Figure 2

Phenotypic and chemical analyses of P. fluorescens strain SS101, and its clpA mutant. (A) Drop collapse assay with cell suspensions of wild-type strain SS101, clpA plasposon mutant, clpA mutant + pME6031 (empty vector control) and clpA mutant + pME6031-clpA. Bacterial cultures grown for 2 days at 25°C on KB agar plates were suspended in sterile water to a final density of 1x10¹⁰ cells/ml. 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) RP-HPLC chromatograms of cell-free culture extracts of the wild-type strain SS101, clpA plasposon mutant, clpA + pME6031 (empty vector control) and clpA + pME6031-clpA as described in panel 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. (C) Swarming motility of the wild-type strain SS101, clpA plasposon mutant, clpA mutant + pME6031 (empty vector control) and clpA mutant + pME6031-clpA on soft (0.6% wt/vol) agar plates. Five microliter (1×10¹⁰ cells/ml) of washed cells from overnight cultures was spot-inoculated in the center of a soft agar plate and incubated for 48–72 h at 25°C. (D) Growth of the wild-type SS101 strain, clpA plasposon mutant, clpA mutant + pME6031 (empty vector control), clpA mutant + pME6031-clpA and clpP site-directed mutagenesis mutant in liquid medium at 25°C. The optical density of the cell cultures was measured spectrophotometrically (600 nm) at different time points. Mean values of four biological replicates are given; the error bars represent the standard error of the mean.