Zhoukun Li

Involvement of the Cytochrome P450 System EthBAD in the N-Deethoxymethylation of Acetochlor by Rhodococcus sp. Strain T3-1

ABSTRACT Acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)-acetamide] is a widely applied herbicide with potential carcinogenic properties. N-Deethoxymethylation is the key step in acetochlor biodegradation. N-Deethoxymethylase is a multicomponent enzyme that catalyzes the conversion of acetochlor to 2′-methyl-6′-ethyl-2-chloroacetanilide (CMEPA). Fast detection of CMEPA by a two-enzyme (N-deethoxymethylase–amide hydrolase) system was established in this research. Based on the fast detection method, a three-component enzyme was purified from Rhodococcus sp. strain T3-1 using ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular masses of the components of the purified enzyme were estimated to be 45, 43, and 11 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the results of peptide mass fingerprint analysis, acetochlor N-deethoxymethylase was identified as a cytochrome P450 system, composed of a cytochrome P450 oxygenase (43-kDa component; EthB), a ferredoxin (45 kDa; EthA), and a reductase (11 kDa; EthD), that is involved in the degradation of methyl tert-butyl ether. The gene cluster ethABCD was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). Resting cells of a recombinant E. coli strain showed deethoxymethylation activity against acetochlor. Subcloning of ethABCD showed that ethABD expressed in E. coli BL21(DE3) has the activity of acetochlor N-deethoxymethylase and is capable of converting acetochlor to CMEPA.

Biodegradation of propyzamide by Comamonas testosteroni W1 and cloning of the propyzamide hydrolase gene camH.

Propyzamide is a widely used benzamide herbicide for controlling weeds in lettuce, soybeans, cotton and other crops. An efficient propyzamide-degrading strain W1 was firstly isolated from activated sludge and identified as Comamonas testosteroni. A metabolite of propyzamide by strain W1 was firstly identified. The novel gene camH encoding a hydrolase that catalyzed the amide bond cleavage of propyzamide was cloned from strain W1. The gene contained an open reading frame of 1452 bp, the deduced amino acid sequence showed low identity with other amidases. The recombinant enzyme CamH was expressed in Escherichia coli BL21 and purified. CamH displayed the highest activity at 30°C and pH 8.0 with propyzamide as the substrate. These results provide important knowledge on the fate of propyzamide in the biodegradation, and elucidate the biodegradation mechanism of propyzamide by the strain W1.

Characterization of three cryptic plasmids from Lactobacillus plantarum G63 that was isolated from Chinese pickle.

Three plasmids from Lactobacillus plantarum G63, pG6301, pG6302 and pG6303, were sequenced and have molecular sizes of 3516-bp, 9112-bp and 10047-bp, respectively. We determined the replicons of these plasmids. The pG6301 plasmid carried a replication gene that functioned by the rolling-circle replication mechanism. The Rep protein of pG6302 shared extremely low similarity with a reported plasmid, pLME300, which replicated by a bi-directional mechanism. Both the Rep protein and OriV analyses indicated a similar replication mechanism in pG6302. Conversely, we found neither the Rep protein nor OriV when we analyzed the whole sequence of pG6303. This finding may illustrate a novel replication mechanism. Additionally, the transposon, a mobilization element, was analyzed and compared to a similar insertion sequence (IS) element. A predicted lysozyme gene, pG6303 guhA, was heterologously expressed, but no activity was detected. The pG6302 and pG6303 plasmids contain new replicons and may be useful vector candidates for future molecular manipulation of L. plantarum.

Metabolic pathway involved in 6-chloro-2-benzoxazolinone degradation by Pigmentiphaga sp. strain DL-8 and identification of the novel metal-dependent hydrolase CbaA

ABSTRACT 6-Chloro-2-benzoxazolinone (CDHB) is a precursor of herbicide, insecticide, and fungicide synthesis and has a broad spectrum of biological activity. Pigmentiphaga sp. strain DL-8 can transform CDHB into 2-amino-5-chlorophenol (2A5CP), which it then utilizes as a carbon source for growth. The CDHB hydrolase (CbaA) was purified from strain DL-8, which can also hydrolyze 2-benzoxazolinone (BOA), 5-chloro-2-BOA, and benzamide. The specific activity of purified CbaA was 5,900 U · mg protein−1 for CDHB, with Km and k cat values of 0.29 mM and 8,500 s−1, respectively. The optimal pH for purified CbaA was 9.0, the highest activity was observed at 55°C, and the inactive metal-free enzyme could be reactivated by Mg2+, Ni2+, Ca2+, or Zn2+. Based on the results obtained for the CbaA peptide mass fingerprinting and draft genome sequence of strain DL-8, cbaA (encoding 339 amino acids) was cloned and expressed in Escherichia coli BL21(DE3). CbaA shared 18 to 21% identity with some metal-dependent hydrolases of the PF01499 family and contained the signature metal-binding motif Q127XXXQ131XD133XXXH137. The conserved amino acid residues His288 and Glu301 served as the proton donor and acceptor. E. coli BL21(DE3-pET-cbaA) resting cells could transform 0.2 mM CDHB into 2A5CP. The mutant strain DL-8ΔcbaA lost the ability to degrade CDHB but retained the ability to degrade 2A5CP, consistent with strain DL-8. These results indicated that cbaA was the key gene responsible for CDHB degradation by strain DL-8. IMPORTANCE 2-Benzoxazolinone (BOA) derivatives are widely used as synthetic intermediates and are also an important group of allelochemicals acting in response to tissue damage or pathogen attack in gramineous plants. However, the degradation mechanism of BOA derivatives by microorganisms is not clear. In the present study, we reported the identification of CbaA and metabolic pathway responsible for the degradation of CDHB in Pigmentiphaga sp. DL-8. This will provide microorganism and gene resources for the bioremediation of the environmental pollution caused by BOA derivatives.

Metabolic pathway involved in 2-methyl-6-ethylaniline degradation by Sphingobium sp. strain MEA3-1 and cloning of the novel flavin-dependent monooxygenase system meaBA.

ABSTRACT 2-Methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the chloroacetanilide herbicides acetochlor and metolachlor. Sphingobium sp. strain MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol compounds as sole carbon and energy sources for growth. We isolated the mutant strain MEA3-1Mut, which converts MEA only to 2-methyl-6-ethyl-hydroquinone (MEHQ) and 2-methyl-6-ethyl-benzoquinone (MEBQ). MEA may be oxidized by the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA), which can be hydrolytically spontaneously deaminated to MEBQ or MEHQ. The MEA microbial metabolic pathway was reconstituted based on the substrate spectra and identification of the intermediate metabolites in both the wild-type and mutant strains. Plasmidome sequencing indicated that both strains harbored 7 plasmids with sizes ranging from 6,108 bp to 287,745 bp. Among the 7 plasmids, 6 were identical, and pMEA02′ in strain MEA3-1Mut lost a 37,000-bp fragment compared to pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and protein mass fingerprinting (PMF) showed that MEA3-1Mut lost the two-component flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded by a gene in the lost fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with oxygenase components of some TC-FDMs, whereas MeaB showed no sequence identity with the reductase components of those TC-FDMs. Complementation with meaBA in MEA3-1Mut and heterologous expression in Pseudomonas putida strain KT2440 resulted in the production of an active MEHQ monooxygenase.

Characterization of a novel GH36 α-galactosidase from Bacillus megaterium and its application in degradation of raffinose family oligosaccharides

A novel α-galactosidase gene (agaB) from Bacillus megaterium 3-7 was cloned and expressed in Escherichia coli. The gene coded for a protein with 741 amino acids and a calculated molecular mass of 85.4kDa. The native structure of the recombined AgaB was determined to be a homotrimer. AgaB showed the highest identity of 57% with the characterized glycosyl hydrolase family 36 α-galactosidase from Clostridium stercorarium F-9. The enzyme exhibited a specific activity of 362.6U/mg at 37°C and pH 6.8. The enzyme showed strong resistance to proteases and great tolerance to galactose (Ki=12.5mM). AgaB displayed wide substrate specificity toward pNPGal, melibiose, raffinose and stachyose, with a Km of 0.42, 12.1, 17.0 and 25.4mM, respectively. Furthermore, AgaB completely hydrolyzed raffinose and stachyose present in soybean milk at 37°C within 4h when combined with trypsin. These favorable properties make AgaB a potential candidate for applications in the food and feed industries.

Purification, cloning, expression, and biochemical characterization of a monofunctional catalase, KatP, from Pigmentiphaga sp. DL-8.

Catalases are essential components of the cellular equipment used to cope with oxidative stress. The monofunctional catalase KatP was purified from Pigmentiphaga sp. using ammonium sulfate precipitation (ASP), diethylaminoethyl ion exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC). The purified catalase formed polymer with an estimated monomer molecular mass of 54kDa, which were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. KatP exhibited a specific catalytic activity of 73,000U/mg, which was higher than that of catalase-1 of Comamonas terrigena N3H (55,900U/mg). Seven short tryptic fragments of this catalase were obtained by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS/MS), and the gene, katP, was cloned by PCR amplification and overexpressed in Escherichia coli BL21 (DE3). Based on the complete amino acid sequence, KatP was identified as a clade 3 monofunctional catalase. The specific activities of recombinant KatP for hydrogen peroxide (690,000U/mg) increased 9-fold over that of the parent strain. The Km and Vmax of recombinant KatP were 9.48mM and 81.2mol/minmg, respectively. The optimal pH and temperature for KatP were 7.0 and 37°C, respectively, and the enzyme displayed abroad pH-stable range of 4.0-11.0. The enzyme was inhibited by Zn(2+), Cu(2+), Cr(2+), and Mn(2+), whereas Fe(3+) and Mg(2+) stimulated KatP enzymatic activity. Interestingly, the catalase activity of recombinant KatP displayed high stability under different temperature and pH conditions, suggesting that KatP is a potential candidate for the production of catalase.

Effect of ethylene and 1-MCP on post-harvest physiology and on expression of the ethylene receptor genes PpETR3 and PpERS2 in pear (Pyrus pyrifolia Nakai 'Kikusui') fruit.

Summary Exogenous ethylene and 1-MCP were investigated for their potential effects on fruit quality, post-harvest physiology, and expression of the ethylene receptor genes PpETR3 and PpERS2 in ‘Kikusui’ pear fruit. The results showed that exogenous ethylene treatment promoted a reduction in fruit firmness and soluble solids content (SSC), an increase in the activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) at a later stage of fruit storage, improved the rates of respiration and ethylene release, and up-regulated expression of the PpERS2 gene from 0 – 9 d after harvest, but did not affect expression of PpETR3. However, the effects of 1-MCP on fruit quality and post-harvest physiology were contrary to those of ethylene. 1-MCP up-regulated expression of the PpETR3 gene from 0 – 9 d and down-regulated expression of PpERS2 from 6 – 15 d after harvest. These data indicate that exogenous ethylene or 1-MCP could accelerate or inhibit pear fruit ripening during storage, respectively. At different stages of storage, ethylene and 1-MCP had different effects on the expression of PpETR3 and PpERS2. Moreover, the expression patterns of PpETR3 and PpERS2 during fruit ripening suggest that these two ethylene receptors have important functions during ethylene signal transduction.

The Regulation of para-Nitrophenol Degradation in Pseudomonas putida DLL-E4

Pseudomonas putida DLL-E4 can efficiently degrade para-nitrophenol and its intermediate metabolite hydroquinone. The regulation of para-nitrophenol degradation was studied, and PNP induced a global change in the transcriptome of P. putida DLL-E4. When grown on PNP, the wild-type strain exhibited significant downregulation of 2912 genes and upregulation of 845 genes, whereas 2927 genes were downregulated and 891 genes upregulated in a pnpR-deleted strain. Genes related to two non-coding RNAs (ins1 and ins2), para-nitrophenol metabolism, the tricarboxylic acid cycle, the outer membrane porin OprB, glucose dehydrogenase Gcd, and carbon catabolite repression were significantly upregulated when cells were grown on para-nitrophenol plus glucose. pnpA, pnpR, pnpC1C2DECX1X2, and pnpR1 are key genes in para-nitrophenol degradation, whereas pnpAb and pnpC1bC2bDbEbCbX1bX2b have lost the ability to degrade para-nitrophenol. Multiple components including transcriptional regulators and other unknown factors regulate para-nitrophenol degradation, and the transcriptional regulation of para-nitrophenol degradation is complex. Glucose utilization was enhanced at early stages of para-nitrophenol supplementation. However, it was inhibited after the total consumption of para-nitrophenol. The addition of glucose led to a significant enhancement in para-nitrophenol degradation and up-regulation in the expression of genes involved in para-nitrophenol degradation and carbon catabolite repression (CCR). It seemed that para-nitrophenol degradation can be regulated by CCR, and relief of CCR might contribute to enhanced para-nitrophenol degradation. In brief, the regulation of para-nitrophenol degradation seems to be controlled by multiple factors and requires further study.

Impact of pnpR, a LysR-type regulator-encoding gene, on the cellular processes of Pseudomonas putida DLL-E4.

LysR-type transcriptional regulators (LTTRs) regulate various cellular processes in bacteria. pnpR is an LTTR-encoding gene involved in the regulation of hydroquinone (HQ) degradation, and its effects on the cellular processes of Pseudomonas putida DLL-E4 were investigated at the physiological, biochemical and molecular levels. Reverse transcription polymerase chain reaction revealed that pnpR positively regulated its own expression and that of the pnpC1C2DECX1X2 operon; additionally, pnpR partially regulated the expression of pnpA when P. putida was grown on para-nitrophenol (PNP) or HQ. Strains DLL-E4 and DLL-ΔpnpR exhibited similar cellular morphologies and growth rates. Transcriptome analysis revealed that pnpR regulated the expression of genes in addition to those involved in PNP degradation. A total of 20 genes were upregulated and 19 genes were downregulated by at least 2-fold in strain DLL-ΔpnpR relative to strain DLL-E4. Bioinformatic analysis revealed putative PnpR-binding sites located in the upstream regions of genes involved in PNP degradation, carbon catabolite repression and other cellular processes. The utilization of L-aspartic acid, L-histidine, L-pyroglutamic acid, L-serine, γ-aminobutyric acid, D,L-lactic acid, D-saccharic acid, succinic acid and L-alaninamide was increased at least 1.3-fold in strain DLL-ΔpnpR as shown by BIOLOG assays, indicating that pnpR plays a potential negative regulation role in the utilization of carbon sources.