Forward genetics

Finding genes in Thiomicrospira crunogena that are necessary for growth

under low CO2 conditions

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

Random mutagenesis of Thiomicrospira crunogena

Finding genes responsible for ‘CO2 vacuuming’

Thiomicrospira crunogena

–Hydrothermal vent chemolithoautotroph–.g-proteobacterium–Oxidizes sulfur cpds for energy–RAPID growth rate–Erratic environment

Bright and Scott, 1998

The ‘CO2 vacuum’

Can “trap” high conc’ns of bicarbonate inside their cells

0

2

4

6

8

10

12

0 0.05 0.1 0.15 0.2 0.25

Extracellular DIC (mM)

Intr

ac

ellu

lar

DIC

(m

M)

Dobrinski, Longo, and Scott, 2005J. Bact. 187: 5741-5766.

One possible vacuum ‘mechanism’HCO3

-

HCO3-

HCO3-

CO2

biomass

CA

Ru biscoCO2

Which genes encode the components?

CO2-vacuuming genes via knockouts•Knockout mutagenesis–Mate w/E. coli–Interrupt genes at random with a transposon–Screen for loss of CO2-vacuuming ability

Larsen, Metcalf et al., 2002

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

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

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 )

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

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

Screen T. crunogena mutants for CO2 sensitivity

• Have any of them lost their ‘CO2-vacuuming’ ability?unable to grow under low-CO2 conditions

Recap: Forward genetics to find genes responsible for CO2 vacuuming

• Mate transposon into T. crunogena• Collect transconjugant T. crunogena– Each has one interrupted gene

• Screen transconjugants for CO2 sensitivity