1. Forward genetics Finding genes in Thiomicrospira crunogena that are necessary for growth under low CO 2 conditions

2. Terminologies Forward genetics: – Start with a phenotype. – Find the genes responsible for a phenotype. Reverse genetics: – Start with a/some gene(s) – Figure out the traits they confer

3. Random mutagenesis of Thiomicrospira crunogena Finding genes responsible for ‘CO 2 vacuuming’

4. Thiomicrospira crunogena – Hydrothermal vent chemolithoautotroph –.  -proteobacterium – Oxidizes sulfur cpds for energy – RAPID growth rate – Erratic environment Bright and Scott, 1998

5. The ‘CO 2 vacuum’ Can “trap” high conc’ns of bicarbonate inside their cells Dobrinski, Longo, and Scott, 2005 J. Bact. 187: 5741-5766.

6. One possible vacuum ‘mechanism’ HCO 3 - CO 2 biomass CA Rubisco CO 2

7. Which genes encode the components?

8. CO 2 -vacuuming genes via knockouts Knockout mutagenesis – Mate w/E. coli – Interrupt genes at random with a transposon – Screen for loss of CO 2 - vacuuming ability Larsen, Metcalf et al., 2002

9. More details on the E. coli host and pRL27 Host E. coli BW20767 genome encodes: – transfer functions necessary for pRL 27 transfer – Transposase inhibitor – Phage genes necessary for oriR6K replication (pir) pRL27 encodes: – oriT for transfer – oriR6K for theta replication in appropriate host – Tnp transposase OUTSIDE of transposon – aph = kan resistance

10. Steps Grow T. crunogena and E. coli separately Allow them to mate <3 <3 <3 Cultivate T. crunogena transconjugants on recovery plates (+ kanamycin) Larsen, Metcalf et al., 2002

11. Mating E. coli and T. crunogena Mix suspensions of both types of cell Pipette onto solid growth medium to create biofilm Let mate overnight ‘Mating medium’: keep both E. coli and T. crunogena happy overnight – No antibiotic (T. crunogena ) – Thiosulfate (T. crunogena ) – Low salt (E. coli ) – Yeast extract, tryptone (E. coli ) – 32-34°C (E. coli, T. crunogena )

12. Selection for T. crunogena transconjugants Scrape mating biofilms off mating plates Wash cells to remove tryptone and yeast extract Spread cells on selective medium – Seawater salt concentrations, thiosulfate T. crunogena, E. coli  – Does not contain tryptone and yeast extract T. crunogena, E. coli  – Contains kanamycin T. crunogena wild type , T. crunogena mutants

13. 1-2 wks later: Isolating kanamycin- resistant knockout mutants Pick colonies from mating plates to isolate them Screen them to make sure they’re T. crunogena and not E. coli – Acid production from thiosulfate

14. Screen T. crunogena mutants for CO 2 sensitivity Have any of them lost their ‘CO 2 -vacuuming’ ability?  unable to grow under low-CO 2 conditions

15. Recap: Forward genetics to find genes responsible for CO 2 vacuuming Mate transposon into T. crunogena Collect transconjugant T. crunogena – Each has one interrupted gene Screen transconjugants for CO 2 sensitivity