Curr Protoc Microbiol [JOURNAL]
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200 papers
Genetic Manipulation of Corynebacterium diphtheriae and Other Corynebacterium Species.
Chang C, Nguyen MT, Ton-That H
Curr Protoc Microbiol · 2020 Sep · PMID 32865881 · Full text
This article describes several established approaches for genetic manipulation of Corynebacterium diphtheriae, the causative agent of diphtheria that is known to have provided key evidence for Koch's postulates on the ge...
This article describes several established approaches for genetic manipulation of Corynebacterium diphtheriae, the causative agent of diphtheria that is known to have provided key evidence for Koch's postulates on the germ theory. First, it includes a detailed gene deletion method that generates nonpolar, in-frame, markerless deletion mutants, utilizing the levansucrase SacB as a counter-selectable marker. Second, it provides a thorough protocol for rescuing deletion mutants using Escherichia coli-Corynebacterium shuttle vectors. Finally, a Tn5 transposon mutagenesis procedure is described. In principle, these protocols can be used for other Corynebacterium species, including Corynebacterium glutamicum and Corynebacterium matruchotii. © 2020 Wiley Periodicals LLC Basic Protocol 1: Gene deletion in Corynebacterium diphtheriae Basic Protocol 2: Complementation of a mutant strain Basic Protocol 3: Tn5 transposon mutagenesis of Corynebacterium diphtheriae.
A Low-Cost Tebuconazole-Based Screening Test for Azole-Resistant Aspergillus fumigatus.
Brackin AP, Shelton JMG, Abdolrasouli A … +2 more , Fisher MC, Sewell TR
Curr Protoc Microbiol · 2020 Sep · PMID 32857921 · Publisher ↗
The global emergence of azole resistance in Aspergillus fumigatus is resulting in health and food security concerns. Rapid diagnostics and environmental surveillance methods are key to understanding the distribution and...
The global emergence of azole resistance in Aspergillus fumigatus is resulting in health and food security concerns. Rapid diagnostics and environmental surveillance methods are key to understanding the distribution and prevalence of azole resistance. However, such methods are often associated with high costs and are not always applicable to laboratories based in the least-developed countries. Here, we present and validate a low-cost screening protocol that can be used to differentiate between azole-susceptible "wild-type" and azole-resistant A. fumigatus isolates. © 2020 The Authors. Basic Protocol 1: Preparation of Tebucheck multi-well plates Basic Protocol 2: Inoculation of Tebucheck multi-well plates.
The Propagation, Quantification, and Storage of Vesicular Stomatitis Virus.
Abdelmageed AA, Ferran MC
Curr Protoc Microbiol · 2020 Sep · PMID 32833351 · Full text
Vesicular stomatitis virus (VSV) is the prototypical member of the Rhabdoviridae family of negative-sense single-stranded RNA viruses. This virus has been used as a powerful model system for decades and is currently bein...
Vesicular stomatitis virus (VSV) is the prototypical member of the Rhabdoviridae family of negative-sense single-stranded RNA viruses. This virus has been used as a powerful model system for decades and is currently being used as a vaccine platform and an oncolytic agent. Here, we present methods to propagate, quantitate, and store VSV. We also review the proper safety protocol for the handling of VSV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation, purification, and storage of vesicular stomatitis virus stocks Basic Protocol 2: Quantification of vesicular stomatitis virus by plaque assay Support Protocol: Propagation of Vero cells.
Corynebacterium diphtheriae Virulence Analyses Using a Caenorhabditis elegans Model.
Chen YW, Ton-That H
Curr Protoc Microbiol · 2020 Sep · PMID 32658384 · Full text
Corynebacterium diphtheriae is the leading cause of pharyngeal diphtheria, a respiratory disease characterized by formation of a pseudomembrane at the site of infection. Although outbreaks of C. diphtheriae infections ar...
Corynebacterium diphtheriae is the leading cause of pharyngeal diphtheria, a respiratory disease characterized by formation of a pseudomembrane at the site of infection. Although outbreaks of C. diphtheriae infections are rare nowadays, the emergence of multidrug-resistant C. diphtheriae strains is one of the most significant public health concerns worldwide. Although C. diphtheriae has been studied for more than a century and diphtheria toxin and pili have been identified as major virulence factors, little is known about factors involved in bacterial colonization and development of disease. Here, we describe the utilization of Caenorhabditis elegans as a cost-effective, versatile model of infection to evaluate C. diphtheriae virulence. We provide detailed protocols for nematode synchronization and for evaluation of nematode survival and formation of a deformed anal region induced by C. diphtheriae infection. These protocols will permit future high-throughput screenings of virulence factors in C. diphtheriae and advance our knowledge of C. diphtheriae pathogenesis. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synchronization of nematodes Basic Protocol 2: Assay for nematode survival following C. diphtheriae infection Basic Protocol 3: Assays for bacterial colonization and formation of deformed anal region.
Human Bocavirus 1 Infection of Well-Differentiated Human Airway Epithelium.
Yan Z, Deng X, Qiu J
Curr Protoc Microbiol · 2020 Sep · PMID 32639683 · Full text
Human bocavirus 1 (HBoV1) is a small DNA virus that belongs to the Bocaparvovirus genus of the Parvoviridae family. HBoV1 is a common respiratory pathogen that causes mild to life-threatening acute respiratory tract infe...
Human bocavirus 1 (HBoV1) is a small DNA virus that belongs to the Bocaparvovirus genus of the Parvoviridae family. HBoV1 is a common respiratory pathogen that causes mild to life-threatening acute respiratory tract infections in children and immunocompromised individuals, infecting both the upper and lower respiratory tracts. HBoV1 infection causes death of airway epithelial cells, resulting in airway injury and inflammation. In vitro, HBoV1 only infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI), but not any dividing human cells. A full-length HBoV1 genome of 5543 nucleotides has been cloned from DNA extracted from a human nasopharyngeal swab into a plasmid called HBoV1 infectious clone pIHBoV1. Transfection of pIHBoV1 replicates efficiently in human embryonic kidney 293 (HEK293) cells and produces virions that are highly infectious. This article describes protocols for production of HBoV1 in HEK293 cells, generation of HAE-ALI cultures, and infection with HBoV1 in HAE-ALI. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Human bocavirus 1 production in HEK293 cells Support Protocol 1: HEK293 cell culture and transfection Support Protocol 2: Quantification of human bocavirus 1 using real-time quantitative PCR Basic Protocol 2: Differentiation of human airway cells at an air-liquid interface Support Protocol 3: Expansion of human airway epithelial cell line CuFi-8 Support Protocol 4: Expansion of human airway basal cells Support Protocol 5: Coating of plastic dishes and permeable membranes of inserts Support Protocol 6: Transepithelial electrical resistance measurement Basic Protocol 3: Human bocavirus 1 infection in human airway epithelium cultured at an air-liquid interface Support Protocol 7: Isolation of infected human airway epithelium cells from inserts Basic Protocol 4: Transduction of airway basal cells with lentiviral vector.
Improved Method for Transformation of Vibrio vulnificus by Electroporation.
Jayakumar JM, Shapiro OH, Almagro-Moreno S
Curr Protoc Microbiol · 2020 Sep · PMID 32614522 · Publisher ↗
Vibrio vulnificus, an emergent human pathogen, causes fulminant septicemia with a mortality rate of over 50%. Unlike for other pathogenic Vibrio species, the factors to conclusively indicate the virulence potential of V....
Vibrio vulnificus, an emergent human pathogen, causes fulminant septicemia with a mortality rate of over 50%. Unlike for other pathogenic Vibrio species, the factors to conclusively indicate the virulence potential of V. vulnificus strains remain largely unknown. Understanding the pathogenesis of this bacterium at a molecular level is severely hindered by inefficiencies in transformation, for instance, due to the presence of a periplasmic nuclease, Vvn. Currently, successful transformation of V. vulnificus is nearly impossible due to lack of mobilizable plasmids for the bacterium, requiring (i) very high DNA concentrations, (ii) plasmid linearization, (iii) development of novel V. vulnificus-derived plasmids, or (iv) time-consuming conjugation-based methods. To overcome these limitations, we describe a rapid, efficient, and reproducible electroporation protocol to effectively transform widely available plasmids, with different copy numbers and antibiotic resistances, into phylogenetically distant strains of V. vulnificus. Cells are made competent in high concentrations of sucrose devoid of cations and recovered from electroporation using a high-salinity recovery medium. Compared to existing methods for transformation of V. vulnificus, significantly higher efficiencies are obtained using this improved protocol. Rapid and effective transformations can markedly improve molecular analyses of V. vulnificus leading to a greater understanding of its virulence potential. This is crucial to develop rapid detection methods which have the potential to prevent future outbreaks. The electroporation protocol described here may be particularly useful for optimizing transformation of other nuclease-producing bacteria. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent cells Basic Protocol 2: Transformation of cells by electroporation.
An In Vitro Microneutralization Assay for SARS-CoV-2 Serology and Drug Screening.
Amanat F, White KM, Miorin L … +10 more , Strohmeier S, McMahon M, Meade P, Liu WC, Albrecht RA, Simon V, Martinez-Sobrido L, Moran T, García-Sastre A, Krammer F
Curr Protoc Microbiol · 2020 Sep · PMID 32585083 · Full text
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the city of Wuhan, Hubei Province, China, in late 2019. Since then, the virus has spread globally and caused a pandemic. Assays that can measure...
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the city of Wuhan, Hubei Province, China, in late 2019. Since then, the virus has spread globally and caused a pandemic. Assays that can measure the antiviral activity of antibodies or antiviral compounds are needed for SARS-CoV-2 vaccine and drug development. Here, we describe in detail a microneutralization assay, which can be used to assess in a quantitative manner if antibodies or drugs can block entry and/or replication of SARS-CoV-2 in vitro. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Microneutralization assay to test inhibition of virus by antibodies (purified antibodies or serum/plasma) Basic Protocol 2: Screening of anti-SARS-CoV-2 compounds in vitro Support Protocol: SARS-CoV-2 propagation.
Genetic Manipulation and Virulence Assessment of Fusobacterium nucleatum.
Peluso EA, Scheible M, Ton-That H … +1 more , Wu C
Curr Protoc Microbiol · 2020 Jun · PMID 32539234 · Full text
Considered a commensal, the Gram-negative anaerobe Fusobacterium nucleatum is a key member of the oral microbiome due to its wide range of interactions with many oral microbes. While the periodontal pathogenic properties...
Considered a commensal, the Gram-negative anaerobe Fusobacterium nucleatum is a key member of the oral microbiome due to its wide range of interactions with many oral microbes. While the periodontal pathogenic properties of this organism have widely been examined, its connotation with extra-oral infections, including preterm birth and colorectal cancer, has now become apparent. Nonetheless, little is known about the mechanisms of pathogenicity and the associated virulence factors of F. nucleatum, most likely due to limited genetic tools and facile methodology. Here, we describe molecular techniques for the genetic manipulation of F. nucleatum, including markerless, nonpolar gene deletion, complementation, and Tn5 transposon mutagenesis. Further, we provide methodology to assess virulence potential of F. nucleatum using a mouse model of preterm birth. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of a galK mutant strain Basic Protocol 2: Complementation of a mutant strain Basic Protocol 3: Tn5 transposon mutagenesis of F. nucleatum Basic Protocol 4: Mouse model of preterm birth.
Giardia lamblia: Laboratory Maintenance, Lifecycle Induction, and Infection of Murine Models.
Fink MY, Shapiro D, Singer SM
Curr Protoc Microbiol · 2020 Jun · PMID 32515871 · Full text
Giardia lamblia is a protozoan parasite that is found ubiquitously throughout the world and is a major contributor to diarrheal disease. Giardia exhibits a biphasic lifestyle existing as either a dormant cyst or a vegeta...
Giardia lamblia is a protozoan parasite that is found ubiquitously throughout the world and is a major contributor to diarrheal disease. Giardia exhibits a biphasic lifestyle existing as either a dormant cyst or a vegetative trophozoite. Infections are typically initiated through the consumption of cyst-contaminated water or food. Giardia was first axenized in the 1970s and can be readily maintained in a laboratory setting. Additionally, Giardia is one of the few protozoans that can be induced to complete its complete lifecycle using laboratory methods. In this article, we outline protocols to maintain Giardia and induce passage through its lifecycle. We also provide protocols for infecting and quantifying parasites in an animal infection model. © 2020 Wiley Periodicals LLC. Basic Protocol 1: In vitro maintenance and growth of Giardia trophozoites Basic Protocol 2: In vitro encystation of Giardia cysts Basic Protocol 3: In vivo infections using Giardia trophozoites.
Vibrio fischeri: Laboratory Cultivation, Storage, and Common Phenotypic Assays.
Christensen DG, Visick KL
Curr Protoc Microbiol · 2020 Jun · PMID 32497392 · Full text
Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species that can be readily grown and stored with common laboratory equipment. In this article, protocols for routine growth, storage, and phenotyp...
Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species that can be readily grown and stored with common laboratory equipment. In this article, protocols for routine growth, storage, and phenotypic assessment of V. fischeri, as well as recipes for useful media, are included. Specifically, this article describes procedures and considerations for growth of this microbe in complex and minimal media. It also describes assays for biofilm formation, motility, and bioluminescence, three commonly assessed phenotypes of V. fischeri. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth of V. fischeri from frozen stocks Basic Protocol 2: Growth of V. fischeri in rich, undefined liquid medium Alternate Protocol 1: Growth of V. fischeri in minimal medium Basic Protocol 3: Storage of V. fischeri in frozen stocks Basic Protocol 4: Biofilm assay on solid agar Alternate Protocol 2: Biofilm assay in shaking liquid culture Alternate Protocol 3: Biofilm assay in static liquid culture Basic Protocol 5: Motility assay Basic Protocol 6: Luminescence assay.
Two Detailed Plaque Assay Protocols for the Quantification of Infectious SARS-CoV-2.
Mendoza EJ, Manguiat K, Wood H … +1 more , Drebot M
Curr Protoc Microbiol · 2020 Jun · PMID 32475066 · Full text
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causal agent of COronaVIrus Disease-19 (COVID-19), an atypical pneumonia-like syndrome that emerged in December 2019. While SARS-CoV...
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causal agent of COronaVIrus Disease-19 (COVID-19), an atypical pneumonia-like syndrome that emerged in December 2019. While SARS-CoV-2 titers can be measured by detection of viral nucleic acid, this method is unable to quantitate infectious virions. Measurement of infectious SARS-CoV-2 can be achieved by tissue culture infectious dose-50 (TCID ), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen. However, this method only provides a qualitative infectious virus titer. Plaque assays are a quantitative method of measuring infectious SARS-CoV-2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. As such, plaque assays remain the gold standard in quantifying concentrations of replication-competent lytic virions. Here, we describe two detailed plaque assay protocols to quantify infectious SARS-CoV-2 using different overlay and staining methods. Both methods have several advantages and disadvantages, which can be considered when choosing the procedure best suited for each laboratory. These assays can be used for several research purposes, including titration of virus stocks produced from infected cell supernatant and, with further optimization, quantification of SARS-CoV-2 in specimens collected from infected animals. © 2019 The Authors. Basic Protocol: SARS-CoV-2 plaque assay using a solid double overlay method Alternate Protocol: SARS-CoV-2 plaque assay using a liquid overlay and fixation-staining method.
Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes.
Porter SS, McBride AA
Curr Protoc Microbiol · 2020 Jun · PMID 32378811 · Full text
This protocol describes the production of human papillomavirus (HPV)-derived quasiviruses. Quasiviruses are infectious particles that are produced in 293TT packaging cells and contain a complete viral genome. We describe...
This protocol describes the production of human papillomavirus (HPV)-derived quasiviruses. Quasiviruses are infectious particles that are produced in 293TT packaging cells and contain a complete viral genome. We describe methods for infection of primary human keratinocytes with HPV quasiviruses, as well as assays to measure early viral DNA replication and transcription. Published 2020. U.S. Government. Basic Protocol 1: Transfection, harvest, and isolation of HPV quasiviruses Alternate Protocol 1: Packaging HPV DNA replicated in 293TT cells Alternate Protocol 2: Production of higher-purity quasivirus using the "Ripcord" method Support Protocol 1: Production of HPV minicircles Support Protocol 2: Production of recircularized HPV genomes Support Protocol 3: Screening of fractions for viral proteins Support Protocol 4: Screening of fractions for viral DNA Support Protocol 5: Measuring viral titer Support Protocol 6: Quantitation of quasivirions Basic Protocol 2: Infection of primary human foreskin keratinocytes with quasivirus Basic Protocol 3: HPV quasivirus transcription assay Basic Protocol 4: HPV quasivirus replication assay.
SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup.
Stadlbauer D, Amanat F, Chromikova V … +13 more , Jiang K, Strohmeier S, Arunkumar GA, Tan J, Bhavsar D, Capuano C, Kirkpatrick E, Meade P, Brito RN, Teo C, McMahon M, Simon V, Krammer F
Curr Protoc Microbiol · 2020 Jun · PMID 32302069 · Full text
In late 2019, cases of atypical pneumonia were detected in China. The etiological agent was quickly identified as a betacoronavirus (named SARS-CoV-2), which has since caused a pandemic. Several methods allowing for the...
In late 2019, cases of atypical pneumonia were detected in China. The etiological agent was quickly identified as a betacoronavirus (named SARS-CoV-2), which has since caused a pandemic. Several methods allowing for the specific detection of viral nucleic acids have been established, but these only allow detection of the virus during a short period of time, generally during acute infection. Serological assays are urgently needed to conduct serosurveys, to understand the antibody responses mounted in response to the virus, and to identify individuals who are potentially immune to re-infection. Here we describe a detailed protocol for expression of antigens derived from the spike protein of SARS-CoV-2 that can serve as a substrate for immunological assays, as well as a two-stage serological enzyme-linked immunosorbent assay (ELISA). These assays can be used for research studies and for testing in clinical laboratories. © 2020 The Authors. Basic Protocol 1: Mammalian cell transfection and protein purification Basic Protocol 2: A two-stage ELISA for high-throughput screening of human serum samples for antibodies binding to the spike protein of SARS-CoV-2.
Using Direct RNA Nanopore Sequencing to Deconvolute Viral Transcriptomes.
Depledge DP, Wilson AC
Curr Protoc Microbiol · 2020 Jun · PMID 32255550 · Full text
The genomes of DNA viruses encode deceptively complex transcriptomes evolved to maximize coding potential within the confines of a relatively small genome. Defining the full range of viral RNAs produced during an infecti...
The genomes of DNA viruses encode deceptively complex transcriptomes evolved to maximize coding potential within the confines of a relatively small genome. Defining the full range of viral RNAs produced during an infection is key to understanding the viral replication cycle and its interactions with the host cell. Traditional short-read (Illumina) sequencing approaches are problematic in this setting due to the difficulty of assigning short reads to individual RNAs in regions of transcript overlap and to the biases introduced by the required recoding and amplification steps. Additionally, different methodologies may be required to analyze the 5' and 3' ends of RNAs, which increases both cost and effort. The advent of long-read nanopore sequencing simplifies this approach by providing a single assay that captures and sequences full length RNAs, either in cDNA or native RNA form. The latter is particularly appealing as it reduces known recoding biases whilst allowing more advanced analyses such as estimation of poly(A) tail length and the detection of RNA modifications including N -methyladenosine. Using herpes simplex virus (HSV)-infected primary fibroblasts as a template, we provide a step-by-step guide to the production of direct RNA sequencing libraries suitable for sequencing using Oxford Nanopore Technologies platforms and provide a simple computational approach to deriving a high-quality annotation of the HSV transcriptome from the resulting sequencing data. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Productive infection of primary fibroblasts with herpes simplex virus Support Protocol: Cell passage and plating of primary fibroblasts Basic Protocol 2: Preparation and sequencing of dRNA-seq libraries from virus-infected cells Basic Protocol 3: Processing, alignment, and analysis of dRNA-seq datasets.
Aseptic Technique.
Bykowski T, Stevenson B
Curr Protoc Microbiol · 2020 Feb · PMID 32150342 · Publisher ↗
This article describes common laboratory procedures that can reduce the risk of culture contamination (sepsis), collectively referred as "aseptic technique." Two major strategies for aseptic work are described: using a B...
This article describes common laboratory procedures that can reduce the risk of culture contamination (sepsis), collectively referred as "aseptic technique." Two major strategies for aseptic work are described: using a Bunsen burner and using a laminar flow hood. Both methods are presented in the form of general protocols applicable to a variety of laboratory tasks such as pipetting and dispensing aliquots, preparing growth media, and inoculating, passaging, and spreading microorganisms on petri dishes. © 2020 by John Wiley & Sons, Inc.
Laboratory Maintenance and Growth of Talaromyces marneffei.
Andrianopoulos A
Curr Protoc Microbiol · 2020 Jan · PMID 32040264 · Publisher ↗
Talaromyces marneffei is an important opportunistic human pathogen endemic to Southeast Asia. It is one of a number of pathogenic fungi that exhibits thermally controlled dimorphism. At 25°C, T. marneffei grows in a mult...
Talaromyces marneffei is an important opportunistic human pathogen endemic to Southeast Asia. It is one of a number of pathogenic fungi that exhibits thermally controlled dimorphism. At 25°C, T. marneffei grows in a multicellular, filamentous hyphal form that can differentiate to produce dormant spores called conidia. These conidia are the likely infectious agent. At 37°C, T. marneffei grows as a uninucleate yeast that divides by fission. The yeast cells are the pathogenic form of this fungus. The protocols described here explain how to grow T. marneffei in the two vegetative growth forms in vitro, grow yeast cells inside mammalian macrophages, produce conidial stocks, and store strains both short and long term. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Growth of the vegetative hyphal form on solid medium Alternate Protocol 1: Growth of the vegetative hyphal form in liquid suspension Basic Protocol 2: Growth of the vegetative yeast form on solid medium Alternate Protocol 2: Growth of the vegetative yeast form in liquid suspension Basic Protocol 3: Growth for production of dormant conidia Support Protocol: Preparation of Miracloth filter tubes Basic Protocol 4: Growth of Talaromyces marneffei in mammalian macrophages Basic Protocol 5: Storage of Talaromyces marneffei strains Alternate Protocol 3: Lyophilization of Talaromyces marneffei strains.
Step-by-Step Pipeline for the Ecological Analysis of Endophytic Fungi using ITS nrDNA Data.
Montero-Vargas M, Escudero-Leyva E, Díaz-Valerio S … +1 more , Chaverri P
Curr Protoc Microbiol · 2020 Mar · PMID 31910332 · Publisher ↗
The nuclear ribosomal DNA internal transcribed spacer (ITS) is accepted as the genetic marker or barcode of choice for the identification of fungal samples. Here, we present a protocol to analyze fungal ITS data, from qu...
The nuclear ribosomal DNA internal transcribed spacer (ITS) is accepted as the genetic marker or barcode of choice for the identification of fungal samples. Here, we present a protocol to analyze fungal ITS data, from quality preprocessing of raw sequences to identification of operational taxonomic units (OTUs), taxonomic classification, and assignment of functional traits. The pipeline relies on well-established and manually curated data collections, namely the UNITE database and the FUNGuild script. As an example, real ITS data from culturable endophytic fungi were analyzed, providing detailed descriptions for every step, parameter, and downstream analysis, and finishing with a phylogenetic analysis of the sequences and assigned ecological roles. This article constitutes a comprehensive guide for researchers that have little familiarity with bioinformatic analysis of essential steps required in further ecological studies of fungal communities. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Raw sequencing data processing Support Protocol: Building a BLAST database Basic Protocol 2: Obtaining information from databases Basic Protocol 3: Phylogenetic analysis.
Deconstructing and Reconstructing Cheese Rind Microbiomes for Experiments in Microbial Ecology and Evolution.
Cosetta CM, Wolfe BE
Curr Protoc Microbiol · 2020 Mar · PMID 31891451 · Publisher ↗
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 diver...
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 & 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.
Laboratory Cultivation and Storage of Shigella.
Payne SM
Curr Protoc Microbiol · 2019 Dec · PMID 31816179 · Full text
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...
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.
Quantitative Proteomic Profiling of Cryptococcus neoformans.
Ball B, Geddes-McAlister J
Curr Protoc Microbiol · 2019 Dec · PMID 31797572 · Publisher ↗
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 productio...
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.
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