2. Life on Earth Thought to be ~3.8 billion years old
For the first 1.5 billion years, it was all aquatic microbes

3. Diversity of bacteria and archaea
Only ~1% of all microbial species can be cultured
97% of prokaryotic isolates in stock centers are from just 4 phyla:
Proteobacteria (Escherichia, Helicobacter, Pseudomonas)
Firmicutes (Bacillus, Streptococcus, Staphylococcus)
Actinobacteria (Mycobacterium)
Bacteriodetes

4. Unculturable microbes
Hugenholtz P (2002) Genome Biology 3, 1-8

5. Environmental sampling: two approaches
SSU rRNA
Common to all cells
Can be amplified with universal PCR primers
Does not reveal metabolic diversity
Random genomic DNA
Obtained by blindly cloning mixed DNA samples
Difficult to know what you have got!
But this is where the genes underlying metabolic diversity are

6. SSU rRNA Phylogeny
Pace NR (1997) Science 276, 734

7. Genome sequencing
The first microbe (Haemophilus influenzae) was sequenced in 1995
Since then, well over 100 genomes have been completed
Over 500 genome projects are ongoing

8. Diversity of sequenced genomes
Archaea
Bacteria
Eukarya
Importance:
academic
medicinal
agricultural
ecological
industrial
Representation of completed genome sequences over time (x-axis) and size (y-axis, in Mb, logarithmic scale) labeled according to their social impact. Genomes from Archaea (squares), Bacteria (circles) and Eukarya (triangles) are colored according to their academic (blue), medical (pink), agricultural (light green), ecological (dark green) and industrial (black) relevance.
Janssen et al. (2003) Genome Biology 4, 402

9. Genome projects online

10. 60K protein families and counting
clustering of 311,256 proteins from 83 complete genomes
Kunin et al. (2003) Genome Biology 4, 401

11. Shotgun sequencing
Random size-selected clones are sequenced from both ends (mate pairs)
Overlapping sequences are assembled in contigs
Contigs connected by mate pairs are assembled in scaffolds
The total number of bases sequenced divided by the length of the genome is the coverage.
Coverage must be at least 8X to avoid large gaps
contigs &
scaffolds
mate pair
reads
chromosome

12. What if we combined environmental sampling and shotgun sequencing?
How many genomes would be sampled and from what organisms?
How many novel genes would be discovered?
How many genomes could we completely assemble?

13. Current estimates of microbial community diversity
Curtis et al (2002) PNAS 99, estimated that there are up to 160 species in a typical millilitre of seawater while there are somewhere between 6,400 and 38,000 in a typical gram of soil.

14. The Sargasso Sea A sea with no coastline (bounded by ocean currents)
It moves!
Generally between the West Indies and the Azores
Water is very placid (the ‘doldrums’)
Covered by a lens of warm, nutrient-poor water and a vast mat of algae (Sargassum)
As simple a microbial community as is likely to be found in the ocean

15. The Sargasso Sea

16. Sargassum (can you spot the nudibranchs?)
© 2000 by Image Quest 3-D

17. Institute for Biological Energy Alternatives
Founded by J. Craig Venter, who also founded TIGR and Celera
The IBEA
“is dedicated to exploring solutions for carbon sequestration using microbes, microbial pathways, and plants.”
“will develop and use microbial pathways and microbial metabolism to produce fuels with higher energy content in an environmentally sound fashion”.
“will undertake genome engineering to better understand the evolution of cellular life and how these cell components function together in a living system”.
For example, genomics could be applied to enhance the ability of terrestrial and oceanic microbial communities to remove carbon from the atmosphere.

18. Sampling scheme
1700 liters of surface water sampled from four different sites
Most during season of winter nutrient upwelling in February
Some during nutrient-poor season in May
Filters allowed only cells in the micron range
Excluded dissolved DNA and free virus
Excluded most eukaryotes

19. Sampling sites and ocean chlorophyll levels (February)

20. Lots and lots of sequences
2 million cloned fragments 2-6 kbp in size were sequenced
This yielded 1.6 billion base pairs total
1 billion bp non-redundant
For comparison, the human genome is 3 billion bp

21. Assembly issues Organisms differ in
abundance
genome size
Cannot rely on assumption that coverage is uniformly random
Some contigs will have extremely deep coverage, which is a challenge for assembly algorithms
Requires an iterative assembly process with lots of manual intervention and many unassembled sequences

22. Assembly results Assembly was only successful in February sample
64,000 scaffolds, most less than 10 kbp
500,000 clones did not assemble
Of those with 3X or greater coverage
About ½ could be classified taxonomically
21 scaffolds with greater than 14X coverage
SNPs occur at 1/10 kbp, suggesting genetic diversity within ‘species’
Only two genomes were fully assembled, and then only with the aid of an existing reference sequence for both

23. Genome diversity in Prochlorococcus
outer circle: Prochlorococcus marinus
colors represent role categories
There are up to four structurally distinct
scaffolds aligning with a given
interval on the reference genome

24. Unexpected sequences
Relatives of Burkholderia and Shewanella, typical of much more nutrient-rich environments, possibly living off of ‘marine snow’
At least two abundant Archaeal organisms, typical of much greater depths (200 meters)
At least 10 mega-plasmids, many with genes related to trace metal utilization or toxicity
Not too surprising
Some phage genomes, presumably integrated
About 70 different eukaryote species (based mainly on the presence of 18S rDNA)

25. Homology of four scaffolds to reference crenarchaeal sequence 4B7

26. How many new genes are discovered?
1.2 million genes identified
Equal to the number of genes submitted to the Swissprot/TrEMBL database from the last 8 years!
Interesting findings
Ammonium oxidation in Archaea, which was previously unknown
Widespread presence of genes allowing unconventional forms of phosphate uptake
Only~37 Rubisco sequences were found, but ~800 proteorhodopsin-like genes

27. Can we estimate the relative abundance of the taxa?
How good of a marker is rDNA?
Universal primers may miss some taxa
Genomes vary in rDNA copy number
Examined five other gene markers to measure relative abundance of different taxa

29. How many species are there?
Number of distinct SSU genes
1164 in February
248 in May
Dominated by proteobacteria
148 are new phylotypes (at 97% identity)
Can we estimate how many remain to be sampled?
From a model of assembly completeness, one can estimate 1,800 to 48,000 species in the combined sample
With 5-10 fold deeper coverage, one can estimate that 50 genomes could be fully assembled

30. Patchiness
Patchiness is well documented in marine macro-organisms in the open ocean, but is not known for microbial communities
Of the species represented by assemblies with more than 50 fragments, more than half differed in abundance among sites

31. Proteorhodopsin
Proteorhodopsin was recently discovered in an uncultured lineage of planktonic Gammaproteobacteria
Related to the light-driven proton pump bacteriorhodopsins
Is part of a non-chlorophyll based photosynthetic pathway
About 70 sequences are known, mostly from proteobacteria
~800 proteorhodopsins were sequenced
Can be grouped into 13 subfamilies
Only four of which are found in cultured organisms
Seven are only known from the Sargasso Sea

32. Rhodopsin-like sequences

33. Is this the future? Cost of sequencing decreases steadily
The more reference sequences exist, the easier data analysis will become
Many proteins can be expressed even when organisms cannot be cultured
How else could we sample the genetic diversity of unculturable microbes?