Transcriptional organization of the region encoding the synthesis of the flagellar filament in Pseudomonas fluorescens (original) (raw)
Related papers
2003
The single polar flagellum of Pseudomonas aeruginosa is an important virulence and colonization factor of this opportunistic pathogen. In this study, the annotation of the genes belonging to the fla regulon was updated and their organization was analysed in strains PAK and PAO1, representative type-a and type-b strains of P. aeruginosa respectively. The flagellar genes are clustered in three non-contiguous regions of the chromosome. A polymorphic locus flanked by flgJ and fleQ in Region I contains a glycosylation island in PAK. The expression and ordered assembly of the complex multicomponent flagellum is intricately regulated. Dedicated flagellar genes fleQ , fleS , fleR , fliA , flgM and fleN encode proteins that participate in the regulation of the flagellar transcriptional circuit. In addition, expression of the flagellum is coordinately regulated with other P. aeruginosa virulence factors by the alternative sigma factor s s s s 54 , encoded by rpoN. In order to gain insight into the hierarchical regulation of flagellar genes, deletion mutations were constructed in fleQ , fleR , fliA and rpoN. The transcriptional impact of these mutations was examined by transcriptional profiling using a P. aeruginosa whole genome microarray. Analysis of the transcriptomes generated for each of these mutants indicates a four-tiered (Classes I-IV) hierarchy of transcriptional regulation. Class I genes are constitutively expressed and include the transcriptional regulator fleQ and the alternative sigma factor fliA (s s s s 28). Class II genes including fleSR , encoding a two-component regulatory system require FleQ and RpoN (s s s s 54) for their transcriptional activation. Class III genes are positively regulated by the activated response regulator FleR in concert with RpoN. The transcription of Class IV genes is dependent on the availability of free FliA following the export of the FliA specific antisigma factor FlgM through the basal body rod-hook structure (assembled from Class II and III gene products). Two previously uncharacterized genes, which are coordinately regulated with known flagellar genes have been identified by genome-wide analysis and their role in flagellar biogenesis was analysed.
fleN , a Gene That Regulates Flagellar Number in Pseudomonas aeruginosa
Journal of Bacteriology, 2000
The single polar flagellum of Pseudomonas aeruginosa plays an important role in the pathogenesis of infection by this organism. However, regulation of the assembly of this organelle has not been delineated. In analyzing the sequence available at the Pseudomonas genome database, an open reading frame (ORF), flanked by flagellar genes flhF and fliA , that coded for a protein (280 amino acids) with an ATP-binding motif at its N terminus was found. The ORF was inactivated by inserting a gentamicin cassette in P. aeruginosa PAK and PAO1. The resulting mutants were nonmotile on motility agar plates, but under a light microscope they exhibited random movement and tumbling behavior. Electron microscopic studies of the wild-type and mutant strains revealed that the mutants were multiflagellate, with three to six polar flagella per bacterium as rather than one as in the wild type, indicating that this ORF was involved in regulating the number of flagella and chemotactic motility in P. aerugin...
Molecular microbiology, 2001
The first flagellar assembly checkpoint of Caulobacter crescentus couples assembly of the early class II components of the basal body complex to the expression of class III and IV genes, which encode extracytoplasmic structures of the flagellum. The transcription of class III/IV flagellar genes is activated by the response regulator factor, FlbD. Gain of function mutations in flbD, termed bfa, can bypass the transcriptional requirement for the assembly of class II flagellar structures. Here we show that the class II flagellar gene fliX encodes a transacting factor that couples flagellar assembly to FlbD-dependent transcription. We show that the overexpression of fliX can suppress class III/IV gene expression in both wild-type and flbD-bfa cells. Introduction of a bfa allele of flbD into cells possessing a deletion in fliX restores motility indicating that FliX is not a structural component of the flagellum, but rather a transacting factor. Furthermore, extragenic motile suppressors which arise in DfliX cells map to the flbD locus. These results indicate that FlbD functions downstream of FliX in activating class III/IV transcription. b-Lactamase fusions to FliX and analysis of cellular fractions demonstrate that FliX is a cytosolic protein that demonstrates some peripheral association with the cytoplasmic membrane. In addition, we have isolated a mutant allele of fliX that exhibits a bfa-like phenotype, restoring flbD-dependent class III/IV transcription in strains that contain mutations in class II flagellar structural genes. Taken together, these results indicated both a positive and negative regulatory function for FliX in coupling the assembly of class II basal body components to gene expression.
Transductional analysis of the flagellar genes in Pseudomonas aeruginosa
Journal of bacteriology, 1983
Complementation in bacteriophage E79 tv-l-mediated transduction and the phenotypic properties of the flagellar genes in Pseudomonas aeruginosa PAO were investigated by using 195 flagellar mutants of this organism. A total of 15 fla. 1 mot, and 2 che cistrons were identified. At least 5 fla cistrons (fla V to flaZ) and one mot cistron resided in one region, and at least 10 fla cistrons (flaA to flaJ) and two che cistrons (cheA and cheB) resided in another. The flaC mutants exhibited cistron-specific leakiness on motility agar plates. The flaE cistron may be the structural gene for the component protein of the flagellar filament. The cheA mutations, which resulted in pleiotropic phenotypes for flagellar formation, motility, and taxis, belonged to the same complementation group as the flaF mutations; that is, we inferred that cheA and flaF are synonymous.
Journal of Bacteriology, 2001
Flagellar number in Pseudomonas aeruginosa is controlled by FleN, a putative ATP/GTP binding protein. Disruption of fleN results in multiflagellation of the otherwise monoflagellate strains PAK and PAO1 and is associated with a chemotactic defect. We propose that flagellar number is maintained by the antiactivator FleN, which downregulates flagellar genes by binding to their transcriptional activator, FleQ, an enhancer binding protein belonging to the NifA subfamily. In this report we demonstrate direct interaction of FleN and FleQ in the yeast two-hybrid system. Mutagenesis of the putative ATP/GTP binding motif in FleN 24K→Q and truncation of FleN at either the N or C terminus abrogates this interaction. FleN does not inhibit the DNA binding ability of FleQ in vitro, thus indicating that it probably utilizes another mechanism(s) to serve as a FleQ antiactivator.
Regulation cascade of flagellar expression in Gram-negative bacteria
FEMS Microbiology Reviews, 2003
Flagellar motility helps bacteria to reach the most favourable environments and to successfully compete with other micro-organisms. These complex organelles also play an important role in adhesion to substrates, biofilm formation and virulence process. In addition, because their synthesis and functioning are very expensive for the cell (about 2% of biosynthetic energy expenditure in Escherichia coli) and may induce a strong immune response in the host organism, the expression of flagellar genes is highly regulated by environmental conditions. In the past few years, many data have been published about the regulation of motility in polarly and laterally flagellated bacteria. However, the mechanism of motility control by environmental factors and by some regulatory proteins remains largely unknown. In this respect, recent experimental data suggest that the master regulatory protein-encoding genes at the first level of the cascade are the main target for many environmental factors. This mechanism might require DNA topology alterations of their regulatory regions. Finally, despite some differences the polar and lateral flagellar cascades share many functional similarities, including a similar hierarchical organisation of flagellar systems. The remarkable parallelism in the functional organisation of flagellar systems suggests an evolutionary conservation of regulatory mechanisms in Gram-negative bacteria. ß
Journal of Bacteriology, 2002
In Pseudomonas aeruginosa, flagellar genes are regulated in a cascade headed by FleQ, an NtrC/NifA-type activator. FleQ and RpoN positively regulate expression of flhA, fliE, fliL, and fleSR genes, among others. Direct interaction of FleQ with flhA, fliE, fliL, and fleSR promoters was demonstrated by gel shift assay, along with experiments to conclusively determine the specificity of its binding. DNase I footprinting was performed to determine the FleQ binding sites on flhA, fliE, fliL, and fleSR promoters. No sequence conservation among these binding sites was observed. Primer extension analysis revealed the transcription start sites (TSSs) to be localized above the FleQ binding sites in flhA, fliE, and fliL promoters. Analysis of the above data revealed FleQ binding to be in the leader sequence of these promoters, whereas FleQ binding was 67 bp upstream of the TSS in the fleSR promoter. Mutagenesis of the FleQ binding site in the flhA promoter confirmed its functionality in vivo. ...
Ordering of the flagellar genes in Pseudomonas aeruginosa by insertions of mercury transposon Tn501
Journal of Bacteriology
The flagellar genes of Pseudomonas aeruginosa PAO cluster on the chromosome at two distinct regions, region I and region II. The order of the flagellar cistrons in this organism was established by using transducing phage G101 and plasmids FP5 and R68.45. A method to insert transposon TnSOl near thefla genes was devised. We obtained two strains in which TnSOl was inserted at sites close to the flagellar cistrons in region II. We isolated Flamutants in which the chromosomal segment between the two TnSOI insertion sites was deleted. Using TnSOl-encoded mercury resistance as an outside marker, we determined the order of 9 of the 11 flagellar cistrons in region II as follows: puuF-region I-flaG-flaC-flal-flaH-flaD-flaB-flaA-flaF-flaE-pur-67. By using phage G101-mediated transduction, the mutation converting monoflagellated bacteria into the multiflagellated (mfl) form was closely linked to the five fla cistrons in region I. Using mfl as an outside marker, we determined the order of the five cistrons as follows: puuF-flaV-flaZ-flaW-flaX-flaY-region II. The mfl mutation was shown to be either located within the flaV cistron or linked very closely to this cistron. No linkage was observed in transductions between any of thefla cistrons in region I and any of the fla cistrons in region II.
Molecular microbiology, 2001
Biogenesis of the single polar flagellum of Caulobacter crescentus is regulated by a complex interplay of cell cycle events and the progression of flagellum assembly. The expression of class III/IV flagellar genes requires the assembly of an early flagellar basal body structure, encoded by class II genes, and is activated by the transcription factor FlbD. Previous experiments indicated that the class II flagellar gene, flbE, encoded a transacting factor that was required for FlbD activity. Here, using mutant alleles of flbE we have determined that FlbE is either a structural component of the flagellum or is required for flagellar assembly and does not, as originally proposed, function as a transacting factor. We also demonstrate that two deleted derivatives of flbE have a dominant negative effect on the transcriptional activation of class III/IV flagellar genes that can be relieved by a gain-of-function mutation in flbD called bfa. This same mutation in flbD has been shown to restore class III/IV transcription in the absence of early class II flagellar assembly. These deleted mutants of flbE also exhibited a filamentous cell phenotype that was indistinguishable from that previously observed in class II flagellar mutants. Introduction of a flbD-bfa mutation into these cells expressing the deleted alleles of flbE, as well as several class II mutant strains, restored normal cell division and FtsZ localization. These results suggest that class III/IV transcription and a step in cell division are coupled to flagellar assembly by the same genetic pathway.