Isolation of phloridzin-degrading,IAA-producing bacterium Ochrobactrum haematophilum and its effects on the apple replant soil environment

We isolated and identified a bacterium that could produce IAA and degrade phloridzin in the rhizosphere soil of healthy replanted apple(the rootstock is M9T337 and the scion is Yanfu 3),providing a theoretical basis for reducing the obstacles associated with apple replant disease(ARD).Isolates were screened using Salkowski colorimetry and screening medium for phloridzin.The isolate of interest(W6)was identified as Ochrobactrum haematophilum based on morphological analysis,physiological and biochemical tests,and 16S rDNA sequencing.In a laboratory experiment,W6 produced auxin and promoted the growth of Arabidopsis thaliana roots,and its degradation rate of 100 mg.L^(-1 )phloridzin was 62.0%.In a pot experiment,W6 significantly reduced the phenolic acid contents of replanted soil,lowered the abundance of the harmful fungus Fusarium solani,and increased soil enzyme activities,thereby improving the micro-ecological environment of replant soil.W6 increased the root antioxidant enzyme activity and leaf photosynthetic pigment content of replanted Malus hupehensis Rehd.seedlings,effectively alleviating the decrease in net photosynthetic rate,transpiration rate and stomatal conductance caused by ARD.In a field experiment,W6 also promoted the growth of replanted apple(the rootstock is M9T337 and the scion is Yanfu 3)saplings.Therefore,W6 can promote apple growth and degrade phenolic acids,and it can be used as an effective treatment for the reduction of ARD.

Cloning of multicopper oxidase gene from <i>Ochrobactrum sp</i>. 531 and characterization of its alkaline laccase activity towards phenolic substrates

A 1602 bp fragment was cloned from a soil bacterium Ochrobactrum sp. 531. It contained an open reading frame (ORF) of 1092 bp which was identified as a multicopper oxidase (MCO) with potential laccase activity. After inserting the cloned gene into the expression vector pET23a, it was expressed in E. coli BL21(DE3)pLysS, and its product was purified to homogeneity through chromatography. The Ochrobactrum sp. 531 MCO, consisting of 533 amino acids with a molecular mass of 57.8 kDa, was quite stable in neutral pH and showed laccase-like activity oxidizing 2,6-dimethoxyphenol (DMP), 2,2’-azino-bis(3-ethylbe- nzthiazolinesulfonic acid) (ABTS), and syringaldazine (SGZ). The enzyme showed optimum activity towards DMP, ABTS, and SGZ at the pH 8.0, 3.6, and 7.5 respectively. Kinetic studies gave this enzyme Km, kcat and kcat//Km values of: 0.09 mM, 7.94 s–1, and 88.22 s–1?mM–1 for DMP;0.072 mM, 2.95 s–1, and 40.97 s–1.mM–1 for ABTS;and 0.015 mM, 2.4 s–1, and 160 s–1.mM–1 for SGZ. Our results demonstrate that Ochrobactrum sp. 531 MCO is a bacterial laccase which oxidized phenolic substrates DMP and SGZ effectively under alkaline conditions. These unusual properties make the enzyme an interesting biocatalyst in applications for which classical laccases are unsuitable.

First Isolation and Characterization of Ochrobactrum anthropi from Pig

One Gram-negative Bacillus was isolated from a brain sample of a pig with neurological symptoms.Pathological examination showed meningitis at necropsy. Ochrobactrum anthropi (O. anthropi) was successfully isolated from the brain sample and was confirmed by biochemical reaction results (API 20 NE) and gene sequencing. The strain was highly resistant to b-lactam antibiotics. Mice were experimentally infected with O. anthropi and showed typical meningitis. This is the first report on O. anthropi isolated from a pig, and indicates that O. anthropi may have a broader host spectrum of infection.

Ochrobactrum intermedium培养条件优化及其对含蜡原油作用效果分析

利用微生物对原油蜡组分的降解作用,解决含蜡原油开采与输送过程中的蜡沉积问题。从大庆油田受原油污染土壤中筛选出一株Ochrobactrum intermedium H6,考察菌H6对含蜡原油的作用效果。实验结果表明,菌H6最优培养条件为温度34℃、培养基初始pH为6。菌H6可产生脂肽类生物表面活性剂,在与大庆原油作用10 d后,原油蜡含量降低5.74%,当温度为菌株最优培养温度34℃时,降黏率达到49.37%。菌H6可对原油蜡组分进行降解,具有在油田清防蜡过程中发挥作用的潜力。

Isolation of marine benzo[a]pyrene-degrading Ochrobactrum sp. BAP5 and proteins characterization

A bacterial strain BAP5 with a relatively high degradation ability of benzo[a]pyrene (BaP) was isolated from marine sediments of Xiamen Western Sea, China and identified as Ochrobactrum sp. according to 16S rRNA gene sequence as well as Biolog microbial identification system. Strain BAP5 could grow in mineral salt medium with 50 mg/L of BaP and degrade about 20% BaP after 30 d of incubation. Ochrobactrum sp. BAP5 was able to utilize other polycyclic aromatic hydrocarbons (PAHs) (such as phenanthrene, pyrene and fluoranthene) as the sole carbon source and energy source, suggesting its potential application in PAHs bioremediation. The profile of total soluble protein from Ochrobactrum sp. BAP5 was also investigated. Some over- and special-expressed proteins of strain BAP5 when incubated with the presence of BaP were detected by two-dimensional polyacrylamide gel electrophoresis, and found to be related with PAHs metabolism, DNA translation, and energy production based on peptide fingerprint analysis through matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Characterization and optimization of EPSproducing and diesel oil-degrading Ochrobactrum anthropi MP3 isolated from refinery wastewater

Petroleum refinery wastewater(PRW) containing hydrocarbon is highly toxic to the environment and the surrounding ecosystem. Proper treatment of the PRW effluent is necessary to remove the pollutants before discharge. Bioremediation is considered to be a promising approach as it is ecofriendly and efficient. The exopolysaccharide(EPS) produced by the O. anthropi acts as a bioemulsifier and showed the highest emulsification activity of 60% on diesel. An EPS yield of about 0.42 g/L was obtained under optimized conditions. The carbohydrate and protein content of the EPS was found to be 71.1% and 19.7% respectively, showing the glycoprotein nature. The structural properties of EPS were analyzed by FT-IR and 1H NMR. The batch degradation of oil in PRW by O. anthropi was studied gravimetrically, and showed about 53% degradation in 7 days, indicating the strong ability of the isolated strain to degrade the hydrocarbons in PRW.

苍白杆菌(Ochrobactrum sp.)FP1对芳香烃化合物降解特性及相关基因研究

文章对分离的一株高效石油降解菌生理特性及芳香烃化合物降解特性、降解相关基因进行了研究。用细菌生理生化和16S rDNA序列分析初步鉴定FP1为苍白杆菌(Ochrobactrum sp.)。将菌株在含不同种类混合芳烃基础培养基中培养7 d后,用GC-MS分析发现菌株对芳烃类化合物有较好利用和降解能力,对苯、甲苯、乙苯、二甲苯、萘和联苯的降解率分别为100.00%、60.11%、66.00%、57.72%、67.94%、77.72%。芳香烃化合物降解相关基因有31个,而参与萘、甲苯、乙苯、二甲苯降解的基因占芳香烃化合物降解基因总数的38.72%。研究结果为芳烃污染土壤的微生物修复提供了基础。

Identification and characterization of the chromium(Ⅵ) responding protein from a newly isolated Ochrobactrum anthropi CTS-325

A Gram-negative, chromium(Ⅵ) tolerant and reductive strain CTS-325, isolated from a Chinese chromate plant, was identified as Ochrobactrum anthropi based on its biochemical properties and 16S rDNA sequence analysis. It was able to tolerate up to 10 mmol/L Cr(Ⅵ) and completely reduce 1 mmol/L Cr(Ⅵ) to Cr(Ⅲ) within 48 h. When the strain CTS-325 was induced with Cr(Ⅵ), a protein increased significantly in the whole cell proteins. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed that this protein was a superoxide dismutase (SOD) homology. The measured superoxide dismutase activity was 2694 U/mg after three steps of purification. The SOD catalyzes the dismutation of the superoxide anion (O2.-) into hydrogen peroxide and molecular oxygen. This protein is considered to be one of the most important anti-oxidative enzymes for O. anthropi as it allows the bacterium to survive high oxygen stress environments, such as the environment produced during the reduction process of Cr(Ⅵ).

COLONIZATION OF VIGNA RADIATA ROOTS BY CHROMIUM RESISTANT BACTERIAL STRAINS OF OCHROBACTRUM INTERMEDIUM,BACILLUS CEREUS AND BREVIBACTERIUM SP.

The present study deals with colonization potential of plant growth promoting bacterial strains (Ochrobactrum intermedium, Bacillus cereus and Brevibacterium sp.) on Vigna radiata roots. The roots were heavily colonized with O. intermedium and B. cereus as compared to Brevibacterium sp. O. intermedium mainly colonized rhizoplane while B. cereus occurred both on the rhizoplane and near root zone. O. intermedium and B. cereus were found to be present both on the rhizoplane and near root zone, while Brevibacterium only in the rhizosphere in the form of groups. The cells of B. cereus were found more in the sites where root exudates were existed. From the above results it was observed that the number of O. intermedium cells were large at root exudate site. Fig 2, Tab 1, Ref

Effect of Different Energy Sources on Ochrobactrum anthropi Bacteria Chromium Reduction

Ochrobactrum anthropi CTS-325 isolated from a chromium-contaminated site had better resistance to Cr(Ⅵ) in LB medium under aerobic condition.Meanwhile,it was found that the reduction of Cr(Ⅵ) is not complete during the experimental process.Therefore,a series of small molecule energy sources including nitrogen and carbon sources were added into the LB medium in the bacterial stationary phase to promote the chromium reducibility.The result showed that the bacterial growth was positively correlated with the chromium reduction.SDS-PAGE analysis indicated that the protein groups were changed when the bacteria were stimulated by the chromium.Additionally,it was revealed that O.anthropi CTS-325 could utilize the cheaper alternative of sugar(sucrose residue leaching solution) well for further growth and restart the chromium reduction,which offered a new method for practical appli-cations.

响应面法优化羽毛降解菌Ochrobactrum intermedium JY-25的降解培养基

【目的】作为构成羽毛主要成分的角蛋白,因其存在大量相互交联的二硫键、氢键和盐键等化学键,导致羽毛结构稳定难以被常规手段降解。相较于物理、化学方法降解废弃羽毛,利用微生物产生的角蛋白酶使废弃羽毛变成可再生资源,具有高效、环保的优点。课题组前期从家禽养殖场的土壤中分离、筛选到一株羽毛降解菌,经鉴定该菌株为中间苍白杆菌JY-25。研究旨在进一步提高中间苍白杆菌JY-25的羽毛降解率,为今后工业化降解废弃羽毛提供依据。【方法】依次采用单因素试验、Plackett-Burman试验设计和响应面法对中间苍白杆菌JY-25的羽毛降解培养基进行优化。【结果】通过单因素试验确定了在降解培养基中外加碳源(糊化淀粉)和氮源(玉米粉)时,都能提高羽毛的降解率。Plackett-Burman试验设计发现降解培养基的8种成分中,糊化淀粉、硫酸亚铁和羽毛量是影响中间苍白杆菌JY-25对羽毛降解率的显著因素。最后通过响应面法确定了最佳降解培养基的组分为:羽毛2 g/L、糊化淀粉15 g/L、玉米粉10 g/L、硫酸亚铁0.02 g/L、磷酸氢二钾1.5 g/L、氯化钠0.3 g/L、硫酸镁0.025 g/L、氯化钙0.025 g/L,在该优化的培养基配方下,菌株JY-25的羽毛降解率高达76.31%,相较于初始的降解率提高了24.4%。【结论】经过多种优化方式,显著地提高了中间苍白杆菌JY-25的羽毛降解率,为菌株用于工业化降解废弃羽毛提供了理论基础。

高效降酚菌株Ochrobactrum sp. CH10生长动力学和苯酚降解特性的研究

菌株Ochrobactrum sp. CH10是从北京元大都城垣遗址处的人工湿地筛选到的高效苯酚降解菌.以苯酚为唯一碳源和能源对其进行了生长和苯酚降解特性的研究.该菌生长和降解苯酚的适宜条件为30℃、初始pH 7.0、接种量为5%.在该条件下,初始苯酚浓度为400 mg·L-1,24 h时苯酚完全被降解;初始苯酚浓度为900 mg·L-1时,44 h的降解率为92.3%;初始苯酚浓度为1 000 mg·L-1,48 h时的降解率为82.2%.对该菌株苯酚降解动力学过程进行模拟,符合基质抑制型的Haldane模型,各参数分别为:υmax(最大比降解速率)0.126 h-1,KS(半饱和常数)23.53 mg·L-1,KI(抑制常数)806.1 mg·L-1.该菌在苯酚中的生长动力学符合Andrews模型,表现出与苯酚降解相似的趋势.该菌为目前所发现的Ochrobactrum菌属中苯酚降解能力最强的菌株.该菌株在高效处理含酚废水方面具有广阔的应用前景.

麦草畏降解菌株Ochrobactrum sp.3-3的分离鉴定及其降解特性

麦草畏是理想的抗除草剂转基因工程的靶标除草剂;发掘新的麦草畏高效降解菌株和基因具有非常重要的理论和应用价值.从南京土壤样品中分离到一株麦草畏高效降解菌株,命名为3-3.根据生理生化特征和16S r DNA序列相似性分析,将其初步鉴定为苍白杆菌属(Ochrobactrum sp.).菌株3-3在48 h内完全降解100 mg/L的麦草畏.该菌株降解麦草畏的最适温度为30℃,最适p H为7.0.代谢产物高效液相和质谱鉴定结果表明该菌株降解麦草畏的起始步骤是脱甲基,形成没有除草活性的3,6-二氯水杨酸(DCSA).菌株粗酶液只在NADH存在时才有麦草畏脱甲基酶活性.PCR扩增和该菌基因组生物信息学分析表明该菌株没有已报道的麦草畏脱甲基酶基因DMO、Mtv及Dmt或其同源序列.总之,本研究首次分离筛选到苍白杆菌属的麦草畏降解菌,且该菌可能存在一个新的氧化酶类麦草畏脱甲基酶基因.

喹啉降解菌Ochrobactrum sp.的好氧降解特性及其在焦化废水中的生物强化作用

喹啉是焦化废水中的难降解有机物之一,以喹啉为唯一碳氮源从某焦化废水处理厂活性污泥中分离出1株喹啉降解菌KDQ3,16S r DNA序列分析表明KDQ3为Ochrobactrum sp.,其对喹啉降解的最适条件为37℃和初始pH 7.0~8.0,喹啉降解动力学符合Haldane模型.KDQ3能在10.4 mg·L^(-1)Cr(Ⅵ)存在时降解200 mg·L^(-1)喹啉.此外,KDQ3能在实际好氧池焦化废水环境中降解喹啉和提高COD去除率,说明该菌具有生物强化焦化废水的应用潜力.

一株分离自地层水的石油降解菌的特性研究:Ochrobactrum intermedium环境分离菌株与临床分离菌株的比较基因组分析（英文）

目的:对一株地层水分离的石油降解菌Ochrobactrum intermedium 2745-2进行生理生化特性的研究、全基因组测序以及比较基因组研究。创新点:首次对一株分离自地层水的石油降解菌O.intermedium 2745-2进行了生理生化特性研究以及基因组测序,从基因组角度解释菌株2745-2对石油的降解能力。通过菌株2745-2与同种其他临床分离菌株的比较基因组学分析,表明2745-2仍具有多种与致病性相关的基因。方法:通过微生物富集培养的方法从油井的地层水中分离石油降解微生物,通过聚合酶链反应(PCR)扩增16S核糖体RNA(r RNA)序列进行比较和分析确定菌株的分类地位属于O.intermedium(图1)。采用Illumina Hi Seq2000对菌株2745-2进行高通量测序,采用Velvet 1.2.07和RAST server分别进行数据组装和注释(表1)。PHAST寻找基因组中的噬菌体相关序列(图4和表2)。通过BLAST+和BRIG对环境分离菌株(2745-2)和临床分离菌株(M86、229E和LMG3301T)进行基因组比较(表3、表4和图5)。结论:首次对一株环境分离的O.intermedium(2745-2)进行全基因组测序,揭示具有多种与石油降解相关的基因。通过环境分离菌株(2745-2)与临床分离菌株(M86,229E和LMG3301T)的基因组比较分析,表明2745-2仍具有多种致病性相关的基因。

三烯霉素J——来自深海细菌Ochrobactrum sp.的新安莎霉素

目的探究深海来源细菌Ochrobactrum sp.OUCMDZ-2164中的细胞毒活性代谢产物。方法利用硅胶柱色谱、凝胶柱色谱和高效液相色谱等手段对发酵产物进行分离纯化,运用紫外、红外、质谱、核磁共振及圆二色谱等方法鉴定化合物的结构,采用MTT和细胞计数试剂盒-8(CCK-8)法分别评价化合物对乳腺癌MCF-7细胞和肺癌A549细胞及慢性髓性白血病K562细胞的增殖抑制活性。结果从细菌Ochrobactrum sp.OUCMDZ-2164的发酵产物中分离获得了4个化合物,分别鉴定为3-O-去甲基三烯霉醇(1)、flazin(2)、flazin-3-carboxylicacid(3)和胸腺嘧啶(4)。化合物1在10μmol/L浓度下对MCF-7细胞的增殖抑制率为61.5%。结论化合物1为新的安沙霉素类化合物,命名为三烯霉素J,其具有一定的乳腺癌细胞毒活性。

煤泥不同预处理方式对其微生物降解过程的影响研究

利用超声波、硝酸及超声波→硝酸3种方式分别对煤泥进行预处理,得到超声波预处理煤泥(CMN)、硝酸预处理煤泥(XMN)和超声波→硝酸预处理煤泥(CXMN),再采用苍白杆菌属对预处理前后的煤泥进行微生物降解,研究煤泥预处理方式对煤泥微生物降解过程的影响。实验结果表明:经过预处理的煤泥微生物降解效果均比未经预处理的煤泥(MN)微生物降解效果好,其中CXMN的降解率最高,为30.03%,超声波→硝酸预处理方式中超声波所用功率为200 W,时间为2 min,硝酸的浓度为8 mol/L;通过傅立叶红外光谱仪(FTIR)对原煤泥及不同预处理方式后的煤泥进行表征,发现经过预处理的煤泥芳香度、芳香环缩合度均降低,含氧官能团含量均增加,说明超声波和硝酸可以破坏煤泥中的芳香环结构、降低芳香环缩合度、增加煤泥表面含氧官能团含量;扫描电镜分析结果发现,CMN出现裂缝,XMN出现层状结构,CXMN呈粉末状,不同方式预处理后煤泥结构的改变有利于煤泥与微生物接触,从而提高煤泥的降解率;氮吸附分析结果发现,CMN与MN相比,孔径减小、孔体积增大、孔隙率增加,XMN孔体积和孔径较MN孔体积和孔径变化不大,CXMN孔体积和孔径较CMN孔体积和孔径均增大;采用气相色谱-质谱联用仪对不同预处理方式煤泥降解的液相产物进行表征,发现苍白杆菌属降解不同方式预处理后煤泥液相产物中的化合物组成相似,都包含烷烃类、酯类、醛酮类、羧酸类等物质。

氧化胁迫诱导苍白杆菌JP1形成VBNC状态及其特征

石油降解菌在各种有害环境因素作用下会进入活的非可培养(viable but non-culturable,VBNC)状态,从而影响其生长及石油降解率。为了研究有害环境因素对石油降解菌生长及石油降解率的影响,采用分光光度法、荧光染色-激光共聚焦显微镜观测H 2O 2胁迫下苍白杆菌(Ochrobactrum sp.)JP1细胞的生长及VBNC状态形成情况。结果表明,不同浓度H 2O 2对其生长有一定抑制作用,当培养液中H 2O 2浓度为75.0 mmol/L时,可有效抑制苍白杆菌JP1生长,处理12 h后苍白杆菌JP1进入VBNC状态。VBNC状态的苍白杆菌JP1细胞缩小变成球体,周质间隙增大;在适宜条件下,VBNC状态苍白杆菌JP1能够复苏为可培养状态,添加丙酮酸钠能够促进VBNC状态细菌细胞的复苏。复苏后的苍白杆菌RJP1具有良好的环境适应性和石油降解能力,为石油污染生物修复的菌种筛选及应用提供了新的策略。

中间苍白杆菌尼古丁降解相关基因ocnH的克隆及功能分析

通过PCR扩增获得中间苍白杆菌SCUEC4菌株中与尼古丁降解相关的ocnH基因,构建重组质粒pET28a(+)-ocnH,转化大肠杆菌BL21(DE3)菌株进行异源表达,并对表达条件进行优化,用高效液相色谱法测定重组表达菌株的马来酸异构酶活性.结果表明:ocnH基因大小为585 bp,编码蛋白分子量约为20.6 kDa.重组表达菌株在IPTG浓度为0.6 mmol·L^(-1)、诱导温度为25℃、诱导表达时间为20 h时,OcnH蛋白的表达量较高.转入pET28a(+)-ocnH的大肠杆菌BL21(DE3)菌株具有将马来酸转化为富马酸的功能.