噬菌体434单链阻遏蛋白的高亲和力DNA结合序列的设计和筛选

一． 设计和筛选单链阻遏蛋白的高亲和力DNA结合序列 　　单链阻遏蛋白RRTRES是噬菌体434阻遏蛋白的衍生物，它是噬菌体434阻 遏蛋白的N端DBD(1-69位氨基酸)组成的共价二聚体。这个单链分子有两个DBD，一个是野生型噬菌体434的DBD-R，另一个是突变了的DBD - RTRES，二者用重组接头以头接尾的方式连接起来。在RTRES的α3-螺旋中．1、1、2、5位氨基酸与DNA识别紧密相关，它们分别为T、R、E、S。为了筛选出突变的RTRES的DNA结合位点，设计了核心序列为CATACAAGAAAGNNNNNNTTTATG随机DNA库，通过RRTRES与随机DNA库的体外结合和循环筛选。将筛选到的群体克隆并测序。通过与单链阻遏蛋白RRTRES的亲和力测定，对每一个筛选到的序列进行特性分析。结果表明，当结合位点（上述划线部分）为TTAC或TTCC时为最适操纵区序列。它们与单链阻遏蛋白RRTRES的亲和力很高，Kd值在1-10pM的范围。其中随机部分为TTTACG的操纵区与RRTRES的亲和力最高，Kd值约为lpM;当结合位点为TTAC时，平均Kd值为3pM：当结合位点为 TTCC时，Kd值在5-lOpM之间。天然噬菌体434阻遏蛋白与其操纵区的亲和力的Kd值在nM数量级，与之相比，所筛选操纵区的亲和力明显提高。此外，亲和力大小还受到结合位点两侧的碱基的影响，特别是5'位碱基的影响。 　　表达纯化同源双突变的单链阻遏蛋白RTRESRTRE'根据RTRES的以上识别特一点，设计了一系列新的操纵区序列，它们的共有序列为GTAAGAAARNTTACN，或GGAAGAAARNTTCCN，并测定它们与RTRES RTRE之间的结合特异性。结果表明，它们可被RTRES RTRES特异识别，且亲和力也很高，Kd值在5-40pM之间。其中GTAAGAAAGTTTACG与RTRES RTRES之间结合的Kd值约为5pM。同样，表达了异源双突变的单链阻遏蛋白R*RTRES，然而它与 设计的相关操纵区的亲和力并不很高，Kd值约为lOOpM。利用本工作中的随机筛选和合理设计的原则，得到了新的具有特异性识别和高亲和力的蛋白一DNA相互作用。这个方法可望用于其他DBP的新的结合特异性的筛选。 二． 非同位素的方法筛选单链阻遏蛋白的最佳DNA结合序列初探 　　克隆和表达了带半胱氨酸尾的单链阻遏蛋白，利用已包被了马来酰胺的活性板可以与自由巯基结合的特性，将蛋白固定在活性板表面。体外筛选RTRES RTRES的最佳DNA结合序列，得到了一些与RTRES RTRES结合的序列，但Kd值nM数量级。此方法需进一步优化。

全球变化的植被——环境相关性：中国和东北样带（NECT）研究

全球变化与陆地生态系统(GCTE)的研究一直是国际地圈一生物圈计划(IGBP)．全球变化研究的焦点之一，其中植被与环境，尤其是气候的关系研究，虽然古老却又包含许多新的涵义，而陆地样带(Terrestrial Transect)则是近年来发起于GCTE，并拓展到IGBP其它核心项目的研究热点．博士后研究的主要工作有：将全球变化作为主线，以单个树种青冈(Cyclobalanopsisgtauco)为对象，研究其过去、现在和将来的地理分布与气候的关系;分别利用气温和降水指标，拓展Kira指标形成生物热量指数和干湿度指数，以及建立水热积指数，在宏观尺度上研究了中国植被与气候的关系;结合生物群区和植物功能型概念的发展以及生物多样性的研究，进行了以生物多样性保育为目的的我国生态地理区划;在熟悉IGBP陆地样带的科学计划并总结其最新发展动态的基础上，分析了中国东北样带的基本生态地理特征;最后，粗略分析r生物性多样保育与自然保护区建设和管理的关系问题。 1.植被一环境（气候）分类：指标、系统和模型 植被一气候关系是一个古老的命题，但在当今的全球变化研究中成为最基础和最具活力的工作。从单一因子的或多因子简单组合的分类指标，如Koppen，Box指标等，到以可能蒸散为基础的综合分类指标，如Penman，Thornthwaite, Holdridge，Budyko，Kira指标等，科学家们发展了众多的植被一气候分类系统和模型，如Koppen．Thornthwaite，Holdridge. Kira. Box. Neilson.Woodward, Budyko, Prentice系统，以及Holdridge，Uvardy，Matthews. Olson. Bailey．Woodward.Prentice. Box模型，为现今植被一气候关系的研究以及全球变化对陆地生态系统的影响，和大气C02增加对潜在植被变化的响应预测奠定了良好的基础。 2.基于物种的植被一气候关系研究：中国青冈的地理分布与气候的关系 在广泛收集青冈[Cyclobalanopsis glauca (Thunb.) Oerst.]地理分布资料的基础上，利用目前国际上比较流行的研究植被与气候相互关系的指标和方法，包括Kira的水热指标、Penman的公式、Thomthwaite的指标和气候分类、Holdridge的生命地带分类系统指标， 以及年平均气温(TEMP). 1月均温(Tl)、’7月均温(T7)、极端最高气温(TMAX)、极端最低气温(TMIN).≥10℃积温(AT)和年降水量(PREC)，研究了青冈在中国的地理分布与气候的关系，讨论了青冈垂直分布的上限、下限以及北界的Kira热量指标状况。根据孢粉资料和历史文献，探讨r历史时期青冈在中国大陆的分布与变迁及其与气候的关系，并利用Holdridge生命地带分类系统指标预测了C02浓度倍增条件下中国青冈分布区的可能变化。 3.宏观尺度上的中国植被一气候关系 1）用气温、降水指标研究中国植被一气候关系 能够在气象台站直接、方便地测试到的年平均气温、降水量指标，与其它水热气候因子有显著的相关性，用它们来研究中国植被与气候的关系是可行且有用的。利用全国689个气象站点的气象记录，计算得出了中国各植被地带、亚地带的年平均气温，年降水量指标和温雨系数，利用生态信息系统EIS作出了各气候指标在中国的分布，并将年平均气温和降水量作散点图，均较好地表现了中国各植被类型与气候指标的关系和格局。总结可得中国各植被地带的气候指标范围及界限。综合孢粉，古生物等资料信息，前人确定在全新世中期中国存在一个大暖期，其中稳定暖湿的鼎盛阶段在7.2—6,0 Ka．B.P.，其时中国境内大部分地区的年平均气温比现在高2℃左右，年降水平均高于现在100 mm，通过数学处理，利用生态信息系统恢复重建了全新世大暖期中国大陆的气温和降水分布状况．参考孢粉、古植物和他人研究资料及现代植被气候关系，恢复编制了大暖期鼎盛阶段中国大陆的植被区划图，与现代植被区划相比较，东部各个植被带在大暖期盛时表现出明显的北迁，温带植被的迁移幅度大于亚热带和热带植被;西部的植被带出现了经向西迁，西北部的草原范围扩张，荒漠缩小;青藏高原地区高寒半荒漠和荒漠植被的范围大大缩小，而且植被带仍有不同程度向北迁移的表现。这可为预测与阐明未来的气候变化和植被变迁提供有力的证据． 2）KIRA指标的拓展及其在中国植被与气候关系研究中的应用 根据Kira以月平均气温5℃为界的热量指数和干湿度指数概念，提出了以月平均气温10℃为界的生物热量指数，包括生物温暖指数BWI和生物寒冷指数BCI，并修正其干湿度指数为生物干湿度指数BK。利用中国689个标准气象台站的资料，分析我国主要植被类型分布与热量因子和干湿度因子的关系，得出两者之间有较好的相关性，生物温暖指数、寒冷指数和干湿度指数的散点图，较好地表现了中国各植被类型与气候指标的关系和格局。并得出中国各植被地带的气候指标范围及界限，以1 0℃为界的生物温暖指数不仅对我国森林植被的地理分布和温度气候带的划分具有较好的指示作用，而且对西南部高山、亚高山地区的植被与气候关系指示性较强;生物寒冷指数则对亚热带和热带的指示性很好，能够较好区分亚热带南部及热带地区;由热量指数和降水量综合得出的生物干湿度指数，对中国西北部干旱、半干旱区以至全国的植被分布与水分、热量因子的关系分析有较好的应用价值。 3）水热积指数的估算及其在中国植被与气候关系研究中的应用 试图利用大气年平均气温、年降水量、可能蒸散和土壤水分平衡之间的关系建立一个水热积指数，并应用年平均气温．水分盈亏值和水热积指数三个气候变量来限定植物群落组合，构成一个圆形的生命-气候图式．根据全国689个标准气象台站的气候资料，计算了中国8个植被地带和26个亚地带的年平均气温、年水分盈亏和水热积指数，绘制了各气候指标在中国的分布图及散点图，较好表现了中国各植被类型与气候指标的关系和格局，包括寒温带针叶林、冷温带针阔叶混交林、暖温带落叶阔叶林、亚热带常绿阔叶林、热带雨林和季雨林、温带草原、温带荒漠、青藏高原高寒植被，并得到了中国各植被地带的气候指标范围及界限。通过分析可以看出，年平均气温的等值线较好地反映了中国大陆的热量梯度，经度和纬度方向的区分均较明显;土壤水分盈亏曲线的等值线则比较零乱;综合了热量和水分差异的水热积指数，其等值线与热量梯度和水分梯度均有一定的对应性，与植被类型的对应也较好。这是在宏观尺度上进行的植被与气候关系研究的一种尝试，有待于增加机理性的内容，使其得到进一步的改进。 4.生物多样性保育和全球变化研究中的陆地生物群区类型 Biome(生物群区)是当今生物多样性保育和全球变化研究中的一个重要概念，根据此概念及植物功能型概念的发展，评述了9个重要的世界陆地生物群区分类系统，并根据中国的植被分类和区划，尝试划分了在中国的生物多样性保育和全球变化研究中所需要的陆地生物群区类型。 5.中国生物多样性的生态地理区划 利用各种生态地理因子，包括气候指标如与植物耐寒性有关的绝对最低温度(TMrN)，最冷月平均气温(TJAN)，最冷月日平均温度的最大值(MXT)和最小值(MIT);与需热性有关的植物生长季积温(AT);年降水量的季节分配，包括最冷月降水(PJAN)，最热月降水(PJUL)，年降水量，年降水的统计标准差(PSD)和变异系数(年变率PCV);植被指标如植被类型(VEGET)、，植被区划类型(VEGED)、植被的净第一性生产力(NPP)、植物区系类型(FLORA)、动物区系类型(FA UNA).植物特有属的丰富度(EDGENUS)以及度量植物多样性的植物种丰富度(属数GENUS、种数SPECIES);土壤指标如土壤类型(SOILT)，土壤理化性质如土壤酸碱度(SOILPH)、土壤表层阳离子交换量(SOILEXC)等;地形和地貌特征如经度(LONG)、纬度(LAT)和海拔高度(ALT)，利用模糊聚类的手段，综合进行了中国生物多样性的生态地理区划。采用四级区划，即：生物大区(biodomain) -生物亚区(subbiodomain) -生物群区(biome) -生物区(bioregion).全国划分为5个生物大区，7个生物亚区和1 8个生物群区。 I北方森林大区 I A欧亚北方森林亚区 I Al南泰加山地寒温针叶林 IA2北亚针阔叶混交林 II北方草原荒漠大区 II B欧亚草原亚区 II Bl内亚温带高草草原 II B2黄土高原森林草原（灌木草原） il C亚非荒漠亚区 II Cl中亚温带荒漠 ⅡC2蒙古／内亚温带荒漠 III东亚大区 III D东亚落叶阔叶林亚区 m DI东亚落叶阔叶林 III E东亚常绿阔叶林亚区 III El东亚落叶•常绿阔叶混交林 III E2东亚常绿阔叶林 ⅡI E3东亚季风常绿阔叶林 III E4西部山地常绿阔叶林 IV旧热带大区 IV F印度一马来热带森林亚区 IV Fl北热带雨林、季雨林 IV F2热带海岛植被 V亚洲高原大区 vG青藏高原亚区V Gl青藏高寒灌丛草甸V G2青藏高寒草原V G3青藏高寒荒漠V G4青藏温性草原V Gs青藏温性荒漠IGBP陆地样带：科学计划与最新进展 作为国际地圈一生物圈计划(IGBP)的交叉项目(Interproject)的陆地样带(Terrestrial Transect)，已成为IGBP的全球变化研究中最引人重视的发展和新研究方法之一。它以一系列综合性的全球变化研究计划为基础，是由沿着一个主要全球变化驱动因素（如温度、降水、土地利用强度等）的梯度上的一系列研究站点所构成研究区域，并配合以模型模拟和综合分析，其地理范围为1000 km或更大的长度，数百公里的宽度，以涵盖大气环流模型(GCM)运作的最小单元。本节论述了IGBP陆地样带的概念和研究的意义，样带的类型、一般设计和选择标准，国际上IGBP样带的初步设置，包括①经受土地利用变化的潮湿热带系统;②从北方森林到冻原的高纬度地区;③从干旱森林到灌丛的半干旱热带地区;④从森林或灌丛过渡到草地的中纬度半干旱地区，以及其它的一些样带和PAGES核心计划中的PEP样带，主要内容有样带设置的原因、研究内容和主要样带特点等，并总结了样带的最新研究进展和动态． 6.中国东北样带(N ECT)的生态地理特征分析 陆地样带研究已成为国际地圈-生物圈计划(IGBP):全球变化研究的重要手段与热点。中国东北森林-草原样带(NECT)已被列为IGBP国际全球变化陆地样带之一。该样带在东经1120与130030’之间沿北纬43030’设置，长约l 600 km，是一条中纬度温带以降水为驱动因素的梯度，具有由温带针阔叶混交林向温带草原的3个亚地带：草甸草原、典型草原与荒漠草原过渡的空间系列。本文给出了样带的基本生态地理特征及其梯度分析，包括其地理位置、设置意义、地形地貌、气候梯度、土壤类型、土地利用格局、植被类型、主要优势种和群落类型的生态地理特征以及全新世适宜期（大暖期）的植被分布格局。NECT将成为我国全球变化与陆地生态系统(GCTE)与其它IGBP核心项目研究的前沿阵地。 7.自然保护区的作用、建设和管理及其与生物多样性的关系 一般而言，“就地保护”是保护生物多样性的主要措施和最根本的途径，生境的“就地保护”是生物多样性保护最为有力和最为高效的保护方法，而就地保护的措施就是建立自然保护区，通过对自然保护区的建设和有效管理，使生物多样性得到切实有效的人为保护。从自然保护区定义和类型划分及生物多样性的定义本身可以看出，自然保护区的主要保护对象是世界上丰富多彩的生物多样性，自然保护区是生物多样性就地保护的重要基地，是物种多样性的基因库，是留给野生动植物的宝贵栖息地，应把保护区的建设和生物多样性的保护与持续利用密切结合起来，合理开发利用自然资源，促进生物多样性的可持续发展。

母鸡输卵管子宫部初级感觉神经元的CB-HRP法定位研究

应用CB-HRP溶液注入母鸡输卵管子宫部浆膜下,四甲基联苯胺(TMB)组织化学呈色,以探察母鸡输卵管子宫部初级感觉神经元的定位。结果表明:母鸡输卵管子宫部的初级感觉神经元位于双侧T1-LS13脊神经节、颈静脉神经节和结状神经节。标记细胞数分布不均,在体左侧多于体右侧,在脊神经节多于颈静脉神经节和结状神经节。在脊神经节内标记细胞有T5-LS1和LS8-LS11前后两个相对集中区,峰值分别在T7和LS11。说明尽管母鸡输卵管子宫部是单侧发育成熟脏器,但其感觉沿双侧脊神经和迷走神经传入中枢,以体左侧传入为主;且

Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays all important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. Coli Under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1. excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His-Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.

Isolation and identification of a novel WSSV nucleocapsid protein by cDNA phage display using an scFv antibody

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A I with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A I scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the prefer-red selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv. (c) 2006 Elsevier B.V. All rights reserved.

Context and conformation dictate function of a transcription antitermination switch

In bacteriophage, transcription elongation is regulated by the N protein, which binds a nascent mRNA hairpin ( termed boxB) and enables RNA polymerase to read through distal terminators. We have examined the structure, energetics and in vivo function of a number of N boxB complexes derived from in vitro protein selection. Trp18 fully stacks on the RNA loop in the wild-type structure, and can become partially or completely unstacked when the sequence context is changed three or four residues away, resulting in a recognition interface in which the best binding residues depend on the sequence context. Notably, in vivo antitermination activity correlates with the presence of a stacked aromatic residue at position 18, but not with N boxB binding affinity. Our work demonstrates that RNA polymerase responds to subtle conformational changes in cis-acting regulatory complexes and that approximation of components is not sufficient to generate a fully functional transcription switch.

Identification and molecular characterization of a peritrophin-like protein from fleshy prawn (Fenneropenaeus chinensis)

Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) (Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048 bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes. (c) 2005 Elsevier Ltd. All rights reserved.

RinA controls phage-mediated packaging and transfer of virulence genes in gram-positive bacteria

Phage-mediated transfer of microbial genetic elements plays a crucial role in bacterial life style and evolution. In this study, we identify the RinA family of phage-encoded proteins as activators required for transcription of the late operon in a large group of temperate staphylococcal phages. RinA binds to a tightly regulated promoter region, situated upstream of the terS gene, that controls expression of the morphogenetic and lysis modules of the phage, activating their transcription. As expected, rinA deletion eliminated formation of functional phage particles and significantly decreased the transfer of phage and pathogenicity island encoded virulence factors. A genetic analysis of the late promoter region showed that a fragment of 272 bp contains both the promoter and the region necessary for activation by RinA. In addition, we demonstrated that RinA is the only phage-encoded protein required for the activation of this promoter region. This region was shown to be divergent among different phages. Consequently, phages with divergent promoter regions carried allelic variants of the RinA protein, which specifically recognize its own promoter sequence. Finally, most Gram-postive bacteria carry bacteriophages encoding RinA homologue proteins. Characterization of several of these proteins demonstrated that control by RinA of the phage-mediated packaging and transfer of virulence factor is a conserved mechanism regulating horizontal gene transfer.

Genetic analysis of bacteriophages from clinical and environmental samples

Bacteriophages, viruses infecting bacteria, are uniformly present in any location where there are high numbers of bacteria, both in the external environment and the human body. Knowledge of their diversity is limited by the difficulty to culture the host species and by the lack of the universal marker gene present in all viruses. Metagenomics is a powerful tool that can be used to analyse viral communities in their natural environments. The aim of this study was to investigate diverse populations of uncultured viruses from clinical (a sputum of patient with cystic fibrosis, CF) and environmental samples (a sludge from a dairy food wastewater treatment plant) containing rich bacterial populations using genetic and metagenomic analyses. Metagenomic sequencing of viruses obtained from these samples revealed that the majority of the metagenomic reads (97-99%) were novel when compared to the NCBI protein database using BLAST. A large proportion of assembled contigs were assignable as novel phages or uncharacterised prophages, the next largest assignable group being single-stranded eukaryotic virus genomes. Sputum from a cystic fibrosis patient contained DNA typical of phages of bacteria that are traditionally involved in CF lung infections and other bacteria that are part of the normal oral flora. The only eukaryotic virus detected in the CF sputum was Torque Teno virus (TTV). A substantial number of assigned sequences from dairy wastewater could be affiliated with phages of bacteria that are typically found in the soil and aquatic environments, including wastewater. Eukaryotic viral sequences were dominated by plant pathogens from the Geminiviridae and Nanoviridae families, and animal pathogens from the Circoviridae family. Antibiotic resistance genes were detected in both metagenomes suggesting phages could be a source for transmissible antimicrobial resistance. Overall, diversity of viruses in the CF sputum was low, with 89 distinct viral genotypes predicted, and higher (409 genotypes) in the wastewater. Function-based screening of a metagenomic library constructed from DNA extracted from dairy food wastewater viruses revealed candidate promoter sequences that have ability to drive expression of GFP in a promoter-trap vector in Escherichia coli. The majority of the cloned DNA sequences selected by the assay were related to ssDNA circular eukaryotic viruses and phages which formed a minority of the metagenome assembly, and many lacked any significant homology to known database sequences. Natural diversity of bacteriophages in wastewater samples was also examined by PCR amplification of the major capsid protein sequences, conserved within T4-type bacteriophages from Myoviridae family. Phylogenetic analysis of capsid sequences revealed that dairy wastewater contained mainly diverse and uncharacterized phages, while some showed a high level of similarity with phages from geographically distant environments.

Mutational and molecular analyses of lactococcal bacteriophages TP901-1 and Tuc2009

Bacteriophages belonging to the Siphoviridae family represent viruses with a noncontractile tail that function as extremely efficient bacterium-infecting nanomachines. The Siphoviridae phages TP901-1 and Tuc2009 infect Lactococcus lactis, and both belong to the so-called P335 species. As P335 phages are typically capable of a lytic and lysogenic life cycle, a number of molecular tools are available to analyse their virions. This doctoral thesis describes mutational and molecular analyses of TP901-1 and Tuc2009, with emphasis on the role of their tail-associated structural proteins. Several novel and intriguing findings discovered during the course of this study on the nature of Siphoviridae phages furthers a basic molecular understanding of their virions, and the role of their virion proteins, during the initial stages of infection. While Siphoviridae virions represent complex quaternary structures of multiple proteins and subunits thereof, mutagenic analysis represents an efficient mechanism to discretely characterize the function of individual proteins, and constituent amino acids, in the assembly of the phage structure and their biological function. However, as always, more research is required to delve deeper into the mechanisms by which phages commence infection. This is important to advance our understanding of this intricate process and to facilitate application of such findings to manipulate phage infections. On the one hand, we may want to prevent phages from infecting starter cultures used in the dairy industry, while on the other hand it may be desirable to optimize viral infection for the application of phages as bacterial parasites and therapeutic agents.

The biodiversity, control and genetic characterisation of the lactococcal 936 group phages

Phages belonging to the 936 group represent one of the most prevalent and frequently isolated phages in dairy fermentation processes using Lactococcus lactis as the primary starter culture. In recent years extensive research has been carried out to characterise this phage group at a genomic level in an effort to understand how the 936 group phages dominate this particular niche and cause regular problems during large scale milk fermentations. This thesis describes a large scale screening of industrial whey samples, leading to the isolation of forty three genetically different lactococcal phages. Using multiplex PCR, all phages were identified as members of the 936 group. The complete genome of thirty eight of these phages was determined using next generation sequencing technologies which identified several regions of divergence. These included the structural region surrounding the major tail protein, the replication region as well as the genes involved in phage DNA packing. For a number of phages the latter genomic region was found to harbour genes encoding putative orphan methyltransferases. Using small molecule real time (SMRT) sequencing and heterologous gene expression, the target motifs for several of these MTases were determined and subsequently shown to actively protect phage DNA from restriction endonuclease activity. Comparative analysis of the thirty eight phages with fifty two previously sequenced members of this group showed that the core genome consists of 28 genes, while the non-core genome was found to fluctuate irrespective of geographical location or time of isolation. This study highlights the continued need to perform large scale characterisation of the bacteriophage populations infecting industrial fermentation facilities in effort to further our understanding dairy phages and ways to control their proliferation.

Antisense Gene Silencing and Bacteriophages as Novel Disinfection Processes for Engineered Systems

The growth and proliferation of invasive bacteria in engineered systems is an ongoing problem. While there are a variety of physical and chemical processes to remove and inactivate bacterial pathogens, there are many situations in which these tools are no longer effective or appropriate for the treatment of a microbial target. For example, certain strains of bacteria are becoming resistant to commonly used disinfectants, such as chlorine and UV. Additionally, the overuse of antibiotics has contributed to the spread of antibiotic resistance, and there is concern that wastewater treatment processes are contributing to the spread of antibiotic resistant bacteria.

Due to the continually evolving nature of bacteria, it is difficult to develop methods for universal bacterial control in a wide range of engineered systems, as many of our treatment processes are static in nature. Still, invasive bacteria are present in many natural and engineered systems, where the application of broad acting disinfectants is impractical, because their use may inhibit the original desired bioprocesses. Therefore, to better control the growth of treatment resistant bacteria and to address limitations with the current disinfection processes, novel tools that are both specific and adaptable need to be developed and characterized.

In this dissertation, two possible biological disinfection processes were investigated for use in controlling invasive bacteria in engineered systems. First, antisense gene silencing, which is the specific use of oligonucleotides to silence gene expression, was investigated. This work was followed by the investigation of bacteriophages (phages), which are viruses that are specific to bacteria, in engineered systems.


For the antisense gene silencing work, a computational approach was used to quantify the number of off-targets and to determine the effects of off-targets in prokaryotic organisms. For the organisms of Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv the mean number of off-targets was found to be 15.0 + 13.2 and 38.2 + 61.4, respectively, which results in a reduction of greater than 90% of the effective oligonucleotide concentration. It was also demonstrated that there was a high variability in the number of off-targets over the length of a gene, but that on average, there was no general gene location that could be targeted to reduce off-targets. Therefore, this analysis needs to be performed for each gene in question. It was also demonstrated that the thermodynamic binding energy between the oligonucleotide and the mRNA accounted for 83% of the variation in the silencing efficiency, compared to the number of off-targets, which explained 43% of the variance of the silencing efficiency. This suggests that optimizing thermodynamic parameters must be prioritized over minimizing the number of off-targets. In conclusion for the antisense work, these results suggest that off-target hybrids can account for a greater than 90% reduction in the concentration of the silencing oligonucleotides, and that the effective concentration can be increased through the rational design of silencing targets by minimizing off-target hybrids.

Regarding the work with phages, the disinfection rates of bacteria in the presence of phages was determined. The disinfection rates of E. coli K12 MG1655 in the presence of coliphage Ec2 ranged up to 2 h-1, and were dependent on both the initial phage and bacterial concentrations. Increasing initial phage concentrations resulted in increasing disinfection rates, and generally, increasing initial bacterial concentrations resulted in increasing disinfection rates. However, disinfection rates were found to plateau at higher bacterial and phage concentrations. A multiple linear regression model was used to predict the disinfection rates as a function of the initial phage and bacterial concentrations, and this model was able to explain 93% of the variance in the disinfection rates. The disinfection rates were also modeled with a particle aggregation model. The results from these model simulations suggested that at lower phage and bacterial concentrations there are not enough collisions to support active disinfection rates, which therefore, limits the conditions and systems where phage based bacterial disinfection is possible. Additionally, the particle aggregation model over predicted the disinfection rates at higher phage and bacterial concentrations of 108 PFU/mL and 108 CFU/mL, suggesting other interactions were occurring at these higher concentrations. Overall, this work highlights the need for the development of alternative models to more accurately describe the dynamics of this system at a variety of phage and bacterial concentrations. Finally, the minimum required hydraulic residence time was calculated for a continuous stirred-tank reactor and a plug flow reactor (PFR) as a function of both the initial phage and bacterial concentrations, which suggested that phage treatment in a PFR is theoretically possible.

In addition to determining disinfection rates, the long-term bacterial growth inhibition potential was determined for a variety of phages with both Gram-negative and Gram-positive bacteria. It was determined, that on average, phages can be used to inhibit bacterial growth for up to 24 h, and that this effect was concentration dependent for various phages at specific time points. Additionally, it was found that a phage cocktail was no more effective at inhibiting bacterial growth over the long-term than the best performing phage in isolation.

Finally, for an industrial application, the use of phages to inhibit invasive Lactobacilli in ethanol fermentations was investigated. It was demonstrated that phage 8014-B2 can achieve a greater than 3-log inactivation of Lactobacillus plantarum during a 48 h fermentation. Additionally, it was shown that phages can be used to protect final product yields and maintain yeast viability. Through modeling the fermentation system with differential equations it was determined that there was a 10 h window in the beginning of the fermentation run, where the addition of phages can be used to protect final product yields, and after 20 h no additional benefit of the phage addition was observed.

In conclusion, this dissertation improved the current methods for designing antisense gene silencing targets for prokaryotic organisms, and characterized phages from an engineering perspective. First, the current design strategy for antisense targets in prokaryotic organisms was improved through the development of an algorithm that minimized the number of off-targets. For the phage work, a framework was developed to predict the disinfection rates in terms of the initial phage and bacterial concentrations. In addition, the long-term bacterial growth inhibition potential of multiple phages was determined for several bacteria. In regard to the phage application, phages were shown to protect both final product yields and yeast concentrations during fermentation. Taken together, this work suggests that the rational design of phage treatment is possible and further work is needed to expand on this foundation.

Contribution of bacterial outer membrane vesicles to innate bacterial defense.

BACKGROUND: Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria throughout growth and have proposed roles in virulence, inflammation, and the response to envelope stress. Here we investigate outer membrane vesiculation as a bacterial mechanism for immediate short-term protection against outer membrane acting stressors. Antimicrobial peptides as well as bacteriophage were used to examine the effectiveness of OMV protection. RESULTS: We found that a hyper-vesiculating mutant of Escherichia coli survived treatment by antimicrobial peptides (AMPs) polymyxin B and colistin better than the wild-type. Supplementation of E. coli cultures with purified outer membrane vesicles provided substantial protection against AMPs, and AMPs significantly induced vesiculation. Vesicle-mediated protection and induction of vesiculation were also observed for a human pathogen, enterotoxigenic E. coli (ETEC), challenged with polymyxin B. When ETEC with was incubated with low concentrations of vesicles concomitant with polymyxin B treatment, bacterial survival increased immediately, and the culture gained resistance to polymyxin B. By contrast, high levels of vesicles also provided immediate protection but prevented acquisition of resistance. Co-incubation of T4 bacteriophage and OMVs showed fast, irreversible binding. The efficiency of T4 infection was significantly reduced by the formation of complexes with the OMVs. CONCLUSIONS: These data reveal a role for OMVs in contributing to innate bacterial defense by adsorption of antimicrobial peptides and bacteriophage. Given the increase in vesiculation in response to the antimicrobial peptides, and loss in efficiency of infection with the T4-OMV complex, we conclude that OMV production may be an important factor in neutralizing environmental agents that target the outer membrane of Gram-negative bacteria.

Ocean acidification shows negligible impacts on high-latitude bacterial community structure in coastal pelagic mesocosms

The impact of ocean acidification and carbonation on microbial community structure was assessed during a large-scale in situ costal pelagic mesocosm study, included as part of the EPOCA 2010 Arctic campaign. The mesocosm experiment included ambient conditions (fjord) and nine mesocosms with pCO(2) levels ranging from similar to 145 to similar to 1420 mu atm. Samples for the present study were collected at ten time points (t-1, t1, t5, t7, t12, t14, t18, t22, t26 to t28) in seven treatments (ambient fjord (similar to 145), 2x similar to 185, similar to 270, similar to 685, similar to 820, similar to 1050 mu atm) and were analysed for "small" and "large" size fraction microbial community composition using 16S rRNA (ribosomal ribonucleic acid) amplicon sequencing. This high-throughput sequencing analysis produced similar to 20 000 000 16S rRNA V4 reads, which comprised 7000OTUs. The main variables structuring these communities were sample origins (fjord or mesocosms) and the community size fraction (small or large size fraction). The community was significantly different between the unenclosed fjord water and enclosed mesocosms (both control and elevated CO2 treatments) after nutrients were added to the mesocosms, suggesting that the addition of nutrients is the primary driver of the change in mesocosm community structure. The relative importance of each structuring variable depended greatly on the time at which the community was sampled in relation to the phytoplankton bloom. The sampling strategy of separating the small and large size fraction was the second most important factor for community structure. When the small and large size fraction bacteria were analysed separately at different time points, the only taxon pCO(2) was found to significantly affect were the Gammaproteobacteria after nutrient addition. Finally, pCO(2) treatment was found to be significantly correlated (non-linear) with 15 rare taxa, most of which increased in abundance with higher CO2.

The major tegumental antigen of Fasciola hepatica contains repeated elements

In order to provide a better understanding of the interaction between the liver fluke (Fasciola hepatica) and the immune system of its mammalian host immunoreactive ? bacteriophage clones containing F. hepatica cDNA have been isolated. Plasmids from these clones were sequenced and found to encode a family of proteins containing certain common elements. All the clones contained a coding repeating sequence (RRRXCA) which is conserved at the nucleic acid level followed by a non-repeating element coding for the C terminal used by the proteins which shows conservation of amino acids at certain positions. Antisera raised against a ß-galactosidase fusion protein with one of these sequences as a terminal extension was used to localize the immunoreactive antigens. Binding was predominantly in the tegument of the juvenile fluke but was reduced in the adult tegument. The wall of the uterus showed strong reactivity in the adult. Rats immunized with the ß-galactosidase fusion protein showed enhanced resistance to challenge infections. The role of these antigens in the host response to infection by F. hepatica is discussed.