Pyrethroid-degrading Sphingobium sp. JZ-2 and the purification and characterization of a novel pyrethroid hydrolase

A pyrethroid-degrading bacterium strain JZ-2 was isolated from activated sludge treating pyrethroid-manufacturing wastewater. Based on the morphological, physiological and biochemical characterization, and phylogenetic analysis of the 16S rRNA gene sequence, the strain was identified as Sphingobium sp. Strain JZ-2 was capable of degrading fenpropathrin, cypermethrin, permethrin, cyhalothrin, deltamethrin, fenvalerate and bifenthrin. This strain degraded fenpropathrin by hydrolysis of the carboxylester linkage to yield 3-phenoxybenzaldehyde and 2,2,3,3-tetramethylcyclopropanecarboxylic acid. 3-Phenoxybenzaldehyde, 3-phenoxybenzoate, protocatechuate and catechol are the intermediates of fenpropathrin degradation. Protocatechuate and catechol were further oxidized by ortho-cleavage pathway. A novel pyrethroid hydrolase from cell-free extract was purified 108.5-fold to apparent homogeneity with a 10.2% overall recovery. It was a monomer with a molecular mass of 31 ± 1 kDa, a pI of 4.85. The optimal pH and temperature were 7.5 and 40 °C, respectively. No cofactors or coenzymes were required for the pyrethroid-hydrolysis activity. The enzyme was strongly inhibited by many irons (Ag⁺, Cu²⁺, Hg²⁺ and Zn²⁺), SDS, p-chloromercuribenzoic acid, phenylmethylsulfonyl fluoride and malathion.     

Screening of a beta-cypermethrin-degrading bacterial strain <Emphasis Type="Italic">Brevibacillus parabrevis</Emphasis> BCP-09 and its biochemical degradation pathway

A novel beta-cypermethrin (Beta-CP)-degrading strain isolated from activated sludge was identified as Brevibacillus parabrevis BCP-09 based on its morphological and physio-biochemical characteristics, and 16S rRNA gene analysis. Strain BCP-09 could effectively degrade Beta-CP at pH 5.0–9.0, 20–40 °C, and 10–500 mg L^?1 Beta-CP. Under optimal conditions (pH 7.41, 38.9 °C, 30.9 mg L^?1 Beta-CP), 75.87% Beta-CP was degraded within 3 days. Beta-CP degradation (half-life, 33.45 h) and strain BCP-09 growth were respectively described using first-order-kinetic and logistic-kinetic models. Seven metabolites were detected by high-performance liquid chromatography and gas chromatography-mass spectrometry- methyl salicylate, catechol, phthalic acid, salicylic acid, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzaldehyde, and 3-phenoxybenzoic acid (3-PBA). The major Beta-CP metabolite, 3-PBA was further degraded into phenol, benzoic acid, and 4-methylhexanoic acid. BCP-09 also degraded aromatic compounds such as phenol, catechol, and protocatechuic acid. Beta-CP appears to be mainly degraded into 3-PBA, which is continuously degraded into smaller benzene or chain compounds. Thus, strain BCP-09 could form a complete degradation system for Beta-CP and might be considered a promising strain for application in the bioremediation of environments and agricultural products polluted by Beta-CP.     

Degradation pathways of cyclic alkanes in Rhodococcus sp. NDKK48

The degradation pathways for cyclic alkanes (c-alkanes) in Rhodococcus sp. NDKK48 were investigated. Strain NDKK48 used dodecylcyclohexane as a sole carbon and energy source, and five metabolites in the dodecylcyclohexane degradation pathway were detected by gas-chromatography/mass spectra. The metabolites were identified as cyclohexanecarboxylic acid, cyclohexylacetic acid, 1-cyclohexene-1-acetic acid, 4-dodecylcyclohexanol, and 4-dodecylcyclohexanone. The strain degrades dodecylcyclohexane via a ring oxidation pathway and an alkyl side chain oxidation pathway. Cyclohexanecarboxylic acid was further oxidized to muconic acid via 1-cyclohexene-1-carboxylic acid and benzoic acid, and the muconic acid was finally used by strain NDKK48 for growth. Methylcyclohexane and cyclohexane were co-oxidized with hexadecane by strain NDKK48. Methylcyclohexane was degraded via a ring oxidation pathway, and the degradation pathway contained part of the Baeyer-Villiger oxidation for ring cleavage. Cyclohexane was also degraded by the same pathway as methylcyclohexane. Thus, strain NDKK48 has two pathways for the complete degradation of c-alkanes.     

Biodegradation of deltamethrin and its hydrolysis product 3-phenoxybenzaldehyde by a newly isolated Streptomyces aureus strain HP-S-01

A newly isolated actinomycete strain HP-S-01 from activated sludge could effectively degrade deltamethrin and its major hydrolysis product 3-phenoxybenzaldehyde. Based on the morphological, cultural, physio-biochemical characteristics, and 16S rDNA sequence analysis, strain HP-S-01 was identified as Streptomyces aureus. Strain HP-S-01 was also found highly efficient in degrading cyfluthrin, bifenthrin, fenvalerate, fenpropathrin, permethrin, and cypermethrin. Strain HP-S-01 rapidly degraded deltamethrin without a lag phase over a wide range of temperature (18~38°C) and pH (5~10), and metabolized to produce α-hydroxy-3-phenoxy-benzeneacetonitrile and 3-phenoxybenzaldehyde by hydrolysis of the carboxylester linkage. The 3-phenoxybenzaldehyde was further oxidized to form 2-hydroxy-4-methoxy benzophenone resulting in its detoxification. No persistent accumulative product was detected by gas chromatography-mass spectrometry (GC/MS) analysis. Response surface methodology was used to optimize degradation conditions. Strain HP-S-01 completely removed 50~300 mg L⁻¹ deltamethrin within 7 days under the optimal degradation conditions. Furthermore, the biodegradation kinetics corresponded with the first-order model. Therefore, strain HP-S-01 is suitable for the efficient and rapid bioremediation of pyrethroid-contaminated environment.     

Microbial detoxification of bifenthrin by a novel yeast and its potential for contaminated soils treatment

Bifenthrin is one the most widespread pollutants and has caused potential effect on aquatic life and human health, yet little is known about microbial degradation in contaminated regions. A novel yeast strain ZS-02, isolated from activated sludge and identified as Candida pelliculosa based on morphology, API test and 18S rDNA gene analysis, was found highly effective in degrading bifenthrin over a wide range of temperatures (20-40 °C) and pH (5-9). On the basis of response surface methodology (RSM), the optimal degradation conditions were determined to be 32.3 °C and pH 7.2. Under these conditions, the yeast completely metabolized bifenthrin (50 mg · L(-1)) within 8 days. This strain utilized bifenthrin as the sole carbon source for growth as well as co-metabolized it in the presence of glucose, and tolerated concentrations as high as 600 mg · L(-1) with a q(max), K(s) and K(i) of 1.7015 day(-1), 86.2259 mg · L(-1) and 187.2340 mg · L(-1), respectively. The yeast first degraded bifenthrin by hydrolysis of the carboxylester linkage to produce cyclopropanecarboxylic acid and 2-methyl-3-biphenylyl methanol. Subsequently, 2-methyl-3-biphenylyl methanol was further transformed by biphenyl cleavage to form 4-trifluoromethoxy phenol, 2-chloro-6-fluoro benzylalcohol, and 3,5-dimethoxy phenol, resulting in its detoxification. Eventually, no persistent accumulative product was detected by gas chromatopraphy-mass spectrometry (GC-MS) analysis. This is the first report of a novel pathway of degradation of bifenthrin by hydrolysis of ester linkage and cleavage of biphenyl in a microorganism. Furthermore, strain ZS-02 degraded a variety of pyrethroids including bifenthrin, cyfluthrin, deltamethrin, fenvalerate, cypermethrin, and fenpropathrin. In different contaminated soils introduced with strain ZS-02, 65-75% of the 50 mg · kg(-1) bifenthrin was eliminated within 10 days, suggesting the yeast could be a promising candidate for remediation of environments affected by bifenthrin. Finally, this is the first described yeast capable of degrading bifenthrin.     

Oxidized low density lipoprotein is resistant to cathepsins and accumulates within macrophages

The rate of uptake of oxidized low density lipoprotein (LDL) by mouse peritoneal macrophages is similar to that of acetyl LDL; but only approximately 50% of the internalized oxidized LDL is ultimately degraded, in contrast to the near-complete degradation seen with acetyl LDL. The objectives of this study were to determine if this was due to increased surface binding of oxidized LDL, different uptake pathways for oxidized LDL and acetyl LDL, lysosomal dysfunction caused by oxidized LDL, or resistance of oxidized LDL to hydrolysis by lysosomal proteinases. LDL binding studies at 4 degrees C showed that the increased cell association with oxidized LDL could not be explained by differences in cell-surface binding. Immunofluorescence microscopy confirmed intracellular accumulation of apoB-immunoreactive material in macrophages incubated with oxidized LDL, but not with acetyl LDL. The scavenger receptor ligand polyinosinic acid inhibited both the cell association and degradation of oxidized LDL in macrophages by greater than 75%, suggesting a common uptake pathway for degraded LDL and nondegraded LDL. Studies in THP-1 cells also did not reveal more than one specific uptake pathway for oxidized LDL. LDL derivatized by incubation with oxidized arachidonic acid (under conditions that prevented oxidation of the LDL itself) showed inefficient degradation, similar to oxidized LDL. When macrophages were incubated with oxidized LDL together with acetyl 125I-LDL, the acetyl LDL was degraded normally, excluding lysosomal dysfunction as the explanation for the accumulation of oxidized LDL. Generation of trichloroacetic acid-soluble products from oxidized 125I-LDL by exposure to cathepsins B and D was less than that observed with native 125I-LDL. LDL modified by exposure to reactive products derived from oxidized arachidonic acid was also degraded more slowly than native 125I-LDL by cathepsins. In contrast, acetyl 125I-LDL was degraded more rapidly by cathepsins than native 125I-LDL, and aggregated LDL and malondialdehyde-modified LDL were degraded at the same rate as native 125I-LDL. It is concluded that the intracellular accumulation of oxidized LDL in macrophages can be explained at least in part by the resistance of oxidatively modified apolipoprotein B to cathepsins. This resistance to cathepsins does not appear to be due to aggregation of oxidized LDL, but may be a consequence of modification of apolipoprotein B by lipid peroxidation products.     

Cometabolic biotransformation of fenpropathrin by Clostridium species strain ZP3

A novel bacterial strain capable of degrading the pyrethroid pesticide fenpropathrin was isolated from mixed wastewater and sludge samples. Phylogenetic analysis of the 16S rDNA sequence revealed that the organism belongs to the genus Clostridium. The organism can co-metabolically transform fenpropathrin at 100?mg?l(-1) at 35°C and pH 7.5 in 12?days. Metabolic products of fenpropathrin from strain ZP3 were examined by gas chromatography/mass spectrometry, and the results showed that the organism degraded fenpropathrin with an oxidization process to yield benzyl alcohol, benzenemethanol, 3,5-dimethylamphetamine. Analyses of cell-free extracts from this strain showed that the optimal degrading conditions for degrading fenpropathrin were 35°C and pH 7.5, and degradation efficiency was 20.0?mg?l(-1)?day(-1), and it might be potential using for rapid treating fenpropathrin, for example, on the surface of fruits and vegetables.     

Degradation Mechanism of Poly(vinyl alcohol) by Successive Reactions of Secondary Alcohol Oxidase and β-Diketone Hydrolase from Pseudomonas sp.

The secondary alcohol oxidase from Pseudomonas sp. catalyzed the oxidation of various vinyl alcohol oligomers with the molecular weight of 220 to 1500 and of β-ketols such as 5-hydroxy-3-heptanone, 4-hydroxy-2-nonanone, 3-hydroxy-5-nonanone, 6-hydroxy-4-nonanone, 7-hydroxy-5-dodecanone, and 8-hydroxy-6-tridecanone. β-Diketone hydrolase from the same strain catalyzed the hydrolysis of various aliphatic β-diketones and some aromatic β-diketones such as 1-phenyl-1,3-butanedione and 1-phenyl-2,4-pentanedione. 4,6-Nonanediol, used as a low molecular weight model of poly(vinyl alcohol) (PVA), was oxidized to 4,6-nonanedione by way of 6-hydroxy-4-nonanone by secondary alcohol oxidase. 4,6-Nonanedione was hydrolyzed to 2-pentanone and n-butyric acid by β-diketone hydrolase. These reactions were stoichiometric.

The presence of the β-diketone structure in PVA oxidized by secondary alcohol oxidase was confirmed by spectral experiments. The absorption due to β-diketone structure in the oxidized PVA decreased as it was hydrolyzed by β-diketone hydrolase. The ratio of the amount of carboxyl groups in the degraded PVA to that of carbonyl groups in the oxidized PVA became more than 0.5. A pathway for the enzymatic degradation of PVA was proposed.     

Biodegradation of fenvalerate and 3-phenoxybenzoic acid by a novel Stenotrophomonas sp. strain ZS-S-01 and its use in bioremediation of contaminated soils

A bacterial strain ZS-S-01, newly isolated from activated sludge, could effectively degrade fenvalerate and its hydrolysis product 3-phenoxybenzoic acid (3-PBA). Based on the morphology, physiological biochemical characteristics, and 16 S rDNA sequence, strain ZS-S-01 was identified as Stenotrophomonas sp. Strain ZS-S-01 could also degrade and utilize deltamethrin, beta-cypermethrin, beta-cyfluthrin, and cyhalothrin as substrates for growth. Strain ZS-S-01 was capable of degrading fenvalerate rapidly without a lag phase over a wide range of pH and temperature, even in the presence of other carbon sources, and metabolized it to yield 3-PBA, then completely degraded it. No persistent accumulative product was detected by HPLC and GC/MS analysis. Studies on biodegradation in various soils showed that strain ZS-S-01 demonstrated efficient degradation of fenvalerate and 3-PBA (both 50 mg·kg⁻¹) with a rate constant of 0.1418–0.3073 d⁻¹, and half-lives ranged from 2.3 to 4.9 days. Compared with the controls, the half-lives for fenvalerate and 3-PBA reduced by 16.9–156.3 days. These results highlight strain ZS-S-01 may have potential for use in bioremediation of pyrethroid-contaminated environment.     

cDNA cloning and expression of Xenopus sperm-specific basic nuclear protein 5 (SP5) gene

As part of our continuing program to understand the molecular mechanisms controlling the synthesis of sperm-specific nuclear proteins (SPs1–6) during spermatogenesis in Xenopus, we report here on the isolation of a cDNA clone for SP5, the partial sequencing of the amino acids in the SPs, and the expression of the mRNA for SP5. A cDNA clone (pXSP633) was isolated from a cDNA library, previously prepared from poly (A)⁺ mRNA obtained from Xenopus round spermatids. Determination of the amino acid sequence of the N-terminal regions of all the SPs(1–6) suggested that pXSP633 encodes SP5, whereas SPs3, 4, and 6 are derived from a second mRNA species, and SPs1 and 2 from a third mRNA species. Thus it seems likely that the six SPs are derived from three different mRNA species. Northern blot analyses of RNA, extracted from primary spermatocytes and round spermatids, was performed with oligonucleotide probes specific for SPs4 and 5 mRNAs. The results showed that whereas both SPs4 and 5 mRNAs are expressed in primary spermatocytes, the amount of SP5 mRNA is only about one-fifth of that of SP4 mRNA. However, both mRNA species undergo a similar size change in the length of their poly (A) tracts during spermatogenesis: the size of the mRNA in cultured round spermatids on day 0 was longer than that in primary spermatocytes, but the size of the mRNA in round spermatids on day 6 was shorter than that in round spermatids on day 0. © 1994 Wiley-Liss, Inc.     

An ubiquitin ligase recognizing a protein oxidized by iron: implications for the turnover of oxidatively damaged proteins

Protein oxidation is a natural consequence of aerobic metabolism in cells. Oxidative modification of amino acid residues of proteins causes to lose activity or function of proteins. Organisms have thus developed pathways to remove oxidized proteins by rapid protein degradation. These pathways are important components in cellular quality control mechanisms. It has been suggested that oxidized proteins are degraded by the proteasome. However, whether ubiquitylation is necessary for the degradation of oxidized proteins remains a controversial issue. We have recently identified HOIL-1 (heme-oxidized IRP2 ubiquitin ligase-1) as an E3 ligase that recognizes a protein that has been oxidized by iron. This review describes the recent progress made in understanding the ubiquitin-proteolytic pathway and the regulation of iron metabolism. The process involved in eliminating oxidized proteins and the possible roles that HOIL-1 ubiquitin ligase may play in these processes are discussed.     

Coarse-Grained Simulations of Topology-Dependent Mechanisms of Protein Unfolding and Translocation Mediated by ClpY ATPase Nanomachines

Clp ATPases are powerful ring shaped nanomachines which participate in the degradation pathway of the protein quality control system, coupling the energy from ATP hydrolysis to threading substrate proteins (SP) through their narrow central pore. Repetitive cycles of sequential intra-ring ATP hydrolysis events induce axial excursions of diaphragm-forming central pore loops that effect the application of mechanical forces onto SPs to promote unfolding and translocation. We perform Langevin dynamics simulations of a coarse-grained model of the ClpY ATPase-SP system to elucidate the molecular details of unfolding and translocation of an α/β model protein. We contrast this mechanism with our previous studies which used an all-α SP. We find conserved aspects of unfolding and translocation mechanisms by allosteric ClpY, including unfolding initiated at the tagged C-terminus and translocation via a power stroke mechanism. Topology-specific aspects include the time scales, the rate limiting steps in the degradation pathway, the effect of force directionality, and the translocase efficacy. Mechanisms of ClpY-assisted unfolding and translocation are distinct from those resulting from non-allosteric mechanical pulling. Bulk unfolding simulations, which mimic Atomic Force Microscopy-type pulling, reveal multiple unfolding pathways initiated at the C-terminus, N-terminus, or simultaneously from both termini. In a non-allosteric ClpY ATPase pore, mechanical pulling with constant velocity yields larger effective forces for SP unfolding, while pulling with constant force results in simultaneous unfolding and translocation.     

Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium

Aims:  The metabolism of phenanthrene and anthracene by a moderate thermophilic Nocardia otitidiscaviarum strain TSH1 was examined.
Methods and Results:  When strain TSH1 was grown in the presence of anthracene, four metabolites were identified as 1,2-dihydroxy-1,2-dihydroanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, 2,3-dihydroxynaphthalene and benzoic acid using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). Degradation studies with phenanthrene revealed 2,2'-diphenic acid, phthalic acid, 4-hydroxyphenylacetic acid, o -hydroxyphenylacetic acid, benzoic acid, a phenanthrene dihydrodiol, 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid and 1-hydroxy-2-naphthoic acid (1H2NA), as detectable metabolites.
Conclusions:  Strain TSH1 initiates phenanthrene degradation via dioxygenation at the C-3 and C-4 or at C-9 and C-10 ring positions. Ortho -cleavage of the 9,10-diol leads to formation of 2,2'-diphenic acid. The 3,4-diol ring is cleaved to form 1H2NA which can subsequently be degraded through o -phthalic acid pathway. Benzoate does not fit in the previously published pathways from mesophiles. Anthracene metabolism seems to start with a dioxygenation at the 1 and 2 positions and ortho -cleavage of the resulting diol. The pathway proceeds probably through 2,3-dicarboxynaphthalene and 2,3-dihydroxynaphthalene. Degradation of 2,3-dihydroxynaphthalene to benzoate and transformation of the later to catechol is a possible route for the further degradation of anthracene.
Significance and Impact of the Study:  For the first time, metabolism of phenanthrene and anthracene in a thermophilic Nocardia strain was investigated.     

Construction of a novel secretion expression system guided by native signal peptide of PhoD in Zymomonas mobilis

In the current study, three native signal peptides (SPs) from PhoC, PhoD, and ZMO0331were investigated and compared to construct novel secretion expression systems in Zymomonas mobilis. The secretion expression of target protein, α-amylase from Bacillus amyloliquefaciens (BAA), guided by PhoD’s SP resulted in more hydrolysis of starch than that by the other two SPs. Extracellular and intracellular α-amylase activities of the strain containing PhoD’s SP were also higher than the other two strains containing PhoC or ZMO0331’s SP. In addition, the evidence by alcohol dehydrogenase activity assay further confirmed that the starch hydrolysis was resulted from the secretion expression of BAA rather than the breakage of cells. Our results indicated that the SP of PhoD is able to serve as a promising candidate to assist secretion expression of heterogeneous genes in Z. mobilis. This will contribute to development of engineered Z. mobilis strains converting starch into ethanol.     

Antigenic polysaccharides of bacteria. 35. Establishment of the structure of polysaccharide chains of lipopolysaccharides of Pseudomonas cepacia IMV 4176 and IMV 4202 (Serotype 3)

On mild acid degradation of the Pseudomonas cepacia strain IMV 4176 lipopolysaccharide, two polysaccharides were obtained, one of which is a homopolymer of N-acetyl-D-galactosamine and the other is composed of equal amounts of N-acetyl-D-galactosamine and D-ribose. Partial hydrolysis with aqueous oxalic acid caused depolymerization of the heteropolysaccharide, and the homopolysaccharide was isolated in the individual state. On the basis of methylation and 13C NMR analysis, it was concluded that both polysaccharides are built up of disaccharide repeating units having the following structures: ----4)-alpha-D-GalpNAc-(1----4)-beta-D-GalpNAc-(1---- and ----4)-alpha-D-GalpNAc-(1----2)-beta-D-Ribf-(1----. The heteropolysaccharide from P. cepacia strain 4176 is identical by the structure of the repeating unit to the O-specific polysaccharide of P. cepacia strain IMV 4202 (serotype 3), Pseudomonas aeruginosa O12 and Serratia marcescens O14.     

Microbial metabolism of quinoline and related compounds. V. Degradation of 1H-4-oxoquinoline by Pseudomonas putida 33/1

A bacterial strain was isolated with the ability to use 1H-4-oxoquinoline as the sole source of carbon, nitrogen and energy. On the basis of its physiological properties, this isolate was classified as Pseudomonas putida. 1H-3-Hydroxy-4-oxoquinoline, N-formylanthranilic acid, anthranilic acid and catechol were identified as intermediates in the degradation pathway. The latter was further degraded by ortho-cleavage. The enzymatic conversion of 1H-4-oxoquinoline into 1H-3-hydroxy-4-oxoquinoline requires oxygen and NADH. Experiments with 18O2 showed that the oxygen consumed in this enzymatic reaction is derived from the atmosphere.     

Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation

HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.     

A <Emphasis Type="Italic">sirA</Emphasis>-like gene, <Emphasis Type="Italic">sirA2</Emphasis>, is essential for 3-succinoyl-pyridine metabolism in the newly isolated nicotine-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HZN6 strain

A novel nicotine-degrading Pseudomonas sp. strain, HZN6, was isolated from a pesticide-wastewater treatment facility in Hangzhou. The strain could grow on nicotine as its sole source of carbon, nitrogen, and energy. The strain’s main intermediate metabolites were determined to be pseudooxynicotine, 3-succinoyl-pyridine (SP), and 6-hydroxy-3-succinoyl-pyridine (HSP). A Tn5 transposon mutant was generated in which the degradation pathway was blocked at the SP. A 4,583-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR and analyzed. The mutant gene orfC displays 89% deduced amino acid sequence identity with the sirA-like gene (sirA2, a sulfurtransferase homologue gene) of Pseudomonas stutzeri A1501. The orfC-disrupted strain lost the ability to degrade SP, and the complementation strains with the orfC from the Pseudomonas sp. HZN6 and the sirA2 (PP_1233) from Pseudomonas putida KT2440 recovered the degradation ability. Though the orfC-disrupted strain also lost the xanthine dehydrogenase activity, the effects of tungsten on the degradation of SP and hypoxanthine revealed that the hydroxylation of SP to HSP was not a xanthine dehydrogenase type. These results demonstrated that the orfC gene was essential for the SP metabolism involved in the nicotine metabolic pathway in the Pseudomonas sp. HZN6 strain. This study might advance the understanding of the nicotine metabolic mechanism in Pseudomonas.     

Bacterial degradation of tert-amyl alcohol proceeds via hemiterpene 2-methyl-3-buten-2-ol by employing the tertiary alcohol desaturase function of the Rieske nonheme mononuclear iron oxygenase MdpJ

Tertiary alcohols, such as tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA) and higher homologues, are only slowly degraded microbially. The conversion of TBA seems to proceed via hydroxylation to 2-methylpropan-1,2-diol, which is further oxidized to 2-hydroxyisobutyric acid. By analogy, a branched pathway is expected for the degradation of TAA, as this molecule possesses several potential hydroxylation sites. In Aquincola tertiaricarbonis L108 and Methylibium petroleiphilum PM1, a likely candidate catalyst for hydroxylations is the putative tertiary alcohol monooxygenase MdpJ. However, by comparing metabolite accumulations in wild-type strains of L108 and PM1 and in two mdpJ knockout mutants of strain L108, we could clearly show that MdpJ is not hydroxylating TAA to diols but functions as a desaturase, resulting in the formation of the hemiterpene 2-methyl-3-buten-2-ol. The latter is further processed via the hemiterpenes prenol, prenal, and 3-methylcrotonic acid. Likewise, 3-methyl-3-pentanol is degraded via 3-methyl-1-penten-3-ol. Wild-type strain L108 and mdpJ knockout mutants formed isoamylene and isoprene from TAA and 2-methyl-3-buten-2-ol, respectively. It is likely that this dehydratase activity is catalyzed by a not-yet-characterized enzyme postulated for the isomerization of 2-methyl-3-buten-2-ol and prenol. The vitamin requirements of strain L108 growing on TAA and the occurrence of 3-methylcrotonic acid as a metabolite indicate that TAA and hemiterpene degradation are linked with the catabolic route of the amino acid leucine, including an involvement of the biotin-dependent 3-methylcrotonyl coenzyme A (3-methylcrotonyl-CoA) carboxylase LiuBD. Evolutionary aspects of favored desaturase versus hydroxylation pathways for TAA conversion and the possible role of MdpJ in the degradation of higher tertiary alcohols are discussed.