Jiandong Jiang

Molecular characterization of the enzymes involved in the degradation of a brominated aromatic herbicide

Dehalogenation is the key step in the degradation of halogenated aromatics, while reductive dehalogenation is originally thought to rarely occur in aerobes. In this study, an aerobic strain of Comamonas sp. 7D‐2 was shown to degrade the brominated aromatic herbicide bromoxynil completely and release two equivalents of bromides under aerobic conditions. The enzymes involved in the degradation of bromoxynil to 4‐carboxy‐2‐hydroxymuconate‐6‐semialdehyde, including nitrilase, reductive dehalogenase (BhbA), 4‐hydroxybenzoate 3‐monooxygenase and protocatechuate 4,5‐dioxygenase, were molecularly characterized. The novel dehalogenase BhbA was shown to be a complex of a respiration‐linked reductive dehalogenase (RdhA) domain and a NAD(P)H‐dependent oxidoreductase domain and to have key features of anaerobic respiratory RdhAs, including two predicted binding motifs for [4Fe‐4S] clusters and a close association with a hydrophobic membrane protein (BhbB). BhbB was confirmed to anchor BhbA to the membrane. BhbA was partially purified and found to use NAD(P)H as electron donors. Full‐length bhbA homologues were found almost exclusively in marine aerobic proteobacteria, suggesting that reductive dehalogenation occurs extensively in aerobes and that bhbA is horizontally transferred from marine microorganisms. The discovery of a functional reductive dehalogenase and ring‐cleavage oxygenases in an aerobe opens up possibilities for basic research as well as the potential application for bioremediation.

Adsorption and degradation of triazophos, chlorpyrifos and their main hydrolytic metabolites in paddy soil from Chaohu Lake, China

Triazophos and chlorpyrifos are organophosphorus pesticides (OPs), and their primary hydrolytic metabolites are 1-phenyl-3-hydroxy-1,2,4-triazole (BZC) and 3,5,6-trichloro-2-pyridinol (TCP). In this study, the adsorption and degradation of triazophos, chlorpyrifos, BZC and TCP were investigated in paddy soil from Chaohu Lake, China. Adsorption tests demonstrated that the adsorption of these compounds to soils could be described by the Freundlich equation. Moreover, chlorpyrifos displayed the highest affinity for adsorption, followed by triazophos, BZC and TCP. Degradation of these compounds in non-sterile soil followed first-order exponential decay kinetics, and the half-life (t(1/2)) of these contaminants ranged from 8.40 to 44.34 d. Sterilization of soil decreased the degradation rate, indicating that microorganisms played a significant role in the degradation of these compounds. The values of t(1/2) and K(oc) were fitted to obtain models that could predict the leaching potential of the contaminants from soil. Compared to their parent compounds, BZC and TCP showed high potential for leaching into groundwater. The inoculation of OPs-degrading bacterium (Diaphorobacter sp. GS-1) removed 95.38%, 100% and 100% of triazophos, chlorpyrifos and BZC in paddy soil after 21 d, respectively. The pollution risk of triazophos, chlorpyrifos and BZC could be greatly decreased by inoculating soil with Diaphorobacter sp. GS-1, which decreases the t(1/2) of the contaminants.

Lysobacter ruishenii sp. nov., a chlorothalonil-degrading bacterium isolated from a long-term chlorothalonil-contaminated soil.

An aerobic, Gram-negative bacterial strain, designated CTN-1(T), capable of degrading chlorothalonil was isolated from a long-term chlorothalonil-contaminated soil in China, and was subjected to a polyphasic taxonomic investigation. Strain CTN-1(T) grew at 15-37 °C (optimum 28-30 °C) and at pH 6.0-9.0 (optimum pH 7.0-7.5). The G+C content of the total DNA was 67.1 mol%. Based on 16S rRNA gene sequence analysis, strain CTN-1(T) was related most closely to Lysobacter daejeonensis DSM 17634(T) (97.1  % similarity), L. soli DCY21(T) (95.7  %), L. concretionis Ko07(T) (95.5  %), L. gummosus LMG 8763(T) (95.3 %) and L. niastensis DSM 18481(T) (95.2  %). The novel strain showed less than 95.0 % 16S rRNA gene sequence similarity to the type strains of other Lysobacter species. The major cellular fatty acids of strain CNT-1(T) were iso-C₁₆:₀ (23.0  %), iso-C₁₅:₀ (21.4  %) and iso-C₁₇:₁ω9c (15.3  %). The major isoprenoid quinone was Q-8 (99 %), and the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. These chemotaxonomic data supported the affiliation of strain CTN-1(T) to the genus Lysobacter. Levels of DNA-DNA relatedness between strain CTN-1(T) and L. daejeonensis DSM 17634(T) were 34.6-36.1  %. Phylogenetic analysis based on 16S rRNA gene sequences, DNA-DNA hybridization data and biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiation of strain CTN-1(T) from recognized species of the genus Lysobacter. Strain CTN-1(T) is therefore considered to represent a novel species of the genus Lysobacter, for which the name Lysobacter ruishenii sp. nov. is proposed. The type strain is CTN-1(T) (=DSM 22393(T) =CGMCC 1.10136(T)).

A Novel Hydrolytic Dehalogenase for the Chlorinated Aromatic Compound Chlorothalonil

Dehalogenases play key roles in the detoxification of halogenated aromatics. Interestingly, only one hydrolytic dehalogenase for halogenated aromatics, 4-chlorobenzoyl-coenzyme A (CoA) dehalogenase, has been reported. Here, we characterize another novel hydrolytic dehalogenase for a halogenated aromatic compound from the 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil)-degrading strain of Pseudomonas sp. CTN-3, which we have named Chd. Chd catalyzes a hydroxyl substitution at the 4-chlorine atom of chlorothalonil. The metabolite of the Chd dehalogenation, 4-hydroxy-trichloroisophthalonitrile, was identified by reverse-phase high-performance liquid chromatography (HPLC), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR). Chd dehalogenates chlorothalonil under anaerobic and aerobic conditions and does not require the presence of cofactors such as CoA and ATP. Chd contains a putative conserved domain of the metallo-beta-lactamase superfamily and shows the highest identity with several metallohydrolases (24 to 29%). Chd is a monomer (36 kDa), and the isoelectric point (pI) of Chd is estimated to be 4.13. Chd has a dissociation constant (K(m)) of 0.112 mM and an overall catalytic rate (k(cat)) of 207 s(-1) for chlorothalonil. Chd is completely inhibited by 1,10-phenanthroline, diethyl pyrocarbonate, and N-bromosuccinic acid. Site-directed mutagenesis of Chd revealed that histidines 128 and 157, serine 126, aspartates 45, 130 and 184, and tryptophan 241 were essential for the dehalogenase activity. Chd differs from other reported hydrolytic dehalogenases based on the analysis of amino acid sequences and catalytic mechanisms. This study provides an excellent dehalogenase candidate for mechanistic study of hydrolytic dehalogenation of halogenated aromatic compound.

Dehalogenation of chlorobenzenes, dichlorotoluenes, and tetrachloroethene by three Dehalobacter spp.

Three enrichment cultures containing Dehalobacter spp. were developed that dehalogenate each of the dichlorobenzene (DCB) isomers to monochlorobenzene (MCB), and the strains using 1,2-DCB (12DCB1) or 1,3-DCB (13DCB1) are now considered isolated, whereas the strain using 1,4-DCB (14DCB1) is considered highly enriched. In this study, we examined the dehalogenation capability of each strain to use chlorobenzenes with three or more chlorines, tetrachloroethene (PCE), or dichlorotoluene (DCT) isomers. Strain 12DCB1 preferentially dehalogenated singly flanked chlorines, but not doubly flanked or unflanked chlorines. It dehalogenated pentachlorobenzene to MCB with little buildup of intermediates. Strain 13DCB1, which could use either 1,3-DCB or 1,2-DCB, demonstrated the widest dehalogenation spectrum of electron acceptors tested, and dehalogenated every chlorobenzene isomer except 1,4-DCB. Notably, strain 13DCB1 dehalogenated the recalcitrant 1,3,5-trichlorobenzene isomer to MCB, and qPCR of 16S rRNA genes indicated that strain 13DCB1 grew. Strain 14DCB1 exhibited the narrowest range of substrate utilization, but was the only strain to dehalogenate para-substituted chlorines. Strains 12DCB1 and 13DCB1 dehalogenated PCE to cis-dichloroethene, and all strains dehalogenated 3,4-DCT to monochlorotoluene. These findings show that Dehalobacter spp., like Dehalococcoides spp., are versatile dehalogenators and should be considered when determining the fate of chlorinated organics at contaminated sites.

Novel Three-Component Rieske Non-Heme Iron Oxygenase System Catalyzing the N-Dealkylation of Chloroacetanilide Herbicides in Sphingomonads DC-6 and DC-2

ABSTRACT Sphingomonads DC-6 and DC-2 degrade the chloroacetanilide herbicides alachlor, acetochlor, and butachlor via N-dealkylation. In this study, we report a three-component Rieske non-heme iron oxygenase (RHO) system catalyzing the N-dealkylation of these herbicides. The oxygenase component gene cndA is located in a transposable element that is highly conserved in the two strains. CndA shares 24 to 42% amino acid sequence identities with the oxygenase components of some RHOs that catalyze N- or O-demethylation. Two putative [2Fe-2S] ferredoxin genes and one glutathione reductase (GR)-type reductase gene were retrieved from the genome of each strain. These genes were not located in the immediate vicinity of cndA. The four ferredoxins share 64 to 72% amino acid sequence identities to the ferredoxin component of dicamba O-demethylase (DMO), and the two reductases share 62 to 65% amino acid sequence identities to the reductase component of DMO. cndA, the four ferredoxin genes, and the two reductases genes were expressed in Escherichia coli, and the recombinant proteins were purified using Ni-affinity chromatography. The individual components or the components in pairs displayed no activity; the enzyme mixture showed N-dealkylase activities toward alachlor, acetochlor, and butachlor only when CndA-His6 was combined with one of the four ferredoxins and one of the two reductases, suggesting that the enzyme consists of three components, a homo-oligomer oxygenase, a [2Fe-2S] ferredoxin, and a GR-type reductase, and CndA has a low specificity for the electron transport component (ETC). The N-dealkylase utilizes NADH, but not NADPH, as the electron donor.

Horizontal transfer of dehalogenase genes involved in the catalysis of chlorinated compounds: evidence and ecological role

Horizontal gene transfer (HGT) of dehalogenase genes is considered an important mechanism of genomic evolution, the metabolic resilience of biotopes, and microbial community adaptation in chlorinated compound-contaminated ecosystems. In this review, we summarize the current evidence for the HGT of dehalogenase genes involved in the catalysis of various chlorinated compounds, such as chlorinated alkanes and alkanoic acids, chlorinated ethenes, chlorinated herbicide, and chlorinated aromatics. We also highlight the ecological role of HGT as it relates to the contribution to the diversification of dehalogenating microorganisms and the resulting facilitation of rapid microbial community adaptation to ecosystem contaminated with chlorinated compounds.

Facilitation of bacterial adaptation to chlorothalonil-contaminated sites by horizontal transfer of the chlorothalonil hydrolytic dehalogenase gene.

ABSTRACT Horizontal transfer of the chlorothalonil hydrolytic dehalogenase gene (chd) is proposed based on the high conservation of the chd gene and its close association with a novel insertion sequence, ISOcsp1, in 16 isolated chlorothalonil-dechlorinating strains belonging to eight different genera. The ecological role of horizontal gene transfer is assumed to facilitate bacterial adaptation to chlorothalonil-contaminated sites, through detoxification of chlorothalonil to less toxic 2,4,5-trichloro-6-hydroxybenzene-1,3-dicarbonitrile.

Biodegradation of Pentachloronitrobenzene by Labrys portucalensis pcnb-21 Isolated from Polluted Soil

Abstract A bacterial strain, pcnb-21, capable of degrading pentachloronitrobenzene (PCNB) under aerobic and anoxic conditions, was isolated from a long-term PCNB-polluted soil by an enrichment culture technique and identified as Labrys portucalensis based upon its morphological, physiological and biochemical properties, as well as 16S rRNA gene sequence analysis. Effects of different factors, such as temperature and pH, on PCNB biodegradation were studied. Strain pcnb-21 efficiently degraded PCNB at temperatures from 20 to 30 °C and initial pH values from 4 to 7, which might be the first time that a Labrys strain was found capable of efficiently degrading PCNB. The degradation of PCNB was affected by oxygen, and the degradation decreased with increasing aeration. Exogenous electron donors such as glucose, lactic acid and succinic acid promoted the biodegradation of PCNB, while electron acceptors such as sodium nitrite, sodium sulfate, sodium nitrate and sodium sulfate inhibited PCNB biodegradation. The degradation of PCNB in sterile and non-sterile soils by a green fluorescent protein (GFP)-labeled strain, pcnb-21- gfp , was also studied. Cells of pcnb-21- gfp efficiently degraded 100 mg kg −1 PCNB in sterile and non-sterile soils and could not be detected after 42 days. Strain pcnb-21 might be useful in bioremediating PCNB-polluted soils and environment.

Co-metabolism of DDT by the newly isolated bacterium, Pseudoxanthomonas sp. wax

Microbial degradation of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) is the most promising way to clean up DDT residues found in the environment. In this paper, a bacterium designated as wax, which was capable of co-metabolizing DDT with other carbon sources, was isolated from a long-term DDT-contaminated soil sample by an enrichment culture technique. The new isolate was identified as a member of the Pseudoxanthomonas sp., based on its morphological, physiological and biochemical properties, as well as by 16S rRNA gene analysis. In the presence of 100 mg l-1 glucose, the wax strain could degrade over 95% of the total DDT, at a concentration of 20 mg l-1, in 72 hours, and could degrade over 60% of the total DDT, at a concentration of 100 mg l-1, in 144 hours. The wax strain had the highest degradation efficiency among all of the documented DDT-degrading bacteria. The wax strain could efficiently degrade DDT at temperatures ranging from 20 to 37oC, and with initial pH values ranging from 7 to 9. The bacterium could also simultaneously co-metabolize 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), 2,2-bis(p-chlorophenyl)-1,1-dichlorethylene (DDE), and other organochlorine compounds. The wax strain could also completely remove 20 mg kg-1 of DDT from both sterile and non-sterile soils in 20 days. This study demonstrates the significant potential use of Pseudoxanthomonas sp. wax for the bioremediation of DDT in the environment.