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BIOACTIVE COMPOUNDS FROM CYANOBACTERIA: A BIOCHEMICAL AND MOLECULAR CHARACTERIZATION
Maria Estela Silva-Stenico1; Luiz Alberto Beraldo de Moraes2; Augusto Etchegaray3; Caroline Souza Pamplona da Silva1; Omar Teschke4; Marli de Fátima Fiore1
1Centro de Energia Nuclear na Agricultura, CENA/USP, Lab. Biologia Celular e Molecular, C.P. 96, 13400-970, Piracicaba-SP;
2Universidade de São Paulo - Departamento de Química - Av. Bandeirantes, 3900 Ribeirão Preto SP;
3Faculdade de Química, PUC-Campinas, Caixa Postal 317, 13012-970 Campinas SP; 4Laboratório de Nanoestruturas e Interfaces, Instituto de Física, UNICAMP, 13083-970, Campinas, SP, Brazil.
The increasing bacterial resistance to antibiotics has intensified the search for novel antimicrobial compounds. Cyanobacteria produce numerous and structurally diverse secondary metabolites, in particular nonribosomal peptide (NRP) and polyketide (PK) structure and some may prove useful for development into commercial drugs. The aim of this work was to investigate the presence of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes in cyanobacteria and characterize their biosynthetic products. These large multifunctional enzymes are known to be involved in the biosynthesis of a number of antibiotics in other bacteria and fungi. Cyanobacterial strains were selected by the presence of NRPS and PKS genes. After genomic DNA extraction from several cyanobacterial strains from CENA culture collection, PCR amplification was carried out using specific primers for NRPS (MTF/MTR) and PKS (KSF/KSR). In this way, two cyanobacterial strains, Microcystis aeruginosa NPCD-1 and Synechococcus sp PCC7942, were selected for further studies. These cyanobacteria were inoculated in specific liquid media for 30 d at 23oC under continuous illumination (40 µmol photons m-2 s-1). Metabolites were extracted from the culture media using ethyl acetate and chloroform followed by extracts concentration. Intracellular compounds were obtained with methanol extraction after cell disruption. Organic extracts were investigated for the presence of bioactive compounds, including siderophores and antibiotics. The results showed inhibitory activity against Salmonella typhimurium, Bacillus subtilis and Candida kruzeii and the production of siderophore by Synechococcus sp PCC7942. Analysis using thin layer chromatography (TLC) revealed the presence of bioactive compounds. Their molar masses were achieved by mass spectrometry analysis and revealed peptides of 680 Da for NPCD-1 and 609 Da for PCC7942. According to the database these peptides are classified as different variants of aeruginosin. The peptide effect against B. subtilis cells was analyzed by atomic force microscopy (AFM). The AFM images showed that the cyanobacterial extract containing bioactive compound caused damages in the B. subtilis cell wall.
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