Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium <Emphasis Type="Italic">Novosphingobium pentaromativorans</Emphasis> US6-1

Novosphingobium pentaromativorans US6-1 is a Gram-negative halophilic marine bacterium able to utilize several polycyclic aromatic hydrocarbons such as phenanthrene, pyrene, and benzo[a]pyrene. In this study, using transmission electron microscopy, we confirmed that N. pentaromativorans US6-1 produces outer membrane vesicles (OMVs). N. pentaromativorans OMVs (hereafter OMV_Novo) are spherical in shape, and the average diameter of OMV_Novo is 25–70 nm. Proteomic analysis revealed that outer membrane proteins and periplasmic proteins of N. pentaromativorans are the major protein components of OMV_Novo. Comparative proteomic analysis with the membrane-associated protein fraction and correlation analysis demonstrated that the outer membrane proteins of OMV_Novo originated from the membrane- associated protein fraction. To the best of our knowledge, this study is the first to characterize OMV purified from halophilic marine bacteria.     

Complete and integrated pyrene degradation pathway in Mycobacterium vanbaalenii PYR-1 based on systems biology

Mycobacterium vanbaalenii PYR-1 was the first bacterium isolated by virtue of its ability to metabolize the high-molecular-weight polycyclic aromatic hydrocarbon (PAH) pyrene. We used metabolic, genomic, and proteomic approaches in this investigation to construct a complete and integrated pyrene degradation pathway for M. vanbaalenii PYR-1. Genome sequence analyses identified genes involved in the pyrene degradation pathway that we have proposed for this bacterium. To identify proteins involved in the degradation, we conducted a proteome analysis of cells exposed to pyrene using one-dimensional gel electrophoresis in combination with liquid chromatography-tandem mass spectrometry. Database searching performed with the M. vanbaalenii PYR-1 genome resulted in identification of 1,028 proteins with a protein false discovery rate of <1%. Based on both genomic and proteomic data, we identified 27 enzymes necessary for constructing a complete pathway for pyrene degradation. Our analyses indicate that this bacterium degrades pyrene to central intermediates through o-phthalate and the beta-ketoadipate pathway. Proteomic analysis also revealed that 18 enzymes in the pathway were upregulated more than twofold, as indicated by peptide counting when the organism was grown with pyrene; three copies of the terminal subunits of ring-hydroxylating oxygenase (NidAB2, MvanDraft_0817/0818, and PhtAaAb), dihydrodiol dehydrogenase (MvanDraft_0815), and ring cleavage dioxygenase (MvanDraft_3242) were detected only in pyrene-grown cells. The results presented here provide a comprehensive picture of pyrene metabolism in M. vanbaalenii PYR-1 and a useful framework for understanding cellular processes involved in PAH degradation.     

Study of Biochemical Pathways and Enzymes Involved in Pyrene Degradation by Mycobacterium sp. Strain KMS

Pyrene degradation is known in bacteria. In this study, Mycobacterium sp. strain KMS was used to study the metabolites produced during, and enzymes involved in, pyrene degradation. Several key metabolites, including pyrene-4,5-dione, cis-4,5-pyrene-dihydrodiol, phenanthrene-4,5-dicarboxylic acid, and 4-phenanthroic acid, were identified during pyrene degradation. Pyrene-4,5-dione, which accumulates as an end product in some gram-negative bacterial cultures, was further utilized and degraded by Mycobacterium sp. strain KMS. Enzymes involved in pyrene degradation by Mycobacterium sp. strain KMS were studied, using 2-D gel electrophoresis. The first protein in the catabolic pathway, aromatic-ring-hydroxylating dioxygenase, which oxidizes pyrene to cis-4,5-pyrene-dihydrodiol, was induced with the addition of pyrene and pyrene-4,5-dione to the cultures. The subcomponents of dioxygenase, including the alpha and beta subunits, 4Fe-4S ferredoxin, and the Rieske (2Fe-2S) region, were all induced. Other proteins responsible for further pyrene degradation, such as dihydrodiol dehydrogenase, oxidoreductase, and epoxide hydrolase, were also found to be significantly induced by the presence of pyrene and pyrene-4,5-dione. Several nonpathway-related proteins, including sterol-binding protein and cytochrome P450, were induced. A pyrene degradation pathway for Mycobacterium sp. strain KMS was proposed and confirmed by proteomic study by identifying almost all the enzymes required during the initial steps of pyrene degradation.     

Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24

Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.     

Multiplicity of genes for aromatic ring-hydroxylating dioxygenases in Mycobacterium isolate KMS and their regulation

Mycobacterium sp. strain KMS has bioremediation potential for polycyclic aromatic hydrocarbons (PAHs), such as pyrene, and smaller ring aromatics, such as benzoate. Degradation of these aromatics involves oxidation catalyzed by aromatic ring-hydroxylating dioxygenases. Multiple genes encoding dioxygenases exist in KMS: ten genes encode large-subunits with homology to phenylpropionate dioxygenase genes, sixteen pairs of adjacent genes encode alpha- and beta-subunits of dioxygenase and two genes encode beta-subunits. These genes include orthologs of nid genes essential for degradation of multi-ring PAHs in M. vanbaalenii isolate PYR-1. The multiplicity of genes in part is explained by block duplication that results in two or three copies of certain genes on the chromosome, a linear plasmid, and a circular plasmid within the KMS genome. Quantitative real-time PCR showed that four dioxygenase beta-subunit nid genes from operons with almost identical promoter sequences otherwise unique in the genome were induced by pyrene to similar extents. No induction occurred with benzoate. Unlike isolate PYR-1, isolate KMS has an operon specifying benzoate catabolism and the expression of the alpha-subunit dioxygenase gene was activated by benzoate but not pyrene. These studies showed that isolate KMS had a genome well adapted to utilization of different aromatic compounds.     

Identification and Functional Analysis of Two Aromatic-Ring-Hydroxylating Dioxygenases from a Sphingomonas Strain That Degrades Various Polycyclic Aromatic Hydrocarbons

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [¹⁴C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1_a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.     

Metabolic engineering of Arabidopsis for remediation of different polycyclic aromatic hydrocarbons using a hybrid bacterial dioxygenase complex

The widespread presence of polycyclic aromatic hydrocarbons (PAHs) and their potential harm to various organisms has generated interest in efficiently eliminating these compounds from the environment. Phytoremediation is an efficient technology for cleaning up pollutants. However, unlike microorganisms, plants lack the catabolic pathway for complete degradation of these dangerous groups of compounds. One way to enhance the potential of plants for remediation of these compounds is by transferring genes involved in xenobiotic degradation from microbes to plants. In this paper, four genes, namely nidA and nidB (encoding the large and small subunits of naphthalene dioxygenase of Mycobacterium vanbaalenii PYR-1) as well as NahAa and NahAb (encoding flavoprotein reductase and ferredoxin of the electron-transport chain of the Pseudomonas putida G7 naphthalene dioxygenase system), were transferred and ectopically expressed in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing the heterozygous naphthalene dioxygenase system exhibited enhanced tolerance toward 2–4 rings PAHs. Transgenic plants assimilated PAHs from the culture media faster and accumulated less in vivo than wild-type plants. Furthermore, examination of metabolic intermediates by gas chromatography–mass spectrometry revealed that the naphthalene metabolic pathway in transgenic plants mainly involves the dioxygenase pathway. Taken together, our findings suggest that grafting the naphthalene dioxygenase complex into plants is a possible strategy to breed PAH-tolerant plants to efficiently degrade PAHs in the environment.     

Characterization of polycyclic aromatic hydrocarbons degradation and arsenate reduction by a versatile Pseudomonas isolate

A Pseudomonas isolate, designated PAHAs-1, was found capable of reducing arsenate and degrading polycyclic aromatic hydrocarbons (PAHs) independently and simultaneously. This isolate completely reduced 1.5 mM arsenate within 48 h and removed approximately 100% and 50% of 60 mg l⁻¹ phenanthrene and 20 mg l⁻¹ pyrene within 60 h, respectively. Using PAHs as the sole carbon source, however, this isolate showed a slow arsenate reduction rate (4.62 μM h⁻¹). The presence of arsenic affected cell growth and concurrent PAHs removal, depending on PAH species and arsenic concentration. Adding sodium lactate to the medium greatly enhanced the arsenate reduction and pyrene metabolism. The presence of the alpha subunit of the aromatic ring-hydroxylating dioxygenase (ARHD) gene, arsenate reductase (arsC) and arsenite transporter (ACR3(2)) genes supported the dual function of the isolate. The finding of latter two genes indicated that PAHAs-1 possibly reduced arsenate via the known detoxification mechanism. Preliminary data from hydroponic experiment showed that PAHAs-1 degraded the majority of phenanthrene (>60%) and enhanced arsenic uptake by Pteris vittata L. (from 246.7 to 1187.4 mg kg⁻¹ As in the fronds). The versatile isolate PAHAs-1 may have potentials in improving the bioremediation of PAHs and arsenic co-contamination using the plant-microbe integrated strategy.     

Pleiotropic and Epistatic Behavior of a Ring-Hydroxylating Oxygenase System in the Polycyclic Aromatic Hydrocarbon Metabolic Network from Mycobacterium vanbaalenii PYR-1

Despite the considerable knowledge of bacterial high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) metabolism, the key enzyme(s) and its pleiotropic and epistatic behavior(s) responsible for low-molecular-weight (LMW) PAHs in HMW PAH-metabolic networks remain poorly understood. In this study, a phenotype-based strategy, coupled with a spray plate method, selected a Mycobacterium vanbaalenii PYR-1 mutant (6G11) that degrades HMW PAHs but not LMW PAHs. Sequence analysis determined that the mutant was defective in pdoA2, encoding an aromatic ring-hydroxylating oxygenase (RHO). A series of metabolic comparisons using high-performance liquid chromatography (HPLC) analysis revealed that the mutant had a lower rate of degradation of fluorene, anthracene, and pyrene. Unlike the wild type, the mutant did not produce a color change in culture media containing fluorene, phenanthrene, and fluoranthene. An Escherichia coli expression experiment confirmed the ability of the Pdo system to oxidize biphenyl, the LMW PAHs naphthalene, phenanthrene, anthracene, and fluorene, and the HMW PAHs pyrene, fluoranthene, and benzo[a]pyrene, with the highest enzymatic activity directed toward three-ring PAHs. Structure analysis and PAH substrate docking simulations of the Pdo substrate-binding pocket rationalized the experimentally observed metabolic versatility on a molecular scale. Using information obtained in this study and from previous work, we constructed an RHO-centric functional map, allowing pleiotropic and epistatic enzymatic explanation of PAH metabolism. Taking the findings together, the Pdo system is an RHO system with the pleiotropic responsibility of LMW PAH-centric hydroxylation, and its epistatic functional contribution is also crucial for the metabolic quality and quantity of the PAH-MN.     

石油污染土壤自然衰减过程多环芳烃降解基因动态特征

新疆石油污染土壤中微生物多环芳烃(polycyclic aromatic hydrocarbons,PAHs)降解功能基因研究甚少,且环境因子和功能基因之间相关性仍不清楚。【目的】揭示新疆石油污染砂质土壤自然衰减过程中多环芳烃降解关键基因结构和变化规律。【方法】以新疆准东油田为研究区,分析同一采油区不同石油污染年限土壤理化因子和多环芳烃含量变化,采用扩增子测序研究石油自然衰减过程中多环芳烃降解酶基因结构变化规律,利用Mental检验探讨其环境驱动因子。【结果】石油污染时间1年和3年的土壤中有多项理化指标与背景土存在显著性差异,而污染5年土壤与背景土之间仅2项指标具有显著性差异,随石油自然衰减逐渐恢复至正常。石油污染1年的土壤中16种多环芳烃除苊烯和?以外,其余14种多环芳烃均高于石油污染3年和5年土壤,多环芳烃总量和含油率污染1年土壤均显著高于污染3年和5年的土壤,多环芳烃会在污染后短时间内迅速被降解。扩增子测序结果显示,萘双加氧酶基因分类操作单元(operational taxonomic units,OTUs)序列随污染年限延长逐渐增多;芳环羟化双加氧酶基因OTUs序列BLAST(...     

Comparison of the substrate specificity of two ring-hydroxylating dioxygenases from Sphingomonas sp. VKM B-2434 to polycyclic aromatic hydrocarbons

The genes of two ring-hydroxylating dioxygenases (RHDs) of Sphingomonas sp. VKM B-2434 were cloned and expressed in Escherichia coli. The relative values of the RHD specificity constants were estimated for six polycyclic aromatic hydrocarbons (PAHs) based on the kinetics of PAH mixture conversion by the recombinant strains. The substrate specificity profiles of the enzymes were found to be very different. Dioxygenase ArhA was the most specific to acenaphthylene and showed a low specificity to fluoranthene. Dioxygenase PhnA was the most specific to anthracene and phenanthrene and showed a considerable specificity to fluoranthene. Knockout derivatives of Sphingomonas sp. VKM B-2434 lacking ArhA, PhnA, and both dioxygenases were constructed. PAH degradation by the single-knockout mutants was in agreement with the substrate specificity of the RHD remaining intact. Double-knockout mutant lacking both enzymes was unable to oxidize PAHs. A mutant form of dioxygenase ArhA with altered substrate specificity was described.     

Simultaneous Biodegradation of Polyaromatic Hydrocarbons by a <Emphasis Type="Italic">Stenotrophomonas</Emphasis> sp: Characterization of <Emphasis Type="Italic">nid</Emphasis> Genes and Effect of Surfactants on Degradation

A polyaromatic hydrocarbon degrading bacterium was isolated from a petroleum contaminated site and designated as Stenotrophomonas sp. strain IITR87. It was found to utilize pyrene, phenanthrene and benzo(a)pyrene as sole carbon source, but not anthracene, chrysene and fluoranthene. Gas chromatography and mass spectroscopy analysis resulted in identification of pyrene metabolites namely monohydroxypyrene, 4-oxa-pyrene-5-one, dimethoxypyrene and monohydroxyphenanthrene. Southern hybridization using naphthalene dioxygenase gene (nidA) as probe against the DNA of strain IITR87 revealed the presence of nidA gene. PCR analysis suggests dispersed occurrence of nid genes in the genome instead of a cluster as reported in a PAH-degrading Mycobacterium vanbaalenii PYR-1. The nid genes in strain IITR87, dioxygenase large subunit (nidA), naphthalene dioxygenase small subunit (nidB) and aldehyde dehydrogenase gene (nidD) showed more than 97 % identity to the reported nid genes from Mycobacterium vanbaalenii PYR-1. Most significantly, the biodegradation of PAHs was enhanced 25–60 % in the presence of surfactants rhamnolipid and Triton X-100 due to increased solubilization and bioavailability. These results could be useful for the improved biodegradation of high-molecular-weight PAHs in contaminated habitats.     

Cinnamic Acid, an Autoinducer of Its Own Biosynthesis, Is Processed via Hca Enzymes in Photorhabdus luminescens

Photorhabdus luminescens, an entomopathogenic bacterium and nematode symbiont, has homologues of the Hca and Mhp enzymes. In Escherichia coli, these enzymes catalyze the degradation of the aromatic compounds 3-phenylpropionate (3PP) and cinnamic acid (CA) and allow the use of 3PP as sole carbon source. P. luminescens is not able to use 3PP and CA as sole carbon sources but can degrade them. Hca dioxygenase is involved in this degradation pathway. P. luminescens synthesizes CA from phenylalanine via a phenylalanine ammonia-lyase (PAL) and degrades it via the not-yet-characterized biosynthetic pathway of 3,5-dihydroxy-4-isopropylstilbene (ST) antibiotic. CA induces its own synthesis by enhancing the expression of the stlA gene that codes for PAL. P. luminescens bacteria release endogenous CA into the medium at the end of exponential growth and then consume it. Hca dioxygenase is involved in the consumption of endogenous CA but is not required for ST production. This suggests that CA is consumed via at least two separate pathways in P. luminescens: the biosynthesis of ST and a pathway involving the Hca and Mhp enzymes.     

Functional characterization of a gene cluster involved in gentisate catabolism in Rhodococcus sp. strain NCIMB 12038

Rhodococcus sp. strain NCIMB 12038 utilizes naphthalene as a sole source of carbon and energy, and degrades naphthalene via salicylate and gentisate. To identify the genes involved in this pathway, we cloned and sequenced a 12-kb DNA fragment containing a gentisate catabolic gene cluster. Among the 13 complete open reading frames deduced from this fragment, three (narIKL) have been shown to encode the enzymes involved in the reactions of gentisate catabolism. NarI is gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate, NarL is a mycothiol-dependent maleylpyruvate isomerase which catalyzes the isomerization of maleylpyruvate to fumarylpyruvate, and NarK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The narX gene, which is divergently transcribed with narIKL, has been shown to encode a functional 3-hydroxybenzoate 6-monooxygenase. This led us to discover that this strain is also capable of utilizing 3-hydroxybenzoate as its sole source of carbon and energy. Both NarL and NarX were purified to homogeneity as His-tagged proteins, and they were determined by gel filtration to exist as a trimer and a monomer, respectively. Our study suggested that the gentisate degradation pathway was shared by both naphthalene and 3-hydroxybenzoate catabolism in this strain.     

嗜麦芽寡养单胞菌PX1的降解芘特性及定殖效能

为丰富多环芳烃降解菌菌种库、降低农作物的污染风险,本研究对一株可高效降解多环芳烃(PAHs)的植物内生菌进行筛选鉴定,并初步探究其降解途径以及定殖效能。结果表明: 菌株PX1为嗜麦芽寡养单胞菌。该菌株对多环芳烃的降解具有广谱性,7 d几乎可彻底降解PAH无机盐培养基中的萘,在分别含有50.0 mg·L^-1菲、20.0 mg·L^-1芘、20.0 mg·L^-1荧蒽和10.0 mg·L^-1苯并[a]芘的培养体系中,对菲、芘、荧蒽、苯并[a]芘的降解率分别为72.6%、50.7%、31.9%和12.9%。选取芘作为PAHs模型研究菌株PX1的降解特性。酶活性试验表明,芘可诱导菌株PX1体内邻苯二甲酸双加氧酶、邻苯二酚-1,2-双加氧酶和邻苯二酚-2,3-双加氧酶的活性。在芘降解过程中检测到4,5-环氧化芘、4,5-二羟基芘、龙胆酸/原茶儿酸、水杨酸、顺-己二烯二酸/2-羟粘糠酸半醛、顺-2′-羧基苯丙酮酸、1-羟基-2-萘甲酸、水杨醛等中间产物。浸种定殖试验表明,菌株PX1可高效定殖到空心菜和小麦体内,显著促进空心菜和小麦生长,并能够将空心菜、小麦体内及其生长基质中的芘浓度分别降低29.8%～50.7%、52.4%～67.1%和8.0%～15.3%。表明菌株PX1主要通过“水杨酸途径”和“邻苯二甲酸途径”降解芘,且可以定殖到植物体内,促进植物生长。     

Biodegradation of phenanthrene and pyrene by Ganoderma lucidum

Biodegradation of two polycyclic aromatic hydrocarbons (PAHs), phenanthrene and pyrene, by a white rot fungus, Ganoderma lucidum, in broth cultures was investigated. It was found that the biomass of the organism decreased with the increase of PAH concentration in the cultures. In the cultures with 2 to 50 mg l⁻¹ PAHs, the degradation rate constants (k₁) increased with the PAH concentration, whereas, at the level of 100 mg l⁻¹, the degradation rate constants decreased. In the presence of 20 mg l⁻¹ PAHs, the highest degradation rates of both PAHs occurred in cultures with an initial pH of 4.0 at 30 °C. The addition of CuSO₄, citric acid, gallic acid, tartaric acid, veratryl alcohol, guaiacol, 2,2′-azino-bis-(3- ethylbenzothazoline-6-sulfonate) (ABTS) enhanced the degradation of both PAHs and laccase activities; whereas the supplement of oxalate, di-n-butyl phthalate (DBP), and nonylphenol (NP) decreased the degradation of both PAHs and inhibited laccase production. In conclusion, G. lucidum is a promising white rot fungus to degrade PAHs such as phenanthrene and pyrene in the environment.     

Isolation of pyrene degrading <Emphasis Type="Italic">Achromobacter xylooxidans</Emphasis> and characterization of metabolic product

Polycyclic aromatic hydrocarbons (PAHs) are common ubiquitous pollutants existing in nature with high recalcitrance and toxicity. In this study a bacterium capable of aerobic degradation of high molecular weight PAHs (with special reference to pyrene) was isolated by selective enrichment culture technique from oil refinery effluent sludge. The isolate was characterized as Achromobacter xylooxidans by 16S rRNA gene sequence analysis technique. For the first time it is hereby reported a bacterium capable of effectively degrading pyrene (up to 80%), as evident by reverse phase high performance liquid chromatographic analysis (RP-HPLC). After incubation of Achromobacter xylooxidans in minimal salt medium (MSM) containing pyrene, at concentration of 200 mg/L, as sole source of carbon and energy, there was decrease in pyrene concentration concomitant with increase in bacterial cell protein concentration. RP-HPLC analysis revealed that pyrene was degraded into three metabolites viz. I, II and III. The RP-HPLC eluent fraction were collected from 2.5 to 32.0 min by repeated injection of degraded sample, concentrated and analyzed on gas chromatography mass spectroscopy (GC-MS) for metabolite identification. The fraction shows 1-hydroxypyrene, 1-hydroxy-6-methoxypyrene and 1,6dimethoxypyrene as metabolic product of pyrene degradation, on the basis of their m/z values. On contrary to the reported PAH degradation with reference to pyrene by different isolates till date; the efficient degradation, as evident by RP-HPLC, by this isolate holds a promising potential for planning of bioremediation strategies of contaminated sites.     

Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation were investigated in the pyrene-degrading Mycobacterium sp. strain 6PY1. [(14)C]pyrene mineralization experiments showed that bacteria grown with either pyrene or phenanthrene produced high levels of pyrene-catabolic activity but that acetate-grown cells had no activity. As a means of identifying specific catabolic enzymes, protein extracts from bacteria grown on pyrene or on other carbon sources were analyzed by two-dimensional gel electrophoresis. Pyrene-induced proteins were tentatively identified by peptide sequence analysis. Half of them resembled enzymes known to be involved in phenanthrene degradation, with closest similarity to the corresponding enzymes from Nocardioides sp. strain KP7. The genes encoding the terminal components of two distinct ring-hydroxylating dioxygenases were cloned. Sequence analysis revealed that the two enzymes, designated Pdo1 and Pdo2, belong to a subfamily of dioxygenases found exclusively in gram-positive bacteria. When overproduced in Escherichia coli, Pdo1 and Pdo2 showed distinctive selectivities towards PAH substrates, with the former enzyme catalyzing the dihydroxylation of both pyrene and phenanthrene and the latter preferentially oxidizing phenanthrene. The catalytic activity of the Pdo2 enzyme was dramatically enhanced when electron carrier proteins of the phenanthrene dioxygenase from strain KP7 were coexpressed in recombinant cells. The Pdo2 enzyme was purified as a brown protein consisting of two types of subunits with M(r)s of about 52,000 and 20,000. Immunoblot analysis of cell extracts from strain 6PY1 revealed that Pdo1 was present in cells grown on benzoate, phenanthrene, or pyrene and absent in acetate-grown cells. In contrast, Pdo2 could be detected only in PAH-grown cells. These results indicated that the two enzymes were differentially regulated depending on the carbon source used for growth.     

一株芘降解菌的分离鉴定及其降解效果

以芘为唯一碳源,采用平板升华法,从徐州市卧牛山焦化厂周围污染土壤中分离得到一株芘降解菌SE12.经形态观察、生理生化试验和16S rDNA鉴定,该菌株属于分枝杆菌属(Mycobacterium sp.)菌株,与快速生长型非致病性南非分枝杆菌(M.austroa fricanum ATCC33464)的同源性达到98%.SE12降解芘的最适pH和温度为pH9和30℃.当土壤芘初始含量为100和200mg.kg-1,SE12接种量为107CFU.g-1时,30℃培养28d后土壤芘降解率分别达到97%和99%.利用双加氧酶基因的同源序列引物nidAF/nidAR和nidBF/nidBR进行扩增,得出了该菌株编码双加氧酶大亚基和小亚基的基因片段,它们与已知降解芘的分枝杆菌的双加氧酶基因具有高度同源性.     

几株新鞘氨醇杆菌的趋化性及其相关基因组成特点

【目的】选择了几株隶属于新鞘氨醇杆菌属(Novosphingobium)的有和没有多环芳烃(PAHs)降解能力的细菌,在基因组水平比较分析它们的趋化通路,并探究其中一些菌株对芳香化合物和三羧酸(TCA)循环中间代谢产物的趋化性。【方法】通过基因组的比较分析,研究了几株新鞘氨醇杆菌的趋化蛋白组成和趋化基因分布,并采用滴定法和游动平板法检测了相关菌对芳香族化合物和TCA循环中间代谢产物的趋化性。【结果】N.pentaromativorans US6-1、N.pentaromativorans F2、Novosphingobium indicum K13、Novosphingobium stygium DSM 12445、Novosphingobium sp.C2AC等5株新鞘氨醇杆菌对芳香族化合物和TCA循环中间代谢物具有不同程度的趋化性;所选的7株已完成基因组测序的新鞘氨醇杆菌N.pentaromativorans F2、N.pentaromativorans US6-1、Novosphingobium sp.PP1Y、Novosphingobium sp.AP12、Novosphingobium sp.Rr 2-17,Novosphingobium sp.B-7和Novosphingobium sp.DSM 19370均含有趋化蛋白MCP、CheW、CheA、CheB、CheR和CheY,且亲缘关系最近的US6-1、F2和PP1Y 3株菌的che基因簇中的基因排列一致,为che W-Y-D-B-R-A-(X);新鞘氨醇杆菌的趋化系统属Fla类型。【结论】几株新鞘氨醇杆菌都具有较为完整的趋化通路,且对多种芳烃及其代谢物的趋化性各不相同,其中菌株US6-1趋化现象最明显。