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From Biosphere to Molecule via

From Biosphere to Molecule via The Petri Dish M. J. Larkin The QUESTOR Centre and Biological Sciences The Queen’s University of Belfast. The lab and acknowledgements Overview - the planet and its microbial biomass

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From Biosphere to Molecule via

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  1. From Biosphere to Molecule via The Petri DishM. J. Larkin The QUESTOR Centre and Biological Sciences The Queen’s University of Belfast

  2. 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 Summary

  3. 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 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 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 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

  4. The Queen’s University Environmental Science and TechnolOgyResearch Centre Jim Swindall Wilson McGarel

  5. SCOPE OF THE INTERDISCIPLINARY RESEARCH EFFORT FROM THE FIELD AND LABORATORY MICROCOSM TO MOLECULAR MICROBIOLOGY; ENZYMES, CELLS AND GENE EVOLUTION AND DIVERSITY

  6. 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

  7. Microorganisms - the root of diversity. Oxidised Atmosphere Aerobic life Plants & Animals Eubacteria Archaea O2 from water Reduced Anoxic Atmosphere Anaerobic life 3.5 billion years

  8. 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!

  9. 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 The potential of One gram of soil…

  10. Traditional approach: Millions of chemicals 10 x 106Chemicals 8 x 106Xenobiotic 1 x 106Recalcitrant 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 Philosophy of the laboratory mission

  11. "Multi pertransibunt et augebitur scientia“ (Many will pass through and knowledge will be increased). Book of Daniel(chapter 12, verse 4) 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.”

  12. 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

  13. 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?

  14. 1- Chlorobutane degradation by Rhodococcussp NCIMB13064 CH2-OH CH2 CH2 CH3 H2O HCl CH2-Cl CH2 CH2 CH3 CHO CH2 CH2 CH3 COOH CH2 CH2 CH3 X XH2 Y +H2O YH2 DhaA AdhA AldA IS2112 invA dhaR dhaA adhA aldA 1 Kb Order of genes on pRTL1 (approx 100 Kbp plasmid)

  15. GlobalDhaA spread in bacterial isolates Gerrit J. Poelarends, MarjanZandstra, TjibbeBosma, 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.

  16. Spread of dhaAamongst strains world-wide NCIMB13064 UK TB2 USA m15-3 JAPAN GJ70 THE NETHERLANDS HA1 SWITZERLAND Y2 UK IS2112 invA invA dhaR dhaR dhaA dhaA adhA adhA aldA aldA dhaA (100%) also in Pseudomonas pavonaceae 170 The Netherlands (1,3-dichloropropene) Poelarends Janssen et al 1999 Appl.Environ. Microbiol. 64:2931-2936

  17. dhaA: Recombinationsacross species Mycobacterium sp GP1 (1,2-dibromoethane) dhaAf intM invA dhaR Rhodococcus sp NCIMB 13064 intP dhaA tnpA IS1071 P .pavonaceae 170 (1,3-dichlrororopene) IS2112 invA dhaR dhaA adhA aldA

  18. Mycobacterium sp GP1 (1,2-dibromoethane) P .pavonaceae 170 (1,3-dichlrororopene) Rhodococcus sp NCIMB 13064: UK TB2: USA m15-3: JAPAN dhaAf intM invA dhaR IS2112 dhaR dhaA adhA aldA invA Rhodococcus sp GJ70:THE NETHERLANDS HA1: SWITZERLAND Y2: UK invA dhaR dhaA adhA aldA intP dhaA tnpA IS1071 Genetic Recombinations and Global Distribution of Dehalogenases - Summary

  19. 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.

  20. Methyl transferase activity – not halohydrolase Methanethiol HS-

  21. 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 Role of Oxygen in the biosphere

  22. 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.

  23. Activation of Oxygen enzymatically Not common in catabolism Very common in oxygenases

  24. 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

  25. RING HYDROXYLATING DIOXYGENASE RING OPENINING DIOXYGENASES Scheme for naphthalene catabolism in bacteria

  26. Bioavailability Solubility Cellular metabolism DIOXYGENASE BOTTLENECK Substrate fit Natural vs pollutant Affinity and rate Provision of electrons Provision of oxygen EnvironmentAffinity and rate What are the potential rate-limiting steps?

  27. Rhodococcus 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

  28. Comparison of  and  components of ISPNAR (Rhodococcus NDO) and ISPNAH (Pseudomonas NDO) *Analogous Rhodococcus ISPNAR **Pseudomonas ISPNAH Napthalene Pyruvate Salicylate Reductase Ferredoxin ISP NAP NAP NAP NAD + (OX) (OX) (OX) O 2 O H O H Ferredoxin ISP Reductase NADH  55KD NAP NAP NAP + H + (RED) (RED) (RED)  23KD       NO significant DNA HOMOLOGY: Amino acid similarity  (31%)  (39%)

  29. 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.

  30. Diversity of Bacterial NDO alpha subunits Moser and Stahl, 1999

  31. STRUCTURE OF Rhodococcus NDO

  32. nidA B C D oxiA narAa Ab B C rub1 narR1 R2 narK I24 P400 rub1 narR1,R2 narK narAa Ab B C P200 NCBI12038 rub1 narR1,R2 rub2 narK narAa Ab B ΔC orf1 – 3 orf4 – 6 CIR2 Transcription induced by growth on Naphthalene: orf1 rnoA1 A2 A3 A4 rnoB narR1, R2 rub2 narK narAa, Ab, B narC orf1 – 3 orf4 – 6 Organisation of naphthalene degradation genes in Rhodococcus

  33. A B NarB NarB e- NarK NarAa NarAb NarAa NarK O2 O2 NarAb A novel mechanism for electron transfer....

  34. 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

  35. You are here… ‘Universal’ phylogenetic tree - based on 16S rRNA sequence data Halobacteriales Prokaryotes Eukaryotes Animals

  36. Haloarcula marismortui rrnB 97 Haloarcula vallismortis 92 97 Haloarcula argentinensis 100 Haloarcula sp. D1 100 Haloarcula hispanica 100 Haloarcula sp. DSW7 63 Haloarcula marismortui rrnA 28 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 Haloferax volcanii 99 Haloferax sp. D1227 100 100 Haloferax mediterranei Halogeometricum borinquense Halobaculum gomorrense 75 Halorubrum distributum 96 100 Halorubrum saccharovorum 100 Halorubrum lacusprofundi Halorubrum sp. E4 91 Methanospirillum hungatei Novel ‘extremely halophilicArchaea’ Growing on Aromatic substrates

  37. Aerobic growth of Haloarcula sp. D1 benzoic & 4-hydroxybenzoic acids : Gentisate 1,2-dioxygenase: Aromatic substrates ? ? ?

  38. Concentration (mM) Time (hours) Accumulation of gentisic acid from 4-hydroxybenzoic acid in Haloarcula sp. D1 cell suspensions

  39. Intramolecular carboxyl-group migration / ‘NIH-shift’ ? Synthesised 2,6-dideutero-4-hydroxybenzoic acid : 4-Hydroxybenzoate pathway

  40. Authentic (non-deuterated) standard 6 4 3 Deuterated (methyl)gentisate 6 4 3 1H-NMR spectra

  41. Aromatic catabolism in Archaea 4-Hydroxybenzoate pathway – NIH-shift not reported in the Archaea before

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

  43. Nasty toxic environment!

  44. Contaminated Ground water!

  45. Sand 2 GAC 2 Sand 1 GAC 2 Sand 2 Sand 1 Sand 3 ZVI GAC 1 GAC 1 Sand 3 ZVI Input Input Interceptor Interceptor Extent of contamination at SEREBAR remediation site

  46. PRESUMPTIVE PHYLOGENETIC IDENTIFICATION OF EUBACTERIAL 16s rDNA CLONES FROM DIRECT SOIL DNA SAMPLES

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

  48. Benzene Phenol 2,4-Dimethylphenol LABORATORY MICROCOSM REACTIVE BARRIER - removal of key pollutants – aromatic compounds

  49. Onlookers 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 When he goes back to his mobile phone, that's when it'sBack to the lab again yoThis whole rhapsodyHe better go capture this moment and hope it don't pass him….. Eminem - Lose Yourself Interceptor and inlet Microbiological sample points – SEREBAR

  50. Population diversity in the PRB

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