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FleN, a gene that regulates flagellar number in Pseudomonas aeruginosa.

ABSTRACT: 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. aeruginosa. The ORF was named fleN. An intact copy of fleN on a plasmid complemented the mutant by restoring motility and monoflagellate status. The beta-galactosidase activities of eight flagellar operon or gene promoters in the wild-type and fleN mutant strains revealed a direct correlation between six promoters that were upregulated in the fleN mutant (fliLMNOPQ, flgBCDE, fliEFG, fliDS orf126, fleSR, and fliC) and positive regulation by FleQ, an NtrC-like transcriptional regulator for flagellar genes. Based on these results, we propose a model where FleN influences FleQ activity (directly or indirectly) in regulating flagellar number in P. aeruginosa.

SUBMITTER: Dasgupta N

PROVIDER: S-EPMC94283 | biostudies-literature | 2000 Jan

REPOSITORIES: biostudies-literature

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fleN, a gene that regulates flagellar number in Pseudomonas aeruginosa.

Dasgupta N N Arora S K SK Ramphal R R

Journal of bacteriology 20000101 2

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 ca ...[more]

PMID: 10629180

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Project description:Pseudomonas aeruginosa frequently encounters microbes that produce ethanol. Low concentrations of ethanol reduced P. aeruginosa swim zone area by up to 45% in soft agar. The reduction of swimming by ethanol required the flagellar motor proteins MotAB and two PilZ domain proteins (FlgZ and PilZ). PilY1 and the type 4 pilus alignment complex (comprising PilMNOP) were previously implicated in MotAB regulation in surface-associated cells and were required for ethanol-dependent motility repression. As FlgZ requires the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) to represses motility, we screened mutants lacking genes involved in c-di-GMP metabolism and found that mutants lacking diguanylate cyclases SadC and GcbA were less responsive to ethanol. The double mutant was resistant to its effects. As published previously, ethanol also represses swarming motility, and the same genes required for ethanol effects on swimming motility were required for its regulation of swarming. Microscopic analysis of single cells in soft agar revealed that ethanol effects on swim zone area correlated with ethanol effects on the portion of cells that paused or stopped during the time interval analyzed. Ethanol increased c-di-GMP in planktonic wild-type cells but not in ΔmotAB or ΔsadC ΔgcbA mutants, suggesting c-di-GMP plays a role in the response to ethanol in planktonic cells. We propose that ethanol produced by other microbes induces a regulated decrease in P. aeruginosa motility, thereby promoting P. aeruginosa colocalization with ethanol-producing microbes. Furthermore, some of the same factors involved in the response to surface contact are involved in the response to ethanol.IMPORTANCE Ethanol is an important biologically active molecule produced by many bacteria and fungi. It has also been identified as a potential marker for disease state in cystic fibrosis. In line with previous data showing that ethanol promotes biofilm formation by Pseudomonas aeruginosa, here we report that ethanol reduces swimming motility using some of the same proteins involved in surface sensing. We propose that these data may provide insight into how microbes, via their metabolic byproducts, can influence P. aeruginosa colocalization in the context of infection and in other polymicrobial settings.

Project description:Previous work has demonstrated that fleR, the gene for a transcriptional activator belonging to the NtrC subfamily of response regulators, is involved in the regulation of mucin adhesion and flagellar expression by Pseudomonas aeruginosa. This report describes the identification and characterization of fleQ, the gene for another transcriptional regulator which also regulates mucin adhesion and motility in this organism. The complete nucleotide sequence of the fleQ gene was determined on both DNA strands, and an open reading frame (ORF) consisting of 1,493 nucleotides was identified. This ORF coded for a gene product of predicted molecular weight, as confirmed by the overexpression of the fleQ gene as a fusion protein under an inducible promoter. The fleQ gene is flanked by a flagellar operon, fliDSorf126, at the 5' end and the fleSR operon on the 3' end. FleQ also had striking homology to a number of proteins belonging to the NtrC subfamily of response regulators, which work in concert with the alternate sigma factor RpoN (sigma54) to activate transcription. However, FleQ lacks the residues corresponding to Asp-54 and Lys-104 of the NtrC protein which are conserved in most of the members belonging to this subfamily of regulators. In addition, unlike some of the other transcriptional activators of this group, FleQ does not appear to have a cognate sensor kinase. A chromosomal insertional mutation in the fleQ gene abolished mucin adhesion and motility of P. aeruginosa PAK and PAK-NP. Both of these functions were regained by providing the complete fleQ gene on a multicopy plasmid. The location of fleQ immediately upstream of the fleSR operon, which is also necessary for the same process, suggested that these regulators may interact in some way. We therefore examined the regulation of the fleSR operon by fleQ and vice versa. Promoter fusion experiments showed that the fleSR operon was regulated by RpoN and FleQ. On the other hand, the fleQ promoter was independent of RpoN and FleR. FleQ, thus, adds another level of regulation to motility and adhesion in P. aeruginosa, above that of fleSR. We therefore propose the existence of a regulatory cascade which consists of at least two transcriptional regulators, FleQ and FleR, in the control of motility and adhesion in P. aeruginosa.

Project description:Pseudomonas aeruginosa binds to eukaryotic cells via both pilus and nonpilus adhesins, while binding of P. aeruginosa to mucin is pilus independent. To characterize genes involved in non-pilus-mediated adherence, transposon mutants of the nonpiliated strain P. aeruginosa PAK-NP that are unable to bind to cells or mucins were isolated. Two such mutants, P. aeruginosa B164 and P. aeruginosa RR18, were identified previously as deficient in binding to eukaryotic cells or mucins as well as nonmotile. The transposon insertion in each of these strains was mapped to the same gene. Sequence analysis of both DNA flanking the transposons and plasmids that could complement the mutations indicated that this open reading frame encodes a putative protein homolog of both Escherichia coli FliO and Erwinia carotovora subsp. atroseptica MopB. The transposons in both of these mutants are nonpolar, since the addition of the P. aeruginosa fliO gene in trans restored adherence to both cells and mucins to these mutants. The cloned fliO gene also complemented the motility defect of both B164 and RR18. A 1.6-kb KpnI fragment from the PAK chromosome that contained the fliO gene was sequenced. The fliO gene appears to be part of an operon with a complete open reading frame upstream of the FliO homolog encoding a putative protein homolog of FliN of both E. coli and Salmonella typhimurium. The partial open reading frame downstream of fliO encodes a putative homolog of both E. coli and S. typhimurium FliP. The fliN gene is flanked on its 5'-end by the 3'-end of a homolog of a fliM gene. The P. aeruginosa FliN protein was identified with a T7 expression system, while all attempts to identify the P. aeruginosa FliO protein were unsuccessful. Homologs of P. aeruginosa FliO are involved in the biosynthesis of flagella, but the function of FliO in this biosynthetic process remains unknown. Further study should reveal the precise role of P. aeruginosa FliO in non-pilus-mediated adherence, which could include regulation of expression or localization of a nonpilus adhesin.

Project description:Binding of Pseudomonas aeruginosa strain PAK to mucin has been shown to be mediated by the flagellar cap protein, product of the fliD gene. Since the flagellar cap is very likely an exposed structure, the FliD polypeptide should be recognized by the host immune system, analogous to the recognition of dominant epitopes located in the exposed parts of the flagellin polypeptide within the assembled flagellum. In P. aeruginosa, a number of distinct flagellin variants are made, and these variable sequences presumably allow the newly infected P. aeruginosa to escape recognition by the antibody induced during a previous infection. Since similar mechanisms may direct the selection of FliD variants, we examined the extent of sequence heterogeneity among various FliD sequences among a selected group of P. aeruginosa. The results of PCR and nucleotide sequencing of the fliD region of eight different P. aeruginosa strains (laboratory strains PAK, PAO1, and PA103; clinical strains 1244, CS2, and CS32; cystic fibrosis strains CS29 and MDR) suggested that there were two distinct types of FliD in P. aeruginosa, which we named A type and B type. The results of Western blotting using the polyclonal antibodies raised against the purified FliD of A type (PAK) or B type (PAO1) further confirmed the existence of two distinct antigenic types of FliD proteins, with no cross-reactivity between the two serotypes. Further Western immunoblot analysis of the same strains using polyclonal FliC antibody showed that the strains with A-type FliD possessed a-type FliC and those with B-type FliD had b-type FliC. Similar Western blot analyses of 50 more P. aeruginosa strains obtained from varied sources revealed that all strains contained either A-type or B-type FliD, suggesting the existence of only two types of FliD in P. aeruginosa and indicating that fliC and fliD were coinherited. This limited diversity of FliC and FliD serotypes seems to be a unique feature of flagellar proteins. A chromosomal mutant having an insertion in the fliD gene of P. aeruginosa PAO1 was constructed. The motility defect of this mutant and a previously constructed PAK fliD mutant was better complemented with the fliD gene of the homologous types.

Project description:UnlabelledThe opportunistic pathogen Pseudomonas aeruginosa utilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in a fliC mutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis of P. aeruginosa.ImportanceThe inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogen P. aeruginosa produces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particular P. aeruginosa strain elicits inflammasome activation.

Dataset Information

FleN, a gene that regulates flagellar number in Pseudomonas aeruginosa.

Publications

fleN, a gene that regulates flagellar number in Pseudomonas aeruginosa.

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