AGBT 2010 - Penny Chisholm - MIT

From Single Cells to Global Metagenomics: What Prochlorococcus and its Phage Can Teach Us About Life.

It does not cause disease... but all our lives depend on it. Not enough time to discuss both of them.

World view:

* Information genome architecture

* Cellular machinery and physiology

* Population and community dynamics

* Biogenochemistry and Physics (Global Biosphereic Process)

First 2 are How, last 2 are why.

Our goal: Study a single microbe at all scales of organization

What is procholorcoccus

* smallest and most abundant photosynthetic on earth.

* cyanobacteria

* .6-.8 um diameter

* single species, - less than 3% difference in 16S rRNA

* very small and simple

* discovered in 1985

Account for 25-70% of photosynthesis in the oceans.

* sequesters up to 5 billion Volkswagen weights worth of CO2.

Light optimum for growth differs among strains

Also come in temperature optimized strains

Can map different ecotypes at different depths of oceans, as well as latitude of the earth.

Prochlorococcus has the smallest gennome that can make life from scratch.

* (requires no organic compounds)

* has 2000 genes.

* complete self-sufficiency

* Core genes: saturates ate 1250

* Optional genes: up to 5736

What's the global pan-genome?

* has a lot.. and is always changing. (lots of gene exchange)

A lot of data from global ocean survey (thanks Craig Venter!)

[Nice graph with location of core/optional genes along genome. They tend to form clusters in variable numbers.] -- Island regions

80% of island genes are not of cyanobacterial origin. (Probably phage involved in that)

Lots of phage in culture - lots of genome.

* Phage actually carry photosynthesis gene, among others.

2 vignettes:

1. hunt for NO3 utilizing chorlococcus

* problem: synechococcus has a common ancestor, which can use all 3 forms of nitrogens

* prochlorococcus: some use ammonia, some use No2, none use NO3

* confirmed that cells did not contain NO3 reductase.... (used seqencing)

* However, cells were all cultured on HN3+ media...

Went back to database from global ocean survey

* recruit all fragments that had NO3 reductase gene (narB) (Seems to use a cloned library?)

* Find those that also have known Prochlorococus gene.

* were able to find and identify them.

* not distributed everywhere, however, these only are distributed in some regions.

2. modeling the fitness consequence of loss of NO3- utilization in the global ocean.

* used global physical chemical model from Mick Follows (Darwin Project)

[WOW... incredible false color model movie of earth and nutrient concentrations and populations of phytoplankton!!!!!!!!!!]

Where in the oceans are the NO3- loss mutants least disadvantaged to relative to null?

Answer: Least disadvantaged in tropical pacific.

* Agrees with the global survey data.

[That is cool...]

Next:

* directed isolations for NO3 processing prochlorococcus

* single cell sequencing - using cell sorting.

* would like to know what other genes go along with the NO3 processing gene (narB)

Sea water is loaded with extraneous DNA... doing sorting (twice) reduces contaminating DNA.

Normalization reuces unevenness in sequencing coverage

* Genome recovery as high as 99.6% with reference guided assembly (done with cultured cells.)

Next Topic:

Do prochlorococcus populations in the atlantic and pacific have the same genetic composition?

* metagenomic sequencing with 454 FLX

* Recruit prochlorococcus reads

* analyze frequency of genes - tells you about selective pressures.

Results:

* only major difference is.... P- acquisition! (Lots of genes fit here)

* same things happens for a different bacteria Pelagibacter - it's all phosphorus genes, but different ones.

Cyanophage genes are also carying phosphorus genes in the pacific.

Thus, simple system with a clear answer.

Would like to do this more, now using Illumina. Gives same results as 454. Will increase throughput.

Last topic: Choreography of cellular metabolism

* cell cycle synthesizes to light dark cycle.

* transcription is dynamic: 80% of genes are cycled through light/dark cycle.

* Proteome some are dynamic, others are not.

* Many genes cycle, but do not change protein levels much!

Smoking gun: co-evolution between host and phage.... details about calvin cycle, but no time to get into it.

Conclusion:

* we now have a global understand every level ("integrated systems biology")

* genome to global understanding