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

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.

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.

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

Past applications of this concept

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

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

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

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.

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.

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.

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

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

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

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

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?

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)

Questions Or suggesttions