59 results on '"Larkin MJ"'
Search Results
2. Haloalkane-Utilizing Rhodococcus Strains Isolated from Geographically Distinct Locations Possess a Highly Conserved Gene Cluster Encoding Haloalkane Catabolism
- Author
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Poelarends, GJ, Bosma, T, Kulakov, LA, Larkin, MJ, Marchesi, [No Value], Weightman, AJ, Janssen, DB, Kulakov, Leonid A., Larkin, Michael J., Marchesi, Julian R., Weightman, Andrew J., Groningen Biomolecular Sciences and Biotechnology, Biotechnology, Molecular Microbiology, Biopharmaceuticals, Discovery, Design and Delivery (BDDD), and Medicinal Chemistry and Bioanalysis (MCB)
- Subjects
DNA, Bacterial ,DEHALOGENASE ,Hydrolases ,Molecular Sequence Data ,HALIDOHYDROLASE ,Biology ,SEQUENCE ,Microbiology ,Conserved sequence ,Plasmid ,RNA, Ribosomal, 16S ,Alkanes ,Gene cluster ,Rhodococcus ,ARTHROBACTER SP ,2-DICHLOROETHANE ,Molecular Biology ,Gene ,Conserved Sequence ,Southern blot ,Dehalogenase ,Genetics ,PURIFICATION ,ERYTHROPOLIS ,Base Sequence ,Hydrocarbons, Halogenated ,Sequence Analysis, RNA ,16S RIBOSOMAL-RNA ,Chromosome Mapping ,DEGRADATION ,Molecular biology ,RNA, Bacterial ,Genes, Bacterial ,Multigene Family ,Horizontal gene transfer ,HORIZONTAL TRANSFER ,Population Genetics and Evolution ,1,2-DICHLOROETHANE ,Haloalkane dehalogenase - Abstract
The sequences of the 16S rRNA and haloalkane dehalogenase ( dhaA ) genes of five gram-positive haloalkane-utilizing bacteria isolated from contaminated sites in Europe, Japan, and the United States and of the archetypal haloalkane-degrading bacterium Rhodococcus sp. strain NCIMB13064 were compared. The 16S rRNA gene sequences showed less than 1% sequence divergence, and all haloalkane degraders clearly belonged to the genus Rhodococcus . All strains shared a completely conserved dhaA gene, suggesting that the dhaA genes were recently derived from a common ancestor. The genetic organization of the dhaA gene region in each of the haloalkane degraders was examined by hybridization analysis and DNA sequencing. Three different groups could be defined on the basis of the extent of the conserved dhaA segment. The minimal structure present in all strains consisted of a conserved region of 12.5 kb, which included the haloalkane-degradative gene cluster that was previously found in strain NCIMB13064. Plasmids of different sizes were found in all strains. Southern hybridization analysis with a dhaA gene probe suggested that all haloalkane degraders carry the dhaA gene region both on the chromosome and on a plasmid (70 to 100 kb). This suggests that an ancestral plasmid was transferred between these Rhodococcus strains and subsequently has undergone insertions or deletions. In addition, transposition events and/or plasmid integration may be responsible for positioning the dhaA gene region on the chromosome. The data suggest that the haloalkane dehalogenase gene regions of these gram-positive haloalkane-utilizing bacteria are composed of a single catabolic gene cluster that was recently distributed worldwide.
- Published
- 2000
3. Characterization of IS2112, a new insertion sequence from Rhodococcus, and its relationship with mobile elements belonging to the IS110 family
- Author
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Kulakov, LA, Poelarends, GJ, Janssen, DB, Larkin, MJ, Kulakov, Leonid A., Larkin, Michael J., Groningen Biomolecular Sciences and Biotechnology, Biotechnology, Molecular Microbiology, Biopharmaceuticals, Discovery, Design and Delivery (BDDD), and Medicinal Chemistry and Bioanalysis (MCB)
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Transposable element ,Inverted repeat ,Hydrolases ,genome rearrangements ,Molecular Sequence Data ,TRANSPOSABLE ELEMENT ,Transposases ,HALIDOHYDROLASE ,STREPTOMYCES-COELICOLOR A3(2) ,Microbiology ,haloalkane dehalogenase ,Evolution, Molecular ,Species Specificity ,NUCLEOTIDE-SEQUENCE ,Rhodococcus ,Amino Acid Sequence ,Insertion sequence ,RHODOCHROUS NCIMB13064 ,Genetics ,biology ,ERYTHROPOLIS ,Sequence Homology, Amino Acid ,MINI-CIRCLE ,insertion element ,Gene rearrangement ,Rhodococcus rhodochrous ,Sequence Analysis, DNA ,DEGRADATION ,PLASMIDS ,biology.organism_classification ,Molecular biology ,GENE ,Composite transposon ,DNA Transposable Elements ,Haloalkane dehalogenase - Abstract
A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064. The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences. IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116, Two copies of IS2112 were found in R, rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains. There were no sequences homologous to IS2112 found in the l-haloalkane-degrading 'Pseudomonas pavonaceae' 170 and Rhodococcus sp, HA1 or in several Rhodococcus strains which do not utilize haloalkanes, IS2112 was originally found in plasmid pRTL1 of R. rhodochrous NCIMB 13064 which harbours genes encoding utilization of l-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA), Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization. An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements. IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.
- Published
- 1999
4. PRS23 THE POTENTIAL COST IMPACT OF USING A PEG HYDROGEL SEALANT TO PREVENT AIR LEAKS AFTER LUNG RESECTION SURGERY IN THE UNITED KINGDOM
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Minshall, ME, primary, Larkin, MJ, additional, and Ott, M, additional
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- 2010
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5. Spectroscopic Characterisation of the Naphthalene Dioxygenase from Rhodococcus sp. Strain NCIMB12038.
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Baratto MC, Lipscomb DA, Larkin MJ, Basosi R, Allen CCR, and Pogni R
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- Dithionite chemistry, Electron Spin Resonance Spectroscopy, Hydrogen Peroxide chemistry, Oxidation-Reduction, Biodegradation, Environmental, Dioxygenases metabolism, Environmental Pollutants metabolism, Multienzyme Complexes metabolism, Naphthalenes metabolism, Rhodococcus enzymology, Rhodococcus metabolism
- Abstract
Polycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are potential health risks due to their carcinogenic and mutagenic effects. Bacteria from the genus Rhodococcus are able to metabolise a wide variety of pollutants such as alkanes, aromatic compounds and halogenated hydrocarbons. A naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038 has been characterised for the first time, using electron paramagnetic resonance (EPR) spectroscopy and UV-Vis spectrophotometry. In the native state, the EPR spectrum of naphthalene 1,2-dioxygenase (NDO) is formed of the mononuclear high spin Fe(III) state contribution and the oxidised Rieske cluster is not visible as EPR-silent. In the presence of the reducing agent dithionite a signal derived from the reduction of the [2Fe-2S] unit is visible. The oxidation of the reduced NDO in the presence of O
2 -saturated naphthalene increased the intensity of the mononuclear contribution. A study of the "peroxide shunt", an alternative mechanism for the oxidation of substrate in the presence of H2 O2 , showed catalysis via the oxidation of mononuclear centre while the Rieske-type cluster is not involved in the process. Therefore, the ability of these enzymes to degrade recalcitrant aromatic compounds makes them suitable for bioremediative applications and synthetic purposes.- Published
- 2019
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6. Analysis of viral and bacterial communities in groundwater associated with contaminated land.
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Costeira R, Doherty R, Allen CCR, Larkin MJ, and Kulakov LA
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- Bacteria classification, Groundwater virology, Northern Ireland, Viruses classification, Bacteria genetics, Groundwater microbiology, Metagenome, Microbiota, Soil Pollutants analysis, Viruses genetics
- Abstract
This work aimed at the comprehensive analysis of total microbial communities inhabiting a typical hydrocarbon-polluted site, where chemical characteristics of the groundwater were readily available. To achieve this, a joint metagenomic characterization of bacteria and viruses surrounding a contaminant plume was performed over a one-year period. The results presented demonstrated that both potential hydrocarbon degraders and their bacteriophages were dominant around the plume, and that the viral and bacterial diversities found at the site were probably influenced by the pH of the groundwater. Niche-specific and dispersed associations between phages and bacteria were identified. The niche phage-host associations were found at the edge of the site and at the core of the plume where pH was the highest (9.52). The identified host populations included several classes of bacteria (e.g. Clostridia and Proteobacteria). Thirty-six viral generalists were also discovered, with BGW-G9 having the broadest host range across 23 taxa, including Pseudomonas, Polycyclovorans, Methylocaldum and Candidatus Magnetobacterium species. The phages with broad host ranges are presumed to have significant effects on prokaryotic production and horizontal gene transfer, and therefore impact the biodegradation processes conducted by various bacteria of the environment studied. This study for the first time characterized the phages and their bacterial hosts associated with a contaminant plume., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)
- Published
- 2019
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7. Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis.
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Harrington C, Del Casale A, Kennedy J, Neve H, Picton BE, Mooij MJ, O'Gara F, Kulakov LA, Larkin MJ, and Dobson ADW
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- Animals, Bacteriophages classification, Bacteriophages isolation & purification, Bacteriophages physiology, DNA, Bacterial metabolism, DNA, Viral genetics, Molecular Sequence Data, Phylogeny, Porifera genetics, RNA, Ribosomal, 16S metabolism, Seawater chemistry, Seawater microbiology, Seawater virology, Bacteria genetics, Bacteriophages genetics, DNA, Bacterial genetics, Gene Transfer, Horizontal, Metagenome, Porifera virology, RNA, Ribosomal, 16S genetics
- Abstract
Marine sponges have never been directly examined with respect to the presence of viruses or their potential involvement in horizontal gene transfer. Here we demonstrate for the first time, to our knowledge, the presence of viruses in the marine sponge Hymeniacidon perlevis. Moreover, bacterial 16S rDNA was detected in DNA isolated from these viruses, indicating that phage-derived transduction appears to occur in H. perlevis. Phylogenetic analysis revealed that bacterial 16S rDNA isolated from sponge-derived viral and total DNA differed significantly, indicating that not all species are equally involved in transduction.
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- 2012
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8. Extent and variation of phage-borne bacterial 16S rRNA gene sequences in wastewater environments.
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Del Casale A, Flanagan PV, Larkin MJ, Allen CC, and Kulakov LA
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- Molecular Sequence Data, Phylogeny, Sequence Alignment, Waste Disposal, Fluid, Bacteria virology, Bacteriophages genetics, Bacteriophages isolation & purification, RNA, Ribosomal, 16S genetics, Sewage microbiology, Water Microbiology
- Abstract
Phage metagenomes isolated from wastewater over a 12-month period were analyzed. The results suggested that various strains of Proteobacteria, Bacteroidetes, and other phyla are likely to participate in transduction. The patterns of 16S rRNA sequences found in phage metagenomes did not follow changes in the total bacterial community.
- Published
- 2011
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9. Analysis of transduction in wastewater bacterial populations by targeting the phage-derived 16S rRNA gene sequences.
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Del Casale A, Flanagan PV, Larkin MJ, Allen CC, and Kulakov LA
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- Bacteria classification, Bacteria isolation & purification, DNA, Bacterial genetics, DNA, Viral genetics, Denaturing Gradient Gel Electrophoresis, Gene Library, Gene Transfer, Horizontal, Genes, Bacterial, Waste Disposal, Fluid, Water Microbiology, Bacteria genetics, Bacteriophages genetics, Metagenome, Phylogeny, RNA, Ribosomal, 16S genetics, Transduction, Genetic
- Abstract
Bacterial 16S rRNA genes transduced by bacteriophages were identified and analyzed in order to estimate the extent of the bacteriophage-mediated horizontal gene transfer in the wastewater environment. For this purpose, phage and bacterial DNA was isolated from the oxidation tank of a municipal wastewater treatment plant. Phylogenetic analysis of the 16S rRNA gene sequences cloned from a phage metagenome revealed that bacteriophages transduce genetic material in several major groups of bacteria. The groups identified were as follows: Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Actinomycetales and Firmicutes. Analysis of the 16S rRNA gene sequences in the total bacterial DNA from the same sample revealed that several bacterial groups found in the oxidation tank were not present in the phage metagenome (e.g. Deltaproteobacteria, Nitrospira, Planctomycetes and many Actinobacteria genera). These results suggest that transduction in a wastewater environment occurs in several bacterial groups; however, not all species are equally involved into this process. The data also showed that a number of distinctive bacterial strains participate in transduction-mediated gene transfer within identified bacterial groupings. Denaturing gradient gel electrophoresis analysis confirmed that profiles of the transduced 16S rRNA gene sequences and those present in the whole microbial community show significant differences., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)
- Published
- 2011
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10. Genomes of "phiKMV-like viruses" of Pseudomonas aeruginosa contain localized single-strand interruptions.
- Author
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Kulakov LA, Ksenzenko VN, Shlyapnikov MG, Kochetkov VV, Del Casale A, Allen CC, Larkin MJ, Ceyssens PJ, and Lavigne R
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- Consensus Sequence, DNA, Viral genetics, DNA Breaks, Single-Stranded, Genome, Viral, Podoviridae genetics, Pseudomonas aeruginosa virology
- Abstract
The "phiKMV-like viruses" comprise an important genus of T7 related phages infecting Pseudomonas aeruginosa. The genomes of these bacteriophages have localized single-strand interruptions (nicks), a distinguishing genomic trait previously thought to be unique for T5 related coliphages. Analysis of this feature in the newly sequenced phage phikF77 shows all four nicks to be localized on the non-coding DNA strand. They are present with high frequencies within the phage population and are introduced into the phage DNA at late stages of the lytic cycle. The general consensus sequence in the nicks (5'-CGACxxxxxCCTAoh pCTCCGG-3') was shown to be common among all phiKMV-related phages.
- Published
- 2009
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11. Resolving genetic functions within microbial populations: in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization.
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Huang WE, Ferguson A, Singer AC, Lawson K, Thompson IP, Kalin RM, Larkin MJ, Bailey MJ, and Whiteley AS
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- Comamonadaceae isolation & purification, Comamonadaceae metabolism, DNA, Bacterial chemistry, DNA, Bacterial genetics, Molecular Sequence Data, Pseudomonas fluorescens isolation & purification, Pseudomonas fluorescens metabolism, Pseudomonas putida isolation & purification, Pseudomonas putida metabolism, Sequence Analysis, DNA, Water Microbiology, Carbon Isotopes analysis, In Situ Hybridization, Fluorescence methods, Naphthalenes metabolism, RNA, Messenger genetics, RNA, Ribosomal genetics, Staining and Labeling methods
- Abstract
Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope ((13)C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 muM. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment.
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- 2009
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12. Microbial analysis of soil and groundwater from a gasworks site and comparison with a sequenced biological reactive barrier remediation process.
- Author
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Ferguson AS, Huang WE, Lawson KA, Doherty R, Gibert O, Dickson KW, Whiteley AS, Kulakov LA, Thompson IP, Kalin RM, and Larkin MJ
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- Bacteria genetics, Bacteria growth & development, Biodegradation, Environmental, Polycyclic Aromatic Hydrocarbons, Polymerase Chain Reaction, Bacteria classification, Ecosystem, Soil Microbiology, Soil Pollutants metabolism, Water Microbiology
- Abstract
Aims: To investigate the distribution of a polymicrobial community of biodegradative bacteria in (i) soil and groundwater at a former manufactured gas plant (FMGP) site and (ii) in a novel SEquential REactive BARrier (SEREBAR) bioremediation process designed to bioremediate the contaminated groundwater., Methods and Results: Culture-dependent and culture-independent analyses using denaturing gradient gel electrophoresis (DGGE) and polymerase chain reaction (PCR) for the detection of 16S ribosomal RNA gene and naphthalene dioxygenase (NDO) genes of free-living (planktonic groundwater) and attached (soil biofilm) samples from across the site and from the SEREBAR process was applied. Naphthalene arising from groundwater was effectively degraded early in the process and the microbiological analysis indicated a dominant role for Pseudomonas and Comamonas in its degradation. The microbial communities appeared highly complex and diverse across both the sites and in the SEREBAR process. An increased population of naphthalene degraders was associated with naphthalene removal., Conclusion: The distribution of micro-organisms in general and naphthalene degraders across the site was highly heterogeneous. Comparisons made between areas contaminated with polycyclic aromatic hydrocarbons (PAH) and those not contaminated, revealed differences in the microbial community profile. The likelihood of noncultured bacteria being dominant in mediating naphthalene removal was evident., Significance and Impact of the Study: This work further emphasizes the importance of both traditional and molecular-based tools in determining the microbial ecology of contaminated sites and highlights the role of noncultured bacteria in the process.
- Published
- 2007
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13. Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera.
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Fairley DJ, Wang G, Rensing C, Pepper IL, and Larkin MJ
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- Benzoates metabolism, Cloning, Molecular, DNA, Archaeal genetics, Gene Expression Profiling, Haloarcula enzymology, Haloferax enzymology, Molecular Sequence Data, Sequence Alignment, Dioxygenases genetics, Genes, Archaeal, Haloarcula genetics, Haloferax genetics
- Abstract
Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain.
- Published
- 2006
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14. Biodegradation by members of the genus Rhodococcus: biochemistry, physiology, and genetic adaptation.
- Author
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Larkin MJ, Kulakov LA, and Allen CC
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- Adaptation, Physiological, Biodegradation, Environmental, Oxygenases genetics, Phylogeny, Plasmids genetics, Recombination, Genetic, Rhodococcus classification, Rhodococcus genetics, Rhodococcus metabolism, Species Specificity, Gene Expression Regulation, Bacterial, Gene Transfer, Horizontal, Genome, Bacterial, Oxygenases metabolism, Rhodococcus physiology
- Published
- 2006
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15. Structure and increased thermostability of Rhodococcus sp. naphthalene 1,2-dioxygenase.
- Author
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Gakhar L, Malik ZA, Allen CC, Lipscomb DA, Larkin MJ, and Ramaswamy S
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- Amino Acid Sequence, Binding Sites, Circular Dichroism, Dioxygenases, Enzyme Stability, Models, Molecular, Molecular Sequence Data, Multienzyme Complexes genetics, Oxygenases genetics, Protein Conformation, Pseudomonas enzymology, Sequence Homology, Amino Acid, Temperature, Transition Temperature, Multienzyme Complexes chemistry, Multienzyme Complexes metabolism, Oxygenases chemistry, Oxygenases metabolism, Rhodococcus enzymology
- Abstract
Rieske nonheme iron oxygenases form a large class of aromatic ring-hydroxylating dioxygenases found in microorganisms. These enzymes enable microorganisms to tolerate and even exclusively utilize aromatic compounds for growth, making them good candidates for use in synthesis of chiral intermediates and bioremediation. Studies of the chemical stability and thermostability of these enzymes thus become important. We report here the structure of free and substrate (indole)-bound forms of naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038. The structure of the Rhodococcus enzyme reveals that, despite a approximately 30% sequence identity between these naphthalene dioxygenases, their overall structures superpose very well with a root mean square deviation of less than 1.6 A. The differences in the active site of the two enzymes are pronounced near the entrance; however, indole binds to the Rhodococcus enzyme in the same orientation as in the Pseudomonas enzyme. Circular dichroism spectroscopy experiments show that the Rhodococcus enzyme has higher thermostability than the naphthalene dioxygenase from Pseudomonas species. The Pseudomonas enzyme has an apparent melting temperature of 55 degrees C while the Rhodococcus enzyme does not completely unfold even at 95 degrees C. Both enzymes, however, show similar unfolding behavior in urea, and the Rhodococcus enzyme is only slightly more tolerant to unfolding by guanidine hydrochloride. Structure analysis suggests that the higher thermostability of the Rhodococcus enzyme may be attributed to a larger buried surface area and extra salt bridge networks between the alpha and beta subunits in the Rhodococcus enzyme.
- Published
- 2005
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16. Analysis of genes involved in methyl halide degradation in Aminobacter lissarensis CC495.
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Warner KL, Larkin MJ, Harper DB, Murrell JC, and McDonald IR
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- Alphaproteobacteria enzymology, Amino Acid Sequence, Bacterial Proteins chemistry, Biodegradation, Environmental, Cloning, Molecular, Conserved Sequence, DNA, Bacterial chemistry, DNA, Bacterial genetics, Gene Order, Hyphomicrobium genetics, Molecular Sequence Data, Multigene Family, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Synteny, Alphaproteobacteria genetics, Alphaproteobacteria metabolism, Bacterial Proteins genetics, Genes, Bacterial, Hydrocarbons, Brominated metabolism, Methyl Chloride metabolism
- Abstract
Aminobacter lissarensis CC495 is an aerobic facultative methylotroph capable of growth on glucose, glycerol, pyruvate and methylamine as well as the methyl halides methyl chloride and methyl bromide. Previously, cells grown on methyl chloride have been shown to express two polypeptides with apparent molecular masses of 67 and 29 kDa. The 67 kDa protein was purified and identified as a halomethane:bisulfide/halide ion methyltransferase. This study describes a single gene cluster in A. lissarensis CC495 containing the methyl halide utilisation genes cmuB, cmuA, cmuC, orf 188, paaE and hutI. The genes correspond to the same order and have a high similarity to a gene cluster found in Aminobacter ciceronei IMB-1 and Hyphomicrobium chloromethanicum strain CM2 indicating that genes encoding methyl halide degradation are highly conserved in these strains.
- Published
- 2005
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17. Aminobacter ciceronei sp. nov. and Aminobacter lissarensis sp. nov., isolated from various terrestrial environments.
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McDonald IR, Kämpfer P, Topp E, Warner KL, Cox MJ, Hancock TLC, Miller LG, Larkin MJ, Ducrocq V, Coulter C, Harper DB, Murrell JC, and Oremland RS
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- Alphaproteobacteria genetics, Alphaproteobacteria physiology, Bacterial Typing Techniques, Biodegradation, Environmental, California, Canada, Carbamates metabolism, Culture Media, DNA, Bacterial analysis, Fagus, Genes, rRNA, Herbicides metabolism, Hydrocarbons, Brominated metabolism, Methyl Chloride metabolism, Molecular Sequence Data, Northern Ireland, Pesticides metabolism, Phylogeny, RNA, Ribosomal, 16S genetics, Triazines metabolism, Agriculture, Alphaproteobacteria classification, Alphaproteobacteria isolation & purification, Soil Microbiology, Trees
- Abstract
The bacterial strains IMB-1(T) and CC495(T), which are capable of growth on methyl chloride (CH(3)Cl, chloromethane) and methyl bromide (CH(3)Br, bromomethane), were isolated from agricultural soil in California fumigated with CH(3)Br, and woodland soil in Northern Ireland, respectively. Two pesticide-/herbicide-degrading bacteria, strains ER2 and C147, were isolated from agricultural soil in Canada. Strain ER2 degrades N-methyl carbamate insecticides, and strain C147 degrades triazine herbicides widely used in agriculture. On the basis of their morphological, physiological and genotypic characteristics, these four strains are considered to represent two novel species of the genus Aminobacter, for which the names Aminobacter ciceronei sp. nov. (type strain IMB-1(T)=ATCC 202197(T)=CIP 108660(T)=CCUG 50580(T); strains ER2 and C147) and Aminobacter lissarensis sp. nov. (type strain CC495(T)=NCIMB 13798(T)=CIP 108661(T)=CCUG 50579(T)) are proposed.
- Published
- 2005
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18. Biodegradation and Rhodococcus--masters of catabolic versatility.
- Author
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Larkin MJ, Kulakov LA, and Allen CC
- Subjects
- Biodegradation, Environmental, Conserved Sequence, Cytochrome P-450 Enzyme System metabolism, Dioxygenases metabolism, Environmental Pollutants metabolism, Evolution, Molecular, Gene Expression Regulation, Bacterial, Gene Transfer, Horizontal, Rhodococcus metabolism, Genome, Bacterial, Rhodococcus genetics
- Abstract
The genus Rhodococcus is a very diverse group of bacteria that possesses the ability to degrade a large number of organic compounds, including some of the most difficult compounds with regard to recalcitrance and toxicity. They achieve this through their capacity to acquire a remarkable range of diverse catabolic genes and their robust cellular physiology. Rhodococcus appear to have adopted a strategy of hyper-recombination associated with a large genome. Notably, they harbour large linear plasmids that contribute to their catabolic diversity by acting as 'mass storage' for a large number of catabolic genes. In addition, there is increasing evidence that multiple pathways and gene homologues are present that further increase the catabolic versatility and efficiency of Rhodococcus.
- Published
- 2005
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19. Web-type evolution of rhodococcus gene clusters associated with utilization of naphthalene.
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Kulakov LA, Chen S, Allen CC, and Larkin MJ
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- Amino Acid Sequence, Bacterial Proteins metabolism, Base Sequence, Biodegradation, Environmental, Dioxygenases, Gene Expression Regulation, Bacterial, Molecular Sequence Data, Multienzyme Complexes metabolism, Multigene Family, Oxygenases metabolism, Rhodococcus classification, Rhodococcus enzymology, Sequence Analysis, DNA, Transcription, Genetic, Bacterial Proteins genetics, Evolution, Molecular, Genes, Bacterial, Multienzyme Complexes genetics, Naphthalenes metabolism, Oxygenases genetics, Rhodococcus genetics
- Abstract
Clusters of genes which include determinants for the catalytic subunits of naphthalene dioxygenase (narAa and narAb) were analyzed in naphthalene-degrading Rhodococcus strains. We demonstrated (i) that in the region analyzed homologous gene clusters are separated from each other by nonhomologous DNA, (ii) that there are various degrees of homology between related genes, and (iii) that nar genes are located on plasmids in strains NCIMB12038 and P400 and on a chromosome in P200. These observations suggest that genetic exchange and reshuffling of genetic modules, as well as vertical descent of the genetic information, were the main routes in the evolution of naphthalene degradation in Rhodococcus. These conclusions were supported by studies of transcription patterns in the region analyzed. It was found that the nar region is not organized into a single operon but there are several transcription units which differ in the strains investigated. The narA and narB genes were found to be transcribed as a single unit in all strains analyzed, and their transcription was induced by naphthalene. The putative aldolase gene (narC) was found on the same transcript only in strains P200 and P400. In NCIMB12038 transcription of two more gene clusters was induced by growth on naphthalene. Transcription start sites for narA and narB were found to be different in all of the strains studied. Putative regulatory genes (narR1 and narR2) were transcribed as a single mRNA in naphthalene-induced cells. At the same time, a number of the genes known to be essential for naphthalene catabolism in gram-negative bacteria were not found in the region analyzed.
- Published
- 2005
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20. Toxicity assessment of a former manufactured gas plant.
- Author
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Ferguson AS, Doherty R, Larkin MJ, Kalin RM, Irvine V, and Ofterdinger US
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- Biological Assay, Carbon analysis, Carbon toxicity, Cyanides analysis, Cyanides toxicity, Soil Microbiology, Vibrio drug effects, Water Microbiology, Fossil Fuels toxicity, Industry, Soil Pollutants toxicity, Water Pollutants, Chemical toxicity
- Published
- 2003
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21. Multiple inverted DNA repeats of Bacteroides fragilis that control polysaccharide antigenic variation are similar to the hin region inverted repeats of Salmonella typhimurium.
- Author
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Patrick S, Parkhill J, McCoy LJ, Lennard N, Larkin MJ, Collins M, Sczaniecka M, and Blakely G
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Bacteroides fragilis metabolism, Base Sequence, DNA Nucleotidyltransferases genetics, DNA Nucleotidyltransferases metabolism, DNA, Bacterial chemistry, DNA, Bacterial genetics, Gene Expression Regulation, Bacterial, Humans, Molecular Sequence Data, Polysaccharides, Bacterial chemistry, Polysaccharides, Bacterial genetics, Salmonella typhimurium genetics, Antigenic Variation, Bacteroides fragilis genetics, Polysaccharides, Bacterial metabolism, Recombination, Genetic, Repetitive Sequences, Nucleic Acid
- Abstract
The important opportunistic pathogen Bacteroides fragilis is a strictly anaerobic Gram-negative bacterium and a member of the normal resident human gastrointestinal microbiota. Our earlier studies indicated that there is considerable within-strain variation in polysaccharide expression, as detected by mAb labelling. Analysis of the genome sequence has revealed multiple invertible DNA regions, designated fragilis invertible (fin) regions, seven of which are upstream of polysaccharide biosynthesis loci and are approximately 226 bp in size. Using orientation-specific PCR primers and sequence analysis with populations enriched for one antigenic type, two of these invertible regions were assigned to heteropolymeric polysaccharides with different sizes of repeating units, as determined by PAGE pattern. The implication of these findings is that inversion of the fin regions switches biosynthesis of these polysaccharides off and on. The invertible regions are bound by inverted repeats of 30 or 32 bp with striking similarity to the Salmonella typhimurium H flagellar antigen inversion cross-over (hix) recombination sites of the invertible hin region. It has been demonstrated that a plasmid-encoded Hin invertase homologue (FinB), present in B. fragilis NCTC 9343, binds specifically to the invertible regions and the recombination sites have been designated as fragilis inversion cross-over (fix) sites.
- Published
- 2003
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22. Crystallization and preliminary X-ray diffraction analysis of naphthalene dioxygenase from Rhodococcus sp. strain NCIMB 12038.
- Author
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Malik ZA, Allen CC, Gakhar L, Lipscomb DA, Larkin MJ, and Ramaswamy S
- Subjects
- Crystallization, Crystallography, X-Ray, Dioxygenases, Protein Conformation, Multienzyme Complexes chemistry, Oxygenases chemistry, Rhodococcus enzymology
- Abstract
The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by Rhodococcus sp. strain NCIMB 12038. The terminal oxygenase component (naphthalene 1,2-dioxygenase) that catalyzes this reaction belongs to the aromatic ring hydroxylating dioxygenase family and has been crystallized. These enzymes utilize a mononuclear non-heme iron centre to catalyze the addition of dioxygen to their respective substrates. In this reaction, two electrons, two protons and a dioxygen molecule are consumed. The Rhodococcus enzyme has only 33 and 29% sequence identity to the corresponding alpha- and beta-subunits of the NDO system of Pseudomonas putida NCIMB 9816-4, for which the tertiary structure has been reported. In order to determine the three-dimensional structure of the Rhodococcus NDO, diffraction-quality crystals have been prepared by the hanging-drop method. The crystals belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 87.5, b = 144, c = 185.6 A, alpha = beta = gamma = 90 degrees, and diffract to 2.3 A resolution.
- Published
- 2002
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23. Aerobic metabolism of 4-hydroxybenzoic acid in Archaea via an unusual pathway involving an intramolecular migration (NIH shift).
- Author
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Fairley DJ, Boyd DR, Sharma ND, Allen CC, Morgan P, and Larkin MJ
- Subjects
- 2,2'-Dipyridyl metabolism, Aerobiosis, Biotransformation, Haloarcula growth & development, Hydroxybenzoates metabolism, Magnetic Resonance Spectroscopy, Gentisates, Haloarcula metabolism, Parabens metabolism
- Abstract
A novel haloarchaeal strain, Haloarcula sp. strain D1, grew aerobically on 4-hydroxybenzoic acid (4HBA) as a sole carbon and energy source and is the first member of the domain Archaea reported to do so. Unusually, D1 metabolized 4HBA via gentisic acid rather than via protocatechuic acid, hydroquinone, or catechol. Gentisate was detected in 4HBA-grown cultures, and gentisate 1,2-dioxygenase activity was induced in 4HBA-grown cells. Stoichiometric accumulation of gentisate from 4HBA was demonstrated in 4HBA-grown cell suspensions containing 2,2'-dipyridyl (which strongly inhibits gentisate 1,2-dioxygenase). To establish whether initial 1-hydroxylation of 4HBA with concomitant 1,2-carboxyl group migration to yield gentisate occurred, 2,6-dideutero-4HBA was synthesized and used as a substrate. Deuterated gentisate was recovered from cell suspensions and identified as 3-deutero-gentisate, using gas chromatography-mass spectrometry and proton nuclear magnetic resonance spectroscopy. This structural isomer would be expected only if a 1,2-carboxyl group migration had taken place, and it provides compelling evidence that the 4HBA pathway in Haloarcula sp. strain D1 involves a hydroxylation-induced intramolecular migration. To our knowledge, this is the first report of a pathway which involves such a transformation (called an NIH shift) in the domain Archaea.
- Published
- 2002
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24. Tandem enzyme-catalysed oxidations of alkyl phenyl sulfides and alkyl benzenes: enantiocomplementary routes to chiral phenols.
- Author
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Boyd DR, Sharma ND, Ljubez V, Byrne BE, Shepherd SD, Allen CC, Kulakov LA, Larkin MJ, and Dalton H
- Subjects
- Benzene chemistry, Catalysis, Isomerism, Oxidation-Reduction, Sulfides chemistry, Benzene metabolism, Catechols chemistry, Catechols metabolism, Oxidoreductases metabolism, Oxygenases metabolism, Sulfides metabolism
- Abstract
Dioxygenase-catalysed trioxygenation of alkyl phenyl sulfides and alkyl benzenes yields enantiopure cis-dihydrodiol sulfoxides and triols respectively; naphthalene cis-dihydrodiol dehydrogenase-catalysed aromatisation of these diastereoisomers gives enantiopure catechols of either configuration.
- Published
- 2002
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25. Analysis of bacteria contaminating ultrapure water in industrial systems.
- Author
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Kulakov LA, McAlister MB, Ogden KL, Larkin MJ, and O'Hanlon JF
- Subjects
- Colony Count, Microbial, Genes, rRNA, Gram-Negative Bacteria genetics, Gram-Negative Bacteria isolation & purification, Industry, Microscopy, Fluorescence, Molecular Sequence Data, Oxidoreductases genetics, Phylogeny, Polymerase Chain Reaction, RNA, Ribosomal, 16S genetics, Semiconductors, Sequence Analysis, DNA, Universities, Water Purification instrumentation, Equipment Contamination, Gram-Negative Bacteria classification, Water Microbiology, Water Purification methods, Water Supply
- Abstract
Bacterial populations inhabiting ultrapure water (UPW) systems were investigated. The analyzed UPW systems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and other industries. Bacteria present in the polishing loop of the UPW systems were enumerated by both plate counts and epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy (cyanotolyl tetrazolium chloride [CTC] and DAPI [4',6'-diamidino-2-phenylindole] staining) showed significantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by plate counts. A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give a positive signal with CTC stain. Bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for all of the UPW production systems studied. These included Ralstonia pickettii, Bradyrhizobium sp., Pseudomonas saccharophilia, and Stenotrophomonas strains. These bacteria constituted a significant part of the total number of isolated strains (>or=20%). Two sets of primers specific to R. pickettii and Bradyrhizobium sp. were designed and successfully used for the detection of the corresponding bacteria in the concentrated UPW samples. Unexpectedly, nifH gene sequences were found in Bradyrhizobium sp. and some P. saccharophilia strains isolated from UPW. The widespread use of nitrogen gas in UPW plants may be associated with the presence of nitrogen-fixing genes in these bacteria.
- Published
- 2002
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26. Studies of Microthrix parvicella in situ and in laboratory culture: production and use of specific antibodies.
- Author
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Connery N, Thompson AS, Patrick S, and Larkin MJ
- Subjects
- Actinobacteria immunology, Antibodies, Monoclonal, Population Dynamics, Actinobacteria isolation & purification, Antibodies, Bacterial analysis, Sewage microbiology, Waste Disposal, Fluid
- Abstract
Physiological studies on M. parvicella have been conducted to determine the rate of growth of this organism in pure culture. The organism displayed a doubling time of 128 days despite its profuse abundance in a local Wastewater Treatment Plant (WWTW). An extensive survey has been ongoing since February 2000 into the extent of M. parvicella in the WWTW. A suite of monoclonal and polyclonal antibodies has been developed to detect and quantify M. parvicella.
- Published
- 2002
27. Re-examination of the role of within-compound associations in the retrospective revaluation of causal judgements.
- Author
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Aitken MR, Larkin MJ, and Dickinson A
- Subjects
- Adult, Female, Food Hypersensitivity psychology, Food Preferences psychology, Humans, Male, Software, Association Learning, Attention, Problem Solving
- Abstract
We investigated blocking and retrospective revaluation of causal judgements using a scenario in which food cues acted as potential causes of an allergic reaction as the outcome. In the blocking contingency, the treatment cues were either paired or unpaired with the outcome prior to a second stage in which sequential compounds of treatment and target cues were paired with the outcome. The order of this compound and treatment training was reversed in retrospective revaluation contingencies. When the interstimulus interval between the treatment and target cues was unfilled on compound trials (Experiments 1 and 3), both blocking and retrospective revaluation were observed in that the target cue trained in compound with the paired treatment cue attracted lower causal ratings than the target cue trained in compound with the unpaired treatment cue. By contrast, performing a mental arithmetic task using numerals presented during the interstimulus interval had no effect on the magnitude of blocking but rendered retrospective revaluation unreliable (Experiments 2 and 3). These results provide further support for accounts of revaluation based upon within-compound associations.
- Published
- 2001
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28. The diversity of extradiol dioxygenase (edo) genes in cresol degrading rhodococci from a creosote-contaminated site that express a wide range of degradative abilities.
- Author
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Irvine VA, Kulakov LA, and Larkin MJ
- Subjects
- Base Sequence, Biodegradation, Environmental, DNA Primers, Oxygenases metabolism, Plasmids, Polymerase Chain Reaction, Pseudomonas classification, Rhodococcus classification, Soil Microbiology, Creosote, Cresols metabolism, Genetic Variation, Oxygenases genetics, Pseudomonas enzymology, Pseudomonas genetics, Rhodococcus enzymology, Rhodococcus genetics, Soil Pollutants
- Abstract
Analysis of the bacterial population of soil surface samples from a creosote-contaminated site showed that up to 50% of the culturable micro-organisms detected were able to utilise a mixture of cresols. From fifty different microbial isolates fourteen that could utilise more than one cresol isomer were selected and identified by 16S rRNA analysis. Eight isolates were Rhodococcus strains and six were Pseudomonas strains. In general, the Rhodococcus strains exhibited a broader growth substrate range than the Pseudomonas strains. The distribution of various extradiol dioxygenase (edo) genes, previously associated with aromatic compound degradation in rhodococci, was determined for the Rhodococcus strains by PCR detection and Southern-blot hybridization. One strain, Rhodococcus sp. II exhibited the broadest growth substrate range and possessed five different edo genes. Gene disruption experiments indicated that two genes (edoC and edoD) were associated with isopropylbenzene and naphthalene catabolism respectively. The other Rhodococcus strains also possessed some of the edo genes and one (edoB) was present in all of the Rhodococcus strains analysed. None of the rhodococcal edo genes analysed were present in the Pseudomonas strains isolated from the site. It was concluded that individual strains of Rhodococcus possess a wide degradative ability and may be very important in the degradation of complex mixtures of substrates found in creosote.
- Published
- 2000
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29. Roles of horizontal gene transfer and gene integration in evolution of 1,3-dichloropropene- and 1,2-dibromoethane-degradative pathways.
- Author
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Poelarends GJ, Kulakov LA, Larkin MJ, van Hylckama Vlieg JE, and Janssen DB
- Subjects
- Allyl Compounds metabolism, Amino Acid Sequence, Base Sequence, Biodegradation, Environmental, Conserved Sequence, DNA Transposable Elements, DNA-Binding Proteins metabolism, Environmental Pollutants metabolism, Ethylene Dibromide metabolism, Gene Expression Regulation, Bacterial, Hydrocarbons, Brominated, Hydrocarbons, Chlorinated, Integrases genetics, Molecular Sequence Data, Mycobacterium enzymology, Mycobacterium genetics, Pseudomonas enzymology, Pseudomonas genetics, Rhodococcus enzymology, Rhodococcus genetics, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Trans-Activators metabolism, Escherichia coli Proteins, Evolution, Molecular, Gene Transfer, Horizontal, Genes, Bacterial, Hydrocarbons, Halogenated metabolism, Hydrolases genetics, Recombination, Genetic
- Abstract
The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The dhaA gene of the 1-chlorobutane-degrading strain NCIMB13064 was found to reside within a 1-chlorobutane catabolic gene cluster, which also encodes a putative invertase (invA), a regulatory protein (dhaR), an alcohol dehydrogenase (adhA), and an aldehyde dehydrogenase (aldA). The latter two enzymes may catalyze the oxidative conversion of n-butanol, the hydrolytic product of 1-chlorobutane, to n-butyric acid, a growth substrate for many bacteria. The activity of the dhaR gene product was analyzed in Pseudomonas sp. strain GJ1, in which it appeared to function as a repressor of dhaA expression. The 1,2-dibromoethane-degrading strain GP1 contained a conserved DNA segment of 2.7 kb, which included dhaR, dhaA, and part of invA. A 12-nucleotide deletion in dhaR led to constitutive expression of dhaA in strain GP1, in contrast to the inducible expression of dhaA in strain NCIMB13064. The 1, 3-dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little more than the coding region of the dhaA gene. In strains 170 and GP1, a putative integrase gene was found next to the conserved dhaA segment, which suggests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase-dependent gene acquisition were the key mechanisms for the evolution of catabolic pathways for the man-made chemicals 1, 3-dichloropropene and 1,2-dibromoethane.
- Published
- 2000
- Full Text
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30. Super-learning of causal judgements.
- Author
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Aitken MR, Larkin MJ, and Dickinson A
- Subjects
- Adult, Causality, Cues, Female, Food Hypersensitivity prevention & control, Food Hypersensitivity psychology, Humans, Male, Probability Learning, Association Learning, Conditioning, Classical, Judgment, Overlearning
- Abstract
In three experiments, participants learned which of a variety of foods were capable of causing an allergic reaction in a hypothetical patient during training in which a compound of a treatment and a target food cue was paired with the reaction. In Experiment 1 the causal ratings of the target cue were increased if the treatment cue was pretrained as a preventative cause of the reaction. Experiments 2 and 3 demonstrated that the magnitude of this super-learning is unaffected by the order of compound and treatment cue training. The final study also showed that forward super-learning is not induced solely by simple exposure to the treatment cue prior to compound training but, rather, depends upon training the treatment cue as a preventative cause, whereas retrospective super-learning may be due merely to exposure of the treatment cue. These results are problematic for contingency-based accounts of causal induction but accord with modified and extended associative theories.
- Published
- 2000
- Full Text
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31. Cloning and characterization of a novel cis-naphthalene dihydrodiol dehydrogenase gene (narB) from Rhodococcus sp. NCIMB12038.
- Author
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Kulakov LA, Allen CC, Lipscomb DA, and Larkin MJ
- Subjects
- Alcohol Oxidoreductases chemistry, Amino Acid Sequence, Biodegradation, Environmental, Cell Fractionation, Cloning, Molecular, Culture Media, Genes, Bacterial, Molecular Sequence Data, Phylogeny, Rhodococcus growth & development, Sequence Alignment, Sequence Analysis, DNA, Alcohol Oxidoreductases genetics, Alcohol Oxidoreductases metabolism, Naphthalenes metabolism, Oxidoreductases, Oxidoreductases Acting on CH-CH Group Donors, Rhodococcus enzymology, Rhodococcus genetics
- Abstract
Rhodococcus sp. NCIMB112038 can utilize naphthalene as its sole carbon and energy source. The gene encoding cis-naphthalene dihydrodiol dehydrogenase (narB) of this strain has been cloned and sequenced. Expression of NCIMB12038 cis-naphthalene dihydrodiol dehydrogenase was demonstrated in Escherichia coli cells. narB encodes a putative protein of 271 amino acids and shares 39% amino acid identity with the cis-naphthalene dihydrodiol dehydrogenase from Pseudomonas putida G7. Comparison of NarB with some putative cis-dihydrodiol dehydrogenases from Rhodococcus species revealed significant differences between these proteins. NarB together with two other proteins forms a new group of cis-dihydrodiol dehydrogenases.
- Published
- 2000
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32. Isotopic composition of inorganic carbon as an indicator of benzoate degradation by pseudomonas putida: temperature, growth rate and pH effects.
- Author
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Barth JA, Kalin RM, Larkin MJ, Hall JA, and Fitzgerald U
- Subjects
- Hydrogen-Ion Concentration, Mass Spectrometry, Pseudomonas putida growth & development, Solubility, Temperature, Benzoates chemistry, Carbon analysis, Pseudomonas putida chemistry
- Abstract
Degradation experiments of benzoate by Pseudomonas putida resulted in enzymatic carbon isotope fractionations. However, isotopic temperature effects between experiments at 20 and 30 degrees C were minor. Averages of the last three values of the CO(2) isotopic composition (delta(13)C(CO2(g))) were more negative than the initial benzoate delta(13)C value (-26.2 per thousand Vienna Pee Dee Belenite (VPDB)) by 3.8, 3.4 and 3.2 per thousand at 20, 25 and 30 degrees C, respectively. Although the maximum isotopic temperature difference found was only 0.6 per thousand, more extreme temperature variations may cause larger isotope effects. In order to understand the isotope effects on the total inorganic carbon (TIC), a better measure is to calculate the proportions of the inorganic carbon species (CO(2)(g), CO(2)(aq) and HCO(3)(-)) and to determine their cumulative delta(13)C(TIC). In all three experiments delta(13)C(TIC) was more positive than the initial isotopic composition of the benzoate at a pH of 7. This suggests an uptake of (12)C in the biomass in order to match the carbon balance of these closed system experiments., (Copyright 2000 John Wiley & Sons, Ltd.)
- Published
- 2000
- Full Text
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33. Purification and characterization of a novel naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038.
- Author
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Larkin MJ, Allen CC, Kulakov LA, and Lipscomb DA
- Subjects
- Amino Acid Sequence, Conserved Sequence, Dioxygenases, Molecular Sequence Data, Rhodococcus enzymology, Sequence Homology, Amino Acid, Electron Transport Complex III, Iron-Sulfur Proteins genetics, Multienzyme Complexes genetics, Oxygenases genetics, Rhodococcus genetics
- Abstract
We report here the characterization of the catalytic component (ISP(NAR)) of a new naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038. The genes encoding the two subunits of ISP(NAR) are not homologous to their previously characterized counterparts in Pseudomonas. The deduced amino acid sequences have only 33 and 29% identity with the corresponding subunits in Pseudomonas putida NCIB 9816-4, for which the tertiary structure has been reported.
- Published
- 1999
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34. Halomethane:bisulfide/halide ion methyltransferase, an unusual corrinoid enzyme of environmental significance isolated from an aerobic methylotroph using chloromethane as the sole carbon source.
- Author
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Coulter C, Hamilton JT, McRoberts WC, Kulakov L, Larkin MJ, and Harper DB
- Subjects
- Amino Acid Sequence, Enzyme Activation, Enzyme Stability, Hydrogen-Ion Concentration, Isoelectric Point, Kinetics, Methyltransferases metabolism, Molecular Sequence Data, Molecular Weight, Sulfides metabolism, Temperature, Methyl Chloride metabolism, Methylobacterium enzymology, Methyltransferases isolation & purification
- Abstract
A novel dehalogenating/transhalogenating enzyme, halomethane:bisulfide/halide ion methyltransferase, has been isolated from the facultatively methylotrophic bacterium strain CC495, which uses chloromethane (CH(3)Cl) as the sole carbon source. Purification of the enzyme to homogeneity was achieved in high yield by anion-exchange chromatography and gel filtration. The methyltransferase was composed of a 67-kDa protein with a corrinoid-bound cobalt atom. The purified enzyme was inactive but was activated by preincubation with 5 mM dithiothreitol and 0.5 mM CH(3)Cl; then it catalyzed methyl transfer from CH(3)Cl, CH(3)Br, or CH(3)I to the following acceptor ions (in order of decreasing efficacy): I(-), HS(-), Cl(-), Br(-), NO(2)(-), CN(-), and SCN(-). Spectral analysis indicated that cobalt in the native enzyme existed as cob(II)alamin, which upon activation was reduced to the cob(I)alamin state and then was oxidized to methyl cob(III)alamin. During catalysis, the enzyme shuttles between the methyl cob(III)alamin and cob(I)alamin states, being alternately demethylated by the acceptor ion and remethylated by halomethane. Mechanistically the methyltransferase shows features in common with cobalamin-dependent methionine synthase from Escherichia coli. However, the failure of specific inhibitors of methionine synthase such as propyl iodide, N(2)O, and Hg(2+) to affect the methyltransferase suggests significant differences. During CH(3)Cl degradation by strain CC495, the physiological acceptor ion for the enzyme is probably HS(-), a hypothesis supported by the detection in cell extracts of methanethiol oxidase and formaldehyde dehydrogenase activities which provide a metabolic route to formate. 16S rRNA sequence analysis indicated that strain CC495 clusters with Rhizobium spp. in the alpha subdivision of the Proteobacteria and is closely related to strain IMB-1, a recently isolated CH(3)Br-degrading bacterium (T. L. Connell Hancock, A. M. Costello, M. E. Lidstrom, and R. S. Oremland, Appl. Environ. Microbiol. 64:2899-2905, 1998). The presence of this methyltransferase in bacterial populations in soil and sediments, if widespread, has important environmental implications.
- Published
- 1999
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35. Contrasting effects of a nonionic surfactant on the biotransformation of polycyclic aromatic hydrocarbons to cis-dihydrodiols by soil bacteria.
- Author
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Allen CC, Boyd DR, Hempenstall F, Larkin MJ, and Sharma ND
- Subjects
- Biodegradation, Environmental, Gram-Negative Aerobic Rods and Cocci enzymology, Naphthalenes metabolism, Octoxynol pharmacology, Oxygenases metabolism, Phenanthrenes metabolism, Pseudomonas enzymology, Pseudomonas growth & development, Gram-Negative Aerobic Rods and Cocci metabolism, Hydrocarbons, Aromatic metabolism, Pseudomonas metabolism, Soil Microbiology, Surface-Active Agents pharmacology
- Abstract
The biotransformation of the polycyclic aromatic hydrocarbons (PAHs) naphthalene and phenanthrene was investigated by using two dioxygenase-expressing bacteria, Pseudomonas sp. strain 9816/11 and Sphingomonas yanoikuyae B8/36, under conditions which facilitate mass-transfer limited substrate oxidation. Both of these strains are mutants that accumulate cis-dihydrodiol metabolites under the reaction conditions used. The effects of the nonpolar solvent 2,2,4, 4,6,8,8-heptamethylnonane (HMN) and the nonionic surfactant Triton X-100 on the rate of accumulation of these metabolites were determined. HMN increased the rate of accumulation of metabolites for both microorganisms, with both substrates. The enhancement effect was most noticeable with phenanthrene, which has a lower aqueous solubility than naphthalene. Triton X-100 increased the rate of oxidation of the PAHs with strain 9816/11 with the effect being most noticeable when phenanthrene was used as a substrate. However, the surfactant inhibited the biotransformation of both naphthalene and phenanthrene with strain B8/36 under the same conditions. The observation that a nonionic surfactant could have such contrasting effects on PAH oxidation by different bacteria, which are known to be important for the degradation of these compounds in the environment, may explain why previous research on the application of the surfactants to PAH bioremediation has yielded inconclusive results. The surfactant inhibited growth of the wild-type strain S. yanoikuyae B1 on aromatic compounds but did not inhibit B8/36 dioxygenase enzyme activity in vitro.
- Published
- 1999
- Full Text
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36. Degradation of 1,3-dichloropropene by pseudomonas cichorii 170.
- Author
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Poelarends GJ, Wilkens M, Larkin MJ, van Elsas JD, and Janssen DB
- Subjects
- Alkanes metabolism, Biodegradation, Environmental, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Genes, Bacterial, Hydrocarbons, Chlorinated, Hydrolases genetics, Hydrolases isolation & purification, Mutation, Plasmids genetics, Pseudomonas genetics, Pseudomonas growth & development, Soil Microbiology, Allyl Compounds metabolism, Hydrolases metabolism, Insecticides metabolism, Pseudomonas metabolism
- Abstract
The gram-negative bacterium Pseudomonas cichorii 170, isolated from soil that was repeatedly treated with the nematocide 1, 3-dichloropropene, could utilize low concentrations of 1, 3-dichloropropene as a sole carbon and energy source. Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-n-alkanes. This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for cis-3-chloroacrylic acid and the other specific for trans-3-chloroacrylic acid. The haloalkane dehalogenase and the trans-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the cis-3-chloroacrylic acid dehalogenase was inducible. The presence of these enzymes indicates that 1, 3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid. The latter compound is then dehalogenated, probably forming malonic acid semialdehyde. The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studied dhaA gene of the gram-positive bacterium Rhodococcus rhodochrous NCIMB13064. Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth. PCR analysis showed that these mutants had lost at least part of the dhaA gene.
- Published
- 1998
- Full Text
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37. Applied aspects of Rhodococcus genetics.
- Author
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Larkin MJ, De Mot R, Kulakov LA, and Nagy I
- Subjects
- Biotechnology, Gene Expression Regulation, Bacterial, Gene Transfer Techniques, Genes, Reporter, Genetic Vectors, Plasmids, Recombination, Genetic, Rhodococcus genetics
- Abstract
Eubacteria of the genus Rhodococcus are a diverse group of microorganisms commonly found in many environmental niches from soils to seawaters and as plant and animal pathogens. They exhibit a remarkable ability to degrade many organic compounds and their economic importance is becoming increasingly apparent. Although their genetic organisation is still far from understood, there have been many advances in recent years. Reviewed here is the current knowledge of rhodococci relating to gene transfer, recombination, plasmid replication and functions, cloning vectors and reporter genes, gene expression and its control, bacteriophages, insertion sequences and genomic rearrangements. Further fundamental studies of Rhodococcus genetics and the application of genetic techniques to the these bacteria will be needed for their continued biotechnological exploitation.
- Published
- 1998
- Full Text
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38. Cloning of new Rhodococcus extradiol dioxygenase genes and study of their distribution in different Rhodococcus strains.
- Author
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Kulakov LA, Delcroix VA, Larkin MJ, Ksenzenko VN, and Kulakova AN
- Subjects
- Amino Acid Sequence, Base Sequence, Catechol 2,3-Dioxygenase, Molecular Sequence Data, Polymerase Chain Reaction, Rhodococcus genetics, Sequence Alignment, Sequence Analysis, DNA, Bacterial Proteins genetics, Dioxygenases, Genes, Bacterial genetics, Oxygenases genetics, Rhodococcus enzymology
- Abstract
Four extradiol dioxygenase genes which encode enzymes active against catechol and substituted catechols were cloned from two different Rhodococcus strains, and their nucleotide sequences were determined. A catechol 2,3-dioxygenase gene (edoC) was shown to be identical to the previously described ipbC gene from the isopropylbenzene operon of Rhodococcus erythropolis. Amino acid sequences deduced from the three other genes (edoA, edoB and edoD) were shown to have various degrees of homology to different extradiol dioxygenases. The EdoA and EdoB dioxygenases were classified as belonging to the third family of type I oxygenases and represented two new subfamilies, whereas the EdoD dioxygenase was a type II enzyme. Analysis of six Rhodococcus strains revealed a wide distribution of the above dioxygenase genes. Rhodococcus sp. 11 was shown to harbour all four of the analysed dioxygenase genes. Nucleotide sequences homologous to the edoB gene were present in all of the strains, including R. erythropolis NCIMB 13065, which did not utilize any of the aromatic compounds analysed. The latter finding points to the existence of a silent pathway(s) for degradation of aromatic compounds in this Rhodococcus strain.
- Published
- 1998
- Full Text
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39. Metabolism of Naphthalene, 1-Naphthol, Indene, and Indole by Rhodococcus sp. Strain NCIMB 12038.
- Author
-
Boyd C, Larkin MJ, Reid KA, Sharma ND, and Wilson K
- Abstract
The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.
- Published
- 1997
- Full Text
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40. Cryptic plasmid pKA22 isolated from the naphthalene degrading derivative of Rhodococcus rhodochrous NCIMB13064.
- Author
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Kulakov LA, Larkin MJ, and Kulakova AN
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Base Sequence, Biodegradation, Environmental, Cloning, Molecular, DNA Replication genetics, DNA, Bacterial genetics, DNA-Directed DNA Polymerase genetics, Hydrocarbons, Chlorinated metabolism, Molecular Sequence Data, Open Reading Frames, Phenotype, Recombination, Genetic genetics, Repetitive Sequences, Nucleic Acid, Rhodococcus metabolism, Sequence Alignment, Sequence Homology, Amino Acid, Naphthalenes metabolism, Plasmids genetics, Rhodococcus genetics
- Abstract
Cryptic plasmids were found in Rhodococcus rhodochrous NCIMB13064 derivatives which had lost the ability to utilize short-chain 1-chloroalkanes (chain length C3-C10) and had acquired the ability to degrade naphthalene. The reversions of these derivatives to the original phenotype were accompanied by the loss of the cryptic plasmids. The 4969-bp pKA22 plasmid was cloned in Escherichia coli and sequenced. This plasmid encodes a putative 33,200-Da protein which contains motifs typical of theta replicase proteins and shows a high degree of similarity to a putative theta replicase from Brevibacterium linens plasmid pRBL1 and to a putative protein encoded by ORF1 of the plasmid pAL5000 from Mycobacterium fortuitum. Two sets of long direct repeats were found in pKA22 which may be involved in the replication of the plasmid and recombination processes.
- Published
- 1997
- Full Text
- View/download PDF
41. The plasmid-located haloalkane dehalogenase gene from Rhodococcus rhodochrous NCIMB 13064.
- Author
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Kulakova AN, Larkin MJ, and Kulakov LA
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, Conserved Sequence, Escherichia coli genetics, Gene Expression Regulation, Bacterial, Gram-Negative Aerobic Bacteria enzymology, Gram-Negative Aerobic Bacteria genetics, Hydrolases biosynthesis, Molecular Sequence Data, Mutation, Nucleic Acid Hybridization, Recombinant Proteins biosynthesis, Rhodococcus enzymology, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Species Specificity, Substrate Specificity, Genes, Bacterial, Hydrolases genetics, Plasmids genetics, Rhodococcus genetics
- Abstract
The haloalkane dehalogenase (dhaA) gene from Rhodococcus rhodochrous NCIMB 13064 was cloned and sequenced. Its comparison with the previously studied dhlA gene from Xanthobacter autotrophicus GJ10 did not show homology. However, the amino acid sequences of the products of these genes showed approximately 30% identity and several of the catalytic amino acid residues were conserved in the NCIMB 13,064 dehalogenase. A high level of dhaA expression was demonstrated in Escherichia coli cells and this gene was shown to encode a dehalogenase with the activity against chloroalkanes of chain length C3-C10. Also, some dehalogenase activity against 1,2-dichloroethane encoded by the cloned dhaA gene was detected. The analysis of NCIMB 13,064 derivatives lacking dehalogenase activity showed that the dhaA gene was located on the 100 kbp pRTL1 plasmid. It was also found that reversible rearrangements of DNA in the dhaA region may be responsible for the control of expression of haloalkane dehalogenase in R. rhodochrous NCIMB 13064. A number of repeated and inverted sequences which may cause genetic instability at the locus were found in the haloalkane dehalogenase gene region.
- Published
- 1997
- Full Text
- View/download PDF
42. Isolation of Rhodococcus rhodochrous NCIMB13064 derivatives with new biodegradative abilities.
- Author
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Kulakova AN, Reid KA, Larkin MJ, Allen CC, and Kulakov LA
- Subjects
- Pyruvic Acid metabolism, Hydrocarbons, Chlorinated metabolism, Naphthalenes metabolism, Rhodococcus isolation & purification, Rhodococcus metabolism, Soil Microbiology
- Abstract
Rhodococcus rhodochrous NCIMB13064 can dehalogenate and utilise a number of halogenated aliphatic compounds as sole carbon and energy source. Mutants of NCIMB13064 can be easily isolated with an enlarged range of 1-chloroalkane utilising ability. Dehalogenation of 1-chlorononane, 1-chlorodecane and short-chain 1-chloroalkanes (C3-C8) is encoded by the same plasmid pRTL1. However, a different genetic element(s) is required for the dehalogenation of 3-chloropropionic acid. Two derivatives (P200 and P400) of R. rhodochrous NCIMB13064 were isolated which had acquired the ability to utilise naphthalene as sole carbon and energy source. Both strains lost the ability to utilise short-chain 1-chloroalkanes and underwent some rearrangements associated with pRTL1 plasmid.
- Published
- 1996
- Full Text
- View/download PDF
43. The purification and properties of phosphonoacetate hydrolase, a novel carbon-phosphorus bond-cleavage enzyme from Pseudomonas fluorescens 23F.
- Author
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McGrath JW, Wisdom GB, McMullan G, Larkin MJ, and Quinn JP
- Subjects
- Alkaline Phosphatase, Amino Acid Sequence, Enzyme Activation, Enzyme Stability, Hydrogen-Ion Concentration, Hydrolysis, Metals pharmacology, Molecular Sequence Data, Phosphoric Monoester Hydrolases antagonists & inhibitors, Phosphoric Monoester Hydrolases metabolism, Substrate Specificity, Temperature, Carbon metabolism, Phosphoric Monoester Hydrolases isolation & purification, Phosphorus metabolism, Pseudomonas fluorescens enzymology
- Abstract
A novel, inducible, carbon-phosphorus bond-cleavage enzyme, phosphonoacetate hydrolase, was purified from cells of Pseudomonas fluorescens 23F grown on phosphonoacetate. The native enzyme had a molecular mass of approximately 80 kDa and, upon SDS/PAGE, yielded a homogenous protein band with an apparent molecular mass of about 38 kDa. Activity of purified phosphonoacetate hydrolase was Zn2+ dependent and showed pH and temperature optima of approximately 7.8 and 37 degrees C, respectively. The purified enzyme had an apparent Km of 1.25 mM for its sole substrate phosphonoacetate, and was inhibited by the structural analogues 3-phosphonopropionate and phosphonoformate. The NH2-terminal sequence of the first 19 amino acids displayed no significant similarity to other databank sequences.
- Published
- 1995
- Full Text
- View/download PDF
44. Immunological detection of Bacteroides fragilis in clinical samples.
- Author
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Patrick S, Stewart LD, Damani N, Wilson KG, Lutton DA, Larkin MJ, Poxton I, and Brown R
- Subjects
- Animals, Antibodies, Monoclonal immunology, Antigens, Bacterial immunology, Bacteremia microbiology, Bacteroides Infections diagnosis, Bacteroides Infections drug therapy, Bacteroides fragilis immunology, Fluorescent Antibody Technique, Humans, Immune Sera immunology, Polysaccharides, Bacterial immunology, Suppuration microbiology, Antigens, Bacterial analysis, Bacteroides Infections microbiology, Bacteroides fragilis isolation & purification
- Abstract
A monospecific polyclonal antiserum, prepared against Bacteroides fragilis common polysaccharide antigen purified by polyacrylamide gel immunoblot detected B. fragilis, B. thetaiotaomicron, B. ovatus and Prevotella melaninogenica in pus samples from various anatomical sites by immunofluorescence microscopy of the pus. With standard clinical laboratory culture methods, 36% of 147 samples were positive for one or more of the above bacteria. Of these, B. fragilis accounted for 33%. By immunofluorescent labelling of pus with the common antigen antiserum the detection of these bacteria in the samples increased to 50%. All nine of the blood cultures in which B. fragilis was detected by culture contained bacteria positive for the common antigen. Immunofluorescent labelling of pus samples with a selection of monoclonal antibodies specific for surface polysaccharides which are known to be antigenically variable in culture in vitro and in an animal model of infection showed that these polysaccharides are also variable in natural infection. The results indicate that the common polysaccharide antigen, in contrast to the variable surface polysaccharides, is a suitable target for the immunodetection of B. fragilis in clinical samples from a range of anatomical sites.
- Published
- 1995
- Full Text
- View/download PDF
45. Plasmid pRTL1 controlling 1-chloroalkane degradation by Rhodococcus rhodochrous NCIMB13064.
- Author
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Kulakova AN, Stafford TM, Larkin MJ, and Kulakov LA
- Subjects
- Biotransformation, Crosses, Genetic, Genetic Markers, Mitomycin pharmacology, Phenotype, Rhodococcus drug effects, Species Specificity, Structure-Activity Relationship, Genes, Bacterial, Hydrocarbons, Chlorinated metabolism, Plasmids isolation & purification, Rhodococcus genetics, Rhodococcus metabolism
- Abstract
Rhodococcus rhodochrous NCIMB13064 can dehalogenate and use a wide range of 1-haloalkanes as sole carbon and energy source. The 1-chloroalkane degradation phenotype may be lost by cells spontaneously or after treatment with Mitomycin C. Two laboratory derivatives of the original strain exhibited differing degrees of stability of the chloroalkane degradation marker. Plasmids of approximately 100 kbp (pRTL1) and 80 kbp (pRTL2) have been found in R. rhodochrous NCIMB13064. pRTL1 was shown to be carrying at least some genes for the dehalogenation of 1-chloroalkanes with short chain lengths (C3 to C9). However, no connection was found between the utilization of 1-chloroalkanes with longer chain lengths (C12 to C18) and the presence of pRTL1. Three separate events were observed to lead to the inability of NCIMB13064 to dehalogenate the short-chain 1-chloroalkanes; the complete loss of pRTL1, the integration of pRTL1 into the chromosome, or the deletion of a 20-kbp fragment in pRTL1. High-frequency transfer of the 1-chloroalkane degradation marker associated with pRTL1 has been demonstrated in bacterial crosses between different derivatives of R. rhodochrous NCIMB13064.
- Published
- 1995
- Full Text
- View/download PDF
46. Myocardial revascularization using the "no-touch" technique, with mild systemic hypothermia, in patients with a calcified ascending aorta.
- Author
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Dietl CA, Madigan NP, Laubach CA, Chapman JH, Bering JP, Holcomb PH, Larkin MJ, and Menapace FJ
- Subjects
- Aged, Aged, 80 and over, Angina, Unstable surgery, Aorta surgery, Arteries transplantation, Cardiopulmonary Bypass methods, Emergencies, Female, Humans, Male, Middle Aged, Saphenous Vein transplantation, Surgical Flaps, Aortic Diseases surgery, Arteriosclerosis surgery, Calcinosis surgery, Coronary Artery Bypass methods, Hypothermia, Induced methods
- Abstract
Modifications in the standard technique for coronary artery bypass grafting are recommended in presence of a calcified ascending aorta, to avoid clamp injury or atheroembolism. Between January 1991 and August 1994, we used a "no-touch" technique in 18 patients undergoing myocardial revascularization, who had a heavily calcified and atherosclerotic ascending aorta. Their mean age was 76.1 years (range 63 to 82 years). Cardiopulmonary bypass with mild systemic hypothermia (32 degrees C) was employed in 16 patients; 2 other patients were operated upon without cardiopulmonary bypass. The "no-touch" technique avoids all types of clamps in the aorta. No cardioplegia was given, and no grafts were anastomosed to the aorta. Fifty-two distal anastomoses (mean: 2.9 per patient) were performed, using 37 pedicled arterial grafts (22 internal mammary and 15 gastroepiploic arteries), and 15 free grafts, which were anastomosed proximally to the internal mammary artery. There were no postoperative cerebrovascular accidents. Three patients died (16.7% overall mortality): 1 died of pneumonia, one patient with a large left ventricular aneurysm died in congestive heart failure, and one patient with associated aortic insufficiency died in low cardiac output. Our experience suggests that using pedicled arterial grafts for myocardial revascularization is safe and effective to avoid clamp injury or atheroembolism in patients with a calcified aorta. Deep hypothermia is not necessary when using the "no-touch" technique.
- Published
- 1995
47. Metabolic activity of pathogenic bacteria during semicontinuous anaerobic digestion.
- Author
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Kearney TE, Larkin MJ, and Levett PN
- Subjects
- Adenosine Monophosphate metabolism, Anaerobiosis, Bacteriological Techniques, Campylobacter jejuni metabolism, Colony Count, Microbial, Culture Media, Enterobacteriaceae growth & development, Escherichia coli metabolism, Listeria monocytogenes metabolism, Salmonella typhimurium metabolism, Thymidine metabolism, Time Factors, Yersinia enterocolitica metabolism, Enterobacteriaceae metabolism
- Abstract
In natural environments such as anaerobic digesters, bacteria are frequently subjected to the stress of nutrient fluxes because of the continual changes in the flow of nutrients, and to survive, they must be capable of adapting readily to nutrient changes. In this study, the metabolic activities of Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes, and Campylobacter jejuni were studied within culture bags (Versapor-200 filters, 0.22-microns pore size) in laboratory anaerobic digesters. The metabolic activity of these bacteria was indicated by their adenylate energy charge (EC) ratios and their ability to incorporate [3H]thymidine, which was related to the respective changes in viable numbers within the culture bags during anaerobic digestion. Fluctuations in the adenylate EC ratios, the uptake of [3H]thymidine, and the viable numbers of E. coli, S. typhimurium, Y. enterocolitica, and L. monocytogenes cells were probably due to constant changes in the amount of available nutrients within the anaerobic digesters. The viability of S. typhimurium increased quickly after a fresh supply of nutrients was added to the system as indicated by the uptake of [3H]thymidine and an increase in the adenylate EC ratios. The viable numbers of E. coli, S. typhimurium, Y. enterocolitica, and L. monocytogenes organisms declined rapidly from 10(7) to 10(8) CFU/ml to 10(3) to 10(4) CFU/ml and remained at this level for an indefinite period. The decimal reduction time calculated during the period of exponential decline ranged from 0.8 to 1.2 days for these bacteria. C. jejuni had the greatest mean decimal reduction time value (3.6 days).(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1994
- Full Text
- View/download PDF
48. Haloalkane degradation and assimilation by Rhodococcus rhodochrous NCIMB 13064.
- Author
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Curragh H, Flynn O, Larkin MJ, Stafford TM, Hamilton JT, and Harper DB
- Subjects
- Biodegradation, Environmental, Butanes metabolism, Energy Metabolism, Fatty Acids analysis, Hydrocarbons, Chlorinated metabolism, Industrial Waste, Rhodococcus genetics, Rhodococcus isolation & purification, Soil Microbiology, Soil Pollutants metabolism, Substrate Specificity, Alkanes metabolism, Hydrocarbons, Halogenated metabolism, Rhodococcus metabolism
- Abstract
The bacterium Rhodococcus rhodochrous NCIMB 13064, isolated from an industrial site, could use a wide range of 1-haloalkanes as sole carbon source but apparently utilized several different mechanisms simultaneously for assimilation of substrate. Catabolism of 1-chlorobutane occurred mainly by attack at the C-1 atom by a hydrolytic dehalogenase with the formation of butanol which was metabolized via butyric acid. The detection of small amounts of gamma-butyrolactone in the medium suggested that some oxygenase attack at C-4 also occurred, leading to the formation of 4-chlorobutyric acid which subsequently lactonized chemically to gamma-butyrolactone. Although 1-chlorobutane-grown cells exhibited little dehalogenase activity on 1-chloroalkanes with chain lengths above C10, the organism utilized such compounds as growth substrates with the release of chloride. Concomitantly, gamma-butyrolactone accumulated to 1 mM in the culture medium with 1-chlorohexadecane as substrate. Traces of 4-hydroxybutyric acid were also detected. It is suggested that attack on the long-chain chloroalkane is initiated by an oxygenase at the non-halogenated end of the molecule leading to the formation of an omega-chlorofatty acid. This is degraded by beta-oxidation to 4-chlorobutyric acid which is chemically lactonized to gamma-butyrolactone which is only slowly further catabolized via 4-hydroxybutyric acid and succinic acid. However, release of chloride into the medium during growth on long-chain chloroalkanes was insufficient to account for all the halogen present in the substrate. Analysis of the fatty acid composition of 1-chlorohexadecane-grown cells indicated that chlorofatty acids comprised 75% of the total fatty acid content with C14:0, C16:0, C16:1 and C18:1 acids predominating. Thus the incorporation of 16-chlorohexadecanoic acid, the product of oxygenase attack directly into cellular lipid represents a third route of chloroalkane assimilation. This pathway accounts at least in part for the incomplete mineralization of long-chain chloroalkane substrates. This is the first report of the coexistence of a dehalogenase and the ability to incorporate long-chain haloalkanes into the lipid fraction within a single organism and raises important questions regarding the biological treatment of haloalkane containing effluents.
- Published
- 1994
- Full Text
- View/download PDF
49. Survival of pathogenic bacteria during mesophilic anaerobic digestion of animal waste.
- Author
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Kearney TE, Larkin MJ, Frost JP, and Levett PN
- Subjects
- Animal Husbandry methods, Animals, Cattle microbiology, Chickens microbiology, Escherichia coli physiology, Salmonella typhimurium physiology, Swine microbiology, Vegetables, Yersinia enterocolitica physiology, Anaerobiosis, Campylobacter jejuni physiology, Enterobacteriaceae physiology, Feces microbiology, Listeria monocytogenes physiology, Refuse Disposal
- Abstract
The survival of pathogenic bacteria was investigated during the operation of a full-scale anaerobic digester which was fed daily and operated at 28 degrees C. The digester had a mean hydraulic retention time of 24 d. The viable numbers of Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes and Campylobacter jejuni were reduced during mesophilic anaerobic digestion. Escherichia coli had the smallest mean viable numbers at each stage of the digestion process. Its mean T90 value was 76.9 d. Yersinia enterocolitica was the least resistant to the anaerobic digester environment; its mean T90 value was 18.2 d. Campylobacter jejuni was the most resistant bacterium; its mean T90 value was 438.6 d. Regression analysis showed that there were no direct relationships between the slurry input and performance of the digester and the decline of pathogen numbers during the 140 d experimental period.
- Published
- 1993
- Full Text
- View/download PDF
50. The effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria.
- Author
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Kearney TE, Larkin MJ, and Levett PN
- Subjects
- Anaerobiosis, Animals, Cattle, Colony Count, Microbial, Escherichia coli growth & development, Salmonella typhimurium growth & development, Temperature, Yersinia enterocolitica growth & development, Campylobacter jejuni growth & development, Enterobacteriaceae growth & development, Listeria monocytogenes growth & development, Manure microbiology
- Abstract
The decline in viable numbers of Salmonella typhimurium, Yersinia enterocolitica and Listeria monocytogene in beef cattle slurry is temperature-dependent; they decline more rapidly at 17 degrees C than at 4 degrees C. Mesophilic anaerobic digestion caused an initial rapid decline in the viable numbers of Escherichia coli, Salm. typhimurium, Y. enterocolitica and L. monocytogenes. This was followed by a period in which the viable numbers were not reduced by 90%. The T90 values of E. coli, Salm. typhimurium and Y. enterocolitica ranged from 0.7 to 0.9 d during batch digestion and 1.1 to 2.5 d during semi-continuous digestion. Listeria monocytogenes had a significantly higher mean T90 value during semi-continuous digestion (35.7 d) than batch digestion (12.3 d). Anaerobic digestion had little effect in reducing the viable numbers of Campylobacter jejuni.
- Published
- 1993
- Full Text
- View/download PDF
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