Curr Protoc Microbiol [JOURNAL]
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Šlapeta J
Curr Protoc Microbiol
· 2017 Feb · PMID 28166384
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Cryptosporidium spp. are obligate protozoan parasites of the gastrointestinal tract of vertebrates, including humans. In the majority of human cases, the diarrheal disease cryptosporidiosis is caused by either the human-...
Cryptosporidium spp. are obligate protozoan parasites of the gastrointestinal tract of vertebrates, including humans. In the majority of human cases, the diarrheal disease cryptosporidiosis is caused by either the human-adapted species Cryptosporidium hominis or the zoonotic Cryptosporidium parvum 'bovine genotype' (also known as Cryptosporidium pestis). The infectious stage, environmentally resilient Cryptosporidium oocysts, are shed by the infected host. Cryptosporidium parasites are transmitted by the fecal-oral route and are one of the major water-borne pathogens. The cryptic nature of the microscopic Cryptosporidium oocysts coupled with the existence of several host-adapted and zoonotic species requires molecular tools to identify Cryptosporidium spp. in either fecal or environmental samples. This unit describes methods for Cryptosporidium identification and typing using genotyping based on nuclear loci. We also provide a protocol for morphological confirmation of Cryptosporidium oocysts based on antibody labeling of the Cryptosporidium oocyst wall and a protocol for purification of oocysts from fecal material. © 2017 by John Wiley & Sons, Inc.
Fang Y, Cui L, Gu B
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, Arredondo F, Tyler BM
Curr Protoc Microbiol
· 2017 Feb · PMID 28166383
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Phytophthora is a filamentous fungus-like microorganism, but belongs to the oomycetes, in the kingdom Stramenopila. Phytophthora species are notorious as plant destroyers, causing multibillion-dollar damage to agricultur...
Phytophthora is a filamentous fungus-like microorganism, but belongs to the oomycetes, in the kingdom Stramenopila. Phytophthora species are notorious as plant destroyers, causing multibillion-dollar damage to agriculture and natural ecosystems worldwide annually. For a long time, genome editing has been unattainable in oomycetes, because of their extremely low rate of homologous recombination. The recent implementation of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system in the soybean pathogen Phytophthora sojae, an experimental model for oomycetes, has opened up a powerful new research capability for the oomycete community. Here, we describe a detailed protocol for CRISPR/Cas9-mediated genome editing in P. sojae, including single guide RNA (sgRNA) design and construction, efficient gene replacement, and mutant-screening strategies. This protocol should be generally applicable for most culturable oomycetes. We also describe an optimized transformation method that is useful for other Phytophthora spp. including P. capsici and P. parasitica. © 2017 by John Wiley & Sons, Inc.
Dhakal D, Pokhrel AR, Jha AK
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, Thuan NH, Sohng JK
Curr Protoc Microbiol
· 2017 Feb · PMID 28166382
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Saccharopolyspora spp. are aerobic, Gram-positive, non-acid-fast, and non-motile actinomycetes. Various species of the genus Saccharopolyspora have been reported with an ability to produce various bioactive compounds for...
Saccharopolyspora spp. are aerobic, Gram-positive, non-acid-fast, and non-motile actinomycetes. Various species of the genus Saccharopolyspora have been reported with an ability to produce various bioactive compounds for pharmaceutical and agricultural uses. This unit includes general protocols for the laboratory maintenance of Saccharopolyspora species, including growth in liquid medium, growth on solid agar, long-term storage, and generation of a higher producer strain by mutagenesis. Saccharopolyspora spinosa ATCC 49460 is used as a prototype for explaining the considerations for efficient laboratory maintenance of Saccharopolyspora spp. Saccharopolyspora spinosa is a producer of spinosad, a prominent insecticide with selective activity against various insects. © 2017 by John Wiley & Sons, Inc.
Van Doorslaer K, Chen Z, McBride AA
Curr Protoc Microbiol
· 2016 Nov · PMID 27858973
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Pathology departments routinely process and store formalin-fixed, paraffin-embedded (FFPE) tissue samples for clinical diagnosis. These collections often contain decades' worth of samples and represent a treasure trove o...
Pathology departments routinely process and store formalin-fixed, paraffin-embedded (FFPE) tissue samples for clinical diagnosis. These collections often contain decades' worth of samples and represent a treasure trove of specimens that can be analyzed for retrospective epidemiological studies, diagnostics, and pathogen discovery. Accurate amplification and sequencing of DNA from these samples is critical for the usability of these FFPE samples. Here we present a collection of protocols that describe extraction of DNA from FFPE tissues, PCR amplification of human papillomavirus DNA, and subsequent genotyping of the infecting virus. © 2016 by John Wiley & Sons, Inc.
O'Brien JP, Malvankar NS
Curr Protoc Microbiol
· 2016 Nov · PMID 27858972
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Anaerobic microorganisms play a central role in several environmental processes and regulate global biogeochemical cycling of nutrients and minerals. Many anaerobic microorganisms are important for the production of bioe...
Anaerobic microorganisms play a central role in several environmental processes and regulate global biogeochemical cycling of nutrients and minerals. Many anaerobic microorganisms are important for the production of bioenergy and biofuels. However, the major hurdle in studying anaerobic microorganisms in the laboratory is the requirement for sophisticated and expensive gassing stations and glove boxes to create and maintain the anaerobic environment. This appendix presents a simple design for a gassing station that can be used readily by an inexperienced investigator for cultivation of anaerobic microorganisms. In addition, this appendix also details the low-cost assembly of bioelectrochemical systems and outlines a simplified procedure for cultivating and analyzing bacterial cell cultures and biofilms that produce electric current, using Geobacter sulfurreducens as a model organism. © 2016 by John Wiley & Sons, Inc.
Stensvold CR, Clark CG
Curr Protoc Microbiol
· 2016 Nov · PMID 27858971
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Several typing methods have been used in studies aiming to unravel the molecular epidemiology of Blastocystis, which is one of the most common intestinal parasites in human and many non-human hosts. Such studies have the...
Several typing methods have been used in studies aiming to unravel the molecular epidemiology of Blastocystis, which is one of the most common intestinal parasites in human and many non-human hosts. Such studies have the potential to add to knowledge on Blastocystis transmission, host specificity, phylogeography, and clinical and public health significance, but rely on robust, standardized methods by which data can be generated and compared directly between studies. One of the most used methods is "barcoding,", which involves single-round PCR amplification and sequencing of partial small subunit ribosomal RNA genes of the parasites. Recently, a publicly available online facility was developed for quick and standardized identification of subtypes (ribosomal lineages) and subtype alleles (variation within subtypes) based on sequence data obtained by barcoding PCR. Moreover, a modified barcoding approach is now available using nested PCR, which enables detection of mixed subtype infections. © 2016 by John Wiley & Sons, Inc.
Clark CG, Stensvold CR
Curr Protoc Microbiol
· 2016 Nov · PMID 27858970
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Blastocystis is an intestinal parasite that is very easily isolated in culture from fresh stool samples. In fact, the parasite grows so readily in culture that short-term in vitro culture is sometimes used as a diagnosti...
Blastocystis is an intestinal parasite that is very easily isolated in culture from fresh stool samples. In fact, the parasite grows so readily in culture that short-term in vitro culture is sometimes used as a diagnostic tool in the absence of DNA-based methods. While axenizing Blastocystis cultures remains a significant challenge, the parasite can be propagated for several months in the presence of metabolically active bacteria (xenic culture). Hence, culture can be used for maintaining live Blastocystis strain libraries. This enables the production of a stable resource of reference material, which for instance can be used for DNA-based assays and research. Blastocystis isolates can also be cryopreserved with a view to reestablishing them in culture. Here, we provide protocols for xenic in vitro culture and cryopreservation of Blastocystis. © 2016 by John Wiley & Sons, Inc.
Agbulos DS, Barelli L, Giordano BV
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, Hunter FF
Curr Protoc Microbiol
· 2016 Nov · PMID 27858969
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Zika virus (ZIKV), belonging to the family Flaviviridae, genus Flavivirus, is an arthropod-borne virus that was first discovered from the Zika forest in Uganda in 1947. Recent outbreaks in South America have linked ZIKV...
Zika virus (ZIKV), belonging to the family Flaviviridae, genus Flavivirus, is an arthropod-borne virus that was first discovered from the Zika forest in Uganda in 1947. Recent outbreaks in South America have linked ZIKV to cases of microcephaly and Guillain-Barré syndrome in humans. With the increased interest in ZIKV, protocols must be established to facilitate proper research. Here we describe the laboratory techniques required to quantify, propagate, and store ZIVK. We also review the proper safety protocol for the handling of ZIKV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention. © 2016 by John Wiley & Sons, Inc.
Woodman ME, Savage CR, Arnold WK
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, Stevenson B
Curr Protoc Microbiol
· 2016 Aug · PMID 27517337
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This protocol describes an efficient method for screening intact bacteria for the presence of desired DNA sequences using the polymerase chain reaction (PCR). This method is commonly referred to as colony PCR. © 2016 by...
This protocol describes an efficient method for screening intact bacteria for the presence of desired DNA sequences using the polymerase chain reaction (PCR). This method is commonly referred to as colony PCR. © 2016 by John Wiley & Sons, Inc.
Garcia EC, Cotter PA
Curr Protoc Microbiol
· 2016 Aug · PMID 27517336
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Burkholderia thailandensis is a nonpathogenic Gram-negative bacterium found in tropical soils. Closely related to several human pathogens, its ease of genetic manipulation, rapid growth in the laboratory, and low virulen...
Burkholderia thailandensis is a nonpathogenic Gram-negative bacterium found in tropical soils. Closely related to several human pathogens, its ease of genetic manipulation, rapid growth in the laboratory, and low virulence make B. thailandensis a commonly used model organism. This unit describes the fundamental protocols for in vitro growth and maintenance of B. thailandensis in the laboratory. © 2016 by John Wiley & Sons, Inc.
Geessien Kroon E, Santos Abrahão J, de Souza Trindade G
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, Pereira Oliveira G, Moreira Franco Luiz AP, Barbosa Costa G, Teixeira Lima M, Silva Calixto R, de Oliveira DB, Drumond BP
Curr Protoc Microbiol
· 2016 Aug · PMID 27517335
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Natural infections of Vaccinia virus (VACV)-the prototype species of the Orthopoxvirus genus, from the family Poxviridae and subfamily Chordopoxvirinae-cause an occupational emergent zoonotic disease that is primarily as...
Natural infections of Vaccinia virus (VACV)-the prototype species of the Orthopoxvirus genus, from the family Poxviridae and subfamily Chordopoxvirinae-cause an occupational emergent zoonotic disease that is primarily associated with the handling of infected dairy cattle. In humans, VACV infection is characterized by skin lesions, primarily on the hands, and accompanied by systemic symptoms such as fever, myalgia, headache, and lymphadenopathy. The diagnosis of VACV is usually performed according to the methods described for other orthopoxviruses. This unit describes the methods utilized to obtain clinical samples, the serological and molecular techniques used for diagnosis, and the isolation methods and techniques used for molecular and biological characterization of the viruses. © 2016 by John Wiley & Sons, Inc.
Stone BL, Brissette CA
Curr Protoc Microbiol
· 2016 Aug · PMID 27517334
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Borrelia miyamotoi is a relapsing fever tick-borne pathogen found in Ixodes spp. (hard) ticks. In vitro culturing has proven difficult despite initial reports of cultures maintained in Barbour-Stoenner-Kelly-II (BSK-II)...
Borrelia miyamotoi is a relapsing fever tick-borne pathogen found in Ixodes spp. (hard) ticks. In vitro culturing has proven difficult despite initial reports of cultures maintained in Barbour-Stoenner-Kelly-II (BSK-II) medium. The ability to culture in vitro opens many avenues for investigating the genetics and physiology of bacterial species. This unit describes methods for the maintenance and cultivation of B. miyamotoi in liquid medium. © 2016 by John Wiley & Sons, Inc.
Maresca JA, Keffer JL, Miller KJ
Curr Protoc Microbiol
· 2016 May · PMID 27153387
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Ion-pumping rhodopsins transfer ions across the microbial cell membrane in a light-dependent fashion. As the rate of biochemical characterization of microbial rhodopsins begins to catch up to the rate of microbial rhodop...
Ion-pumping rhodopsins transfer ions across the microbial cell membrane in a light-dependent fashion. As the rate of biochemical characterization of microbial rhodopsins begins to catch up to the rate of microbial rhodopsin identification in environmental and genomic sequence data sets, in vitro analysis of their light-absorbing properties and in vivo analysis of ion pumping will remain critical to characterizing these proteins. As we learn more about the variety of physiological roles performed by microbial rhodopsins in different cell types and environments, observing the localization patterns of the rhodopsins and/or quantifying the number of rhodopsin-bearing cells in natural environments will become more important. Here, we provide protocols for purification of rhodopsin-containing membranes, detection of ion pumping, and observation of functional rhodopsins in laboratory and environmental samples using total internal reflection fluorescence microscopy. © 2016 by John Wiley & Sons, Inc.
Ma Y, Liu P, Majerciak V
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, Zhu J, Zheng ZM
Curr Protoc Microbiol
· 2016 May · PMID 27153386
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Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus, is etiologically linked to the development of several malignancies, mainly Kaposi's sarcoma. Expressed as an early viral protein, KSHV ORF57 is e...
Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus, is etiologically linked to the development of several malignancies, mainly Kaposi's sarcoma. Expressed as an early viral protein, KSHV ORF57 is essential for lytic replication and virion production. ORF57 selectively binds to a subset of viral RNA and affects nearly all aspects of viral RNA processing. To globally identify all viral and host RNA associated with KSHV ORF57 in the infected cells, we have utilized UV cross-linking and immunoprecipitation (CLIP) of KSHV ORF57 combined with high-throughput RNA sequencing (CLIP-seq) to identify ORF57-binding RNA in BCBL-1 cells at genome-wide level. This unit provides step-by-step details on this new method that is applicable for any pathogen or host RNA-binding proteins by slight modification. © 2016 by John Wiley & Sons, Inc.
Abrahão JS, Oliveira GP, Ferreira da Silva LC
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, Dos Santos Silva LK, Kroon EG, La Scola B
Curr Protoc Microbiol
· 2016 May · PMID 27153385
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The aim of this protocol is to describe the replication, purification, and titration of mimiviruses. These viruses belong to the Mimiviridae family, the first member of which was isolated in 1992 from a cooling tower wat...
The aim of this protocol is to describe the replication, purification, and titration of mimiviruses. These viruses belong to the Mimiviridae family, the first member of which was isolated in 1992 from a cooling tower water sample collected during an outbreak of pneumonia in a hospital in Bradford, England. In recent years, several new mimiviruses have been isolated from different environmental conditions. These giant viruses are easily replicated in amoeba of the Acanthamoeba genus, its natural host. Mimiviruses present peculiar features that make them unique viruses, such as the particle and genome size and the genome's complexity. The discovery of these viruses rekindled discussions about their origin and evolution, and the genetic and structural complexity opened up a new field of study. Here, we describe some methods utilized for mimiviruses replication, purification, and titration. © 2016 by John Wiley & Sons, Inc.
Ni T, Majerciak V, Zheng ZM
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, Zhu J
Curr Protoc Microbiol
· 2016 May · PMID 27153384
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Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncovirus linked to the development of several malignancies in immunocompromised patients. Like other herpesviruses, KSHV has a large DNA genome encoding more tha...
Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncovirus linked to the development of several malignancies in immunocompromised patients. Like other herpesviruses, KSHV has a large DNA genome encoding more than 100 distinct gene products. Despite being transcribed and processed by cellular machinery, the structure and organization of KSHV genes in the virus genome differ from what is observed in cellular genes from the human genome. A typical feature of KSHV expression is the production of polycistronic transcripts initiated from different promoters but sharing the same polyadenylation site (pA site). This represents a challenge in determination of the 3' end of individual viral transcripts. Such information is critical for generation of a virus transcriptional map for genetic studies. Here we present PA-seq, a high-throughput method for genome-wide analysis of pA sites of KSHV transcripts in B lymphocytes with latent or lytic KSHV infection. Besides identification of all viral pA sites, PA-seq also provides quantitative information about the levels of viral transcripts associated with each pA site, making it possible to determine the relative expression levels of viral genes at various stages of infection. Due to the indiscriminate nature of PA-seq, the pA sites of host transcripts are also concurrently mapped in the testing samples. Therefore, this technology can simultaneously estimate the expression changes of host genes and RNA polyadenylation upon KSHV infection. © 2016 by John Wiley & Sons, Inc.
Lee D, Norby K, Hayes M
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, Chiu YF, Sugden B, Lambert PF
Curr Protoc Microbiol
· 2016 May · PMID 27153383
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Human papillomaviruses (HPVs) are small double-stranded DNA viruses that are associated with greater than 95% of cervical cancers and 20% of head and neck cancers. These cancers arise from persistent infections in which...
Human papillomaviruses (HPVs) are small double-stranded DNA viruses that are associated with greater than 95% of cervical cancers and 20% of head and neck cancers. These cancers arise from persistent infections in which there is continued expression of the HPV E6 and E7 oncogenes, often as a consequence of integration of HPV DNA into the host genome. Such cancers represent "dead ends" for the virus as integration disrupts the viral genome and because the cancers are defective in normal epithelial differentiation, which is required for production of progeny papillomavirus. In order to study the full viral life cycle, from the establishment to maintenance to productive stages, our lab makes use of the organotypic epithelial tissue culture system. This system allows us to mimic the three-dimensional structure of epithelia whose differentiation is tightly linked to the completion of the HPV viral life cycle. In this chapter we describe how various aspects of the HPV life cycle are monitored in raft cultures making use of an immortalized keratinocyte cell line. © 2016 by John Wiley & Sons, Inc.
Griffin H, Doorbar J
Curr Protoc Microbiol
· 2016 May · PMID 27153382
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Molecular events during the papillomavirus life cycle can be mapped in infected tissue biopsies using antibodies to viral and cellular gene products, or by in situ hybridization approaches that detect viral DNA or viral...
Molecular events during the papillomavirus life cycle can be mapped in infected tissue biopsies using antibodies to viral and cellular gene products, or by in situ hybridization approaches that detect viral DNA or viral transcription products. For proteins, ease of immunodetection depends on antibody specificity and antigen availability. Epitopes in formalin-fixed paraffin-embedded (FFPE) samples are often masked by crosslinking and must be exposed for immunodetection. RNA in FFPE material is often degraded, and such tissue must be handled carefully to optimize detection. Viral proteins and viral genomic DNA are both well preserved in routinely processed FFPE samples, with sensitive detection methodologies allowing the simultaneous detection of multiple markers. The combined visualization of nucleic acid and (viral) protein targets, when coupled with image analysis approaches that allow correlation with standard pathology diagnosis, have allowed us to understand the molecular changes required for normal HPV life-cycle organization as well as deregulation during cancer progression. © 2016 by John Wiley & Sons, Inc.
Melton-Kreft R, Spirk T
Curr Protoc Microbiol
· 2016 Feb · PMID 26855284
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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 b...
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.
Günther T, Theiss JM, Fischer N
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, Grundhoff A
Curr Protoc Microbiol
· 2016 Feb · PMID 26855283
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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., herpesvirus...
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.