the limits to life under dual extremities of high ... · therapies (imct) biomolecular &...

The limits to life under dual extremities of high

temperature and salinity

Bharat PatelSchool of Biomolecular & Biomedical Sciences (BBS), Microbial Gene Research & Resources Facility (MGRRF) andEskitis Institute for Molecular & Cellular Therapies (IMCT), Brisbane, Australia

I. Advantages & disadvantages of molecule choice for phylogenetic reconstruction – a biologists perspective:

•Alignments of gene / protein sequences vs•Alignments of protein sequences with inclusion of structure & functions (3-D protein structures)

II. 16S rRNA gene phylogeny vs genome wide (partial) phylogeny:

•What is the limit to life under two extremities (high temperature & salinity) – Halothermothix orenii, a model bacterium•Do hyperhalothermophiles exist in nature? Should time be spent in searching for such microbes?

III. Structure-Function based phylogeny

CONTENT

PART I

Advantages & disadvantages of molecule choice for phylogenetic reconstruction – a biologists perspective:

• Alignments of gene / protein sequences vs

• Alignments of protein sequences with inclusion of structure & functions (3-D protein structures)

A biologists perspective: Bias in Single Gene Phylogenies & Phylogenomics

• Gene / protein sequences are from the:o Most easily culturable microbeso Most readily available microbeso Most isolated microbes from nature

• Novel gene sequences diluted out / not included

• Alignments are usually generated using orthologous sequences with some possible erroneous annotation in databases & hypotheticals

• The choice of algorithms (estimated vs calculated):o Mutation rates

o Mutation frequencyNot a criticism but “best choice given what is in front of us!!”

Exponential growth of sequence data (great) but annotation errors, redundancy & hypotheticals (not so good!)

3D protein databases- structural and functional attributes known, meticulously curated (great), not

much data (not good) and rarely used for phylogeny

PART II

16S rRNA gene phylogeny vs genome wide (partial) phylogeny:

• What is the limit to life under two extremities (high temperature & salinity) – Halothermothix orenii, a model bacterium

• Do hyperhalothermophiles exist in nature? Should time be spent in searching for such microbes?

8Microbial Gene Research & Resources Facility (MGRRF)Institute for Molecular & Cellular Therapies (IMCT)Biomolecular & Biomedical Sciences (BBS)

In “normal” organisms

High temperatures denature and inactivate important biomolecules.

High salt concentrations aggregate biomolecules & results in the loss of turgor pressure within the cell


Cellular & molecular adaptation strategies extensively studied.

Have evolved specialised adaptations to unique (“extreme”) habitats

Thermophiles to high temperature habitats, halophiles to high salt habitats

Extremophiles


THERMOPHILESStructural Adaptations

Lipid Bilayer StructureCellular Adaptations

Molecular ChaperonesHistone-like DNA Binding Proteins

Molecular AdaptationsExcess glutamate, valine, tyrosine, & proline residuesSalt-bridges, packing density etc.

HALOPHILESStructural Adaptations

Lipid Bilayer StructureCellular Adaptations

Salt-in Cytoplasm StrategyCompatible Solute Strategy

Molecular AdaptationsExcess acidic amino acids

HalothermophilesDo they exist, than their limits to

life??What Adaptation Strategies??

What Adaptation mechanisms??


The surface (exterior) of proteins of halophiles, mesophiles & thermophiles show individual specific AA composition but the AA composition of the core (interior) is similar•Halophiles – surface posses excess acidic amino

acid residues (Asp & Glu)

•Thermophiles – surface posses equal concentrations of acidic & basic AA (Arg, His, Lys)


The thermohalophilic extermophiles

Only two truly halothermophiles known to date:

1Halothermothrix orenii: (optimum 60oC + 10% NaCl; <65oC + < 13% NaCl )

2Thermohalobacter berrensis: (optimum 65oC + 5% NaCl; 70oC with 15% NaCl )

1 Cayol, J-L, Ollivier, B., Patel, B.K.C., Prensier, G., Guezennec, J. and Garcia, J-L (1994). Isolation and characterization of Halothermothrix orenii gen. nov. sp. nov., a halophilic, thermophilic, fermentative strictly anaerobic bacterium. Int. J. of Bacteriol. 44:534-540

2 Cayol, J.-L.,, Ducerf, S., Patel, B.K.C., Garcia, J.-L., Thomas, P. and Ollivier, B. (2000). Thermohalobacter berrensis gen. nov., sp. nov., a thermophilic, strictly halophilic bacterium from a solar saltern. Int. J. Syst. Evol. Microbiol. 50:559–564.

{5M NaCl=~25%=saturated}


Halothermophiles Ph

ylu

m F

irmic

ute

s, d

om

ain

Bacte

ria

Halothermophile phylogeny


Question:

Does H. orenii have an adaptation strategy to counteract halophilic conditions?

Does it posses a “salt in” (protein structure) or a “salt out” (metabolic pathways) strategy?


A Reminder on the adaptive strategies adopted by halophiles

Salt out- biomolecular structures maintained by

cytoplasmic solutes

Salt In- structures maintained by excess acidic

aa in biomolecules


I. A broad genome sequence based approach to answer the question suggests that H. orenii does not use a “salt in” adaptation strategy


1. Constructed a genomic library from H. orenii DNA

2. Sequenced tags of randomly selected clones.

3. Determined ORFs using BLAST against GenBank database

4. Determined aa composition from ORFs

5. Does the aa have an excess acidic amino acidic charge? If yes, than the strategy is “salt in”

Methods


4000 clone genomic library constructed

Estimated bases sequenced: 85 kb

Number of DNA sequencing reactions performed: 172

Number of sequence tags with high scoring matches: 81

Number of sequence tags matching known proteins: 66

Number of sequence tags matching hypothetical conserved proteins: 15

Results from the H. orenii genomic DNA library


Gene Description Organism Length of ORF Score (bits)Intermediate metabolismacetolactate synthase Methanobacterium thermoautotrophicum 92 113acetolactate synthase Synechocystis sp. 89 81.5

adenylosuccinate synthetase Bacillus subtilis 125 158alpha-glucan phosphorylase Pyrococcus abyssi 197 104

aminomethyltransferase Pyrococcus horikoshii 107 100ATP-dependent protease La Bacillus brevis 144 138

C-terminal fumarate hydratase, class I Aquifex aeolicus 80 102diapophytoene dehydrogenase CrtN Heliobacillus mobilis 182 157

esterase* Thermotoga maritima 97 94.4formamidopyrimidine-DNA glycosylase Bacillus firmus 136 129

galactokinase 1 Homo sapiens 174 155gamma-glutamylcysteine synthetase Lycopersicon esculentum 146 79.6

glutamyl-tRNA amidotransferase subunit B Aquifex aeolicus 58 72.6glycerate dehydrogenase Pyrococcus abyssi 153 173

glycerol kinase 2 Thermotoga maritima 54 69.9imidazoleglycerol-phosphate dehydratase Arabidopsis thaliana 134 136imidazoleglycerol-phosphate dehydratase Aquifex aeolicus 114 143

integrase Streptomyces coelicolor 199 95.9lipoate-protein ligase A Escherichia coli 63 71.4

methylcobamide:CoM methyltransferase isozyme A Methanosarcina barkeri 160 74.5Peptidase Bacillus subtilis 120 139

phosphoribosyl formimino-5-aminoimidazole isomerase Thermoanaerobacter ethanolicus 231 136phosphoribosylamine-glycine ligase Bacillus subtilis 148 100

pyrimidine-nucleoside phosphorylase PynP Bacillus stearothermophilus 178 220Pz-Peptidase Bacillus licheniformis 150 133

serine O-acetyltransferase Bacillus stearothermophilus 120 186sucrose phosphate synthase Synechocystis sp. 72 80.4

tryptophanase 1 Symbiobacterium thermophilum 41 60.9Information pathways

aspartyl-tRNA synthetase Bacillus subtilis 175 182DNA gyrase subunit B Clostridium acetobutylicum 209 209

exonuclease SbcD homolog Bacillus subtilis 177 127helicase Bacillus subtilis 148 218

histidyl-tRNA synthetase Aquifex aeolicus 146 68.7histidyl-tRNA synthetase Staphylococcus aureus 150 156m4C-methyltransferase Thermotoga maritima 91 72.6

recombination protein RecF Bacillus subtilis 185 172replicative DNA helicase Deinococcus radiodurans 164 120

Bioenergeticshydrogenase accessory protein HypA Aquifex aeolicus 64 107hydrogenase maturation protein HypF Azotobacter chroococcum 95 84.7

hydrogenase-1 Clostridium thermocellum 200 181periplasmic (Ni-Fe-Se) hydrogenase Desulfomicrobium baculatum 149 102periplasmic (Ni-Fe-Se) hydrogenase Desulfovibrio baculatus 160 101periplasmic (Ni-Fe-Se) hydrogenase Desulfomicrobium baculatum 66 68.3

V-ATPase A subunit* Desulfurococcus sp. SY 150 257Transmembrane transport

manganese transport system membrane protein Methanobacterium thermoautotrophicum 177 108Signal transduction

acid-inducible protein Borrelia burgdorferi 101 61.6chemotaxis response regulator CheY Listeria monocytogenes 33 44.1

chemotaxis sensor histidine kinase CheA Thermotoga maritima 181 141sensor protein DegS Brevibacillus brevis 169 119

transcription regulatory protein RegA Clostridium acetobutylicum 142 113transcriptional repressor CytR Escherichia coli 118 101

two-component sensor histidine kinase YesN Bacillus subtilis 152 89.3vegetative protein CodY Bacillus subtilis 179 123

Cell structure and processesflagellar basal-body rod protein FlgC Thermotoga maritima 102 117

flagellar biosynthetic protein FliP Bacillus subtilis 88 109flagellar hook-associated protein 1 FlgK Treponema pallidum 124 65.6

flagellar motor switch protein FliM Borrelia burgdorferi 156 144flagellor hook basal-body protein FlgG Aquifex aeolicus 195 224

flagellor motor switch protein FliY Treponema pallidum 42 58.2heat shock protein DnaJ Clostridium acetobutylicum 170 170heat shock protein DnaK Bacillus stearothermophilus 131 214heat shock protein GrpE Bacillus stearothermophilus 171 125

penicillin binding protein DacF Bacillus subtilis 177 136RHSD protein precursor Escherichia coli 398 71.4

UTP-glucose-1-phosphate uridylyltransferase Pyrococcus abyssi 72 73


1. H. orenii proteins lack excess acidic amino acids, a hall mark of the “salt in” halophilic adaptation

strategists

-40

-20

0

20

40

60

80

100

120

140

H. orenii

H. praevalens

H. volcanii

A. aeolicusE

xcess A

cid

ic A

min

o

Acid

s

(fr

eq

/ 1

000

)

HaloanaerobialesBacteria

Halophilic Archaea

Deepest member of Bacteria- thermophile


Genus Number of matching

clones

Genome Sequence

Avaliable

Bacillus 25 yes

Aquifex 7 yes

Thermotoga 6 yes

Pyrococcus 5 yes

Synechocystis 5 yes

Clostridium 4 no

Distribution of H. orenii Sequence Tag Matches Amongst Different Genera

2. Instead, the gene sequences of H. orenii closely matches the sequences of true thermophilic and

mesophilic organisms.

???


(A) (B) (C)

3. Structural evidence also points to a lack of surface excess acidic aa on the exposed surface of the H. orenii extracellular -amylase


II. Identification of a novel partial putative operon with a cluster of 6 genes


Direct repeat 1Direct repeat 2

RskA RegA AmyBSpsA ORF2 ORF1

Putative role Sucrose phosphate synthesis

Fructokinase

Regulatory Protein

-Amylase Hypothetical Protein

Hypothetical Protein

Experimental Confirmation

Yes No No Yes No No

Subcloned Yes Yes Yes Yes No No

Overexpressed

Yes Yes Yes Yes No No

Crystallized Yes Current Current Yes No No

X-ray data Yes No No Yes No No

Synchrotron Current No No Yes No No

Structural

Stability Current No No Current No No

New folds Unknown Unknown No No Unknown Unknown

Figure 1: A putative partial operon of H. orenii involved in sugar metabolism and sucrose synthesis.

Genom

e sequencin g at D

OE

-JGI


Operon analysis suggests a “salt out” strategy in H. orenii: Sucrose is a likely

osmoprotectant•A sucrose phosphate synthase (SPS) gene is present

•SPS is unique and not found in 200 microbial genomes sequenced to date

•SPS is similar to plant sequences & not cyanobacteria.

Syn sp.001

Act chi014

H.o SPS032

Cit uns023

Cit uns017

Tri aes028

Mus acu016

Act chi015

Bet vul022

Dau car025

Gly max026

Pha aur021

Hor vul020

Ara tha031

Ory sat019

Zea may018

Pis sat030

Hor vul024

Ory sat029

Ory sat027

Sac off013

Sac off004

Vic fab009

Bet vul008

Sol tub006

Cra pla010

Cit uns007

Ory sat003

Cra pla012

Spi ole005

Ory sat011

Zea may002


Criteria for the selection of a suitable environment for isolating new thermohalophiles and metagenomic studies:

• An easily accessible site• A stable environment• Microbial ecology studies in situ• Total community population census is known (rRNA)• Pure cultures have been isolated• Polyphasic studies on isolates completed• Data will assist in phylogeny and determining the origin,

evolution and adaptaion of halothermophiles• Do hyperhalothermophiles exist and will we search for these.

Future Directions


Hyperthermophiles

Mesophiles & psychrophiles

Thermophiles

Do hyperhalothermophiles exist in nature?

So far there is no evidence.

An inverse relationship exists between halophilicity and thermophilicity


Acknowledgments:Griffith University, Brisbane:

•Ben Mijts, PhD- 2003•Fred Huynh, BSc (Hons)- 2004•Chakka Naga, MSc (Hons)– current•Philip Pope, PhD- current

National University of Singapore:•Sivakumar Neelamegam, PhD- submitted •Tan Tien Chye, PhD– current


The 8th International Conference on Thermophiles(http://www.griffith.edu.au/conference/thermophile05/)

The scientific program:•Astrobiology, Origin of Life & Evolutionary Ecology •Phylogeny, Diversity & Biogeography •Proteomics, Genomics & Comparative Genomics •Molecular Biology & Biochemistry•Biotechnology & Bioprospecting


Gold Coast, Surfers Paradise, Australia

the limits to life under dual extremities of high ... · therapies (imct) biomolecular &...

Documents