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Projeto de investigação
Fighting Staphylococcus aureus - Peptidoglycan amidation as a new target
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DES hidrofóbicos à base de mentol e ácidos orgânicos naturais para utilização em tintas náuticas antifouling
Publication . Valente, Sara Azedo; Duarte, Ana; Gaudêncio, Susana
A bioincrustação marinha tem afetado negativamente a indústria náutica e o meio ambiente tem sofrido com esta ação. Até ao momento, não existem soluções sustentáveis, económicas e ambientalmente benignas para lidar com este fenómeno. Os avanços alcançados em solventes verdes promovem o surgimento de solventes eutécticos (DES), que possuem excelentes aplicações em diversos campos e podem ser revolucionários neste sector da indústria náutica. Neste estudo, o principal objetivo foi investigar o potencial de DES hidrofóbicos à base de mentol e ácidos orgânicos naturais para utilização em tintas náuticas antifouling. A nossa estratégia resultou na caracterização físico-química de diferentes formulações, o que permitiu identificar a razão molar mais adequada e as interações intermoleculares por trás das formações bem-sucedidas dos DES. A miscibilidade destes DES na tinta náutica foi avaliada e bem-sucedida. O desempenho biológico dos sistemas foi estudado em termos da sua atividade antimicrobiana contra um painel de microrganismos. O sistema Men:OL (1:1) demonstrou ser mais promissor, tendo sido o escolhido nos testes subsequentes. Os seus benefícios em termos de custo-produção também contribuíram para esta escolha. O perfil citoxicológico deste DES foi avaliado para células HaCat e demonstrou não promover citotoxicidade relevante. Além disso, foi estudada a aplicação do sistema incorporado nas tintas referidas em estruturas náuticas, recorrendo a espécies marinhas para avaliação do seu efeito antifouling. O DES foi promissor no combate ao macrofouling marinho e potencializou as propriedades das tintas náuticas convencionais, fornecendo indicações sobre a sua utilidade como forte candidato para aplicação em tintas náuticas antifouling. Os ensaios bioquímicos realizados nas espécies marinhas demonstraram que o DES não induz stress oxidativo nas espécies testadas.
Screening of exopolysaccharides (EPS) and polyhydroxyalkanoates (PHA) from sediments from the Azores Archipelago, Canay Islands and Estremadura Spur
Publication . Batista, Maria Rita Nicolau; Torres, Cristiana; Gaudêncio, Susana
Oceans are still largely unexplored as a source of new and interesting biomolecules.
Among these, exopolysaccharides (EPS) and polyhydroxyalkanoates (PHA) are being widely
studied for their interesting biotechnological applications in various industrial fields. EPS are
produced by bacteria as a defense mechanism against biotic or abiotic stress, like low
temperatures or high salinity. PHA are also produced as a survival mechanism, but against
carbon shortage, they accumulate as intracellular granules, in order to be used as energy source
in the absence of some nutrients.
In this work, bacteria were isolated from sediments from the Azores Archipelago, Canary
Islands and Estremadura Spur and screened for the production of EPS and PHA. Growth assays
were performed in glucose, glycerol, fructose, xylose and lactose, as well as 16°C and 30°C, two
different nitrogen concentrations were also tested. The majority of the cultures grew better at
30°C, but produced more EPS at 16°C. All cultures presented fluorescence under the light
microscope, meaning they all produce PHA. Different cultures showed different preferences of
carbon source, and also the bacteria that produced more biomass, were not the one that
produced more EPS.
Three of the cultures with the most interesting results were cultivated in a bioreactor, at
standard operating conditions. From all of the bacteria, C3 showed the most balanced results of
EPS and PHA production, reaching biomass concentration of 2.013 g/L, a specific growth rate
of 0.370 h-1 and volumetric productivities of 78.89 mgEPS/L.h-1 and 40.83 mgPHA/L.h-1. Az4
was taxonomically identified a Bacillus sp., and presented similar growth results to culture C3,
but with lower biopolymer production. Az7 produced the highest biomass, but both productions
of polymer were low, also to notice that this culture produced a bright orange pigment, which
we could not yet identify.
Novel metabolite madeirone and neomarinone extracted from Streptomyces aculeoletus as marine antibiofilm and antifouling agents
Publication . Wissner, Julian L.; Almeida, Joana R.; Grilo, Inês R.; Oliveira, Jhenifer F.; Brízida, Carolina; Escobedo-Hinojosa, Wendy; Pissaridou, Panayiota; Vasquez, Marlen I.; Cunha, Isabel; Sobral, Rita G.; Vasconcelos, Vítor; Gaudêncio, Susana P.; UCIBIO - Applied Molecular Biosciences Unit; Frontiers Media
Introduction: Biofouling poses a significant economic threat to various marine industries, leading to financial losses that can reach billions of euros annually. This study highlights the urgent need for effective alternatives to traditional antifouling agents, particularly following the global ban on organotin compounds. Material and methods: Streptomyces aculeolatus PTM-346 was isolated from sediment samples on the shores of the Madeira Archipelago, Portugal. The crude extract was fractionated using silica flash chromatography and preparative HPLC, resulting in two isolated marinone compounds: madeirone (1), a novel marinone derivative discovered in this study, and neomarinone (2). The antifouling activities of these compounds were tested against five marine bacterial species and the larvae of the mussel Mytilus galloprovincialis. Additionally, in silico and in vivo environmental toxicity evaluations of madeirone (1) and neomarinone (2) were conducted. Results: Madeirone (1) demonstrated significant antibiofilm efficacy, inhibiting Phaeobacter inhibens by up to 66%, Marinobacter hydrocarbonoclasticus by up to 60%, and Cobetia marina by up to 40%. Neomarinone (2) also exhibited substantial antibiofilm activity, with inhibition rates of up to 41% against P. inhibens, 40% against Pseudo-oceanicola batsensis, 56% against M. hydrocarbonoclasticus, 46% against C. marina, and 40% against Micrococcus luteus. The growth inhibition activity at the same concentrations of these compounds remained below 20% for the respective bacteria, highlighting their effectiveness as potent antibiofilm agents without significantly affecting bacterial viability. Additionally, both compounds showed potent effects against the settlement of Mytilus galloprovincialis larvae, with EC50 values of 1.76 µg/mL and 0.12 µg/mL for compounds (1) and (2), respectively, without impairing the viability of the targeted macrofouling species. In silico toxicity predictions and in vivo toxicity assays both support their potential for further development as antifouling agents. Conclusion: The newly discovered metabolite madeirone (1) and neomarinone (2) effectively inhibit both micro- and macrofouling. This distinct capability sets them apart from existing commercial antifouling agents and positions them as promising candidates for biofouling prevention. Consequently, these compounds represent a viable and environmentally friendly alternative for incorporation into paints, primers, varnishes, and sealants, offering significant advantages over traditional copper-based compounds.
Analysis of a Cell Wall Mutant Highlights Rho-Dependent Genome Amplification Events in Staphylococcus aureus
Publication . Portela, Raquel; Faria, Nuno A.; Miragaia, Maria; Mwangi, Michael; de Lencastre, Hermínia; Tomasz, Alexander; Sobral, Rita G.; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; Molecular, Structural and Cellular Microbiology (MOSTMICRO); Instituto de Tecnologia Química e Biológica António Xavier (ITQB); American Society for Microbiology
In a study of antibiotic resistance in Staphylococcus aureus, specific cell wall mutants were previously generated for the peptidoglycan biosynthesis gene murF, by the insertion of an integrative plasmid. A collection of 30 independent mutants was obtained, and all harbored a variable number of copies of the inserted plasmid, arranged in tandem in the chromosome. Of the 30 mutants, only 3, F9, F20 and F26, with a lower number of plasmid copies, showed an altered peptidoglycan structure, lower resistance to β-lactams and a different loss-of-function mutation in rho gene, that encodes a transcription termination factor. The rho mutations were found to correlate with the level of oxacillin resistance, since genetic complementation with rho gene reestablished the resistance and cell wall parental profile in F9, F20 and F26 strains. Furthermore, complementation with rho resulted in the amplification of the number of plasmid tandem repeats, suggesting that Rho enabled events of recombination that favored a rearrangement in the chromosome in the region of the impaired murF gene. Although the full mechanism of reversion of the cell wall damage was not fully elucidated, we showed that Rho is involved in the recombination process that mediates the tandem amplification of exogeneous DNA fragments inserted into the chromosome
The diversity, metabolomics profiling, and the pharmacological potential of actinomycetes isolated from the estremadura spur pockmarks (Portugal)
Publication . Pinto-Almeida, António; Bauermeister, Anelize; Luppino, Luca; Grilo, Inês R.; Oliveira, Juliana; Sousa, Joana R.; Petras, Daniel; Rodrigues, Clara F.; Prieto-Davó, Alejandra; Tasdemir, Deniz; Sobral, Rita G.; Gaudêncio, Susana P.; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; MDPI - Multidisciplinary Digital Publishing Institute
The Estremadura Spur pockmarks are a unique and unexplored ecosystem located in the North Atlantic, off the coast of Portugal. A total of 85 marine-derived actinomycetes were isolated and cultured from sediments collected from this ecosystem at a depth of 200 to 350 m. Nine genera, Streptomyces, Micromonospora, Saccharopolyspora, Actinomadura, Actinopolymorpha, Nocardiopsis, Saccharomonospora, Stackebrandtia, and Verrucosispora were identified by 16S rRNA gene sequencing analyses, from which the first two were the most predominant. Non-targeted LC-MS/MS, in combination with molecular networking, revealed high metabolite diversity, including several known metabolites, such as surugamide, antimycin, etamycin, physostigmine, desferrioxamine, ikarugamycin, piericidine, and rakicidin derivatives, as well as numerous unidentified metabolites. Taxonomy was the strongest parameter influencing the metabolite production, highlighting the different biosynthetic potentials of phylogenetically related actinomycetes; the majority of the chemical classes can be used as chemotaxonomic markers, as the metabolite distribution was mostly genera-specific. The EtOAc extracts of the actinomycete isolates demonstrated antimicrobial and antioxidant activity. Altogether, this study demonstrates that the Estremadura Spur is a source of actinomycetes with potential applications for biotechnology. It highlights the importance of investigating actinomycetes from unique ecosystems, such as pockmarks, as the metabolite production reflects their adaptation to this habitat.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/BIA-MIC/31645/2017