Preferential Attachment of Escherichia coli to Different Particle Size Fractions of an Agricultural Grassland Soil

This study reports on the attachment preference of a faecally derived bacterium, Escherichia coli, to soil particles of defined size fractions. In a batch sorption experiment using a clay loam soil it was found that 35% of introduced E. coli cells were associated with soil particulates >2 μm diameter. Of this 35%, most of the E. coli (14%) were found to be associated with the size fraction 15–4 μm. This was attributed to the larger number of particles within this size range and its consequently greater surface area available for attachment. When results were normalised with respect to estimates of the surface area available for bacterial cell attachment to each size fraction, it was found that E. coli preferentially attached to those soil particles within the size range 30–16 μm. For soil particles >2 μm, E. coli showed at least 3.9 times more preference to associate with the 30–16 μm than any other fraction. We report that E. coli can associate with different soil particle size fractions in varying proportions and that this is likely to impact on the hydrological transfer of cells through soil and have clear implications for our wider understanding of the attachment dynamics of faecally derived bacteria in soils of different compositions.     

黄土高原水蚀风蚀交错带水分和密度对人工草地土壤呼吸的影响

为研究黄土高原水蚀风蚀交错带不同水分与密度处理的人工苜蓿和沙打旺草地土壤呼吸特征,分析了人工草地土壤呼吸对水分和密度的响应及其与环境因子的关系。结果表明：水分增加能够显著促进黄土高原水蚀风蚀交错带人工草地土壤呼吸速率（P〈0.01）,土壤呼吸对不同密度处理的响应特征因草地植被类型不同而异,土壤呼吸速率的季节变化对水分增加和密度响应不敏感。土壤水分是土壤呼吸的限制因子,土壤温度与土壤呼吸速率在6-7月份（旱季后期）相关性不明显,8月份后（雨季）相关性增强。土壤呼吸与表层土壤水分和温度有着显著的耦合关系,加水减弱了苜蓿草地土壤呼吸对水分和温度的依赖,而未改变沙打旺草地土壤呼吸对表层土壤水分和温度的依赖。     

Effects of Feral Pig (Sus scrofa) Exclusion on Enterococci in Runoff from the Forested Headwaters of a Hawaiian Watershed

Water, Air, &; Soil Pollution - The role feral pigs (Sus scrofa) as a source of fecal contamination in Pacific Island ecosystems is not well understood. This study investigated the effects of...     

希拉穆仁草原退化评价及地表风蚀颗粒表征

经过对希拉穆仁草原围封、自由放牧草地及旅游区进行实地调查,结合内蒙古自治区第5次荒漠化和沙化监测实施细则,根据植被盖度和生物量等指标对草原进行了退化诊断与评价,并在此基础上研究了不同退化程度的地表粗粒组成。结果表明:希拉穆仁草原围封草地以轻度退化为主,自由放牧区以中度退化为主,但在坡顶出现重度退化,旅游区退化程度最为严重,均为重度退化区;可以以土壤颗粒0.84mm的百分比含量作为草原退化的判别标准,当其含量在82.22%~89.04%范围内为重度退化,含量在74.01%~79.53%为中度退化,含量在56.05%~66.11%为轻度退化。该指标能够直观、实时地反映草原的退化状况,且具有简便、快速诊断的生产应用性。     

Manure as a Source of Antibiotic-Resistant Escherichia coli and Enterococci: a Case Study of a Wisconsin, USA Family Dairy Farm

The spread of antibiotic-resistant bacteria in the environment is raising serious public health concerns, and manure is being increasingly recognized as a major source of antibiotic-resistant bacteria. In this research, we isolated Escherichia coli and enterococci from manure produced in a Wisconsin, USA family dairy farm to determine their resistance to six representative antibiotics. The average densities for E. coli and enterococci were 6.37(±4.38)?×?10⁷ colony formation units (CFU)?g^?1 and 1.60(±1.57)?×?10⁴ CFU g^?1, respectively. The E. coli isolates were found to be resistant to cephalothin, ampicillin, tetracycline, and erythromycin. In addition to these four antibiotics, the Enterococcus isolates were also resistant to gentamicin and ciprofloxacin. Additionally, we examined the survival and growth of E. coli and enterococci in dairy manure over a period of ~3 days. While the densities of enterococci remained stable over the study period, the concentrations of E. coli on average increased by 1.5 log10 units. Further tests of the bacterial antibiotic resistance over time showed no significant changes in the prevalence of antibiotic resistance. This result indicated that slightly aged manure could represent a larger source of antibiotic-resistant E. coli than fresh manure and the accumulation of antibiotic-resistant E. coli and enterococci in the agricultural fields must be accounted for in the modeling of the spread of antibiotic-resistant bacteria in the environment.     

In situ Evidence for the Association of Total Coliforms and Escherichia coli with Suspended Inorganic Particles in an Australian Reservoir

The results of a field experiment that investigated the in situ association of total coliforms (TC) and E. coli (EC) with suspended inorganic particles in a drinking water reservoir are presented. The experimental program measured TC and EC at various locations and at multiple depths in a medium sized Australian reservoir subject to continuous inflow forcing. Particle concentrations and size distributions were measured using a LISST (Laser In Situ Scattering Transmissometer) profiler. Correlations between the particle measurements and the bacteria were calculated to provide in situ evidence for the association of TC and EC with suspended inorganic particles. Both TC and EC correlated most strongly with fine particles between 3.2 and 4.5 μm. Development of a simple Lagrangian model of the inflow for particles and microbes additionally provided insight into the relative roles of dilution, sedimentation, and inactivation for the bacteria. The model results and the correlations support the theory that high association with the small particles was due to their high number concentration and the effective surface area available for attachment. It was also evident that the majority of bacteria (> 80%) were physically associated with the suspended particles and allowed a quantitative estimate of the net sedimentation rate. Bacterial attachment and the concomitant increase in settling should be considered when modeling bacterial dynamics, during design of monitoring programs and when implementing pathogen risk management strategies.     

棉铃虫组织蛋白酶B酶原在大肠杆菌中的表达及纯化

用合成的基因特异性引物通过RT-PCR扩增，获得棉铃虫(Helicoverpa armigera)组织蛋白酶B基因(HCB)，将基因克隆到pGEX-4T-1载体，在大肠杆菌(Escherichia coli)DH5α内进行扩增，经过PCR筛选获得阳性克隆并提取质粒和测序验证后，再转化大肠杆菌BL21进行诱导表达。表达产物为组织蛋白酶B-谷胱甘肽S转移酶的融合蛋白，分子量在63kD左右。表达产物形成了包涵体，经过对包涵体变性和复性处理，得到了可溶性的融合蛋白，可被Glutathione Sepharose 4B柱亲和纯化，用凝血酶裂解融合蛋白后，经SDS-PAGE分离到37kD左右的棉铃虫组织蛋白酶B酶原。N-末端氨基酸测序鉴定表达产物为棉铃虫组织蛋白酶B。     

鸡MxA基因的克隆及其在大肠杆菌中的表达

MxA是由Ⅰ型干扰素诱导宿主细胞所产生的抗病毒蛋白家族的成员之一。采用RT-PCR方法从鸡胚成纤维细胞(CEF)中扩增MxA,将其克隆至载体pMD-T18中,筛选阳性克隆后回收目的片段,将其克隆入原核表达质粒pGEX-6p-1,构建其重组表达质粒pGEX-MxA,以IPTG诱导表达,经SDS-PAGE、鸡胚新城疫病毒增殖干扰实验和VSV-CEF微量细胞抑制实验进行分析、鉴定。结果表明,经RT-PCR扩增获得的MxA序列与GenBank报道的序列一致,SDS-PAGE和干扰实验证实重组质粒可以表达出相应分子量为45kD的蛋白,与GST-MxA融合蛋白分子量一致,影像分析系统分析显示表达的融合蛋白约占菌体蛋白的30%。     

猪源抗菌肽Protegrin-1基因在大肠杆菌中的融合表达

为了使猪源抗菌肽Protegrin-1(PG-1)在原核表达载体中获得高效表达,在不改变PG-1氨基酸的基础上,根据大肠杆菌偏好密码子表,替换PG-1部分密码子,设计两段互补的引物,利用搭桥PCR技术扩增完整的PG-1成熟肽基因,重组至融合表达载体pGEX-4T-1中,构建成抗菌肽PG-1基因融合表达载体pGEX4T-PG-1,转化至大肠杆菌(Escherichia coli)BL21(DE3)plyS中,筛选得到阳性克隆,并用1.0 mmol/L的IPTG进行诱导表达.SDS-PAGE电泳图谱显示在28 kD处有特异性的蛋白条带出现,经Western blot检测表明重组子已经成功地在大肠杆菌中表达了融合蛋白.纯化得到的GST-PG-1用凝血酶切割后,经用亲合层析得到分子量约为2 kD抗菌肽PG-1蛋白1.38 mg/L.对大肠杆菌和金黄色葡萄球菌(Staphylococcus aureus)抑菌试验结果表明,重组抗菌肽PG-1具有明显的抑菌效果.     

白颈长尾雉圈养种群大肠杆菌耐药性及其耐药基因

对白颈长尾雉圈养条件下的38个样品进行大肠杆菌分离及PCR检定,并采用肠杆菌基因间重复共有序列PCR指纹法（ERIC）剔除各个样品的重叠分离株,检测获得的170个大肠杆菌分离株对9种抗生素的耐药性、Ⅰ型整合子携带率及其可变区抗性基因,结果显示：（1）来自白颈长尾雉的分离株对实验用的9种抗生素的抗性比率和多重耐药性远高于环境源和人源者（来自白颈长尾雉的分离株100%耐受3种及以下的抗生素,而环境源者为50.7%,人源者66.7%）;（2）来自白颈长尾雉的分离株的Ⅰ型整合子携带率（92%）高于环境源（87%）和人源（78%）;（3）来自白颈长尾雉和来自人的大肠杆菌分离株的Ⅰ型整合子可变区抗生素抗性基因检出率相同（36%）,但高于环境源（24%）;（4）携带Ⅰ型整合子的分离株对实验用的抗生素的抗性百分率一般高于不携带者,只有个别种类抗生素这种差异为非显著性差异;（5）Ⅰ型整合子可变区基因盒的基因为3类,即aadA、dfrA和未知功能的orfF;aadA、dfrA的频率相同;3类基因均以基因盒形式存在,分别是dfrA17-aadA5、dfrA12-ofrF-aadA2、dfrA12-aadA2。     

黄土丘陵沟壑区典型草被产流产沙特征及其影响因素

退耕还林还草是黄土高原水土流失重要治理措施之一。为探讨自然降雨条件下,不同种类草被种植对坡面产流产沙特征的影响,采用定位观测方法,基于罗玉沟水土保持监测站6种不同草被(小冠花、红豆草、沙打旺、紫花苜蓿、红三叶、鹰咀紫云英)覆盖坡面的野外观测数据,分析了不同降雨条件下各草被覆盖径流小区的产流产沙规律。结果表明：(1)各场次降雨中,小冠花覆盖下的坡面产流量及产沙量均较大,沙打旺和鹰咀紫云英覆盖坡面均较小;(2)除沙打旺和鹰咀紫云英以外的草被覆盖小区产流产沙量均与降雨量和最大降雨强度呈显著或极显著关系;(3)侵蚀性降水依据降雨量、降雨历时和最大降雨强度分为3种类型：Ⅰ雨型(降雨量大、降雨历时长、最大雨强大)、Ⅱ雨型(降雨量、降雨历时、最大雨强均居中)、Ⅲ雨型(降雨量较小、降雨历时短、最大雨强小)。6种草被在不同雨型下产流产沙特性不同,沙打旺和鹰咀紫云英覆盖坡面在不同雨型下产流量及产沙量均较少,小冠花覆盖坡面在不同雨型下产流量及产沙量均较大。综上,不同草被覆盖下的坡面产流产沙规律不尽相同,对不同降雨特征的响应程度也存在差异,小冠花覆盖小区的抗侵蚀能力较差;沙打旺和鹰咀紫云英覆盖小区的抗侵蚀能力...     

大肠杆菌分子伴侣表达的辅助载体构建及应

异源蛋白在大肠杆菌不能正确折叠表达成包涵体,分子伴侣能在细胞内帮助异源蛋白折叠,改善蛋白的聚集。本研究以大肠杆菌(Escherichia coli)DH5α菌株DNA为模板,利用PCR技术扩增出6个分子伴侣编码基因groEL、groES、dnaK、dnaJ、grpE和clpB,分别将groEL、groES和grpE(Ⅰ组)插入pCDFDuet-1载体,将dnaK、dnaJ和clpB(Ⅱ组)基因插入pRSFDuet-1,构建辅助载体pR-GESP和pC-DJKL,每个重组载体中含有一个人工操纵子,每个基因上游含有T7启动子。SDS-PAGE结果显示,诱导后的重组大肠杆菌上清除了DnaJ外其它5个蛋白明显表达。SDS-PAGE分析了共表达分子伴侣对玉米(Zea mays)四吡咯分子合成的谷氨酸-1-半醛氨基转移酶、尿卟啉原Ⅲ脱羧酶和西罗叶绿三酸:铁螯合酶在大肠杆菌的折叠和聚集影响,结果显示,GroEL、GroES和GrpE能防止玉米西罗叶绿三酸:铁螯合酶在大肠杆菌的聚集,但DnaK、DnaJ和ClpB分子伴侣对该酶则没有作用;GroEL、GroES和GrpE能部分抑制玉米谷氨酸-1-半醛氨基转移酶在大肠杆菌的聚集...     

大肠杆菌K-12中PFL家族基因的生物信息学分析

从生物信息学角度，利用生物学数据库(GenBank、Swiss-Prot、PDB)和分子生物学分析软件Vector NTI8．0，对大肠杆菌K—12中的多个丙酮酸甲酸裂解酶(pyruvate formate-lyase，PFL)进行序列的比较分析和结构预测，进而分析其活性位点、相关保守位点和特殊结构域及序列的二级结构，为利用理性设计改造丙酮酸甲酸裂解酶提供一定的信息。     

Identification of Escherichia coli from shellfish and related environments by automicrobic system

A total of 463 fecal coliform positive isolates obtained from shellfish and related samples gave typical Escherichia coli IMViC reactions. E. coli identifications for 458 (99%) of these isolates were confirmed using a combination of the Automicrobic System (AMS) and the API 20E system (reference system). The AMS (test system) identified 433 isolates as E. coli; the remaining 25 (5%) isolates were identified as E. hermanii by the test system and as E. coli by the reference system. Additional tests performed on the isolates identified as E. hermanii confirmed those AMS identifications to be incorrect.     

玫瑰黄链霉菌几丁质酶基因的克隆及拼接表达

应用PCR扩增克隆到玫瑰黄链霉菌(Streptomyces roseoflavus)的几丁质酶基因chiC的整个催化域部分,将其与包含信号肽区、纤维素结合区、几丁质结合区三个结构功能域的链霉菌(S.lividans)chiC基因片段相连接,插入pET23b( )质粒上,构建表达载体pLCH,用来转化大肠杆菌(Escherichia coli)JM109(DE3),转化子进行破壁之后在胶体几丁质酶培养基上表现活性。     

克雷伯氏菌1,3-丙二醇氧化还原酶基因在大肠杆菌中的克隆与表达

以基因组DNA为模板,利用PCR技术从克雷伯氏菌(Klebsiella pneumoniae)中扩增得到含有1,3-丙二醇氧化还原酶基因(dhaT)的DNA片段,将其定向连接到克隆质粒pGEM-3xf上,得到重组质粒pGEM-dhaT。将此重组质粒转化到受体菌Escherichia coli DH5α中,通过蓝白斑鉴定挑选出阳性菌株。DNA序列分析表明其基因全长为1185bp。将该片段插入表达载体pSE380,构建成重组子pSE-dhaT,并在E．coli JM109中获得表达,经SDS-PAGE检测,表达产物分子质量与天然纯1,3-丙二醇氧化还原酶(DHAT)相同,约为43ku。     

退耕还林(草)对北洛河上游水沙变化的影响

为探究黄土高原地区退耕还林（草）政策的水沙响应问题,以植被恢复较好的北洛河上游为研究区域,根据其控制站吴起水文站1971—2010年4—10月实测水沙资料,定量分析降水量、径流量和输沙量三个水文要素的变化趋势和发生突变的年份。结果表明:1971—2010年,降水量并未发生显著的趋势性变化,径流量和输沙量呈显著减小趋势;综合四种突变点检验方法结果,并结合水土保持措施资料,确定径流量和输沙量的突变年份为2002年;利用双累积曲线定量分析降水和以退耕还林（草）为主的人类活动对径流量与输沙量的影响,相对于1971—2002年,2003—2010年降水和人类活动对径流量的影响程度分别为70.8%和29.2%,对输沙量的影响程度分别为34.0%和66.0%。植被的减沙作用明显大于减水作用,以退耕还林（草）为主的人类活动是北洛河上游流域泥沙减少的主要驱动力。     

近50年半干旱典型草原区域不同时间尺度上的降水、径流研究

以内蒙古锡林河流域典型草原区域为研究区,采用1963—2015年气温、相对湿度、降水量、风速、日照时数、蒸发量、径流日数据,应用统计分析、趋势检验和Spearman相关分析等方法对研究区区域7项水文气象要素的平均最高值、平均值、平均最低值突变前后变化及突变后停滞特征进行了分析,结果表明;（1）内蒙古锡林河流域典型草原区域的降水量最大月（7月）与径流量最大月（4月）不同,降水天数明显小于径流天数,且降水量远大于径流深,多年丰水季降水总量近似于枯水季的3倍,而多年枯水季径流总量是丰水季的2倍;（2）径流量多年来呈现显著减少的趋势,而降水的整体变化趋势不太明显,存在1998—2011年处于由丰变枯阶段,2011年后有由枯转丰的迹象;（3）年、丰水季、枯水季径流发生突变年份均为2004,同一时期发生突变,年、枯水季径流天数发生突变年份为2007年、2004年,丰水季在此期间未发生突变;（4）该区年径流天数枯水季所占比例居多,径流量年、丰、枯水季突变年限一致,造成突变的驱动因数接近,枯水季径流天数相对于年径流天数突变年份较早,枯水季对引起突变的驱动因数更为敏感;（5）影响该区降水大小的主要气象因素依次为日照时数 > 相对湿度 > 平均风速 > 平均气温 > 降水量=蒸发,影响该区径流大小的主要气象因素依次为蒸发 > 相对湿度 > 平均风速 > 平均气温=降水量 > 日照时数。     

烟夜蛾泛素延伸蛋白基因的克隆与表达

应用RT-PCR技术，从烟夜蛾(Helicoverpa assulta)幼虫脂肪体组织总RNA中反转录扩增泛素延伸蛋白基因的cDNA片段，扩增得到的片段全长390bp，编码一个长为129个氨基酸残基的蛋白质，预测分子量14.8kDa。同源性分析表明，此cDNA序列为ubiquitin-53aa extension protein(ubi-53，UBE)基因，在泛素蛋白后融合了一个核糖体L40蛋白(ribosomal L40 protein)。使用Clustal W软件，对cDNA编码的氨基酸序列进行了同源性分析，分析表明：烟夜蛾的泛素延伸蛋白氨基酸序列与其他真核生物泛素延伸蛋白氨基酸序列高度同源（90%－98%），与棉铃虫核多角体病毒(HaSNPV)泛素的同源性为69%。将烟夜蛾的UBE基因克隆到原核表达载体pGEX-4T-2上，转化大肠杆菌BL21(DE3)，经IPTG进行诱导表达，电泳检测到一条约40kDa大小的外源蛋白，用鼠抗GST抗体进行Western blot检测表明原核表达蛋白是目的蛋白。     

费氏弧菌β-内切葡聚糖酶基因在大肠杆菌中的表达及酶学分析

以费氏弧菌（Vibrio fischeri）EM17基因组DNA为模板，通过PCR方法克隆了该菌的β-内切葡聚糖酶基因（GenBank Accession Number : EF533943）。将其插入到表达载体pGEX-6p-1中，转化大肠杆菌BL21（DE3），诱导表达。测序结果经DNAMAN软件分析，该基因与GeneBank中费氏弧菌 ES114基因组预测的β-内切葡聚糖酶基因同源性达到97.1％，但与GeneBank中其他β-内切葡聚糖酶基因同源性较低。酶学性质研究表明，该酶的最适反应温度为60℃，最适反应pH值为9.0。同时研究了6种金属离子对该酶活性的影响，结果显示该酶活性依赖金属离子的存在 Mg2+，Ca2+对酶有激活作用，Mn2+, Pb2+则严重抑制该酶的活性。