Actinomadura graeca sp. nov.: A novel producer of the macrocyclic antibiotic zelkovamycin.

As part of a screening programme for antibiotic-producing bacteria, a novel Actinomadura species was discovered from a soil sample collected in Santorini, Greece. Preliminary 16S rRNA gene sequence comparisons highlighted Actinomadura macra as the most similar characterised species. However, whole-genome sequencing revealed an average nucleotide identity (ANI) value of 89% with A. macra, the highest among related species. Further phenotypic and chemotaxonomic analyses confirmed that the isolate represents a previously uncharacterised species in the genus Actinomadura, for which the name Actinomadura graeca sp. nov. is proposed (type strain 32-07T). The G+C content of A. graeca 32-07 is 72.36%. The cell wall contains DL-diaminopimelic acid, intracellular sugars are glucose, ribose and galactose, the predominant menaquinone is MK-9(H6), the major cellular lipid is phosphatidylinositol and fatty acids consist mainly of hexadecanoic acid. No mycolic acid was detected. Furthermore, A. graeca 32-07 has been confirmed as a novel producer of the non-ribosomal peptide antibiotic zelkovamycin and we report herein a provisional description of the unique biosynthetic gene cluster.

Bacterial diversity from benthic mats of Antarctic lakes as a source of new bioactive metabolites.

During the MICROMAT project, the bacterial diversity of microbial mats growing in the benthic environment of Antarctic lakes was accessed for the discovery of novel antibiotics. In all, 723 Antarctic heterotrophic bacteria belonging to novel and/or endemic taxa in the α-, ß- and γ-subclasses of the Proteobacteria, the Bacteroidetes branch, and of the high and low percentage G+C Gram-positives, were isolated, cultivated in different media and at different temperatures, and then screened for the production of antimicrobial activities. A total of 6348 extracts were prepared by solid phase extraction of the culture broths or by biomass solvent extraction. 122 bacteria showed antibacterial activity against the Gram-positives Staphylococcus aureus and to a lower extent Enterococcus faecium, and versus the Gram-negative Escherichia coli. Few of these strains showed also some antifungal activity against Cryptococcus neoformans, Aspergillus fumigatus and to a lower extent Candida albicans. LC-MS fractionation of extracts from a subset of strains (hits) that exhibited relatively potent antibacterial activities evidenced a chemical novelty that was further investigated. Two strains of Arthrobacter agilis produced potent antibacterial compounds with activity against Gram-positives and possibly related to novel cyclic thiazolyl peptides. To our knowledge, this is the first report of new antibiotics produced by bacteria from benthic microbial mats from Antarctic lakes. With no doubts these microbial assemblages represent an extremely rich source for the isolation of new strains producing novel bioactive metabolites with the potential to be developed as antibiotic compounds.

Diversity and pharmaceutical screening of fungi from benthic mats of Antarctic lakes.

During the MICROMAT project, the fungal diversity of microbial mats growing in the benthic environment of Antarctic lakes was accessed for the discovery of novel antibiotics and anticancers. In all, 160 filamentous fungi belonging to fifteen different genera and 171 yeasts were isolated from 11 lakes, classified and cultivated in different media and at different temperatures. Filamentous fungi were then screened to discover novel antimicrobial and cytotoxic compounds. A total of 1422 extracts were prepared by solid phase extraction of the culture broths or by biomass solvent extraction. 47 (29%) filamentous fungi showed antimicrobial activity; most of them inhibited the growth of gram-positive Staphyloccus aureus (14%), gram-negative E. coli (10%), and of yeasts Candida albicans (11%) and Cryptococcus neoformans (8%). Less activity was detected against representatives of enterobacteria and filamentous fungi. The most productive in terms of bioactivities were cold-tolerant cosmopolitan hyphomycetes such as Penicillium, Aspergillus, Beauveria and Cladosporium. Two bioactive bis-anthraquinones (rugulosin and skyrin) were identified by LC-MS as the main products in a strain of Penicillium chrysogenum isolated from a saline lake in the Vestfold Hills. LC-MS fractionation of extracts from two diverse species of Aspergillus, that exhibited relatively potent antimicrobial activities, evidenced a chemical novelty that was further investigated. To our knowledge, this is the first report of new antibiotics produced by fungi from benthic microbial mats from Antarctic lakes. It can be concluded that these microbial assemblages represent an extremely rich source for the isolation of new strains producing novel bioactive metabolites with the potential to be developed as drugs.

Influence of leucine and valine on ramoplanin production by Actinoplanes sp. ATCC 33076.

Ramoplanin is a glycolipodepsipeptide antibiotic obtained by fermentation of the Actinoplanes sp. ATCC 33076, isolated as a complex of three closely related components A1, A2 and A3, which differ in their fatty acid moiety. We have investigated the influence of L-leucine and L-valine, the biosynthetic precursors of the fatty acids in A2 and A3 factors, on the complex composition and antibiotic productivity. Addition of 5 g/litre of L-leucine at the time of inoculation increases antibiotic production and improves the production of A2 factor, which represents the active principle component under clinical development. Addition of L-valine in the same conditions modifies the composition of the complex towards the A3 factor but does not improve total antibiotic productivity. A possible explanation for the different actions of the two amino acids is presented.