Publications

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    ABSTRACT: Arthrobacter protophormiae RKJ100 was previously characterized for its ability to tolerate extremely high concentrations of o-nitrobenzoate (ONB), a toxic xenobiotic environmental pollutant. The physiological responses of strain RKJ100 to ≥30 mM ONB indicated towards a resistance mechanism manifested via alteration of cell morphology and cell wall structure. In this study, we aim to characterize gene(s) involved in the resistance of strain RKJ100 towards extreme concentrations (i.e. 150 mM) of ONB. Transposon mutagenesis was carried out to generate a mutant library of strain RKJ100, which was then screened for ONB-sensitive mutants. A sensitive mutant was defined and selected as one that could not tolerate ≥30 mM ONB. Molecular and biochemical characterization of this mutant showed that the disruption of endo-β-N-acetylglucosaminidase (ENGase) gene caused the sensitivity. ENGase is an important enzyme for oligosaccharide processing and cell wall recycling in bacteria, fungi, plants and animals. Previous reports have already indicated several possible roles of this enzyme in cellular homeostasis. Results presented here provide the first evidence for its involvement in bacterial resistance towards extreme concentrations of a toxic xenobiotic compound and also suggest that strain RKJ100 employs ENGase as an important component in osmotic shock response for resisting extreme concentrations of ONB.
    Extremophiles 02/2014; · 2.20 Impact Factor
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    ABSTRACT: The metabolism of volatile signal molecules by odorant degrading enzymes (ODEs) is crucial to the ongoing sensitivity and specificity of chemoreception in various insects, and a few specific esterases, cytochrome P450s, glutathione S-transferases (GSTs) and UDP-glucosyltransferases (UGTs) have previously been implicated in this process. Significant progress has been made in characterizing ODEs in Lepidoptera but very little is known about them in Diptera, including in D. melanogaster, a major insect model. We have therefore carried out a transcriptomic analysis of the antennae of D.melanogaster in order to identify candidate ODEs. Virgin male and female and mated female antennal transcriptomes were determined by RNAseq. As with the Lepidoptera, we found that many esterases, cytochrome P450 enzymes, GSTs and UGTs are expressed in D. melanogaster antennae. As olfactory genes generally show selective expression in the antennae, a comparison to previously published transcriptomes for other tissues has been performed, showing preferential expression in the antennae for one esterase, JHEdup, one cytochrome P450, CYP308a1, and one GST, GSTE4. These largely uncharacterized enzymes are now prime candidates for ODE functions. Jhedup was expressed heterologously and found to have high catalytic activity against a chemically diverse group of known ester odorants for this species. This is a finding consistent with an ODE although it might suggest a general role in clearing several odorants rather than a specific role in clearing a particular odorant. Our findings do not preclude the possibility of odorant degrading functions for other antennally expressed esterases, P450s, GSTs and UGTs but, if so, they suggest that these enzymes also have additional functions in other tissues.
    Insect Biochemistry and Molecular Biology. 01/2014;
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    ABSTRACT: Organochlorine insecticide hexachlorocyclohexane (HCH) has recently been classified as a 'Persistent Organic pollutant' by the Stockholm Convention. The LinB haloalkane dehalogenase is a key upstream enzyme in the recently evolved Lin pathway for the catabolism of HCH in bacteria. Here we report a sequence-structure-function analysis of ten naturally occurring and thirteen synthetic mutants of LinB. One of the synthetic mutants was found to have ∼80 fold more activity for β- and δ-hexachlorocyclohexane. Based on detailed biophysical calculations, molecular dynamics and ensemble docking calculations, we propose that the latter variant is more active because of alterations to the shape of its active site and increased conformational plasticity.
    PLoS ONE 01/2014; 9(7):e103632. · 3.53 Impact Factor
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    ABSTRACT: Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified.
    Biodegradation 06/2013; · 2.17 Impact Factor
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    ABSTRACT: Insect carboxylesterases from the αEsterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcαE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcαE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical α/β-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal α-helix that serves as a membrane anchor. Soaking of LcαE7 crystals in OPs led to the capture of a crystallographic snapshot of LcαE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcαE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (∼10(6) M(-1) s(-1)) indicative of a natural substrate.
    Proceedings of the National Academy of Sciences 06/2013; · 9.81 Impact Factor
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    ABSTRACT: Ralstonia sp. strain GA3-3 is a hexachlorocyclohexane (HCH)-degrading bacterial strain isolated from suburban soil in Canberra, Australia. The genome of strain GA3-3 was sequenced to investigate its ability to degrade α-HCH. Here, we report the annotated genome sequence of this strain.
    Genome announcements. 01/2013; 1(4).
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    ABSTRACT: Pandoraea sp. strain SD6-2 is a δ-hexachlorocyclohexane-degrading bacterial strain isolated from lindane-contaminated soil in Queensland, Australia. The genome of SD6-2 was sequenced to investigate its ability to degrade δ-hexachlorocyclohexane. Here we report the annotated genome sequence of this strain.
    Genome announcements. 01/2013; 1(4).
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    ABSTRACT: Aerobic microbial degradation of p-nitrophenol (PNP) has been classically shown to proceed via 'Hydroquinone (HQ) pathway' in Gram-negative bacteria, whereas in Gram-positive PNP degraders it proceed via 'Benzenetriol (BT) pathway'. These pathways are characterized by the ring cleavage of HQ and BT as terminal aromatic intermediates respectively. Earlier reports on PNP degradation have indicated these pathways to be mutually exclusive. We report involvement of both 'HQ' and 'BT' ring cleavage pathways in PNP degradation by Burkholderia sp. strain SJ98. Genetic characterization of an ~41 Kb DNA fragment harboring PNP degradation gene cluster cloned and sequenced from strain SJ98 showed presence of multiple orfs including pnpC and pnpD which corresponded to previously characterized 'benzenetriol-dioxygenase (BtD)' and 'maleylacetate reductase (MaR)' respectively. This gene cluster also showed presence of pnpE1 and pnpE2, which shared strong sequence identity to cognate sub-units of 'hydroquinone dioxygenase' (HqD). Heterologous expression and biochemical characterization ascertained the identity of PnpE1 and PnpE2. In in vitro assay reconstituted heterotetrameric complex of PnpE1 and PnpE2 catalyzed transformation of hydroquinone (HQ) into corresponding hydroxymuconic semialdehyde (HMS) in a substrate specific manner. Together, these results clearly establish branching of PNP degradation in strain SJ98. We propose that strain SJ98 presents a useful model system for future studies on evolution of microbial degradation of PNP.
    AMB Express. 06/2012; 2(1):30.
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    ABSTRACT: A 6-chloronicotinic acid mineralizing bacterium was isolated from enrichment cultures originating from imidacloprid-contaminated soil samples. This Bradyrhizobiaceae, designated strain SG-6C, hydrolytically dechlorinated 6-chloronicotinic acid to 6-hydroxynicotinic acid, which was then further metabolised via the nicotinic acid pathway. This metabolic pathway was confirmed by growth and resting cell assays using HPLC and LC-MS studies. A candidate for the gene encoding the initial dechlorination step, named cch2 (for 6-chloronicotinic acid chlorohydrolase), was identified using genome sequencing and its function was confirmed using resting cell assays on E. coli heterologously expressing this gene. The 464 amino acid enzyme was found to be a member of the metal dependent hydrolase superfamily with similarities to the TRZ/ATZ family of chlorohydrolases. We also provide evidence that cch2 was mobilized into this bacterium by an Integrative and Conjugative Element (ICE) that feeds 6-hydroxynicotinic acid into the existing nicotinic acid mineralization pathway.
    PLoS ONE 01/2012; 7(11):e51162. · 3.53 Impact Factor
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    ABSTRACT: Burkholderia sp. strain SJ98 is known for its chemotaxis towards nitroaromatic compounds (NACs) that are either utilized as sole sources of carbon and energy or co-metabolized in the presence of alternative carbon sources. Here we test for the chemotaxis of this strain towards six chloro-nitroaromatic compounds (CNACs), namely 2-chloro-4-nitrophenol (2C4NP), 2-chloro-3-nitrophenol (2C3NP), 4-chloro-2-nitrophenol (4C2NP), 2-chloro-4-nitrobenzoate (2C4NB), 4-chloro-2-nitrobenzoate (4C2NB) and 5-chloro-2-nitrobenzoate (5C2NB), and examine its relationship to the degradation of such compounds. Strain SJ98 could mineralize 2C4NP, 4C2NB and 5C2NB, and co-metabolically transform 2C3NP and 2C4NB in the presence of an alternative carbon source, but was unable to transform 4C2NP under these conditions. Positive chemotaxis was only observed towards the five metabolically transformed CNACs. Moreover, the chemotaxis was induced by growth in the presence of the metabolisable CNAC. It was also competitively inhibited by the presence of nitroaromatic compounds (NACs) that it could metabolise but not by succinate or aspartate. Burkholderia sp. strain SJ98 exhibits metabolic transformation of, and inducible chemotaxis towards CNACs. Its chemotactic responses towards these compounds are related to its previously demonstrated chemotaxis towards NACs that it can metabolise, but it is independently inducible from its chemotaxis towards succinate or aspartate.
    BMC Microbiology 01/2012; 12:19. · 2.98 Impact Factor
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    01/2012;
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    ABSTRACT: Strain SG-6C (DSM 23264, CCM 7827) is a chemolithoautotrophic bacterium of the family Bradyrhizobiaceae. It can also grow heterotrophically under appropriate environmental conditions. Here we report the annotated genome sequence of this strain in a single 4.3-Mb circular scaffold.
    Journal of bacteriology 07/2011; 193(18):5057. · 3.94 Impact Factor
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    ABSTRACT: Here, we compare the evolutionary routes by which bacteria and insects have evolved enzymatic processes for the degradation of four classes of synthetic chemical insecticide. For insects, the selective advantage of such degradative activities is survival on exposure to the insecticide, whereas for the bacteria the advantage is simply a matter of access to additional sources of nutrients. Nevertheless, bacteria have evolved highly efficient enzymes from a wide variety of enzyme families, whereas insects have relied upon generalist esterase-, cytochrome P450- and glutathione-S-transferase-dependent detoxification systems. Moreover, the mutant insect enzymes are less efficient kinetically and less diverged in sequence from their putative ancestors than their bacterial counterparts. This presumably reflects several advantages that bacteria have over insects in the acquisition of new enzymatic functions, such as a broad biochemical repertoire from which new functions can be evolved, large population sizes, high effective mutation rates, very short generation times and access to genetic diversity through horizontal gene transfer. Both the insect and bacterial systems support recent theory proposing that new biochemical functions often evolve from 'promiscuous' activities in existing enzymes, with subsequent mutations then enhancing those activities. Study of the insect enzymes will help in resistance management, while the bacterial enzymes are potential bioremediants of insecticide residues in a range of contaminated environments.
    Evolutionary Applications 01/2011; 4(2):225-248. · 4.15 Impact Factor
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    ABSTRACT: Here we report specific activities of all seven naturally occurring LinA variants towards three different isomers, α, γ and δ, of a priority persistent pollutant, hexachlorocyclohexane (HCH). Sequence-structure-function differences contributing to the differences in their stereospecificity for α-, γ-, and δ-HCH and enantiospecificity for (+)- and (-)-α -HCH are also discussed. Enzyme kinetic studies were performed with purified LinA variants. Models of LinA2(B90A) A110T, A111C, A110T/A111C and LinA1(B90A) were constructed using the FoldX computer algorithm. Turnover rates (min(-1)) showed that the LinAs exhibited differential substrate affinity amongst the four HCH isomers tested. α-HCH was found to be the most preferred substrate by all LinA's, followed by the γ and then δ isomer. The kinetic observations suggest that LinA-γ1-7 is the best variant for developing an enzyme-based bioremediation technology for HCH. The majority of the sequence variation in the various linA genes that have been isolated is not neutral, but alters the enantio- and stereoselectivity of the encoded proteins.
    PLoS ONE 01/2011; 6(9):e25128. · 3.53 Impact Factor
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    ABSTRACT: Free-enzyme bioremediation is a recently developed technology that allows rapid detoxification of pesticide residues in surface waters, such as irrigation tail water, and potentially from other wettable materials such as soil and the surfaces of commodities. Here we consider the advantages of this technology compared with other pesticide bioremediation strategies, as well as its current limitations and challenges for the future. We exemplify the development of free-enzyme bioremediants with a case study, the Landguard (TM) OP-A organophosphate bioremediant, highlighting the enzymatic, physical and toxicological properties of the enzyme that predispose it to be an effective and efficient environmental bioremediant and the applications explored for it to date.
    Pesticide Mitigation Strategies for Surface Water Quality. 01/2011; 1075:155-174.
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    ABSTRACT: An efficient 3,4-dichloroaniline (3,4-DCA)-mineralizing bacterium has been isolated from enrichment cultures originating from a soil sample with a history of repeated exposure to diuron, a major metabolite of which is 3,4-DCA. This bacterium, Bacillus megaterium IMT21, also mineralized 2,3-, 2,4-, 2,5- and 3,5-DCA as sole sources of carbon and energy. These five DCA isomers were degraded via two different routes. 2,3-, 2,4- and 2,5-DCA were degraded via previously unknown dichloroaminophenol metabolites, whereas 3,4- and 3,5-DCA were degraded via dichloroacetanilide.
    Microbiology 12/2010; 157(Pt 3):721-6. · 3.06 Impact Factor
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    ABSTRACT: Quantum chemistry calculations have been used alongside experimental kinetic analysis to investigate the competition between S(N)2 and E2 mechanisms for the dechlorination of hexachlorocyclohexane isomers, revealing that enzyme specificity reflects the intrinsic reactivity of the various isomers.
    Chemical Communications 11/2010; 47(3):976-8. · 6.38 Impact Factor
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    ABSTRACT: Bt cotton was the first genetically modified crop approved for use in India. However, only a few studies have been conducted to assess the feasibility of its commercial application. Bt cotton is genetically modified to express a proteinaceous endotoxin (Cry) encoded by cry gene of Bacillus thuringiensis that has specific insecticidal activity against bollworms. Therefore, the amount of pesticides used for growing Bt cotton is postulated to be considerably low as compared to their non-Bt counterparts. Alternatively, it is also speculated that application of a genetically modified crop may alter the bio-geochemical balance of the agriculture field(s). Microbial community composition and dynamics is an important descriptor for assessment of such alterations. In the present study, we have assessed the culturable and non-culturable microbial diversities in Bt cotton and non-Bt cotton soils to determine the ecological consequences of application of Bt cotton. The analyses of microbial community structures indicated that cropping of Bt cotton did not adversely affect the diversity of the microbial communities.
    Current Microbiology 08/2010; 61(2):118-24. · 1.52 Impact Factor
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    ABSTRACT: Microbial degradation studies have pointed toward the occurrence of two distinct PNP catabolic pathways in Gram positive and Gram negative bacteria. The former involves 4-nitrocatechol (4-NC), 1,2,4-benzenetriol (BT), and maleylacetate (MA) as major degradation intermediates, whereas the later proceeds via formation of 1,4-benzoquinone (BQ) and hydroquinone (HQ). In the present study we identified a Gram negative organism viz. Burkholderia sp. strain SJ98 that degrades PNP via 4NC, BT, and MA. A 6.89 Kb genomic DNA fragment of strain SJ98 that encompasses seven putatively identified ORFs (orfA, pnpD, pnpC, orfB, orfC, orfD, and orfE) was cloned. PnpC is benzenetriol dioxygenase belonging to the intradiol dioxygenase superfamily, whereas PnpD is identified as maleylacetate reductase, a member of the Fe-ADH superfamily showing NADH dependent reductase activity. The in vitro activity assays carried out with purified pnpC and pnpD (btd and mar) gene products transformed BT to MA and MA to beta-ketoadipate, respectively. The cloning, sequencing, and characterization of these genes along with the functional PNP degradation studies ascertained the involvement of 4-NC, BT, and MA as degradation intermediates of PNP pathway in this strain. This is one of the first conclusive reports for 4-NC and BT mediated degradation of PNP in a Gram negative organism.
    Environmental Science and Technology 04/2010; 44(9):3435-41. · 5.48 Impact Factor
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    ABSTRACT: Lindane, the gamma-isomer of hexachlorocyclohexane (HCH), is a potent insecticide. Purified lindane or unpurified mixtures of this and alpha-, beta-, and delta-isomers of HCH were widely used as commercial insecticides in the last half of the 20th century. Large dumps of unused HCH isomers now constitute a major hazard because of their long residence times in soil and high nontarget toxicities. The major pathway for the aerobic degradation of HCH isomers in soil is the Lin pathway, and variants of this pathway will degrade all four of the HCH isomers although only slowly. Sequence differences in the primary LinA and LinB enzymes in the pathway play a key role in determining their ability to degrade the different isomers. LinA is a dehydrochlorinase, but little is known of its biochemistry. LinB is a hydrolytic dechlorinase that has been heterologously expressed and crystallized, and there is some understanding of the sequence-structure-function relationships underlying its substrate specificity and kinetics, although there are also some significant anomalies. The kinetics of some LinB variants are reported to be slow even for their preferred isomers. It is important to develop a better understanding of the biochemistries of the LinA and LinB variants and to use that knowledge to build better variants, because field trials of some bioremediation strategies based on the Lin pathway have yielded promising results but would not yet achieve economic levels of remediation.
    Microbiology and molecular biology reviews: MMBR 03/2010; 74(1):58-80. · 12.59 Impact Factor

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