Genetic analysis of the AdnA regulon in Pseudomonas fluorescens: nonessential role of flagella in adhesion to sand and biofilm formation

Abstract

AdnA is a transcription factor in Pseudomonas fluorescens that affects flagellar synthesis, biofilm formation, and sand adhesion. To identify the AdnA regulon, we used a promoterless Tn5-lacZ element to study the phenotypes of insertion mutants in the presence and absence of AdnA. Of 12,000 insertions, we identified seven different putative open reading frames (ORFs) activated by AdnA (named aba for activated by AdnA). aba120 and aba177 showed homology to flgC and flgI, components of the basal body of the flagella in Pseudomonas aeruginosa. Two other insertions, aba18 and aba51, disrupted genes affecting chemotaxis. The mutant loci aba160 (possibly affecting lipopolysaccharide synthesis) and aba175 (unknown function) led to loss of flagella. The mutant bearing aba203 became motile when complemented with adnA, but the mutated gene showed no similarity to known genes. Curiously, aba18, aba51, aba160, and aba203 mutants formed biofilms even in the absence of AdnA, suppressing the phenotype of the adnA deletion mutant. The combined findings suggest that flagella are nonessential for sand attachment or biofilm formation. Sequence and promoter analyses indicate that AdnA affects at least 23 ORFs either directly or by polar effects. These results support the concept that AdnA regulates cell processes other than those directly related to flagellar synthesis and define a broader cadre of genes in P. fluorescens than that described so far for its homolog, FleQ, in P. aeruginosa.

FIG. 1.

Chemotaxis and swimming response of P. fluorescens Pf0-1. (A) Chemotactic response to Casamino Acids in a modified swarm plate assay. Pf0-1 was inoculated in the center of soft agar MMO and incubated at 28°C. The movement of inoculum was measured in millimeters. (B) Swimming phenotypes of P. fluorescens and aba insertion mutants. Plates containing 0.3% agar were inoculated and incubated for 24 to 48 h at 28°C. The control consists of an adnA-complemented transposon insertion which is not affected by AdnA; the response of the control is similar to that of adnA-complemented Pf0-2x in which adnA had been deleted.

FIG. 2.

Biofilm formation of P. fluorescens Pf0-1 and aba mutants on different substrates. Crystal violet rings were dissolved in ethanol, and A₆₀₀ was read. Experiments were performed at least three times. Strains in which adnA had been deleted (white bars) and strains where adnA was carried on a plasmid or in wild-type Pf0-1 (black bars) are shown. Pf0-1 (0-1) and Pf0-2x (2x) were used as controls.