Discovery of Novel Metabolic Types of Bacterial Microcompartments
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Transcript of Discovery of Novel Metabolic Types of Bacterial Microcompartments
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Shilpa NadimpalliShiho Tanaka
Todd Yeates, PhD.; UCLA
SoCal BSI 2008
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Introduction /Background
Info
Purpose / Benefits
Project Overview
Methodologies
Results
Future Directions
Acknowledgements / References
1 2
3
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6 7
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Membrane-bound organelles
NUCLEUS
NO significant compartmentalization…
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Phormidium uncinatum(cyanobacteria)
Drews, G. & Niklowitz, W. Beiträge zur Cytologie der Blaualgen. II. Zentroplasma und granulare Einschlüsse von Phormidium uncinatum. Arch. Mikrobiol. 24, 147-162 (1956).
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ICOSAHEDRON
Proteinaceous shell
Functionally related enzymes
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Better understanding of various metabolic types of BMCs
Applications in protein engineering (medical purposes)
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α-carboxysome
β-carboxysome
ethanolamine utilization (eut) microcompartment
propanediol utilization (pdu) microcompartment
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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
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RuBisCO
CA
3-PGA
RuBP
CO2H2O
HCO3-
H+
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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
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α-carboxysome
β-carboxysome
EUT microcompartment
PDU microcompartment
S S3 B Bcbb L cso S2 orf A csoS1 C A
Nccm K4 K3 K2 K1 L M O rbc S X Lcca
A
eut S P Q T D M N E J H A B C Lmez tnpA R orf-79 hemFKG
pocR pdu A B C D E G H J LK M N O P Q S T U V WpduF X
Halothiobacillus neapolitanus
Synechocystis sp. PCC6803
Salmonella typhimurium
Salmonella enterica
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Identify metagenomes to search
Identify metagenomes to search
Search for occurrences of BMC domain / shell proteins
Search for shell proteins
Analyze neighboring
peptides
Group neighbors by
function
Look at close-by / neighboring peptides (to shell protein)
Group neighbor proteins by function
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Metagenome Database
BLAST
RPSBLAST
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Global Ocean Sampling (GOS) Database
Human Gut Database
J. Craig Venter Institute Made from oceanic
microbes (collected during the Sorcerer II expedition)
Available March 2007 WELL ANNOTATED
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17,386,449461
181
Peptides with a match to a BMC
shell protein
Neighboring peptides
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Count PFAM ID Description
73 00016 Ribulose biphosphate carboxylase large chain, catalytic domain
29 00936 BMC domain
21 03319 Ethanolamine utilisation protein EutN/carboxysome structural protein CcmL
11 00171 Aldehyde dehydrogenase family
11 00361 NADH-Ubiquinone/plastoquinone (complex 1), various chains
9 ??? Hypothetical protein
7 01329 Pterin 4 alpha carbinolamine dehydratase
7 00871 Acetokinase family
3 01656 CobQ/CobB/MinD/ParA nucleotide binding domain
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Count PFAM ID Description
3 00210 Ferritin-like domain
1 00120 Glutamine synthetase, catalytic domain
1 02866 Iactate/malate dehydrogenase, alpha/beta C-terminal domain
1 01210 NAD-dependent glycerol-3-phosphate dehydrogenase N-terminus
1 01512 Respiratory-chain NADH dehydrogenase 51 Kd subunit
1 03949 Malic enzyme, NAD binding domain
1 00230 Major intrinsic protein
1 00455 Bacterial regulatory proteins, deoR family
181 total
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Human Gut Database University of Tokyo Whole genome shotgun
(WGS) sequencing project 13 human samples Available December 2007 NO ANNOTATIONS
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386,608(~17,000 bp each)
99
Peptides with a match to a BMC
shell protein
99
0 A T G C G . . . . . . . . . . A T A 165890 C C T G A . . . . . . . . . . C G C 178340 G T A A A . . . . . . . . . . T A G 155300 T G A C A . . . . . . . . . . G G T 189040 A A T C A . . . . . . . . . . G A C 169130 C G T G C . . . . . . . . . . T A T 130120 C G T A C . . . . . . . . . . A T C 9500 C T G A C . . . . . . . . . . A A A 1498
Sequences containing AT LEAST ONE BMC region
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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
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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
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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)
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Carboxy-some
Ethanol-amine
Utilization
Propanediol Utilization
?
ALL reads with a BMC
domain
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Anaerobic Glucose Metabolism Pyruvate formate lyase (Pfl1, Pfl2,
PflA) Pyruvate + coenzyme-A → formate +
acetyl-CoA
Respiratory/Electron Transport Chain?
RnfC – NADH:ubiquinone oxidoreductase
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Neighbor Cluster Algorithm
GOS
Human GutFresh
Water Lake
Farm Soil
Hot Spring
Coastal Microbia
Carboxysome
Pdu
Eut
Cobalamin transfer
Electron Transport
?
?
?
?
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Ronnie ChengProgram Coordinator
Dr. Wendie JohnstonProfessional Development
Dr. Sandy SharpMolecular Life Sciences
Dr. Jamil MomandProgram Director
Dr. Nancy Warter-PerezPython Programming
Dr. Beverly KrilowiczProgram Evaluator
Dr. Silvia HeubachStatistics
Dr. Jennifer FaustBioethics
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Todd Yeates, PhD; Shiho Tanaka; Neil King
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National Science Foundation (NSF)
National Institutes of Health (NIH)
Economic & Workforce Development
United States Department of Energy (DOE)
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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).
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