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.

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

“…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.…”

MorA Defines a New Class of Regulators Affecting Flagellar Development and Biofilm Formation in Diverse Pseudomonas Species

“…We used a genetic approach to identify negative regulators of swimming motility. Based on our hypothesis that knockout of such a gene should lead to enhanced motility, WT Pp transposon mutants (Table 1) were screened for enhanced swimming motility through 0.4% (wt/vol) soft agar (18). Two transposon insertion mutant strains of P. putida, C3H and B12H (Table 1), which exhibited over 2.5-fold-enhanced motility, were identified (data not shown).…”

“…To study swimming motility, semisolid LB agar plates (0.4% [wt/vol] Bacto Agar) were inoculated with 2 l of an overnight bacterial culture. After incubation for 18 h at 30°C, the movement of the bacteria away from the inoculation point was determined relative to the movement of WT Pp , as previously described (18). To study chemotaxis, the chemotaxis assay was adapted from the assay described by Shi et al (24).…”

Screening for Bacillus Isolates in the Broiler Gastrointestinal Tract

“…Formation of biofilm was tested as described by Robleto et al (57) with modifications. Essentially, 2 to 3 ml of Sterlini-Mandelstam medium (16) was inoculated with an exponential-phase culture grown in the same medium to an optical density at 600 nm of Ϸ0.01.…”