From Biosphere to Molecule

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From Biosphere to Molecule via The Petri
Dish M. J. Larkin  The QUESTOR Centre and
Biological Sciences The Queens University of
Belfast
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Summary

• 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

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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 OReilly (Derek
Boyd, Chemistry) Joe Vyle (Chemistry) Peter
Coyle (RVL) Stephen Allen (Chem Eng) Andrew
Ferguson Asa Moyce Derek Fairley Emma
Frew Dave Lipscomb Peter Gray Helen
Irvine Andrew Fraser Ros
Andserson Kathryn Lawson Andrew Lee Veronique
Durocq Nichola Connery Chen Shenchang Andrew
Mudd Tim Gilfedder Harpinder Mundi Paul
Flanagan Antonio de Casale Osa Osalador Jose
Argudo
Ian Thompson, Andrew Whitely, Wei Huang,
Oxford Dick Janssen, Gerrit Poelarends GRONINGEN A
ndy Weightman, Julian Marchesi CARDIFF Andrei
Filonov, Vladimir Ksenzenko PUSHCHINO David
Gibson, Ramaswamy, Rebecca Parales U of IOWA Ian
Pepper and Chris Rensing, John OHanlon Water
Quality Center, U of ARIZONA VISITORS Samera
Alwadi KUWAIT Susheela Carroll U of
Arizona Sebastian Sorensen GEUS Denmark Monika
Knoppova ICT Prague
FUNDING SOURCES INDUSTRYQUESTOR Centre Exxon
ICIDuPont ESB Shell BP SRIF ECFW4EC TDP
PEACE II Centres of Excellence INTAS BBSRC
DTI LINK EPSRC NERC DEL CAST Prospect Globe
Award TALENT Kuwait Government
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The Queens University Environmental Science and
TechnolOgy Research Centre Jim Swindall Wilson
McGarel
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SCOPE OF THE INTERDISCIPLINARY RESEARCH EFFORT
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RESEARCH 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

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Microorganisms - the root of diversity.
Oxidised Atmosphere Aerobic life
O2 from water
Reduced Anoxic Atmosphere Anaerobic life
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Where are they found?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 up to 2 m layer over planet!

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The potential of One gram of soil

• 1 x 1010 microbial cells (typical clay loam)
• 4 x 103 microbial species
• lt 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

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Philosophy 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.
• 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

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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. 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.
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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 18681882
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Fate of chloroalkanes 1- 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?

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1- Chlorobutane degradation by Rhodococcus sp
NCIMB13064
Order of genes on pRTL1 (approx 100 Kbp plasmid)
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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. 1822725-2731.
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Spread of dhaA amongst strains world-wide
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dhaA Recombinations across species
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Genetic Recombinations and Global Distribution
of Dehalogenases - Summary
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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) HalomethaneBisulfide/Halid
e 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 43014312.
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Methyl transferase activity not halohydrolase
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Role 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

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The reactivity of Oxygen

• 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.
• 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.

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Activation of Oxygen enzymatically
Not common in catabolism
Very common in oxygenases
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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
• 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

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Scheme for naphthalene catabolism in bacteria
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What are the potential rate-limiting steps?
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Rhodococcus NDO characterisation

• NCIMB 12038
• Enzyme components purified
• Novel Naphthalene dioxygenase (NDO)
• N-terminal sequences
• DNA and amino acid sequences
• Key active site aas conserved
• Present in other strains

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Comparison of ? and ? components of ISPNAR
(Rhodococcus NDO) and ISPNAH (Pseudomonas NDO)
NO significant DNA HOMOLOGY Amino acid
similarity ? (31) ? (39)
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Conservation of the key amino acids in ?
sub-units of NDOs from Rhodococcus and
Pseudomonas.
Asp205 is probably important for electron
transfer (12) and is essential for activity (18)
Pro118 (as well as Trp211) is from the
catalytic domain.
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Diversity of Bacterial NDO alpha subunits
Moser and Stahl, 1999
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STRUCTURE OF Rhodococcus NDO
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Organisation of naphthalene degradation genes in
Rhodococcus
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A novel mechanism for electron transfer....
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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

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Universal phylogenetic tree - based on 16S rRNA
sequence data
Halobacteriales
Prokaryotes
Eukaryotes
Animals
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Novel extremely halophilic Archaea Growing
on Aromatic substrates
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Aromatic substrates
? ? ?
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Accumulation of gentisic acid from
4-hydroxybenzoic acid in Haloarcula sp. D1 cell
suspensions
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4-Hydroxybenzoate pathway
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1H-NMR spectra
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Aromatic catabolism in Archaea 4-Hydroxybenzoate
pathway NIH-shift not reported in the Archaea
before
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Gasworks Sites Best source of aromatic catabolic
diversity!
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Nasty toxic environment!
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Contaminated Ground water!
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Extent of contamination at SEREBAR remediation
site
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PRESUMPTIVE PHYLOGENETIC IDENTIFICATION OF
EUBACTERIAL 16s rDNA CLONES FROM DIRECT SOIL DNA
SAMPLES
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PRESUMPTIVE PHYLOGENETIC IDENTIFICATION OF
EUBACTERIAL 16s rDNA CLONES FROM DIRECT
GROUNDWATER DNA SAMPLES
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LABORATORY MICROCOSM REACTIVE BARRIER - removal
of key pollutants aromatic compounds
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Microbiological sample points SEREBAR
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Population diversity in the PRB
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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
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Utlilisation of 12C and 13C - naphthalene by
groundwater bacteria in microcosms
Figure 1. Degradation of 12C- and 13C-naphthalene
(3.8 µM) in laboratory microcosm flasks
inoculated with groundwater.
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DGGE and 16s rDNA sequence identification
Acidovorax sp related to Comamonas spp
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NDO ?- subunit expression RT-PCR of 13C-RNA
RT-PCR of 13C-RNA fractions
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Concentration effect on dominant 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
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FISH images show microbial degraders in
groundwater sample.
Red Acidovorax sp Green Pseudomonas sp Purple
overall eubacteria
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Raman micro-spectroscopy analysis of single
cells
Stable isotope based analysis of phylogenetic
identity, functional transcripts and metabolic
activity in natural microbial populations Wei E.
Huang1,2?, 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_
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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
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PERCEPTIONS
Jimi Hendrix ALL ALONG THE WATCHTOWER By Bob
Dylan