Figure 2.

Phenotypic and chemical analyses of P. fluorescens strain SS101 and single or double mutants disrupted in rsmY, rsmZ, gacS or gacA.A. Drop collapse assay with cell cultures of wild-type strain SS101, ΔrsmY, ΔrsmZ, ΔrsmYZ, ΔgacS and ΔgacA mutants. Bacterial cultures grown for 2 days at 25°C on KB agar plates were suspended in sterile water to a final density of 1 × 10¹⁰ cells ml⁻¹, and 10-μl droplets were spotted on parafilm, and crystal violet was added to the droplets to facilitate visual assessment. A flat droplet is a highly reliable proxy for the production of the surface-active lipopeptide massetolide A.B. Reversed phase-high-performance liquid chromatography chromatograms of cell-free culture extracts of wild-type strain SS101, ΔrsmY, ΔrsmZ, ΔrsmYZ, ΔgacS and ΔgacA mutants as described in A. The wild-type strain SS101 produces massetolide A (retention time of approximately 23–25 min) and various other derivatives of massetolide A (minor peaks with retention times ranging from 12 to 18 min) which differ from massetolide A in the amino acid composition of the peptide moiety. AU stands for absorbance unit.C. Swarming motility of wild-type strain SS101, ΔrsmY, ΔrsmZ, ΔrsmYZ, ΔgacS and ΔgacA mutants on soft [0.6% (wt/vol)] agar plates. Five microlitres (1 × 10¹⁰ cells ml⁻¹) of washed overnight cultures of wild-type SS101 or mutants were spot inoculated in the centre of a soft agar plate and incubated for 48 to 72 h at 25°C.D. Growth of wild-type strain SS101, ΔrsmY, ΔrsmZ, ΔrsmYZ, ΔgacS and ΔgacA mutants in liquid broth at 25°C. At different time points, the optical density of the cell cultures was measured spectrophotometrically (600 nm). Mean values of four biological replicates are given; the error bars represent the standard error of the mean.