Current Protocols in Microbiology provides detailed, step-by-step instructions for analyzing bacteria, animal and plant viruses, fungi, protozoans and other microbes. It offers updated coverage of emerging technologies and concepts, such as biofilms, quorum sensing and quantitative PCR, as well as proteomic and genomic methods. It is the first comprehensive source of high-quality microbiology protocols that reflects and incorporates the new mandates and capabilities of this robust and rapidly evolving discipline.

Current Protocols in Microbiology offers detailed, step-by-step instructions for analyzing bacteria, animal and plant viruses, fungi, protozoans, and other microbes. It provides updated coverage of emerging technologies and concepts, such as biofilms, quorum sensing, and quantitative PCR, as well as proteomic and genomic methods. It is the first comprehensive source of high-quality microbiology protocols that reflects and incorporates the new mandates and capabilities of this robust and rapidly evolving discipline.

《Current Protocols in Microbiology》提供了详细的逐步指导，帮助研究人员分析细菌、动物和植物病毒、真菌、原生动物及其他微生物。期刊涵盖了新兴技术和概念的最新进展，如生物膜、群体感应和定量PCR，以及蛋白质组学和基因组学方法。它是首个全面的高质量微生物学协议来源，反映并融入了这一快速发展的学科的新要求和能力。

Current Protocols in Microbiology

Abstract Many bacteria can become naturally competent to take up extracellular DNA across their outer and inner membranes by a dedicated competence apparatus. Whereas some studies show that the DNA delivered to the cytoplasm may be used for genome repair or for nutrition, it can also be recombined onto the chromosome by homologous recombination: a process called natural transformation. Along with conjugation and transduction, natural transformation represents a mechanism for horizontal transfer of genetic material, e.g., antibiotic resistance genes, which can confer new beneficial characteristics onto the recipient bacteria. Described here are protocols for quantifying the frequency of transformation for the human pathogen Vibrio cholerae , one of several Vibrio species recently shown to be capable of natural transformation. © 2014 by John Wiley &amp; Sons, Inc.

Samit S. Watve

Eryn E. Bernardy

Brian K. Hammer

Vibrio cholerae: Measuring Natural Transformation Frequency

霍乱弧菌：自然转化频率的测定

Rift Valley fever virus (RVFV) is an arthropod-borne, zoonotic disease endemic to sub-Saharan Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever have had up to 100% mortality rates in fetal and neonatal sheep. Upon infection of ruminant and human hosts alike, RVFV infection causes an at times severe hepatitis and pathology in many other organs. The enveloped virion contains a tripartite, predominantly negative-sense, single-stranded RNA genome, which codes for the proteins the virus needs to replicate both in mammalian hosts and insect vectors. Endemic countries often use attenuated RVFV strains for vaccination of livestock but there are no commercially licensed vaccines for humans or livestock in non-endemic areas. In the laboratory, RVFV can be readily propagated and manipulated in vitro using cell culture systems. Presented in this article are techniques routinely used in RVFV research that have proven successful in our laboratories. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Propagation of Rift Valley fever virus in mammalian cells Basic Protocol 2: Quantification of Rift Valley fever virus by plaque assay Basic Protocol 3: Quantification of Rift Valley fever virus by 50% tissue culture infectious dose (TCID50 ) assay Basic Protocol 4: Quantification of Rift Valley fever virus by focus-forming assay Basic Protocol 5: Storage and disinfection Alternate Protocol 1: Propagation of Rift Valley fever virus in MRC-5 cells Alternate Protocol 2: Propagation of RVFV in mosquito-derived cells Alternate Protocol 3: TCID50 detection using fluorescence visualization Support Protocol 1: Calculation of the amount of virus needed to infect a flask at a chosen multiplicity of infection Support Protocol 2: Calculation of the virus titer by plaque assay or focus-forming assay Support Protocol 3: Calculation of the TCID50 titer by the method of Reed and Muench Support Protocol 4: Calculation of the antibody volume for the focus-forming assay.

MaRyka R. Smith

Erin E. Schirtzinger

William C. Wilson

A. Sally Davis

Rift Valley Fever Virus: Propagation, Quantification, and Storage

裂谷热病毒：繁殖、定量与储存

Shigella species, which are closely related to Escherichia coli, can easily be maintained and stored in the laboratory. This article includes protocols for preparation of routine growth conditions and media, for storage of the bacteria, and for monitoring of the presence of the virulence plasmid. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Growth of S. flexneri from frozen stocks or agar stabs Basic Protocol 2: Growth of S. flexneri in rich liquid medium Alternate Protocol 1: Growth of S. flexneri in rich defined medium Alternate Protocol 2: Growth of S. flexneri in minimal medium Basic Protocol 3: Storage of S. flexneri in frozen stocks Alternate Protocol 3: Storage of S. flexneri in agar stabs.

Shelley M. Payne

Laboratory Cultivation and Storage of Shigella

志贺氏菌的实验室培养与保存

Cryptococcus neoformans is an opportunistic human fungal pathogen commonly associated with infection in immunocompromised individuals (e.g., patients with HIV/AIDS). Important virulence determinants include the production of a polysaccharide capsule, melanin, and extracellular enzymes, as well as the ability to grow at 37°C. C. neoformans controls a plethora of host defense and evasion mechanisms to survive during infection and to proliferate within the host, causing meningoencephalitis and death. Traditionally, characterization of C. neoformans under different environmental conditions and stresses has relied on genetic and phenotypic analyses, as well as biochemical assays. However, advances in mass spectrometry instrumentation, sample preparation protocols, and bioinformatic tools and databases promote comprehensive profiling of fungal cellular processes, secretion or protein release into the extracellular environment, and vesicle contents. Moreover, proteomics provides insight into regulatory mechanisms influencing signal transduction cascades and protein complexes or networks through profiling of post-translational modifications and protein-protein interactions. Given the medical impact of C. neoformans infections and the recent emergence of antifungal-resistant strains, defining proteins produced in response to unique environments provides an opportunity to uncover antivirulence strategies and alternative therapeutic options to combat infection. Here, we describe culturing and sample preparation of C. neoformans and outline protocols for comprehensively profiling changes in protein abundance within the cellular proteome and secretome. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Growth and sample preparation of Cryptococcus neoformans Basic Protocol 2: Protein extraction from supernatant Basic Protocol 3: Protein extraction from cell pellet Basic Protocol 4: Proteomic profiling and bioinformatics.

Brianna Ball

Jennifer Geddes‐McAlister

Quantitative Proteomic Profiling of Cryptococcus neoformans

新型隐球菌的定量蛋白质组学分析

Abstract Cheese rind microbiomes are useful model systems for identifying the mechanisms that control microbiome diversity. Here, we describe the methods we have optimized to first deconstruct in situ cheese rind microbiome diversity and then reconstruct that diversity in laboratory environments to conduct controlled microbiome manipulations. Most cheese rind microbial species, including bacteria, yeasts, and filamentous fungi, can be easily cultured using standard lab media. Colony morphologies of taxa are diverse and can often be used to distinguish taxa at the phylum and sometimes even genus level. Through the use of cheese curd agar medium, thousands of unique community combinations or microbial interactions can be assessed. Transcriptomic experiments and transposon mutagenesis screens can pinpoint mechanisms of interactions between microbial species. Our general approach of creating a tractable synthetic microbial community from cheese can be easily applied to other fermented foods to develop other model microbiomes. © 2019 by John Wiley &amp; Sons, Inc. Basic Protocol 1 : Isolation of cheese rind microbial communities Support Protocol 1 : Preparation of plate count agar with milk and salt Basic Protocol 2 : Identification of cheese rind bacterial and fungal isolates using 16S and ITS sequences Basic Protocol 3 : Preparation of experimental glycerol stocks of yeasts and bacteria Basic Protocol 4 : Preparation of experimental glycerol stocks of filamentous fungi Basic Protocol 5 : Reconstruction of cheese rind microbial communities in vitro Support Protocol 2 : Preparation of lyophilized and powdered cheese curd Support Protocol 3 : Preparation of 10% cheese curd agar plates and tubes Basic Protocol 6 : Interaction screens using responding lawns Support Protocol 4 : Preparation of liquid 2% cheese curd Basic Protocol 7 : Experimental evolution Basic Protocol 8 : Measuring community function: pH/acidification Basic Protocol 9 : Measuring community function: Pigment production Basic Protocol 10 : RNA sequencing of cheese rind biofilms

Casey M. Cosetta

Benjamin E. Wolfe

Deconstructing and Reconstructing Cheese Rind Microbiomes for Experiments in Microbial Ecology and Evolution

解构与重构奶酪皮微生物群落：微生物生态与进化实验

Abstract Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1 : sgRNA design Basic Protocol 2 : Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors Basic Protocol 3 : Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria Basic Protocol 4 : ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales Support Protocol 1 : Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2 : Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3 : Transformation of E. coli by electroporation Support Protocol 4 : Transformation of CaCl 2 ‐competent E. coli

Amy B. Banta

Ryan D. Ward

Jennifer S. Tran

Emily E. Bacon

Jason M. Peters

Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi

使用移动CRISPRi在多种细菌中实现可编程基因敲降

Abstract Vibrio parahaemolyticus is a Gram‐negative, halophilic bacterium and opportunistic pathogen of humans and shrimp. Investigating the mechanisms of V. parahaemolyticus infection and the multifarious virulence factors it employs requires procedures for bacterial culture, genetic manipulation, and analysis of virulence phenotypes. Detailed protocols for growth assessment, generation of mutants, and phenotype assessment are included in this article. © 2020 Wiley Periodicals LLC. Basic Protocol 1 : Assessment of growth of V. parahaemolyticus Alternate Protocol 1 : Assessment of growth of V. parahaemolyticus using a plate reader Basic Protocol 2 : Swimming/swarming motility assay Basic Protocol 3 : Genetic manipulation Alternate Protocol 2 : Natural transformation Basic Protocol 4 : Secretion assay and sample preparation for mass spectrometry analysis Basic Protocol 5 : Invasion assay (gentamicin protection assay) Basic Protocol 6 : Immunofluorescence detection of intracellular V. parahaemolyticus Basic Protocol 7 : Cytotoxicity assay for T3SS2

Suneeta Chimalapati

Alexander E. Lafrance

Luming Chen

Kim Orth

Vibrio parahaemolyticus: Basic Techniques for Growth, Genetic Manipulation, and Analysis of Virulence Factors

副溶血性弧菌：生长、遗传操作及毒力因子分析的基本技术

Dimorphic fungi in the genera Blastomyces, Histoplasma, Coccidioides, and Paracoccidioides are important human pathogens that affect human health in many countries throughout the world. Understanding the biology of these fungi is important for the development of effective treatments and vaccines. Gene editing is a critically important tool for research into these organisms. In recent years, gene targeting approaches employing RNA‐guided DNA nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease 9 (Cas9), have exploded in popularity. Here, we provide a detailed description of the steps involved in applying CRISPR/Cas9 technology to dimorphic fungi, with Blastomyces dermatitidis in particular as our model fungal pathogen. We discuss the design and construction of single guide RNA and Cas9‐expressing targeting vectors (including multiplexed vectors) as well as introduction of these plasmids into Blastomyces using Agrobacterium‐mediated transformation. Finally, we cover the outcomes that may be expected in terms of gene‐editing efficiency and types of gene alterations produced. © 2020 Wiley Periodicals LLC.

Gregory C. Kujoth

Thomas D. Sullivan

Bruce S. Klein

Gene Editing in Dimorphic Fungi Using CRISPR/Cas9

二态真菌中的基因编辑使用CRISPR/Cas9

Abstract Human papillomavirus (HPV) infection occurs in differentiating epithelial tissues. Cancers caused by high‐risk types (e.g., HPV16 and HPV18) typically occur at oropharyngeal and anogenital anatomical sites. The HPV life cycle is differentiation‐dependent, requiring tissue culture methodology that is able to recapitulate the three‐dimensional (3D) stratified epithelium. Here we report two distinct and complementary methods for growing differentiating epithelial tissues that mimic many critical morphological and biochemical aspects of in vivo tissue. The first approach involves growing primary human epithelial cells on top of a dermal equivalent consisting of collagen fibers and living fibroblast cells. When these cells are grown at the liquid‐air interface, differentiation occurs and allows for epithelial stratification. The second approach uses a rotating wall vessel bioreactor. The low‐fluid‐shear microgravity environment inside the bioreactor allows the cells to use collagen‐coated microbeads as a growth scaffold and self‐assemble into 3D cellular aggregates. These approaches are applied to epithelial cells derived from HPV‐positive and HPV‐negative oral and cervical tissues. The second part of the article introduces potential downstream applications for these 3D tissue models. We describe methods that will allow readers to start successfully culturing 3D tissues from oral and cervical cells. These tissues have been used for microscopic visualization, scanning electron microscopy, and large omics‐based studies to gain insights into epithelial biology, the HPV life cycle, and host‐pathogen interactions. © 2020 Wiley Periodicals LLC. Basic Protocol 1 : Establishing human primary cell–derived 3D organotypic raft cultures Support Protocol 1 : Isolation of epithelial cells from patient‐derived tissues Support Protocol 2 : Growth and maintenance of primary human epithelial cells in monolayer culture Support Protocol 3 : PCR‐based HPV screening of primary cell cultures Basic Protocol 2 : Establishing human 3D cervical tissues using the rotating wall vessel bioreactor Support Protocol 4 : Growth and maintenance of human A2EN cells in monolayer culture Support Protocol 5 : Preparation of the slow‐turning lateral vessel bioreactor Support Protocol 6 : Preparation of Cytodex‐3 microcarrier beads Basic Protocol 3 : Histological assessment of 3D organotypic raft tissues Basic Protocol 4 : Spatial analysis of protein expression in 3D organotypic raft cultures Basic Protocol 5 : Immunofluorescence imaging of RWV‐derived 3D tissues Basic Protocol 6 : Ultrastructural visualization and imaging of RWV‐derived 3D tissues Basic Protocol 7 : Characterization of gene expression by RT‐qPCR

Robert Jackson

Jason D. Maarsingh

Melissa M. Herbst‐Kralovetz

Koenraad Van Doorslaer

3D Oral and Cervical Tissue Models for Studying Papillomavirus Host‐Pathogen Interactions

研究乳头瘤病毒宿主-病原体相互作用的3D口腔和宫颈组织模型

Abstract The fungus Zymoseptoria tritici is one of the most devastating pathogens of wheat. Aside from its importance as a disease‐causing agent, this species has emerged as a powerful model system for evolutionary genetic studies of crop‐infecting fungal pathogens. Z. tritici exhibits exceptionally high levels of genetic and phenotypic diversity as well as morphological plasticity, which can make experimental studies and comparability of results obtained in different laboratories, e.g., from infection assays, challenging. Therefore, standardized experimental methods are crucial for research on Z. tritici biology and the interaction of this fungus with its wheat host. Here, we describe a suite of well‐tested and optimized protocols ranging from isolation of Z. tritici field specimens to analyses of virulence assays under controlled conditions. Several biological and technical aspects of working with Z. tritici under laboratory conditions are considered and carefully described in each protocol. © 2020 The Authors. Basic Protocol 1 : Purification of Z. tritici field isolates from leaf material Basic Protocol 2 : Molecular identification of Z. tritici isolates Support Protocol 1 : Rapid extraction of Z. tritici genomic DNA Support Protocol 2 : Extraction of high‐quality Z. tritici genomic DNA Basic Protocol 3 : In vitro culture and long‐term storage of Z. tritici isolates Basic Protocol 4 : Analysis of Z. tritici virulence in wheat Support Protocol 3 : Preparation of Z. tritici inoculum

Wagner C. Fagundes

Janine Haueisen

Eva H. Stukenbrock

Dissecting the Biology of the Fungal Wheat Pathogen Zymoseptoria tritici: A Laboratory Workflow

剖析真菌小麦病原体Zymoseptoria tritici的生物学：实验室工作流程

ISSN	1934-8525, 1934-8533
h-index	47

Publication Information	Publisher: John Wiley & Sons Inc.，Publishing cycle: ，Journal Type: journal，Open Access Journals: No
Basic data	Year of publication: 2005，Proportion of original research papers: 0.00%，Self Citation Rate:N.A.， Gold OA Rate: 0.00%

Current Protocols in Microbiology

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