Identification of chemotaxis sensory proteins for amino acids in Pseudomonas fluorescens Pf0-1 and their involvement in chemotaxis to tomato root exudate and root colonization

Abstract

Pseudomonas fluorescens Pf0-1 showed positive chemotactic responses toward 20 commonly-occurring l-amino acids. Genomic analysis revealed that P. fluorescens Pf0-1 possesses three genes (Pfl01_0124, Pfl01_0354, and Pfl01_4431) homologous to the Pseudomonas aeruginosa PAO1 pctA gene, which has been identified as a chemotaxis sensory protein for amino acids. When Pf01_4431, Pfl01_0124, and Pfl01_0354 were introduced into the pctA pctB pctC triple mutant of P. aeruginosa PAO1, a mutant defective in chemotaxis to amino acids, its transformants showed chemotactic responses to 18, 16, and one amino acid, respectively. This result suggests that Pf01_4431, Pfl01_0124, and Pfl01_0354 are chemotaxis sensory proteins for amino acids and their genes were designated ctaA, ctaB, and ctaC, respectively. The ctaA ctaB ctaC triple mutant of P. fluorescens Pf0-1 showed only weak responses to Cys and Pro but no responses to the other 18 amino acids, indicating that CtaA, CtaB, and CtaC are major chemotaxis sensory proteins in P. fluorescens Pf0-1. Tomato root colonization by P. fluorescens strains was analyzed by gnotobiotic competitive root colonization assay. It was found that ctaA ctaB ctaC mutant was less competitive than the wild-type strain, suggesting that chemotaxis to amino acids, major components of root exudate, has an important role in root colonization by P. fluorescens Pf0-1. The ctaA ctaB ctaC triple mutant was more competitive than the cheA mutant of P. fluorescens Pf0-1, which is non-chemotactic, but motile. This result suggests that chemoattractants other than amino acids are also involved in root colonization by P. fluorescens Pf0-1.

Fig. 1

Representation of 20 commonly-occurring l-amino acids detected by chemotaxis sensory proteins. This Venn diagram is constructed based on chemotactic responses by FL0124, FL0354, and FL4431 (Table 3).

Fig. 2

Chemotactic responses to tomato root exudate by P. fluorescens Pf0-1 (closed circles), FL0124 (open circles), FL0354 (open squares), FL4431 (closed squares), and FLD3 (open triangles). Digital image processing was used to count the number of bacteria around the mouth of a capillary containing exudates and 1% (w/v) agarose. One videotape frame was analyzed at each time point. The chemotactic response is presented as the normalized cell number. The normalized cell number was calculated by dividing the number of bacteria at each time point by that at the initiation of the observation. Vertical bars represent the standard deviations of measurements done in triplicate experiments.

Fig. 3

Tomato root tip colonization by P. fluorescens strains, (A) alone and (B) in competition with Rif ^r mutants. After 7 days, root systems were sampled. Bar indicates standard deviation. The total CFU numbers at the root tip in Fig. 3B were the same level as in Fig. 3A. There were significant (P<0.05) differences in colonization between Pf0-1Rif and ΔcheA, Pf0-1Rif and FLD3, FLD3Rif and ΔcheA, FLD3Rif and Pf0-1, and FLD3Rif and FL0124.