iTRAQ-based proteomic profiling of a Microbacterium sp. strain during benzo(a)pyrene removal under anaerobic conditions

This study focused on the protein expression of a Microbacterium sp. strain that utilized various concentrations of benzo(a)pyrene (BaP) as the sole source of carbon and energy under anaerobic conditions. A total of 1539 protein species were quantified by isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS. GO, COG, and pathway enrichment analysis showed that most proteins demonstrated catalytic and binding functions and were mainly involved in metabolic processes, cellular processes, and single-organism processes. Sixty-two proteins were found in their abundances in BaP-stress conditions different from normal conditions. These proteins function in the metabolic pathways; the biosynthesis of secondary metabolites, the biosynthesis of antibiotics, microbial metabolism in diverse environments, carbon metabolism, and the biosynthesis of amino acids were markedly altered. Furthermore, enoyl-CoA hydratase was proposed to be a key protein during BaP removal of the Microbacterium sp. strain. This study provides a powerful platform for the further exploration of BaP removal, and the differentially expressed proteins provide insight into the mechanism of the BaP removal pathway.

     

Phosphate removal under denitrifying conditions by Brachymonas sp. strain P12 and Paracoccus denitrificans PP15

In this study, we used the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12 to investigate the enhanced biologic phosphorus-removal (EBPR) mechanism involved with polyhydroxybutyrate (PHB), glycogen, and phosphorus uptake in the presence of acetate under anoxic or aerobic conditions. The results showed that excess acetate concentration and aerobic cultivation can enhance PHB formation efficiency and that PHB formation might be stimulated by glycogenolysis of the cellular glycogen. The efficiency of the uptake of anoxic phosphorus was greater when PHB production was lower. The EBPR mechanism of Brachymonas sp. strain P12 for PHB, phosphorus, and glycogen was similar to the conventional anaerobic-aerobic (or anaerobic-anoxic) EBPR models, but these models were developed under anoxic or aerobic conditions only, without an anaerobic stage. The anoxic or aerobic log phase of growth is divided into two main phases: the early log phase, in which acetate and glycogen are consumed to supply enough energy and reducing power for PHB formation and cell growth (phosphorus assimilation), and the late log phase, which ends the simultaneous degradation of PHB and remaining acetate for polyphosphate accumulation. Glycogenolysis plays a significant role in the alternate responses between PHB formation and phosphorus uptake under anoxic or aerobic conditions. After the application of the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12, aerobic cultivation increases the level of PHB production, and anoxic cultivation further increases phosphorus uptake.     

Biodegradation of benzo(a)pyrene by a newly isolated Fusarium sp

Benzo(a)pyrene (BaP) is a five-ring polycyclic aromatic hydrocarbon produced by the incomplete combustion of organic materials. It is one of the priority pollutants listed by the US Environmental Protection Agency. This study describes a fungal isolate that is able to biodegrade benzo(a)pyrene. The filamentous fungus, isolated from leaves of Pterocarpus macrocarpus Kurz., was identified as a Fusarium sp. (strain E033). Fusarium sp. E033 was able to survive in the presence of benzo(a)pyrene concentrations up to 1.2 mM (300 mg L(-1)). Biodegradation experiments using 0.4 mM (100 mg L(-1)) benzo(a)pyrene demonstrated that Fusarium sp. E033 was able to degrade 65-70% of the initial benzo(a)pyrene provided, and two transformation products, a dihydroxy dihydro-benzo(a)pyrene and a benzo(a)pyrene-quinone, were detected within 30 days of incubation at 32 degrees C. The factors affecting biodegradation efficiency were also investigated. While increasing aeration promoted better fungal growth and benzo(a)pyrene biodegradation, increasing the glucose concentration from 5 to 50 mM had an adverse effect on biodegradation. Ethanol and methanol, provided at 5 mM to increase benzo(a)pyrene water solubility, increased the fungal biomass yield but did not promote degradation. The Fusarium sp. E033 isolated in this study can tolerate and degrade relatively high concentrations of benzo(a)pyrene, suggesting its potential application in benzo(a)pyrene bioremediation.     

模拟淀粉条件下乳杆菌吸附苯并芘的能力

【目的】研究了模拟淀粉条件下嗜酸乳杆菌(Lactobacillus acidophilus)NCFM、植物乳杆菌(Lactobacillus plantarum)121以及戊糖乳酸菌(Lactobacillus pentosus)ML32吸附苯并芘的能力,为利用乳杆菌去除苯并芘提供一定的理论指导。【方法】基于苯并芘的HPLC检测方法,考察了淀粉含量及类型、培养时间和p H等因素对乳杆菌吸附苯并芘能力的影响,研究了淀粉水解产物及菌体活性影响乳杆菌吸附苯并芘的效果。【结果】淀粉含量在2%–10%的范围内,乳杆菌吸附苯并芘的能力与淀粉含量的增加呈正相关性,且与淀粉种类关系不大,但经糊化处理的淀粉可以促进菌体吸附苯并芘。在模拟淀粉体系中,培养前4 h时乳杆菌吸附苯并芘的效率增长快,此后其吸附率增加缓慢。淀粉经酸性(p H为3–4)和碱性(p H为8–9)处理,乳杆菌吸附苯并芘的能力提升。淀粉的水解产物麦芽糖和葡萄糖都能显著改善乳杆菌吸附苯并芘的能力。与活细胞相比,经灭活处理后乳杆菌细胞吸附苯并芘的能力降低。【结论】在淀粉体系中,乳杆菌依然表现出良好的苯并芘吸附能力,且一定范围内淀粉含量增多、糊化作用以及麦芽糖和葡萄糖的存在可促进其吸附苯并芘的能力。因此,本研究中的乳杆菌或许可以用作生物脱除剂来减少淀粉食物中的苯并芘。     

Isolation and characterization of a xanthan-degrading Microbacterium sp. strain XT11 from garden soil

AIMS: Isolation and characterization of the xanthan-degrading Microbacterium sp. XT11. METHODS AND RESULTS: The bacterial isolate XT11, capable of fragmenting xanthan, has been isolated from soil sample. Morphological and biochemical analyses, as well as 16S rRNA gene sequence comparisons, demonstrated that strain XT11 should be grouped in the genus Microbacterium, and represented a new member in this family. Xanthan could be degraded by the xanthan-degrading enzyme released from strain XT11. It has been shown that xantho-oligosaccharides fragmented from xanthan had both elicitor activity and antibacterial effect against Xanthomonas campestris pv. campestris. CONCLUSIONS: The xanthan-degrading enzyme produced by the newly isolated XT11 could fragment xanthan to form oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: Xanthan-degrading products would be useful for potential application in the control of black rot of cruciferous plants caused by X. campestris pv. campestris and, as an oligosaccharide elicitor, in making these plants resistant to disease.     

Purification and characterization of a thermostable uricase from Microbacterium sp. strain ZZJ4-1

In order to study the properties of a thermostable uricase produced by Microbacterium sp. strain ZZJ4-1, the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange, hydrophobic and molecular sieve chromatography. The molecular mass of the purified enzyme was estimated to be 34 kDa by SDS-PAGE. The enzyme was stable between pH 7.0 and 10.00. The optimal reaction temperature of the enzyme was 30 °C at pH 8.5. The K _m and K _cat of the enzyme were 0.31 mM and 3.01 s⁻¹, respectively. Fe³⁺ could enhance the enzyme activity, whereas Ag⁺, Hg²⁺, o-phenanthroline and SDS inhibited the activity of the enzyme considerably. After purification, the enzyme was purified 19.7-fold with 31% yield. As compared with uricases from other microbial sources, the purified enzyme showed excellent thermostability and other unique characteristics. The results of this work showed that strains of Microbacterium could be candidates for the production of a thermostable uricase, which has the potential clinical application in measurement of uric acid.     

Metabolism of both 4-chlorobenzoate and toluene under denitrifying conditions by a constructed bacterial strain.

T1, a dentrifying bacterium originally isolated for its ability to grow on toluene, can also metabolize 4-hydroxybenzoate and other aromatic compounds under denitrifying conditions. A cosmid clone carrying the three genes that code for the 4-chlorobenzoate dehalogenase enzyme complex isolated from the aerobic bacterium Pseudomonas sp. strain CBS3 was successfully conjugated into strain T1. The cloned enzyme complex catalyzes the hydrolytic dechlorination of 4-chlorobenzoate to 4-hydroxybenzoate. Since molecular oxygen is not required for the dehalogenation reaction, the transconjugate strain of T1 (T1-pUK45-10C) was able to grow on 4-chlorobenzoate in the absence of O2 under denitrifying conditions. 4-Chlorobenzoate was dehalogenated to 4-hydroxybenzoate, which was then further metabolized by strain T1. The dehalogenation and metabolism of 4-chlorobenzoate were nitrate dependent and were coupled to the production of nitrite and nitrogen gas. 4-Bromobenzoate was also degraded by this strain, while 4-iodobenzoate was not. Additionally, when T1-pUK45-10C was presented with a mixture of 4-chlorobenzoate and toluene, simultaneous degradation of the compounds was observed. These results illustrate that dechlorination and degradation of aromatic xenobiotics can be mediated by a pure culture in the absence of oxygen. Furthermore, it is possible to expand the range of xenobiotic substrates degradable by an organism, and it is possible that concurrent metabolism of these substrates can occur.     

微杆菌属ZZJ4-1菌株的耐热尿酸氧化酶基因的克隆及重组酶性质

为了研究微杆菌Microbacterium sp.ZZJ4-1菌株的耐热尿酸氧化酶(Uox)的性质,克隆其基因(uox),得到1个894 bp的开放阅读框。该基因与多数已报道的uox无明显同源性,仅与球形节杆菌Arthrobacterglobiformis的uox有72%的同源性。将基因插入质粒pET-15b构成pET-15b-uox表达载体,转化至Escherichiacoli BL21(DE3)中诱导表达。对重组Uox的主要理化性质研究表明:该酶由大小约为35 kDa的亚基组成;其最佳反应温度和pH分别为30℃和7.5;在65℃以下和pH 8.5～11.0范围内稳定;以尿酸为底物的Km值为0.22 mmol/L;Ag+、Zn2+、Cu2+和SDS均能完全抑制酶活,Tween 20、Tween 80和Triton X-100对酶活有一定的促进作用。该重组酶的耐热性是目前报道的重组Uox中最好的,这一特性有利于其在诊断治疗中的开发应用。     

乳杆菌吸附苯并芘的特性

[目的]探讨植物乳杆菌(Lactobacillus plantarum)121和戊糖乳杆菌(Lactobacillus pentosus)ML32的苯并芘吸附作用与机制.[方法]采用高效液相色谱检测菌体对苯并芘的吸附率.[结果]菌株121和ML32对苯并芘的吸附率分别为65.9%和64.9%,这种吸附特性与菌体活力无关,随培养时间延长、温度提高以及细胞浓度的上升而增加.菌株121和ML32的吸附率在pH 4和5时达到最大,分别为87.6%和89.0%.当培养液中Ca2+或Mg2+浓度大于0.05mol/L时,菌体吸附率与盐离子浓度呈正相关.苯洗脱会导致乳杆菌所吸附的苯并芘减少90%.经碱性蛋白酶、中性蛋白酶、溶菌酶及TCA和SDS等方法处理后,菌体吸附率上升,且不易被苯去除.在胆盐及胃酸环境下,两株菌的吸附率均提高至70%以上,而胰蛋白酶的存在仅对菌株121的吸附率有较大影响.[结论]两株乳杆菌可以通过吸附作用从环境中清除苯并芘,其吸附效果与细菌细胞壁的结构和组成有关.     

Modification of norfloxacin by a Microbacterium sp. strain isolated from a wastewater treatment plant

Antimicrobial residues found in municipal wastewater may increase selective pressure on microorganisms for development of resistance, but studies with mixed microbial cultures derived from wastewater have suggested that some bacteria are able to inactivate fluoroquinolones. Medium containing N-phenylpiperazine and inoculated with wastewater was used to enrich fluoroquinolone-modifying bacteria. One bacterial strain isolated from an enrichment culture was identified by 16S rRNA gene sequence analysis as a Microbacterium sp. similar to a plant growth-promoting bacterium, Microbacterium azadirachtae (99.70%), and a nematode pathogen, "M. nematophilum" (99.02%). During growth in medium with norfloxacin, this strain produced four metabolites, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) analyses as 8-hydroxynorfloxacin, 6-defluoro-6-hydroxynorfloxacin, desethylene norfloxacin, and N-acetylnorfloxacin. The production of the first three metabolites was enhanced by ascorbic acid and nitrate, but it was inhibited by phosphate, amino acids, mannitol, formate, and thiourea. In contrast, N-acetylnorfloxacin was most abundant in cultures supplemented with amino acids. This is the first report of defluorination and hydroxylation of a fluoroquinolone by an isolated bacterial strain. The results suggest that some bacteria may degrade fluoroquinolones in wastewater to metabolites with less antibacterial activity that could be subject to further degradation by other microorganisms.     

Reactivity with DNA of three pyrenofuran analogues of benzo(a)pyrene and benzo(e)pyrene

Three pyrenofurans, the pyreno[1,2-b]furan (FP1), the pyreno[2,1-b] furan (FP2) and the pyreno[4,5-b]furan (FP3) have been synthesized as analogues of the mutagenic and carcinogenic benzo(a)pyrene (FP1 and FP2) and of its non-carcinogenic isomer benzo(e)pyrene (FP3). For each of the pyrenofurans, the reactivity with DNA has been tested in presence of liver microsomes of rats induced with 3-methylcholanthrene. Fluorescence spectroscopy showed that only FP2 and FP3 which possess a "bay region" react with DNA. In both cases, metabolites bound to DNA have a fluorescence emission comparable to that of the "bay region" dihydrodiols obtained after the "in vitro" metabolism of initial molecules. FP2 is shown to react similarly to benzo(a)pyrene whereas the reactivity of FP3 is different from that of benzo(e)pyrene, in spite of their structural similarities. This is probably due to reasons of three-dimensional space configuration. The peculiar reactivity of FP3 is predicted by calculations of the bond order values.     

Nitrate removal by a novel autotrophic denitrifier (Microbacterium sp.) using Fe(II) as electron donor

A novel denitrifying bacterium was isolated using bicarbonate as the sole carbon source in a defined medium. Strain W3 was isolated from deep sediments of East Lake (Wuhan, China). In this study, analysis of 16S rRNA genes showed that strain W3 was affiliated with Microbacterium sp. When using Fe2+ as the only electron donor, this strain could convert 88.6 % of NO3 −-N to N2, corresponding to an Fe2+ oxidation rate of 80 %. Meanwhile, neither NO2 −-N nor NH4 +-N was accumulated after the experiment. In similar experiments with Fe(II)-EDTA, cell encrustations did not occur and supplementary substrates were consumed. The accumulated NO2 −-N was below 2.5 mg L−1. In addition, PCR revealed five kinds of key denitrifying genes: narG, napA, nirS, norB and nosZ. These results indicated that strain W3 could be used as an alternative autotrophic denitrifier for the treatment of groundwater and low C/N ratio wastewater.     

Reduction of Cr(VI) by Desulfovibrio vulgaris and Microbacterium sp.

Many industrial wastes contain Cr(VI), a carcinogen and mutagen, the toxicity of which can be ameliorated by reduction to Cr(III). Microbacterium sp. NCIMB 13776 andDesulfovibrio vulgaris NCIMB 8303 reduced Cr(VI) to Cr(III) anoxically using 25 mM sodium citrate buffer (pH 7), with 25 mM sodium acetate and 25 mM sodium formate as electron donors at 30 °C, under which conditions the rates of reduction of 500 M sodium chromate were 77 and 6 nmol h^–1 mg dry cell wt for D. vulgaris and Microbacterium sp., respectively, these being increased to 127 and 17 nmol h^–1 mg dry cell wt in the presence of 20 mM MOPS/NaOH buffer.     

Continuous removal of aromatic hydrocarbons by an AF reactor under denitrifying conditions

Removal of three typical aromatic hydrocarbons, benzene, biphenyl and naphthalene by an anaerobic filter (AF) reactor under continuous mode and denitrifying conditions was studied. Results showed that the AF reactor could degrade these aromatic hydrocarbons effectively under above-mentioned conditions. When influent wastewater contained 900 mg COD/l and about 60 mg (total aromatic hydrocarbons)/l, 90% and 84% removal efficiency could be achieved for them respectively. When COD/NO₃ ⁻-N ratio (C/N) was in the range 5–30, the removal of benzene was slightly influenced by C/N and it remained stable at about 90%. However, degradation of naphthalene, biphenyl and total COD was greatly influenced by C/N, and highest removal was achieved at C/N = 15, it was 90%, 85% and 82% for COD, naphthalene and biphenyl, respectively. Degradation of these three aromatic hydrocarbons followed the order: benzene > naphthalene > biphenyl.     

Biodegradation of pyrene by Candida sp. S1 under high salinity conditions

Polycyclic aromatic hydrocarbon is a toxic recalcitrant environmental pollutant and its removal from the environment is very essential. In this study, a novel S1 strain isolated from the tropical rain forest was identified as Candida species based on 18S rRNA. The pyrene biodegradation was performed by Candida sp. S1. Pyrene was 35% degraded in 15 days. The percentage of pyrene biodegradation increased up to 75% with 24 g L⁻¹ of sodium chloride and decreased along with increasing salinity. Under the acidic condition, the biodegradation was increased up to 60% at pH 5. It was also found that the increasing glucose concentration of more than 10 g L⁻¹ had no significant effect on pyrene biodegradation, while agitation proved to have greater influence. There was a positive relationship between biomass growth and biodegradation rate of pyrene. One pyrene metabolite was identified from the extract solution and analyzed by a thin-layer chromatography, UV–visible absorption and gas chromatography–mass spectrometry. The metabolite found in the pyrene degradation was benzoic acid. Suitable conditions must be found to promote a successful microbial augmentation in liquid culture.

     

Metabolism of benzo(a)pyrene by duck liver microsomes

The metabolism of benzo(a)pyrene [BP], a model carcinogenic PAH, by hepatic microsomes of two duck species, mallard (Anas platyrhynchos) and common merganser (Mergus merganser americanus) collected from chemically-contaminated and relatively non-contaminated areas was investigated. The rate of metabolism of BP by liver microsomes of common merganser and mallard collected from polluted areas (2,650 +/- 310 and 2,200 +/- 310 pmol/min per mg microsomal protein, respectively) was significantly higher than that obtained with liver microsomes of the two species collected from non-polluted areas (334 +/- 33 and 231 +/- 30 pmol/min per mg microsomal protein, respectively). The level of cytochrome P-450 1A1 was significantly higher in the liver microsomes of both duck species from the polluted areas as compared to the ducks from the non-polluted areas. The major BP metabolites, including BP-9, 10-diol, BP-4, 5-diol, BP-7, 8-diol, BP-1, 6-dione, BP-3, 6-dione, BP-6, 12-dione, 9-hydroxy-BP and 3-hydroxy-BP, formed by liver microsomes of both duck species from polluted and non-polluted areas, were qualitatively similar. However, the patterns of these metabolites were considerably different from each other. Liver microsomes of ducks from the polluted areas produced a higher proportion of benzo-ring dihydrodiols than the liver microsomes of ducks from the non-polluted areas, which converted a greater proportion of BP to BP-phenols. The predominant enantiomer of BP-7,8-diol formed by hepatic microsomes of the two duck species had an (-)R,R absolute stereochemistry. The data suggest that duck and rat liver microsomal enzymes have different regioselectivity but similar stereoselectivity in the metabolism of BP.     

The microsomal metabolism of benzo(a) pyrene phenols.

Analysis of repetitive scan difference spectra of incubation mixtures containing rat liver microsomes, 3- or 9-hydroxybenzo(a)pyrene, oxygen, and NADPH shows the formation of products with absorbance in the 400–450 nm region. Based on the chromatographic retention time, absorbance, and fluorescence spectra, the two major products of 9-hydroxybenzo(a)pyrene metabolism may be diphenols. The existence of spectral intermediates which resemble phenols rather than quinones during the steady-state metabolism of 3-hydroxybenzo(a)pyrene strongly indicates that either the major product is a diphenol which slowly oxidizes to yield 3,6-quinone and/or that an active quinone reductase exists in liver microsomes.     

Quantitative proteomic analysis of ibuprofen-degrading Patulibacter sp. strain I11

Ibuprofen is the third most consumed pharmaceutical drug in the world. Several isolates have been shown to degrade ibuprofen, but very little is known about the biochemistry of this process. This study investigates the degradation of ibuprofen by Patulibacter sp. strain I11 by quantitative proteomics using a metabolic labelling strategy. The whole-genome of Patulibacter sp. strain I11 was sequenced to provide a species-specific protein platform for optimal protein identification. The bacterial proteomes of actively ibuprofen-degrading cells and cells grown in the absence of ibuprofen was identified and quantified by gel based shotgun-proteomics. In total 251 unique proteins were quantitated using this approach. Biological process and pathway analysis indicated a number of proteins that were up-regulated in response to active degradation of ibuprofen, some of them are known to be involved in the degradation of aromatic compounds. Data analysis revealed that several of these proteins are likely involved in ibuprofen degradation by Patulibacter sp. strain I11.