1. Project 1: Experimental evolution Methylobacterium –Non-pathogenic, easy to culture, genetics, genome, metabolic & biochemical knowledge –Have fluorescence-based fitness assays –Transfers only every other day –My lab studies it – can lead to ‘real’ work…

2. Methylotrophy (aerobic) Key issue: Efficient growth requires high flux through formaldehyde while maintaining a pool below toxic concentrations – and partition carbon appropriately into assimilatory and dissimilatory metabolism CH 3 -R HCHO CO 2 biomass Methylotrophy (growth on C 1 ) –C 1 compounds oxidized to formaldehyde –Oxidation of formaldehyde to CO 2 –Assimilation of formaldehyde into cell material

3. Methylotrophy (aerobic) Key issue: Efficient growth requires high flux through formaldehyde while maintaining a pool below toxic concentrations – and partition carbon appropriately into assimilatory and dissimilatory metabolism CH 3 -R HCHO CO 2 biomass Methylotrophy (growth on C 1 ) –C 1 compounds oxidized to formaldehyde –Oxidation of formaldehyde to CO 2 –Assimilation of formaldehyde into cell material “If the consumption of cytoplasmic formaldehyde were inhibited, the cytoplasmic formaldehyde concentration would increase to about 100 mM in less than 1 min.” (Vorholt et al., 2000, J Bacteriol)

4. Methylotrophy and HGT 1.Methylotrophy has arisen multiple, independent times in different lineages 2.HGT major force in enabling this specialized metabolism Proteo- bacteria Gram +      16S rDNA Tree of Bacteria Methylotrophs (Kalyuzhnaya et al., 2005, J Bacteriol)

5. Multiple C 1 modules for each role

6. Methylotrophs possess multiple combinations of C 1 modules HCOOH CO 2 CH 4 sMMO pMMO2 pMMO1 Methylococcus capsulatus Bath MDH RuMP assim. H 4 MPT FDH1FDH2 CBB CH 3 OH HCHO MDH FDH2FDH1 CBB Xanthobacter autotrophicus H 4 MPT HCOOH CH 3 OH HCHO CO 2 Methylobacterium extorquens AM1 Glyoxylate regeneration serine cycle PHB TCA MDH MaDH CH 3 OH HCHO HCOOH CO 2 H 4 MPT FDH1FDH2 FDH3 H4FH4F CH 2 =H 4 F CH 3 NH 2 HCOOH Methylobacillus flagellatus KT MDH MaDH H 4 MPT FDH2FDH1 Oxidation HCHO CH 3 OHCH 3 NH 2 CO 2 RuMP assim.

7. Model system: Methylobacterium  -proteobacterium, plant epiphyte Grows on limited number of multi-C compounds –Of cultured methylotrophs, nearly all highly specialized –Suggest consistent tradeoff? Ecological or physiological? –Leading a consortium to analyze sequence of 6 more Methylobacterium genomes (JGI) C 1 and multi-C growth are fundamentally different: succinatemethanol TCA cycle serine cycle C 1 transfers CO 2 succinate biomass energy TCA cycle serine cycle C 1 transfers CO 2 methanol biomass energy

8. Methylotrophy in M. extorquens AM1 1. Oxidation of C 1 substrates to formaldehyde CH 3 OH HCHO MDHMaDH H4FH4F H 4 MPT Fae CH 2 =H 4 FCH 2 =H 4 MPT MtdA, MtdBMtdA CHO-H 4 F HCOOH Fch Fhc Mch FtfL spont. CH 3 NH 2 HCHO CH=H 4 F CHO-H 4 MPT CO 2 spont. BIOMASS serine cycle FDHs CH=H 4 MPT NADPH H 4 F, ATP H 4 MPT H2OH2O H2OH2O NADH NAD(P)H H 2 O, 2e - H 2 O, NH 3, 2e - cytoplasm periplasm H2OH2O H2OH2O H2OH2O H2OH2O

9. CH 3 OH HCHO MDH MaDH H4FH4F H 4 MPT Fae CH 2 =H 4 FCH 2 =H 4 MPT MtdA, MtdBMtdA CHO-H 4 F HCOOH Fch Fhc Mch FtfL spont. CH 3 NH 2 HCHO CH=H 4 F CHO-H 4 MPT CO 2 spont. BIOMASS serine cycle FDHs CH=H 4 MPT NADPH H 4 F, ATP H 4 MPT H2OH2O H2OH2O NADH NAD(P)H H 2 O, 2e - H 2 O, NH 3, 2e - cytoplasm periplasm H2OH2O H2OH2O H2OH2O H2OH2O 2. Condensation of formaldehyde with H 4 F or H 4 MPT Methylotrophy in M. extorquens AM1

10. CH 3 OH HCHO MDH MaDH H4FH4F H 4 MPT Fae CH 2 =H 4 FCH 2 =H 4 MPT MtdA, MtdB MtdA CHO-H 4 F HCOOH Fch Fhc Mch FtfL spont. CH 3 NH 2 HCHO CH=H 4 F CHO-H 4 MPT CO 2 spont. BIOMASS serine cycle FDHs CH=H 4 MPT NADPH H 4 F, ATP H 4 MPT H2OH2O H2OH2O NADH NAD(P)H H 2 O, 2e - H 2 O, NH 3, 2e - cytoplasm periplasm H2OH2O H2OH2O H2OH2O H2OH2O 3. Oxidation of CH 2 =H 4 MPT to formate Methylotrophy in M. extorquens AM1

11. CH 3 OH HCHO MDH MaDH H4FH4F H 4 MPT Fae CH 2 =H 4 FCH 2 =H 4 MPT MtdA, MtdBMtdA CHO-H 4 F HCOOH Fch Fhc Mch FtfL spont. CH 3 NH 2 HCHO CH=H 4 F CHO-H 4 MPT CO 2 spont. BIOMASS serine cycle FDHs CH=H 4 MPT NADPH H 4 F, ATP H 4 MPT H2OH2O H2OH2O NADH NAD(P)H H 2 O, 2e - H 2 O, NH 3, 2e - cytoplasm periplasm H2OH2O H2OH2O H2OH2O H2OH2O 4. Oxidation of formate to CO 2 Methylotrophy in M. extorquens AM1

12. CH 3 OH HCHO MDH MaDH H4FH4F H 4 MPT Fae CH 2 =H 4 FCH 2 =H 4 MPT MtdA, MtdBMtdA CHO-H 4 F HCOOH Fch Fhc Mch FtfL spont. CH 3 NH 2 HCHO CH=H 4 F CHO-H 4 MPT CO 2 spont. BIOMASSserinecycle FDHs CH=H 4 MPT NADPH H 4 F, ATP H 4 MPT H2OH2O H2OH2O NADH NAD(P)H H 2 O, 2e - H 2 O, NH 3, 2e - cytoplasm periplasm H2OH2O H2OH2O H2OH2O H2OH2O 5. Assimilation of CH 2 =H 4 F by serine cycle Methylotrophy in M. extorquens AM1

13. CH 3 OH HCHO MDH MaDH H4FH4F H 4 MPT Fae CH 2 =H 4 FCH 2 =H 4 MPT MtdA, MtdB MtdA CHO-H 4 F HCOOH Fch Fhc Mch FtfL spont. CH 3 NH 2 HCHO CH=H 4 F CHO-H 4 MPT CO 2 spont. BIOMASS serine cycle FDHs CH=H 4 MPT NADPH H 4 F, ATP H 4 MPT H2OH2O H2OH2O NADH NAD(P)H H 2 O, 2e - H 2 O, NH 3, 2e - cytoplasm periplasm H2OH2O H2OH2O H2OH2O H2OH2O 6. Interconversion of CH 2 =H 4 F and formate Methylotrophy in M. extorquens AM1

14. Primary hub of C 1 metabolism: CH 3 -R HCHO CO 2 biomass What happened to simplicity???:

15. Model system: C 1 metabolism in Methylobacterium 1. 3. 2. Topologically, any 2 of the 3 pathways leading to biomass or CO 2 should be sufficient… Mutants defective in pathway 2. or 3. are C 1 -

16. Why are both C 1 transfer pathways needed? 3. 2. 2. & 3. “redundant” for dissimilation? 3. 1. 1. & 3. “redundant” for assimilation? assimilationdissimilation

17. nmol min -1 mL -1 OD 600 Dynamics of transition from S to M Measured fluxes through hub nmol min -1 mL -1 OD 600 (Marx et al., 2005, PLoS Biology)

18. nmol min -1 mL -1 OD 600 ? Developed kinetic model of central C 1 hub Do we really understand this? (Marx et al., 2005, PLoS Biology)

19. Switch from long to direct assimilation Model prediction qualitatively recapitulated the phenomenon… Experimental dataModel predictions (Marx et al., 2005, PLoS Biology)

20. Competitor #1 acclimate Competitor #2 mix day 0 day 1 W = P W > P growth W w > W p -80°C Living fossil record –Examine through time & across replicates Assay competitive fitness: Experimental evolution of laboratory populations

21. Competitor #1 acclimate Competitor #2 mix day 0 day 1 W = P W > P growth W w > W p -80°C Living fossil record –Examine through time & across replicates Assay competitive fitness: What this looked like before… Relative fitness of Venus/no Venus: W M = 1.00001 ±.000352 W S = 1.00016 ±.000154 No Venus Venus (fancy YFP) What it looks like now… Average CV: 5.7 ± 3.1% (David Chou)

22. Project 1: Experimental evolution What we can assay: –Fitness –Growth –In selected and other environments… –Diversity in colony morphology –For some projects, sequence candidate loci

23. Project 1: Experimental evolution Project possibilities –Need to be relatively easy to passage, but hopefully somewhat interesting… –Will present 10 projects – can pick one, modify one, or come up with your own –Each group will write a brief description of plans Will discuss further on Wednesday (and due 2/12) Next Monday we will discuss these further and groups will revise plan and consult with David and I (before 2/14) If all goes well, initiate transfers on Wednesday, 2/14, go over protocol and sign-up for transfer days…

24. Option #1 – Diversification in still medium Similar adaptive diversification as seen w/ P. fluorescens? –Try more than one genotype (lab strain, wild isolate, an evolved isolate) –Try more than one medium (rich vs. minimal, different substrates) –Tradeoff w/ growth in shaken medium? –Assay both diversity in colony morphology and fitness ?

25. Option #2 – Adaptation to solid surface Tradeoffs with growth in liquid? Diversity due to spatial heterogeneity? Changes in biofilm structure? (Initiate with fluorescent strains)

26. Option #3 – Adaptation to poor substrates Are either the dynamics of adaptation or tradeoffs experienced more extreme with poor substrates? –Try more than one genotype (lab strain, an evolved isolate) –Try substrates such as formate, glycerol, ethanol, acetate (compared to methanol or succinate)… TCA cycle serine cycle C 1 transfers CO 2 methanol biomass energy formate ethanol acetate glycerol succinate

27. Option #4 – Adaptation to rich medium Does adaptation to rich medium lead to a diverse community? –Look for potential diversity and frequency-dep. fitness effects between community members –Also can look at tradeoffs in minimal medium TCA cycle serine cycle C 1 transfers CO 2 biomass energy Rich medium

28. Option #5 – Evolve on formaldehyde Can cells balance need to grow with toxicity? –Wild-type is very poor at using formaldehyde directly –May need to supplement early growth with methanol –Another very poor substrate –Look at tradeoffs w/ other C 1 substrates –May unlock secret of formaldehyde transport… ???

29. Option #6 – Evolve on increasing concentrations of methanol Push boundary of physiological capacities Tradeoffs with normal concentration? Can try w/ multiple genotypes –pre-evolved to M –strain w/ engineered foreign formaldehyde oxidation pathway Can step up concentration as they improve… ???

30. Option #7 – Alternate between media lacking C, or N Make PHB (a biodegradable plastic) as storage product Force storage and efficient reutilization? Tradeoffs with normal growth? ancestor CtransferN C N C N

31. Option #8 – Select for growth upon a novel substrate All internal pathways present – only transport appears to be missing… Supplement growth with another compound to get them started, then wean them off? Tradeoffs with current substrates? TCA cycle serine cycle C 1 transfers CO 2 glucose, fructose biomass energy citrate

32. Option #9 – Long-term incubation for growth advantage in stationary phase Donner Party for microbes… Can try both shaken and still environments Tradeoffs between GASP and normal growth? Same molecular targets (ex: rpoS) as seen in E. coli? Lead to cheating?

33. Option #10 – Evolve new, compensatory functions Start with cells lacking a key enzyme and re- evolve growth Supplement initially and then wean? Risky, but could be very interesting (start multiple genotypes and examine those that ‘work’)

34. Many possibilities… 1.Diversification in still medium 2.Adaptation on solid surface 3.Adaptation to poor substrates 4.Adaptation to rich medium 5.Evolve on formaldehyde 6.Evolve on increasing concentrations of methanol 7.Alternate between medium lacking C, or N 8.Select for growth on a novel substrate 9.Long-term incubation for GASP 10.Evolve new, compensatory functions