Beyond uniformity: Investigation pyomelanin?s structural complexity and its significance in UV protection

DIAZ APELLA, MATEO; OPEZZO, OSCAR; KOLENDER, ADRIANA; LOPEZ, NANCY; TRIBELLI, PAULA

Congreso; XVIII Congreso Argentino de Microbiología General, SAMIGE 2023; 2023

Sociedad Argentina de Microbiología General

Melanin is a widespread polymeric pigment synthesized by microorganisms. InPseudomonas, a melanin type called pyomelanin is produced via disruption of the tyrosine degradation pathway. This biosynthesis has been correlated to advantageous traits,including increased resistance to oxidative agents and UV radiation persistence. Despite sharing a common synthesis pathway, pyomelanin's chemical structure remains undefined due to its heterogeneous polymer nature. This leads us to hypothesize that pyomelanin composition varies across even closely related species. Thus, such chemical diversity might correspond to distinct biological roles in microorganisms, opening avenues for biotechnological applications. Our study aims to analyze structural features of pyomelanin from Pseudomonas species with diverse lifestyles: a Crispr-Cas9-engineered P. aeruginosa mutant (PAO1 hmgA*), which is an opportunistic human pathogen, and a P. extremaustralis Tn5 mutant (PexM), an extremophile from Antarctica. Furthermore, we seek to determine if such structural differences impact the pigment's physiological function, specifically its role in UVC radiation persistence. The methodology involved purifying pigments from 24-houraerobic LB cultures of PAO1 hmgA* and PexM through acid precipitation and lyophilization.Subsequent UV-Vis, FTIR (ATR), and NMR (1H and 13C) analyses were performed. First, it was revealed distinct UV spectra: the pigment of PAO1 hmgA* presents dual peaks at 254 and 222 nm, while the one of PexM displays a single peak at 221 nm. FTIR shows changes in phenolic content between strains through intensity ratios of the 1219 (Ph-OH) and 1055(C-OH alcohol) cm-1 bands. PAO1 hmgA* exhibits a near 1:1 ratio, whereas PexM displays a dominant phenol band. Complex NMR spectra suggest polymers of phenolic aromatic rings, carboxylic acids, and alkyl groups substituted with oxygen and nitrogen, highlighting inter-pigment disparities. Moreover, UVC persistence (254 nm) was assessed by irradiatingsamples of wild type and pyomelanin mutant cells adjusted to 0.3 OD in PBS solution, featuring concentrations of 0 or 0.2 mg/ml of purified pyomelanin. Thus, such samples were exposed to a fluence rate of 0,98 W/m2 within 135 and 360 sec for samples without or with melanin, respectively. In the case of PAO1 hmgA*, the addition of pyomelanin reduced the effective fluence rate received to 42% and in PexM to 35%, which was reflected in higher viable cell counts. Notably, even without the addition of pyomelanin, the PAO1 mutant strainshowed heightened persistence, potentially due to intracellular quinone accumulation from disrupted hmgA gene. These findings reveal different pyomelanin subgroups based on structure, elucidating varied impacts on UV radiation protection. These distinctions, previously unnoticed in a seemingly uniform pigment across different organisms, hold substantial importance due to variable physiological effects in producing species.