fleQ , the Gene Encoding the Major Flagellar Regulator of Pseudomonas aeruginosa , Is σ ⁷⁰ Dependent and Is Downregulated by Vfr, a Homolog of Escherichia coli Cyclic AMP Receptor Protein

Abstract

Flagellar biogenesis inPseudomonas aeruginosa is an intricate process in which various transcriptional activators (FleQ, FleR) and alternative sigma factors (RpoN/ 54 , RpoF/ 28 ) participate in the transcriptional regulation of the flagellar genes and operons (3,23,31,33). Of the two transcriptional activators identified in our laboratory, FleQ appears to be the highest-level positive regulator of flagellar biogenesis in P. aeruginosa.FleQ is homologous to the NtrC group of response regulators, which are part of a two-component signal transduction system, that works in concert with the alternative sigma factor RpoN (3). Typically, a two-component system consists of a phosphorelay between a sensor kinase and a response regulator pair (5). The sensor kinase is activated by autophosphorylation when an environmental stimulus is sensed, and the response regulator is subsequently phosphorylated at the receiver domain. The response regulators are usually DNA binding proteins, and phosphorylation activates them to promote activation or repression of the downstream genes (5). However, FleQ and several of its homologs that regulate flagellar biogenesis in other bacterial species, including FlrA of Vibrio cholerae (16), FlaK of Vibrio parahaemolyticus (15), and FlgR of Helicobacter pylori (30), do not contain the hallmark residues in their receiver domains for phosphorylation and do not have a cognate sensor kinase encoded as a part of the same operon. Therefore, phosphorelay signaling does not appear to be the modulating mechanism that regulates the activities of these response regulators. Attempts to detect phosphorylation of FleQ in vitro were not successful in our laboratory, thus raising further doubts about the existence of a putative sensor kinase for FleQ (unpublished data). One of the mechanisms employed to accomplish posttranslational modulation of FleQ activity in P. aeruginosa involves direct protein-protein interactions with FleN, an antiactivator that represses FleQ-dependent transcriptional activation (8). Whether fleQ is subject to transcriptional activation or repression in addition to posttranslational modulation has been a question. Preliminary data have demonstrated that FleQ synthesis occurs from the early log phase to the stationary phase of growth in shaken cultures of P. aeruginosa PAK (unpublished data), which suggests that constitutive expression occurs under these conditions. Whether the constitutive expression observed was driven by a constitutively expressed activator or merely by the housekeeping RNA polymerase holoenzyme is not clear.The flagellar regulon is subject to hierarchical regulation in most of the flagellated bacteria. Among the flagellar regulators of monoflagellate bacteria, flgR, the fleQ homolog of H. pylori,

Influence of the regulatory protein RsmA on cellular functions in Pseudomonas aeruginosa PAO1, as revealed by transcriptome analysis

“…Crp is activated by binding cAMP and then binds to a specific sequence in the promoter of Crp-controlled genes. In P. aeruginosa, Vfr has been implicated in the regulation of twitching motility, QS, pyocyanin production, repression of flagellar biosynthesis and the expression of numerous proteins secreted by the type II pathway, including exotoxin A (Albus et al, 1997;Beatson et al, 2002;Dasgupta et al, 2002;West et al, 1994). As mentioned previously, toxA expression was decreased approximately 2?8-fold in the rsmA mutant compared to PAO1.…”

Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads

“…FleQ is a c-di-GMPresponsive transcriptional regulator, and in P. aeruginosa PA01 it controls the hierarchical regulatory cascade for flagella biosynthesis and the repression of the PEL biosynthesis genes [88][89]. AlgR and AmrZ (also referred to as AlgZ) are transcription factors involved in the regulation of a number of systems including alginate biosynthesis and twitching motility [79,90].…”

Global Regulatory Pathways and Cross-talk ControlPseudomonas aeruginosaEnvironmental Lifestyle and Virulence Phenotype

“…FleQ was previously shown to repress expression of the pel operon (Dasgupta, 2002). Despite the absence of characterized c-di-GMP binding domains, FleQ bound radiolabeled c-di-GMP in vitro, and c-di-GMP binding inhibited FleQ-binding to the pel promoter transcript (Hickman, 2008).…”

“…A consensus Vfr binding sequence has been proposed (Kanack, 2006), and direct binding of Vfr to DNA sequences within target promoters of several genes including those encoding ToxA (toxA), LasR (las quorum-sensing regulator), FleQ, RegA and PtxR (toxA expression regulators) and CpdA (a cAMP phosphodiesterase) has been demonstrated (Albus, 1997;Dasgupta, 2002;Kanack, 2006;Ferrel, 2008;Fuchs, 2010b). Fuchs et al further identified both cAMP-dependent and -independent Vfr DNA binding mechanisms (Fuchs, 2010a).…”

“…Vfr coordinately controls the expression of over 200 genes (Suh, 2002;Wolfgang, 2003b) and positively regulates the production of virulence factors important for acute P. aeruginosa infections, including: Tfp (West, 1994;Beatson, 2002;Wolfgang, 2003b), the T3SS (Wolfgang, 2003b), the T2SS, secreted toxins, degradative enzymes (West, 1994;Beatson, 2002;Wolfgang, 2003b;Ferrel, 2008), and the las and rhl QS systems, which control hundreds of additional genes, including many virulence factors (Albus, 1997;Croda-García, 2011). In contrast, Vfr negatively regulates the expression of flagellar genes by repressing expression of fleQ, which encodes the master regulator of flagellar biogenesis (Dasgupta, 2002).…”