Download dhaA - Queen`s University Belfast

From Biosphere to Molecule
via
The Petri Dish
M. J. Larkin
The QUESTOR Centre and Biological Sciences
The Queen’s University of Belfast
©M J Larkin 2008.
Summary
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The lab and acknowledgements
Overview - the planet and its microbial biomass
The methodological approach - four examples of
research done – pure linked to applied.....
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©M J Larkin 2008.
Chloroalkane degradation - chlorobutane and methyl
chloride
Oxygenases in biodegradation
Archaea and oxidative catabolism – extreme environments
Bioremediation and microbial diversity
ACKNOWLEDGEMENTS - Microbiology Laboratory
QUESTOR Centre – Collaborators - current and recent
inmates
Leoinid Kulakov and Chris Allen
John Quinn
Sheila 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
Derek Fairley
Dave Lipscomb
Helen Irvine
Ros Andserson
Andrew Lee
Nichola Connery
Andrew Mudd
Harpinder Mundi
Antonio de Casale
Jose Argudo
©M J Larkin 2008.
Ian Thompson, Andrew Whitely,
Wei Huang, Oxford
Dick Janssen, Gerrit Poelarends
GRONINGEN
Andy Weightman, Julian Marchesi
CARDIFF
Andrei Filonov, Vladimir Ksenzenko
PUSHCHINO
David Gibson, Ramaswamy, Rebecca
Parales: U of IOWA
Ian Pepper and Chris Rensing, John
O’Hanlon: Water Quality Center, U of
ARIZONA
VISITORS:
Samera Alwadi; KUWAIT
Susheela Carroll; U of Arizona
Sebastian Sorensen; GEUS Denmark
Monika Knoppova; ICT Prague
Asa Moyce
Emma Frew
Peter Gray
Andrew Fraser
Kathryn Lawson
Veronique Durocq
Chen Shenchang
Tim Gilfedder
Paul Flanagan
Osa Osalador FUNDING SOURCES:
INDUSTRY:QUESTOR Centre: Exxon: ICI:DuPont:
ESB; Shell: BP
SRIF: ECFW4:EC TDP: PEACE II Centres of Excellence:
INTAS: BBSRC: DTI: LINK: EPSRC; NERC: DEL CAST:
Prospect Globe Award; TALENT; Kuwait Government
The Queen’s University
Environmental Science and
TechnolOgy Research Centre
Jim Swindall
Wilson McGarel
©M J Larkin 2008.
SCOPE OF THE INTERDISCIPLINARY
RESEARCH EFFORT
FROM THE FIELD AND
LABORATORY MICROCOSM
TO MOLECULAR MICROBIOLOGY; ENZYMES, CELLS
AND GENE EVOLUTION AND DIVERSITY
BH7-7 BH2
abcd abcd M
A
B
Figure 3: 2D-PAGE gels of regions selected from the ‘other
fraction’ – not containing the NarAa and Ab components – but
containing putative reductase and ferredoxin components - from a
MonoQ column. A– pyruvate –grown cells without induction of
naphthalene associated genes; B- naphthalene- grown cells with
induction of naphthalene associated genes. Examples of proteins
associated with naphthalene degradation are indicated with
arrows.
©M J Larkin 2008.
RESEARCH AREAS
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©M J Larkin 2008.
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
Microorganisms
- the root of diversity.
Eubacteria
3.5 billion years
©M J Larkin 2008.
Plants &
Animals
Archaea
Where are they found?
Biomass on the planet.
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©M J Larkin 2008.
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 up to 2 m layer over planet!
The potential of One gram of soil…
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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 mission
• Traditional approach: Millions of chemicals
– 10 x 106 Chemicals
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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.
– Cultivate – Research and Publish
• 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:,
Instauratio Magna
(1620) Francis Bacon
which contained his
Novum Organon
“On the state of Sciences that is neither prosperous
nor far advanced…
Men (sic) seem to have no good sense of either
their resources or their power: but to exaggerate
the former and underrate the latter.
"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.
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 Hercules to
the Sciences; for they are not stirred by the desire
or hope of going further.”
Aerobic biodegradability of some common pollutants
From: Dick B. Janssen, Inez J. T.
Dinkla, Gerrit J. Poelarends and Peter
Terpstra
Bacterial degradation of xenobiotic
compounds: evolution and distribution
of novel enzyme activities.
Environmental Microbiology (2005) 7:
1868–1882
©M J Larkin 2008.
Fate of chloroalkanes:
1- Chlorobutane and Chloromethane
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©M J Larkin 2008.
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?
1- Chlorobutane degradation by Rhodococcus
sp NCIMB13064
CH2-Cl H O HCl CH2-OH
2
X
XH2
CH2
CH2
CH2
CH2
CH3
DhaA
CH3
AdhA
CHO
COOH
Y +H2O YH2
CH2
CH2
CH2
CH2
CH3
CH3
AldA
Order of genes on pRTL1 (approx 100 Kbp plasmid)
IS2112
invA
dhaR
dhaA
adhA
1 Kb
©M J Larkin 2008.
aldA
Global Dha A 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 dhaA amongst strains world-wide
NCIMB13064
TB2
m15-3
UK
USA
JAPAN
IS2112
invA
GJ70
HA1
Y2
dhaR
dhaA
adhA
aldA
THE NETHERLANDS
SWITZERLAND
UK
invA
dhaR
dhaA
adhA
aldA
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.
dhaA : Recombinations across species
Mycobacterium sp GP1
(1,2-dibromoethane)
intM
invA
dhaR
dhaAf
Rhodococcus sp NCIMB 13064
IS2112
P .pavonaceae 170
(1,3-dichlrororopene)
©M J Larkin 2008.
invA
intP
dhaR
dhaA
adhA
aldA
dhaA
tnpA
IS1071
Genetic Recombinations and Global
Distribution of Dehalogenases - Summary
Mycobacterium sp GP1
(1,2-dibromoethane)
intM
P .pavonaceae 170
(1,3-dichlrororopene)
Rhodococcus sp
NCIMB 13064: UK
TB2: USA
m15-3: JAPAN
Rhodococcus sp
GJ70:THE NETHERLANDS
HA1: SWITZERLAND
Y2: UK
©M J Larkin 2008.
invA
dhaR
intP
IS2112
invA
invA
dhaAf
dhaA
dhaR
dhaR
dhaA
dhaA
tnpA
adhA
adhA
IS1071
aldA
aldA
Isolation of chloromethane degrader
CC495 Aminobacter
lissarensis
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 Unusual
Corrinoid Enzyme of Environmental Significance Isolated from
an Aerobic Methylotroph Using Chloromethane
as the Sole Carbon Source. APPLIED AND
ENVIRONMENTAL MICROBIOLOGY, 65: 4301–4312.
©M J Larkin 2008.
Methyl transferase activity – not halohydrolase
Methanethiol
HS-
©M J Larkin 2008.
Role of Oxygen in the biosphere
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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.
The reactivity of Oxygen
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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 FORBIDDEN.
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©M J Larkin 2008.
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.
Activation of Oxygen enzymatically
Not common in catabolism
Very common in oxygenases
©M J Larkin 2008.
Microbial Oxygenases and
Oxygen
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
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©M J Larkin 2008.
This is facilitated by oxygenases
Two types
Mono- add one -OH group
Di- add TWO -OH groups
The “corner-stone” of the C and O cycle in nature.
Naphthalene dioxygenase NDO - well studied in Pseudomonas
The current paradigm
NarA and NarB
in Rhodococcus
Naphthalene
NahA
H
NADH + O2 + H+
OH
NAD+
OH
H
OH
NAD+
Scheme for
naphthalene
catabolism in bacteria
OH
NahB
NADH + H+
1,2-dihydroxynaphthalene
cis-naphthalene dihydrodiol
O2
NahC
OH
O
COOH
NahD
COOH
OH
O
cis-o-hydroxybenzalpyruvate
2-hydroxychromene-2-carboxylate
H2O
NahE
CH3COCOOOH
NAD+
NADH + H+
CHO
NahF
salicylaldehyde
OH
H+, NADH,O2
NADH,O2,ATP,CoA
COOH
salicylate
NAD+
S1H
RING OPENINING
DIOXYGENASES
NAD+
S5H
OH
HO
OH
COOH
OH
catechol
C23O
CHO
COOH
OH
2-hydroxymuconic
semialdehyde
©M J Larkin 2008.
RING HYDROXYLATING
DIOXYGENASE
O2
O2
gentisate
C12O
O2
HOOC
G12O
HOOC
O
OH
HOOC
cis,cis-muconic acid
HOOC
maleylpyruvate
What are the potential rate-limiting steps?
Bioavailability
Solubility
Substrate fit
Natural vs pollutant
Affinity and rate
Cellular
metabolism
DIOXYGENASE
BOTTLENECK
Provision of oxygen
EnvironmentAffinity
and rate
©M J Larkin 2008.
Provision of
electrons
Rhodococcus NDO characterisation
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©M J Larkin 2008.
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
Comparison of  and  components of ISPNAR
(Rhodococcus NDO) and ISPNAH (Pseudomonas NDO)
*Analogous Rhodococcus ISPNAR
Napthalene
Pyruvate
Salicylate
 55KD
 23KD
NAD+
**Pseudomonas ISPNAH
Reductase
NAP
Ferredoxin
(OX)
NAP
(OX)
ISP NAP
(OX)
O2
NADH
+ H+
Reductase
FerredoxinNAP
NAP
(RED)
(RED)

 


ISP
NAP
(RED)

NO significant DNA HOMOLOGY: Amino acid similarity  (31%)  (39%)
©M J Larkin 2008.
OH
OH
Conservation of the key amino acids in  subunits of NDOs from Rhodococcus and
Pseudomonas.
Rieske Centre
Active site
Putative Ferredoxin
Electron
transfer
Region of unknown
binding region
(Rieske-Act. Site)
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 subunits
Moser and Stahl, 1999
©M J Larkin 2008.
STRUCTURE OF Rhodococcus NDO
©M J Larkin 2008.
Organisation of naphthalene degradation
genes in Rhodococcus
I24
nidA
B
C
narAa
Ab
B
Ab
B
D
P400
rub1
narR1 R2
narK
C
oxiA
P200
rub1
narR1,R2
rub1
narR1,R2
narK
narAa
narK
narAa
NCBI12038
rub2
Ab
C
B
ΔC
orf1 – 3
orf4 – 6
CIR2
orf1
rnoA1
A2 A3
A4
rnoB
Transcription induced by growth on Naphthalene:
narR1, R2
©M J Larkin 2008.
rub2
narK
narAa, Ab, B
narC
orf1 – 3
orf4 – 6
A novel mechanism for electron transfer....
B
H
OH
OH
H
OH
OH
OH
OH
H
H
NarB
eNarAa
O2
©M J Larkin 2008.
OH
OH
H
H
OH
OH
NarB
NADH + H+
NAD+
NarK
NarAa
NarAb
O2
NarK
NarAb
Extremophiles – BIODEGRADATION UNDER
EXTREME 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
compounds
• ICI and Water Quality Centre – University of Arizona
©M J Larkin 2008.
‘Universal’
phylogenetic tree based on 16S rRNA
sequence data
Halobacteriales
You are here…
©M J Larkin 2008.
Animals
97
Haloarcula vallismortis
92
97
Novel ‘extremely
Haloarcula argentinensis
100
Haloarcula sp. D1
100
halophilic Archaea’
Haloarcula hispanica
100
Haloarcula sp. DSW7
63
Growing on
Haloarcula marismortui rrnA
28
Aromatic substrates
Haloarcula marismortui rrnB
Halorhabdus utahensis
Halobacterium salinarum
21
Halococcus morrhuae
Nantronococcus occultus
24
100
Nantronobacterium gregoryi
51
Natrialba magadii
59
Natrinema versiforme
89
100
Haloterrigena thermotolerans
Nantronomonas pharaonis
100
99
Haloferax volcanii
Haloferax sp. D1227
100
100
Haloferax mediterranei
Halogeometricum borinquense
75
Halobaculum gomorrense
96
Halorubrum distributum
100
100
91
Halorubrum saccharovorum
Halorubrum lacusprofundi
Halorubrum sp. E4
Methanospirillum hungatei
©M J Larkin 2008.
Aromatic substrates
COOH
COOH
Aerobic growth of Haloarcula sp. D1
benzoic & 4-hydroxybenzoic acids :
OH
COOH
COOH
Gentisate 1,2dioxygenase:
©M J Larkin 2008.
HO
+O2
OH
GDO
O
HOOC
OH
???
Accumulation of gentisic acid from
4-hydroxybenzoic acid in Haloarcula
sp. D1 cell suspensions
O
HOOC
OH
2.5
2
COOH
OH
1.5
COOH
OH
Concentration
(mM)
OH
1
0 .5
0
0
10
20
Time (hours)
©M J Larkin 2008.
COOH
COOH
HO
30
OH
4-Hydroxybenzoate pathway
Intramolecular carboxyl-group
migration / ‘NIH-shift’ ?
OH
COOH
COOH
+ 1/2O2
OH
OH
Synthesised 2,6dideutero-4hydroxybenzoic acid :
HO
COOH
D
OH
D
COOH
D
A
+ 1/2O2
OH
OH
D
COOH
OH
B
©M J Larkin 2008.
O
MeO
1
H
1H-NMR
spectra
OH
2
6
3
5
HO
H
4
H
6
7.4
4
7.2
7.3
3
7.1
ppm
36.406
7.5
Authentic (nondeuterated) standard
O
MeO
1
H
3
5
HO
OH
2
6
4
D
H
©M J Larkin 2008.
6.8
3 ppm
0.795
4
6.9
2.071
6 7.0
8.626
7.1
8.680
7.2
Deuterated
(methyl)gentisate
Aromatic catabolism in Archaea
4-Hydroxybenzoate pathway – NIH-shift not
reported in the Archaea before
HO
COOH
D
OH
D
COOH
D
A
+ 1/2O2
OH
OH
D
COOH
OH
B
©M J Larkin 2008.
Gasworks Sites: Best source of
aromatic catabolic diversity!
©M J Larkin 2008.
Nasty toxic environment!
©M J Larkin 2008.
Contaminated Ground water!
©M J Larkin 2008.
Extent of contamination at
SEREBAR remediation site
prb1
BGBH11
91700
prb2
prb3
91650
prb21
prb5
91600
prb12
prb8
prb7
prb13
prb10
91550
prb9
prb14
prb23
BGBH10 prb11
91500
prb20
prb16
91450
prb18
prb17
292100
©M J Larkin 2008.
292150
292200
292250
292300
CLONE
PHYLOGENY
V1
V2
15F
16F
17F
18F
ß-PROTEOBACTERIA
 -PROTEOBACTERIA;
GEOBACTERIACEAE
FIRMICUTES; LACTOB ACILLACEAE
ß-PROTEOBACTERIA
ß-PROTEOBACTERIA; RHODOCYCLUS
ß-PROTEOBACTERIA;
COMAMONADACEAE
ß-PROTEOBACTERIA; NITROSOLOBUS
ß-PROTEOBACTERIA;
COMAMONADACEAE
 -PROTEOBACTERIA;
GEOBACTERIACEAE
ß-PROTEOBACTERIA; RHODOCYCLUS
UNKNOWN
ß-PROTEOBACTERIA; RHODOCYCLUS
ß-PROTEOBACTERIA; RHODOCYCLUS
-PROTEOBACTERIA; PSEUDOMONAS
ß-PROTEOBACTERIA; RHODOCYCLUS
ß-PROTEOBACTERIA; ALCALIGENACEAE
ß-PROTEOBACTERIA;
COMAMONADACEAE
ß-PROTEOBACTERIA
ß-PROTEOBACTERIA
ß-PROTEOBACTERIA; BURKHOLDERA
-PROTEOBACTERIA; PSEUDOMONAS
19F
20F
UNKNOWN
ß-PROTEOBACTERIA
V4
V5
V7
V8
2F
3F
4F
5F
6F
7F
9F
10F
11F
13F
14F
©M J Larkin 2008.
PRESUMPTIVE
PHYLOGENETIC
IDENTIFICATION OF
EUBACTERIAL 16s rDNA
CLONES FROM DIRECT
SOIL DNA SAMPLES
©M J Larkin 2008.
CLONE
PHYLOGENY
1G
2G
3G
4G
7G
8G
12G
13G
17G
20G
22G
23G
26G
27G
28G
30G
33G
34G
35G
37G
43G
44G
45G
46G
50G
51G
55G
57G
59G
60G
61G
62G
63G
-PROTEOBACTERIA; PSEUDOMONAS
-PROTEOBACTERIA; METHYLOCOCCACEAE
-PROTEOBACTERIA; PSEUDOMONAS
UNKNOWN
-PROTEOBACTERIA; METHYLOCOCCACEAE
UNKNOWN
UNKNOWN
UNKNOWN
UNKNOWN
-PROTEOBACTERIA; XANTHOMONAS
UNKNOWN
UNKNOWN
-PROTEOBACTERIA; XANTHOMONAS
-PROTEOBACTERIA; PSEUDOMONAS
UNKNOWN
UNKNOWN
-PROTEOBACTERIA; ENTEROBACTERIACEAE
-PROTEOBACTERIA; XANTHOMONAS
-PROTEOBACTERIA; PSEUDOMONAS
UNKNOWN
UNKNOWN
UNKNOWN
-PROTEOBACTERIA; ENTEROBACTERIACEAE
UNKNOWN
UNKNOWN
UNKNOWN
UNKNOWN
UNKNOWN
-PROTEOBACTERIA; PSEUDOMONAS
ß-PROTEOBACTERIA; RHODOCYCLUS
UNKNOWN
-PROTEOBACTERIA; PSEUDOMONAS
-PROTEOBACTERIA; METHYLOCOCCACEAE
PRESUMPTIVE
PHYLOGENETIC
IDENTIFICATION
OF EUBACTERIAL
16s rDNA
CLONES FROM
DIRECT
GROUNDWATER
DNA SAMPLES
LABORATORY MICROCOSM REACTIVE BARRIER
- removal of key pollutants – aromatic compounds
Benzene Profile 11-5-01
Benzene
-20.00
Phenol Profile (11-5-01)
0.00
20.00
40.00
60.00
80.00
100.00
120.00
Phenol
-20.00
0
0.00
5000
20.00
2,4-Dimethylphenol
10000 15000 20000 Profile
25000 11-5-01
30000 35000
40.00
(ug/L)
60.00
80.00
100.00
120.00
2,4-Dimethylphenol
-20.00
0
0.00
4000
20.00
0
©M J Larkin 2008.
8000 12000 16000 20000 24000 28000 32000
(ug/L)
40.00
60.00
80.00
100.00
60000
120000 180000 240000 300000 360000
(ug/L)
120.00
Microbiological sample points – SEREBAR
Onlookers
Tool-box
When he goes back to his
mobile phone, that's when
it's
Back to the lab again yo
This whole rhapsody
He better go capture this
moment and hope it don't
pass him…..
Eminem - Lose Yourself
©M J Larkin 2008.
Look, if you had
one shot, one
opportunity
To seize
everything you
ever wanted-One
moment
Interceptor and
Would you capture
inlet
it or just let it slip?
….
Eminem - Lose
Yourself
Population diversity in the PRB
©M J Larkin 2008.
Which organisms are the main degraders ?
- Stable Isotope Probing (SIPS) – to detect
PAH degraders - naphthalene
Using 13C labelled naphthalene
Amplification –
Sequence analysis
= taxonomic and
phylogenetic
information
Functional genes
©M J Larkin 2008.
Napthalene Concentration (µM)
Utlilisation of 12C and 13C - naphthalene by
groundwater bacteria in microcosms
5
4
3
2
1
0
0
10
20
30
40
50
60
70
80
Time (hours)
Figure 1. Degradation of 12C- and 13C-naphthalene (3.8 µM)
in laboratory microcosm flasks inoculated with
groundwater.
©M J Larkin 2008.
DGGE and 16s rDNA sequence identification
T=0 hr factions 6-15
T=36 hr fraction 6-15
M1 M2
6 7 8 9 10 11 12 13 14 15
6 7 8 9 10 11 12 13 14 15
A
GW with 3.8 µM
12C-Naph
GW with 3.8 µM
13C-Naph
GW only
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
Acidovorax sp – related to Comamonas spp
©M J Larkin 2008.
NDO - subunit expression –
RT-PCR of 13C-RNA
RT-PCR of 13C-RNA fractions
Ladder
©M J Larkin 2008.
C
6
7
8
9
10
11
12
C
Concentration effect on dominant
degraders.......
GW
3µM
30µM
60µM
300µM
600µM
+c
-c1
-c2
NDO  - subunit
Pseudomonas P. putida G7 type
NDO  - subunit
Commonas Ralstonia U2 type
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
groundwater sample.
Red Acidovorax sp
Green Pseudomonas sp
Purple overall eubacteria
©M J Larkin 2008.
13
C content per cell (Atom%)
Raman micro-spectroscopy analysis of single cells
120
100
Acidovorax
Pseudomonas
80
20
19
60
40
40
10
20
0
3.8 µm
13
30 µm
C Naphthalene concentration
Stable isotope based analysis of phylogenetic identity, functional transcripts and metabolic activity in natural
microbial populations
Wei 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.
Acknowledgements
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.
PERCEPTIONS
Jimi Hendrix
ALL ALONG THE WATCHTOWER
By Bob Dylan
©M J Larkin 2008.