes are indicated by arrows on the left of the figure. (A) Different amount of each RelB-like protein (3.75 µM, 7.5 µM, and 15 µM ) interacts with their promoter DNA. (B) The concentration of Rv1247c remains constant at 7.5 µM. The interaction between various ratio of RelE/RelB (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (C) The concentration of RelF remains constant at 7.5 µM. The interaction between different ratio of RelF/RelG (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (D) The concentration of RelJ remains constant at 3.75 µM. The interaction between different ratio of RelK/RelJ (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (E) Competitive assays. Three non-labeled promoter DNA substrates (5-fold, 10-fold or 50-fold) were used to compete with their corresponding labeled DNA substrates. The species of promoter DNA was indicated on top of each panel in the figure.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2871789&req=5

pone-0010672-g002: Self-interactions of three M. tuberculosis relBE-like modules.Electrophoretic mobility shift assays (EMSA) were used to detect the binding of RelBE proteins with their operon promoters. A fixed amount of 32P-labeled DNA substrate was incubated with various amounts of proteins in a total volume of 15 µL of an EMSA buffer. Electrophoresis was performed and gels were exposed to a storage-phosphor screen overnight as described in the “Materials and Methods”. The images were acquired by Typhoon Scanner (GE Healthcare). Both DNA substrate and protein/DNA complexes are indicated by arrows on the left of the figure. (A) Different amount of each RelB-like protein (3.75 µM, 7.5 µM, and 15 µM ) interacts with their promoter DNA. (B) The concentration of Rv1247c remains constant at 7.5 µM. The interaction between various ratio of RelE/RelB (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (C) The concentration of RelF remains constant at 7.5 µM. The interaction between different ratio of RelF/RelG (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (D) The concentration of RelJ remains constant at 3.75 µM. The interaction between different ratio of RelK/RelJ (8∶1, 4∶1, 2∶1, 1∶1, 1∶2, 1∶4) and a fixed amount of DNA substrate. (E) Competitive assays. Three non-labeled promoter DNA substrates (5-fold, 10-fold or 50-fold) were used to compete with their corresponding labeled DNA substrates. The species of promoter DNA was indicated on top of each panel in the figure.
Mentions: Promoter DNA (described as 1247p, 2865p, and 3357p below) was used as substrate to further investigate the in vitro association of RelBE with promoters. As shown in Fig. 2A, of the three RelB-like proteins (from 3.75 to 15 µM), only RelJ associated strongly with its promoter as a singular protein and produced a substantial shifted protein/DNA complex band (Fig. 2A). In contrast, no complex was observed for either the single RelB or RelF antitoxin proteins under similar experimental conditions.

Bottom Line: RelBE modules appear to be autoregulated in an atypical manner compared to other TA systems; however, the molecular mechanisms and potential interactions between different RelBE modules remain to be elucidated.The RelBE-like modules exerted complex cross-regulation effects on mycobacterial growth.Conversely, relF enhanced the toxicity of the relE toxin gene, while relB increased the toxicity of relK.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.

ABSTRACT
RelBE represents a typical bacterial toxin-antitoxin (TA) system. Mycobacterium tuberculosis H37Rv, the pathogen responsible for human tuberculosis, contains three RelBE-like modules, RelBE, RelFG, and RelJK, which are at least partly expressed in human macrophages during infection. RelBE modules appear to be autoregulated in an atypical manner compared to other TA systems; however, the molecular mechanisms and potential interactions between different RelBE modules remain to be elucidated. In the present study, we characterized the interaction and cross-regulation of these Rel toxin-antitoxin modules from this unique pathogen. The physical interactions between the three pairs of RelBE proteins were confirmed and the DNA-binding domain recognized by three RelBE-like pairs and domain structure characteristics were described. The three RelE-like proteins physically interacted with the same RelB-like protein, and could conditionally regulate its binding with promoter DNA. The RelBE-like modules exerted complex cross-regulation effects on mycobacterial growth. The relB antitoxin gene could replace relF in cross-neutralizing the relG toxin gene. Conversely, relF enhanced the toxicity of the relE toxin gene, while relB increased the toxicity of relK. This is the first report of interactions between different pairs of RelBE modules of M. tuberculosis.

Show MeSH
Related in: MedlinePlus