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1.  Draft Genome Sequence of Ralstonia sp. Strain GA3-3, Isolated from Australian Suburban Soil 
Genome Announcements  2013;1(4):e00414-13.
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.
doi:10.1128/genomeA.00414-13
PMCID: PMC3703592  PMID: 23833131
2.  Draft Genome Sequence of Pandoraea sp. Strain SD6-2, Isolated from Lindane-Contaminated Australian Soil 
Genome Announcements  2013;1(4):e00415-13.
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.
doi:10.1128/genomeA.00415-13
PMCID: PMC3703593  PMID: 23833132
3.  Cloning of a Novel 6-Chloronicotinic Acid Chlorohydrolase from the Newly Isolated 6-Chloronicotinic Acid Mineralizing Bradyrhizobiaceae Strain SG-6C 
PLoS ONE  2012;7(11):e51162.
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.
doi:10.1371/journal.pone.0051162
PMCID: PMC3511419  PMID: 23226482
4.  Branching of the p-nitrophenol (PNP) degradation pathway in burkholderia sp. Strain SJ98: Evidences from genetic characterization of PNP gene cluster 
AMB Express  2012;2:30.
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.
doi:10.1186/2191-0855-2-30
PMCID: PMC3485097  PMID: 22681853
P-nitrophenol; Hydroquinone dioxygenase; PNP pathway; Burkholderia sp SJ98
5.  Genome Sequence of the Newly Isolated Chemolithoautotrophic Bradyrhizobiaceae Strain SG-6C ▿  
Journal of Bacteriology  2011;193(18):5057.
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.
doi:10.1128/JB.05647-11
PMCID: PMC3165654  PMID: 21742875
6.  Chemotaxis of Burkholderia sp. Strain SJ98 towards chloronitroaromatic compounds that it can metabolise 
BMC Microbiology  2012;12:19.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2180-12-19
PMCID: PMC3293717  PMID: 22292983
7.  The evolution of new enzyme function: lessons from xenobiotic metabolizing bacteria versus insecticide-resistant insects 
Evolutionary Applications  2011;4(2):225-248.
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.
doi:10.1111/j.1752-4571.2010.00175.x
PMCID: PMC3352558
carbamates; hydrolases; organochlorines; organophosphates; pyrethroids
8.  Biochemistry of Microbial Degradation of Hexachlorocyclohexane and Prospects for Bioremediation 
Summary: Lindane, the γ-isomer of hexachlorocyclohexane (HCH), is a potent insecticide. Purified lindane or unpurified mixtures of this and α-, β-, and δ-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.
doi:10.1128/MMBR.00029-09
PMCID: PMC2832351  PMID: 20197499
9.  Cloning and Biochemical Characterization of a Novel Carbendazim (Methyl-1H-Benzimidazol-2-ylcarbamate)-Hydrolyzing Esterase from the Newly Isolated Nocardioides sp. Strain SG-4G and Its Potential for Use in Enzymatic Bioremediation▿ †  
A highly efficient carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC)-mineralizing bacterium was isolated from enrichment cultures originating from MBC-contaminated soil samples. This bacterium, Nocardioides sp. strain SG-4G, hydrolyzed MBC to 2-aminobenzimidazole, which in turn was converted to the previously unknown metabolite 2-hydroxybenzimidazole. The initial steps of this novel metabolic pathway were confirmed by growth and enzyme assays and liquid chromatography-mass spectrometry (LC-MS) studies. The enzyme responsible for carrying out the first step was purified and subjected to N-terminal and internal peptide sequencing. The cognate gene, named mheI (for MBC-hydrolyzing enzyme), was cloned using a reverse genetics approach. The MheI enzyme was found to be a serine hydrolase of 242 amino acid residues. Its nearest known relative is an uncharacterized hypothetical protein with only 40% amino acid identity to it. Codon optimized mheI was heterologously expressed in Escherichia coli, and the His-tagged enzyme was purified and biochemically characterized. The enzyme has a Km and kcat of 6.1 μM and 170 min−1, respectively, for MBC. Radiation-killed, freeze-dried SG-4G cells showed strong and stable MBC detoxification activity suitable for use in enzymatic bioremediation applications.
doi:10.1128/AEM.02990-09
PMCID: PMC2863442  PMID: 20228105
10.  Diversity of ‘benzenetriol dioxygenase’ involved in p-nitrophenol degradation in soil bacteria 
Indian Journal of Microbiology  2008;48(2):279-286.
Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic machinery of microbial degradation of several mono- phenolic and biphenolic compounds including nitrophenols. Assessment of the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great significance from basic as well as applied study point of view. In the present study we have evaluated the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils. The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the taxonomic ranks of the host microorganisms.
doi:10.1007/s12088-008-0038-x
PMCID: PMC3450173  PMID: 23100721
Benzenetriol dioxygenase; p-Nitrophenol; Phylogenetic analysis
11.  The enzymatic basis for pesticide bioremediation 
Indian Journal of Microbiology  2008;48(1):65-79.
Enzymes are central to the biology of many pesticides, influencing their modes of action, environmental fates and mechanisms of target species resistance. Since the introduction of synthetic xenobiotic pesticides, enzymes responsible for pesticide turnover have evolved rapidly, in both the target organisms and incidentally exposed biota. Such enzymes are a source of significant biotechnological potential and form the basis of several bioremediation strategies intended to reduce the environmental impacts of pesticide residues. This review describes examples of enzymes possessing the major activities employed in the bioremediation of pesticide residues, and some of the strategies by which they are employed. In addition, several examples of specific achievements in enzyme engineering are considered, highlighting the growing trend in tailoring enzymatic activity to a specific biotechnologically relevant function.
doi:10.1007/s12088-008-0007-4
PMCID: PMC3450202  PMID: 23100701
Bacterial enzymes; Bioremediation; Pesticides; Xenobiotics
12.  Bacterial Chemotaxis toward Environmental Pollutants: Role in Bioremediation† 
Applied and Environmental Microbiology  2002;68(12):5789-5795.
doi:10.1128/AEM.68.12.5789-5795.2002
PMCID: PMC134409  PMID: 12450797

Results 1-12 (12)