Qing Hong

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.

Isolation of a methyl parathion-degrading strain Stenotrophomonas sp. SMSP-1 and cloning of the ophc2 gene

A rod-shaped, gram-negative bacterium Stenotrophomonas sp. SMSP-1 was isolated from the sludge of a wastewater treating system of a pesticide manufacturer. Strain SMSP-1 could hydrolyze methyl parathion to p-nitrophenol (PNP) and dimethyl phosphorothioate but could not degrade PNP further. Strain SMSP-1 was able to hydrolyze other organophosphate pesticides, including fenitrothion, ethyl parathion, fenthion, and phoxim, but not chlorpyrifos. A 4395-bp DNA fragment, including an organophosphorus hydrolase encoding gene ophc2, was cloned from the chromosome of strain SMSP-1 using the shotgun technique. Its sequence analysis showed that ophc2 was associated with a typical mobile element ISPpu12 consisting of tnpA (encoding a transposase), lspA (encoding a lipoprotein signal peptidase), and orf1 (encoding a CDF family heavy metal/H+ antiporter). The ophc2 gene was effectively expressed in E. coli. This is the second report of cloning the ophc2 gene and the first report of this gene from the genus of Stenotrophomonas.

Rhizobium petrolearium sp. nov., isolated from oil- contaminated soil

Two Gram-negative, aerobic, rod-shaped bacteria, designated strains SL-1(T) and F11, which had the ability to decompose polycyclic aromatic hydrocarbons (PAHs), were isolated from soil samples contaminated by oil. The cells were motile by polar or lateral flagella. According to comparison of 16S rRNA gene sequences, strains SL-1(T) and F11 were identical and showed the greatest degree of similarity (96.8%) to both Rhizobium oryzae Alt505(T) and Rhizobium mesosinicum CCBAU 25010(T); however, only Rhizobium oryzae with SL-1(T) and F11 formed a separate clade. There were low similarities (<90%) between the atpD and recA sequences of the two strains and those of the genus of Rhizobium. The bacteria grew at temperatures of 10-40 °C with an optimum of 30 °C. The pH range for growth was 6.0-10.0 and optimum pH was 7.0-8.0. Growth occurred at NaCl concentrations up to 3.0% (w/v). They were catalase- and oxidase-positive. The main cellular fatty acids were summed feature 8 (18:1ω7c and/or 18:1ω6c) and 16:0. The DNA G+C content was 62.2 mol%. Strain SL-1(T) showed 29 and 0% DNA-DNA relatedness, respectively, with the most related strains R. oryzae Alt505(T) and R. mesosinicum CCBAU 25010(T) according to phylogenic analysis of the 16S rRNA gene. According to physiological and biochemical characteristics and genotypic data obtained in this work, the bacteria represent a novel species of the genus Rhizobium, and the name Rhizobium petrolearium is proposed. The type strain is SL-1(T) (u200a=u200aACCC 11238(T)u200a=u200aKCTC 23288(T)) and it could nodulate Medicago sativa in nodulation tests.

A gene linB2 responsible for the conversion of β-HCH and 2,3,4,5,6-pentachlorocyclohexanol in Sphingomonas sp. BHC-A

Commercial formulations of hexachlorocyclohexane (HCH) consist of a mixture of four isomers: α, β, γ, and δ. All four isomers are toxic and recalcitrant pollutants. β-HCH is more problematic due to its longer persistence in the environment. Sphingomonas sp. BHC-A was able to degrade not only α-, γ-, and δ-HCH but also β-HCH. To clone a gene responsible for the degradation of β-HCH, a Tn5 mutation was introduced into BHC-A, and one mutant BHC-A45 defective in β-HCH degradation was selected. Sequencing analysis showed this mutant had a Tn5 insertion at the site of one haloalkane dehalogenase gene, designated linB2. linB2 was overexpressed in Escherichia coli and the 32-kDa product LinB2 showed the conversion activity of not only β-HCH to β-2,3,4,5,6-pentachlorocyclohexanol (β-PCHL) but also β-PCHL to β-2,3,5,6-tetrachloro-1,4-cyclohexanediol.

Extracellular polymeric substances enhanced mass transfer of polycyclic aromatic hydrocarbons in the two-liquid-phase system for biodegradation

The objective was to elucidate the role of extracellular polymeric substances (EPS) in biodegradation of polycyclic aromatic hydrocarbons in two-liquid-phase system (TLPs). Therefore, biodegradation of phenanthrene (PHE) was conducted in a typical TLPs—silicone oil–water—with PHE-degrading bacteria capable of producing EPS, Sphingobium sp. PHE3 and Micrococcus sp. PHE9. The results showed that the presence of both strains enhanced mass transfer of PHE from silicone oil to water, and that biodegradation of PHE mainly occurred at the interfaces. The ratios of tightly bound (TB) proteins to TB polysaccharides kept almost constant, whereas the ratios of loosely bound (LB) proteins to LB polysaccharides increased during the biodegradation. Furthermore, polysaccharides led to increased PHE solubility in the bulk water, which resulted in an increased PHE mass transfer. Both LB-EPS and TB-EPS (proteins and polysaccharides) correlated with PHE mass transfer in silicone oil, indicating that both proteins and polysaccharides favored bacterial uptake of PHE at the interfaces. It could be concluded that EPS could facilitate microbial degradation of PHE in the TLPs.

Burkholderia zhejiangensis sp. nov., a methyl-parathion-degrading bacterium isolated from a wastewater-treatment system

The taxonomic status of a methyl-parathion-degrading strain, OP-1(T), isolated from a wastewater-treatment system in China, was determined using a polyphasic approach. The rod-shaped cells were Gram-staining-negative, non-spore-forming and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel strain belonged to the genus Burkholderia, as it appeared closely related to Burkholderia glathei ATCC 29195(T) (97.4 % sequence similarity), Burkholderia sordidicola KCTC 12081(T) (96.5 %) and Burkholderia bryophila LMG 23644(T) (96.3 %). The major cellular fatty acids, C(16:0), C(17:0) cyclo and C(18:1)ω7c, were also similar to those found in established members of the genus Burkholderia. The genomic DNA G+C content of strain OP-1(T) was 59.4 mol%. The level of DNA-DNA relatedness between the novel strain and the closest recognized species, Burkholderia glathei ATCC 29195(T), was only 30 %. Based on the phenotypic, genotypic and phylogenetic evidence, strain OP-1(T) represents a novel species of the genus Burkholderia, for which the name Burkholderia zhejiangensis sp. nov. is proposed. The type strain is OP-1(T) ( = CCTCC AB 2010354(T) = KCTC 23300(T)).

Sphingobium qiguonii sp. nov., a carbaryl- degrading bacterium isolated from a wastewater treatment system

A Gram-staining-negative, catalase-positive, carbaryl-degrading, non-spore-forming, non-motile, rod-shaped bacterium, designated strain X23(T), was isolated from a wastewater treatment system. Phylogenetic analysis based on 16S rRNA gene sequence indicated that the strain belongs to the genus Sphingobium. The highest 16S rRNA gene sequence similarity observed for the isolate was 96.6u200a% with the type strain of Sphingobium amiense. Chemotaxonomic data [major ubiquinone: Q-10; major polar lipids: diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, sphingoglycolipid, phosphatidylethanolamine and unknown aminolipids and phospholipids; major fatty acids: summed feature 7 (C(18u200a:u200a1)ω7c, C(18u200a:u200a1)ω9t and/or C(18u200a:u200a1)ω12t), C(16u200a:u200a1)ω5c, C(14u200a:u200a0) 2-OH and C(16u200a:u200a0) 2-OH] as well as the inability to reduce nitrate and the presence of spermidine as the major polyamine supported the affiliation of the strain to the genus Sphingobium. Based on the phylogenetic analysis, whole-cell fatty acid composition and biochemical characteristics, the strain could be separated from all recognized species of the genus Sphingobium. Strain X23(T) should be classified as a novel species of the genus Sphingobium, for which the name Sphingobium qiguonii sp. nov. is proposed, with strain X23(T) (=CCTCC AB 208221(T) =DSM 21541(T)) as the type strain.

Expression of Methyl Parathion Hydrolase in Pichia pastoris

In the Pichia pastoris expression system, increasing the copy number of the expression cassette often has the effect of increasing the amount of protein expressed. To improve the expression level of methyl parathion hydrolase (MPH), we constructed two integration vectors with four and eight direct repeats of the expression cassette using an in vitro multimerization approach. After two successive integrations, at least 12 copies of the MPH expression cassette were integrated into the P.xa0pastoris chromosome. Under shake-flask conditions, over 55xa0mg active MPH/l was secreted into the medium by the multicopy clones. The extracellular enzyme activity was about 10-fold higher for the multicopy clones than for clones containing a single copy of the gene. Further investigations revealed that the multicopy MPH expression cassette could remain stably integrated and functional over five generations. Note that the expression vector pRF constructed in our study can be not only used to construct multiple copies of the expression cassette in vitro, but also integrated into the P.xa0pastoris genome without introducing any antibiotic resistance gene, which is desirable for production of biotherapeutic proteins.

SulE, a sulfonylurea herbicide de-esterification esterase from Hansschlegelia zhihuaiae S113.

ABSTRACT De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification, was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag+, Cd2+, Zn2+, methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ΔsulE, was constructed by insertion mutation. ΔsulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments.

Construction of a stable genetically engineered rhamnolipid-producing microorganism for remediation of pyrene-contaminated soil

One rhamnolipid-producing bacterial strain named Pseudomonas aeruginosa BSFD5 was isolated and characterized. Its rhlABRI cassette including necessary genes for rhamnolipid synthesis was cloned and transformed into the chromosome of P. putida KT2440 by a new random transposon vector without introducing antibiotic-resistance marker, generating a genetically engineered microorganism named P. putida KT2440-rhlABRI, which could stably express the rhlABRI cassette and produce rhamnolipid at a yield of 1.68xa0gxa0l−1. In experiments using natural soil, it was shown that P. putida KT2440-rhlABRI could increase the dissolution of pyrene and thus promote its degradation by indigenous microorganisms. P. putida KT2440-rhlABRI thus demonstrated potential for enhancing the remediation of soils contaminated with polycyclic aromatic hydrocarbons.