Melanins are heterogenous biopolymers produced by many macro and microorganisms. They enhance the ecological fitness of the producer organisms by improving their virulence and protecting them from radiation, osmotic and heat stresses. Fungi synthesise either DOPA or DHN melanin and deposit them on their cell walls. Some fungal isolates produce water soluble melanin (pyomelanin) which is excerted out of the hyphae into the surrounding environment. Pyomelanin, a polymer of homogentisate, exhibits antimicrobial, UV screening, antioxidative, and anti-inflammatory activities and recent studies also show that pyomelanin could find use in space travel as radiation shield. However, scant literature is available on fungi with ability to produce pyomelanin. We report for the first time that Amorosia littoralis occurs as a root endophyte in mangrove species including Avicennia marina, Bruguiera cylindrica and Bruguiera gymnorhiza and produces pyomelanin. Considering the various technological uses of pyomelanin, this study underscores the need to explore fungi of different habitats to identify hyperproducers and to obtain chemically diverse pyomelanin.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis (CF) and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals; pigment production has been reported to enable Bcc strains to overcome the host cell oxidative burst. In this work, we investigated the role of pyomelanin in resistance to oxidative stress and virulence in strains J2315 and K56-2, two epidemic CF isolates belonging to the Burkholderia cenocepacia ET-12 lineage. We previously reported that a single amino acid change from glycine to arginine at residue 378 in homogentisate 1,2-dioxygenase (HmgA) affects the pigment production phenotype: pigmented J2315 has an arginine at position 378, while non-pigmented K56-2 has a glycine at this position. Herein, we performed allelic exchange to generate isogenic non-pigmented and pigmented strains of J2315 and K56-2, respectively, and tested these to determine whether pyomelanin contributes to the protection against oxidative stress in vitro as well as in a respiratory infection in CGD mice in vivo. Our results indicate that the altered pigment phenotype does not significantly impact these strains\' ability to resist oxidative stress with H2O2 and NO in vitro and did not change the virulence and infection outcome in CGD mice in vivo suggesting that other factors besides pyomelanin are contributing to the pathophysiology of these strains.IMPORTANCEThe Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria that are often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals and overcoming the host cell oxidative burst. We investigated the role of pyomelanin in Burkholderia cenocepacia strains J2315 (pigmented) and K56-2 (non-pigmented) and performed allelic exchange to generate isogenic non-pigmented and pigmented strains, respectively. Our results indicate that the altered pigment phenotype does not significantly impact these strains\' ability to resist H2O2 or NO in vitro and did not alter the outcome of a respiratory infection in CGD mice in vivo. These results suggest that pyomelanin may not always constitute a virulence factor and suggest that other features are contributing to the pathophysiology of these strains.

Microorganisms in the waterline zone can secrete pigments to avoid damage caused by ultraviolet radiation, some of which have corrosive effects. In this work, we found that the secretion of pyomelanin by P3 strain of Pseudoalteromonas lipolytica significantly increases under strong lighting conditions, accelerating the corrosion of the material. Molecular mechanisms indicate that strong light, as a stressful environmental factor, enhances the expression of melanin secretion-related genes to prevent bacteria from being damaged by ultraviolet radiation. Therefore, this work proposes a new corrosion mechanism in the waterline zone, pigment-producing microorganisms are also involved in the waterline corrosion process.

Considering the increasing interest in understanding the biotic component of methane removal from our atmosphere, it becomes essential to study the physiological characteristics and genomic potential of methanotroph isolates, especially their traits allowing them to adapt to elevated growth temperatures. The genetic signatures of Methylocaldum species have been detected in many terrestrial and aquatic ecosystems. A small set of representatives of this genus has been isolated and maintained in culture. The genus is commonly described as moderately thermophilic, with the growth optimum reaching 50 °C for some strains. Here, we present a comparative analysis of genomes of three Methylocaldum strains-two terrestrial M. szegediense strains (O-12 and Norfolk) and one marine strain, Methylocaldum marinum (S8). The examination of the core genome inventory of this genus uncovers significant redundancy in primary metabolic pathways, including the machinery for methane oxidation (numerous copies of pmo genes) and methanol oxidation (duplications of mxaF, xoxF1-5 genes), three pathways for one-carbon (C1) assimilation, and two methods of carbon storage (glycogen and polyhydroxyalkanoates). We also investigate the genetics of melanin production pathways as a key feature of the genus.

Helicobacter pylori (H. pylori) infection is the most common cause of chronic gastritis, peptic ulcers and gastric cancer. Successful colonization of the stomach by H. pylori is related to the complex interactions of these bacteria and its components with host cells. The growing antibiotic resistance of H. pylori and various mechanisms of evading the immune response have forced the search for new biologically active substances that exhibit antibacterial properties and limit the harmful effects of these bacteria on gastric epithelial cells and immune cells. In this study, the usefulness of pyomelanin (PyoM) produced by Pseudomonas aeruginosa for inhibiting the metabolic activity of H. pylori was evaluated using the resazurin reduction assay, as well as in vitro cell studies used to verify the cytoprotective, anti-apoptotic and pro-regenerative effects of PyoM in the H. pylori LPS environment. We have shown that both water-soluble (PyoMsol) and water-insoluble (PyoMinsol) PyoM exhibit similar antibacterial properties against selected reference and clinical strains of H. pylori. This study showed that PyoM at a 1 μg/mL concentration reduced H. pylori-driven apoptosis and reactive oxygen species (ROS) production in fibroblasts, monocytes or gastric epithelial cells. In addition, PyoM enhanced the phagocytosis of H. pylori. PyoMsol showed better pro-regenerative and immunomodulatory activities than PyoMinsol.

Melanins are complex pigments with various biological functions and potential applications in space exploration and biomedicine due to their radioprotective properties. Aspergillus niger, a fungus known for its high radiation resistance, is widely used in biotechnology and a candidate for melanin production. In this study, we investigated the production of fungal pyomelanin (PyoFun) in A. niger by inducing overproduction of the pigment using L-tyrosine in a recombinant ΔhmgA mutant strain (OS4.3). The PyoFun pigment was characterized using three spectroscopic methods, and its antioxidant properties were assessed using a DPPH-assay. Additionally, we evaluated the protective effect of PyoFun against non-ionizing radiation (monochromatic UV-C) and compared its efficacy to a synthetically produced control pyomelanin (PyoSyn). The results confirmed successful production of PyoFun in A. niger through inducible overproduction. Characterization using spectroscopic methods confirmed the presence of PyoFun, and the DPPH-assay demonstrated its strong antioxidant properties. Moreover, PyoFun exhibited a highly protective effect against radiation-induced stress, surpassing the protection provided by PyoSyn. The findings of this study suggest that PyoFun has significant potential as a biological shield against harmful radiation. Notably, PyoFun is synthesized extracellularly, differing it from other fungal melanins (such as L-DOPA- or DHN-melanin) that require cell lysis for pigment purification. This characteristic makes PyoFun a valuable resource for biotechnology, biomedicine, and the space industry. However, further research is needed to evaluate its protective effect in a dried form and against ionizing radiation.

Bacteria are the source of many bioactive compounds, including polymers with various physiological functions and the potential for medical applications. Pyomelanin from Pseudomonas aeruginosa, a nonfermenting Gram-negative bacterium, is a black-brown negatively charged extracellular polymer of homogentisic acid produced during L-tyrosine catabolism. Due to its chemical properties and the presence of active functional groups, pyomelanin is a candidate for the development of new antioxidant, antimicrobial and immunomodulatory formulations. This work aimed to obtain bacterial water-soluble (Pyosol), water-insoluble (Pyoinsol) and synthetic (sPyo) pyomelanin variants and characterize their chemical structure, thermosensitivity and biosafety in vitro and in vivo (Galleria mallonella). FTIR analysis showed that aromatic ring connections in the polymer chains were dominant in Pyosol and sPyo, whereas Pyoinsol had fewer Car-Car links between rings. The differences in chemical structure influence the solubility of various forms of pyomelanins, their thermal stability and biological activity. Pyosol and Pyoinsol showed higher biological safety than sPyo. The obtained results qualify Pyosol and Pyoinsol for evaluation of their antimicrobial, immunomodulatory and proregenerative activities.

BACKGROUND: Antarctica has one of the most extreme environments in the world. This region is inhabited by specifically adapted microorganisms that produce various unique secondary metabolites (e.g. pigments) enabling their survival under the harsh environmental conditions. It was already shown that these natural, biologically active molecules may find application in various fields of biotechnology.
RESULTS: In this study, a cold-active brown-pigment-producing Pseudomonas sp. ANT_H4 strain was characterized. In-depth genomic analysis combined with the application of a fosmid expression system revealed two different pathways of melanin-like compounds biosynthesis by the ANT_H4 strain. The chromatographic behavior and Fourier-transform infrared spectroscopic analyses allowed for the identification of the extracted melanin-like compound as a pyomelanin. Furthermore, optimization of the production and thorough functional analyses of the pyomelanin were performed to test its usability in biotechnology. It was confirmed that ANT_H4-derived pyomelanin increases the sun protection factor, enables scavenging of free radicals, and interacts with the iron from minerals. Moreover, it was shown for the first time that pyomelanin exhibits priming properties toward Calendula officinalis hairy roots in in vitro cultures.
CONCLUSIONS: Results of the study indicate the significant biotechnological potential of ANT_H4-derived pyomelanin and open opportunities for future applications. Taking into account protective features of analyzed pyomelanin it may be potentially used in medical biotechnology and cosmetology. Especially interesting was showing that pyomelanin exhibits priming properties toward hairy roots, which creates a perspective for its usage for the development of novel and sustainable agrotechnical solutions.

Shewanella oneidensis is the best understood model microorganism for the study of diverse cytochromes (cytos) c that support its unparallel respiratory versatility. Although RNA chaperone Hfq has been implicated in regulation of cyto c production, little is known about the biological pathways that it affects in this bacterium. In this study, from a spontaneous mutant that secretes pyomelanin and has a lowered cyto c content, we identified Hfq to be the regulator that critically associates with both phenotypes in S. oneidensis. We found that expression of the key genes in biosynthesis and degradation of heme is differentially affected by Hfq at under- and overproduced levels, and through modulating heme levels, Hfq influences the cyto c content. Although Hfq in excess results in overproduction of the enzymes responsible for both generation and removal of homogentisic acid (HGA), the precursor of pyomelanin, it is compromised activity of HmgA that leads to excretion and polymerization of HGA to form pyomelanin. We further show that Hfq mediates HmgA activity by lowering intracellular iron content because HmgA is an iron-dependent enzyme. Overall, our work highlights the significance of Hfq-mediated posttranscriptional regulation in the physiology of S. oneidensis, unraveling unexpected mechanisms by which Hfq affects cyto c biosynthesis and pyomelanin production. IMPORTANCE In bacteria, Hfq has been implicated in regulation of diverse biological processes posttranslationally. In S. oneidensis, Hfq affects the content of cytos c that serve as the basis of its respiratory versatility and potential application in bioenergy and bioremediation. In this study, we found that Hfq differentially regulates heme biosynthesis and degradation, leading to altered cyto c contents. Hfq in excess causes a synthetic effect on HmgA, an enzyme responsible for pyomelanin formation. Overall, the data presented manifest that the biological processes in a given bacterium regulated by Hfq are highly complex, amounting to required coordination among multiple physiological aspects to allow cells to respond to environmental changes promptly.

4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of β-triketone herbicides in plants. This enzyme, involved in the tyrosine pathway, is also present in a wide range of living organisms, including microorganisms. Previous studies, focusing on a few strains and using high herbicide concentrations, showed that β-triketones are able to inhibit microbial HPPD. Here, we measured the effect of agronomical doses of β-triketone herbicides on soil bacterial strains. The HPPD activity of six bacterial strains was tested with 1× or 10× the recommended field dose of the herbicide sulcotrione. The selected strains were tested with 0.01× to 15× the recommended field dose of sulcotrione, mesotrione, and tembotrione. Molecular docking was also used to measure and model the binding mode of the three herbicides with the different bacterial HPPD. Our results show that responses to herbicides are strain-dependent with Pseudomonas fluorescens F113 HPPD activity not inhibited by any of the herbicide tested, when all three β-triketone herbicides inhibited HPPD in Bacillus cereus ATCC14579 and Shewanella oneidensis MR-1. These responses are also molecule-dependent with tembotrione harboring the strongest inhibitory effect. Molecular docking also reveals different binding potentials. This is the first time that the inhibitory effect of β-triketone herbicides is tested on environmental strains at agronomical doses, showing a potential effect of these molecules on the HPPD enzymatic activity of non-target microorganisms. The whole-cell assay developed in this study, coupled with molecular docking analysis, appears as an interesting way to have a first idea of the effect of herbicides on microbial communities, prior to setting up microcosm or even field experiments. This methodology could then largely be applied to other family of pesticides also targeting an enzyme present in microorganisms.