Genomics of cold-adapted microorganisms

Some like it cold What can microbial genomes tell us about life's extremes? Neil Saunders School 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 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 ● Karl et al. (2001). Nature 409: 507-510

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 °C mesophile 20 - 45 °C thermophile > 45 °C hyperthermophile > 80 °C

Archaeal isolates from Antarctica Moderately saline ● Perennially 1-2 °C ● Methane-saturated ● Ace Lake, Vestfold Hills, Antarctica Isolates Topt Tmin ● Methanogenium frigidum 15 °C < 0 °C ● Methanococcoides burtonii 23 °C < 0 °C ● Halorubrum lacusprofundi

Shotgun sequencing of archaeal genomes Sequencing centres Joint Genome Institute ● ● Molecular Dynamics/Genome Applications ● AGRF Statistics M. frigidum ~ 2x coverage, 10 000 reads M. 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, 2002 www.the-scientist.com

Computational infrastructure for genomics Hardware Biological Analysis ● Workstation? objects (limitless) ● Cluster? Genome Sequence analysis Assembly Regulatory motifs Computational objects Gene sequence Structural modeling Protein sequence Phylogeny Protein structure Comparative genomics Pathway Pathway reconstruction Software ● Linux ● Databases Key points ● Web servers ● Linux! ● Toolkits/libraries ● Perl/BioPerl ● Scripts/compiled ● Free, open-source ● Open source ● Many tools + “glue” ● Never-ending...

“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 Archaea Bacteria 27 organisms 52 organisms 62 338 ORFs 165 192 ORFs Amino acid frequency (bioperl) data matrix organisms (rows) x composition (columns) PCA principal components (R stats package)

Statistical analysis of amino acid composition Archaea Amino acid composition v. PC2 27 organisms Asp 0.66 His 0.53 PC1 v. GC -0.95 Leu -0.91 PC2 v. OGT -0.94 Gln 0.61 Ser 0.57 Thr 0.72 Trp -0.68

Statistical analysis of amino acid composition Bacteria Amino acid composition v. PC2 52 organisms Asp 0.71 Glu -0.74 PC1 v. GC 0.96 His 0.56 PC2 v. OGT -0.81 Leu -0.41 Met 0.55 Gln 0.55 Ser 0.67 Thr 0.74

Protein structure homology modeling Archaea Bacteria BLAST v. PDB 27 organisms 52 organisms select templates 62 338 ORFs 165 192 ORFs PROSPECT modeller script MODELLER 5 513 raw models 20 785 raw models ProCheck 3 383 models 13 966 models g-factor > -0.5 DSSP 3 207 models 13 035 models For the set of models from each organism, calculate fraction 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 -0.41 Asp -0.63 Asp -0.73 Ser -0.62 His -0.41 Thr -0.85 Ser -0.38 Thr -0.46 Trp 0.40 Tyr 0.39

Proteins: summary Psychrophiles, mesophiles and thermophiles can be distinguished by ● the amino acid composition of the proteome Composition In 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 Is tRNA GC content related to OGT? stems Use tRNAScan to find tRNA in ● archaeal genomes % GC Calculate mean GC content for ● each organism all bases OGT (°C) GC content becomes significant only above ~ 60 °C Flexibility and nucleoside modification M. 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? ● Protein sequences PROSPECT thread v. CSD folds MODELLER d1sro__ M. burtonii YP_564958 structural 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 with ● a 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 UAG ● is pyrrolysine - the 22nd genetically-encoded amino acid. Hao et al. (2002). Science 296: 1459.

What's expressed at 4 °C? LC MS/MS 528 proteins identified ● ~ 23% of the proteome ● DNA replication/processing Energy Proteins annotated and classified production/conversion ● Transposases Carbon fixation/carbohydrate Cell division/chromosome metabolism partitioning by (1) biological process, (2) Nucleotide metabolism Defense mechanisms Amino acid metabolism RNA synthesis/processing genome organisation Coenzyme metabolism Signal transduction Unassigned Motility Protein synthesis/processing Goodchild et al. (2004a). J. Prot. Res. 3: 1164 Protein 135 hypothetical/conserved PTM/degradation/folding ● Cell envelope hypothetical proteins analysed Transport Methanogenesis separately Goodchild et al. (2004a). J. Prot. Res. 3: 1164 Some key processes Putative exosome/proteasome components Expression of 2 transposases ● Protein folding (chaperones, ● chaperonins, isomerases) RNA and protein processing ● (exosome/proteasome superoperon) Our predicted CSD-like proteins are ● Koonin et al. (2001). Genome Res. 11: 240 part of the putative exosome

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 Biological inferences Analyses 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 Physics Rick Cavicchioli Mark Raftery Paul Curmi Sohail Siddiqui Mike Guilhaus Torsten Thomas Amber Goodchild Laura Giaquinto Dominic Burg Lily Ting Davide de Francisci Charmaine Ng Marilyn Katrib Sequencing Centres CSIRO Joint Genome Institute, CA, USA Peter Franzmann Genomics Applications, CA, USA Venter Institute/Moore Foundation, MD, USA AGRF, Brisbane

Methanogenesis + (CH3)3NH Na+ MttP Mtr CoM-SH H4-MPT (CH3)3NH+ MttC MttB H4-MPT-CH3 F420 CH3 + CoM-SH (CH3)2HNH Mer F420H2 MtbB MtbA MtbC H4-MPT=CH2 CoM-S-CH3 F420 CH3H2NH+ Mtd MtmC MtmB CoB-SH F420H2 Mcr NH4+ H4-MPT≡CH CH4 MF Fol/Ftr CoM-S-S-CoB H4-MPT CHO-MF Fwd Na+ CoM-SH + CoB-SH CO2 + 2H+ + MF ATP ADP + Pi F420 F420H2 A1AO F420H2 MP F420 Frh Fpo Hdr MPH2 H+ 2H+ H2 2H+ 2H+

Genomics of cold-adapted microorganisms

by Neil Saunders

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