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.

FIG. 1

Schematic representation of the fleN locus based on the P. aeruginosa PAO1 sequence of contig 53 from the Pseudomonas genome database. Bold arrows indicate locations of the ORFs (flhA, flhF, fleN, fliA, and cheY) in this region, and their respective orientations; ● indicates the presence of a ς⁵⁴ consensus sequence; ♦ denotes the location of the fliA promoter. The position of the unique EcoRV site in fleN, used for inserting the gentamicin resistance gene, is shown. Arrowheads show positions and orientations of primers used in this study.

FIG. 2

(A) Motility phenotype of P. aeruginosa wild-type strains PAK and PAO (PAO1) and their fleN mutants, PAK-N and PAO-N. (B) Motility phenotype of PAK-N containing plasmid constructs pPZ375-fleN, pET-fleN, and their vector controls, pPZ375 and pET15bVP, respectively. The strains were freshly grown on LB plates with appropriate antibiotics; cells were transferred to 0.3% agar plates with a sterile toothpick and incubated at 37°C for 8 h (A) or 12 h (B).

FIG. 3

Electron micrographs of wild-type PAK (A) and mutant PAK-N (B). The wild-type cell has a long single polar flagellum, as opposed to the polar tuft of shorter flagella present on the mutant bacteria visible in this field.

FIG. 4

Electron micrographs of mutant PAK-N containing plasmid constructs pPZ375 (A), pPZ375-fleN (B), pET15bVP (C), and pET-fleN (D). The vector controls (A and C) retain the multiple polar flagella of the mutant, whereas most of the cells in panel B, with pPZ375-fleN, are nonflagellated. pET-fleN (D) restores the wild-type monoflagellate status to the mutant, as most of the cells are seen to possess a single polar flagellum.

FIG. 5

PILEUP- and PRETTYBOX-generated alignment of the deduced amino acid sequence of FleN from P. aeruginosa (paeflen) and those of its positional homologues OrfC of P. putida (ppuorfc), Orf313 of V. cholerae (vchorf313), YlxH of B. burgdorferi (bboylxh), Orf304 of T. pallidum (tpaorf304), YlxH of H. pylori (hpyylxh), Orf298 of B. subtilis (bsuorf298), and MinD-1 of A. aeolicus (aaemind-1), using Wisconsin Package (version 10.0; Genetics Computer Group, Madison, Wis.). Dark shading denotes identical amino acid residues, and shades of gray represent conserved substitutions; ∼ represents gaps introduced at the beginning or end of the sequence, and dots denote gaps introduced within the sequence for optimal alignment. A consensus sequence was generated based on the presence of the same amino acid in at least four sequences. The ATP-binding motif corresponding to residues 18 to 25 of paeflen is conserved except in ppuorfc.