Some like it cold

What can microbial genomes tell us about life's extremes?

Neil SaundersSchool of Molecular and Microbial Sciences, UQ

We live on a cold planet

80% of the biosphere is permanently < 5 °C

Microbial diversity in cold environments

Karl et al. (2001). Nature 409: 507-510

● Estimates 1.3 x 1028 archaeal cells, 3.1 x 1028 bacterial cells in the oceans

● Other cold environments: polar and alpine regions, permafrost, subsurface

● Non-terrestrial environments?

● Ecological significance: carbon cycle, methane

● Biotechnological potential: enzymes

Archaea and extremophiles

● Archaea are the third domain of life, distinct from Bacteria and Eukarya

● Many (but not all) Archaea are extremophiles

Temperature extremes

psychrophile < 20 °Cmesophile 20 - 45 °Cthermophile > 45 °Chyperthermophile > 80 °C

Archaeal isolates from Antarctica

Isolates Topt

Tmin

● Methanogenium frigidum 15 °C < 0 °C● Methanococcoides burtonii 23 °C < 0 °C● Halorubrum lacusprofundi

Ace Lake, Vestfold Hills, Antarctica

● Moderately saline

● Perennially 1-2 °C

● Methane-saturated

Shotgun sequencing of archaeal genomes

Sequencing centres● Joint Genome Institute● Molecular Dynamics/Genome Applications● AGRF

StatisticsM. frigidum ~ 2x coverage, 10 000 readsM. burtonii ~ 12x coverage, 50 000 reads

Computational infrastructure for genomics

"So what new skills will postdocs need to ensure that they don't become science relics? The answer is math,statistics, and knowledge of a scripting language for computers."

-The Scientist, "Bioinformatics Knowledge Vital to Careers"Volume 16 | Issue 17 | 53 | Sep. 2, 2002www.the-scientist.com

Computational infrastructure for genomics

Key points● Linux!● Perl/BioPerl● Free, open-source● Many tools + “glue”● Never-ending...

Genome

Assembly

Gene sequence

Protein sequence

Protein structure

Pathway

Computationalobjects

Hardware● Workstation?● Cluster?

Software● Linux● Databases● Web servers● Toolkits/libraries● Scripts/compiled● Open source

Biologicalobjects

Analysis(limitless)

Comparative genomics

Pathway reconstruction

Phylogeny

Structural modeling

Sequence analysis

Regulatory motifs

“Global” genomic features of cold-adapted prokaryotes

Is there anything “obviously different” about genes and proteins from psychrophilic prokaryotes?

● Amino acid composition and protein structure

● Novel gene products

● Structural RNA features

Amino acid composition of the proteome

Archaea27 organisms62 338 ORFs

Bacteria52 organisms165 192 ORFs

Amino acid frequency(bioperl)

PCAprincipal components

(R stats package)

data matrixorganisms (rows) x

composition (columns)

Statistical analysis of amino acid composition

Archaea Amino acid composition v. PC2

27 organisms Asp 0.66His 0.53

PC1 v. GC -0.95 Leu -0.91PC2 v. OGT -0.94 Gln 0.61

Ser 0.57Thr 0.72Trp -0.68

Statistical analysis of amino acid composition

Bacteria Amino acid composition v. PC2

52 organisms Asp 0.71Glu -0.74

PC1 v. GC 0.96 His 0.56PC2 v. OGT -0.81 Leu -0.41

Met 0.55Gln 0.55Ser 0.67Thr 0.74

Protein structure homology modeling

Archaea27 organisms62 338 ORFs

Bacteria52 organisms165 192 ORFs

BLAST v. PDBselect templates

PROSPECTmodeller script

MODELLER5 513 raw models 20 785 raw models

ProCheckg-factor > -0.53 383 models 13 966 models

DSSP3 207 models 13 035 models

For the set of models from each organism, calculatefraction of each residue that is solvent-accessible

Analyse using LDA

Analysis of homology models

Archaea Bacteria

LD1 v. OGT 0.89 LD1 v. OGT 0.84

Ala -0.78 Ala -0.41Asp -0.63 Asp -0.73Ser -0.62 His -0.41Thr -0.85 Ser -0.38

Thr -0.46Trp 0.40Tyr 0.39

Proteins: summary

● Psychrophiles, mesophiles and thermophiles can be distinguished by the amino acid composition of the proteome

CompositionIn the direction thermophile psychrophile we see:

● increase in non-charged polar (Gln, Ser, Thr), His and Asp● decrease in hydrophobic (Leu, Trp) and Glu

Accessible surface● The 3 thermal classes of organism can also be distinguished by the

degree to which certain residues are solvent-accessible● In general, Asp, Ala, Ser and Thr are more exposed in proteins from

psychrophiles versus thermophiles

Biological rationales● Thermal denaturation: Gln (deamidation), Thr (peptide cleavage)● Thermostability: Glu (surface salt bridges), hydrophobic core● Low temperature function: increased global/local flexibility?

surface destabilisation (hydrophobic) ?avoid aggregation (polar non-charged) ?

Analysis of structural RNA

OGT (°C)%

GC

Is tRNA GC content related to OGT?

● Use tRNAScan to find tRNA in archaeal genomes

● Calculate mean GC content for each organism

stems

all bases

GC content becomes significant only above ~ 60 °C

Flexibility and nucleoside modificationM. burtonii tRNA contains > 1 dihydrouridine/molecule(Noon et al. 2003, J. Bact. 185: 5483)

Cold shock protein in M. frigidum

● First CSP identified in a psychrophilic archaeon

● Contains all conserved residues for RNA binding

● Is being functionally and structurally characterised

CSD-like proteins in M. burtonii● No CSP homologue identified in M. burtonii

● csp mutants of E. coli can be complemented by proteins with a CSD-fold

● Does M. burtonii express novel CSD-like proteins?

d1sro__ M. burtonii YP_564958

Protein sequences

PROSPECTthread v. CSD folds

MODELLERstructural model

Proteomic studies of M. burtonii

● What's expressed at 4 °C ?

● What's different at 4 °C versus 23 °C ?

● Protein identification is easy witha genome sequence!

● Work performed by Amber Goodchild at the BMSF, UNSW

● 2D-PAGE and LC MS/MS both employed

What's different between 4 °C and 23 °C?

● 237 spots analysed● 21 spots more intense at 4 °C● 33 spots more intense at 23 °C● 19/21 and 24/33 identified

Upregulated 4 °C● RNAP subunit E● Methanogenesis● Acetate -> amino acid biosynthesis● CheY-like response regulator● Peptidyl prolyl cis/trans isomerase

Upregulated 23 °C● DnaK/HSP70

Goodchild et al. (2004b). Mol. Microbiol. 53: 309

Protein modifications and new amino acids● Several spot patterns indicate PTM● Trimethylamine methyltransferase

(TMA-MT) maps to 2 ORFs● This results from read-through of an

in-frame amber UAG codon

● The amino acid incorporated at the UAGis pyrrolysine - the 22nd genetically-encodedamino acid.

Hao et al. (2002). Science 296: 1459.

What's expressed at 4 °C? LC MS/MS

Goodchild et al. (2004a). J. Prot. Res. 3: 1164

● 528 proteins identified● ~ 23% of the proteome● Proteins annotated and classified

by (1) biological process, (2) genome organisation

● 135 hypothetical/conserved hypothetical proteins analysed separately

DNA replication/processing

Transposases

Cell division/chromosome partitioningDefense mechanisms

RNA synthesis/processingSignal transduction

Motility

Protein synthesis/processingProtein PTM/degradation/folding

Cell envelopeTransport

Methanogenesis

Energy production/conversion

Carbon fixation/carbohydrate metabolism

Nucleotide metabolismAmino acid metabolism

Coenzyme metabolismUnassigned

Some key processes● Expression of 2 transposases● Protein folding (chaperones,

chaperonins, isomerases)● RNA and protein processing

(exosome/proteasome superoperon)● Our predicted CSD-like proteins are

part of the putative exosome

Goodchild et al. (2004a). J. Prot. Res. 3: 1164

Putative exosome/proteasome components

Koonin et al. (2001). Genome Res. 11: 240

Conclusions: the biology● Cold physiology is a complex process; no “gene for cold adaptation”

● Features of psychrophilic archaea include:

➢ Higher proportion of polar non-charged amino acids

➢ More hydrophobic, less charged solvent-accessible surface

➢ Modified structural RNAs for increased flexibility

➢ Membrane lipid unsaturation

➢ Complex transcriptional and translational regulatory networks

➢ Metabolic regulation: energy production v. biosynthesis

➢ Mechanisms to promote proper protein folding

➢ Coupled regulation of RNA/protein synthesis and turnover

Conclusions: the computers

Biological system Biological objects

Computational objects

AnalysesBiological inferences

Generic approach to biological problems

Future directions

● M. burtonii Genome closed, released April 2006

● M. frigidum High coverage draft planned (JCVI)

● H. lacusprofundi Scheduled for sequencing (JGI)

Other UNSW projects

● Sphingopyxis alaskensis Genome closed, due for release

● Marine and environmental microbiology

Pseudoalteromonas tunicata JCVI Vibrio angustum JCVI Roseobacter gallaeciensis JCVI LAS-degrading consortium (3 organisms) JGI

Acknowledgements

UNSW BABS UNSW BMSF UNSW PhysicsRick Cavicchioli Mark Raftery Paul CurmiSohail Siddiqui Mike GuilhausTorsten ThomasAmber GoodchildLaura GiaquintoDominic BurgLily TingDavide de FrancisciCharmaine NgMarilyn Katrib

Sequencing Centres CSIROJoint Genome Institute, CA, USA Peter FranzmannGenomics Applications, CA, USAVenter Institute/Moore Foundation, MD, USAAGRF, Brisbane

Methanogenesis(CH

3)

3NH+

MttP

(CH3)

3NH+

MttB

MtbB

MtmB

MttC

MtbC

MtmC

CoM-SH

CoM-S-CH3

CoB-SHMcr

CH4

CoM-S-S-CoB

(CH3)

2HNH+

Hdr

A1AO

CH3H

2NH+

Fpo

MP

MPH2

CoM-SH + CoB-SH

F420H2

F420

2H+ 2H+

MtrCoM-SH H4-MPT

H4-MPT-CH3

NH4

+

Mer

H4-MPT=CH2

Mtd

H4-MPT≡CH

Fol/Ftr

H+

ADP + Pi

ATP

Na+

MF

H4-MPT

CHO-MF

Fwd

CO2 + 2H+ + MF

F420

F420

F420H2

F420H2

FrhF420

F420H2

2H+ H2

Na+

MtbA

CH3