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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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Related in: MedlinePlus

Effects of Ms6479 and c-di-GMP synthesis on the expression of lipid transport and metabolism related genes in M. smegmatis. qRT-PCR assays for determining the relative expression levels of lipid transport and metabolism genes were performed as described in ‘Materials and Methods’ section. Expression levels of genes were normalized using the sigA gene as an invariant transcript and an unrelated Ms6021 gene was used as a negative control. Error bars represent the variant range of the data derived from three biological replicates. The P-values of the relative expression data were calculated by unpaired two-tailed Student’s t-test using GraphPad Prism 5. The P-values of the results (<0.05) are indicated by an asterisk (*) on top of the column in the figure. Relative expression levels of target genes in Msm and Msm/ΔMs6479 (A), in Msm/pMV261 and Msm/pMV261-Ms6479 (B), and in Msm/pMV261-b1535 mut and Msm/pMV261-b1535 wt (C) strains were assayed. b1535wt encodes a diguanylate cyclase that can elevate c-di-GMP levels in M. smegmatis (42). b1535 mut contained mutations that abolished the diguanylate cyclase activity of the wild-type protein. As a positive control, total DNA of each strain was used as template for PCR amplification. The cDNA of the mutant strains and the recombinant strain containing an empty pMV261 vector was used as template in the negative controls. no RT represents genomic DNA contamination control. Data were analyzed using the 2ΔΔCt method (38,47).
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gks923-F6: Effects of Ms6479 and c-di-GMP synthesis on the expression of lipid transport and metabolism related genes in M. smegmatis. qRT-PCR assays for determining the relative expression levels of lipid transport and metabolism genes were performed as described in ‘Materials and Methods’ section. Expression levels of genes were normalized using the sigA gene as an invariant transcript and an unrelated Ms6021 gene was used as a negative control. Error bars represent the variant range of the data derived from three biological replicates. The P-values of the relative expression data were calculated by unpaired two-tailed Student’s t-test using GraphPad Prism 5. The P-values of the results (<0.05) are indicated by an asterisk (*) on top of the column in the figure. Relative expression levels of target genes in Msm and Msm/ΔMs6479 (A), in Msm/pMV261 and Msm/pMV261-Ms6479 (B), and in Msm/pMV261-b1535 mut and Msm/pMV261-b1535 wt (C) strains were assayed. b1535wt encodes a diguanylate cyclase that can elevate c-di-GMP levels in M. smegmatis (42). b1535 mut contained mutations that abolished the diguanylate cyclase activity of the wild-type protein. As a positive control, total DNA of each strain was used as template for PCR amplification. The cDNA of the mutant strains and the recombinant strain containing an empty pMV261 vector was used as template in the negative controls. no RT represents genomic DNA contamination control. Data were analyzed using the 2ΔΔCt method (38,47).

Mentions: In order to characterize the function of LtmA, an LtmA-deleted mutant strain of M. smegmatis was generated by a gene replacement strategy (40) (Supplementary Figure S8). We then carried out qRT–PCR analysis to compare the expression levels of lipid transport and metabolism genes in both wild-type and LtmA-deleted mutant M. smegmatis strains to assess the regulatory role of the c-di-GMP-responsive regulator. As shown in Figure 6A and Supplementary Figure S9, expression of most of the tested genes, indicated by an asterisk (*) on top of the column in the figure, was found to be significantly down-regulated (P < 0.05) in the mutant strain compared to the wild-type. In contrast, expression level of the negative control gene Ms6021 was not significantly different between the wild-type and the mutant strains. These results indicate that LtmA functions as a positive regulator in M. smegmatis and are consistent with the results of our LtmA overexpression assays. As shown in Figure 6B and Supplementary Figure S9, compared with the wild-type strain, the expressions of many target genes, indicated by an asterisk (*) on top of the column in the figure, were significantly up-regulated (P < 0.05) when LtmA was overexpressed using a pMV261-derived recombinant plasmid. Importantly, expression level of Ms6021, an unrelated gene, did not change upon overexpression of LtmA. Based on these results, we conclude that LtmA functions as a positive regulator of lipid transport and metabolism genes in M. smegmatis.Figure 6.


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 and c-di-GMP synthesis on the expression of lipid transport and metabolism related genes in M. smegmatis. qRT-PCR assays for determining the relative expression levels of lipid transport and metabolism genes were performed as described in ‘Materials and Methods’ section. Expression levels of genes were normalized using the sigA gene as an invariant transcript and an unrelated Ms6021 gene was used as a negative control. Error bars represent the variant range of the data derived from three biological replicates. The P-values of the relative expression data were calculated by unpaired two-tailed Student’s t-test using GraphPad Prism 5. The P-values of the results (<0.05) are indicated by an asterisk (*) on top of the column in the figure. Relative expression levels of target genes in Msm and Msm/ΔMs6479 (A), in Msm/pMV261 and Msm/pMV261-Ms6479 (B), and in Msm/pMV261-b1535 mut and Msm/pMV261-b1535 wt (C) strains were assayed. b1535wt encodes a diguanylate cyclase that can elevate c-di-GMP levels in M. smegmatis (42). b1535 mut contained mutations that abolished the diguanylate cyclase activity of the wild-type protein. As a positive control, total DNA of each strain was used as template for PCR amplification. The cDNA of the mutant strains and the recombinant strain containing an empty pMV261 vector was used as template in the negative controls. no RT represents genomic DNA contamination control. Data were analyzed using the 2ΔΔCt method (38,47).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks923-F6: Effects of Ms6479 and c-di-GMP synthesis on the expression of lipid transport and metabolism related genes in M. smegmatis. qRT-PCR assays for determining the relative expression levels of lipid transport and metabolism genes were performed as described in ‘Materials and Methods’ section. Expression levels of genes were normalized using the sigA gene as an invariant transcript and an unrelated Ms6021 gene was used as a negative control. Error bars represent the variant range of the data derived from three biological replicates. The P-values of the relative expression data were calculated by unpaired two-tailed Student’s t-test using GraphPad Prism 5. The P-values of the results (<0.05) are indicated by an asterisk (*) on top of the column in the figure. Relative expression levels of target genes in Msm and Msm/ΔMs6479 (A), in Msm/pMV261 and Msm/pMV261-Ms6479 (B), and in Msm/pMV261-b1535 mut and Msm/pMV261-b1535 wt (C) strains were assayed. b1535wt encodes a diguanylate cyclase that can elevate c-di-GMP levels in M. smegmatis (42). b1535 mut contained mutations that abolished the diguanylate cyclase activity of the wild-type protein. As a positive control, total DNA of each strain was used as template for PCR amplification. The cDNA of the mutant strains and the recombinant strain containing an empty pMV261 vector was used as template in the negative controls. no RT represents genomic DNA contamination control. Data were analyzed using the 2ΔΔCt method (38,47).
Mentions: In order to characterize the function of LtmA, an LtmA-deleted mutant strain of M. smegmatis was generated by a gene replacement strategy (40) (Supplementary Figure S8). We then carried out qRT–PCR analysis to compare the expression levels of lipid transport and metabolism genes in both wild-type and LtmA-deleted mutant M. smegmatis strains to assess the regulatory role of the c-di-GMP-responsive regulator. As shown in Figure 6A and Supplementary Figure S9, expression of most of the tested genes, indicated by an asterisk (*) on top of the column in the figure, was found to be significantly down-regulated (P < 0.05) in the mutant strain compared to the wild-type. In contrast, expression level of the negative control gene Ms6021 was not significantly different between the wild-type and the mutant strains. These results indicate that LtmA functions as a positive regulator in M. smegmatis and are consistent with the results of our LtmA overexpression assays. As shown in Figure 6B and Supplementary Figure S9, compared with the wild-type strain, the expressions of many target genes, indicated by an asterisk (*) on top of the column in the figure, were significantly up-regulated (P < 0.05) when LtmA was overexpressed using a pMV261-derived recombinant plasmid. Importantly, expression level of Ms6021, an unrelated gene, did not change upon overexpression of LtmA. Based on these results, we conclude that LtmA functions as a positive regulator of lipid transport and metabolism genes in M. smegmatis.Figure 6.

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