Shilpa Nadimpalli Shiho Tanaka Todd Yeates, PhD.; UCLA SoCal BSI 2008.

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).