AUTHOR=Zhang Qingxia , Xing Chenglin , Kong Xiangwei , Wang Cheng , Chen Xijun 

TITLE=ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.667637

DOI=10.3389/fmicb.2021.667637

ISSN=1664-302X

ABSTRACT=<p>Many <italic>Pseudomonas protegens</italic> 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 <italic>P. protegens</italic> 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 <italic>P. protegens</italic> FD6. The consensus <italic>P. protegens</italic> Vfr binding site predicted from nucleotide sequence alignment is TCACA. The qPCR data showed that Vfr positively regulates the expression of <italic>phlF</italic> and <italic>phlG</italic>, and the expression of these genes was characterized in detail. The purified recombinant Vfr bound to an approximately 240-bp fragment within the <italic>phlF</italic> and <italic>phlG</italic> 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 <italic>phlF</italic> and <italic>phlG</italic>, 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 <italic>phlF</italic> and <italic>phlG</italic> genes. However, unlike other Vfr-targeted genes, Vfr binding to <italic>P. protegens</italic> FD6 does not require an intact binding consensus motif. Furthermore, we demonstrated that <italic>vfr</italic> expression is autoregulated in this bacterium. These results provide novel insights into the regulatory role of Vfr in the biocontrol agent <italic>P. protegens</italic>.</p>