Cometabolism of 1,1-Dichloro-2,2-Bis(4-Chlorophenyl)Ethylene by Pseudomonas acidovorans M3GY Grown on Biphenyl

1,1-Dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), a toxic breakdown product of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), has traditionally been viewed as a dead-end metabolite: there are no published reports detailing enzymatic ring fission of DDE by bacteria in either soil or pure culture. In this study, we investigated the ability of Pseudomonas acidovorans M3GY to transform DDE and its unchlorinated analog, 1,1-diphenylethylene (DPE). While strain M3GY could grow on DPE, cells grown on DPE as a sole carbon source could not degrade DDE. Cells grown on biphenyl, however, did degrade DDE. Mass balance analysis of [¹⁴C]DDE showed transformation of more than 40% of the recoverable radioactivity. Nine chlorinated metabolites produced from DDE were identified by gas chromatography-mass spectrometry–Fourier-transform infrared spectrometry (GC-MS-FTIR) from cultures grown on biphenyl. Recovery of these metabolites demonstrates that biphenyl-grown cells degrade DDE through a meta-fission pathway. This study provides a possible model for biodegradation of DDE in soil by biphenyl-utilizing bacteria.     

Isolation of Terrabacter sp. strain DDE-1, which metabolizes 1, 1-dichloro-2,2-bis(4-chlorophenyl)ethylene when induced with biphenyl

Terrabacter sp. strain DDE-1, able to metabolize 1,1-dichloro-2, 2-bis(4-chlorophenyl)ethylene (DDE) in pure culture when induced with biphenyl, was enriched from a 1-1-1-trichloro-2, 2-bis(4-chlorophenyl)ethane residue-contaminated agricultural soil. Gas chromatography-mass spectrometry analysis of culture extracts revealed a number of DDE catabolites, including 2-(4'-chlorophenyl)-3,3-dichloropropenoic acid, 2-(4'-chlorophenyl)-2-hydroxy acetic acid, 2-(4'-chlorophenyl) acetic acid, and 4-chlorobenzoic acid.     

Characterization of the 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) degradation system in Janibacter sp. TYM3221

Bacterial degradation of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) has been previously reported, however, its degradation enzyme system has not been characterized. In this study, a DDE-degrading bacterium, Janibacter sp. TYM3221, was isolated and characterized. Transformation of DDE was demonstrated by TYM3211 resting cells grown in LB in the presence and absence of biphenyl. Gas chromatography-mass spectrometry analysis revealed five metabolites of DDE containing a meta-ring cleavage product and 4-chlorobenzoic acid, suggesting that TYM3221 degrades DDE to 4-chlorobenzoic acid via a meta-ring cleavage product. A gene cluster, bphAaAbAcAd, which codes for biphenyl dioxygenase subunits, was cloned from TYM3221. A mutant strain with a bphAa-gene inactivation did not grow on biphenyl, and showed no DDE degradation activity. These results indicate that in strain TYM3221, the bphAa-coded biphenyl dioxygenase is involved not only in the metabolism of biphenyl but also in the degradation of DDE.     

Characterization of the 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) degradation system in Janibacter sp. TYM3221

Bacterial degradation of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) has been previously reported, however, its degradation enzyme system has not been characterized. In this study, a DDE-degrading bacterium, Janibacter sp. TYM3221, was isolated and characterized. Transformation of DDE was demonstrated by TYM3211 resting cells grown in LB in the presence and absence of biphenyl. Gas chromatography–mass spectrometry analysis revealed five metabolites of DDE containing a meta-ring cleavage product and 4-chlorobenzoic acid, suggesting that TYM3221 degrades DDE to 4-chlorobenzoic acid via a meta-ring cleavage product. A gene cluster, bphAaAbAcAd, which codes for biphenyl dioxygenase subunits, was cloned from TYM3221. A mutant strain with a bphAa-gene inactivation did not grow on biphenyl, and showed no DDE degradation activity. These results indicate that in strain TYM3221, the bphAa-coded biphenyl dioxygenase is involved not only in the metabolism of biphenyl but also in the degradation of DDE.     

Isolation of Acetobacterium sp. Strain AG,Which Reductively Debrominates Octa- and Pentabrominated Diphenyl Ether Technical Mixtures

Polybrominated diphenyl ethers (PBDEs) are a class of environmental pollutants that have been classified as persistent organic pollutants since 2009. In this study, a sediment-free enrichment culture (culture G) was found to reductively debrominate octa- and penta-BDE technical mixtures to less-brominated congeners (tetra-, tri-, and di-BDEs) via a para-dominant debromination pattern for the former and a strict para debromination pattern for the latter. Culture G could debrominate 96% of 280 nM PBDEs in an octa-BDE mixture to primarily tetra-BDEs in 21 weeks. Continuous transferring of culture G with octa-/penta-BDEs dissolved in n-nonane or trichloroethene (TCE) yielded two strains (Acetobacterium sp. strain AG and Dehalococcoides sp. strain DG) that retained debromination capabilities. In the presence of lactate but without TCE, strain AG could cometabolically debrominate 75% of 275 nM PBDEs in a penta-BDE mixture in 33 days. Strain AG shows 99% identity to its closest relative, Acetobacterium malicum. In contrast to strain AG, strain DG debrominated PBDEs only in the presence of TCE. In addition, 18 out of 19 unknown PBDE debromination products were successfully identified from octa- and penta-BDE mixtures and revealed, for the first time, a comprehensive microbial PBDE debromination pathway. As an acetogenic autotroph that rapidly debrominates octa- and penta-BDE technical mixtures, Acetobacterium sp. strain AG adds to the still-limited understanding of PBDE debromination by microorganisms.     

Characterization of the 450-kb Linear Plasmid in a Polychlorinated Biphenyl Degrader, Rhodococcus sp. Strain RHA1

A strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, has diverse biphenyl/PCB degradative genes and harbors huge linear plasmids, including pRHL1 (1,100 kb), pRHL2 (450 kb), and pRHL3 (330 kb). The diverse degradative genes are distributed mainly on the pRHL1 and pRHL2 plasmids. In this study, the structural and functional characteristics of pRHL2 were determined. We constructed a physical map of pRHL2, and the degradative enzyme genes, including bphB2, etbD2, etbC, bphDEF, bphC2, and bphC4, were localized in three regions. Conjugal transfer of pRHL2 between RHA1 mutant derivatives was observed at a frequency of 7.5 × 10⁻⁵ transconjugant per recipient. These results suggested that the linear plasmid is a possible determinant of propagation of the diverse degradative genes in rhodococci. The termini of pRHL2 were cloned and sequenced. The left and right termini of pRHL2 had 3-bp perfect terminal inverted repeats and were not as similar to each other (64% identity) as the known actinomycete linear replicons are. Southern hybridization analysis with pRHL2 terminal probes suggested that the right terminus of pRHL2 is similar to pRHL1 and pRHL3 termini. Retardation of both terminal fragments in the gel shift assay indicated that each terminus of pRHL2 is linked to a protein. We suggest that pRHL2 has invertron termini, as has been reported previously for Streptomyces linear replicons.     

Isolation and Characterization of Pseudoxanthomonas sp. Strain YP1 Capable of Denitrifying Phosphorus Removal (DPR)

A bacterial strain (designated as YP1) was isolated from an aerobic granular sequence batch reactor (SBR) performing simultaneous nitrogen and phosphorus removal. Based on the morphological, biochemical characteristics, and phylogenetic analysis of 16S rRNA gene sequence, YP1 was identified as Pseudoxanthomonas sp. strain. Strain YP1 was confirmed to have the ability to conduct denitrifying phosphorus removal (DPR). The optimal conditions for YP1 were pH 8.0, phosphorus (PO₄^3?-P) concentration of 8.0 mg/L, sodium citrate as carbon source, and nitrate nitrogen (NO₃^?-N) concentration of 30 mg/L. The functional genes including ppk and ppx, narG and narA, nirS and nirK were amplified for understanding the DPR pathways. The results provide more information about denitrifying polyphosphate-accumulating organisms (DPAOs) in aerobic granular sludge (AGS) and lay the foundations for full-scale DPR.     

Two Nearly Identical Aromatic Compound Hydrolase Genes in a Strong Polychlorinated Biphenyl Degrader, Rhodococcus sp. Strain RHA1

The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes, etbD1 and etbD2, were cloned from a strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, and their nucleotide sequences were determined. The etbD2 gene was located in the vicinity of bphA gene homologs and encoded an enzyme whose amino-terminal sequence was very similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1. Using the etbD2 gene fragment as a probe, we cloned the etbD1 gene encoding the purified HOHD hydrolase by colony hybridization. Both genes encode a product having 274 amino acid residues and containing the nucleophile motif conserved in α/β hydrolase fold enzymes. The deduced amino acid sequences were quite similar to the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as dmpD, cumD, todF, and xylF, and not very similar to the amino acid sequences of the products of bphD genes from PCB degraders, including RHA1. The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene, bphD, were expressed in Escherichia coli, and their relative enzymatic activities were examined. The product of bphD was very specific to HPDA, and the products of etbD1 and etbD2 were specific to HOHD. All of the gene products exhibited poor activities against the meta-cleavage product of catechol. These results agreed with the results obtained for BphD and EtbD1 hydrolases purified from RHA1. The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridization analysis and in a reporter gene assay when a promoter probe vector was used. They were induced by biphenyl, ethylbenzene, benzene, toluene, and ortho-xylene. Strain RCD1, an RHA1 mutant strain lacking both the bphD gene and the etbD2 gene, grew well on ethylbenzene. This result suggested that the etbD1 gene product is involved in the meta-cleavage metabolic pathway of ethylbenzene.     

Isolation and Characterization of a Hydrogen Sulfide-Removing Bacterium,Pseudomonas sp. Strain DO-1

Five microbial strains that removed hydrogen sulfide (H₂S) or methylmercaptan (CH₃SH) gas were newly isolated from soil samples. Strain DO-1, one of the isolates, was identified as a member of Pseudomonas sp., and it’s immobilized cells removed 1 or 10 ppm of H₂S gas within 2 hours. When strain DO-1 was cultured aerobically in a flask containing nutrient broth medium, the deodorizing activity increased, depending on the growth of the culture, and the maximum activity was obtained after 48 hours. Even though the immobilized cells were stored at 4 or 25°C in sealed bottles for 6 months, the deodorizing activity remained. Throughout this study, strain DO-1 removed H₂ S gas without preliminary feeding or exposure to sulfur com-pounds as growth substrates or inducers. These characteristics are advantageous for the deodorization of the malodorous gases surrounding us in daily life.     

Metabolism of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene by Alcaligenes denitrificans

Metabolism of 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE), a persistent metabolite of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), by an Alcaligenes denitrificans was optimal under `non-shaking' conditions, was accelerated by adding 1 g glucose l^–1, and inhibited by 1 g sodium acetate l^–1 or 1 g sodium succinate l^–1. Addition of biphenyl, in the vapor form, to the reaction mixture did not enhance DDE metabolism. During the reaction, accumulation of conventional metabolites, 1-chloro-2,2-bis(4-chlorophenyl)ethylene (DDMU) and 4-chlorobenzoate, was not observed.     

Isolation and Characterization of Novosphingobium sp. Strain MT1, a Dominant Polychlorophenol-Degrading Strain in a Groundwater Bioremediation System

A high-rate fluidized-bed bioreactor has been treating polychlorophenol-contaminated groundwater in southern Finland at 5 to 8°C for over 6 years. We examined the microbial diversity of the bioreactor using three 16S ribosomal DNA (rDNA)-based methods: denaturing gradient gel electrophoresis, length heterogeneity-PCR analysis, and restriction fragment length polymorphism analysis. The molecular study revealed that the process was dependent on a stable bacterial community with low species diversity. The dominant organism, Novosphingobium sp. strain MT1, was isolated and characterized. Novosphingobium sp. strain MT1 degraded the main contaminants of the groundwater, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, at 8°C. The strain carried a homolog of the pcpB gene, coding for the pentachlorophenol-4-monooxygenase in Sphingobium chlorophenolicum. Spontaneous deletion of the pcpB gene homolog resulted in the loss of degradation ability. Phenotypic dimorphism (planktonic and sessile phenotypes), low growth rate (0.14 to 0.15 h⁻¹), and low-copy-number 16S rDNA genes (single copy) were characteristic of strain MT1 and other MT1-like organisms isolated from the bioreactor.     

Cloning and Characterization of Benzoate Catabolic Genes in the Gram-Positive Polychlorinated Biphenyl Degrader Rhodococcus sp. Strain RHA1

Benzoate catabolism is thought to play a key role in aerobic bacterial degradation of biphenyl and polychlorinated biphenyls (PCBs). Benzoate catabolic genes were cloned from a PCB degrader, Rhodococcus sp. strain RHA1, by using PCR amplification and temporal temperature gradient electrophoresis separation. A nucleotide sequence determination revealed that the deduced amino acid sequences encoded by the RHA1 benzoate catabolic genes, benABCDK, exhibit 33 to 65% identity with those of Acinetobacter sp. strain ADP1. The gene organization of the RHA1 benABCDK genes differs from that of ADP1. The RHA1 benABCDK region was localized on the chromosome, in contrast to the biphenyl catabolic genes, which are located on linear plasmids. Escherichia coli cells containing RHA1 benABCD transformed benzoate to catechol via 2-hydro-1,2-dihydroxybenzoate. They transformed neither 2- nor 4-chlorobenzoates but did transform 3-chlorobenzoate. The RHA1 benA gene was inactivated by insertion of a thiostrepton resistance gene. The resultant mutant strain, RBD169, neither grew on benzoate nor transformed benzoate, and it did not transform 3-chlorobenzoate. It did, however, exhibit diminished growth on biphenyl and growth repression in the presence of a high concentration of biphenyl (13 mM). These results indicate that the cloned benABCD genes could play an essential role not only in benzoate catabolism but also in biphenyl catabolism in RHA1. Six rhodococcal benzoate degraders were found to have homologs of RHA1 benABC. In contrast, two rhodococcal strains that cannot transform benzoate were found not to have RHA1 benABC homologs, suggesting that many Rhodococcus strains contain benzoate catabolic genes similar to RHA1 benABC.     

Mineralization of 4-Chlorodibenzofuran by a Consortium Consisting of Sphingomonas sp. Strain RW1 and Burkholderia sp. Strain JWS

The dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R.-M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) attacks 4-chlorodibenzofuran on the unsubstituted aromatic ring via distal dioxygenation adjacent to the ether bridge to produce 3(prm1)-chloro-2,2(prm1),3-trihydroxybiphenyl, which was identified by nuclear magnetic resonance spectroscopy and mass spectrometry. The compound is subsequently meta cleaved, and the respective intermediate is hydrolyzed to form a C-5 moiety, which is further degraded to Krebs cycle intermediates and to 3-chlorosalicylate. This dead-end product is released into the culture medium. A coculture of strain RW1 and the 3,5-dichlorosalicylate-degrading strain Burkholderia sp. strain JWS (A. Schindowski, R.-M. Wittich, and P. Fortnagel, FEMS Microbiol. Lett. 84:63-70, 1991) is able to completely degrade 4-chlorodibenzofuran with concomitant release of Cl(sup-) and formation of biomass.     

Isolation of Fenitrothion-degrading Strain Burkholderia sp. FDS-1 and Cloning of mpd Gene

A short rod shaped, gram-negative bacterium strain Burkholderia sp. FDS-1 was isolated from the sludge of the wastewater treating system of an organophosphorus pesticides manufacturer. The isolate was capable of using fenitrothion as the sole carbon source for its growth. FDS-1 first hydrolyzed fenitrothion to 3-methyl-4-nitrophenol, which was further metabolized to nitrite and methylhydroquinone. The addition of other carbon source and omitting phosphorus source had little effect on the hydrolysis of fenitrothion. The gene encoding the organophosphorus hydrolytic enzyme was cloned and sequenced. The sequence was similar to mpd, a gene previously shown to encode a parathion-methyl-hydrolyzing enzyme in Plesiomonas sp. M6. The inoculation of strain FDS-1 (10⁶ cells g⁻¹) to soil treated with 100 mg fenitrothion emulsion kg⁻¹ resulted in a higher degradation rate than in noninoculated soils regardless of the soil sterilized or nonsterilized. These results highlight the potential of this bacterium to be used in the cleanup of contaminated pesticide waste in the environment. Zhonghui Zhang, Qing Hong: Both authors contributed equally to this work     

Production of (R)-3-Chloro-1,2-Propanediol from Prochiral 1,3-Dichloro-2-Propanol by Corynebacterium sp. Strain N-1074

The production of (R)-3-chloro-1,2-propanediol [(R)-MCP] from prochiral 1,3-dichloro-2-propanol (DCP) was examined with a bacterial strain identified as a Corynebacterium strain. The addition of glycerol as a carbon source or some chlorinated alcohols to a medium was effective for the induction of activity catalyzing the transformation of DCP into MCP. The optimum pH for (R)-MCP production by the resting cell reaction was around 8.0. The optical purity of (R)-MCP formed was improved by keeping the level of DCP in the reaction mixture at a low concentration. (R)-MCP was obtained from 77.5 mM DCP with a 97.3% molar conversion yield and an 83.8% enantiomeric excess of its optical purity by periodic feeding of the substrate.     

1株聚乳酸降解细菌的筛选、鉴定及产酶研究

从污泥中筛选出1株对聚乳酸（poly—L—lacticacid,PLA）具有降解活力的细菌DSL09,该菌株对PLA的乳化液、粉末及薄膜都具有降解作用。通过形态学、16SrDNA比对及生理生化特性的分析,鉴定该菌株属于芽胞杆菌属（Bacillus sp．）。为提高该菌株对PLA的降解活力,对其进行了紫外诱变,获得了稳定遗传的突变株DSL09-60b,该突变株的PLA降解活性提高至原始菌株的1．5倍。对该突变株产PLA降解酶的发酵条件进行了优化,经测定DSL09-60b在初始培养基pH为8．0、0．5％酪蛋白为诱导物、接种量6％（体积比）的条件下37℃培养54h时发酵液酶活性最高。     

Isolation, identification and characterization of a novel triazophos-degrading Bacillus sp. (TAP-1)

A novel triazophos-degrading Bacillus sp., TAP-1, was isolated from sewage sludge in a wastewater treating system of organophosphorus pesticide produced by Funong Group Co. in Jianou, Fujian, southeastern China. The isolate is a gram-positive and rod-shaped bacterium capable of hydrolyzing insecticide triazophos and was identified as a strain of Bacillus using polyphasic taxonomy combined with analysis of the morphological and physio-biochemical properties. TAP-1 could degrade triazophos through co-metabolism. When fed with nutrients such as yeast extract, peptone and glucose, TAP-1 could degrade 98.5% of TAP in the medium (100 mg/l) within 5 days. The optimal pH and temperature for the degradation were 6.5-8 and 32°C, respectively. An enzyme distribution experiment showed that the enzyme responsible for TAP degradation appeared to be intracellular.     

Isolation of Rhodococcus sp. Strain ECU0066, a New Sulfide Monooxygenase-Producing Strain for Asymmetric Sulfoxidation

A new and efficient sulfide monooxygenase-producing strain, ECU0066, was isolated and identified as a Rhodococcus sp. that could transform phenylmethyl sulfide (PMS) to (S)-sulfoxide with 99% enantiomeric excess via two steps of enantioselective oxidations. Its enzyme activity could be effectively induced by adding PMS or phenylmethyl sulfoxide (PMSO) directly to a rich medium at the early log phase (6 h) of fermentation, resulting in over 10-times-higher production of the enzyme. This bacterial strain also displayed fairly good activity and enantioselectivity toward seven other sulfides, indicating a good potential for practical application in asymmetric synthesis of chiral sulfoxides.