Molecular tools in ecology: Sequence analysis and phylogeny German Jurgens University of Helsinki, Finland Molecular tools in ecology: Sequence analysis and phylogeny rRNA approach and non-extreme Archaea 21 February 2006 Uppsala University, Sweden Diversity and Function of Microorganisms in Nature course

Outline WHY we need to study microbes? HOW to study the microbes: rRNA approach cycle - methods and techniques including: - PCR - DGGE - cloning and sequencing - computer methods - phylogeny - FISH Application of rRNA approach for archaeal environmental research 21 February 2006 Uppsala University, Sweden Diversity and Function of Microorganisms in Nature course

Why microorganisms important? Microbes represent more than 50% of the biomass on Earth , almost all rest – plants Microbes lives everywhere: in the air, in the soil, lakes and oceans. Extremely diverse and have different features, that allow them to survive in extreme conditions We can say that functioning of the whole biosphere depends mainly on microbes 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Why microbes are so diverse? Microbes had plenty of time to develop – they are the oldest life form on our planet Microfossil prokaryote from 3,5 billion year old rock 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

What do we know about them? Why we do not know nothing 95-99% of microbes? Most looks similar under light microscope – difficult to group by simple shape criteria Problematic to find suitable growing conditions for different microbes Some can grow slow, some not yet grow at all in lab Those, who grow easily, may not represent the major fraction of the studied community 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Fact and problem Number of bacteria we saw in the microscope usually hundred times exceeds the number of bacteria we can grow in the lab The PROBLEM Microbes are exist in large numbers in the environment Potentially significant for environment Difficult to study them in any systematic manner 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

How to study the microbes? Traditional way – enrichment culture technique - attempts to find media and conditions, selective for desire microbe and counterselective for undesired and then isolating pure culture of the microbe Very important, still developing, but can be difficult and time consuming 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Cells stains Helps obtain number and show dynamics of an ecosystem Fluorescent stain by DAPI which is binding to DNA Viability staining Live/Dead Fluorescence antibodies against cells surface components 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Statement Bacterial ecology needs good methods to study microbial communities which can be combined with traditional cultivation for fast and reliable analysis of complex environmental samples 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Idea of new molecular methods study bacterial communities by retrieving informative molecules – DNA or RNA sequences directly from environmental samples Name rRNA approach comes from small subunit (16S) of ribosomal RNA gene which appeared to be most suitable molecule for identification of microbes 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Secondary structure of 16S rRNA 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Evolutionary Chronometers rRNA features that make them excellent tool Relatively large – 1500 nuc. Functionally constant Universally distributed Contain several conserved and also variable regions SSU (small subunit): 16S or 18S Huge databases exists: RDP, EMBL Carl Woese – pioneer and inventor 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

From cultures to trees - draft Isolation of DNA from sample PCR is used to amplify the gene encoding 16S rRNA from genomic DNA PCR product is sequenced Make alignment of the sequences Generating trees, finding phylogenetic relations of our sequences 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Microbial ecology methods: PCR PCR - Polymerase Chain Reaction –technique that allows quick replication of DNA. With PCR, small quantities of specific genetic material can be amplified millions of times within a few hours. 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Cloning PCR amplification from community generates a single gel band, containing amplified DNA of single size, but this band contain many highly related but not identical genes, because sequences between conserved priming sites can vary as a result of evolutionary changes in the 16S rRNA of different species. So if the goal to sequence amplified genes we need to resolve different genes by cloning this PCR band or... 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Microbial ecology methods:DGGE DGGE/TGGE (denaturing/temperature gradient gel electrophoresis) - separation different rRNA genes of the same size that differ in their melting profile, because of differences in base sequence. Bands then can be excised and sequenced 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Microbial ecology methods RFLP – Restriction Fragment Length Polymorphism – produce rRNA gene fragments patterns that are specific for different communities Dot/Southern hybridization – method for finding DNA fragments with similar to DNA probe sequence Phylogenetic analysis – construction of phylogenetic trees using various calculation methods and different computer programs 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Microbial ecology methods:FISH Using Fluorescent In Situ Hybridization (FISH) technique and specific probes we can : visualize and quantify the number of different microbes directly (in situ) in the original environmental sample 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

?Microbial ecology questions? Methods can be used not only for rRNA gene Microbial ecology try to find answer on the questions: WHO THEY ARE? and WHAT THEY ARE DOING? rRNA approach tries to give answer to first question Using functional genes - WHAT THEY ARE DOING? 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea rRNA approach cycle scheme 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

rRNA approach cycle scheme environmental sample extract nucleic acids by chemical methods Get total nucleic acids of the community - all genes of all microorganisms By PCR and PCR primers, designed specifically for 16S rRNA gene, obtain PCR products – DNA fragments of 16S rRNA gene sequences Environmetal sample Extraction Community nucleic acids DNA RNA PCR Community rRNA genes 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Importance of primers selection rRNA gene of which microorganism(s) we will obtain, depends on specificity of PCR primers Primers can be general - designed for all Archaea, and after PCR we get only archaeal rRNA’s, or very specific, designed for particular group of microorganisms or species 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

rRNA approach cycle scheme Having community of rRNA genes we can: 1. Analyze this multiple "unseparated" PCR products by - DGGE or - RFLP (patterns, specific for community members), so we get ”community fingerprints", or Community rRNA genes DGGE RFLP Community ”fingerprints” 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

rRNA approach cycle scheme Having community of rRNA genes we can also 2. Obtain individual rDNA gene clones by cloning. Each individual clone contain vector plasmid with integrated single fragment of some 16S rRNA gene, which can be sequenced. Community rRNA genes cloning rDNA gene clones sequencing 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

rRNA approach cycle scheme As a result we get rDNA sequence data and, with help of computer programs, can compare new sequences with known 16S rRNA gene sequences, available from sequence databases. rDNA sequences and databases Comparative anaysis 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

rRNA gene sequence information If the obtained sequence is similar to sequence, existing in database - the conclusion would be that the organism (or it’s close relative) is present in the sample Sequences, that are dissimilar from those already known indicate novel microbe group 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

rRNA gene sequence information Comparison of the sequences to ones already present in the databases can : suggest the taxonomic group to which unknown organism belongs to possibly tell us something about its physiology This information may, in turn, provide researchers with a clues about media and enrichment techniques help with cultivation conditions 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Alignment and Important step in the analysis of novel 16S rRNA sequences is processing the sequence data using computers (comparative analysis) 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Evolutionalry distance Corrected ED is statistical correction necessary to account for either back mutations to original phenotype or additional forward mutational the same sate that could have happened 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Phylogenetic trees Construction of phylogenetic trees to best fit 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Phylogenetic trees - conclusion The more closely related two species are, the more similar rRNA gene sequence they have, so it’s possible to arrange species in phylogenetic trees, which graphically illustrate the relationships between sequences and, therefore, between species 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Tree of Life every living organism has rRNA gene in genome by comparing rRNA genes we can compare all known living organisms and construct so called universal 16S rRNA phylogenetic Tree of Life Humans are HERE! Microbes 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

rRNA approach cycle scheme With help of phylogenetic trees, 16S rRNA gene sequence information, databases and computers programs is possible to find (design) short fragments of DNA called “nucleic acid probes”. Nucleic acid probes Probe design rDNA sequences and databases Phylogenetic trees 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

rRNA approach cycle scheme Environmetal sample FISH These probes can be used as labels for specific microorganisms to track by FISH and follow their fate in environment, closing cycle of rRNA approach. Nucleic acid probes 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

rRNA approach cycle scheme Blotting/ hybridization techniques with specific probes can help to quantify the amount of specific microbes in community nucleic acid and community rRNA genes steps. Community nucleic acids DNA RNA Quantitative dot blot Dot/Southern blot Nucleic acid probes Community rRNA genes 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

rRNA approach cycle scheme Dot/colony hybridization with probes can be useful to sort clones and choose right for sequencing, since natural microbial communities are complex and many clones may require sequencing Nucleic acid probes Dot/colony blot rDNA gene clones 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Remarks Description of each of these methods required separate lecture Not all methods are described (SIP etc…) All methods has their own advantages, disadvantages and limitations 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Environmental genomics Other modern genetic approach to the molecular study of microbial communities is environmental genomics – or metagenomics Random shotgun sequencing of total cloned DNA from community (or sequencing of long DNA fragments in BAC/fosmids) – idea is to detect as many genes as possible that encode recognizable proteins together with phylogenetic marker (16S rRNA) 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

rRNA approach and Archaea Susan Barns, Norman Pace’s lab (Indiana University), used rRNA approach to look for diversity of Archaea in hot springs in Yellowstone park, USA. Octopus spring Obsidian pool 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Yellowstone park story In all, Barns has discovered 38 new species of Archaea in the Obsidian Pool, most of which aren’t related closely to any known genus. It was Korarchaeota, pJP27, 78 “There’s twice as much evolutionary distance between these new organisms in this one pool than between us and plants” she says. Korarchaeota 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Archaea in the open ocean Edward DeLong (University of California) and Jed Fuhrman (University of Southern California) used rRNA approach to study microbial community in surface and deep ocean – Marine Crenarcheota Group I (most abundant) and Marine Euryarchaeota Group II were found. 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Marine Archaea 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Abundance of Archaea in oceans FISH results shows that world ocean contains 1.3x1028 crenarchaeal cells (and 3.1x1028 bacterial) Distribution of Archaea and Bacteria with depth 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Archaea everywhere During past 14 years non-extreme Archaea were found in: marine picoplankton and sediment different types of soil (forest soil in Finland) freshwater lake sediments and water (Finland) plant roots fishes, marine sponge tissue, sea cucumber gut deep down in subsurface etc, 50-300 sequences/month (EMBL) 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Current view of Archaeal 16S rRNA gene phylogeny made with ARB Schleper et al. 2005, Nature reviews Microbiology 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

With a little help of my friends… Despite of accumulating knowledge and abundance of Group I Crenarchaeota, indicating that they might have large impact on global energy cycles, none of them have been obtained in pure culture until very recently (predicted by metagenomics and with help of rRNA approach) Molecular studies of AOB (ammonia oxidizing bacteria) in nitrifying systems so far have been limited to Beta and Gamma-proteobacteria 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Known nitrifying bacteria fall into 2 distinct physiological groups: those who oxidize ammonia to nitrite (AOB, in green) and other oxidized nitrite to nitrate (in red ) 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea AMO story All characterized AOB contain AMO – ammonia monooxygenase – enzyme, which oxidized ammonia to hydroxylamine, and then by hydroxylamine oxidoreductase to nitrite. Overall reaction: NH3 +1.5O2 NO-2+H2O+H+ 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Metagenomics prediction In 2004 Treuch et al. Environ. Microbiol. made fosmid metagenomic library from soil and identified two genes, encoding proteins related to AMO. Fragments belonged to Crenarchaeota as it was identified by 16S rRNA marker Also, AMO-related genes were reported in environmental sequences from Sargasso Sea, obtained by shotgun metagenomic approach (Venter et al. Science, 2004) 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Metagenomics prediction These facts give a hint that non-thermophilic Crenarchaeota living in soil and marine environments use ammonia as their primary energy source , and could be chemolitho- autotrophic ammonia oxidizers (nitrifiers) Proteins from family of AMO (amoA) and pmoA (particulate methane monooxygenases of methanotrophs) have been detected before only in Bacteria, so new homologues extend family 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea amoA/pmoA tree 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Ammonia-oxidizing Archaea Last year Könneke (D.Stahl group) (Isolation of autotrophic ammonia-oxidizing marine archaeon, Nature, 2005) after rRNA gene survey of nitrifying environments reported detection of sequences, belonging to marine Crenarchaeota Group I in estuary sediment, and two aquariums - nitrifying filtration systems and marine tropical fish tank 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Ammonia-oxidizing Archaea Then these Crenarchaeota were enriched from ammonia-oxidizing cultures and was found that oxidation rates of ammonia to nitrite corresponded with increasing abundance of Crenarchaeota 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Ammonia-oxidizing Archaea Finally pure culture of Crenarchaeota SCM1 - Nitrosopumilus maritimus was obtained and confirmed by FISH, quantitative PCR and phylogeny 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea Phylogeny: 98% sequence identity with marine Group I, which share only 84% 16S rRNA identity with soil Crenarchaeota and less than 80% with thermophiles 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea

Molecular tools in ecology: rRNA approach and non-extreme Archaea 21 February 2006, german.jurgens@helsinki.fi Molecular tools in ecology: rRNA approach and non-extreme Archaea