Abstract
Bacterial motility plays a crucial role in competitiveness and colonization in the rhizosphere. In this work, Chromatin ImmunoPrecipitation Sequencing (ChIP-seq) analysis has been used to identify genes putatively regulated by the transcriptional regulatory protein FleQ in Pseudomonas fluorescens F113 and Pseudomonas putida KT2440. This protein was previously identified as a master regulator of flagella and biofilm formation in both strains. This work has demonstrated that FleQ from both bacteria are conserved and functionally equivalent for motility regulation. Furthermore, the ChIP-seq analysis has shown that FleQ is a global regulator with the identification of 121 and 103 FleQ putative binding sites in P. fluorescens F113 and P. putida KT2440 respectively. Putative genes regulated by FleQ included, as expected, flagellar and motility-related genes and others involved in adhesion and exopolysaccharide production. Surprisingly, the ChIP-seq analysis also identified iron homeostasis-related genes for which positive regulation was shown by RT-qPCR. The results also showed that FleQ from P. fluorescens F113 shares an important part of its direct regulon with AmrZ, a global regulator also implicated in environmental adaption. Although AmrZ also regulates motility and iron uptake, the overlap occurred mostly with the iron-related genes, since both regulators control a different set of motility-related genes.
Highlights
Flagella biosynthesis in pseudomonads requires more than 50 genes subjected to four levels of hierarchical regulation[1]
We have characterized the direct regulon of the master regulatory protein FleQ in P. fluorescens F113 and P. putida KT2440
Distribution of binding sites is similar in both species and a significant number of orthologues are putatively regulated by FleQ both in F113 and in KT2440
Summary
Flagella biosynthesis in pseudomonads requires more than 50 genes subjected to four levels of hierarchical regulation[1]. The function of FleQ in flagella synthesis regulation has been studied in Pseudomonas aeruginosa[1,2], in P. putida[3,4,5] and in P. fluorescens[6,7,8]. FleQ regulates the biosynthesis of P. aeruginosa exopolysaccharides (EPSs, pel and psl operons) in a c-di-GMP-dependent manner, triggering either the activation or repression of these genes[14,18]. FleQ has been shown to regulate the expression of the cdrA and lapA genes, encoding adhesins required for biofilm formation in P. aeruginosa[23] and P. putida[24] respectively, in a c-di-GMP-dependent way. FleQ has been shown to be essential for biofilm formation in P. putida[25,26] Another central node in environmental adaption in pseudomonads is the transcriptional regulator AmrZ27,28. Considering that FleQ and AmrZ regulate similar traits such as motility, exopolysaccharides production and biofilm formation, the aim of this work was to identify the genes and operons regulated by FleQ in P. fluorescens F113 and P. putida KT2440 by using ChIP-seq and to analyze the possible overlap between the FleQ and AmrZ regulons in P. fluorescens F113
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