Name: generation of null mutants to elucidate the role of bacterial glycosyltransferases in bacterial motility
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Description: Watch this Scientific Journal Video about Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility at JoVE.com

这项工作得到了加拿大国家研究委员会、西班牙国家经济和竞争部计划和加泰罗尼亚政府的支持。

该过程的示意图如图 1所示。
1. 气单胞菌中鞭毛糖基化岛（FGIs）的生物信息学鉴定
<ol><li>要鉴定气单胞菌基因组中的PseAc生物合成簇，请使用NCBI数据库26中的tblastn工具。首先，从测序气单胞菌菌株的数据库中检索拟南芥AH-3的PseC和PseI（识别码为OCA61126.1和OCA61113.1）的直系蛋白，然后使用<e...

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该方法的关键早期步骤是鉴定参与鞭毛和推定GT糖基化的区域，因为这些酶显示出高度同源性并参与许多过程。公共数据库中 气单胞菌 基因组的生物信息学分析表明，该区域与极鞭毛区域2相邻，该区域包含许多菌株中的鞭毛蛋白基因，并含有参与拟南芥酸生物合成的基因27。这使得有可能制定检测 气单胞菌 中鞭毛糖基化岛的指南，该指南可用于识别鞭毛聚糖含有?...

蛋白质糖基化已在革兰氏阳性和革兰氏阴性细菌中均有描述，并且由聚糖与氨基酸侧链1，2的共价连接组成。在原核生物中，该过程通常 通过 两种主要的酶机制发生： O- 和 N-糖基化3。在 O-糖基化中，聚糖连接到丝氨酸（Ser）或苏氨酸（Thr）残基的羟基上。在 N-糖基化中，聚糖附着在三肽序列Asn-X-Ser/Thr中的天冬酰胺（Asn）残基的侧链酰胺氮上，其中X可以是除脯氨酸以外的任何氨基酸。
聚糖可以采用线性或支链结构，由糖苷键共价连接的单糖或多糖组成。在原核生物中，与真核聚糖4相比，聚糖通常显示出糖组成和结构的多样性。此外，已经描述了两种不同的细菌糖基化途径，它们在聚糖如何组装和转移到受体蛋白方面有所不同：序贯和 整体 糖基化5，6。对于顺序糖基化，复合聚糖通过连续添加单糖直接在蛋白质上建立。在 整体 糖基化中，预组装的聚糖通过专门的寡糖转移酶（OTase）从脂质连接的低聚糖转移到蛋白质中。这两种途径已被证明参与 N- 和 O-糖基化过程7。
蛋白质糖基化在调节蛋白质的物理化学和生物学特性方面具有作用。聚糖的存在可以影响蛋白质与其配体的相互作用，从而影响蛋白质的生物活性，但也可以影响蛋白质的稳定性，溶解度，对蛋白水解的易感性，免疫原性和微生物 - 宿主相互作用8，9。然而，一些糖基化参数，如聚糖的数量，聚糖组成，位置和附着机制，也可能影响蛋白质的功能和结构。
糖基转移酶（GTs）是复合聚糖和糖偶联物生物合成的关键酶。这些酶催化来自活化的供体分子和特定底物受体的糖部分之间的糖苷键形成。GT可以使用核苷酸和非核苷酸作为供体分子，并靶向不同的底物受体，例如蛋白质，糖类，核酸和脂质10。因此，在分子水平上了解GT对于确定其作用机制和特异性非常重要，并且还可以了解修饰相关分子的聚糖的糖组成如何与致病性相关。碳水化合物活性酶数据库（CAZy）11 根据其序列同源性对GT进行分类，这提供了一种预测工具，因为在大多数GT家族中，结构折叠和作用机制是不变的。然而，四个原因使得难以预测许多GT的底物特异性：1）在原核生物中没有确定底物特异性的明确序列基序12，2）许多GT和OT酶显示底物混杂性13，14，3）功能性GT难以以重组形式高产地产生;4）供体和受体底物的鉴定很复杂。尽管如此，最近的诱变研究使得在理解催化机制和减去GT结合方面取得了重大进展。
在细菌中，O-糖基化似乎比 N-糖基化更普遍。 O-糖基化位点不显示一致序列，许多 O-糖基化蛋白是分泌的或细胞表面蛋白，如鞭毛，毛利或自转运蛋白1。鞭毛蛋白糖基化显示受体位点数量、聚糖组成和结构的变异性。例如， Burkholderia spp鞭毛虫只有一个受体位点，而在 空肠弯曲杆菌中，鞭毛林具有多达19个受体位点15，16个。此外，对于某些细菌，聚糖是单一单糖，而其他细菌具有由不同单糖破坏形成寡糖的异质聚糖。这种异质性甚至发生在同一物种的菌株中。鞭毛虫杆菌 仅由假氨基酸（PseAc）17修饰，鞭毛 弯曲杆菌 可以通过PseAc（假氨基酸（PseAm）或军团胺酸（LegAm）的乙酰脒形式）和从这些糖中提取的聚糖与乙酰基， N-乙酰葡糖胺或丙酸取代物18，19进行修饰。在 气单胞菌中，鞭毛被聚糖修饰，其组成范围从单个PseAc酸衍生物到杂多糖20，并且聚糖与鞭毛蛋白单体的连接总是 通过 PseAc衍生物。
一般来说，鞭毛的糖基化对于鞭毛细丝组装、运动性、毒力和宿主特异性至关重要。然而，虽然C. jejuni16，H. pylori17和Aeromonas sp的鞭毛。21不能组装成长丝，除非蛋白质单体被糖基化，假单胞菌属和伯克霍尔德菌属。15 不需要糖基化来组装鞭毛。此外，在一些空肠衣原体菌株中，鞭毛聚糖的糖组成的变化会影响细菌 - 宿主的相互作用，并可能在逃避某些免疫反应中发挥作用16。自凝集是另一种表型特征，受与鞭毛林相关的聚糖组成的修饰的影响。较低的自凝集导致形成微菌落和生物膜22的能力降低。在一些细菌中，鞭毛触发促炎反应的能力与鞭毛蛋白糖基化有关。因此，在铜绿假单胞菌中，糖基化的鞭毛蛋白诱导比未烷基化23更高的促炎反应。
气单胞菌是环境中无处不在的革兰氏阴性细菌，这使得它们能够位于所有One Health组件24的界面处。嗜中性气单胞菌具有单一的极性鞭毛，其构成性地产生。超过一半的临床分离物也表达侧鞭毛蛋白，在高粘度介质或平板中诱导。不同的研究将两种鞭毛类型与细菌发病机制的早期阶段联系起来25。虽然迄今为止报道的极性鞭毛在5-8 Ser或Thr残基的中枢免疫原性结构域处呈O-糖基化，但侧鞭毛林在所有菌株中均未O-糖基化。尽管来自不同菌株的极性鞭毛聚糖在其碳水化合物组成和链长20上显示出多样性，但连接糖已被证明是一种假单胺酸衍生物。
本手稿的目的是描述一种在含有GT的特定GT或染色体区域获得零突变体的方法，以分析它们在相关多糖的生物合成和细菌致病性中的参与，以及聚糖本身的作用。例如，我们鉴定并删除了含有 气单胞菌 GT的染色体区域，以确定其参与极性鞭毛蛋白糖基化并分析鞭毛蛋白聚糖的作用。我们展示了如何删除特定的GT以建立其在该聚糖的生物合成中的功能和修饰聚糖的作用。虽然以 气单胞菌 为例，但该原理可用于鉴定和研究其他革兰氏阴性菌的鞭毛糖基化岛，并分析参与其他聚糖（如O-抗原脂多糖）生物合成的GT的功能。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ABI PRISM Big Dye Terminator v. 3.1 Cycle Sequencing Ready Reaction Kit</td>
			<td>Applied Biosystems</td>
			<td>4337455</td>
			<td>Used for sequencing</td>
		</tr>
		<tr>
			<td>AccuPrime Taq DNA Polymerase, high fidelity</td>
			<td>Invitrogen</td>
			<td>12346-086</td>
			<td>Used for amplification of AB, CD and AD fragments</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Conda-Pronadise</td>
			<td>8008</td>
			<td>Used for DNA electrophoresis</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase, calf intestinal (CIAP)</td>
			<td>Promega</td>
			<td>M1821</td>
			<td>Used to remove phosphate at the 5&#8217; end</td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>Becton Dickinson</td>
			<td>214010</td>
			<td>Use for motility analysis</td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>Promega</td>
			<td>R6021</td>
			<td>Used for endonuclease restriction</td>
		</tr>
		<tr>
			<td>BglII</td>
			<td>Promega</td>
			<td>R6081</td>
			<td>Used for endonuclease restriction</td>
		</tr>
		<tr>
			<td>BioDoc-It Imagin System</td>
			<td>UVP</td>
			<td></td>
			<td>Bio-imaging station used for DNA visualization</td>
		</tr>
		<tr>
			<td>Biotaq polymerase</td>
			<td>Bioline</td>
			<td>BIO-21040</td>
			<td>Used for colony screening</td>
		</tr>
		<tr>
			<td>Cesium chloride</td>
			<td>Applichem</td>
			<td>A1126,0100</td>
			<td>Used for flagella purification</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Applichem</td>
			<td>A1806,0025</td>
			<td>Used for triparental mating</td>
		</tr>
		<tr>
			<td>Cytiva illustra GFX PCR DNA and Gel Band Purification Kit</td>
			<td>Cytivia</td>
			<td>28-9034-71</td>
			<td>Used for purification of PCR amplicons and DNA fragments.</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Applichem</td>
			<td>131026.1211</td>
			<td>Used for DNA electrophoresis</td>
		</tr>
		<tr>
			<td>Electroporation cuvettes 2 mm gap</td>
			<td>VWR</td>
			<td>732-1133</td>
			<td>Used for transformation</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Applichem</td>
			<td>A1152,0025</td>
			<td>Use for DNA visualization</td>
		</tr>
		<tr>
			<td>HyperLadder 1 Kb marker</td>
			<td>Bioline</td>
			<td>BIO-33053</td>
			<td>DNA marker</td>
		</tr>
		<tr>
			<td>Invitrogen Easy-DNA gDNA Purification Kit</td>
			<td>Invitrogen</td>
			<td>10750204</td>
			<td>Used for bacterial chromosomal DNA purification</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) Miller agar</td>
			<td>Condalab</td>
			<td>996</td>
			<td>Used for Escherichia coli culture</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) Miller broth</td>
			<td>Condalab</td>
			<td>1551</td>
			<td>Used for Escherichia coli culture</td>
		</tr>
		<tr>
			<td>Nanodrop ND-1000</td>
			<td>NanoDrop Techonologies Inc</td>
			<td></td>
			<td>Spectrophotometer used for DNA quantification</td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Applichem</td>
			<td>A2220,0005</td>
			<td>Used for triparental mating</td>
		</tr>
		<tr>
			<td>SOC Medium</td>
			<td>Invitrogen</td>
			<td>15544034</td>
			<td>Used for electroporation recovery</td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Applichem</td>
			<td>A3834,0005</td>
			<td>Used for triparental mating</td>
		</tr>
		<tr>
			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman</td>
			<td>331362</td>
			<td>Used for flagella purification</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Invitrogen</td>
			<td>15224017</td>
			<td>Used for ligation reaction</td>
		</tr>
		<tr>
			<td>Trypticasein soy agar</td>
			<td>Condalab</td>
			<td>1068</td>
			<td>Used for Aeromonas grown</td>
		</tr>
		<tr>
			<td>Trypticasein soy broth</td>
			<td>Condalab</td>
			<td>1224</td>
			<td>Used for Aeromonas grown</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Condalab</td>
			<td>1612</td>
			<td>Use for motility analysis</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Applichem</td>
			<td>A2264,0500</td>
			<td>Used for DNA electrophoresis and flagella purification</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Applichem</td>
			<td>A4975,0100</td>
			<td>Used for bacterial lysis</td>
		</tr>
		<tr>
			<td>Ultra Clear tubes (14 mm x 89 mm)</td>
			<td>Beckman</td>
			<td>344059</td>
			<td>Used for flagella purification</td>
		</tr>
		<tr>
			<td>Veriti 96 well Thermal Cycler</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>Used for PCR reactions</td>
		</tr>
		<tr>
			<td>Zyppy Plasmid Miniprep II Kit</td>
			<td>Zymmo research</td>
			<td>D4020</td>
			<td>Used for isolation of plasmid DNA</td>
		</tr>
	</tbody>
</table>

generation of null mutants to elucidate the role of bacterial glycosyltransferases in bacterial motility

原核生物糖基化的研究是一个快速增长的领域。细菌在其表面上具有不同的糖基化结构，其聚糖构成菌株特异性条形码。与真核生物相比，相关的聚糖在糖组成和结构上表现出更高的多样性，并且在细菌 - 宿主识别过程和与环境的相互作用中很重要。在致病细菌中，糖蛋白已参与感染过程的不同阶段，聚糖修饰会干扰糖蛋白的特定功能。然而，尽管在理解聚糖组成，结构和生物合成途径方面取得了进展，但对糖蛋白在致病性或与环境相互作用中的作用的理解仍然非常有限。此外，在一些细菌中，蛋白质糖基化所需的酶与其他多糖生物合成途径共享，例如脂多糖和胶囊生物合成途径。糖基化的功能重要性已在几种细菌中阐明，通过突变被认为参与糖基化过程的特定基因，并研究其对目标糖蛋白和修饰聚糖表达的影响。嗜温 性气单胞菌 具有单一和 O-糖基化的极性鞭毛。鞭毛聚糖在 气单胞菌 菌株之间显示出碳水化合物成分和链长的多样性。然而，迄今为止分析的所有菌株都显示假氨基酸衍生物作为修饰丝氨酸或苏氨酸残基的连接糖。假鞭毛组装需要假单胞菌酸衍生物，其损失对粘附、生物膜形成和定植有影响。本文中详述的方案描述了如何使用零突变体的构建来了解含有假定糖基转移酶的基因或基因组区域在鞭毛聚糖的生物合成中的参与。这包括了解所涉及的糖基转移酶的功能和聚糖的作用的潜力。这将通过将缺乏聚糖的突变体与野生型菌株进行比较来实现。

该方法提供了一个有效的系统，可以在 气单胞菌 的基因或染色体区域中产生零突变体，这些突变体可以影响鞭毛糖基化和鞭毛丝的作用（图1）。
该协议从推定的GFI的生物信息学鉴定开始，编码GT的基因存在于该区域。在气单胞菌中，FGIs的染色体定位基于对三种基因的检测：参与拟氨基酸（pseI和pseC）生物合成的基因，极性鞭毛蛋白...

Watch this Scientific Journal Video about Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility at JoVE.com

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

在这里，我们描述了在特定的糖基转移酶或含有糖基转移酶的区域中 构建气单胞菌 的零突变体，进行运动性测定和鞭毛纯化以确定其编码酶在聚糖生物合成中的参与和功能，以及该聚糖在细菌发病机制中的作用。

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

所以神经酸衍生物只存在于致病菌中，弱糖基化的鞭毛含有这种糖。在框架中去除基因中所含的区域可以帮助找到鞭毛糖基化簇。该技术，最新的特定区域或基因，如糖基转移酶，它们之间具有高同源性并且参与不同的过程，并分析其参与鞭毛糖基化过程。该技术还可以帮助发现参与致病细菌中相关多糖生物合成的糖基转移酶的作用，以及参与鞭毛形成的编码蛋白的基因的作用。演示该程序将由我实验室的技术人员Maite Polo完成。设计两对引物，扩增要删除的选定基因或区域的上游和下游的DNA区域。引物A和D需要在五个素数端包括一个内切酶的限制性位点，允许在pDM4中克隆。确保引物B和C位于要删除的基因内或要删除的区域的第一个和最后一个基因内。确保两个引物都在框架中，并且位于基因内的五到六个密码子之间。确保两种引物在五个素端包括21个碱基对互补序列，以加入DNA扩增子AB和CD.使用100纳克纯化的染色体DNA作为模板，在两组不对称PCR中用AB和CD引物，然后在1%琼脂糖凝胶中分析所得产物。从电泳凝胶中纯化和定量切除的扩增子。将100纳克每种扩增子与PCR试剂混合，无需引物。扩展后，加入A和D引物并将其作为单个片段扩增。接下来，如前所述分析，纯化和量化所得产品。用内切酶消化一微克pDM4和400纳克AD扩增子。将消化的pDM4与AD扩增子以摩尔载体结合，插入比为1比4，并制备连接反应。在20摄氏度下孵育过夜。之后，在70摄氏度下灭活T4连接酶20分钟。将大肠杆菌菌株接种到Luria Bertani Miller肉汤中，并在30摄氏度下孵育，以200 RPM振荡，直到观察到0.4至0.6之间的光密度。将细菌以5， 000倍G和4摄氏度离心15分钟进行沉淀培养。将颗粒悬浮在冷却的蒸馏水中，并重复清洁过程两次。将细菌悬浮液转移到微量离心管中，并在14， 000倍G和4摄氏度下重复离心五分钟。将沉淀重悬于冷冻水中后，加入约3.5微升连接混合物并在冰上孵育5分钟。将内容物转移到冷却的两毫米间隙电穿孔比色皿中。电穿孔后加入SOC培养基并将内容物转移到培养管中。在30摄氏度下孵育一小时，以200 RPM振荡。将转化后的细胞接种在补充氯霉素的LB琼脂平板上。使用pDM4克隆侧侧的引物，通过PCR检查将缺失构建体插入pDM4。准备过夜培养物，使受体气单胞菌菌株耐利福平和两种不同的大肠杆菌菌株。具有pDM4重组质粒的供体菌株，以及具有PRK 2073质粒的辅助菌株在30摄氏度。用150微升LB将相同数量的供体，受体和辅助菌株的菌落悬浮在三个LB管中。然后在不含抗生素的LB琼脂平板上将三种细菌悬浮液在接受者与辅助者与供体的比例为五比一的两滴中混合，并将板面朝上孵育在30摄氏度下过夜。通过在LB琼脂平板中加入一毫升LB肉汤来收获细菌。将细菌悬浮液转移到锥形1.5毫升微量离心管中并剧烈涡旋。用利福平和氯霉素在LB琼脂平板上涂抹100微升细菌悬浮液。旋转200微升和600微升样品，将沉淀悬浮在100微升LB肉汤中，并用利福平和氯霉素将平板放在LB琼脂上，然后孵育。进行氧化酶测试以确认菌落是气单胞菌。进一步确认使用E和F引物通过PCR将pDM4重组质粒插入到特定的染色体区域。用10%蔗糖和不含抗生素在30摄氏度下在两毫升LB中生长重组菌落过夜。制备不同的培养稀释液后，将100微升细菌培养物和稀释液放在LB琼脂平板上，用10%蔗糖在30摄氏度下孵育过夜。第二天，拿起菌落并将其转移到含或不带氯霉素的LB平板上。在30摄氏度下孵育过夜后，选择氯霉素敏感菌落。使用EF引物对通过PCR分析敏感菌落，并选择其PCR产物与删除的构建体大小相关的菌落。在盖玻片的四个角上滴一小滴硅胶，然后将过夜生长的气单胞菌培养物安装在盖玻片上。将挖掘出的载玻片放在盖玻片上，然后将滑梯倒置。使用光学显微镜通过光学显微镜分析游泳运动，然后将三微升培养物转移到软琼脂板的中心。将板面朝上孵育在25至30摄氏度之间过夜。在孵育结束时使用尺子，测量细菌通过琼脂从板中心向外围的迁移。培养气单胞菌在25至30摄氏度之间的900毫升TSB中过夜。离心后，将沉淀悬浮在pH为7.8的100毫摩尔三氯化氢缓冲液的20毫升中。为了去除锚定的鞭毛，将悬浮液在带有玻璃棒的漩涡中三到四分钟，然后重复通过21号注射器。通过四摄氏度的两次连续离心沉淀细菌，并将上清液收集到单独的管中。超速离心上清液后，将沉淀悬浮在150微升含有两毫摩尔EDTA缓冲液的100毫摩尔三氯化氢中。将150微升富集的鞭毛部分转移到薄壁超透明管中，并用12毫升氯化铯溶液填充。在摆动的桶形转子中以60， 000倍G在4摄氏度下超速离心管48小时。用注射器将鞭毛带收集到氯化铯梯度中，并透析100毫摩尔三氯化氢加两毫摩尔EDTA，然后通过电泳和考马斯蓝色染色或质谱分析。在下图中，通道 WT 是 Aeromonas piscicola AH3。泳道1至8显示第二种重组的氯霉素敏感菌落。ST 段是膀胱亢进 1KB 标志物。泳道1至3和5至8显示具有野生型FGI4基因的菌落作为泳道WT。泳道四显示了一个具有已删除FGI4基因的集落。光学显微镜检测表明，极性或侧鞭毛修饰仅导致迁移直径减小，因此，AH-3 FGI突变体和AH-3 FGI-4突变体仅显示出相对于野生型菌株的运动性降低。该图显示了来自泳道一的AH-3的极性鞭毛，以及泳道二的FGI区域和三车道的FGI-4基因中没有突变体，其中两个突变体都具有分子量低于野生型菌株的极性鞭毛，这表明鞭毛聚糖的改变。重要的是要确保突变不会影响下游基因，并导致鞭毛无法组装或鞭毛细丝无法发育。当鞭毛蛋白的变化不可检测到时，需要使用鞭毛蛋白肽的总蛋白进行质谱分析，以了解聚糖质量。

Research

JoVE Journal

Immunology and Infection

Using Luciferase to Image Bacterial Infections in Mice

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Bioluminescent Bacterial Imaging In Vivo

Bioluminescent Bacterial Imaging In Vivo

生物发光细菌成像<em在体内</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

方法来抑制细菌Pyomelanin生产和确定敏感性氧化应激的影响相应增加

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

抽动运动功能的可视化和表征的角色的<em&gt; PilG</em&gt;在<em&gt;叶缘焦枯病菌</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Vitronectin 在细菌黏附和血清耐药性中作用的实验研究

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...