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Project 1: Experimental evolution

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…. CH 3 -R. HCHO. CO 2. biomass.

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Project 1: Experimental evolution

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  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. CH3-R HCHO CO2 biomass Methylotrophy (aerobic) • Methylotrophy (growth on C1) • C1 compounds oxidized to formaldehyde • Oxidation of formaldehyde to CO2 • Assimilation of formaldehyde into cell material • 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

  3. CH3-R HCHO CO2 biomass Methylotrophy (aerobic) • Methylotrophy (growth on C1) • C1 compounds oxidized to formaldehyde • Oxidation of formaldehyde to CO2 • Assimilation of formaldehyde into cell material • 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 “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. Tree of Bacteria Gram+ Methylotrophs a b 16S rDNA g d Proteo-bacteria e Methylotrophy and HGT • Methylotrophy has arisen multiple, independent times in different lineages • HGT major force in enabling this specialized metabolism (Kalyuzhnaya et al., 2005, J Bacteriol)

  5. Multiple C1 modules for each role

  6. Methylotrophs possess multiple combinations of C1 modules Xanthobacter autotrophicus Methylobacillus flagellatus KT CH3OH CH3OH CH3NH2 MDH MaDH MDH HCHO RuMP assim. HCHO H4MPT HCOOH H4MPT Oxidation FDH1 FDH2 HCOOH CO2 FDH1 FDH2 CO2 CBB Methylococcus capsulatus Bath Methylobacterium extorquens AM1 CH4 CH3OH CH3NH2 pMMO2 sMMO pMMO1 CH3OH MDH MaDH RuMP assim. MDH PHB HCHO HCHO serine cycle Glyoxylate regeneration CH2=H4F H4MPT HCOOH TCA H4MPT H4F FDH1 FDH2 CO2 HCOOH FDH3 FDH1 FDH2 CBB CO2

  7. succinate methanol succinate methanol C1 transfers C1 transfers TCA cycle TCA cycle serine cycle serine cycle energy energy biomass biomass CO2 CO2 Model system: Methylobacterium • a-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) • C1 and multi-C growth are fundamentally different:

  8. CH3OH CH3NH2 CH3NH2 CH3OH Methylotrophy in M. extorquens AM1 MDH MaDH HCHO periplasm H2O, 2e- H2O, NH3, 2e- cytoplasm H4MPT HCHO H4F Fae serine cycle 1. Oxidation of C1 substrates to formaldehyde spont. spont. H2O H2O CH2=H4F CH2=H4MPT MtdA MtdA, MtdB NAD(P)H NADPH BIOMASS CH=H4F CH=H4MPT H2O H2O Mch Fch CHO-H4F CHO-H4MPT H2O FtfL H4MPT H2O Fhc H4F, ATP HCOOH FDHs NADH CO2

  9. Methylotrophy in M. extorquens AM1 CH3OH CH3NH2 MDH MaDH HCHO CH3NH2 CH3OH periplasm H2O, 2e- H2O, NH3, 2e- cytoplasm H4MPT HCHO H4F Fae spont. spont. serine cycle 2. Condensation of formaldehyde with H4F or H4MPT H2O H2O CH2=H4F CH2=H4MPT MtdA MtdA, MtdB NAD(P)H NADPH BIOMASS CH=H4F CH=H4MPT H2O H2O Mch Fch CHO-H4F CHO-H4MPT H2O FtfL H4MPT H2O Fhc H4F, ATP HCOOH FDHs NADH CO2

  10. Methylotrophy in M. extorquens AM1 CH3OH CH3NH2 MDH MaDH HCHO CH3NH2 CH3OH periplasm H2O, 2e- H2O, NH3, 2e- cytoplasm H4MPT HCHO H4F Fae serine cycle 3. Oxidation of CH2=H4MPT to formate spont. spont. H2O H2O CH2=H4F CH2=H4MPT MtdA, MtdB MtdA NAD(P)H NADPH BIOMASS CH=H4F CH=H4MPT H2O H2O Mch Fch CHO-H4F CHO-H4MPT H2O FtfL H4MPT Fhc H2O H4F, ATP HCOOH FDHs NADH CO2

  11. Methylotrophy in M. extorquens AM1 CH3OH CH3NH2 MDH MaDH HCHO CH3NH2 CH3OH periplasm H2O, 2e- H2O, NH3, 2e- cytoplasm H4MPT HCHO H4F Fae serine cycle 4. Oxidation of formate to CO2 spont. spont. H2O H2O CH2=H4F CH2=H4MPT MtdA MtdA, MtdB NAD(P)H NADPH BIOMASS CH=H4F CH=H4MPT H2O H2O Mch Fch CHO-H4F CHO-H4MPT H2O FtfL H4MPT H2O Fhc H4F, ATP HCOOH FDHs NADH CO2

  12. serine cycle Methylotrophy in M. extorquens AM1 CH3OH CH3NH2 MDH MaDH HCHO CH3NH2 CH3OH periplasm H2O, 2e- H2O, NH3, 2e- cytoplasm H4MPT HCHO H4F Fae 5. Assimilation of CH2=H4F by serine cycle spont. spont. H2O H2O CH2=H4F CH2=H4MPT MtdA MtdA, MtdB NAD(P)H NADPH BIOMASS CH=H4F CH=H4MPT H2O H2O Mch Fch CHO-H4F CHO-H4MPT H2O FtfL H4MPT H2O Fhc H4F, ATP HCOOH FDHs NADH CO2

  13. Methylotrophy in M. extorquens AM1 CH3OH CH3NH2 MDH MaDH HCHO CH3NH2 CH3OH periplasm H2O, 2e- H2O, NH3, 2e- cytoplasm H4MPT HCHO H4F Fae serine cycle 6. Interconversion of CH2=H4F and formate spont. spont. H2O H2O CH2=H4F CH2=H4MPT MtdA MtdA, MtdB NAD(P)H NADPH BIOMASS CH=H4F CH=H4MPT H2O H2O Fch Mch CHO-H4F CHO-H4MPT H2O FtfL H4MPT H2O Fhc H4F, ATP HCOOH FDHs NADH CO2

  14. CH3-R HCHO CO2 biomass Primary hub of C1 metabolism: What happened to simplicity???:

  15. Model system: C1 metabolism in Methylobacterium Topologically, any 2 of the 3 pathways leading to biomass or CO2 should be sufficient… 1. 2. 3. Mutants defective in pathway 2. or 3. are C1-

  16. Why are both C1 transfer pathways needed? 1. & 3. “redundant” for assimilation? 2. & 3. “redundant” for dissimilation? 1. 2. 3. 3. assimilation dissimilation

  17. Measured fluxes through hub nmol min-1 mL-1 OD600 nmol min-1 mL-1 OD600 nmol min-1 mL-1 OD600 • Dynamics of transition from S to M (Marx et al., 2005, PLoS Biology)

  18. Do we really understand this? nmol min-1 mL-1 OD600 ? nmol min-1 mL-1 OD600 nmol min-1 mL-1 OD600 • Developed kinetic model of central C1 hub (Marx et al., 2005, PLoS Biology)

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

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

  21. growth transfer growth transfer growth transfer growth transfer growth transfer growth transfer ancestor -80°C No Venus Venus (fancy YFP) Relative fitness of Venus/no Venus: WM = 1.00001 ± .000352 WS = 1.00016 ± .000154 Experimental evolution of laboratory populations What this looked like before… What it looks like now… • Living fossil record • Examine through time & across replicates • Assay competitive fitness: Average CV: 5.7 ± 3.1% day 0 day 1 acclimate Ww > Wp mix growth Competitor #1 Competitor #2 W > P W = P (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. ethanol methanol acetate succinate C1 transfers TCA cycle serine cycle energy biomass CO2 formate glycerol 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)…

  27. Rich medium C1 transfers TCA cycle serine cycle energy biomass CO2 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

  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 C1 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. C transfer N transfer C transfer N transfer C transfer N transfer Option #7 – Alternate between media lacking C, or N ancestor • Make PHB (a biodegradable plastic) as storage product • Force storage and efficient reutilization? • Tradeoffs with normal growth?

  31. glucose, fructose citrate C1 transfers TCA cycle serine cycle energy biomass CO2 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?

  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… • Diversification in still medium • Adaptation on solid surface • Adaptation to poor substrates • Adaptation to rich medium • Evolve on formaldehyde • Evolve on increasing concentrations of methanol • Alternate between medium lacking C, or N • Select for growth on a novel substrate • Long-term incubation for GASP • Evolve new, compensatory functions

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