BEYOND UNIFORMITY: Pyomelanin,s structural complexity impacts on UV shielding in Pseudomonas species with different lifestyles.

Melanin, a polymeric pigment synthesized by various organisms, confers advantageous traits, including heightened resistance to stress agents. In Pseudomonas, disruption of the tyrosine degradation pathway leads to pyomelanin production. Despite a shared synthetic pathway, the chemical structure of pyomelanin remains elusive due to its heterogeneous polymeric nature, suggesting composition variations even among closely related species. Our objective was to analyze pyomelanin structural features across Pseudomonas strains: CRISPR/nCas9-engineered hmgA mutants of PAO1 and PA14, reference strains of the human opportunist pathogen P. aeruginosa; a natural melanogenic mutant (PAM) from a patient; and a Tn5 mutant of the extremophile bacterium P. extremaustralis (PexM). Structural analysis revealed strain-specific differences. UV spectra exhibited dual peaks for PAO1 and PA14 mutants, while PAM and PexM displayed a single peak. FTIR indicated changes in the alcohol content ratio, with PAO1 and PA14 hmgA mutants having a near 1:1 ratio, PexM a dominant phenol band, and PAM a predominance of the alcohol band. Complex NMR spectra suggested non-linear polymers composed by substituted phenolic rings, carboxylic acids, and alkyl chains, highlighting inter-pigment disparities. UVC (254 nm) susceptibility assessment showed increased survival with pyomelanin addition, correlating with the attenuation of the incoming radiation due to absorption in the culture medium. Moreover, survival to UVC of P. extremaustralis was different depending on the melanin source being the most protective pyomelanin obtained from PAO1. These findings reveal distinct pyomelanin subgroups based on structure among strains, elucidating varied physiological effects.