Microbial marine communities diversity : from culture to function

For cultivation of picocyanobacteria from the North Sea, ASN-3 medium was modified by lowering the level of nitrate to 20% of the original receipe and add 0.2 mM sodium-silicate. For the Baltic Sea a mixture of ASN-3 and BG-11 was used to obtain a salinity of 0.6%. Nitrate was omitted and in stead 1 mM of ammonium-chloride was added. For enrichment of N2-fixing microorganisms combined nitrogen was omitted.

All the cytotoxic extracts obtained from the fermentation broths of the bacteria isolated during the miracle project were subjected to a workflow that consisted in the following steps: - A mass-spectrometry de-replication. All the active extracts were subjected to 4 different analysis, electrospray positive, electrospray negative, atmospheric pressure chemical ionisation (APCI) positive and APCI negative. The spectra obtained were compared to a database generated with 304 different compounds previously isolated at I. Biomar. All the samples that contained previously isolated compounds were not further studied. The 71 active actinobacteria isolated during the Miracle at I. Biomar were subjected to this de-replication procedure and 44 extracts were discarded for containing known compounds. The compounds detected at this point were: Antimycin (in 29 extracts), Nocardamine (in 15 extracts), Geldanamycin (1 extract), Ikarugamycin (1 extract), Bafilomycin (1 extract) and different types of Steroids (3 extracts). We received 595 strains from our partners in this project and 47 showed activity. This work is still in progress with the last strains received but so far some metabolites have been identified: fatty acids, Isocoumarins, Antimycin, Actinomycin and several Macrolides. - The next step was the scaled-up fermentation of the active strains in which the active compound was not identified. A new mass-spectrometry de-replication was performed at this stage but it was carried out after separation of the solid and liquid phases of the fermentation broth. A different set of solvents is used too. The fermentation was performed in 2 liter flasks containing 250 ml of fermentation broth and with at least two passages of inoculum previously to inoculation of the fermentor. Some other compounds were detected in this second step, that includes Ergosterol, several other Steroids, Genistein, Daidzein, and Antimycins. - The third step was the fermentation at higher scales for the product isolation. 7 strains were subjected to fermentation in higher volumes (typically in multiples of 4 liter) and only two still remain in study. The rest have shown to produce known compounds all of them included in previous reports. Additionally, there is still work with one cyanobacteria that showed good cytotoxic activity. The progress with this kind of organisms is much slower since the growth of enough quantity of cell mass for the purification of the active compound requires very long incubation periods.

The Department of Marine Microbiology (NIOO-KNAW) maintains a collection of marine phototrophic microorganisms. This Culture Collection Yerseke (CCY) contains currently 300 cyanobacteria, 150 diatoms and 50 other algae. The majority of these strains are unique and have been isolated by the members of the Department. Most of the strains are available as axenic (bacteriological pure) cultures. A detailed (genetic) characterization of each of the strains is in progress. An internet webpage including a catalogue of the strains is in preparation. The CCY possesses extensive experience in the isolation and purification of phototrophic microorganisms, optimization of culture conditions and medium composition. For each of the cultrues protocolls are being developed for cryo-preservation of the cultures. The cultures are cryo-preserved in a special containiner in the vapour of liquid nitrogen. The genomes of two of the CCY cultures have been completely sequenced and that of two other strains is currently in progress. All strains are available to the public on request.

Instituto Biomar, S.A. carried out the fermentation of a group of strains isolated either at I. Biomar or by some of the partners. The distribution of the strains among the different collaborating groups was as follows: Instituto Biomar (351 strains), University of Barcelona (79), Plymouth Marine Laboratory (385), University of Bergen (144), University Miguel Hernández (4), and the Netherlands Institute of Ecology (15). The strains were fermented in different media depending on whether they were actinomycetes or not. This first fermentation was carried out in flask at a small scale (30 or 40 ml). All the broths were freeze-dried and then extracted with a solvent mixture design to extract metabolites in a broad range of polarities. Aliquots of these extracts were analysed for their cytotoxic activity while the rest was kept in store for chemical de-replication studies. This extract collection was the starting point for the detection of compounds having potential economic interest. The most relevant activity addressed in the Miracle project was the antitumor (cytotoxic) activity, but other activities will be studied in the future. The antitumor (cytotoxic) activity was assayed against four tumor cell lines. The tumor lines were A549 (lung cancer), H116 (colon), PSN1 (pancreas) and T98G (glyoma). The activity was measured by the MTT proliferation assat.

Micro-Mar is a database for dynamic representation of marine microbial biodiversity. It is an ambitious project that aims to become a virtual and interactive book of marine microbiology (although restricted to prokaryotes by now). We would like above all that it would be useful for the growing community of marine microbiologists and help in integrating all the information that is often published without being properly incorporated into the mainstream of marine microbiology. This database will be justified (like any other) to the extent that it is useful and is used. We can modify it and adjust it as demands and criticisms are raised by users. With a little help from all of you we can just succeed and maybe one day we will start to get the big picture of the oceans of microbes.

This collection of environmental sequences encompasses in total 434 sequences obtained from clone libraries. At this moment 14 clone libraries are still under construction and this work is in progress. In total 280 unique seciences were obtained. From the North Sea 159 sequences of the 16S rRNA gene have been obtained of which 76 were unique and included 53 cyanobacterial sequences and 23 heterotrophic bacteria. From the Ionian Sea (Mediterranean) 129 sequences of the 16S rRNA gene were obtained of which 82 were unique, including 27 from cyanobacteria, 47 from heterotrophic bacteria and 8 from eukaryotic plastids. From the Baltic Sea 49 16S rRNA gene sequences were obtained, all of which were unique and included 42 cyanobacterial sequences and 7 of heterotrophic bacteria. Further, 39 sequences were obtained for the phycoerythrin operon (cpeBA) from the Baltic Sea, all of which were unique and from cyanobacteria. A clone library of cDNA of transcripts of nifH (structural gene of nitrogenase, the N2-fixing enzyme) resulted in 39 sequences, all unique and belonging to cyanobacteria. The sequences will be submitted to GeneBank.

The following set of primers have been used for PCR of environmental DNA samples and for subsequent construction of clone libraries of phototrophic microorganisms. For the 16S rRNA gene and the intergenic transcribed spacer (ITS): Mir-PA8F and Cya23S-58R. For 16S rRNA gene: p16S 621cyanF and p16S 1438CyanR. For ITS-1: Mir-Bac1055F and PITS E Cyan R. For cpeBA either CyaCpe-F1 and CyaCpe-R1 or B3FW and SynA1R (M. Wood). For nifH a nested PCR with Yanni and nif 450 and nif down and nif up (J. Zehr). Primer sequences will be published in the primary literature.

A total of 99 phototrophic microorganisms has been isolated and obtained as pure (axenic), clonal cultures. The cultures have been obtained from the North Sea (20) and from the Baltic Sea (79). From the North Sea 14 picocyanobacteria (Synechococcus) were isolated of which 12 possessed the photopigment phycoerythrin (red coloured strains)and 2 contained only phycocyanin (blue-green coloured strains). In addition 6 picoeukaryotes (pale green) were obtained. From the Baltic Sea 46 picocyanobacteria (Synechococcus) were isolated of which 36 contain phycoerythrin (red strains) and 10 only phycocyanin (blue-green strains). Further, 25 tiny filamentous strains (< 1 um) of the genus Pseudanabaena were isolated. These strains contained phycoerythrin, phycocyanin or both and they appeared in a range of different colours. These strains were capable of complementary chromatic adaptation. In addition 8 picoeukaryotes (pale green) were obtained. Full characterization is in progress. The strains are deposited in the Culture Collection Yerseke (CCY) and publicy available.

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