Gram variability and prodigiosin production in isolate of Serratia marcescens obtained from blackberries

Prodigiosin is a secondary metabolite produced by Gram negative Serratia marcescens (S. marcescens) bacteria and it is characterized by a pyrrolyl pyrromethene skeleton and different alkyl substituents. In its production are involved different enzymes and it is a pigment capable of inducing the apoptosis of several cancer cell lines, so its antioxidant, antifungal, antitumoral and antibiotic potential is very important. Prodigiosin production needs dissolved oxygen, an incubation time of ≈ 60 hours, a solution pH of ≈ 8, a growth temperature of 28°C, and presence of dissolved phosphates. Its production is controlled by a complex regulatory network of both N-acyl-L-homoserine lactone-quorum-sensing-dependent and independent pathways. Prodigiosin can exist in two forms, depending on the hydrogen ion concentration of the solution: in an acid medium, the red pigment exhibits a sharp spectral peak at 535 nm, while in the alkaline medium the pigment is coloured orange-yellow and has a broader spectral curve at 470 nm. In my work, three isolates of Serratia marcescens from blackberry fruit (Rubus fruticosus, cultivar "Polar"), later named D2, D4 and D6, were used, while the isolate Serratia marcescens var kiliensis PCM 550 (designated as PJ), from the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław was used as reference microorganism. The aims of the work were, firstly, to characterize the pigment prodigiosin and, secondly, to verify if the Gram variability of S. marcescens, noticed in previous works, was correlated to prodigiosin production. To understand the spectrophotometric characteristics of the pigment, extraction and purification methods were performed. On the other hand, the Gram variability was studied through growth curve analysis in LB and LB + 1% glucose, which inhibits the prodigiosin production: in this way, slide samples at different timepoints were collected for Gram staining. Further understanding of the phenomenon was proved by HPLC analysis for lipopolysaccharides’ (LPS) carbohydrates, isolated from bacteria grown in shaking conditions, where no prodigiosin production was seen, and stable conditions, where prodigiosin was observed. All these analyses are supported by previous information found in the literature to understand the growing conditions of S. marcescens (e.g., oxygen, glucose, and nutrients influence) and the localization of prodigiosin.