shilpa nadimpalli shiho tanaka todd yeates, phd.; ucla socal bsi 2008
TRANSCRIPT
Shilpa NadimpalliShiho Tanaka
Todd Yeates, PhD.; UCLA
SoCal BSI 2008
Introduction /Background
Info
Purpose / Benefits
Project Overview
Methodologies
Results
Future Directions
Acknowledgements / References
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Membrane-bound organelles
NUCLEUS
NO significant compartmentalization…
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).
ICOSAHEDRON
Proteinaceous shell
Functionally related enzymes
Better understanding of various metabolic types of BMCs
Applications in protein engineering (medical purposes)
α-carboxysome
β-carboxysome
ethanolamine utilization (eut) microcompartment
propanediol utilization (pdu) microcompartment
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
RuBisCO
CA
3-PGA
RuBP
CO2H2O
HCO3-
H+
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
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
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
Metagenome Database
BLAST
RPSBLAST
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
17,386,449461
181
Peptides with a match to a BMC
shell protein
Neighboring peptides
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
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
Human Gut Database University of Tokyo Whole genome shotgun
(WGS) sequencing project 13 human samples Available December 2007 NO ANNOTATIONS
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
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
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
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)
Carboxy-some
Ethanol-amine
Utilization
Propanediol Utilization
?
ALL reads with a BMC
domain
Anaerobic Glucose Metabolism Pyruvate formate lyase (Pfl1, Pfl2,
PflA) Pyruvate + coenzyme-A → formate +
acetyl-CoA
Respiratory/Electron Transport Chain?
RnfC – NADH:ubiquinone oxidoreductase
Neighbor Cluster Algorithm
GOS
Human GutFresh
Water Lake
Farm Soil
Hot Spring
Coastal Microbia
Carboxysome
Pdu
Eut
Cobalamin transfer
Electron Transport
?
?
?
?
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
Todd Yeates, PhD; Shiho Tanaka; Neil King
National Science Foundation (NSF)
National Institutes of Health (NIH)
Economic & Workforce Development
United States Department of Energy (DOE)
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).