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LtmA, a novel cyclic di-GMP-responsive activator, broadly regulates the expression of lipid transport and metabolism genes in Mycobacterium smegmatis.

Li W, He ZG - Nucleic Acids Res. (2012)

Bottom Line: Overexpression of Ms6479 in M. smegmatis significantly reduced the permeability of the cell wall to crystal violet and increased mycobacterial resistance to anti-tuberculosis antibiotics.Interestingly, Ms6479 lacks the previously reported c-di-GMP binding motifs.Our findings introduce Ms6479 (here designated LtmA for lipid transport and metabolism activator) as a new c-di-GMP-responsive regulator.

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 430070, China.

ABSTRACT
In a bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)/transcription factor binding screen, we identified Mycobacterium smegmatis Ms6479 as the first c-di-GMP-responsive transcriptional factor in mycobacteria. Ms6479 could specifically bind with c-di-GMP and recognize the promoters of 37 lipid transport and metabolism genes. c-di-GMP could enhance the ability of Ms6479 to bind to its target DNA. Furthermore, our results establish Ms6479 as a global activator that positively regulates the expression of diverse target genes. Overexpression of Ms6479 in M. smegmatis significantly reduced the permeability of the cell wall to crystal violet and increased mycobacterial resistance to anti-tuberculosis antibiotics. Interestingly, Ms6479 lacks the previously reported c-di-GMP binding motifs. Our findings introduce Ms6479 (here designated LtmA for lipid transport and metabolism activator) as a new c-di-GMP-responsive regulator.

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Assays for the interactions between Ms6479 and its neighboring gene upstream regions and the regulation of c-di-GMP. (A) The Ms6479 gene cluster. Several neighboring non-coding regions, which lengths are longer than 50 bp, are indicated by black arrows. (B) Bacterial one-hybrid assays. A pair of pBXcmT/pTRG plasmids was co-transformed into the reporter strain and its growth was monitored together with that of the self-activation controls on selective medium as described in ‘Materials and Methods’ section. Co-transformants containing the pBX-Rv2031/pTRG-Rv3133 plasmids (37) served as positive controls (CK+) and co-transformants containing the empty vectors pBX and pTRG served as negative controls (CK−). (C) ChIP assays. ChIP using preimmune (P) or immune sera (I) raised against Ms6479. The mycobacterial promoter Ms6021p, which does not contain motif sequence, was used as a negative control. (D) EMSA assays for the specific DNA binding activity of Ms6479 on Ms6484 promoter DNA. 32P-labeled Ms6484 promoter DNA substrate was co-incubated with Ms6479 protein in the absence (lanes 1–4) or presence of c-di-GMP (0.3–1.5 mM) (lanes 5–7) or GTP (1.5 mM) (lane 8). The ability of the unlabeled Ms6484 promoter (lanes 9–12) to compete with binding of the labeled Ms6484 promoter with the Ms6479 protein was examined.
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gks923-F2: Assays for the interactions between Ms6479 and its neighboring gene upstream regions and the regulation of c-di-GMP. (A) The Ms6479 gene cluster. Several neighboring non-coding regions, which lengths are longer than 50 bp, are indicated by black arrows. (B) Bacterial one-hybrid assays. A pair of pBXcmT/pTRG plasmids was co-transformed into the reporter strain and its growth was monitored together with that of the self-activation controls on selective medium as described in ‘Materials and Methods’ section. Co-transformants containing the pBX-Rv2031/pTRG-Rv3133 plasmids (37) served as positive controls (CK+) and co-transformants containing the empty vectors pBX and pTRG served as negative controls (CK−). (C) ChIP assays. ChIP using preimmune (P) or immune sera (I) raised against Ms6479. The mycobacterial promoter Ms6021p, which does not contain motif sequence, was used as a negative control. (D) EMSA assays for the specific DNA binding activity of Ms6479 on Ms6484 promoter DNA. 32P-labeled Ms6484 promoter DNA substrate was co-incubated with Ms6479 protein in the absence (lanes 1–4) or presence of c-di-GMP (0.3–1.5 mM) (lanes 5–7) or GTP (1.5 mM) (lane 8). The ability of the unlabeled Ms6484 promoter (lanes 9–12) to compete with binding of the labeled Ms6484 promoter with the Ms6479 protein was examined.

Mentions: The target promoter of the LtmA transcription factor has not yet been identified. We examined the potential binding of LtmA to the upstream regions of several neighboring gene within the LtmA local gene cluster (Figure 2A). As indicated by arrows in Figure 2A, we chose five longer intergenic sequences than 50 bp within the LtmA local gene cluster—Ms6480p, Ms6482p, Ms6484p, Ms6490p and Ms6492p—to test their interactions with LtmA. Using bacterial one-hybrid assays, these upstream DNA fragments were cloned into the reporter vector pBXcmT (37) and co-transformed with pTRG-LtmA into reporter strains. Only the LtmA+Ms6484p and LtmA+Ms6482p co-transformant strains grew well on the screening medium (Figure 2B). No growth was observed for LtmA+negative control promoter Ms6021p co-transformant strains (Supplementary Figure S3) and for the strains containing either LtmA alone or the intergenic sequence alone. These results established Ms6482p and Ms6484p as the potential target sequences of LtmA.Figure 2.


LtmA, a novel cyclic di-GMP-responsive activator, broadly regulates the expression of lipid transport and metabolism genes in Mycobacterium smegmatis.

Li W, He ZG - Nucleic Acids Res. (2012)

Assays for the interactions between Ms6479 and its neighboring gene upstream regions and the regulation of c-di-GMP. (A) The Ms6479 gene cluster. Several neighboring non-coding regions, which lengths are longer than 50 bp, are indicated by black arrows. (B) Bacterial one-hybrid assays. A pair of pBXcmT/pTRG plasmids was co-transformed into the reporter strain and its growth was monitored together with that of the self-activation controls on selective medium as described in ‘Materials and Methods’ section. Co-transformants containing the pBX-Rv2031/pTRG-Rv3133 plasmids (37) served as positive controls (CK+) and co-transformants containing the empty vectors pBX and pTRG served as negative controls (CK−). (C) ChIP assays. ChIP using preimmune (P) or immune sera (I) raised against Ms6479. The mycobacterial promoter Ms6021p, which does not contain motif sequence, was used as a negative control. (D) EMSA assays for the specific DNA binding activity of Ms6479 on Ms6484 promoter DNA. 32P-labeled Ms6484 promoter DNA substrate was co-incubated with Ms6479 protein in the absence (lanes 1–4) or presence of c-di-GMP (0.3–1.5 mM) (lanes 5–7) or GTP (1.5 mM) (lane 8). The ability of the unlabeled Ms6484 promoter (lanes 9–12) to compete with binding of the labeled Ms6484 promoter with the Ms6479 protein was examined.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC3526308&req=5

gks923-F2: Assays for the interactions between Ms6479 and its neighboring gene upstream regions and the regulation of c-di-GMP. (A) The Ms6479 gene cluster. Several neighboring non-coding regions, which lengths are longer than 50 bp, are indicated by black arrows. (B) Bacterial one-hybrid assays. A pair of pBXcmT/pTRG plasmids was co-transformed into the reporter strain and its growth was monitored together with that of the self-activation controls on selective medium as described in ‘Materials and Methods’ section. Co-transformants containing the pBX-Rv2031/pTRG-Rv3133 plasmids (37) served as positive controls (CK+) and co-transformants containing the empty vectors pBX and pTRG served as negative controls (CK−). (C) ChIP assays. ChIP using preimmune (P) or immune sera (I) raised against Ms6479. The mycobacterial promoter Ms6021p, which does not contain motif sequence, was used as a negative control. (D) EMSA assays for the specific DNA binding activity of Ms6479 on Ms6484 promoter DNA. 32P-labeled Ms6484 promoter DNA substrate was co-incubated with Ms6479 protein in the absence (lanes 1–4) or presence of c-di-GMP (0.3–1.5 mM) (lanes 5–7) or GTP (1.5 mM) (lane 8). The ability of the unlabeled Ms6484 promoter (lanes 9–12) to compete with binding of the labeled Ms6484 promoter with the Ms6479 protein was examined.
Mentions: The target promoter of the LtmA transcription factor has not yet been identified. We examined the potential binding of LtmA to the upstream regions of several neighboring gene within the LtmA local gene cluster (Figure 2A). As indicated by arrows in Figure 2A, we chose five longer intergenic sequences than 50 bp within the LtmA local gene cluster—Ms6480p, Ms6482p, Ms6484p, Ms6490p and Ms6492p—to test their interactions with LtmA. Using bacterial one-hybrid assays, these upstream DNA fragments were cloned into the reporter vector pBXcmT (37) and co-transformed with pTRG-LtmA into reporter strains. Only the LtmA+Ms6484p and LtmA+Ms6482p co-transformant strains grew well on the screening medium (Figure 2B). No growth was observed for LtmA+negative control promoter Ms6021p co-transformant strains (Supplementary Figure S3) and for the strains containing either LtmA alone or the intergenic sequence alone. These results established Ms6482p and Ms6484p as the potential target sequences of LtmA.Figure 2.

Bottom Line: Overexpression of Ms6479 in M. smegmatis significantly reduced the permeability of the cell wall to crystal violet and increased mycobacterial resistance to anti-tuberculosis antibiotics.Interestingly, Ms6479 lacks the previously reported c-di-GMP binding motifs.Our findings introduce Ms6479 (here designated LtmA for lipid transport and metabolism activator) as a new c-di-GMP-responsive regulator.

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 430070, China.

ABSTRACT
In a bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP)/transcription factor binding screen, we identified Mycobacterium smegmatis Ms6479 as the first c-di-GMP-responsive transcriptional factor in mycobacteria. Ms6479 could specifically bind with c-di-GMP and recognize the promoters of 37 lipid transport and metabolism genes. c-di-GMP could enhance the ability of Ms6479 to bind to its target DNA. Furthermore, our results establish Ms6479 as a global activator that positively regulates the expression of diverse target genes. Overexpression of Ms6479 in M. smegmatis significantly reduced the permeability of the cell wall to crystal violet and increased mycobacterial resistance to anti-tuberculosis antibiotics. Interestingly, Ms6479 lacks the previously reported c-di-GMP binding motifs. Our findings introduce Ms6479 (here designated LtmA for lipid transport and metabolism activator) as a new c-di-GMP-responsive regulator.

Show MeSH
Related in: MedlinePlus