Isolation of Strain LW-3 Capable of Degrading Chlorimuron-ethyl and Its Biological Characteristics / 微生物学通报

A bacterium(designated as LW-3), capable of degrading Chlorimuron-ethyl, was isolated from the long-term contaminated soil by Chlorimuron-ethyl. Based on physiological and biochemical analyses and sequences comparison of 16S rDNA, strain LW-3 was identified as Pseudomonas sp. LW-3 could use Chlorimuron-ethyl as sole nitrogen source for growth. The optimum pH and temperature for degradation of Chlorimuron-ethyl were pH 6.5~7.2 and 30?C~35?C, at same temperature the pH change to the Chlorimuron -ethyl degrading influence is large. When the pH and temperature were pH 6.5 and 32?C, 50 mg/L Chlori- muron-ethyl could be degraded to 70%~80% level within 7 days.

Study on the atrazine-degrading genes in Arthrobacter sp. AG1 / 生物工程学报

Atrazine could be used as the sole carbon, nitrogen and energy sources for growth by strain Arthrobacter sp. AG1, and the atrazine-degrading genes of AG1 were found to be the combination of trzN, atzB and atzC. The atrazine chloride hydrolysase gene trzN was cloned by PCR amplification,whose sequence shared 99% identity with that of Norcardioides sp. C190. Two large plasmids were found in AG1,and trzN and atzB were confirmed to be localized on the larger plasmid pAG1 by the method of southern hybridization. Subculture of AG1 in liquid LB for three generations, 34% of the subsequent cells were found to lose degrading activity, however, neither plasmid was lost. PCR amplification results showed that the mutants had only lost the trzN gene instead of atzB and atzC. It was deduced that mutation might be due to the trzN gene deletion from the plasmid. This study provided new evidence that atrazine metabolic genotypes were resulted from horizontal gene transfer between different bacteria under environmental selective pressure.

Recombinant expression and secretion of mpd gene using the promoters and signal peptide-encoding sequences of ytkA and ywoF gene from Bacillus subtilis / 生物工程学报

A shuttle promoter-probe vector pNW33N-mpd was constructed with the E. coli-B. subtilis shuttle vector pNW33N and the mature mpd gene without it' s signal peptide-encoding sequence. The promoter fragments of B. subtilis ytkA and ywoF gene were cloned from plasmid pMPDP3 and pMPDP29 then generated the shuttle expression vector pNYTM and pNYWM. Expression vectors pNYTM and pNYWM were transformed into B. subtilis 1A751 to construct the expression strain 1A751 (pNYTM) and 1A751 (pNYTM), in these strains, under the control of the promoters and signal peptides of ytkA and ywoF gene, mpd gene was expressed and secreted with its biological activity; the result showed that the promoter of ytkA gene is much stronger than that of ywoF gene. Then a new shuttle expression-secretion vector pYNMK was constructed using the ytkA gene promoter and the signal peptide-encoding sequence of B. subtilis nprB gene, the expression of mpd gene achieved a higher level using the B. subtilis WB800 as the host, the methyl parathion hydrolase activity accumulated to a maximum level of 10.40 u/mL after 84 h of cultivation at the late stationary phase, which was 10.8-fold higher than the expression level of the original Plesiomonas strain M6, about 91.4% of the recombinant expression production was secreted into the culture medium.

Construction of multifunctional genetically engineered pesticides-degrading bacteria by homologous recombination / 生物工程学报

Construction of multifunctional pesticides-degrading genetically engineered microorganisms (GEMs) is increasing important in the bioremediation of various pesticides contaminants in environment. However, construction of genetically stable GEMs without any exogenous antibiotic resistance is thought to be one of the bottlenecks in GEMs construction. In this article, homologous recombination vectors with the recipient's 16S rDNA as homologous recombination directing sequence (HRDS) and sacB gene as double crossover recombinants positive selective marker were firstly constructed. The methyl parathion hydroalse gene (mpd) was inserted into the 16S rDNA site of the carbofuran degrading strain Sphingomonas sp. CDS-1 by homologous recombination single crossover in the level of about 3.7 x 10-(7) - 6.8 x 10(-7). Multifunctional pesticides-degrading GEMs with one or two mpd genes inserted into the chromosome without any antibiotic marker were successfully constructed. The homologous recombination events were confirmed by PCR and southern blot methods. The obtained GEMs were genetically stable and could degrade methyl parathion and carbofuran simultaneously. The insertion of mpd gene into rrn site did not have any significant effect on recipient' s physiological and original degrading characteristics. The methyl parathion hydrolase (MPH) was expressed at a relatively high level in the recombinants and the recombinant MPH specific activity in cell lysate was higher than that of original bacterium (DLL-1) in every growth phase tested. The highest recombinant MPH specific activity was 6.22 mu/tg. In this article, we describe a first attempt to use rRNA-encoding regions of Sphingomonas strains as target site for expression of exogenous MPH, and constructed multifunctional pesticides degrading GEMs, which are genetically stable and promising for developing bioremediation strategies for the decontamination of pesticides polluted soils.

Determination of the catalytic structures of methyl parathion hydrolase / 生物工程学报

Methyl parathion hydrolase (MPH) is a novel member of organophosphorus hydrolase. In this study, mpd gene was expressed in Escherichia coli DH5alpha with its native promoter. MPH was purified to homogeneity. Results show that metal-chelating compounds cannot inhabit the enzyme activity. Inductively Coupled Plasma-Atomic Emission Spectrometry analysis showed that MPH is a zinc-containing enzyme, the Zinc to enzyme molar ratio is near 2:1. In order to investigate critical residues related to enzymatic activity of MPH, chemical modification reagents EDC, DEPC, butanedione and pyridoxal were tested. Experiment results suggested that aspartate, glutamate, arginine and lysine are not important for enzyme activity. But DEPC, which can modify histidine residue, inactivate the enzyme activity greatly, and the inactivation rate is 9.6 h(-1). This result reflects that histidine residues are essential for enzyme activity. All these results provide basic data for MPH structure and molecular evolution research.

Screening of Mutants of Carbofuran Degrading Bacterium CFDS-1 by Transposon Tagging / 微生物学通报

In order to mutate a carbofuran degrading strain CFDS-1,transposon of pSC123 was introduced into the genomic DNA of strain CFDS-1 by conjugation with E.coli DH5? (pSC123) as donor and strain CFDS-1 as recipient,6 mutants which lost carbofuran degrading ability were obtained with a kanamycin resistant gene in the middle of transposon and the disappearance of a red compound during the degrading of carbofuran as preliminary selecting marks,they were designated as CFDS-M1～CFDS-M6. UV scanning and GS assay results also proved their mutations. PCR was carried out respectively with primers designed according to the sequence of transposon and genomic DNA of 6 mutants as templates,restriction analysis of PCR products showed that the mutation of carbofuran degrading genes of these mutants was caused by transposon insertion.

Osmoregulation of a Halophilic Bacteria Strain Halomonas sp.BYS-1 / 微生物学通报

Halomonas sp.BYS-1 was a moderately halophilic bacteria strain isolated from activated sludge,It could grow on MM with NaCl concentration from 0.1～2.6 mol/L and phenyl acetic acid as sole carbon souce. When BYS-1 grew in the media with different concentrations of NaCl, there was no obvious change in its intracellular Na+ contents , it accumulated K+, glutamic acid and betaine as osmoprotectants. Its intracellular contents of K+,glutamic acid and betaine increased by 1.9,2.4 and 13.6 times, respectively, when the concentration of NaCl increased from 0.1 mol/L to 2mol/L.

ENZYMOLOGY OF MICROBIAL DEGRADATION OF ORGANOPHOSPHATE CHEMICALS / 微生物学通报

Organophosphate chemicals are widely used as agricultural pesticides and war reagents, their biodegradation is emphasized on the theoretical and practical aspects. Organophosphate hydrolases play important roles in the biodegradation of organophosphate chemicals. Great advancement was achieved recently in the determination of crystal structure and catalytic mechanisms of the hydrolase. This paper reviewed the research progresses in the enzymology, protein structure, catalytic mechanisms and application of the organophosphate hydrolase, and predicted the future research in this field.