Current protocols in microbiology

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ISSN 1934-8533

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    ABSTRACT: Complex regulation of viral transcription patterns and DNA replication levels is a feature of many DNA viruses. This is especially true for those viruses which establish latent or persistent infections (e.g., herpesviruses, papillomaviruses, polyomaviruses, or adenovirus), as long-term persistence often requires adaptation of gene expression programs and/or replication levels to the cellular milieu. A key factor in the control of such processes is the establishment of a specific chromatin state on promoters or replication origins, which in turn will determine whether or not the underlying DNA is accessible for other factors that mediate downstream processes. Chromatin immunoprecipitation (ChIP) is a powerful technique to investigate viral chromatin, in particular to study binding patterns of modified histones, transcription factors or other DNA-/chromatin-binding proteins that regulate the viral lifecycle. Here, we provide protocols that are suitable for performing ChIP-PCR and ChIP-Seq studies on chromatin of large and small viral genomes.© 2016 by John Wiley & Sons, Inc.
    No preview · Article · Feb 2016 · Current protocols in microbiology
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    ABSTRACT: Nucleic acids can be obtained in numerous ways from clinical specimens; however, the quality of the nucleic acid is only as good as the sampling and isolation protocol. While nucleic acids may be extracted they may not be representative of the original source. Large areas of tissue and explanted hardware must be successfully surveyed to reflect the overall clinical picture. Once good sampling technique has been established, successful bacterial nucleic acid isolation is essential. Clinical samples may be difficult to process because of the presence of scar tissue, bone, implants, and bacterial biofilms. The following protocols provide details on sampling techniques and DNA isolation from a variety of clinical samples which can then be used in downstream molecular applications including PCR-MS-ESI-TOF technology. © 2016 by John Wiley & Sons, Inc.
    No preview · Article · Feb 2016 · Current protocols in microbiology
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    ABSTRACT: Nocardia spp. are aerobic, Gram-positive, catalase positive, and non-motile actinomycetes. They are associated with human infections. However, some species produce important natural products, degrade toxic chemicals, and are involved in biotransformation of valuable products. The lack of robust genetic tools has hindered detailed studies and advanced research. This unit describes the major genetic engineering approaches using Nocardia sp. CS682 as a prototype. These methods will certainly help in understanding the basis of their pathogenicity as well as biosynthetic and biotransforming abilities. It can be expected that knowledge of the biochemistry behind their pathogenicity will be crucial in developing effective treatment strategies. These genetic tools can be utilized to develop rational metabolic engineering approaches for crafting host strains with higher production or biotransformation ability. © 2016 by John Wiley & Sons, Inc.
    No preview · Article · Feb 2016 · Current protocols in microbiology
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    ABSTRACT: Papillomaviruses are a family of small, non-enveloped DNA tumor viruses. Knowing a complete transcription map of each papillomavirus genome can provide guidance for various papillomavirus studies. This unit provides detailed protocols to construct a transcription map of human papillomavirus type 18. The same approach can be easily adapted to other transcription map studies of any other papillomavirus genotype due to the high degree of conservation in genome structure, organization, and gene expression among papillomaviruses. The focused methods are 5'- and 3'-rapid amplification of cDNA ends (RACE), which are techniques commonly used in molecular biology to obtain full-length RNA transcript or to map a transcription start site (TSS) or an RNA polyadenylation (pA) cleavage site. Primer walking RT-PCR is a method for studying the splicing junction of RACE products. In addition, RNase protection assay and primer extension are also introduced as alternative methods in the mapping analysis. © 2016 by John Wiley & Sons, Inc.
    No preview · Article · Feb 2016 · Current protocols in microbiology
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    ABSTRACT: Genetic manipulation of obligate intracellular bacteria of the genus Rickettsia is currently undergoing a rapid period of change. The development of viable genetic tools, including replicative plasmids, transposons, homologous recombination, fluorescent protein-encoding genes, and antibiotic selectable markers has provided the impetus for future research development. This unit is designed to coalesce the basic methods pertaining to creation of genetically modified Rickettsia. The unit describes a series of methods, from inserting exogenous DNA into Rickettsia to the final isolation of genetically modified bacterial clones. Researchers working towards genetic manipulation of Rickettsia or similar obligate intracellular bacteria will find these protocols to be a valuable reference. © 2015 by John Wiley & Sons, Inc.
    No preview · Article · Nov 2015 · Current protocols in microbiology
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    ABSTRACT: This unit first describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector to generate a recombinant virus. Methods are also presented for purifying vaccinia virus and for isolating viral DNA, which can be used during transfection. Also presented are selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented.
    No preview · Article · Nov 2015 · Current protocols in microbiology
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    ABSTRACT: This unit is an overview of biosafety compliance and oversight in the United States. Specific attention is given to the oversight of the Institutional Biosafety Committee (IBC) and how the purview of the IBC may overlap with other local committees, such as the Institutional Animal Care and Use Committee (IACUC) for animal research and the Institutional Review Board (IRB) for research on human subjects. Requirements for the Federal Select Agent Program and Dual Use Research of Concern (DURC) are also briefly reviewed for those working with materials and experiments covered under these regulations. This unit serves as a guide for new and established investigators who are navigating the regulatory world and how regulatory oversight applies to their research. © 2015 by John Wiley & Sons, Inc.
    No preview · Article · Nov 2015 · Current protocols in microbiology
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    ABSTRACT: Myxobacteria are a highly social group among the delta proteobacteria that display unique multicellular behaviors during their complex life cycle and provide a rare opportunity to study the boundary between single cells and multicellularity. These organisms are also unusual as their entire life cycle is surface associated and includes a number of social behaviors: social gliding and rippling motility, 'wolf-pack'-like predation, and self-organizing complex biostructures, termed fruiting bodies, which are filled with differentiated environmentally resistant spores. Here we present methods for the growth, maintenance, and storage of Myxococcus xanthus, the most commonly studied of the myxobacteria. We also include methods to examine various developmental and social behaviors (fruiting body and spore formation, predation, and rippling motility). As the myxobacteria, similar to the streptomycetes, are excellent sources of many characterized and uncharacterized antibiotics and other natural products, we have provided a protocol for obtaining natural isolates from a variety of environmental sources. © 2015 by John Wiley & Sons, Inc.
    No preview · Article · Nov 2015 · Current protocols in microbiology
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    ABSTRACT: Nocardia spp. are aerobic, Gram-positive, catalase-positive, non-motile actinomycetes. Various species of the genus Nocardia have attracted attention due to their detrimental effects on human health. Recent discoveries, however, have exposed their importance as producers of bioactive compounds and degraders of complex organic compounds, as well as their involvement in biotransformation into valuable products. This unit includes general protocols for the laboratory maintenance of Nocardia spp., including growth in liquid medium, growth on solid agar, and long-term storage. Nocardia sp. CS682 (KCTC11297BP), isolated from soil collected in Jeonnam, Korea, is used as a prototype for explaining the considerations for efficient laboratory maintenance of Nocardia spp. © 2015 by John Wiley & Sons, Inc.
    No preview · Article · Nov 2015 · Current protocols in microbiology
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    ABSTRACT: The vast majority of surgical biopsy and post-mortem tissue samples are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Using the protocols described here, the full genome of the 1918 virus was determined at high coverage in one high-throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. This basic methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of infectious diseases. © 2015 by John Wiley & Sons, Inc.
    No preview · Article · Sep 2015 · Current protocols in microbiology
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    ABSTRACT: Human papillomaviruses (HPV) infect skin or mucosal epidermis. The simplistic capsid consists of a major capsid protein L1, a minor capsid protein L2, and a double-stranded circular DNA of about 8kB in size. The development of HPV-based vectors [i.e., pseudovirions (PsV)] as tools to study the initial infection has facilitated our understanding of HPV entry. The covalent coupling of fluorescent molecules to these PsV allows following the viruses en route to the nucleus, i.e., the site of replication. In the first section, we describe a facile method to covalently label HPV PsV that retain their infectivity. In this method, fluorophores coupled to a reactive succinimidyl ester are covalently attached to amine residues in the virion in a one-step chemical reaction. In the second section of this unit, several assays are outlined that use the fluorescently labeled virions for entry studies in live and fixed cells. © 2015 by John Wiley & Sons, Inc.
    No preview · Article · Sep 2015 · Current protocols in microbiology
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    ABSTRACT: The prognosis from thyroid cancer subtypes in humans covers a spectrum from "cured at almost 90%" to "100% lethal." Invasive and poorly differentiated forms of thyroid cancer are among the most aggressive human cancers, and there are few effective therapeutic options. Genetically engineered mice, based on mutations observed in patients, can accurately recapitulate the human disease and its progression, providing invaluable tools for the preclinical evaluation of novel therapeutic approaches. This overview details models developed to date as well as their uses for identifying novel anticancer agents. © 2015 by John Wiley & Sons, Inc.
    No preview · Article · Sep 2015 · Current protocols in microbiology
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    ABSTRACT: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging highly pathogenic respiratory virus. Although MERS-CoV only emerged in 2012, we and others have developed assays to grow and quantify infectious MERS-CoV and RNA products of replication in vitro. MERS-CoV is able to infect a range of cell types, but replicates to high titers in Vero E6 cells. Protocols for the propagation and quantification of MERS-CoV are presented. © 2015 by John Wiley & Sons, Inc.
    No preview · Article · Sep 2015 · Current protocols in microbiology
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    ABSTRACT: Epichloë species (including former Neotyphodium species) are endophytic fungi that significantly affect fitness of cool-season grass hosts, potentially by increasing nutrient uptake and resistance to drought, parasitism and herbivory. Epichloë species are obligately biotrophic, living in the intercellular spaces of their plant hosts, and spreading systemically throughout host aerial tissues. The reproduction of Epichloë species is versatile; some strains have both sexual and asexual modes of reproduction, but others are restricted to one or the other mode. The reproduction mode determines the dissemination mechanism, and the asexual species most important to agriculture are strictly seed-borne, cause no signs or symptoms, and are undetectable except by specialized microscopic, molecular or antigenic procedures. These procedures can be used to identify endophytes in a variety of plant tissues. Similar protocols can be modified to detect less common symbionts, such as the penicillate "p-endophytes," when they occur by themselves or together with Epichloë species. © 2015 by John Wiley & Sons, Inc.
    No preview · Article · Aug 2015 · Current protocols in microbiology
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    ABSTRACT: Vibrio cholerae is the agent of cholera, a potentially lethal diarrheal disease that remains a significant threat to populations in developing nations. The infant rabbit model of cholera is the only non-surgical small animal model system that closely mimics human cholera. Following orogastric inoculation, V. cholerae colonizes the intestines of infant rabbits, and the animals develop severe cholera-like diarrhea. In this unit, we provide a detailed description of the preparation of the V. cholerae inoculum, the inoculation process and the collection and processing of tissue samples. This infection model is useful for studies of V. cholerae factors and mechanisms that promote its intestinal colonization and enterotoxicity, as well as the host response to infection. The infant rabbit model of cholera enables investigations that will further our understanding of the pathophysiology of cholera and provides a platform for testing new therapeutics. © 2015 by John Wiley & Sons, Inc.
    No preview · Article · Aug 2015 · Current protocols in microbiology
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    ABSTRACT: Immunization with Human Papillomavirus (HPV) L1 virus-like particles or L2 capsid protein elicits neutralizing antibodies that mediate protection. A high-throughput and sensitive in vitro neutralization assay is therefore valuable for prophylactic HPV vaccine studies. Over several hours during infection of the genital tract, virions take on a distinct intermediate conformation, including a required furin cleavage of L2 at its N-terminus. This intermediate is an important target for neutralization by L2-specific antibody, but it is very transiently exposed during in vitro infection of most cell lines resulting in insensitive measurement for L2, but not L1-specific neutralizing antibodies. To model this intermediate, we describe a protocol to generate furin-cleaved HPV pseudovirions (fc-PsV), which deliver an encapsidated reporter plasmid to facilitate infectivity measurements. We also describe a protocol for use of fc-PsV in a high-throughput in vitro neutralization assay for the sensitive measurement of both L1 and L2-specific neutralizing antibodies. © 2015 by John Wiley & Sons, Inc.
    No preview · Article · Aug 2015 · Current protocols in microbiology