Association of hemolytic activity of Pseudomonas entomophila, a versatile soil bacterium, with cyclic lipopeptide production

Abstract

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C(10)OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.

FIG. 1.

Schematic representation of the entolysin biosynthesis gene clusters and surrounding open reading frames in the P. entomophila genome. Below the genes is the module and domain organization of EtlA, EtlB, and EtlC. Predicted amino acid specificity is indicated below each module. All amino acids are identified by standard three-letter biochemical notation, with D and L referring to their stereochemistry.

FIG. 2.

Hemolytic activity, biosurfactant production, and swarming motility of wild-type P. entomophila and mutants affected in different genes of the entolysin biosynthesis gene clusters. (A) Hemolytic activity as visualized on blood plates. Single colonies were patched onto sheep blood plates and allowed to grow for 24 h at 30°C. (B) Biosurfactant activity was visualized by the drop-collapsing assay, in which 20-μl droplets of each culture supernatant were deposited onto a hydrophobic (Parafilm) substrate. Bromophenol blue was added to stain the supernatants for photographic purposes and had no influence on the shape of the droplets. (C) After drying, the size (in millimeters) of each supernatant droplet was measured. Each value is the mean of five experiments. (D) Swarming motility was measured on minimal medium plates containing 0.5% agar. Plates were inoculated with a toothpick, and bacteria were allowed to grow for 24 h at 30°C. (E) Hemolytic activity of purified entolysins A and B. Five microliters of concentrated products was deposited onto a blood plate, and activity was allowed to develop for 24 h. (F) Surface tension activity of purified entolysins A and B. Two microliters of concentrated product was added to 20 μl of water, which was deposited onto Parafilm.

FIG. 3.

HPLC analysis of the surface active compound(s) of P. entomophila. C₁₈ reverse-phase HPLC profiles of ethyl acetate extracts derived from 6 ml of culture supernatants of wild-type P. entomophila (A), hemolysin-deficient mutant IM3045 (B), and a gacA mutant (C). Chromatograms were obtained through sample analysis at a wavelength of 206 nm. The two main peaks represent entolysin isoforms A and B. AU, arbitrary units.

FIG. 4.

CID tandem mass spectra and fragmentation schemes of the cyclic form of entolysin A (the m/z 1,721 species) (A) and the open-ring form (m/z 1,738 [1,721 + 17] species) (B). (C) Proposed structure of entolysin, based on MALDI MS/MS, fatty acid, and amino acid analyses. Ser, serine; Glu, glutamic acid; Gln, glutamine; Val, valine; Xle, isoleucine or leucine.

FIG. 5.

Regulation of lipopeptide production by the Gac system. (A) Hemolytic activities of mutants affected in the different constituents of the Gac system. (B) Biosurfactant production by these mutants visualized by the drop-collapsing effect and quantified by measuring droplet size (C). (D) Quantification of the β-galactosidase activity of the translational fusion etlB-lacZ (Pseen3045-lacZ) (see Materials and Methods) as a function of bacterial growth in different genetic contexts. Diamonds represent expression of the fusion in the wild-type strain carrying the empty vector pPSV35 (closed diamonds) or overexpressing the gene encoding the translational repressor RsmA2 from the same vector (pPSVrsmA2, open diamonds). Squares represent expression of the fusion in the rsmY rsmZ double mutant carrying the empty vector pPSV35 (opened squares) or overexpressing the rsmY gene from the same vector (pPSVrsmY, closed squares). Cultures were grown with gentamicin and 1 mM IPTG to allow full expression of the genes from the plasmids. Each value is the average of three different cultures, and error bars represent standard deviations. MU, Miller units; OD600, optical density at 600 nm.

FIG. 6.

Entolysin is not involved in the virulence of P. entomophila for Drosophila. Wild-type Oregon^r female flies (closed symbol) and relish mutant female flies (opened symbol) were naturally infected by the P. entomophila wild-type strain (Pe, diamonds), a gacA mutant (ΔgacA, squares), and an entolysin-deficient mutant (IM3045, circles).