Disruption of this
hrpU-like CYT387 molecular weight operon in MFN1032 abolishes cell-associated hemolytic activity , as described for mutations in the T3SS apparatus in P. aeruginosa. Our hypothesis was that the first target of MFN1032 T3SS would probably be eukaryotic cells of the rhizosphere, such as plants or amoebae. To test this hypothesis, we investigated the interactions of MFN1032 and other Pseudomonas strains with red blood cells, plants, amoebae and macrophages. In contrast with environmental Pseudomonas, all of the clinical strains of P. fluorescens tested were cytotoxic for erythrocytes through contact. MFN1032 was unable to induce HR on plants and was cytotoxic for amoebae and macrophages. Disruption of the hrpU-like operon in MFN1032 click here abolished these cytotoxicities that were independent of cyclolipopeptide production. GacS/GacA system seems to be a positive regulator for D. discoideum growth inhibition but not for cell-associated hemolysis or macrophage lysis, suggesting that these processes are not identical. Results P. fluorescens MFN1032 and other clinical strains have cell-associated hemolytic activity but do not induce HR on tobacco leaves We investigated find more the distribution of cell-associated hemolytic activity on a panel of
Pseudomonas strains. Cell-associated hemolytic activity (cHA) was measured by the technique used by Dacheux , adapted as described in methods. We tested cHA at 37°C for MFN1032, many MFY162, MFY70 and MFY63 (clinical isolates of P. fluorescens), MF37 (P. fluorescens strain isolated from raw milk), C7R12 and SBW25 (rhizospheric P. fluorescens strains)
and DC3000 (P. syringae plant pathogen) after growth at 28°C (for strain origin see Table 1). Table 1 Bacterial strains used in this study, origins, growth temperatures and references Species Strains Optimal growth temperature (°C) Origins References Pseudomonas fluorescens SBW25 28°C Field grown-sugar beet  C7R12 Flax rhizosphere  MF37 Milk tank  MFY63 Clinical (urine)  MFY70 Clinical (abscess)  MFY162 Clinical (sputum)  MFN1032 Clinical (sputum)  MFN1030 MFN1032 hrpU-like operon mutant  MFN1030- pBBR1MCS-5 MFN1030 carrying pBBR1MCS-5 This study MFN1030-pBBR-rscSTU MFN1030 carrying rscSTU genes of SBW25 cloned into pBBR1MCS-5 This study MFN1031 MFN1030 revertant  V1 MFN1032 spontaneous gacA mutant  V1gacA V1 carrying the gacA gene (plasmid pMP5565)  V3 MFN1032 Variant group 2 (Cyclolipopeptides -) [9, 14] Pseudomonas syringae DC3000 Tomato  Pseudomonas aeruginosa CHA 37°C Clinical  PA14 Clinical  Klebsiella aerogenes KA Environmental  Only clinical strains had cHA (Figure 1). MFY63 showed the highest level of cHA (80% lysis); MFY70 and MFN1032 displayed significant cHA (70% lysis) and MFY162 a median cHA (40% lysis).