Transcriptional phase variation at the flhB gene of Pseudomonas putida DOT-T1E is involved in response to environmental changes and suggests the participation of the flagellar export system in solvent tolerance

Abstract

Frameshift mutations in a poly(G) track at the flhB gene of Pseudomonas putida DOT-T1E are responsible for the diminished swimming of this strain on semisolid medium, which contrasts with the high swimming ability of P. putida KT2440, which does not exhibit a poly(G) track at the flhB gene. We previously showed that a mutant lacking FlhB was more sensitive to solvents than the wild-type strain (Segura et al., J. Bacteriol., 183:4127-4133, 2001). In this study, we show that swimming ability correlates with solvent tolerance in P. putida DOT-T1E, so that growth conditions favoring a functional flhB gene (growth on semisolid medium) resulted in increased innate tolerance to a sudden toluene shock.

FIG. 1.

Morphology of P. putida DOT-T1E and P. putida KT2440 colonies in soft-agar plates.

FIG. 2.

(A) Alignment of the 5′ ends of P. putida DOT-T1E (upper sequence) and P. putida KT2440 (lower sequence) flhB genes. The deduced protein sequences are also shown. Amino acid differences between the two strains are shown inside squares. Changes in nucleotides are in bold. The eight Gs in P. putida DOT-T1E and the corresponding sequence in P. putida KT2440 are shown in the shaded box. (B) Schematic representation of the sizes of five different polypeptides deduced from the sequences obtained. The length of each deduced protein is shown on the right. The number of Gs within the flhB gene is shown inside the protein representation. aa, amino acids.

FIG. 3.

Survival rate of P. putida DOT-T1E pregrown in liquid LB medium (A), solid LB medium (B), and soft-agar medium (C). Cells were collected very carefully by using a loop, letting them stand for 10 min on LB, and then centrifuging the culture at 2,000 rpm (Desaga MC-2; Sarstedt-Gruppe) for 1 min to pellet the soft agar. Cells were suspended in LB until they reached a density of around 10⁹ CFU per ml. The sample was divided into two halves; one was kept as a control (open symbols), and the other was treated with 0.3% (vol/vol) toluene (closed symbols). At the indicated time, the number of CFU/ml was determined.