Microbial transformation of cadina-4,10(15)-dien-3-one, aromadendr-1(10)-en-9-one and methyl ursolate by Mucor plumbeus ATCC 4740

The sesquiterpenes cadina-4,10(15)-dien-3-one (1) and aromadendr-1(10)-en-9-one (squamulosone) (14) along with the triterpenoid methyl ursolate (21) were incubated with the fungus Mucor plumbeus ATCC 4740. Substrates 1, 14 and ursolic acid (20) were isolated from the plant Hyptis verticillata in large quantities. M. plumbeus hydroxylated 1 at C-12 and C-14. When the iron content of the medium was reduced, however, hydroxylation at these positions was also accompanied by epoxidation of the exocyclic double bond. In total nine new oxygenated cadinanes have been obtained. Sesquiterpene 14 was converted to the novel 2α,13-dihydroxy derivative along with four other metabolites. Methyl ursolate (21) was transformed to a new compound, methyl 3β,7β,21β-trihydroxyursa-9(11),12-dien-28-oate (22). Two other triterpenoids, 3β,28-dihydroxyurs-12-ene (uvaol) (23) and 3β,28-bis(dimethylcarbamoxy)urs-12-ene (24) were not transformed by the micro-organism, however. © 2002 Elsevier Science Ltd. All rights reserved.

The Biotransformation of Pentachlorophenol by Mucor plumbeus: Mechanistic Insights

Dissertation presented to obtain the Ph.D degree in Biochemistry

Effect of fungal colonization on mechanical performance of cork

The industrialization of traditional processes relies on the scientific ability to understand the empirical evidence associated with traditional knowledge. Cork manufacturing includes one operation known as stabilization, where humid cork slabs are extensively colonized by fungi. The implications of fungal growth on the chemical quality of cork through the analysis of putative fungal metabolites have already been investigated. However, the effect of fungal growth on the mechanical properties of cork remains unexplored. This study investigated the effect of cork colonization on the integrity of the cork cell walls and their mechanical performance. Fungal colonization of cork by Chrysonilia sitophila, Mucor plumbeus Penicillium glabrum, P. olsonii, and Trichoderma longibrachiatum was investigated by microscopy. Growth occurred primarily on the surface of the cork pieces, but mycelium extended deeper into the cork layers, mostly via lenticular channels and by hyphal penetration of the cork cell wall. In this first report on cork decay in which specific correlation between fungal colonization and mechanical proprieties of the cork has been investigated, all colonizing fungi except C. sitophila, reduced cork strength, markedly altering its viscoelastic behaviour and reducing its Young’s modulus.

Screening pentachlorophenol degradation ability by environmental fungal strains belonging to the phyla Ascomycota and Zygomycota

Pentachlorophenol (PCP) bioremediation by the fungal strains amongst the cork- colonising community has not yet been analysed. In this paper, the co- and direct metabolism of PCP by each of the 17 fungal species selected from this community were studied. Using hierarchical data analysis, the isolates were ranked by their PCP bioremediation potential. Fifteen isolates were able to degrade PCP under co-metabolic conditions, and surprisingly Chrysonilia sitophila, Trichoderma longibrachiatum, Mucor plumbeus, Penicillium janczewskii and P. glandicola were able to directly metabolise PCP, leading to its complete depletion from media. PCP degradation intermediates are preliminarily discussed. Data emphasise the signiWcance of these fungi to have an interesting potential to be used in PCP bioremediation processes.

Biotransformation using Mucor rouxii for the production of oleanolic acid derivatives and their antimicrobial activity against oral pathogens

The goal of this study is to produce oleanolic acid derivatives by biotransformation process using Mucor rouxii and evaluate their antimicrobial activity against oral pathogens. The microbial transformation was carried out in shake flasks at 30A degrees C for 216 h with shaking at 120 rpm. Three new derivatives, 7 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, 7 beta,21 beta-dihydroxy-3-oxo-olean-12-en-28-oic acid, and 3 beta,7 beta,21 beta-trihydroxyolean-12-en-28-oic acid, and one know compound, 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, were isolated, and the structures were elucidated on the basis of spectroscopic analyses. The antimicrobial activity of the substrate and its transformed products was evaluated against five oral pathogens. Among these compounds, the derivative 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid displayed the strongest activity against Porphyromonas gingivalis, which is a primary etiological agent of periodontal disease. In an attempt to improve the antimicrobial activity of the derivative 21 beta-hydroxy-3-oxo-olean-12-en-28-oic acid, its sodium salt was prepared, and the minimum inhibitory concentration against P. gingivalis was reduced by one-half. The biotransformation process using M. rouxii has potential to be applied to the production of oleanolic acid derivatives. Research and antimicrobial activity evaluation of new oleanolic acid derivatives may provide an important contribution to the discovery of new adjunct agents for treatment of dental diseases such as dental caries, gingivitis, and periodontitis.

The isolation and characterization of cytochrome c nitrite reductase subunits (NrfA and NrfH) from Desulfovibrio desulfuricans ATCC 27774

Eur. J. Biochem. 270, 3904–3915 (2003)

Cytochrome c nitrite reductase from desulfovibrio desulfuricans ATCC 27774

The Journal of Biological Chemistry Vol. 278, No. 19, Issue of May 9, pp. 17455–17465, 2003

Spectroscopic characterization of a novel 2 /[4Fe /4S] ferredoxin isolated from Desulfovibrio desulfuricans ATCC 27774

Inorganica Chimica Acta 356 (2003) 215-221

Estudo de novos centros contendo ferro: isolamento e caracterização de proteínas de desulfovibrio desulfuricans ATCC 27774

Dissertação apresentada para obtenção do Grau de Doutor em Bioquímica, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Development of electrochemical nitrite biosensors using cytochrome c nitrite reductase from Desulfovibrio desulfuricans ATCC 27774

Dissertação apresentada para a obtenção do Grau de Doutor em Química Sustentável pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Jul 1;64(Pt 7):593-5

EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

J Biol Inorg Chem (2006) 11: 609–616 DOI 10.1007/s00775-006-0110-0

The isolation and characterization of cytochrome c nitrite reductase subunits (NrfA and NrfH) from Desulfovibrio desulfuricans ATCC 27774

Eur. J. Biochem. 270, 3904–3915 (2003) doi:10.1046/j.1432-1033.2003.03772.x

Proteómica microbiana: análise comparativa da bactéria redutora de sulfato Desulfovibrio desulfuricans ATCC 27774 e das actinobactérias Salinispora arenicola e Salinispora pacifica

As bactérias redutoras de sulfato (BRS) são um grupo diversificado de micro-organismos anaeróbios que obtêm energia a partir da redução dissimilativa do sulfato. Algumas espécies de BRS possuem versatilidade a nível respiratório, como é o caso de Desulfovibrio desulfuricans ATCC 27774, devido à utilização de aceitadores finais de eletrões alternativos. Neste contexto, o objetivo da primeira parte desta dissertação consistiu na identificação das ferramentas metabólicas (proteínas) envolvidas na flexibilidade respiratória desta bacteria induzidas por diferentes aceitadores de eletrões (nitrato vs sulfato). Assim, os extratos proteicos totais de células de D. desulfuricans crescidas em meios VMN com nitrato ou sulfato, foram analisados por eletroforese bidimensional (2DE), num gradiente de pH 4 - 7. Nas condições experimentais testadas, foram observados 604 e 519 spots de proteínas nos géis de células crescidas em meio contendo nitrato ou sulfato, respetivamente. Pela avaliação estatística foi possível observar aproximadamente 25 % de spots diferenciais. Os resultados obtidos sugerem que na presença de nitrato, a bactéria não só cresce mais rapidamente e com maior rendimento, como também produz uma maior quantidade de proteínas. Estes dados foram relacionados com a adaptação de D. desulfuricans ao substrato respiratório alternativo. Tal como esperado, nos ensaios das atividades enzimáticas das redutases do nitrito e do nitrato, foi possível observar maior atividade nos extratos das células crescidas em nitrato do que em sulfato. As actinobactérias marinhas do género Salinispora, têm vindo a ser exploradas como fontes de biofármacos naturais. Assim, na segunda parte deste trabalho, realizou-se um estudo preliminar, que pretendeu caracterizar as proteínas envolvidas na biossíntese destes compostos bioactivos em S. arenicola e de S. pacifica. Para tal, foi utilizada uma abordagem proteómica diferencial, baseada em 2DE. Surpreendentemente, os perfis proteicos das duas espécies mostraram-se bastante distintos, tendo-se identificado apenas 37 spots comuns entre os 650 observados no gel de S. arenicola e 510 no gel de S. pacifica.

Real-time antifungal susceptibility testing of Mucor spp., Fusarium spp. and Scedosporium spp. by isothermal microcalorimetry

Objective: Aspergillus species are the main pathogens causing invasive fungal infections but the prevalence of other mould species is rising. Resistance to antifungals among these new emerging pathogens presents a challenge for managing of infections. Conventional susceptibility testing of non-Aspergillus species is laborious and often difficult to interpret. We evaluated a new method for real-time susceptibility testing of moulds based on their of growth-related heat production.Methods: Laboratory and clinical strains of Mucor spp. (n = 4), Scedoporium spp. (n = 4) and Fusarium spp. (n = 5) were used. Conventional MIC was determined by microbroth dilution. Isothermal microcalorimetry was performed at 37 C using Sabouraud dextrose broth (SDB) inoculated with 104 spores/ml (determined by microscopical enumeration). SDB without antifungals was used for evaluation of growth characteristics. Detection time was defined as heat flow exceeding 10 lW. For susceptibility testing serial dilutions of amphotericin B, voriconazole, posaconazole and caspofungin were used. The minimal heat inhibitory concentration (MHIC) was defined as the lowest antifungal concentration, inhbiting 50% of the heat produced by the growth control at 48 h or at 24 h for Mucor spp. Susceptibility tests were performed in duplicate.Results: Tested mould genera had distinctive heat flow profiles with a median detection time (range) of 3.4 h (1.9-4.1 h) for Mucor spp, 11.0 h (7.1-13.7 h) for Fusarium spp and 29.3 h (27.4-33.0 h) for Scedosporium spp. Graph shows heat flow (in duplicate) of one representative strain from each genus (dashed line marks detection limit). Species belonging to the same genus showed similar heat production profiles. Table shows MHIC and MIC ranges for tested moulds and antifungals.Conclusions: Microcalorimetry allowed rapid detection of growth of slow-growing species, such as Fusarium spp. and Scedosporium spp. Moreover, microcalorimetry offers a new approach for antifungal susceptibility testing of moulds, correlating with conventional MIC values. Interpretation of calorimetric susceptibility data is easy and real-time data on the effect of different antifungals on the growth of the moulds is additionally obtained. This method may be used for investigation of different mechanisms of action of antifungals, new substances and drug-drug combinations.