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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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shilpa nadimpalli shiho tanaka todd yeates phd ucla socal bsi 2008
Shilpa Nadimpalli

Shiho Tanaka

Todd Yeates, PhD.; UCLA

SoCal BSI 2008

Discovery of Novel Metabolic Types of Bacterial Microcompartments

what s to come

1

Introduction /

Background Info

Purpose /

Benefits

2

Project Overview

3

What\'s to Come…

4

Methodologies

5

Results

7

6

Future Directions

Acknowledgements / References

what is a bacterial microcompartment bmc

Membrane-bound organelles

Prokaryote

What IS a "Bacterial Microcompartment" (BMC)?

NUCLEUS

Eukaryote

NO significant

compartmentalization…

slide4

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).

slide5

What BMCs really look like…

A

B

C

D

E

F

G

H

β-carboxysomes

Synechocystis sp. PCC 6803 (cyanobacteria)

α-carboxysomes

Halothiobacillusneopolitanus (chemoautotrophs)

slide6

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

slide7

Why do we care?

  • Better understanding of various metabolic types of BMCs
  • Applications in protein engineering (medical purposes)
slide8

Currently Known BMC Types:

α-carboxysome

β-carboxysome

ethanolamine utilization (eut) microcompartment

propanediol utilization (pdu) microcompartment

slide9

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
slide10

Carboxysome Function

HCO3-

RuBP

H+

CA

CO2

H2O

RuBisCO

3-PGA

slide11

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
slide12

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

slide13

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

slide14

Tools to Use

  • Metagenome Database
  • BLAST
  • RPSBLAST
slide15

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
slide16

GOS Database

Peptides with a match to a BMC shell protein

181

Neighboring peptides

17,386,449

461

slide17

1

Results:

Grouping of Neighbors

slide18

2

Results:

Grouping of Neighbors

slide19

Second Metagenome

  • Human Gut Database
  • University of Tokyo
  • Whole genome shotgun (WGS) sequencing project
  • 13 human samples
  • Available December 2007
  • NO ANNOTATIONS
slide20

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)

slide21

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
slide22

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

slide23

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)
slide24

Next Step: Clustering

Propanediol Utilization

Carboxy-some

ALL reads with a BMC domain

?

Ethanol-amine Utilization

slide25

(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
slide26

Future Steps:

Carboxysome

GOS

Pdu

?

Human Gut

Neighbor

Cluster

Algorithm

Fresh Water Lake

Eut

?

Farm Soil

Cobalamin transfer

Hot Spring

?

Coastal Microbia

Electron Transport

?

thanks to socal bsi

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

thanks to ucla

Thanks to UCLA

Todd Yeates, PhD; Shiho Tanaka; Neil King

funding

National Science Foundation (NSF)

National Institutes of Health (NIH)

Funding

Economic & Workforce Development

United States Department of Energy (DOE)

references

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

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