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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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Effects of Ms6479 on M. smegmatis cell and colony morphology. (A) Cells were observed with a light microscope after staining with 2% crystal violet. The Ms6479 overexpression strain Msm/pMV261-Ms6479 (right panel) stained much weaker than the wild-type Msm/pMV261 strain (left panel). (B) Scanning electron microscopy assays of cell morphology. The images were taken at 8000× magnification. Bars, 2 μm. (C) Representative colony morphology of the M. smegmatis spot on 7H10 medium.
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gks923-F5: Effects of Ms6479 on M. smegmatis cell and colony morphology. (A) Cells were observed with a light microscope after staining with 2% crystal violet. The Ms6479 overexpression strain Msm/pMV261-Ms6479 (right panel) stained much weaker than the wild-type Msm/pMV261 strain (left panel). (B) Scanning electron microscopy assays of cell morphology. The images were taken at 8000× magnification. Bars, 2 μm. (C) Representative colony morphology of the M. smegmatis spot on 7H10 medium.

Mentions: Most of the potential target genes identified above have been implicated in the synthesis of mycolic acids (Supplementary Table S6), a major constituent of mycobacterial cell wall (52). This suggested that LtmA could be involved in the regulation of cell wall metabolism. To test this hypothesis, we first examined the sensitivity of mycobacteria to crystal violet staining. Examination of the mycobacterial cell walls under a light microscope revealed that the wild-type M. smegmatis strain was stained easily by crystal violet (Figure 5A, left panel). In contrast, very weak crystal violet staining was observed for the LtmA-overexpressed strain (Figure 5A, right panel), suggesting that LtmA is involved in the regulation of mycobacterial cell wall permeability or cell wall composition. This interpretation is consistent with previous conclusions (52) that mycolic acid metabolism affects the permeability of mycobacterial cell wall and sensitivity to crystal violet. However, no obvious change in cell morphology was observed in the LtmA-overexpressed strain when assayed under SEM (Figure 5B) or under the light microscope (Figure 5A). Interestingly, a significant difference was observed in the spot-colony morphology of the cells on 7H10 agar solid medium between the wild-type and LtmA-overexpressed strains (Figure 5C). The wild-type strain presented a typical rough-edged colony and rugose surface (Figure 5C, left panel). In contrast, the overexpression strain had a relatively smooth-edged colony and lacked rugose surface (Figure 5C, right panel). Taken together, these results strongly suggest that LtmA may regulate the sensitivity to crystal violet staining and colony morphology in mycobacteria.Figure 5.


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)

Effects of Ms6479 on M. smegmatis cell and colony morphology. (A) Cells were observed with a light microscope after staining with 2% crystal violet. The Ms6479 overexpression strain Msm/pMV261-Ms6479 (right panel) stained much weaker than the wild-type Msm/pMV261 strain (left panel). (B) Scanning electron microscopy assays of cell morphology. The images were taken at 8000× magnification. Bars, 2 μm. (C) Representative colony morphology of the M. smegmatis spot on 7H10 medium.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks923-F5: Effects of Ms6479 on M. smegmatis cell and colony morphology. (A) Cells were observed with a light microscope after staining with 2% crystal violet. The Ms6479 overexpression strain Msm/pMV261-Ms6479 (right panel) stained much weaker than the wild-type Msm/pMV261 strain (left panel). (B) Scanning electron microscopy assays of cell morphology. The images were taken at 8000× magnification. Bars, 2 μm. (C) Representative colony morphology of the M. smegmatis spot on 7H10 medium.
Mentions: Most of the potential target genes identified above have been implicated in the synthesis of mycolic acids (Supplementary Table S6), a major constituent of mycobacterial cell wall (52). This suggested that LtmA could be involved in the regulation of cell wall metabolism. To test this hypothesis, we first examined the sensitivity of mycobacteria to crystal violet staining. Examination of the mycobacterial cell walls under a light microscope revealed that the wild-type M. smegmatis strain was stained easily by crystal violet (Figure 5A, left panel). In contrast, very weak crystal violet staining was observed for the LtmA-overexpressed strain (Figure 5A, right panel), suggesting that LtmA is involved in the regulation of mycobacterial cell wall permeability or cell wall composition. This interpretation is consistent with previous conclusions (52) that mycolic acid metabolism affects the permeability of mycobacterial cell wall and sensitivity to crystal violet. However, no obvious change in cell morphology was observed in the LtmA-overexpressed strain when assayed under SEM (Figure 5B) or under the light microscope (Figure 5A). Interestingly, a significant difference was observed in the spot-colony morphology of the cells on 7H10 agar solid medium between the wild-type and LtmA-overexpressed strains (Figure 5C). The wild-type strain presented a typical rough-edged colony and rugose surface (Figure 5C, left panel). In contrast, the overexpression strain had a relatively smooth-edged colony and lacked rugose surface (Figure 5C, right panel). Taken together, these results strongly suggest that LtmA may regulate the sensitivity to crystal violet staining and colony morphology in mycobacteria.Figure 5.

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