1. 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

2. 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

3. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

4. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

5. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

6. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

7. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

8. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

9. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

10. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

11. Secondary structure of 16S rRNA
21 February 2006,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

12. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

13. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

14. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

15. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

16. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

17. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

18. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

19. Molecular tools in ecology: rRNA approach and non-extreme Archaea
21 February 2006,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

20. ?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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

21. Molecular tools in ecology: rRNA approach and non-extreme Archaea
rRNA approach cycle scheme
21 February 2006,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

22. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

23. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

24. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

25. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

26. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

27. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

28. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

29. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

30. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

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

32. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

33. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

34. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

35. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

36. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

37. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

38. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

39. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

40. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

41. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

42. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

43. Molecular tools in ecology: rRNA approach and non-extreme Archaea
Marine Archaea
21 February 2006,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

44. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

45. 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, sequences/month (EMBL)
21 February 2006,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

46. Molecular tools in ecology: rRNA approach and non-extreme Archaea
21 February 2006,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

47. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

48. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

49. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

50. 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 +1.5O NO-2+H2O+H+
21 February 2006,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

51. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

52. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

53. Molecular tools in ecology: rRNA approach and non-extreme Archaea
amoA/pmoA tree
21 February 2006,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

54. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

55. 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

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

57. Molecular tools in ecology: rRNA approach and non-extreme Archaea
Phylogeny: % 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,
Molecular tools in ecology: rRNA approach and non-extreme Archaea

58. Molecular tools in ecology: rRNA approach and non-extreme Archaea
21 February 2006,
Molecular tools in ecology: rRNA approach and non-extreme Archaea