Impact of Aeration Conditions on Brown Melanin-Like Pigment Production in Pseudomonas extremaustralis 14-3B

DIAZ APPELLA MATEO; KOLENDER A; RAIGER IUSMAN LAURA; LOPEZ NANCY I; TRIBELLI PAULA M

Los Cocos, Cordoba

Congreso; XVII Congreso Argentino de Microbiología General; 2021

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 inbacteria involve melanin precursors derived from tyrosine transformations. Briefly, the oxidation andpolymerization of these precursors, such as the compound homogentisate, leads to the productionof 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 thebiotechnologically relevant Pseudomonas extremaustralis 14-3b, was carried out. Such brownpigment-producing strain resulted from the random insertion of the transposon mini-Tn5. Ourhypothesis is that the pigment production biosynthesis will be affected by the availability of O2 andwill alter physiological aspects, such as biofilm formation. Initially, it was determined that themini-Tn5 was inserted into a non-traditional melanin production inducer gene, which codifies adiguanylate cyclase located in the same region of a gene involved in the biosynthesis of aromaticaminoacids. 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 hppdgenes encoding the enzyme that catalyzes the synthesis of homogentisate, and an assay performedwith an Hppd inhibitor showed an inhibition of pigment production, after 48 h exposure at 1mMconcentration. The biofilm and planktonic growth in LB medium at 30°C were studied, as well aspigment production in aerobic (150 rpm) and microaerobic (supplemented with 0.08% KNO3 and noagitation) conditions. Growth followed by OD600nm showed similar results for both mutant and wildtype strain until 30 h of cultivation, however bacterial viable counts showed an important decrease inthe pigmented strain at 24 h, suggesting that high pigment production could affect survival. Nopigment 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 pigmentwas extracted and purified by acid precipitation. As a result, we were able to extract 0.6 mgpigment/mg biomass after 48 h growth under aerobic conditions.The absorption spectrum of thepigment 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, producedwith the involvement of novel genes related to the homogentisate pathway