ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6
Many Pseudomonas protegens strains produce the antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), both of which have antimicrobial properties. The biosynthesis of these metabolites is typically controlled by multiple regulatory factors. Virulence factor regulator (Vfr) is a mul...
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Frontiers Media S.A.
2021-05-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2021.667637/full |
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| author | Qingxia Zhang Qingxia Zhang Chenglin Xing Xiangwei Kong Cheng Wang Xijun Chen |
| author_facet | Qingxia Zhang Qingxia Zhang Chenglin Xing Xiangwei Kong Cheng Wang Xijun Chen |
| author_sort | Qingxia Zhang |
| collection | DOAJ |
| description | Many Pseudomonas protegens strains produce the antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), both of which have antimicrobial properties. The biosynthesis of these metabolites is typically controlled by multiple regulatory factors. Virulence factor regulator (Vfr) is a multifunctional DNA-binding regulator that modulates 2,4-DAPG biosynthesis in P. protegens FD6. However, the mechanism by which Vfr regulates this process remains unclear. In the present study, chromatin immunoprecipitation of FLAG-tagged Vfr and nucleotide sequencing analysis were used to identify 847 putative Vfr binding sites in P. protegens FD6. The consensus P. protegens Vfr binding site predicted from nucleotide sequence alignment is TCACA. The qPCR data showed that Vfr positively regulates the expression of phlF and phlG, and the expression of these genes was characterized in detail. The purified recombinant Vfr bound to an approximately 240-bp fragment within the phlF and phlG upstream regions that harbor putative Vfr consensus sequences. Using electrophoretic mobility shift assays, we localized Vfr binding to a 25-bp fragment that contains part of the Vfr binding region. Vfr binding was eliminated by mutating the TACG and CACA sequences in phlF and phlG, respectively. Taken together, our results show that Vfr directly regulates the expression of the 2,4-DAPG operon by binding to the upstream regions of both the phlF and phlG genes. However, unlike other Vfr-targeted genes, Vfr binding to P. protegens FD6 does not require an intact binding consensus motif. Furthermore, we demonstrated that vfr expression is autoregulated in this bacterium. These results provide novel insights into the regulatory role of Vfr in the biocontrol agent P. protegens. |
| first_indexed | 2024-12-21T10:44:26Z |
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| language | English |
| last_indexed | 2024-12-21T10:44:26Z |
| publishDate | 2021-05-01 |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-3d1030375e5a4cdf8226d747b34ae1ad2022-12-21T19:06:51ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2021-05-011210.3389/fmicb.2021.667637667637ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6Qingxia Zhang0Qingxia Zhang1Chenglin Xing2Xiangwei Kong3Cheng Wang4Xijun Chen5College of Horticulture and Plant Protection, Yangzhou University, Yangzhou, ChinaJoint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, ChinaCollege of Horticulture and Plant Protection, Yangzhou University, Yangzhou, ChinaCollege of Horticulture and Plant Protection, Yangzhou University, Yangzhou, ChinaCollege of Horticulture and Plant Protection, Yangzhou University, Yangzhou, ChinaCollege of Horticulture and Plant Protection, Yangzhou University, Yangzhou, ChinaMany Pseudomonas protegens strains produce the antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), both of which have antimicrobial properties. The biosynthesis of these metabolites is typically controlled by multiple regulatory factors. Virulence factor regulator (Vfr) is a multifunctional DNA-binding regulator that modulates 2,4-DAPG biosynthesis in P. protegens FD6. However, the mechanism by which Vfr regulates this process remains unclear. In the present study, chromatin immunoprecipitation of FLAG-tagged Vfr and nucleotide sequencing analysis were used to identify 847 putative Vfr binding sites in P. protegens FD6. The consensus P. protegens Vfr binding site predicted from nucleotide sequence alignment is TCACA. The qPCR data showed that Vfr positively regulates the expression of phlF and phlG, and the expression of these genes was characterized in detail. The purified recombinant Vfr bound to an approximately 240-bp fragment within the phlF and phlG upstream regions that harbor putative Vfr consensus sequences. Using electrophoretic mobility shift assays, we localized Vfr binding to a 25-bp fragment that contains part of the Vfr binding region. Vfr binding was eliminated by mutating the TACG and CACA sequences in phlF and phlG, respectively. Taken together, our results show that Vfr directly regulates the expression of the 2,4-DAPG operon by binding to the upstream regions of both the phlF and phlG genes. However, unlike other Vfr-targeted genes, Vfr binding to P. protegens FD6 does not require an intact binding consensus motif. Furthermore, we demonstrated that vfr expression is autoregulated in this bacterium. These results provide novel insights into the regulatory role of Vfr in the biocontrol agent P. protegens.https://www.frontiersin.org/articles/10.3389/fmicb.2021.667637/fullPseudomonas protegens24-DAPGbiological controlVfrantibiotic |
| spellingShingle | Qingxia Zhang Qingxia Zhang Chenglin Xing Xiangwei Kong Cheng Wang Xijun Chen ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6 Frontiers in Microbiology Pseudomonas protegens 2 4-DAPG biological control Vfr antibiotic |
| title | ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6 |
| title_full | ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6 |
| title_fullStr | ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6 |
| title_full_unstemmed | ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6 |
| title_short | ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6 |
| title_sort | chip seq analysis of the global regulator vfr reveals novel insights into the biocontrol agent pseudomonas protegens fd6 |
| topic | Pseudomonas protegens 2 4-DAPG biological control Vfr antibiotic |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2021.667637/full |
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