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Discovery of Novel Metabolic Types of Bacterial Microcompartments

Shilpa Nadimpalli Shiho Tanaka Todd Yeates, PhD.; UCLA SoCal BSI 2008. Discovery of Novel Metabolic Types of Bacterial Microcompartments. 1. Introduction / Background Info. Purpose / Benefits. 2. Project Overview. 3. What's to Come…. 4. Methodologies. 5. Results. 7. 6.

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Discovery of Novel Metabolic Types of Bacterial Microcompartments

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  1. Shilpa Nadimpalli Shiho Tanaka Todd Yeates, PhD.; UCLA SoCal BSI 2008 Discovery of Novel Metabolic Types of Bacterial Microcompartments

  2. 1 Introduction / Background Info Purpose / Benefits 2 Project Overview 3 What's to Come… 4 Methodologies 5 Results 7 6 Future Directions Acknowledgements / References

  3. Membrane-bound organelles Prokaryote What IS a "Bacterial Microcompartment" (BMC)? NUCLEUS Eukaryote NO significant compartmentalization…

  4. Compartments in bacteria? Phormidium uncinatum (cyanobacteria) 1956 Drews, G. & Niklowitz, W. Beiträge zur Cytologie der Blaualgen. II. Zentroplasma und granulare Einschlüsse von Phormidium uncinatum. Arch. Mikrobiol. 24, 147-162 (1956).

  5. What BMCs really look like… A B C D E F G H β-carboxysomes Synechocystis sp. PCC 6803 (cyanobacteria) α-carboxysomes Halothiobacillusneopolitanus (chemoautotrophs)

  6. What BMCs really look like… I J K L ICOSAHEDRON Proteinaceous shell P M N O 1,2-propanediol utilization (pdu) microcompartments Salmonella enterica, serovarTyphimurium LT2 ethanolamine utilization (eut) microcompartments Salmonella enterica, serovarTyphimurium LT2 Functionally related enzymes carboxysomes Synechococcus sp. & H. neopolitanus

  7. Why do we care? • Better understanding of various metabolic types of BMCs • Applications in protein engineering (medical purposes)

  8. Currently Known BMC Types: α-carboxysome β-carboxysome ethanolamine utilization (eut) microcompartment propanediol utilization (pdu) microcompartment

  9. Currently Known BMC Types: • Carboxysomes: • Found in some chemoautotrophs and all cyanobacteria • ~1200 Å in diameter • Contain CO2-fixing enzymes (RuBisCO & carbonic anhydrase) • α-type: • Contain Form1A RuBisCO • β-type: • Contain Form1B RuBisCO

  10. Carboxysome Function HCO3- RuBP H+ CA CO2 H2O RuBisCO 3-PGA

  11. Currently Known BMC Types: • Eut & Pdu BMCs: • Found in some heterotrophic bacteria (Salmonella & E. coli) • Larger, more contorted compartments • Involved in cobalamin-dependent degradation of ethanolamine • Involved in B12-dependent catabolism of 1,2-propanediol

  12. Operons encoding BMCs: α-carboxysome Halothiobacillus neapolitanus S S3 B B cbb L cso S2 orf A csoS1 C A β-carboxysome Synechocystis sp. PCC6803 ccm K4 N O rbc S cca A K3 K2 K1 L M X L EUT microcompartment Salmonella typhimurium mez tnpA G K R orf-79 hemF eut S P Q T D M N E J H A B C L PDU microcompartment Salmonella enterica pocR pduF X pdu A B C D E G H J K L M N O P Q S T U V W

  13. How do I discover a new metabolic type of BMC? Identify metagenomes to search Search for shell proteins Analyze neighboring peptides Group neighbors by function Look at close-by / neighboring peptides (to shell protein) Search for occurrences of BMC domain / shell proteins 1 2 3 4 Identify metagenomes to search Group neighbor proteins by function

  14. Tools to Use • Metagenome Database • BLAST • RPSBLAST

  15. Part 1: Identify Metagenomes • Global Ocean Sampling (GOS) Database • J. Craig Venter Institute • Made from oceanic microbes (collected during the Sorcerer II expedition) • Available March 2007 • WELL ANNOTATED • Human Gut Database

  16. GOS Database Peptides with a match to a BMC shell protein 181 Neighboring peptides 17,386,449 461

  17. 1 Results: Grouping of Neighbors

  18. 2 Results: Grouping of Neighbors

  19. Second Metagenome • Human Gut Database • University of Tokyo • Whole genome shotgun (WGS) sequencing project • 13 human samples • Available December 2007 • NO ANNOTATIONS

  20. Human Gut Database Peptides with a match to a BMC shell protein 0 A T G C G . . . . . . . . . . A T A 16589 0 C C T G A . . . . . . . . . . C G C 17834 0 G T A A A . . . . . . . . . . T A G 15530 0 T G A C A . . . . . . . . . . G G T 18904 0 A A T C A . . . . . . . . . . G A C 16913 0 C G T G C . . . . . . . . . . T A T 13012 0 C G T A C . . . . . . . . . . A T C 950 0 C T G A C . . . . . . . . . . A A A 1498 386,608 99 99 Sequences containing AT LEAST ONE BMC region (~17,000 bp each)

  21. How do I find the neighboring peptides? • Approach 1: • Look at 600bp sequences directly before and after matched region • Approach 2: • Find domains from start to end of whole sequences, then look at the neighboring domains

  22. Approach #2: C C A A C G T A C A G T A C A G T A C A A C C G T A C A C A G T A C A C A C A G T T T T G A C A C A C G T G A C A C A T T G A C A C A C G T T G G C A C A C A C A C G T G G T C A C A C G T A C T C T C T A G T A C A G T A C A G T A C A G T A C A G T T T T G A C A C A C G T G A C A C A T T G A C A C A C G T T G G C A C A C A C A C G T G G T C A C A C BMC BMC

  23. Problems with 2nd Approach: • Fragmentation • “Stitching” fragments together to determine full domains is difficult • Fused Domains • “Neighboring” domains may actually be fused to BMC shell proteins = same peptide (no open reading frames annotated)

  24. Next Step: Clustering Propanediol Utilization Carboxy-some ALL reads with a BMC domain ? Ethanol-amine Utilization

  25. (Current) Results • Anaerobic Glucose Metabolism • Pyruvate formate lyase (Pfl1, Pfl2, PflA) • Pyruvate + coenzyme-A → formate + acetyl-CoA • Respiratory/Electron Transport Chain? • RnfC – NADH:ubiquinone oxidoreductase

  26. Future Steps: Carboxysome GOS Pdu ? Human Gut Neighbor Cluster Algorithm Fresh Water Lake Eut ? Farm Soil Cobalamin transfer Hot Spring ? Coastal Microbia Electron Transport ?

  27. Ronnie Cheng Program Coordinator Dr. Wendie Johnston Professional Development Dr. Jamil Momand Program Director Dr. Beverly Krilowicz Program Evaluator Thanks to SoCal BSI Dr. Sandy Sharp Molecular Life Sciences Dr. Silvia Heubach Statistics Dr. Nancy Warter-Perez Python Programming Dr. Jennifer Faust Bioethics

  28. Thanks to UCLA Todd Yeates, PhD; Shiho Tanaka; Neil King

  29. National Science Foundation (NSF) National Institutes of Health (NIH) Funding Economic & Workforce Development United States Department of Energy (DOE)

  30. Cannon, G. C. et al. Microcompartments in prokaryotes: carboxysomes and related polyhedra. Appl Environ Microbiol 67, 5351-61 (2001). Havemann, G. D., Sampson, E. M. & Bobik, T. A. PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2. J Bacteriol 184, 1253-61 (2002). Kofoid, E., Rappleye, C., Stojiljkovic, I. & Roth, J. The 17-gene ethanolamine (eut) operon of Salmonella typhimurium encodes five homologues of carboxysome shell proteins. J Bacteriol 181, 5317-29 (1999). Yeates T. O., Kerfeld C. A., Heinhorst S., Cannon G. C., Shively J. M. Protein-based organelles in bacteria: carboxysomes and related microcompartments. Nat Rev Microbiol (2008). References

  31. Questions?

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