Microbial Biodiversity Detection and Monitoring Using Molecular Biology Tools Teresa Lettieri Institute for Environment and Sustainability 2006 EUR 22600 EN The mission of the Institute for Environment and Sustainability is to provide scientific-technical support to the European Union’s Policies for the protection the environment and sustainable development of the European and global environment. European Commission Directorate-General Joint Research Centre Institute for Environment and Sustainability Contact information Address:via E. Fermi, 1,TP 300 E-mail: [email protected] Tel.: +39-0332789868 Fax: 39-0332789352 http://ies.jrc.cec.eu.int http://www.jrc.cec.eu.int Legal Notice Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of this publication. A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server http://europa.eu.int EUR 22600 EN ISSN 1018-5593 Luxembourg: Office for Official Publications of the European Communities © European Communities, 2006 Reproduction is authorised provided the source is acknowledged Printed in Italy CONTENTS INTRODUCTION 1 MICROBIAL WORLD 3 MOLECULAR BIOLOGY TOOLS 5 DNA Sequencing 5 DNA Cloning System 5 PCR and Real Time PCR 6 Hybridization Techniques 9 Immunofluorescence 10 FROM FIRST MICROBIAL GENOMICS TO MICROBIAL COMMUNITY 12 GENOMICS TOOLS 13 DNA Microarray Technology 13 Mass Spectrometry 16 Single Cell Genomic Sequencing 18 PUBLIC DATABASES 19 MICROBIAL COOMUNITIES: DYNAMIC CHANGES 21 CONCLUSIONS 23 REFERENCES 25 Introduction Micro-organisms are responsible for the most biogeochemical cycles that shape the environment of earth and its oceans. So far, only part of these organisms has been well studied, especially those living on earth and more considered from an anthropogenic perspective, e.g causing human diseases or providing useful products and services. Further the inability to generate pure culture has hampered the possibility to study and understand the metabolic processes of many micro- organisms. Recently, advances in molecular biology and -omics technologies are offering new and more exciting perspectives and knowledge of the microbial world, such as to understand the biological communities and function relationships, to identify the diversity of the population; to monitor the effects of environmental factors on the community structures. In March 2003 J. Craig Venter and coworkers have begun to explore environmental bacteria in a culture-independent manner by isolating DNA from directly the environmental sample, Sargasso Sea and then transforming into large insert clones for sequencing (1). The sequencing revealed the identification of new species. Indeed, molecular approaches have significantly influenced the understanding of the microbial diversity and ecology. In particular, ribosomal RNA (rRNA) gene sequence comparisons have provided a revolutionary approach for interpreting microbial evolutionary relationships (2). In a logical extension of this technique extraction of phylogenetically informative genes directly from naturally occurring represents another important development, in microbiology, opening up the natural microbial world to be closer scrutiny (3, 4 ). 1 Further advances in genome sequencing have had and still having a great impact on microbial biology providing insights into biochemistry, physiology and diversity. So far the full genome sequencing of many microorganisms, especially marine ones, have been completed and many others are underway. Combining the cultivation –independent gene sequences with the genomic approaches (e.g. whole genome shotgun, -omic technologies) it is now possible to investigate a more comprehensive natural microbial communities. These techniques, first applied to marine plankton to characterize uncultivated marine bacterial and archeal species (5) are now becoming a common method to characterize the community structure. Applications include the genome analysis of uncharacterized taxa (6, 7 ) expression of novel genes pathways from uncultured environmental microorganisms, elucidation of community-specific metabolism and comparison of different community gene contents. If from on one hand, we are discovering huge microbial diversity, on the other hand it is increasing concerns about the loss of biodiversity, especially caused by exploitation, pollution and habitat destruction, or indirectly through climate change and related perturbations of ocean biogeochemistry (8-10 ). The review will explore the development, application of the genomics technologies to the microbial biodiversity (microbial ecology) monitoring. I will first introduce the concept of microbial world, what we know so far and then facing with the molecular biology tools the transforming view of microbial diversity the (metagenomics, ecogenomics). The second section is dedicated to the applicability and to the illustration of examples of –omics technologies to the monitoring of microbial biodiversity either in sea/ocean or in freshwaters. This part will be introduced by -omics technologies 2 (DNA Microarray, Mass Spectrometry Maldi-TOFF and single cell genome sequencing) and by the availability of public databases. Microbial World The microbial world contains a highly heterogeneous group and they are distributed in four domains, Bacteria Archea, Eukaryota , and Viruses. In the Eukaryota, the micro-organisms are within the eukaryotic micro-organisms such as protozoa, algae and fungi. Viruses are the most abundant entities on the planet. Although they are not organisms in the sense as Eukaryotes, Archeans and Bacteria, are of considerable biological importance and, like bacteria, help cycle organic matter in the marine food web. In a more global vision we can define three classes in the ecosystem, the producers, decomposers and consumers. The producers are plants, algae, and autotrophic bacteria and archea (cyanobacteria), they acquire nutrients from inorganic sources (carbon dioxide) and water into organic compounds (photosynthesis), and usually it is referred to them as phytoplankton. The decomposers, in contrast, cannot acquire carbon from inorganic sources. But they can transform organic sources supplied by producers, either as exudates or as dead organic matter. They play a key role in the recycling of dissolved organic material (DOM), either transforming organic material into inorganic forms (mineralization, nutrients for producers), or contributing to the food chain since they are eaten by micro-flagellate and ciliate which then, are food for small fishes (zooplankton, first consumer), such process is also known as the microbial loop (Fig.1). In the microbial loop, the role of viruses is still unclear, especially the bacteriophage. The bacteriophages attack the bacteria, so they can influence the loop releasing more 3 dissolved organic matter (DOM) and at the same time, they can affect the bacteria community, then the microbial loop as well as the carbon cycle. Carbon (CO 2) + Nutrients Dissolved Organic Matter DOM ? Micro-flagellates Microbial Loop Viruses Bacteria ? Fig.1 Microbial Loop is shown. The carbon cycle and energy flow is controlled by the microbial world, either for the production (phytoplankton) or for the release of dissolved Organic matter in the cycle (heterotrophic bacteria, bacteria). It is still unknown the role of viruses as well as their influences. They attack bacteria and unicellular algae influencing the total biomass. 4 Molecular Biology Tools The genomic age began in 1977 when a virus that infects Escherichia coli was sequenced and with it the development of molecular biology techniques allowed the cloning of part of genome or single gene to be, then further characterized. At beginning of 1990, the high throughput DNA-sequencing technique and instruments boosted the sequencing of many other organisms opening the era of genomics and related technologies. I will describe in this paragraph technologies which were known before the genomic era. They deserve a separated paragraph since they have been founder of the new technologies as recognised by the scientific communities who awarded the authors with the Nobel Prize. DNA sequencing As mentioned above the basic principle of sequencing was established in the early ’70 by the work of Sanger (11 ) (Nobel Prize in 1980), it took almost twenty years to get an efficient and automatized system capable to use fluorescent dyes to tag the dideoxyribonculeotides with one colour of each of the four nucleotides (12 ). Furthermore faster and cheaper sequencing methods and equipment are continuously developed. For example, the recent pyrosequencing method uses a novel fibre-optic slide of individual wells. This method could sequence 25 millions bases in one 4- hour run with an accuracy of 99.96% (13 ). DNA Cloning System Paul Berg creates the first recombinant DNA molecules (ref. Berg, Science). While studying the actions of isolated genes, he devised methods for splitting DNA 5 molecules at selected sites and attaching the resulting segments to the DNA of a virus or plasmid, which could then enter bacterial or animal cells. Indeed a large variety of cloning systems is now available to accommodate different types and sizes of DNA fragments. For examples, the vectors can be based on plasmids (optimal range of DNA fragments 0.5-10 Kb), part of virus e.g. bacteriophage (7-20 Kb), cosmid or fosmid (35-45 Kb), bacterial artificial chromosome (BAC,