CONICET | Buscador de Institutos y Recursos Humanos

Pigment production in Pseudomonas genus has been associated with numerous advantages for itsfitness. Particularly, some melanin-producing strains have shown an increased resistance to stressagents, among other self-benefit properties. The most widespread pathways for melanin synthesis in bacteria involve melanin precursors derived from tyrosine transformations. Briefly, the oxidation and polymerization of these precursors, such as the compound homogentisate, leads to the production of melanin. Thus, some key genes have been identified as involved in the production of this pigment. In this work, an analysis of the production of a brown melanin-like pigment in a mutant of the biotechnologically relevant Pseudomonas extremaustralis 14-3b, was carried out. Such brown pigment-producing strain resulted from the random insertion of the transposon mini-Tn5. Our hypothesis is that the pigment production biosynthesis will be affected by the availability of O2 and will alter physiological aspects, such as biofilm formation. Initially, it was determined that the miniTn5 was inserted into a non-traditional melanin production inducer gene, which codifies a diguanylate cyclase located in the same region of a gene involved in the biosynthesis of aromatic aminoacids. These genes may be part of an operon according to previous transcriptomic data. Genomic analysis of homogentisate pathway showed that this bacterium has two probable hppd genes encoding the enzyme that catalyzes the synthesis of homogentisate, and an assay performed with an Hppd inhibitor showed an inhibition of pigment production, after 48 h exposure at 1mM concentration. The biofilm and planktonic growth in LB medium at 30°C were studied, as well as pigment production in aerobic (150 rpm) and microaerobic (supplemented with 0.08% KNO3 and no agitation) conditions. Growth followed by OD600nm showed similar results for both mutant and wild type strain until 30 h of cultivation, however bacterial viable counts showed an important decrease in the pigmented strain at 24 h, suggesting that high pigment production could affect survival. No pigment was produced by either strain in microaerobic planktonic conditions. The biofilm production studied by crystal violet assay showed a decrease in the biofilm formation at both 24 h and 48 h in the pigmented strain. No pigment production was detected by the biofilm after 48 h. The pigment was extracted and purified by acid precipitation. As a result, we were able to extract 0.6 mg pigment/mg biomass after 48 h growth under aerobic conditions.The absorption spectrum of the pigment was measured, and showed a maximum range of absorption at the UV region, similar to the ones of some types of melanin. All these results suggest a role of oxygen in the production of this brown pigment. Furthermore, this evidence might indicate that the pigment is melanin, produced with the involvement of novel genes related to the homogentisate pathway.

enviar mensaje