FleQ, the Major Flagellar Gene Regulator in Pseudomonas aeruginosa , Binds to Enhancer Sites Located Either Upstream or Atypically Downstream of the RpoN Binding Site

Abstract

In gram-negative bacteria, the alternate sigma factor 54 , working in concert with transcriptional activators that belong to the NtrC superfamily, activates a variety of genes that are regulated in response to external stimuli. For example, in various bacteria, 54 is required for expression of the enzymatic pathways responsible for nitrogen utilization, dicarboxylate transport, xylene degradation, and hydrogen utilization (7,16,19,21,35).54 -regulated genes may be involved in RNA modification (13), chemotaxis, development, energy transduction, fructose assimilation (21), response to heat and phage shock (33), and expression of alternate sigma factors such as H (rpoH) (23) and S (rpoS) (29). In Pseudomonas aeruginosa, 54 is also involved in the regulation of expression of virulence factors including pilin (14), flagellin (31), and alginate (34). There is no obvious theme in the repertoire of functions carried out by 54 -dependent transcripts. Flagellar biogenesis in P. aeruginosa involves more than 40 genes intertwined in a complex regulatory cascade. Its flagellar hierarchy appears to be different from the FlhDC-dependent Salmonella enterica serovar Typhimurium hierarchy (20) and resembles more closely that in Vibrio cholerae, which involves both 28 -and 54 -dependent genes (24). FleQ, a NifA/NtrCtype 54 -dependent activator, is at the highest level of the flagellar hierarchy in P. aeruginosa. Homologues of FleQ have been identified in Caulobacter crescentus (25), V. cholerae (17), and Helicobacter pylori (28) and play important roles in flagellar protein synthesis and secretion. Structurally FleQ lacks the highly conserved phospho-acceptor Asp54 and instead has a serine residue. Phosphorylation could also occur at the serine residue, but lack of evidence to that effect and absence of any cognate sensor kinase probably indicate that FleQ does not require phosphorylation for its activation (9). This is also observed in NifA of Klebsiella pneumoniae, which lacks an Nterminal phospho-acceptor domain, and FlrA of V. cholerae, which lacks the same aspartate residue.Previous studies showed that FleQ positively regulates many flagellar genes (1, 2, 10, 31). These include flhA and fliLM-NOPQ involved in flagellar export; flhF involved in the localization of the flagellar apparatus; fleSR, a two-component sensor and regulator involved in flagellin synthesis; fliEFG, encoding the flagellar basal body MS ring and motor switch complex; fliDS, encoding the flagellar cap and export proteins; and flgA, involved in P-ring formation of the flagellar basal body (N. Dasgupta, unpublished data). The mechanism by which FleQ activates these flagellar genes has not been elucidated. Also, it is unknown whether FleQ acts in the typical manner as other NtrC-like regulators by binding to consensus upstream activating enhancer elements. In this study we have randomly selected four of the above promoters and identified the binding sites of FleQ in order to derive a consensus, if evident. Four different FleQ binding sites were identified whi...

Analysis of Pseudomonas fluorescens F113 genes implicated in flagellar filament synthesis and their role in competitive root colonization

“…In the mutation affecting the fleQ gene we have found the same morphological and non-motile phenotype. These results are in agreement with the already described function for the FleQ protein, which is the major flagellar regulator in P. aeruginosa (Dasgupta et al, 2002;Jyot et al, 2002). In P. fluorescens Pf0-1, a fleQ homologue gene called adnA encodes a transcriptional factor that affects persistence and spread, also being required for bacterial adhesion and motility Marshall et al, 2001).…”

“…Two of these complete ORFs and the partial ORF show high homology and synteny with the previously described genes fliD, encoding the filament cap protein, fleQ (adnA), encoding a master transcriptional regulator, and fleS, encoding a twocomponent sensor protein, respectively. These genes have been found in every pseudomonad analysed (Arora et al, 2000;Dasgupta et al, 2002Dasgupta et al, , 2003Robleto et al, 2003) and have been thoroughly characterized. Downstream of the fliC gene, there is a small ORF homologous to ORFs with the same gene context in other pseudomonads.…”

Deletion of a previously uncharacterized flagellar-hook-length control gene fliK modulates the σ 54-dependent regulon in Campylobacter jejuni

“…Sigma factor s 54 is involved in flagellar expression in most polar flagellate species. This enables the inclusion of additional checkpoints in flagellar assembly, mediated by transcription activators of the NtrC class and best characterized in Vibrio cholerae (Prouty et al, 2001), Pseudomonas aeruginosa (Dasgupta et al, 2003;Jyot et al, 2002) and Caulobacter crescentus (Mohr et al, 1998).…”

Identification of the binding site of the σ 54 hetero-oligomeric FleQ/FleT activator in the flagellar promoters of Rhodobacter sphaeroides

“…Therefore, activation mediated by the hetero-oligomeric FleQ/FleT complex must occur using similar mechanisms to those of other well-characterized EBP proteins. In contrast, it has been reported that FleQ from P. aeruginosa is able to activate transcription from sites that are adjacent to the promoter sequence (Jyot et al, 2002). Since FleQ from P. aeruginosa is highly similar to FleQ from R. sphaeroides, it appeared possible that these proteins shared this particular property.…”

“…Given the reported location of previously characterized UASs, we envisioned that the UAS for flhAp and flgAp could be contiguous to the promoter, as has been reported for FleQ in Pseudomonas aeruginosa (Jyot et al, 2002), or at the canonical 100 bp distance from each promoter, overlapping the coding sequence of the flhA and flgA operons. To test these possibilities a deletion analysis for each promoter region was carried out, as shown in Figs 2 and 3.…”