Biodegradation of malachite green by strain Pseudomonas sp. K9 and cloning of the tmr2 gene associated with an ISPpu12.

A bacterial strain K9 capable of degrading malachite green was isolated from the sludge of the wastewater treatment system of a chemical plant. It was identified preliminarily as Pseudomonas sp. Strain K9 was also able to degrade other triphenylmethane dyes, such as Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase, was cloned from strain K9, and functionally expressed in E. coli. A 5946-bp DNA fragment including the tmr2 gene was cloned from the genomic DNA of strain K9 by chromosome walking. Its sequence analysis showed that tmr2 was associated with a typical mobile element ISPpu12 consisting of tnpA (encoding a transposase), lspA (encoding a lipoprotein signal peptidase) and orf1 (encoding a putative MerR family regulator), orf2 (encoding a CDF family heavy metal/H(+) antiporter). This association was also found in another malachite green-degrading strain Pseudomonas sp. MDB-1, which indicated that the tmr2 gene might be a horizontally transferable gene.

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.

Biodegradation of ethametsulfuron-methyl by Pseudomonas sp. SW4 isolated from contaminated soil.

A soil bacterium SW4, capable of degrading the sulfonylurea herbicide ethametsulfuron-methyl (ESM), was isolated from the bottom soil of a herbicide factory. Based on physiological characteristics, biochemical tests and phylogenetic analysis of the 16S rRNA gene sequence, the strain was identified as a Pseudomonas sp. The total degradation of ESM in the medium containing glucose was up to 84.6% after 6 days of inoculation with SW4 strain. The inoculation of strain SW4 to soil treated with ESM resulted in a higher degradation rate than in noninoculated soil regardless of the soil sterilized or nonsterilized. Five metabolites of ESM degradation were analyzed by liquid chromatography/mass spectrometry. Based on the identified products, strain SW4 seemed to degrade ESM after two separate and different pathways: one leads to the cleavage of the sulfonylurea bridge, whereas the other to the dealkylation and opening of the triazine ring of ESM.

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.

Isolation and characterization of Pseudomonas sp. strain ONBA-17 degrading o-nitriobenzaldehyde.

Aerobic bacteria degrading o-nitrobenzaldehyde (ONBA) were isolated from activated sludges. One of the isolates, ONBA-17, was identified as Pseudomonas sp. The isolate could grow on ONBA as its sole source of carbon and nitrogen. Further studies demonstrated that the strain was a moderately halophilic bacterium and capable of degrading benzoic acid, 2-nitrophenol, 2-aminophenol, 4-hydroxybenzoic acid, and 4-dimetylaminobenzaldehyde. It could completely degrade 100 mg L(-1) ONBA at a range of pH 6-8 in 48 h at 30 degrees C, and up to 400 mg L(-1) after 288 h. The strain showed potential to be a good candidate for biotreatment of industrial wastewaters containing ONBA due to its salt-tolerance ability, multiresistance to some heavy metals and antibiotics, and the abilities of degradation of aromatic compounds. These findings may help in developing a process for ONBA-containing industrial wastewater treatment.

Isolation and characterization of a carbendazim-degrading Rhodococcus sp. djl-6.

Bacterium djl-6, capable of degrading carbendazim, was isolated by continuous enrichment culture originating from carbendazim-treated soil. The isolate was identified as Rhodococcus sp. according to its phenotypic features, physiologic and biochemical characteristics, and phylogenetic analysis. The strain could use carbendazim as sole carbon or nitrogen source. It showed a high average degradation rate of 55.56 mg . L(-1) . d(-1) in M9 medium amended with carbendazim. High-pressure liquid chromatography-mass spectrometry (HPLC-MS) analysis showed the presence of 2-aminobenzimidazole, benzimidazole, and an unknown metabolite with molecular ions (M(+)) of m/z 104.8 and 118.5. The degradation in the isolate djl-6 seems to be initiated with the cleavage of the methyl carbemate side chain, resulting in the formation of 2-aminobenzimidazole and benzimidazole. This is the first report of the intermediates benzimidazole and 2-aminobenzimidazole found together in the culture filtrate of pure bacterium.

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.

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.

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.

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.

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.

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.