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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 in vivo DNA-binding activity of Ms6479. (A) The IR-containing motif GGACACATGTCC was used to search the intergenic regions between M. smegmatis Open Reading Frames. The conserved sequence is highlighted by black background. The included genes in the same operon were presented in the brackets, however, the entire gene names were provided in Supplementary Table S5 limited by figure space. (B) ChIP assays for the in vivo association of Ms6479 with the promoter sequences of lipid transport and metabolism target genes using preimmune (P) or immune sera (I) raised against Ms6479. DNA recovered from the immunoprecipitates was amplified. Ms6021p was used as a negative control. (C) Logo assays for the protected region. The logos were generated using the MEME software suite (50).
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gks923-F4: Assays for in vivo DNA-binding activity of Ms6479. (A) The IR-containing motif GGACACATGTCC was used to search the intergenic regions between M. smegmatis Open Reading Frames. The conserved sequence is highlighted by black background. The included genes in the same operon were presented in the brackets, however, the entire gene names were provided in Supplementary Table S5 limited by figure space. (B) ChIP assays for the in vivo association of Ms6479 with the promoter sequences of lipid transport and metabolism target genes using preimmune (P) or immune sera (I) raised against Ms6479. DNA recovered from the immunoprecipitates was amplified. Ms6021p was used as a negative control. (C) Logo assays for the protected region. The logos were generated using the MEME software suite (50).

Mentions: We used the 12-bp sequence motif (GGACANNTGTCC) to search for LtmA target genes in the M. smegmatis genome and only two mismatches in the underlined palindromic sequences at the most are allowed. To perform the searching, the fuzznuc program was employed in local machine with the parameter ‘–pattern GGACANNTGTCC–mismatch 0-2’ against the 500-bp upstream of each coding sequences (51), which were extracted from the M. smegmatis genome using Perl script. We identified a total of 229 potential target promoters covering 336 genes (Supplementary Table S5 and Supplementary Figure S6). We further classified these target genes in terms of clusters of orthologous groups categories. We found that 37 of them are lipid transport and metabolism genes, 21 are transcriptional regulators, 13 are cell wall/membrane biogenesis genes and many are transport- and metabolism-related genes (Supplementary Figure S7). Majority of the target genes are involved in mycolic acid metabolism in mycobacteria (Supplementary Table S6) (52). We further used the WebLogo tool (53) to characterize a more general conserved motif sequence for LtmA binding (Figure 4A and B). Furthermore, all 37 target promoters of lipid transport and metabolism genes could be specifically recovered in a ChIP assay using an LtmA antibody (Figure 4C). Importantly, a negative control promoter (Ms6021p) could not be recovered using the LtmA antibody under the same conditions. These results indicate that LtmA can recognize the promoters of diverse target genes including at least 37 lipid transport and metabolism genes.Figure 4.


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 in vivo DNA-binding activity of Ms6479. (A) The IR-containing motif GGACACATGTCC was used to search the intergenic regions between M. smegmatis Open Reading Frames. The conserved sequence is highlighted by black background. The included genes in the same operon were presented in the brackets, however, the entire gene names were provided in Supplementary Table S5 limited by figure space. (B) ChIP assays for the in vivo association of Ms6479 with the promoter sequences of lipid transport and metabolism target genes using preimmune (P) or immune sera (I) raised against Ms6479. DNA recovered from the immunoprecipitates was amplified. Ms6021p was used as a negative control. (C) Logo assays for the protected region. The logos were generated using the MEME software suite (50).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks923-F4: Assays for in vivo DNA-binding activity of Ms6479. (A) The IR-containing motif GGACACATGTCC was used to search the intergenic regions between M. smegmatis Open Reading Frames. The conserved sequence is highlighted by black background. The included genes in the same operon were presented in the brackets, however, the entire gene names were provided in Supplementary Table S5 limited by figure space. (B) ChIP assays for the in vivo association of Ms6479 with the promoter sequences of lipid transport and metabolism target genes using preimmune (P) or immune sera (I) raised against Ms6479. DNA recovered from the immunoprecipitates was amplified. Ms6021p was used as a negative control. (C) Logo assays for the protected region. The logos were generated using the MEME software suite (50).
Mentions: We used the 12-bp sequence motif (GGACANNTGTCC) to search for LtmA target genes in the M. smegmatis genome and only two mismatches in the underlined palindromic sequences at the most are allowed. To perform the searching, the fuzznuc program was employed in local machine with the parameter ‘–pattern GGACANNTGTCC–mismatch 0-2’ against the 500-bp upstream of each coding sequences (51), which were extracted from the M. smegmatis genome using Perl script. We identified a total of 229 potential target promoters covering 336 genes (Supplementary Table S5 and Supplementary Figure S6). We further classified these target genes in terms of clusters of orthologous groups categories. We found that 37 of them are lipid transport and metabolism genes, 21 are transcriptional regulators, 13 are cell wall/membrane biogenesis genes and many are transport- and metabolism-related genes (Supplementary Figure S7). Majority of the target genes are involved in mycolic acid metabolism in mycobacteria (Supplementary Table S6) (52). We further used the WebLogo tool (53) to characterize a more general conserved motif sequence for LtmA binding (Figure 4A and B). Furthermore, all 37 target promoters of lipid transport and metabolism genes could be specifically recovered in a ChIP assay using an LtmA antibody (Figure 4C). Importantly, a negative control promoter (Ms6021p) could not be recovered using the LtmA antibody under the same conditions. These results indicate that LtmA can recognize the promoters of diverse target genes including at least 37 lipid transport and metabolism genes.Figure 4.

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