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- 1. Evolution of the minimal cell Hutchison et al. 2016 Science 2020-10-06 Roy Z Moger-Reischer rzmogerr@indiana.edu
- 2. Acknowledgements • Jay Lennon, Daniel Schwartz Emmi Mueller, Ford Fishman, Pat Wall • Betsy Snider, Kelsey McKenzie, JD French, Danni Boylan • Farrah Bashey-Visser, Jake McKinlay, Matt Hahn • John Glass, Kim Wise, Lijie Sun, Megan Behringer, Mike Lynch • Etienne Nzabarushimana
- 3. Understanding complexity through simplification • Hydrogen • Hardy-Weinberg equilibrium • Escherichia coli and Saccharomyces cerevisiae
- 4. A minimal organism through synthetic biology Hutchison et al. 2016 Science Mycoplasma mycoides JCVI-syn3.0
- 5. Questions and expectations • How does genome minimization affect rate and outcome of evolution? • With removal of DNA repair genes, mutation rate and spectrum may change, in turn affecting evolution • With fewer genetic targets for natural selection, adaptation could be slower • What are the mechanisms and features of (re-)adaptation?
- 6. Questions and expectations • How does genome minimization affect rate and outcome of evolution? • With removal of DNA repair genes, mutation rate and spectrum may change, in turn affecting evolution • With fewer genetic targets for natural selection, adaptation could be slower • What are the mechanisms and features of (re-)adaptation?
- 7. Mutation accumulation experiment Lynch et al. 2016 (Nat. Rev. Genet.)
- 8. • Highest recorded bacterial mutation rate • High rate in wildtype likely reflects small Ne (obligate pathogen, small genome, low GC content) and lack of mismatch repair • Genome minimization had no effect on per-nt mutation rate • Evolution is not limited by availability of new mutations Mutation rate Mutationrate (muts⋅nt-1⋅gen-1) P = 0.54
- 9. Mutation spectrum Single nucleotide mutations (SNM s) Deletions Insertions P = 0.125 No effect of genome minimization of spectrum of mutation types Proportion
- 10. P < 0.0001 Proportion ns ns*** *** *** *** Single-nucleotide mutation spectrum is altered by minimization
- 11. Minimal cell has excessive C -> T mutations ecocyc.org Ung
- 12. Minimal cell has excessive C -> T mutations ecocyc.org Ung Ireton et al. 2002 (J. Mol. Biol.)
- 13. Minimal cell has excessive C -> T mutations ecocyc.org Ung C:G U:G via deamination U:G U:A in subsequent DNA replication U:A T:A in subsequent DNA replication
- 14. Questions and expectations • How does genome minimization affect rate and outcome of evolution? • With removal of DNA repair genes, mutation rate and spectrum may change, in turn affecting evolution • With fewer genetic targets for natural selection, adaptation could be slower • What are the mechanisms of (re-)adaptation?
- 15. Questions and expectations • How does genome minimization affect rate and outcome of evolution? • With removal of DNA repair genes, mutation rate and spectrum may change, in turn affecting the ability to adapt • With fewer genetic targets for natural selection, adaptation could be slower • What are the mechanisms of (re-)adaptation?
- 17. Competition trials measure fitness
- 18. Adaptation experiment: relative fitness 1) Minimal cell is “sick” - ~50 % reduction in fitness compared to wildtype Wildtype Minimal
- 19. Adaptation experiment: relative fitness 1) Minimal cell is “sick” - ~50 % reduction in fitness compared to wildtype 2) After 2000 generations, minimal cell regains ~80 % of this fitness cost 3) In addition, rate of fitness gain in minimal cell is same as that in ancestor Wildtype Minimal
- 20. Genome minimization does not constrain molecular evolution • Calculated dN/dS ratio to test for signatures of constrained molecular evolution. • dN/dS ratio is not lower for the minimal cell.
- 21. Minimization alters route to adaptation • Compared composition of mutations for essential genes between strains • Suggests different genetic pathways to achieve the same fitness gain. PERMANOVA, P = 0.029 Minimal Wildtype
- 22. Questions and expectations • How does genome minimization affect rate and outcome of evolution? • With removal of DNA repair genes, mutation rate and spectrum may change, in turn affecting the ability to adapt • With fewer genetic targets for natural selection, adaptation could be slower • What are the mechanisms of (re-)adaptation?
- 23. Questions and expectations • How does genome minimization affect rate and outcome of evolution? • With removal of DNA repair genes, mutation rate and spectrum may change, in turn affecting the ability to adapt • With fewer genetic targets for natural selection, adaptation could be slower • What are the mechanisms of (re-)adaptation?
- 24. Adaptation experiment: Identify specific significant genes • 100000 simulations to identify genes with more mutations than expected by chance • Observed mutations placed at random, proportional to: • gene length • gene relative mutation rate based on GC content • P = number of simulations in which Msim ≥ Mobs, ÷100000 • Padj = Benjamini-Hochberg corrected P-value (Benjamini and Hochberg 1995 J. R. Stat. Soc. B) PERMANOVA, P = 0.029
- 25. Significant mutations in the wild type (JCVI-syn1.0) ** = not present in minimal cell gene Annotation Category Padj (B-H) ftsZ Cell division protein ftsZ Cell division < 0.00032 dnaA_1 Chromosomal replication initiator protein DNA replication 0.00045 tnpA_1 ** IS1296 transposase protein A Transposition 0.00070 tnpB_1 ** IS1296 transposase protein B Transposition 0.0018 rpoA DNA-directed RNA polymerase, alpha subunit Transcription 0.0070 MMSYN1_0187 ** Transcriptional regulator, GntR family Transcription (regulation) 0.012 lpdA Dihydrolipoyl dehydrogenase Central glucose metabolism < 0.00032 pyk Pyruvate kinase Central metabolism (glucose metabolism) 0.017 MMSYN1_0641 Metal ABC transporter, permease component Membrane transport < 0.00032 MMSYN1_0030 Uncharacterized ABC transporter, ATP-binding protein Membrane transport 0.018 MMSYN1_0339 ** Na+ ABC transporter, ATP-binding component Membrane transport 0.018 MMSYN1_0892 ** Putative membrane protein Membrane transport? 0.017 tetM Tetracycline resistance ribosomal protection Genetic tools 0.018 MMSYN1_0471 ** Hypothetical protein 0.00032 MMSYN1_0751 ** Putative viral A-type inclusion protein 0.016 MMSYN1_0898 ** Conserved hypothetical protein 0.034 Adaptation experiment: target genes
- 26. Significant mutations in minimal cell (JCVI-syn3B) ++ = nonessential Gene Annotation Category Padj (B-H) ftsZ ++ Cell division protein ftsZ Cell division < 0.00022 pyrG CTP synthase DNA metabolism 0.00022 dnaN DNA polymerase III subunit beta DNA replication 0.000073 rpoC DNA-directed RNA polymerase, beta' subunit Transcription (stress response) 0.00097 JCVISYN3A_0430 Uncharacterized DNA-binding protein Transcription? (putative) 0.0024 rpsC 30S ribosomal protein S3 Translation 0.019 atpD F0F1 ATP synthase subunit beta Central metabolism (ATP metabolism) < 0.00022 amiF Oligopeptide ABC transporter ATP-binding Membrane transport 0.0033 fakA Fatty acid kinase subunit A Lipid metabolism 0.0021 lgt Diacylglyceryl transferase Lipid metabolism (lipoprotein biosynthesis) 0.0055 clsA Cardiolipin synthase Lipid metabolism (phospholipid biosynthesis) 0.020 tetM Tetracycline resistance ribosomal protection Genetic tools 0.0035 JCVISYN3A_0373 Uncharacterized protein 0.021 JCVISYN3A_0691 Uncharacterized protein 0.032 Adaptation experiment: target genes
- 27. Minimal: • Transcription • DNA replication • Lipid metabolism Wildtype: • Transcription • Glucose metabolism • Membrane transport Both: • Cell division
- 28. • Cell size is positively correlated with growth rate, one of the most important fitness components • Cell size may be controlled through mechanisms related to the cell division protein FtsZ • ftsZ is the only native gene that is significant for both strains Phenotypic evolution: cell size
- 29. Minimization alters evolution of cell size • Similar ancestral cell sizes • Evolution of cell size in wild type: increased by 100% (consistent w/ Lenski and Travisano 1994, PNAS) • Size of minimal cell did not change over 2000 generations • Suggests minimization may constrain evolution of cell size
- 30. Questions and expectations • How does genome minimization affect rate and outcome of evolution? • With removal of DNA repair genes, mutation rate and spectrum may change, in turn affecting the ability to adapt • With fewer genetic targets for natural selection, adaptation could be slower • What are the mechanisms of (re-)adaptation?
- 31. • To investigate effects of specific mutations, introduce putatively adaptive mutations into clean ancestral strain background (including ftsZ mutations to investigate cell size) Future directions
- 32. • To investigate effects of specific mutations, introduce putatively adaptive mutations into clean ancestral strain background (including ftsZ mutations to investigate cell size) • Evaluate cell size evolution hypotheses Future directions
- 33. • Minimal organisms may have much to teach • Minimal cell can adapt at same rate as wildtype • Different genetic mechanisms and phenotypes, which were likely not due merely to altered availability of mutations Conclusions
- 34. • Implications for synthetic biology problem-solving (desirable and undesirable) • Minimal organisms in nature • Natural selection and evolution as universal properties Implications
- 38. Kept Deleted Hutchison et al. 2016 Science
- 39. Balish 2014 (J. Bacteriol.)
- 40. Turbidity of ancestral and evolved bacterial cultures • Blue: wildtype • Red: minimal • Dashed lines: ancestral values • Symbols: evolved replicates • Error bars: ± 2 SEM • Turbidity of minimal cell cultures was negligible in ancestor. • Minimal cell partially recovered through evolution.
- 44. Wildtype evolved
- 45. Minimal ancestor
- 46. Minimal evolved
Editor's Notes
- For example,in one study they deleted all cytochrome oxidases from E. coli, reducing oxygen uptake by 85%. These mutants, though, evolved to regain oxygen uptake ability by evolved higher expression of ygiN (quinol monooxygenase) and sodAB, regained the ability to ferment glucose to lactate. Ruin glucose metabolism Or NADPH production Then see whether the organisms can evolve to overcome that---the mechanism by which they do it teaches about the possible ways to perform that function
- > I owe a debt of gratitude to everyone who helped manually, intellectually, and with critical thinking.
- HWE is where we start. Then start violating assumptions to see what happens, start to understand that law of evolution. But it is still not great, it is difficult to start accounting for the biology of real organisms And EC and SC are not great either. They are used to model other organisms; we often find mechanisms in EC first, and then use that information to learn about other interesting organisms--- CC, AT, Vcholerae, for example. Or we study yeast because we really want to learn about stem cells, which also divide in an asymmtrcl fashion. But, EC and SC are complex cells with thousands of genes, many of which are still uncharrzd
- >>This has been seen as a crowning achievement of synthetic biology. One thing that was observed: The minimal cell was kind of sick. It’ doubling time was twice as lo g as the WT from which it was derived. This is MAYBE NOT SURPRISING---unlike every other organism in existence, this bacterium has NOT been honed by billions of years of natural selection. It was created by humans, and humans have limited “knowedlge” compared to the achievements of nature. But there’s a natural follow-up question: HOW WOULD IT EVOLVE? So on the one hand it’s a test of this evolution-in-the-simplest-case. And on the other hand it’s an organism with a lot of adapting to do.
- For all of these questions: COMPARE the minimal TO THE WILDTYPE. The other questions are all subquestions or ways of getting at the first, main question General idea that it might constrain evolution. Among those essential proteins, though, might see accelerated evolution by positive selection. And while there is literature on genome streamlining– Moran and the genomes of endosymbionts; Morris’s BQH--- gap exists in the literature when it comes to the effects of genome streamlining on subsequent ability to evolve/adapt.
- For all of these questions: COMPARE the minimal TO THE WILDTYPE. General idea that it might constrain evolution. Among those essential proteins, though, might see accelerated evolution by positive selection. And while there is literature on genome streamlining– Moran and the genomes of endosymbionts; Morris’s BQH--- gap exists in the literature when it comes to the effects of genome streamlining on subsequent ability to evolve/adapt.
- For example,in one study they deleted all cytochrome oxidases from E. coli, reducing oxygen uptake by 85%. These mutants, though, evolved to regain oxygen uptake ability by evolved higher expression of ygiN (quinol monooxygenase) and sodAB, regained the ability to ferment glucose to lactate. Ruin glucose metabolism Or NADPH production Then see whether the organisms can evolve to overcome that---the mechanism by which they do it teaches about the possible ways to perform that function General idea that it might constrain evolution. Among those essential proteins, though, might see accelerated evolution by positive selection. And while there is literature on genome streamlining– Moran and the genomes of endosymbionts; Morris’s BQH--- gap exists in the literature when it comes to the effects of genome streamlining on subsequent ability to evolve/adapt.
- Similarly, we found that the mutational spectrum was very similar between the two strains when we looked at the proportions of insertions, deletions, and single nucleotide mutations. There was some evidence for stronger deletion bias in the wildtype, suggesting that minimal cell may be less tolerant of deletions….especially large ones, but overall, the mutation spectra for the minimal and wildtype cells were statistically indistinguishable from one another Given that approximately 90% of all mutations fell into the category of single nucleotide mutations (SNMs), we wanted to look at these in a bit more detail. (~2600 mutations in total)
- Discuss ung
- In contrast to the previous two slides, we found that the spectrum of single nucleotide mutation (SNMs) was significantly affected by genome reduction. On this slide, I want to draw your attention to the third panel on the right-hand side. We observed that there was a high degree of bias towards AT mutations. Higher than any other bacterium known. In fact, if this to-AT biased mutation went entirely unopposed by selection, wild Mycoplasma mycoides would end up with a genomic GC content of … 3%. The GC content of the genome is very low, 24%, but it’s also clear that there is natural selection against SUCH an extreme genome composition, keeping the gc above 3%! In particular, 85% of all the SNM resulted in AT bias, which is consistent with Mycoplasma mycoides having the highest genome-wide AT content of any bacterium (76%) Are there any explanations for the AT biased mutations? Both the wildtype and minimal cell lack the gene dut which encodes for the enzyme that prevent incorporation of uracil into DNA, which results in pairing with adenine. But there’s still this DIFFERENCE in the minimal, which can be explained mechanistically….
- In contrast to the previous two slides, we found that the spectrum of single nucleotide mutation (SNMs) was significantly affected by genome reduction. On this slide, I want to draw your attention to the third panel on the right-hand side. We observed that there was a high degree of bias towards AT mutations. Higher than any other bacterium known. In fact, if this to-AT biased mutation went entirely unopposed by selection, wild Mycoplasma mycoides would end up with a genomic GC content of … 3%. The GC content of the genome is very low, 24%, but it’s also clear that there is natural selection against SUCH an extreme genome composition, keeping the gc above 3%! In particular, 85% of all the SNM resulted in AT bias, which is consistent with Mycoplasma mycoides having the highest genome-wide AT content of any bacterium (76%) Are there any explanations for the AT biased mutations? Both the wildtype and minimal cell lack the gene dut which encodes for the enzyme that prevent incorporation of uracil into DNA, which results in pairing with adenine. But there’s still this DIFFERENCE in the minimal, which can be explained mechanistically….
- Discuss ung Uracil-DNA glycosylase
- Discuss ung
- Discuss ung So this is all quite mechanistic and just one example, but the idea here is that we’re able to look at the two organisms closely side by side, and come to better understandings of the observed patterns.
- More generally, what were the important things that we learned from the MA? Mutation rate wasn’t change. Some, explainable differences in the proportions of SNM types. We were NOT mutation limited---every nt was getting sampled about 2000 times during the evolution (based ont the rate) The types of mutations were largely similar, with a couple of small differences, and those differences could be explained CT bias)
- For all of these questions: COMPARE the minimal TO THE WILDTYPE. General idea that it might constrain evolution. Among those essential proteins, though, might see accelerated evolution by positive selection. And while there is literature on genome streamlining– Moran and the genomes of endosymbionts; Morris’s BQH--- gap exists in the literature when it comes to the effects of genome streamlining on subsequent ability to evolve/adapt.
- AND WE OBSERVED the same trend when we measured OD. Minimal started out a lot lower. Then, they both improved at the same rate.
- AND WE OBSERVED the same trend when we measured OD. Minimal started out a lot lower. Then, they both improved at the same rate.
- AND WE OBSERVED the same trend when we measured OD. Minimal started out a lot lower. Then, they both improved at the same rate.
- PCoA on Bray-Curtis distances plotted with 95% confidence ellipses Only shared genes were included Bray-Curtis distances evaluated for significance via PERMANOVA on principle coordinates (P = 0.029) Cool/major finding/takehome. What are consequences of this? Complexity even in the simplest of cells! What does this mean for synthetic biology? Evolutionary biology? Can we circle back on our set-up in the Intro?
- For all of these questions: COMPARE the minimal TO THE WILDTYPE. General idea that it might constrain evolution. Among those essential proteins, though, might see accelerated evolution by positive selection. And while there is literature on genome streamlining– Moran and the genomes of endosymbionts; Morris’s BQH--- gap exists in the literature when it comes to the effects of genome streamlining on subsequent ability to evolve/adapt.
- CATEGORIES OF GENES?????????
- Remind some of the expectations Im/exporters? Glucose metabolism? !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
- First thing to notice: HALF of the significant genes are essential in the WT Remind some of the expectations Im/exporters? Glucose metabolism?
- Ask the microbiologists! Why would this be???? This is a work in progress, if u have ideas, let us know Also talk about the function of ftsZ in Mycoplasma species?
- However, the composition of these essential and potentially adaptive mutations are completely distinct. While there is some overlap in general categories, there is no overlap in the identity of the putatively adapted essential genes that were mutated. Suggests, that even in the minimal cell, there are different genetic pathways to achieve the same fitness gains. But keeping in mind the figure is misleading because the blue bracket includes nonessential genes while the blue oval includes only essential genes
- Also talk about the function of ftsZ in Mycoplasma species?
- For all of these questions: COMPARE the minimal TO THE WILDTYPE. General idea that it might constrain evolution. Among those essential proteins, though, might see accelerated evolution by positive selection. And while there is literature on genome streamlining– Moran and the genomes of endosymbionts; Morris’s BQH--- gap exists in the literature when it comes to the effects of genome streamlining on subsequent ability to evolve/adapt.
- So let’s go back to the original problem: We need to use simple systems to understand more complicated ones. I made some predictions about the way a minimal organism should behave during evolution, and I was NOT able to simply predict its behavior from principles. In fact we found…
- /…. …. When you have Darwin’s criteria blah blah blah. We observed some surprising, or perhaps maybe very unsurprising, patterns of natural selection in an entirely unnatural organism. And I think that that drives home the universal nature of natural selection and evolution that will always be occurring wherever you have imperfectly self-replicating populations of entities.
- Mutation spectrum in significant genes in NSE
- “But, in the process of minimization, there is a lot that changes compared to a more typical, more complex cell…” Here, include table of DNA repair/replication genes that were deleted. Remember, THAT IS WHAT THE MICROBIOLOGISTS LOVE.
- “But, in the process of minimization, there is a lot that changes compared to a more typical, more complex cell…” Here, include table of DNA repair/replication genes that were deleted. Remember, THAT IS WHAT THE MICROBIOLOGISTS LOVE.
- Cell division in M. pneumoniae
- s1
- 1_6 evolved
- s1
- 1_1 evolved
- 3b anc
- b_3 evolved