© ©m j larkin 2008. from biosphere to molecule via the petri dish m. j. larkin the questor centre...

©M J Larkin 2008.

From Biosphere to MoleculeFrom Biosphere to Moleculevia via

The Petri DishThe Petri Dish

M. J. Larkin M. J. Larkin

The QUESTOR Centre and Biological SciencesThe QUESTOR Centre and Biological Sciences The Queen’s University of BelfastThe Queen’s University of Belfast

From Biosphere to MoleculeFrom Biosphere to Moleculevia via

The Petri DishThe Petri Dish

M. J. Larkin M. J. Larkin

The QUESTOR Centre and Biological SciencesThe QUESTOR Centre and Biological Sciences The Queen’s University of BelfastThe Queen’s University of Belfast

©M J Larkin 2008.

SummarySummarySummarySummary

• The lab and acknowledgements

• Overview - the planet and its microbial biomass

• The methodological approach - four examples of research done – pure linked to applied.....

• Chloroalkane degradation - chlorobutane and methyl chloride

• Oxygenases in biodegradation

• Archaea and oxidative catabolism – extreme environments

• Bioremediation and microbial diversity

• The lab and acknowledgements

• Overview - the planet and its microbial biomass

• The methodological approach - four examples of research done – pure linked to applied.....

• Chloroalkane degradation - chlorobutane and methyl chloride

• Oxygenases in biodegradation

• Archaea and oxidative catabolism – extreme environments

• Bioremediation and microbial diversity

©M J Larkin 2008.

ACKNOWLEDGEMENTS - Microbiology Laboratory ACKNOWLEDGEMENTS - Microbiology Laboratory QUESTOR Centre – Collaborators - current and recent QUESTOR Centre – Collaborators - current and recent

inmatesinmatesLeoinid Kulakov and Chris AllenJohn QuinnSheila Patrick (Medicine and Dentistry)Johannes Barth, Jim Hall (Bob Kalin, Trevor Elliot, Civ’ Eng’)Cathy Coulter (David Harper, Jack Hamilton , Agriculture)Dave Clarke, Gwen O’Reilly (Derek Boyd, Chemistry)Joe Vyle (Chemistry); Peter Coyle (RVL); Stephen Allen (Chem Eng)

Andrew Ferguson Asa MoyceDerek Fairley Emma FrewDave Lipscomb Peter GrayHelen Irvine Andrew Fraser Ros Andserson Kathryn LawsonAndrew Lee Veronique DurocqNichola Connery Chen ShenchangAndrew Mudd Tim GilfedderHarpinder Mundi Paul FlanaganAntonio de Casale Osa OsaladorJose Argudo

FUNDING SOURCES:INDUSTRY:QUESTOR Centre: Exxon: ICI:DuPont:ESB; Shell: BPSRIF: ECFW4:EC TDP: PEACE II Centres of Excellence: INTAS: BBSRC: DTI: LINK: EPSRC; NERC: DEL CAST: Prospect Globe Award; TALENT; Kuwait Government

Ian Thompson, Andrew Whitely,Wei Huang, OxfordDick Janssen, Gerrit PoelarendsGRONINGENAndy Weightman, Julian MarchesiCARDIFFAndrei Filonov, Vladimir KsenzenkoPUSHCHINODavid Gibson, Ramaswamy, Rebecca Parales: U of IOWAIan Pepper and Chris Rensing, John O’Hanlon: Water Quality Center, U of ARIZONAVISITORS:Samera Alwadi; KUWAITSusheela Carroll; U of ArizonaSebastian Sorensen; GEUS DenmarkMonika Knoppova; ICT Prague

©M J Larkin 2008.

The The QQueen’s ueen’s UUniversity niversity EEnvironmental nvironmental SScience and cience and

TTechnolechnolOOgy gy RResearch Centreesearch Centre

Jim SwindallJim SwindallWilson McGarelWilson McGarel

©M J Larkin 2008.

TO MOLECULAR MICROBIOLOGY; ENZYMES, CELLS TO MOLECULAR MICROBIOLOGY; ENZYMES, CELLS AND GENE EVOLUTION AND DIVERSITYAND GENE EVOLUTION AND DIVERSITY

A

B

BH7-7 BH2 a b c d a b c d M

FROM THE FIELD AND FROM THE FIELD AND LABORATORY MICROCOSMLABORATORY MICROCOSM

SCOPE OF THE INTERDISCIPLINARY SCOPE OF THE INTERDISCIPLINARY RESEARCH EFFORTRESEARCH EFFORT

©M J Larkin 2008.

RESEARCH AREASRESEARCH AREASRESEARCH AREASRESEARCH AREAS

•Molecular Biology/Genetics - Biochemistry of Biodegradation - and Biotransformations.

•Mobile genetic elements – insertion sequences

•Soil bacteria – Rhodococcus - Genetic systems and regulation

•Extremophiles (Salinity/pH)

•Naphthalene dioxygenase - evolution and mechanism

•haloalkane dehalogenases

•Waste water treatment - Sludge bulking and Microthrix

•Contaminated land remediation – isotope probing

©M J Larkin 2008.

Microorganisms Microorganisms - the root of diversity.- the root of diversity.

Microorganisms Microorganisms - the root of diversity.- the root of diversity.

3.5 billion years

Eubacteria

Plants & Animals Archaea

©M J Larkin 2008.

Where are they found?Where are they found?Biomass on the planet.Biomass on the planet.Where are they found?Where are they found?Biomass on the planet.Biomass on the planet.

• Most culturing analysis misses over 99% of the microbial population

•Molecular techniques now reveal hidden diversity

• Heterotrophs 5-20% biomass in sea waters

• Rich bacterial communities in sub-surface strata (600 m deep)

• up to 2 x 1040 tons - more than all flora and fauna

• equivalent upequivalent up to 2 m layer over planet!to 2 m layer over planet!

• Most culturing analysis misses over 99% of the microbial population

•Molecular techniques now reveal hidden diversity

• Heterotrophs 5-20% biomass in sea waters

• Rich bacterial communities in sub-surface strata (600 m deep)

• up to 2 x 1040 tons - more than all flora and fauna

• equivalent upequivalent up to 2 m layer over planet!to 2 m layer over planet!

©M J Larkin 2008.

The potential of One gram of soil…The potential of One gram of soil…

• 1 x 1010 microbial cells (typical clay loam)

• 4 x 103 microbial ‘species’

• < 0.1% can be cultivated in vitro (so far…)

• Many groups known only from DNA sequence data

• Only 1 or 2 cultivated members of some diverse

taxonomic orders are known

©M J Larkin 2008.

Philosophy of the laboratory missionPhilosophy of the laboratory mission

• Traditional approach: Millions of chemicals– 10 x 106 Chemicals

• 8 x 106 Xenobiotic

• 1 x 106 Recalcitrant

• 0.4 x 106 traded at over 50 tonnes per year

• Toxicological/ biodegradative data on only around 5000-6000

– Pick one - get a degrader - define catabolism - look in situ.

– CCultivate – RResearch aand PPublish• Alternative approach

– Look at environment and population diversity - set out to isolate specific dominant groups - define novel catabolism - look for activity in situ

– Rhodococcus – Haloarchaea - Alkaliphiles

©M J Larkin 2008.

Title-page of:, Title-page of:, Instauratio MagnaInstauratio Magna (1620) Francis Bacon (1620) Francis Bacon which contained his which contained his Novum Organon Novum Organon

““On the state of Sciences that is neither prosperous On the state of Sciences that is neither prosperous nor far advancednor far advanced……

Men (sic) seem to have no good sense of either Men (sic) seem to have no good sense of either their resources or their power: but to exaggerate their resources or their power: but to exaggerate the former and underrate the latter.the former and underrate the latter.

Hence either they put an insane value on the Arts which they already have and look no further or, undervaluing themselves, they waste their power on trifles and fail to try out things which go to the heart of the matter.

And so they are like the fatal pillars of Herculespillars of Hercules to the Sciences; for they are not stirred by the desire or hope of going further.”

"Multi pertransibunt et augebitur scientia“

(Many will pass through and knowledge will be increased).

Book of Daniel (chapter 12, verse 4)

©M J Larkin 2008.

From: Dick B. Janssen, Inez J. T. Dinkla, Gerrit J. Poelarends and Peter TerpstraBacterial degradation of xenobiotic compounds: evolution and distribution of novel enzyme activities. Environmental Microbiology (2005) 7: 1868–1882

From: Dick B. Janssen, Inez J. T. Dinkla, Gerrit J. Poelarends and Peter TerpstraBacterial degradation of xenobiotic compounds: evolution and distribution of novel enzyme activities. Environmental Microbiology (2005) 7: 1868–1882

Aerobic biodegradability of some common pollutantsAerobic biodegradability of some common pollutants

©M J Larkin 2008.

Fate of chloroalkanes: Fate of chloroalkanes: 1- Chlorobutane and Chloromethane1- Chlorobutane and Chloromethane

•Chloalkanes very commonly used in industry in a wide range of processes.

•1-chlorobutane a good model substrate to investigate the biodegradation mechanisms possible.

•Chloromethane (CH3Cl): most abundant volatile halocarbon in the atmosphere.

•Amospheric concentration: 600 parts per 1012 : 5 million metric tons.

•Ozone destruction - 15 to 20% - natural origin – not industrial: e.g. wood-rot fungi

•Biodegradative fate only more recently investigated

•Same mechanism as other haloalkanes?

©M J Larkin 2008.

1- Chlorobutane degradation by 1- Chlorobutane degradation by Rhodococcus Rhodococcus sp NCIMB13064sp NCIMB13064

1- Chlorobutane degradation by 1- Chlorobutane degradation by Rhodococcus Rhodococcus sp NCIMB13064sp NCIMB13064

CH2-ClCH2

CH2

CH3

CH2-OHCH2

CH2

CH3

CHOCH2

CH2

CH3

COOHCH2

CH2

CH3

H2O HCl X XH2 Y +H2O YH2

DhaA AldAAdhA

Order of genes on pRTL1 (approx 100 Kbp plasmid)

IS2112 invA dhaR dhaA adhA aldA

1 Kb

©M J Larkin 2008.

GlobalGlobal Dha A Dha A spread in bacterial isolates spread in bacterial isolatesGlobalGlobal Dha A Dha A spread in bacterial isolates spread in bacterial isolates

Gerrit J. Poelarends, Marjan Zandstra, Tjibbe Bosma, Leonid A. Kulakov, Michael J. Larkin, Julian R. Marchesi, Andrew J. Weightman, and Dick B. Janssen (2000)Haloalkane-Utilizing Rhodococcus Strains Isolated from Geographically Distinct

Locations Possess a Highly Conserved Gene Cluster Encoding Haloalkane Catabolism. J.Bacteriol. 182:2725-2731. .

©M J Larkin 2008.

Spread of Spread of dhaA dhaA amongst strains world-wideamongst strains world-wideSpread of Spread of dhaA dhaA amongst strains world-wideamongst strains world-wide

invA dhaR dhaA adhA aldA

GJ70 THE NETHERLANDSHA1 SWITZERLANDY2 UK


NCIMB13064 UKTB2 USAm15-3 JAPAN

dhaA (100%) also in Pseudomonas pavonaceae 170 The Netherlands (1,3-dichloropropene)

Poelarends Janssen et al 1999 Appl.Environ. Microbiol. 64:2931-2936

©M J Larkin 2008.

dhaAdhaA : Recombinations across species : Recombinations across speciesdhaAdhaA : Recombinations across species : Recombinations across species


Rhodococcus sp NCIMB 13064

invA dhaR dhaAfintM

Mycobacterium sp GP1(1,2-dibromoethane)

intP dhaA tnpA IS1071P .pavonaceae 170(1,3-dichlrororopene)

©M J Larkin 2008.

intPintP dhaAdhaA tnpAtnpA IS1071IS1071

invAinvA dhaRdhaR dhaAfdhaAfintMintM

invAinvA dhaRdhaR dhaAdhaA adhAadhA aldAaldA

IS2112IS2112 invAinvA dhaRdhaR dhaAdhaA adhAadhA aldAaldA

Rhodococcus sp NCIMB 13064: UK

TB2: USAm15-3: JAPAN

Mycobacterium sp GP1(1,2-dibromoethane)

P .pavonaceae 170(1,3-dichlrororopene)

Rhodococcus spGJ70:THE NETHERLANDS

HA1: SWITZERLANDY2: UK

Genetic Recombinations and Global Genetic Recombinations and Global Distribution of Dehalogenases - SummaryDistribution of Dehalogenases - Summary

©M J Larkin 2008.

Isolation of chloromethane degraderIsolation of chloromethane degrader

CATHERINE COULTER, JOHN T. G. HAMILTON, W. COLIN MCROBERTS, LEONID KULAKOV,MICHAEL J. LARKIN,AND DAVID B. HARPER (1999) Halomethane:Bisulfide/Halide Ion Methyltransferase, an UnusualCorrinoid Enzyme of Environmental Significance Isolated froman Aerobic Methylotroph Using Chloromethaneas the Sole Carbon Source. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 65: 4301–4312.

CC495 AminobacterCC495 Aminobacterlissarensislissarensis

©M J Larkin 2008.

Methyl transferase activity – not halohydrolaseMethyl transferase activity – not halohydrolase

HSHS--

MethanethiolMethanethiol

©M J Larkin 2008.

Role of Oxygen in the biosphereRole of Oxygen in the biosphereRole of Oxygen in the biosphereRole of Oxygen in the biosphere

• For many compounds to be degraded quickly there needs to be a reaction with Oxygen.

• Known as Oxygen fixation• Mediated in nature my many microorganisms• Enzymes known as oxygenases• Carbon and Oxygen cycle at necessary for life on the

planet• Fortunately molecular Oxygen is not very reactive

• For many compounds to be degraded quickly there needs to be a reaction with Oxygen.

• Known as Oxygen fixation• Mediated in nature my many microorganisms• Enzymes known as oxygenases• Carbon and Oxygen cycle at necessary for life on the

planet• Fortunately molecular Oxygen is not very reactive

©M J Larkin 2008.

• Oxygen in the air is in its "ground“ state - 3O2.

• Outermost pair of electrons have parallel spins (↑↑ ) - "triplet" state.

• This does not allow them to react with most molecules – just as well !!!

– SPIN FORBIDDENSPIN FORBIDDEN.

• However, triplet oxygen can be activated by the addition of energy, and transformed into reactive oxygen species.

• Outermost pair of electrons have antiparallel spins (↓↑ ) - "singlet" state.

The reactivity of OxygenThe reactivity of OxygenThe reactivity of OxygenThe reactivity of Oxygen

©M J Larkin 2008.

Activation of Oxygen enzymaticallyActivation of Oxygen enzymaticallyActivation of Oxygen enzymaticallyActivation of Oxygen enzymatically

Not common in catabolism

Very common in oxygenases

©M J Larkin 2008.

Microbial Microbial Oxygenases and Oxygenases and OxygenOxygen

For most compounds to be degraded they must react with O2

Mediated by bacteria in the environment at low temperature using iron in diverse enzymes

• This is facilitated by oxygenases

• Two types

• Mono- add one -OH group

• Di- add TWO -OH groups

• The “corner-stone” “corner-stone” of the C and O cycle in nature.

• Naphthalene dioxygenase NDO - well studied in Pseudomonas

• The current paradigm

©M J Larkin 2008.

OH

OHH

H

OH

OH

O

OH

COOHOH

O

COOH

OH

CHO

OH

COOH

OH

OH

OHOH

COOH

OH

COOHCHO

HOOC

HOOC OHHOOC

HOOC

O

Naphthalene

cis-naphthalene dihydrodiol1,2-dihydroxynaphthalene

2-hydroxychromene-2-carboxylatecis-o-hydroxybenzalpyruvate

salicylaldehyde

salicylate

NahA

NahB

NahC

NahD

NahE

NahF

NADH + O2 + H+

NAD+

NAD+

NADH + H+

O2

H2O

CH3COCOO-NAD+

NADH + H+

NADH,O2H+,

NAD+

NADH,O2,ATP,CoA

NAD+

O2

O2

O2catechol

gentisate

maleylpyruvate2-hydroxymuconic semialdehyde

cis,cis-muconic acid

S1H S5H

C23O C12O G12O

NarA and NarBin Rhodococcus

Scheme for Scheme for naphthalene naphthalene

catabolism in bacteriacatabolism in bacteria

RING HYDROXYLATINGRING HYDROXYLATINGDIOXYGENASEDIOXYGENASE

RING OPENINING RING OPENINING DIOXYGENASESDIOXYGENASES

©M J Larkin 2008.

What are the potential rate-limiting steps?What are the potential rate-limiting steps?

DIOXYGENASE DIOXYGENASE BOTTLENECKBOTTLENECK

BioavailabilityBioavailabilitySolubilitySolubility

Substrate fitSubstrate fitNatural vs pollutantNatural vs pollutant

Affinity and rateAffinity and rate

Provision of oxygenProvision of oxygenEnvironmentAffinity EnvironmentAffinity

and rateand rate

CellularCellularmetabolismmetabolism

Provision of Provision of electronselectrons

©M J Larkin 2008.

RhodococcusRhodococcus NDO characterisation NDO characterisationRhodococcusRhodococcus NDO characterisation NDO characterisation

• NCIMB 12038

• Enzyme components purified

• Novel Naphthalene dioxygenase (NDO)

• N-terminal sequences

• DNA and amino acid sequences

• Key active site aa’s conserved

• Present in other strains

©M J Larkin 2008.

Comparison of Comparison of and and components of ISP components of ISPNARNAR

((RhodococcusRhodococcus NDO) and ISP NDO) and ISPNAHNAH ( (Pseudomonas Pseudomonas NDONDO))

NO significant DNA HOMOLOGY: Amino acid similarity (31%) (39%) NO significant DNA HOMOLOGY: Amino acid similarity (31%) (39%)

Reductase NAP

(OX)

Reductase NAP

(RED)

Ferredoxin NAP

(OX)

FerredoxinNAP

(RED)

ISP NAP

(OX)

ISP NAP

(RED)

OHOH

O2

NADH + H+

NAD+

**Pseudomonas ISPNAH

23KD

55KD

*Analogous Rhodococcus ISPNAR

Napthalene Pyruvate

Salicylate

©M J Larkin 2008.

Conservation of the key amino acids in Conservation of the key amino acids in sub- sub-units of NDOs from units of NDOs from RhodococcusRhodococcus and and

Pseudomonas.Pseudomonas.

Conservation of the key amino acids in Conservation of the key amino acids in sub- sub-units of NDOs from units of NDOs from RhodococcusRhodococcus and and

Pseudomonas.Pseudomonas.Rieske Centre Active site Putative Ferredoxin

binding region

Electron transfer

(Rieske-Act. Site)

Region of unknown

function

NahAc NarAa NahAc NarAa NahAc NarAa NahAc NarAa NahAc NarAa

C81 C88 N201 N209 K97 S104 W106 W113 T299 T297

C101 C108 H208 H216 G98 H105 V117 V124 V300 V298

H83 H90 H213 H221 V100 R107 R84 R91 F301 F299

H104 H111 D205* D213 Q115 V122 E200 D208 P302 P300

D362 D372 S116 G123 N303 N301

P118** P125**

W211 M219

*Asp205 is probably important for electron transfer (12) and is essential for activity (18); **Pro118 (as well as Trp211) is

from the catalytic domain.

©M J Larkin 2008.

Diversity of Bacterial NDO alpha subunitsDiversity of Bacterial NDO alpha subunitsDiversity of Bacterial NDO alpha subunitsDiversity of Bacterial NDO alpha subunits

Moser and Stahl, 1999

©M J Larkin 2008.

STRUCTURE OF STRUCTURE OF RhodococcusRhodococcus NDO NDO

©M J Larkin 2008.

P400

P200

NCBI12038

I24

CIR2

Transcription induced by growth on Naphthalene:

narR1, R2 rub2 narK narAa, Ab, B narC orf1 – 3 orf4 – 6

rub1 narR1,R2 rub2 narK narAa Ab B ΔC orf1 – 3 orf4 – 6

rnoA1 A2 A3 A4 rnoB orf1

nidA B C D

narR1 R2 narK narAa Ab B Crub1 oxiA

narR1,R2 narK narAa Ab B Crub1

Organisation of naphthalene degradation Organisation of naphthalene degradation genes in genes in RhodococcusRhodococcus

Organisation of naphthalene degradation Organisation of naphthalene degradation genes in genes in RhodococcusRhodococcus

©M J Larkin 2008.

OH

OHH

H

OH

OH

Naphthalene

cis-naphthalene dihydrodiol1,2-dihydroxynaphthalene

NahA

NahB

NADH + O2 + H+

NAD+

NAD+

NADH + H+

NarA and NarB in Rhodococcus

AA

OH

OHOH

OHH

H

ee--

OO22

NarKNarK

NarBNarB

NarAaNarAa NarAbNarAb

OH

OH

OH

OHH

H

OH

OHH

H

NAD+

OO22

NarKNarK

NarBNarB

NarAaNarAa

NarAbNarAb

NADH + H+

BB

A novel mechanism for electron transfer....A novel mechanism for electron transfer....

©M J Larkin 2008.

Extremophiles – BIODEGRADATION UNDER Extremophiles – BIODEGRADATION UNDER EXTREME CONDITIONSEXTREME CONDITIONS

• Many industrial waste and environmental have:• Extremes of pH – often very caustic waste• Extremes of salinity

• Alkaliphile capabilities – • Exxon – Mobil – caustic waste• Halophile capabilities – biodegradation of aromatic Halophile capabilities – biodegradation of aromatic

compoundscompounds• ICI and Water Quality Centre – University of Arizona

©M J Larkin 2008.

HalobacterialesHalobacteriales

You are here…You are here…

‘‘Universal’ Universal’ phylogenetic tree - phylogenetic tree - based on 16S rRNA based on 16S rRNA

sequence datasequence data

AnimalsAnimals

ProkaryotesProkaryotes

EukaryotesEukaryotes

©M J Larkin 2008.

Novel ‘extremely Novel ‘extremely

halophilic Archaea’halophilic Archaea’

Growing onGrowing on

Aromatic substratesAromatic substrates

Haloarcula sp. D1

Haloarcula marismortui rrnB

Halorubrum saccharovorum

Halorubrum distributum

Halorubrum lacusprofundi

Methanospirillum hungatei

Nantronomonas pharaonis

Halobacterium salinarum

Halorhabdus utahensis

Haloarcula marismortui rrnA

Haloarcula sp. DSW7

Halococcus morrhuae

Haloarcula hispanica

Nantronococcus occultus

Halobaculum gomorrense

Halogeometricum borinquense

Nantronobacterium gregoryi

Haloferax mediterranei

Haloarcula argentinensis

Natrialba magadii

Haloarcula vallismortis

Natrinema versiforme

Haloterrigena thermotolerans

Haloferax volcanii

Haloferax sp. D1227

100

51

21

75

28

63

100

100

24

100

100

96 100

97

100

59

100

92

100

89

97

100

99

91 Halorubrum sp. E4

©M J Larkin 2008.

Aromatic substratesAromatic substrates

COOH COOH

OH

Aerobic growth of Haloarcula sp. D1

benzoic & 4-hydroxybenzoic acids :

HOCOOH

OH

COOH

OH

OHOOC

GDO

+O2Gentisate 1,2-

dioxygenase: ? ? ? ? ? ?

©M J Larkin 2008.

Accumulation of gentisic acid from Accumulation of gentisic acid from

4-hydroxybenzoic acid in 4-hydroxybenzoic acid in HaloarculaHaloarcula

sp. D1 cell suspensionssp. D1 cell suspensions

0

0.5

1

1.5

2

2.5

0 10 20 30

Concentration

(mM)

Time (hours)

OH

COOH

OH

OH

COOH

HOCOOH

OH

COOH

OH

OHOOC

©M J Larkin 2008.

4-Hydroxybenzoate pathway4-Hydroxybenzoate pathway

OH

OH

COOH

OH

COOH

+ 1/2O2

Intramolecular carboxyl-group

migration / ‘NIH-shift’ ?

OH

COOH

DD

COOH

DOH

OH

OH

D

OH

COOH

+ 1/2O2

A

B

Synthesised 2,6-

dideutero-4-

hydroxybenzoic acid :

©M J Larkin 2008.

2

3

4

5

61

O

H

OH

H

H

OH

MeO

Authentic (non-deuterated) standard

7.17.27.37.47.5 ppm

36.

40

6

6 4 36 4 3

2

3

4

5

61

O

H

OH

H

OH

MeO

D

Deuterated (methyl)gentisate

6.86.97.07.17.2 ppm0

.795

2.0

71

8.6

26

8.6

80

6 4 36 4 3

11H-NMR spectraH-NMR spectra

©M J Larkin 2008.

Aromatic catabolism in ArchaeaAromatic catabolism in Archaea4-Hydroxybenzoate pathway – NIH-shift not 4-Hydroxybenzoate pathway – NIH-shift not

reported in the Archaea beforereported in the Archaea before

OH

COOH

DD

COOH

DOH

OH

OH

D

OH

COOH

+ 1/2O2

A

B

©M J Larkin 2008.

Gasworks Sites: Best source of Gasworks Sites: Best source of aromatic catabolic diversity!aromatic catabolic diversity!

©M J Larkin 2008.

Interce pto

r

Sand 1ZVI

Sand 3

Sand 2GAC 2

GAC 1 Input

Extent of contamination at Extent of contamination at SEREBAR remediation site SEREBAR remediation site

292100 292150 292200 292250 292300

91450

91500

91550

91600

91650

91700prb1

p rb 10

p rb 11

p rb 12

p rb 13

p rb 14

p rb 16

p rb 17p rb 18

prb2

p rb 20

p rb 21

prb3

prb5

prb7prb8

prb9

p rb 23

BG B H 10

BG B H 11Inter cept or

Sand 1ZVI

Sand 3

Sand 2GAC 2GAC 1 Input

©M J Larkin 2008.

CLONE PHYLOGENY

V1 ß-PROTEOBACTERIA

V2 -PROTEOBACTERIA;GEOBACTERIACEAE

V4 FIRMICUTES; LACTOBACILLACEAE

V5 ß-PROTEOBACTERIA

V7 ß-PROTEOBACTERIA; RHODOCYCLUS

V8 ß-PROTEOBACTERIA;COMAMONADACEAE

2F ß-PROTEOBACTERIA; NITROSOLOBUS

3F ß-PROTEOBACTERIA;COMAMONADACEAE

4F -PROTEOBACTERIA;GEOBACTERIACEAE

5F ß-PROTEOBACTERIA; RHODOCYCLUS

6F UNKNOWN



10F -PROTEOBACTERIA; PSEUDOMONAS


13F ß-PROTEOBACTERIA; ALCALIGENACEAE

14F ß-PROTEOBACTERIA;COMAMONADACEAE

15F ß-PROTEOBACTERIA


17F ß-PROTEOBACTERIA; BURKHOLDERA

18F -PROTEOBACTERIA; PSEUDOMONAS

19F UNKNOWN


PRESUMPTIVE PRESUMPTIVE PHYLOGENETIC PHYLOGENETIC IDENTIFICATION OF IDENTIFICATION OF EUBACTERIAL 16s rDNA EUBACTERIAL 16s rDNA CLONES FROM DIRECTCLONES FROM DIRECT SOIL DNA SAMPLESSOIL DNA SAMPLES

©M J Larkin 2008.

CLONE PHYLOGENY

1G -PROTEOBACTERIA; PSEUDOMONAS2G -PROTEOBACTERIA; METHYLOCOCCACEAE3G -PROTEOBACTERIA; PSEUDOMONAS4G UNKNOWN7G -PROTEOBACTERIA; METHYLOCOCCACEAE8G UNKNOWN

12G UNKNOWN13G UNKNOWN17G UNKNOWN20G -PROTEOBACTERIA; XANTHOMONAS22G UNKNOWN23G UNKNOWN26G -PROTEOBACTERIA; XANTHOMONAS27G -PROTEOBACTERIA; PSEUDOMONAS28G UNKNOWN30G UNKNOWN33G -PROTEOBACTERIA; ENTEROBACTERIACEAE34G -PROTEOBACTERIA; XANTHOMONAS35G -PROTEOBACTERIA; PSEUDOMONAS37G UNKNOWN43G UNKNOWN44G UNKNOWN45G -PROTEOBACTERIA; ENTEROBACTERIACEAE46G UNKNOWN50G UNKNOWN51G UNKNOWN55G UNKNOWN57G UNKNOWN59G -PROTEOBACTERIA; PSEUDOMONAS60G ß-PROTEOBACTERIA; RHODOCYCLUS61G UNKNOWN62G -PROTEOBACTERIA; PSEUDOMONAS63G -PROTEOBACTERIA; METHYLOCOCCACEAE

PRESUMPTIVE PRESUMPTIVE PHYLOGENETIC PHYLOGENETIC IDENTIFICATION IDENTIFICATION OF EUBACTERIAL OF EUBACTERIAL 16s rDNA 16s rDNA CLONES FROM CLONES FROM DIRECT DIRECT GROUNDWATER GROUNDWATER DNA SAMPLESDNA SAMPLES

©M J Larkin 2008.

LABORATORY MICROCOSM REACTIVE BARRIERLABORATORY MICROCOSM REACTIVE BARRIER- removal of key pollutants – aromatic compounds- removal of key pollutants – aromatic compounds

0.00 20.00 40.00 60.00 80.00 100.00 120.00

-20.00

0 5000 10000 15000 20000 25000 30000 35000(ug/L)

B enz en e P ro file 11 -5-01

0.00 20.00 40.00 60.00 80.00 100.00 120.00

-20.00 Benzene

0.00 20.00 40.00 60.00 80.00 100.00 120.00

-20.00

0 4000 8000 12000 16000 20000 24000 28000 32000

(ug/L)

P heno l P rof ile (1 1-5-01)

0.00 20.00 40.00 60.00 80.00 100.00 120.00

-20.00 Phenol

2,4-Dimethylphenol

0.00 20.00 40.00 60.00 80.00 100.00 120.00

-20 .00

0 60000 120000 180000 240000 300000 360000

2,4-Dim eth ylp henol P rofile 11-5-01

(ug /L )

0 .00 20.00 40.00 60.00 80.00 100.00 120.00

-20 .00

0 100 200 300 400 500 600 700 8000

100

200

300

400

5ug / L 25ug / L 45ug / L 65ug / L

©M J Larkin 2008.

Microbiological sample points – SEREBARMicrobiological sample points – SEREBAR

Interceptor and inlet

When he goes back to his mobile phone, that's when

it'sBack to the lab again yo

This whole rhapsodyHe better go capture this moment and hope it don't

pass him…..

Eminem - Lose Yourself

Look, if you had one shot, one

opportunityTo seize

everything you ever wanted-One

momentWould you capture it or just let it slip?

….

Eminem - Lose Yourself

Tool-box

Onlookers

©M J Larkin 2008.

Which organisms are the main degraders ? Which organisms are the main degraders ? - Stable Isotope Probing (SIPS) – to detect - Stable Isotope Probing (SIPS) – to detect PAH degraders - naphthalenePAH degraders - naphthalene

Amplification –

Sequence analysis

= taxonomic and

phylogenetic

information

Functional genes

Using 13C labelled naphthalene

©M J Larkin 2008.

0

1

2

3

4

5

0 10 20 30 40 50 60 70 80

Time (hours)

Nap

thal

ene

Co

nce

ntr

atio

n (

µM

)

Figure 1. Degradation of 12C- and 13C-naphthalene (3.8 µM) in laboratory microcosm flasks inoculated with

groundwater.

Utlilisation of Utlilisation of 1212C and C and 1313C - naphthalene by C - naphthalene by groundwater bacteria in microcosmsgroundwater bacteria in microcosms

©M J Larkin 2008.

T=0 hr factions 6-15 T=36 hr fraction 6-15

GW

with

3.8 µM

12C

-Nap

h

GW

with

3.8 µM

13C

-Nap

h

GW

on

ly

6 7 8 9 10 11 12 13 14 15 6 7 8 9 10 11 12 13 14 15

M1 M2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

A

DGGE and 16s rDNA sequence identificationDGGE and 16s rDNA sequence identification

Acidovorax sp – related to Comamonas spp

©M J Larkin 2008.

NDO NDO - subunit expression – - subunit expression – RT-PCR of RT-PCR of 1313C-RNA C-RNA

RT-PCR of 13C-RNA fractions

Ladder C 6 7 8 9 10 11 12 C

©M J Larkin 2008.

GW 3µM 30µM 300µM 600µM60µM +c -c1 -c2

NDO NDO - subunit - subunitPseudomonas -Pseudomonas -

P. putidaP. putida G7 type G7 type

NDO NDO - subunit - subunitCommonas -Commonas -

Ralstonia Ralstonia U2U2 typetype

Concentration effect on dominant Concentration effect on dominant degraders.......degraders.......

Extensive independent study ..........

Only Pseudomonas and Rhodococcus strains isolated

No Acidovorax or Comamonas related strains cultivated

Comamonas – like NDO -subunit genes amplified from groundwater

©M J Larkin 2008.

FISH images show microbial degraders in FISH images show microbial degraders in groundwater sample. groundwater sample.

Red Red Acidovorax spAcidovorax sp

GGreen reen Pseudomonas spPseudomonas sp

Purple overall eubacteriaPurple overall eubacteria

©M J Larkin 2008.

0

20

40

60

80

100

120

3.8 µm 30 µm

AcidovoraxPseudomonas

13 C

co

nte

nt

per

cel

l (A

tom

%)

13C Naphthalene concentration

40

10

19

20

Raman micro-spectroscopy analysis of single cells Raman micro-spectroscopy analysis of single cells

Stable isotope based analysis of phylogenetic identity, functional transcripts and metabolic activity in natural microbial populationsWei E. Huang1,2*ψ, Andrew Ferguson3,4, Andrew C. Singer2, Kathryn Lawson4,5, Ian P. Thompson2, Robert M. Kalin3, Michael J. Larkin4,5, Mark J. Bailey1 and Andrew S. Whiteley1* - in press

Stable isotope based analysis of phylogenetic identity, functional transcripts and metabolic activity in natural microbial populationsWei E. Huang1,2*ψ, Andrew Ferguson3,4, Andrew C. Singer2, Kathryn Lawson4,5, Ian P. Thompson2, Robert M. Kalin3, Michael J. Larkin4,5, Mark J. Bailey1 and Andrew S. Whiteley1* - in press

13C labelled cells have significant red-shift in spectrum (Huang, W. E., Griffiths, R. I., Thompson, I. P., Bailey, M. J., & Whiteley, A. S. (2004) Anal. Chem. 76, 4452-4458_

©M J Larkin 2008.

AcknowledgementsAcknowledgements

Alan Bull – Cardiff

Martin Day – Cardiff

Werner Arber – Basle

Roger Whittenbury – Warwick

Heinz Saedler – Cologne

Mick Chandler – Toulouse

Simon Baumberg – Leeds

Howard Dalton – Warwick

Gerben Zylstra – Rutgers

Chris Knowles – Oxford

Julian Davies - Vancouver

© ©m j larkin 2008. from biosphere to molecule via the petri dish m. j. larkin the questor centre...

Documents