Characterization of Rhamnolipid Produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolate Bs20

Rhamnolipid produced by Pseudomonas aeruginosa isolate Bs20 is viscous sticky oily yellowish brown liquid with a fruity odor. It showed solubility at aqueous pH > 4 with optimum solubility at pH 7–7.5 and freely soluble in ethyl acetate. This biosurfactant has a very high surface activity as it could lower the surface tension of water to 30 mN/m at about 13.4 mg/L, and it exhibited excellent stabilities at high temperatures (heating at 100°C for 1 h and autoclaving at 121°C for 10 min), salinities (up to 6% NaCl), and pH values (up to pH 13). The produced biosurfactant can be used in the crude form either as cell-free or cell-containing culture broth of the grown bacteria, since both preparations showed high emulsification indices ranged between 59% and 66% against kerosene, diesel, and motor oil. These characters make the test rhamnolipid a potential candidate for use in bioremediation of hydrocarbon-contaminated sites or in the petroleum industry. High-performance thin-layer chromatography densitometry revealed that the extracted rhamnolipid contained the two most active rhamnolipid homologues dirhamno dilipidic rhamnolipid and monorhamno dilipidic rhamnolipid at 44% and 56%, respectively, as compared to 51% and 29.5%, respectively, in a standard rhamnolipid preparation. The nature and ratio of these two rhamnolipid homologues showed to be strain dependent rather than medium-component dependent.     

假单胞菌BS-03代谢产物的组成、结构与抗菌活性

为拓展生物发酵法制取糖脂类化合物在生物医药等方面的应用,该文自筛选经复合诱变的假单胞菌(Pseudonomas sp.)BS-03,以甘油为碳源,利用薄层层析(TLC)法鉴定其发酵液组分,采用傅里叶红外光谱(FTIR),电喷雾质谱(ESI-MS)和核磁共振(NMR)法分析发酵液提取物成分,并推测其化学结构.结果表明:发酵液组分主要由中性脂、糖脂和脂肽组成.糖脂提取物主要由4种鼠李糖脂构成,分别为RhC_(10)、RhC_(10)C_(10)、Rh_2C_(10)和Rh_2C_(10)C_(10),同时还含有多种自由酸前体及少量脱水自由酸前体.鼠李糖脂的抗菌活性试验结果显示鼠李糖脂的抗菌带较宽,对不同时期的微生物作用效果不同,其中霉菌的抑制效果最强,其次是酵母菌,效果最差的是细菌.     

曲拉通（TX-100）对恶臭假单胞菌降解生物膜中双酚

研究非离子型表面活性剂曲拉通（TX-100）对恶臭假单胞菌降解生物膜中双酚A的影响. 结果表明, 低浓度（小于临界胶束浓度, CMC）TX-100抑制生物膜中双酚A的降解, 而高浓度（大于CMC）TX-100可促进生物膜中双酚A的降解, 并且随着生物膜中TX-100浓度的增加, 双酚A的降解率逐渐增大; 同时发现TX-100存在条件下生物膜主要化学组分的选择性萃取分离可导致恶臭假单胞菌降解双酚A的能力下降.     

北京地区2008～2011年实验动物绿脓杆菌检测结果与分析

目的了解SPF级实验动物中绿脓杆菌（Pseudomonas aeruginosa,PA）感染现状和规律,为防止绿脓杆菌感染提供依据。方法参照国家标准,对SPF级小鼠、大鼠、豚鼠和兔进行检测。结果在2008～2011年中检测的SPF级动物中,PA感染的阳性率为8.9%（267/2988）;不同动物感染率无显著差异;不同时间（不同年份和不同季度）PA阳性检出率无显著差异;其中,免疫缺陷动物的PA阳性检出率为32.5%明显高于免疫功能正常动物的7.6%;PA检出率随着送检单位和动物数量的增加逐年升高;不同动物群体间感染率相当,且保持稳定。结论北京地区SPF级实验动物中PA检出率较高,应加强管理。     

Pseudomonas koreensis JDM-2株ACC脱氨酶基因的克隆和表达

应用PCR从杜仲内生Pseudomonas koreensis JDM-2株基因组中扩增出ACC脱氨酶基因acdS,将其克隆到pMD18-T载体并段进行DNA测序,通过BamHⅠ和HindⅢ酶切从重组质粒pMD18ACDS中切下acdS基因序列,将其连接到pQE30质粒的BamHⅠ和HindⅢ位点,构建表达质粒pQE30ACDS,转化大肠杆菌BL21,利用比色法测定ACC脱氨酶活力.测序结果表明,JDM-2菌株acdS基因大小为1 017 bp,与已报道不同菌种acdS基因的同源性在86％～99％之间.SDS-PAGE结果显示在大肠杆菌BL21中表达了分子量为38 kDa的ACC脱氨酶.酶活性检测结果显示重组菌ACC脱氨酶的酶活力为0.214 U/mg.     

纳米银对荧光假单胞菌生物膜形成的影响

分别以空白和0.4%纳米银乙烯醋酸乙烯酯(EVA)塑料片作为载体培养荧光假单胞菌生物膜,通过菌落计数法测定生物膜的变化曲线,分别采用普通显微镜观察生物膜形成并计算覆盖率(BCR),扫描电镜观察生物膜的微观结构,激光共聚焦显微镜观察生物膜中的细菌状态并计算死亡率.结果表明,空白、纳米银EVA表面生物膜分别在12和24 h达到稳定状态,各时间点的纳米银EVA表面菌数及BCR均显著低于空白EVA(p<0.05);在10000倍的视野下,空白EVA表面的荧光假单胞菌通过胞外产物和菌体形成致密生物膜,纳米银EVA表面多为分散菌体;在24,48 h时,纳米银EVA表面的死亡菌体数显著高于空白EVA(p<0.05);纳米银通过抑制荧光假单胞菌生长和减少胞外产物的分泌而影响生物膜的形成.     

绿脓素对降解原油的微生物菌群的影响

铜绿假单胞菌WJ-1是从蒙古林油田油水样中分离得到的一株能够以原油为唯一碳源, 通过合成某些代谢产物增加其环境竞争力和存活率的烃降解菌. 用氯仿从WJ-1LB培养液中萃取绿脓素, 气相色谱-质谱法(Gas chromatograph-Mass Spectrometry, GC-MS)分析得该物质为1-羟基吩嗪. 通过在原油培养基中加入和未加入0.2 g/L绿脓素, 培养32 d来研究绿脓素对烃降解菌群T2的群落结构和原油降解能力的影响. 变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis, DGGE)分析结果表明: 在32 d的原油降解中, 含有绿脓素的培养液中T2菌群多样性减少. 气相色谱和棒薄层层析分析得: 含有绿脓素的培养液中原油的芳香族烃等非烃类和正构烷烃中长链组分的降解率比未加入绿脓素的要低. 超氧化物歧化酶(SOD)和过氧化氢酶(catalase)活性检测结果表明: 不同菌体的过氧化氢酶和超氧化物歧化酶含量与活性不同, 引起对绿脓素抗性的差异. 可见绿脓素对烃降解菌群的结构和原油降解能力有负面影响.     

海洋产灵菌红素细菌的基因分析与鉴定

报道了从大亚湾表面海水中分离到的１株海洋产灵菌红素细菌的分子鉴定结果．通过对该菌株１６ＳｒＲＮＡ基因的测定与分析,并与Ｇｅｎｂａｎｋ的数据进行比较,认为该菌属于假单胞菌属而不是色杆菌属．对于该菌株种的确定尚需进一步实验研究．     

The secretion of lecithinase of Pseudomonas alcaligenes S2 was via type Ⅱ secretion pathway

Strain S2 is a lecithin （or phosphatidylcholine）- solubilizing bacterium, which was isolated from the rice rhizosphere in rural areas of Beijing, China. On the basis of a polyphasic study involving phenotypic tests, physiological and biochemical tests, 16S rDNA sequence analysis, G＋C content determination and DNA-DNA hybridizations analysis, strain S2 was identified as Pseudomonas alcaligenes. R alcaligenes S2 was mutagenized with Tn5 and four mutants showing decreased or increased solubilizing ability of lecithin were isolated based on the halo size around colonies on the solid plate supplemented with egg yolk. To characterize the genes of R alcaligenes S2 involved in solubilization of lecithin, the EcoR I fragments of the chromosomes from the four mutant strains carrying a single transposon were cloned, and the DNA sequences flanking the Tn5 were determined. The Tn5 insertion sites in the mutants M808, M1329 and M1400, showing decreased solubilizing ability of lecithin, were found to be located in the xcpS, xcpX and xcpW , respectively, whose products XcpS, XcpX and XcpW were the components of type Ⅱ secretion pathway. Complementation of xcpS, xcpX and xcpW could restore the corresponding mutants M808, M1329 and M1400 to solubilize lecithin. The data suggested that mutation in one of these xcp genes would lead to the absence of mature lecithinase secretion into the extracellular medium. The data also indicated that the secretion of lecithin-hydrolyzing enzyme of R alcaligenes was via type Ⅱ secretion pathway. In the mutant M20 showing increasing lecithin-hydrolyzing activity, the interrupted gene showed 86% identity with chpA of Pseudomonas aeruginosa PAO1, whose product plays an important role in controlling twitching motility of the bacterial ceils.     

铜绿假单胞菌的LexA蛋白在大肠杆菌中的高效表达与纯化

目的:对铜绿假单胞菌(Pseudomonas aeruginosa,PA)的阻遏蛋白LexA进行基因克隆、重组表达及蛋白纯化.方法:采用PCR法从PAO1株基因组中扩增出1 086 bp的LexA基因,将此基因片段插入到表达载体pET32a( )上,并在大肠杆菌BL21(DE3)中表达.包涵体洗涤后用8 mol/L尿素溶解,以镍离子亲合柱层析作为第1步纯化,Superdex75凝胶过滤层析作为第2步精细纯化,反相高相液相色谱法(HPLC)测定蛋白的浓度.结果:LexA以包涵体形式表达,表达量为45%,经镍离子亲合柱层析纯化后LexA蛋白纯度达到85%以上,回收率大于80%,镍离子亲合柱层析和凝胶过滤层析两步纯化目的蛋白,经HPLC测定,最终目的蛋白的纯度为98.97%.结论:在pET32a( )表达系统中实现了PA的LexA蛋白的高效表达,两步纯化法获得高纯度的目的蛋白,为进一步鉴定LexA靶位点奠定了基础.