Shunpeng Li

Self-formed adaptor PCR: a simple and efficient method for chromosome walking.

ABSTRACT We developed a self-formed adaptor PCR (termed SEFA PCR) which can be used for chromosome walking. Most of the amplified flanking sequences were longer than 2.0 kb, and some were as long as 6.0 kb. SEFA PCR is simple and efficient and should have broad applications in the isolation of unknown sequences in complex genomes.

Cre/lox System and PCR-Based Genome Engineering in Bacillus subtilis

ABSTRACT We have developed a fast and accurate method to engineer the Bacillus subtilis genome that involves fusing by PCR two flanking homology regions with an antibiotic resistance gene cassette bordered by two mutant lox sites (lox71 and lox66). The resulting PCR products were used directly to transform B. subtilis, and then transient Cre recombinase expression in the transformants was used to recombine lox71 and lox66 into a double-mutant lox72 site, thereby excising the marker gene. The mutation process could also be accomplished in 2 days by using a strain containing a cre isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible expression cassette in the chromosome as the recipient or using the lox site-flanked cassette containing both the cre IPTG-inducible expression cassette and resistance marker. The in vivo recombination efficiencies of different lox pairs were compared; the lox72 site that remains in the chromosome after Cre recombination had a low affinity for Cre and did not interfere with subsequent rounds of Cre/lox mutagenesis. We used this method to inactivate a specific gene, to delete a long fragment, to realize the in-frame deletion of a target gene, to introduce a gene of interest, and to carry out multiple manipulations in the same background. Furthermore, it should also be applicable to large genome rearrangement.

High-Level Expression and Secretion of Methyl Parathion Hydrolase in Bacillus subtilis WB800

ABSTRACT The methyl parathion hydrolase (MPH)-encoding gene mpd was placed under the control of the P43 promoter and Bacillus subtilis nprB signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain B. subtilis WB800 as the host.

Cloning of a novel pyrethroid-hydrolyzing carboxylesterase gene from Sphingobium sp. strain JZ-1 and characterization of the gene product.

ABSTRACT A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. strain JZ-1. The gene contained an open reading frame of 840 bp. Sequence identity searches revealed that the deduced enzyme shared the highest similarity with many α/β-hydrolase fold proteins (20 to 24% identities). PytH was expressed in Escherichia coli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography. It was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. PytH was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides, and isomer selectivity was not observed. No cofactors were required for enzyme activity.

mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis

Here, we present a novel method for the directed genetic manipulation of the Bacillus subtilis chromosome free of any selection marker. Our new approach employed the Escherichia coli toxin gene mazF as a counter-selectable marker. The mazF gene was placed under the control of an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible expression system and associated with a spectomycin-resistance gene to form the MazF cassette, which was flanked by two directly-repeated (DR) sequences. A double-crossover event between the linearized delivery vector and the chromosome integrated the MazF cassette into a target locus and yielded an IPTG-sensitive strain with spectomycin-resistance, in which the wild-type chromosome copy had been replaced by the modified copy at the targeted locus. Another single-crossover event between the two DR sequences led to the excision of the MazF cassette and generated a strain with IPTG resistance, thereby realizing the desired alteration to the chromosome without introducing any unwanted selection markers. We used this method repeatedly and successfully to inactivate a specific gene, to introduce a gene of interest and to realize the in-frame deletion of a target gene in the same strain. As there is no prerequisite strain for this method, it will be a powerful and universal tool.

Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by Cupriavidus sp. DT-1

A bacterial strain, Cupriavidus sp. DT-1, capable of degrading chlorpyrifos and 3,5,6-trichloro-2-pyridinol (TCP) and using these compounds as sole carbon source was isolated and characterized. Investigation of the degradation pathway showed that chlorpyrifos was first hydrolyzed to TCP, successively dechlorinated to 2-pyridinol, and then subjected to the cleavage of the pyridine ring and further degradation. The mpd gene, encoding the enzyme responsible for chlorpyrifos hydrolysis to TCP, was cloned and expressed in Escherichia coli BL21. Inoculation of chlorpyrifos-contaminated soil with strain DT-1 resulted in a degradation rate of chlorpyrifos and TCP of 100% and 94.3%, respectively as compared to a rate of 28.2% and 19.9% in uninoculated soil. This finding suggests that strain DT-1 has potential for use in bioremediation of chlorpyrifos-contaminated environments.

Biodegradation of chloroacetamide herbicides by Paracoccus sp. FLY-8 in vitro.

A butachlor-degrading strain, designated FLY-8, was isolated from rice field soil and was identified as Paracoccus sp. Strain FLY-8 could degrade and utilize six chloroacetamide herbicides as carbon sources for growth, and the degradation rates followed the order alachlor > acetochlor > propisochlor > butachlor > pretilachlor > metolachlor. The influence of molecular structure of the chloroacetamide herbicides on the microbial degradation rate was first analyzed; the results indicated that the substitutions of alkoxymethyl side chain with alkoxyethyl side chain greatly reduced the degradation efficiencies; the length of amide nitrogens alkoxymethyl significantly affected the biodegradability of these herbicides: the longer the alkyl was, the slower the degradation efficiencies occurred. The phenyl alkyl substituents have no obvious influence on the degradation efficiency. The pathway of butachlor complete mineralization was elucidated on the basis of the results of metabolite identification and enzyme assays. Butachlor was degraded to alachlor by partial C-dealkylation and then converted to 2-chloro-N-(2,6-dimethylphenyl)acetamide by N-dealkylation, which subsequently transformed to 2,6-diethylaniline, which was further degraded via the metabolites aniline and catechol, and catechol was oxidized through an ortho-cleavage pathway. This study highlights an important potential use of strain FLY-8 for the in situ bioremediation of chloroacetamide herbicides and their metabolite-contaminated environment.

Biodegradation of the sulfonylurea herbicide chlorimuron‐ethyl by the strain Pseudomonas sp. LW3

The chlorimuron-ethyl-degrading bacterium LW3 was isolated from contaminated soil and identified by 16S rRNA gene sequencing as Pseudomonas sp. When chlorimuron-ethyl was provided as the sole nitrogen source, the degradation efficiency in liquid medium was about 81.0% after 7 days of inoculation with strain LW3. The effects of chlorimuron-ethyl concentration and temperature on biodegradation were examined. Two metabolites of degradation were analyzed by LC/MS. Based on the identified products, strain LW3 seemed to be able to degrade chlorimuron-ethyl by cleavage of the sulfonylurea bridge. The inoculation of strain LW3 to chlorimuron-ethyl-treated soil resulted in a higher degradation rate than in uninoculated soil, regardless of the soil being sterilized or nonsterilized. This microbial culture has great potential for the bioremediation of soil contaminated with chlorimuron-ethyl.

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.