2. Growth rates of heterotrophic bacteria determined by 16S rRNA:rDNA ratios
hank you
some ideas and plans
project that I am calling
Tom S. Lankiewicz

3. The microbial loop
Marine bacteria are important because they fill a vital role in marine food webs
Green=FULL HALF of PP
ONE HALF of all the primary production in the ocean
Sole users of DOM and POM.
Better word: Recyclers
When their biomass is consumed they return carbon to the traditional food web than would have other wise been lost.

4. Why do growth rates matter? ½ of population controls
Top-down controls
Removal of cells from population
(grazing and viral lysis)
Bottom-up controls
Addition of new cells to population
(growth)
Resource limitation (organic carbon)
Growth rate determined by extent of resource limitation
Actual rate of cell division = some fraction of organisms max growth rate, based on extent carbon limitation.

5. Ribosomes as a measure of growth
Some very basic assumptions: More ribosomes = more protein synthesis More protein synthesis = more growth
Ribosome make proteins so rapid protein synthesis demanded by growth in turn demands high ribosomes.
One way to measure ribosome in a cell is through quantifying the 16S rRNA sequence fragments in a sample:
Metagenomic-environement
qRT-PCR-culture
Currently has been used in the environment to determined relative growth rates of specific bacterial clades.
Could be used as a more direct quantification of grwoth.
Determine what ratio or ratios correspond to rapid growth rates in isolate?
What correspond to slow?
Useful in comparing across samples

6. Past applications of this concept

7. rRNA:rDNA ratios
# of 16 S rRNA copies # of 16S rDNA copies rRNA: could be quantified with high throughput sequencing or with Q-PCR rDNA: can be inferred from abundances
Definition of rRNA to rDNA ratios
Example of how we could calculate rRNA to rDNA ratios

8. rRNA:rDNA ratios: recent studies
Dave edited the top one: the one that really brought me to the kirchman lab

9. Current limitations in using rRNA:rDNA as a measure of growth
With current knowledge data is relative: X is growing fast compared to other populations of X We don’t know what the growth rate of X is in relation to Y Some unique relationship between rRNA:rDNA ratio and growth rate for each bacterial strain
Ribosome make proteins so rapid protein synthesis demanded by growth in turn demands high ribosomes.
One way to measure ribosome in a cell is through quantifying the 16S rRNA sequence fragments in a sample:
Metagenomic-environement
qRT-PCR-culture
Currently has been used in the environment to determined relative growth rates of specific bacterial clades.
Could be used as a more direct quantification of grwoth.
Determine what ratio or ratios correspond to rapid growth rates in isolate?
What correspond to slow?
Useful in comparing across samples

10. Phase 1: In lab
Will now outline the first part of my prposed work which will be done in the lab using a culturable isolate of SAR11.

11. Hypothesis
Higher rRNA:rDNA ratios are indicative of faster growth within one organism, But there is a unique relationship between rRNA:rDNA and growth rate for each strain.

12. Growth of heterotrophic bacteria in culture
What is the relationship between rRNA:rDNA ratios and growth rate?
Leucine incorporation and growth rate? (easy bulk measurement of growth)  double check
Main questions I will be asking in this first study ….
What specific ratios for this strain are indicative of fast and which are indicative of slow growth
What are incorportaion rates like when grwoing rapidly? when grwoing slwoly?
Are these two things very similar or the same? If not this has intersting implication for storage of carbon for in famine situations.

13. Samples Taken: rRNA (QPCR) Cell abundance (DAPI)
Leucine Inc (bulk incorporation assay)
Organic acid uptake (bulk incorporation)
(Respiration)
Basic growth curve experiments: batch cultures
Defined media; will be able to adjust growth rates
Will ideally determine these measurements for several growth curves 2nd animation
with different rates of increase and carrying capacities
Will be able to determine what
Dilution rate (D) = Growth Rate (μ)
Test several different μ to determine a relationship

14. Complementary to work with heterotrophic bacterial respiration
Will be run in parallel with rRNA:rDNA ratio experiment Respiration, uptake, and growth rates will provide a robust picture of carbon utilization during diff. rates of growth
Matt will be doing experiments on the respired fraction of carbon in this organism et the same time that these experiements will be happening
Will provide a really well rounded picture of what happens to carbon when it is consumed by P. ubique

15. Habitat where isolated
Possible strains
Organism
Clade
Habitat where isolated
Sequenced?
Max growth rate (d-1)
Pelagibacter ubique
SAR11
Oligotrophic
Yes
0.4
Ruegeria pomeroyi
Roseobacter
Copiotrophic
Polaribacter
Flavobacteria .8
HTCC2207
SAR92
2.0
Missing citations

16. Phase 2: Delaware Bay sampling
And study will build off the first and apply new knowledge of rRNA ratios and SAR11 carbon usage to environmental samples
Less well defined
Could deal with growth or carbon storage questions depending on the results of part 1

17. Growth rates of natural populations
Which organisms in the Delaware Bay estuary are growing rapidly and which are growing slowly? Is abundance negatively related to growth rate?

18. Delaware Bay estuary bacterial biogeography
Prime environment for sampling a variety of communities
Where SAR11 is present in different degrees
Where different types of SAR11 are present
Kirchman et al. (2005)

19. Questions
Or suggesttions