MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Microbiology' source. Sat, 10 Oct 2009 19:37:19 +0100 http://www.medworm.com/index.php?rid=2517004&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_9 The need for inhibitors for enzymes linked with microbial infection, specifically the NS3 protease of hepatitis C virus (HCV), inspired us to develop a unique, rapid and easy color-based method described herein. The NS3 serine protease of HCV has a role in processing viral polyprotein and it has been implicated in interactions with various cell constituents, resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for antiviral drugs. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2517004 Sun, 01 Mar 2009 00:00:00 +0100 2517004 http://www.medworm.com/index.php?rid=2517003&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_8 We describe a human cell display strategy to isolate high-affinity single-chain antibody fragments (scFvs) specific for CD22 for the treatment of B-cell malignancies. Our strategy uses flow cytometry and human embryonic kidney 293T (HEK-293T) cells that are widely used for transient protein expression. Flow cytometry enhances the screen’s sensitivity thereby allowing us to isolate high-affinity scFvs. Using human cell display, one could isolate and engineer scFvs, single domains, Fabs, or whole IgGs for increased affinity and other biological functions. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2517003 Sun, 01 Mar 2009 00:00:00 +0100 2517003 http://www.medworm.com/index.php?rid=2517002&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_7 Phage display has the capacity to rapidly isolate recombinant antibodies against protein targets and other molecules of significant size. However, there is no obvious lower limit to the power of the selection methods: this chapter describes how the techniques of phage display can be adapted to allow the isolation of antibodies against very small compounds. Antibodies generated in this way have many uses including the detection and quantitative analysis of the target chemical moiety in samples such as foods, water, and body fluids. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2517002 Sun, 01 Mar 2009 00:00:00 +0100 2517002 http://www.medworm.com/index.php?rid=2517001&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_6 The cartilaginous fish (sharks, skates, and rays) are the oldest phylogenetic group in which a human-type adaptive immune system and immunoglobulins (Igs) have been found. In addition to their conventional (heavy-light chain heterodimeric) isotypes, IgM and IgW, sharks produce the novel isotype, IgNAR, a heavy chain homodimer that does not associate with light chains. Instead, its variable (V) regions act as independent, soluble units in order to bind antigen. In this chapter, we detail our immunization protocol in order to raise a humoral IgNAR response in the nurse shark (Ginglymostoma cirratum) and the subsequent cloning of the single-domain V regions from this isotype in order to select antigen-specific binders by phage display. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2517001 Sun, 01 Mar 2009 00:00:00 +0100 2517001 http://www.medworm.com/index.php?rid=2517000&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_5 This chapter describes the construction and screening of a library of single-chain variable fragments (svFv) derived from patients with autoimmune disease. The methods cover the isolation of mononuclear cells from peripheral blood, preparation of RNA, and recovery of immunoglobulin-coding sequences by polymerase chain reaction (PCR). Cloning into a phage display vector and screening of the scFv display library by a simple panning procedure are described. These methods are applicable to library construction from any patient group or (with alternative primer sets) any mammalian species. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2517000 Sun, 01 Mar 2009 00:00:00 +0100 2517000 http://www.medworm.com/index.php?rid=2516999&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_4 A method for the construction of West Nile virus immune donor antibody repertoires is described. B cells are harvested from a suitable donor and the antibody variable genes are amplified using polymerase chain reaction (PCR). The PCR fragments are cloned in a phage display vector to construct a repertoire that can be used in panning procedures to identify many unique monoclonal antibodies. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516999 Sun, 01 Mar 2009 00:00:00 +0100 2516999 http://www.medworm.com/index.php?rid=2516998&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_3 A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase the frequency of antibody phage particles of interest in the library and allow for efficient isolation monoclonal antibodies with the predefined specificity. (Source: Springer protocols feed by Micr... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516998 Sun, 01 Mar 2009 00:00:00 +0100 2516998 http://www.medworm.com/index.php?rid=2516997&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_2 Diversity—the variability carried by the amino acid sequences of a synthetic antibody library—can be generated by synthetic degenerate oligonucleotides. One can experiment with different diversity designs in the variable domains of light and heavy chains (VH and VL) to generate antibody libraries with different properties. The ability to precisely define the final diversity of a library facilitates the process of isolating, characterizing, and optimizing an antibody lead. Here we describe detailed protocols for the design and construction of phage-displayed synthetic antibody libraries in which diversity is generated in the complementarity determining regions (CDRs) of the VH of a single humanized bivalent Fab scaffold. The example used in the protocol provides a general method... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516997 Sun, 01 Mar 2009 00:00:00 +0100 2516997 http://www.medworm.com/index.php?rid=2516996&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_1 Antibody-based immunologic reagents are useful for identifying, isolating, or eliminating cells with particular characteristics related to different diseases. Phage display is a highly valuable technique for antibody selection related to this purpose. In brief, a diverse group of antibody genes prepared from a patient or generated in vitro are inserted into a phagemid vector or the phage genome so that when the protein is expressed, it becomes anchored on the surface of the phage by fusion to a coat protein. A diverse library of recombinant antibodies is generated in this way and can then be exposed or panned on the antigen of interest, typically, this being a molecule associated with a particular pathological condition. Phage that carry proteins or peptides bind preferentially to the targ... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516996 Sun, 01 Mar 2009 00:00:00 +0100 2516996 http://www.medworm.com/index.php?rid=2516995&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_18 Numerous techniques are available for investigating protein-ligand interactions. The phage display technique is one such method routinely used to identify antibody-antigen interactions and has the benefit of being easily adaptable to high-throughput screening platforms. Once identified, antigen-binding domains on fragment antibodies or single-chain fragment antibodies (scFv) can be expressed and purified for further studies. In this chapter, we describe a method for high-level expression of a phage display-derived scFv in Pichia pastoris. The phage display-derived antibody A33scFv recognizes a cell surface glycoprotein (designated A33) expressed in colon cancer that serves as a target antigen for radioimmunoimaging and/or immunotherapy of human colon cancer. The expression and purification... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516995 Sun, 01 Mar 2009 00:00:00 +0100 2516995 http://www.medworm.com/index.php?rid=2516994&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_17 We present here an alternative procedure that uses cytoplasmic expression of soluble active scFv. This can be accomplished by using either specially engineered E. coli strains or hyperstable scFvs. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516994 Sun, 01 Mar 2009 00:00:00 +0100 2516994 http://www.medworm.com/index.php?rid=2516993&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_16 The isolation of recombinant antibodies by phage display naturally leads to experiments to evaluate their biological and immunological properties. Although crude preparations may have their value in initial studies, the need often exists for highly purified protein that can be tested in vivo. This chapter describes methods to generate high yields of scFv from bacterial cultures and to purify protein to the degree of homogeneity required for the most exacting analysis. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516993 Sun, 01 Mar 2009 00:00:00 +0100 2516993 http://www.medworm.com/index.php?rid=2516992&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_15 We describe procedures for intracellular expression of scFv in eukaryotic cells. Starting from a scFv gene cloned in a phage-display vector, we describe the cloning step into a mammalian expression vector, the transient transfection of a HeLa cell line, and the monitoring of intrabody expression by immunofluorescence staining and FACS analysis. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516992 Sun, 01 Mar 2009 00:00:00 +0100 2516992 http://www.medworm.com/index.php?rid=2516991&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_14 Conventionally, antibody phage display has been used to isolate recombinant antibodies that are monovalent in their interaction with target antigens. These antibodies can be reengineered for expression in mammalian cell culture as full-length, monospecific immunoglobulins. An emerging branch of research has sought to generate bivalent recombinant antibodies by manipulating the length of the linker separating heavy- and light-chain variable domains in single-chain Fv proteins, thereby promoting inter-scFv interaction and the formation of “diabodies.” With careful control, this can generate scFv-based proteins able to bind two very distinct targets, “bispecific diabodies.” Further manipulation enables the assembly of higher-order complexes. (Source: Springer protocols... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516991 Sun, 01 Mar 2009 00:00:00 +0100 2516991 http://www.medworm.com/index.php?rid=2516990&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_13 A method is described for the identification of the antigen recognised by an scFv isolated from an antibody phage display library using selection against a complex mixture of proteins (e.g. intact cells, purified cell surface membranes, and tissue sections). The method takes advantage of a yeast two-hybrid system that additionally allows for reorganization of post-translational modifications to the bait and target proteins. This technique is therefore especially useful for identifying surface-expressed antigens. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516990 Sun, 01 Mar 2009 00:00:00 +0100 2516990 http://www.medworm.com/index.php?rid=2516989&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_12 This chapter outlines a protocol for the selection by phage display of single-chain variable antibody fragments with dual properties-specificity for tumor cells and the ability to be internalized. The protocol is based on a direct incubation of living target cells with antibody phage display libraries under conditions that allow active endocytosis of phage particles by cancer cells as well as recovery of intracellular phage particles that retain infectivity. This “functional” selection helps avoid the isolation of irrelevant phages that may be obtained when selection is performed on heterogeneous material as a source of antigen. Internalizing antibodies recovered from human antibody repertoires are promising reagents for various therapeutic applications including the delivery o... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516989 Sun, 01 Mar 2009 00:00:00 +0100 2516989 http://www.medworm.com/index.php?rid=2516988&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_11 Many phage display techniques drive selection toward the isolation of highly specific antibodies. However, the identification of monoclonal antibodies that are cross-reactive has implications for the development of diagnostics, therapeutics, and vaccines against pathogens or cancer cells that are able to rapidly generate variants and escape mutants. To identify human monoclonal antibodies with high activity against HIV and broad-spectrum activity, we developed a technique termed sequential antigen panning. This methodology could be used to isolated recombinant antibodies against any antigen that shares epitopes with other antigens. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516988 Sun, 01 Mar 2009 00:00:00 +0100 2516988 http://www.medworm.com/index.php?rid=2516987&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_10 We provide procedures for the panning of fully humanized Fab antibodies using guided selection. Human heavy and light chain genes are amplified. A parental light chain is cloned into a phage display vector and combined with the heavy chain library. After several rounds of panning, positive clones are identified and the heavy chain sequences that are recovered are combined with light chains for further selection by phage display. Human Fab antibodies are obtained that bind the same epitope as the parental antibody. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2516987 Sun, 01 Mar 2009 00:00:00 +0100 2516987 http://www.medworm.com/index.php?rid=2387502&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_10 Bacteriophages manipulate bacterial gene expression in order to express their own genes or influence bacterial metabolism. Gene expression can be studied using real-time PCR or microarrays. Either technique requires the prior isolation of high quality RNA uncontaminated by the presence of genomic DNA. We outline the considerations necessary when working with bacteriophage infected bacterial cells. We also give an example of a protocol for extraction and quantification of high quality RNA from infected bacterial cells, using the marine cyanobacterium WH7803 and the phage S-PM2 as a case study. This protocol can be modified to extract RNA from the host/bacteriophage of interest. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2387502 Tue, 28 Oct 2008 04:00:00 +0100 2387502 http://www.medworm.com/index.php?rid=2040770&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_9 A common approach for investigating evolutionary relationships between genes and organisms is to compare extant DNA or protein sequences and infer an evolutionary tree. This methodology is known as molecular phylogenetics and may be the most informative means for exploring phage evolution, since there are few morphological features that can be used to differentiate between these tiny biological entities. In addition, phage genomes can be mosaic, meaning different genes or genomic regions can exhibit conflicting evolutionary histories due to lateral gene transfer or homologous recombination between different phage genomes. Molecular phylogenetics can be used to identify and study such genome mosaicism. This chapter provides a general introduction to the theory and methodology used to recons... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040770 Tue, 28 Oct 2008 04:00:00 +0100 2040770 http://www.medworm.com/index.php?rid=2040769&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_8 Knowledge of the regulatory elements contained within bacteriophage genomes forms the basis for understanding genomic expression and organization. The in silico prediction of promoter and terminator sequences in phage genomes is a first step towards this understanding. In this chapter, a number of programs and resources to identify regulatory elements are listed and discussed. Combining the available web-resources and literature data optimizes these predictions and can thus aid in a more directed experimental identification of these regulatory elements. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040769 Tue, 28 Oct 2008 04:00:00 +0100 2040769 http://www.medworm.com/index.php?rid=2040768&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_7 Tailed-bacteriophage virions contain a single linear dsDNA chromosome which can range in size from about 18 to 500 kbp across the known tailed-phage types. These linear chromosomes can have one of several known types of termini as follows: cohesive ends ( $5^{\prime}$ - or $3^{\prime}$ -single-strand extensions), circularly permuted direct terminal repeats, short or long exact direct terminal repeats, terminal host DNA sequences, or covalently bound terminal proteins. These different types of ends reflect differing DNA replication strategies and especially differing terminase actions during DNA packaging. In general, complete genome sequence determination does not by itself elucidate the nature of these ends, so directed experimental analysis is usually requir... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040768 Tue, 28 Oct 2008 04:00:00 +0100 2040768 http://www.medworm.com/index.php?rid=2040767&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_6 One of the most satisfying aspects of a genome sequencing project is the identification of the genes contained within it.These are of two types: those which encode tRNAs and those which produce proteins. After a general introduction on the properties of protein-encoding genes and the utility of the Basic Local Alignment Search Tool (BLASTX) to identify genes through homologs, a variety of tools are discussed by their creators. These include for genome annotation: GeneMark, Artemis, and BASys; and, for genome comparisons: Artemis Comparison Tool (ACT), Mauve, CoreGenes, and GeneOrder. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040767 Tue, 28 Oct 2008 04:00:00 +0100 2040767 http://www.medworm.com/index.php?rid=2040766&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_5 PCR is a quick and effective way of identifying the presence and ‘affiliation’ of bacteriophages, or phage-encoded genes from environmental samples, bacterial cells or purified viruses. The limitations are that you have to know what you are looking for in order to find it. Although the bacteriophage world does not have the advantage of a conserved gene, present in all members, there are many phage genes that do show nucleotide conservation even between phages which infect fairly divergent taxa. As more sequence data become available through both metagenomic approaches and the sequencing of complete bacteriophage genomes, PCR primers can be further refined and thus it should be an increasingly useful tool for bacteriophage biology. (Source: Springer protocols feed by Microbiolog... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040766 Tue, 28 Oct 2008 04:00:00 +0100 2040766 http://www.medworm.com/index.php?rid=2040765&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_4 The most efficient method to determine the genomic sequence of a dsDNA phage is to use a whole genome shotgun approach (WGSA). Preparation of a library where each genomic fragment has an equal chance of being represented is critical to the success of the WGSA. For many phages, there are regions of the genome likely to be under-represented in the shotgun library, which results in more gaps in the shotgun assembly than predicted by the Poisson distribution. However, as phage genomes are relatively small, this increased number of gaps does not present an insurmountable impediment to using the WGSA. This chapter will focus on construction of a high-quality random library and sequence analysis of this library in a 96-well format. Techniques are described for the mechanical fragmentation of geno... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040765 Tue, 28 Oct 2008 04:00:00 +0100 2040765 http://www.medworm.com/index.php?rid=2040764&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_3 Standard agarose gel electrophoresis is extensively used to resolve DNA fragments from 0.2 to 40–50 kb. Larger fragments of genomic DNA or whole viral genomes can only effectively be resolved by pulsed-field gel electrophoresis (PFGE), which extends the range of molecular separation from 200 bp to 12 Mb. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040764 Tue, 28 Oct 2008 04:00:00 +0100 2040764 http://www.medworm.com/index.php?rid=2040763&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_21 The Internet provides a myriad of useful tools for the phage worker including access to culture collections, specific databases, tools for gene identification, and whole genome comparisons, lecture notes, information on upcoming scientific meetings, books, etc. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040763 Tue, 28 Oct 2008 04:00:00 +0100 2040763 http://www.medworm.com/index.php?rid=2040762&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_20 Techniques developed over the past 20 years for the display of foreign peptides and proteins on the surfaces of filamentous bacteriophages have been a major driving force in the rapid development of recombinant antibody technology in recent years. With phage display of antibodies as one of its key components, recombinant antibody technology has led to the development of an increasing number of therapeutic monoclonal antibodies. Antibody gene libraries are fused to a gene encoding a phage coat protein. Recombinant phage expressing the resulting antibody libraries in fusion with the coat protein are propagated in Escherichia coli. Phage displaying monoclonal antibodies with specificities for target antigens are isolated from the libraries by a process called panning. The genes encoding the d... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040762 Tue, 28 Oct 2008 04:00:00 +0100 2040762 http://www.medworm.com/index.php?rid=2040761&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_2 DNA base compositional analysis is something which is rarely undertaken today, but it is still a useful criterion for phage taxonomy. A variety of techniques are described including hydrolysis of the DNA to the level of bases or nucleosides and separation by paper chromatography or HPLC. Spectroscopic and spectrofluorometric procedures are also outlined. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040761 Tue, 28 Oct 2008 04:00:00 +0100 2040761 http://www.medworm.com/index.php?rid=2040760&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_19 We describe the construction of fragmented whole genome and gene fragment phage display libraries and interaction selection by panning. This strategy allows the identification and characterization of interacting proteins on a genomic scale by screening the fragmented “proteome” against protein baits. Gene fragment libraries allow a more in depth characterization of the protein–protein interaction site by identification of the protein region involved in the interaction. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040760 Tue, 28 Oct 2008 04:00:00 +0100 2040760 http://www.medworm.com/index.php?rid=2040759&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_18 Lysins are phage-encoded, peptidoglycan (cell wall) hydrolases that accumulate in the bacterial cytoplasm during a lytic infection cycle. Late during infection, the lysins undergo holin-mediated translocation across the inner membrane into the peptidoglycan matrix where they cleave cell wall covalent bonds required for wall stability and allow bacterial lysis and progeny phage release. This potent hydrolytic activity is now the foundation of a powerful genetic-based screening process for the identification and analysis of phage lysin proteins. Here, we describe a method for identifying a lysin, PlyG, from a bacteriophage that specifically infects the Gram-positive organism Bacillus anthracis; however, the techniques described can be adapted to clone, express, and analyze lysins from any ph... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040759 Tue, 28 Oct 2008 04:00:00 +0100 2040759 http://www.medworm.com/index.php?rid=2040758&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_17 Phage typing is a rapid, economical, reliable, and reproducible technique, requiring no specialized equipment, for fingerprinting disease-causing agents for epidemiological investigation and surveillance. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040758 Tue, 28 Oct 2008 04:00:00 +0100 2040758 http://www.medworm.com/index.php?rid=2040757&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_16 Current appreciation of the vast expanse of prokaryotic diversity has largely come through molecular phylogenetic exploration of sequence diversity within the universally conserved gene for small subunit ribosomal RNA (16S rDNA). A plethora of methodologies for characterizing the diversity and composition of bacterial communities is based on sequence polymorphisms within this single gene. By comparison, no gene is universally shared among viruses or bacteriophages, which has prevented broad scale characterization of viral diversity within microbial ecosystems. With the reduction in DNA sequencing costs and wide availability of bioinformatics software, the tools of whole genome shotgun sequencing are now beginning to be applied to the characterization of genetic diversity within whole micro... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040757 Tue, 28 Oct 2008 04:00:00 +0100 2040757 http://www.medworm.com/index.php?rid=2040756&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_15 Since most of the phage genomes isolated from natural samples are previously unknown sequences, an isolation-independent approach is necessary to quantify the diversity of natural viral communities. Currently, two different methodological approaches are widely used to obtain genetic fingerprints of natural phage communities. While the separation of different viral genomes with pulsed field gel electrophoresis (PFGE) is based on the size of the genome, denaturing gradient gel electrophoresis (DGGE) uses minor differences in gene base composition to separate fragments of amplified DNA from natural viral communities. Finger printing techniques are a relatively fast and cheap tool to assess the diversity of environmental viruses. Together, PFGE and DGGE provide useful tools to study viral ecol... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040756 Tue, 28 Oct 2008 04:00:00 +0100 2040756 http://www.medworm.com/index.php?rid=2040755&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_14 Current techniques in mass spectrometry (MS) allow sensitive and accurate identification of proteins thanks to the in silico availability of these protein sequences within databases. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040755 Tue, 28 Oct 2008 04:00:00 +0100 2040755 http://www.medworm.com/index.php?rid=2040754&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_13 Concentration and purification of infectious particles are prerequisites for structural and functional characterization of bacteriophages. The methods detailed in the first part of this chapter outline the protocols commonly used to obtain purified phages: the concentration of phage particles by precipitation with polyethylene glycol and their purification by centrifugation in CsCl step gradients and subsequently by equilibrium centrifugation. This sequence of procedures, if carried out as a whole, ensures a purification of high quality, which is well suited for most analytical techniques used to characterize bacteriophage particles. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040754 Tue, 28 Oct 2008 04:00:00 +0100 2040754 http://www.medworm.com/index.php?rid=2040753&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_12 Gene expression microarrays offer the ability to monitor the expression of all phage genes over an infection cycle. However, there are relatively few reports to date of microarrays being used to investigate phage biology. This chapter aims to provide an overview of how to design and implement a microarray experiment to investigate phage biology. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040753 Tue, 28 Oct 2008 04:00:00 +0100 2040753 http://www.medworm.com/index.php?rid=2040752&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_11 Real-time, or quantitative PCR, is a valuable technique useful in bacteriophage research to quantify the abundance of phage or host gene transcripts. It can be used during the infection cycle both to monitor the expression of individual viral transcripts and to compare relative gene expression levels throughout the infection cycle. It is fairly economical to conduct and is useful in bacteria–phage systems where obtaining high yields of RNA is problematic. To perform real-time PCR, it is simply necessary to know the DNA sequence of the genes to be monitored, to have accurately quantified mRNA good quality cDNA, and access to a light-cycler. Although this chapter briefly reviews the basic principles of real-time PCR, the emphasis is on aspects of technique that are specific to the stud... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040752 Tue, 28 Oct 2008 04:00:00 +0100 2040752 http://www.medworm.com/index.php?rid=2040751&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_10 Bacteriophages manipulate bacterial gene expression in order to express their own genes or influence bacterial metabolism. Gene expression can be studied using real-time PCR or microarrays. Either technique requires the prior isolation of high quality RNA uncontaminated by the presence of genomic DNA. We outline the considerations necessary when working with bacteriophage infected bacterial cells. We also give an example of a protocol for extraction and quantification of high quality RNA from infected bacterial cells, using the marine cyanobacterium WH7803 and the phage S-PM2 as a case study. This protocol can be modified to extract RNA from the host/bacteriophage of interest. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040751 Tue, 28 Oct 2008 04:00:00 +0100 2040751 http://www.medworm.com/index.php?rid=2040750&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_1 Preparation of pure bacteriophage DNA used to rely on using CsCl gradients to give high purity or methods that yielded DNA that was either of low recovery or subject to significant genomic contamination. Recently though, new methods have come along that allow the purification of DNA from plate lysates that are not only capable of high yield but also, for all intents and purposes, free of genomic contamination (i.e. no visible genomic contamination on restriction analysis or when used for bacteriophage sequencing). (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2040750 Tue, 28 Oct 2008 04:00:00 +0100 2040750 http://www.medworm.com/index.php?rid=2505909&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-254-4_12 (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2505909 Tue, 30 Sep 2008 23:00:00 +0100 2505909 http://www.medworm.com/index.php?rid=2505908&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-254-4_11 (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2505908 Tue, 30 Sep 2008 23:00:00 +0100 2505908 http://www.medworm.com/index.php?rid=2505907&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-254-4_10 This chapter deals with control charts to monitor the performance of Indirect ELISAs. An Indirect ELISA kit for the detection of antibodies against Brucella is used to demonstrate the methods. Many of the features explained in Chapter 9 are relevant to this chapter; some repetition is intended, as this chapter may be read independently. Figure 1 gives an overview of the indirect ELISA scheme used. The details of the procedure, which involves plotting the data graphically (charting methods), are explained. As a reminder, the objectives of charting data are as follows: 1.To keep a constant record of all data.2.To monitor the assay from plate to plate in any one day's testing.3.To monitor the tests made from day to day, week to week, year to year.4.To allow rapid identification of unacceptabl... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2505907 Tue, 30 Sep 2008 23:00:00 +0100 2505907 http://www.medworm.com/index.php?rid=2505906&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-254-4_1 (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2505906 Tue, 30 Sep 2008 23:00:00 +0100 2505906 http://www.medworm.com/index.php?rid=2028366&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_9 The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. Described here is a drop plaque assay, which, being simpler, faster and more efficient than either the classical overlay or direct plating methods, enhances efficiency in processing large numbers of samples. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028366 Tue, 01 Jul 2008 04:00:00 +0100 2028366 http://www.medworm.com/index.php?rid=2028365&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_8 A method is described for determination of the concentration of infectious phage particles by the direct plating plaque assay, which is simpler and faster than the double agar overlay plaque procedure outlined in the previous chapter. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028365 Tue, 01 Jul 2008 04:00:00 +0100 2028365 http://www.medworm.com/index.php?rid=2028364&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_7 The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. In this chapter the classical overlay protocol is described. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028364 Tue, 01 Jul 2008 04:00:00 +0100 2028364 http://www.medworm.com/index.php?rid=2028363&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_6 We present here the details of procedures that can be used to cultivate previously undetectable viruses that are either comparatively large or aggregation-prone. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028363 Tue, 01 Jul 2008 04:00:00 +0100 2028363 http://www.medworm.com/index.php?rid=2028362&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_5 The detection and isolation of viruses directly from high temperature (>80 $^\circ$ C) acidic (pH<4) hot springs, fumaroles, and soils has long been challenging. These extreme environments tend to have a low biomass (typically <106 cells/ml) and low free viral abundance (103-106particles/ml) compared to eutrophic freshwater lakes (1) or near- shore marine environments (2). Establishing laboratory cultures from these environments pose challenges due to the extreme and poorly defined growth conditions that must be mimicked in the laboratory. Likewise, culture-independent approaches for isolation of viruses are problematic because of our rudimentary knowledge of viral diversity in these environments and the lack of universally conserved signatures that can be used for viru... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028362 Tue, 01 Jul 2008 04:00:00 +0100 2028362 http://www.medworm.com/index.php?rid=2028361&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_4 Cyanophages are a group of viruses which specifically infect cyanobacteria. The cyanobacteria are predominantly aquatic phototrophic bacteria and the two dominant genera Synechococcus and Prochlorococcus contribute significantly to primary production in the oceans. Cyanophages that infect marine cyanobacteria were first isolated in the early 1990s and it is now known that by lysing their host cells they play an important role in the microbial loop and alter biogeochemical cycles. They are also thought to influence the community structure and evolution of the cyanobacteria that they infect. Most recently cyanophages have been shown to carry host photosynthetic genes. It was only by the isolation and purification of cyanophage isolates have these important functions become known. Although th... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028361 Tue, 01 Jul 2008 04:00:00 +0100 2028361 http://www.medworm.com/index.php?rid=2028360&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_3 Most bacterial cells carry prophage genomes either integrated into the host DNA or present as repressed plasmids. Methods are described for the induction of prophages using Mitomycin C, and for the isolation of prophage-cured bacterial cell lines. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028360 Tue, 01 Jul 2008 04:00:00 +0100 2028360 http://www.medworm.com/index.php?rid=2028359&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_25 As interest in lytic phages as antimicrobial therapies or as treatments to reduce environmental contamination with pathogenic bacteria has increased, so has the need to determine if the use of lytic phages may lead to dissemination of virulence factors through generalized transduction, as occurs with temperate phages. Here we describe simple methods we have developed to determine if a lytic phage, rV5, can mediate generalized transduction in Escherichia coli O157:H7. These sensitive methods can be easily adapted to study generalized transduction between virulent and avirulent strains of bacteria. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028359 Tue, 01 Jul 2008 04:00:00 +0100 2028359 http://www.medworm.com/index.php?rid=2028358&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_24 This chapter describes a method for the generation of polyclonal antibodies against bacteriophages and how these may be assayed immunochemically and biologically. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028358 Tue, 01 Jul 2008 04:00:00 +0100 2028358 http://www.medworm.com/index.php?rid=2028357&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_23 Transduction is the process in which bacterial DNA is transferred from one bacterial cell to another by means of a phage particle. There are two types of transduction, generalized transduction and specialized transduction. In this chapter two of the best-studied systems – Escherichia coli-phage P1, and Salmonella enterica-phage P22 – are discussed from theoretical and practical perspectives. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028357 Tue, 01 Jul 2008 04:00:00 +0100 2028357 http://www.medworm.com/index.php?rid=2028356&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_22 The fate of lysogens following prophage induction has assumed added significance with the finding that in many pathogens virulence genes are carried on prophages and, in some, the production and/or release of the virulence factor is under control of the phage lytic regulatory program. We outline a method for identifying and characterizing from a total lysogen population, the subpopulation in which the prophage is induced. The prophage is genetically altered so that on induction it does not go through the lytic pathway, but does express a resolvase that acts at a reporter cassette located at another site on the bacterial chromosome to irreversibly change the resistance of the bacterium from tetracycline to chloramphenicol. Thus, induced derivatives survive and are easily identified even if ... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028356 Tue, 01 Jul 2008 04:00:00 +0100 2028356 http://www.medworm.com/index.php?rid=2028355&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_21 Recombineering is a recently developed method of in vivo genetic engineering used in Escherichia coli and other Gram-negative bacteria. Recombineering can be used to create single-base changes, small and large deletions, and small insertions in phage $\lambda$ as well as in bacterial chromosomes, plasmids, and bacterial artificial chromosomes (BACS). This technique uses the bacteriophage $\lambda$ generalized recombination system, Red, to catalyze homologous recombination between linear DNA and a replicon using short homologies of 50 base pairs. With recombineering, single-stranded oligonucleotides or double-stranded PCR products can be used to directly modify the phage $\lambda$ genome in vivo. It may also be possible to modify the genomes of other b... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028355 Tue, 01 Jul 2008 04:00:00 +0100 2028355 http://www.medworm.com/index.php?rid=2028354&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_20 Recent studies have established that the most abundant life form, that of phages, has had major influence on the biosphere, bacterial evolution, bacterial genome, and lateral gene transmission. Importantly the phages have served and continue to serve as valuable model systems. Such studies have led to a renewed interest and activity in the study of phages and their genomes. In order to determine the details of the involvement of phages in these important processes and activities, it is critical to assign specific functions to the phage gene products. The initial functional and gene assignments can be made by general mutagenesis of the phage genomes and of these specific gene products. A very informative mutagenic protocol that has found renewed interest is that using hydroxylamine. This mu... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028354 Tue, 01 Jul 2008 04:00:00 +0100 2028354 http://www.medworm.com/index.php?rid=2028353&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_2 Classical bacterial enrichment devised by Sergius Winogradsky (1856–1953) and Martinus Beijerinck (1851–1931) can be modified to enrich for bacteria-specific viruses. In this chapter simple protocols are presented for the enrichment of phages from water samples, such as sewage, and soil. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028353 Tue, 01 Jul 2008 04:00:00 +0100 2028353 http://www.medworm.com/index.php?rid=2028352&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_19 The objective is to preserve the initial characteristics of the microorganism and to avoid the genetic drift that occurs when the organism is maintained indefinitely in an actively growing state. The same holds true in phage biology and it is of particular interest when a collection of phages is to be maintained. The aim of this chapter is to provide phage biologists with general procedures to prepare and maintain bacteriophage stocks on a long-term basis. The protocols described below should be considered as general guidelines because although many phages and bacterial strains can be propagated and stored in these conditions, specific media and/or growth and storage conditions must be evaluated for each phage and bacterium. Since it was not the scope of this chapter to provide an exhausti... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028352 Tue, 01 Jul 2008 04:00:00 +0100 2028352 http://www.medworm.com/index.php?rid=2028351&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_18 Bacteriophage growth may be differentiated into sequential steps: (i) phage collision with an adsorption-susceptible bacterium, (ii) virion attachment, (iii) virion nucleic acid uptake, (iv) an eclipse period during which infections synthesize phage proteins and nucleic acid, (v) a “post-eclipse” period during which virions mature, (vi) a virion release step, and (vii) a diffusion-delimited period of virion extracellular search for bacteria to adsorb (1). The latent period begins at the point of virion attachment (ii) and/or nucleic acid uptake (iii) and ends with infection termination, spanning both the eclipse (iv) and the post-eclipse maturation (v) periods. For lytic phages, latent-period termination occurs at lysis, i.e., at the point of phage-progeny release (vi). A secon... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028351 Tue, 01 Jul 2008 04:00:00 +0100 2028351 http://www.medworm.com/index.php?rid=2028350&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_17 Laboratory characterization of bacteriophage growth traditionally is done either in broth cultures or in semisolid agar media. These two environments may be distinguished in terms of their spatial structure, i.e., the degree to which they limit diffusion, motility, and environmental mixing. Well-mixed broth, for example, represents the microbiological ideal of a non-spatially structured environment. Agar, by contrast, imposes significant limitations on phage and bacterial movement and therefore gives rise to spatial structure. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028350 Tue, 01 Jul 2008 04:00:00 +0100 2028350 http://www.medworm.com/index.php?rid=2028349&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_16 Practical methods are described for studying the interaction between bacterial viruses and their surface receptors. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028349 Tue, 01 Jul 2008 04:00:00 +0100 2028349 http://www.medworm.com/index.php?rid=2028348&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_15 Practical methods are described for studying the adsorption rate of bacteriophages to cells and the interaction between these viruses and their surface receptors. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028348 Tue, 01 Jul 2008 04:00:00 +0100 2028348 http://www.medworm.com/index.php?rid=2028347&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_14 The host range of a bacteriophage is defined by what bacterial genera, species and strains it can lyse; it is one of the defining biological characteristics of a particular bacterial virus. Because of host factors such as masking by O antigens that affects injection and the presence of restriction endonucleases, the relative efficiency of plating (EOP), that is, the titer of the phage on a given bacterial cell line compared to the maximum titer observed, may vary considerably. This chapter describes rapid procedures for determining the host range and relative EOP on each host of any phage. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028347 Tue, 01 Jul 2008 04:00:00 +0100 2028347 http://www.medworm.com/index.php?rid=2028346&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_13 Prokaryote viruses include 14 officially accepted families and at least five other potential families awaiting classification. Approximately 5,500 prokaryote viruses have been examined in the electron microscope. Classification has a predictive value and is invaluable to control experimental techniques and results. In describing viruses, the choice of methods depends on structure and taxonomical position of viruses. The study of isometric, filamentous, and pleomorphic viruses requires more detailed investigations than that of tailed species. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028346 Tue, 01 Jul 2008 04:00:00 +0100 2028346 http://www.medworm.com/index.php?rid=2028345&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_12 Negative staining of purified viruses is the most important electron microscopical technique in virology. The principal stains are phosphotungstate and uranyl acetate, both of which have problems and advantages. Particular problems are encountered in photography, calibration of magnification, measurements, and interpretation of artifacts. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028345 Tue, 01 Jul 2008 04:00:00 +0100 2028345 http://www.medworm.com/index.php?rid=2028344&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_11 Rapid identification and enumeration of the numerically important bacteriophages has been till recently a major limitation for studies of virus ecology. The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has changed this. The flow cytometric method allows the detection and discrimination of a wide variety of viruses of different morphology, genome type, and size. The present paper describes an optimized protocol for the enumeration of bacteriophages using a standard benchtop flow cytometer. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028344 Tue, 01 Jul 2008 04:00:00 +0100 2028344 http://www.medworm.com/index.php?rid=2028343&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_10 Determination of virus abundance using epifluorescence microscopy is a rapid and accurate method. The protocol requires the concentration of virus particles by collection on a filter. The nucleic acid in the virus particles is then stained with a fluorescent stain and the sample viewed with an epifluorescence microscope. The method was originally developed to determine the abundance of virus particles in water samples, however the protocol has been adapted for cultures and sediment samples. Although the method provides total counts of all virus-sized particles, regardless of infectivity, the method can be used for rapidly screening samples for further study. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028343 Tue, 01 Jul 2008 04:00:00 +0100 2028343 http://www.medworm.com/index.php?rid=2028342&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-164-6_1 Viruses are omnipresent and extraordinarily abundant in the microbial ecosystems of water, soil, and sediment. In nearly every reported case for aquatic and porous media environments (soils and sediments) viral abundance exceeds that of co-occurring host populations by 10–100-fold. If current estimates based on metagenome DNA sequence data are correct, then viruses represent the largest reservoir of unknown genetic diversity on Earth. Microscopy and molecular genetic tools have been critical in demonstrating that viruses are a dynamic component of microbial ecosystems capable of significantly influencing the productivity and population biology of their host communities. Moreover, these approaches have begun to describe and constrain the immense genetic diversity of viral communities.... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=2028342 Tue, 01 Jul 2008 04:00:00 +0100 2028342 http://www.medworm.com/index.php?rid=1538683&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_18 Whole body biophotonic imaging (BPI) is a technique that has contributed significantly to the way researchers study bacterial pathogens and develop pre-clinical treatments to combat their ensuing infections in vivo. Not only does this approach allow disease profiles and drug efficacy studies to be conducted non-destructively in live animals over the entire course of the disease, but in many cases, it enables investigators to observe disease profiles that could otherwise easily be missed using conventional methodologies. The principles of this technique are that bacterial pathogens engineered to express bioluminescence (visible light) can be readily monitored from outside of the living animal using specialized low-light imaging equipment, enabling their movement, expansion and treatment to ... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538683 Fri, 01 Feb 2008 05:00:00 +0100 1538683 http://www.medworm.com/index.php?rid=1538682&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_16 Salmonella enterica serovars cause a variety of diseases ranging from self-limiting gastroenteritis to severe systemic infections. Virulence of these facultative intracellular pathogens is dependent on their ability to invade and replicate within non-phagocytic cells, and cultured epithelial cell systems have been used extensively to dissect the molecular mechanisms involved. For efficient invasion in vitro, the bacterial cell growth conditions as critical since the invasion associated type III secretion system (T3SS1) must be expressed and functional. The ability of Salmonella to invade, and replicate within, epithelial cells can be easily assessed using a gentamicin protection assay or immunofluorescence microscopy. Here, the protocols used in our laboratory are described in detail. (Sou... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538682 Fri, 01 Feb 2008 05:00:00 +0100 1538682 http://www.medworm.com/index.php?rid=1538681&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_13 Anaplasma phagocytophilum is the etiologic agent of granulocytic anaplasmosis, a tick-borne, zoonotic, emerging infectious disease. A. phagocytophilum is an obligate intracellular pathogen that primarily resides within membrane-bound, cytoplasmic vacuoles of host neutrophils. Closely related to Ehrlichial and Rickettsial organisms, A. phagocytophilum is a small, fragile, Gram-negative bacterium that presents unique challenges for culture, isolation, enumeration, and labeling. This chapter delineates pathogen-specific considerations for culture and labeling of this organism for subsequent use in assays to examine mechanisms of host cell–pathogen interactions. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538681 Fri, 01 Feb 2008 05:00:00 +0100 1538681 http://www.medworm.com/index.php?rid=1538680&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_4 Comparative genomic hybridization analyses have contributed greatly to our understanding of bacterial evolution, population genetics, and pathogenesis. Here, we describe a robust protocol for microarray-based comparison of genome content, which could be applied to any bacterial species of interest. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538680 Fri, 01 Feb 2008 05:00:00 +0100 1538680 http://www.medworm.com/index.php?rid=1538679&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_21 The continued increase in antibiotic resistance among bacterial pathogens, coupled with a decrease in infectious disease research among pharmaceutical companies, has escalated the need for novel and effective antibacterial chemotherapies. While current agents have emerged almost exclusively from whole-cell screening of natural products and small molecules that cause microbial death, recent advances in target identification and assay development have resulted in a flood of target-driven drug discovery methods. Whether genome-based methodologies will yield new classes of agents that conventional methods have been unable to is yet to be seen. At the end of the day, perhaps a synergy between old and new approaches will harvest the next generation of antibacterial treatments. (Source: Springer ... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538679 Fri, 01 Feb 2008 05:00:00 +0100 1538679 http://www.medworm.com/index.php?rid=1538678&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_1 Lipopolysaccharide (LPS) is the signature glycolipid isolated from almost all Gram-negative bacteria. LPSs are well known for their ability to elicit the release of cytokines from eukaryotic cells including macrophages, neutrophils, and epithelial cells. LPS can be isolated free of contaminating nucleic acids and proteins by various techniques. In this review, we outline approaches for the isolation and preparation of LPSs for structural studies as well as preparation of very highly purified material for biological studies. Methods are also provided for the analysis of the purity and the structural composition of the LPSs. Finally, three methods for the isolation of lipid A are described. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538678 Fri, 01 Feb 2008 05:00:00 +0100 1538678 http://www.medworm.com/index.php?rid=1538677&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_6 The lack of a system for genetic manipulation has hindered studies on the molecular pathogenesis of relapsing fever Borrelia. The focus of this chapter is to describe selectable markers, manipulation strategies, and methods to electro-transform and clone wild-type infectious Borrelia hermsii. Preliminary studies suggest that the variable tick protein (Vtp) of B. hermsii is involved in tick-to-mammal transmission. To address this hypothesis, we have developed a system for genetic manipulation and have constructed clones of a Vtp mutant and an isogenic reconstituted strain. The methods described here are applicable for the inactivation of other loci in B. hermsii and should be adaptable for other species of relapsing fever spirochetes. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538677 Fri, 01 Feb 2008 05:00:00 +0100 1538677 http://www.medworm.com/index.php?rid=1538676&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_3 The analysis of the expression of virulence genes and the elucidation of metabolic and regulatory pathways of Staphylococcus aureus provide us with important information about the interaction between the pathogen and its host, mechanisms by which this organism causes diseases, and the resistance to antibiotics. In order to investigate regulatory networks of S. aureus, we analyze the cytoplasmic and extracellular proteome by using two-dimensional (2D) gel analyses combined with matrix-assisted laser ionization–time-of-flight mass spectrometry (MALDI–TOF MS). Gel-based proteomics is an extremely valuable tool in microbial physiology that can, in combination with various visualization and quantitation software packages, very rapidly provide comparative and quantitative data for mu... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538676 Fri, 01 Feb 2008 05:00:00 +0100 1538676 http://www.medworm.com/index.php?rid=1538675&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_20 Humana Press, Totowa, NJ This chapter describes methods for using non-human primates as a model of group A streptococcal (GAS) pharyngitis. This model has been used successfully to study host–pathogen interactions occurring during pharyngeal GAS infections. The protocol as described will compare two different GAS strains for their ability to cause clinical symptoms of pharyngitis. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538675 Fri, 01 Feb 2008 05:00:00 +0100 1538675 http://www.medworm.com/index.php?rid=1538674&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_9 Staphylococcus aureus is a leading cause of human infections worldwide and causes a variety of diseases ranging in severity from mild to life-threatening. The ability of S. aureus to cause disease is based in part on its ability to subvert the innate immune system. Advances in genome-wide analysis of host–pathogen interactions have provided the necessary tools to investigate molecular factors that directly contribute to S. aureus pathogenesis. This chapter describes methods to analyze gene expression in S. aureus during interaction with human neutrophils. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538674 Fri, 01 Feb 2008 05:00:00 +0100 1538674 http://www.medworm.com/index.php?rid=1538673&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_10 We developed PCR assays to detect and quantitate Yersinia pestis, the bacterial agent of plague, in flea vector and mammalian host tissues. Bacterial numbers in fleas, fleabite sites, and infected lymph nodes were determined using real-time PCR with primers and probes for a gene target on a multi-copy plasmid specific to Y. pestis. Tissue-matched standard curves used to determine absolute bacterial numbers in unknown samples were linear over at least five orders of magnitude. The methods were applied to studies of transmission of Y. pestis by the rat flea Xenopsylla cheopis, but should be generally useful to investigate the transmission dynamics of any arthropod-borne disease. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538673 Fri, 01 Feb 2008 05:00:00 +0100 1538673 http://www.medworm.com/index.php?rid=1538672&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_22 The analyses of numerous prokaryotic and eukaryotic genomes have revealed the presence of variable number tandem repeats (VNTRs). VNTR analysis is currently widely used to sub-speciate many bacterial, fungal, and viral pathogens and has facilitated a number of molecular epidemiology studies. In this chapter, we focus on spa typing which is based on sequence analysis of VNTRs in the polymorphic X region of the Staphylococcus aureus protein A gene Staphylococcus aureus. As the specific methods for spa typing, detailed in this chapter, are well-established and routine procedures (e.g., DNA isolation, PCR and DNA sequencing) for most molecular biology laboratories, we highlight the analytic methods used to interpret the genotyping data generated by sequence analysis and their potential applica... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538672 Fri, 01 Feb 2008 05:00:00 +0100 1538672 http://www.medworm.com/index.php?rid=1538671&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_15 Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that has a tropism for cells of the mononuclear phagocyte system. Following internalization, C. burnetii remains in a phagosome that ultimately matures into a vacuole with lysosomal characteristics that supports pathogen replication. Most in vitro investigations of Coxiella –macrophage interactions have employed continuous cell lines. Although these studies have been informative, genetic alterations of immortalized cells may result in attenuated biological responses to infection relative to primary cells. Consequently, primary macrophages are preferred as in vitro model systems. Here, we describe procedures for propagation and isolation of C. burnetii from cell culture and the use of these preparations to... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538671 Fri, 01 Feb 2008 05:00:00 +0100 1538671 http://www.medworm.com/index.php?rid=1538670&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_2 We describe a proteomic approach routinely used in our laboratory to characterize culture supernatant proteins using small-format two-dimensional gel electrophoresis. Proteins are collected after overnight growth of the bacteria in broth media. Compounds that inhibit isoelectric focusing, such as salts, are removed by enzymatic treatment and precipitation with trichloroacetic acid and acetone. Following resuspension in denaturing solution, the proteins are separated by isoelectric focusing using a 7-cm immobilized strip with a pH gradient of 4–7. Subsequently, proteins are further separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained with SYPRO Ruby. The small-gel format requires less time, reagents, and smaller culture volumes co... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538670 Fri, 01 Feb 2008 05:00:00 +0100 1538670 http://www.medworm.com/index.php?rid=1538669&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_12 Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric epithelium and plays a causative role in the development of peptic ulcers and gastric cancer. Phagocytosis is an element of innate defense used by macrophages and neutrophils to engulf microorganisms. We and others have shown that strains of H. pylori that contain the cag pathogenicity island actively retard their entry into phagocytes. Consequently, there is a lag of several minutes between bacterial binding and the onset of engulfment, and relative to other particles and microbes, the rate of internalization is slow. Herein, we describe in detail the use of synchronized phagocytosis and indirect immunofluorescence microscopy to quantify the rate and extent of H. pylori phagocytosis. This method is appropriate for... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538669 Fri, 01 Feb 2008 05:00:00 +0100 1538669 http://www.medworm.com/index.php?rid=1538668&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_8 Exopolysaccharides play a crucial role in the formation of biofilms and biofilm resistance to antimicrobials and innate host defense. Here we describe methods to analyze and quantify polysaccharide intercellular adhesin (PIA), a biofilm exopolysaccharide made of N-acetylglucosamine that is found in staphylococci and many other bacterial biofilm-forming pathogens. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538668 Fri, 01 Feb 2008 05:00:00 +0100 1538668 http://www.medworm.com/index.php?rid=1538667&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_5 Bacteria communicate with other members of their community through the secretion and perception of small chemical cues or signals. The recognition of a signal normally leads to the expression of a large suite of genes, which in some bacteria are involved in the regulation of virulence factors, and as a result, these signaling compounds are key regulatory factors in many disease processes. Thus, it is of interest when studying pathogens to understand the mechanisms used to control the expression of virulence genes so that strategies might be devised for the control of those pathogens. Clearly, the ability to interfere with this process of signaling represents a novel approach for the treatment of bacterial infections. There is a broad range of compounds that bacteria can use for signaling p... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538667 Fri, 01 Feb 2008 05:00:00 +0100 1538667 http://www.medworm.com/index.php?rid=1538666&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_11 Intracellular bacterial pathogens have evolved sophisticated strategies to survive and proliferate within cells of their hosts. Studying their intracellular life cycle is key to understanding virulence and requires methodologies that can identify the compartments in which they localize and characterize the replicative niche they generate. Here, we describe immunofluorescence-based microscopy techniques applied to the intracellular pathogens Brucella abortus and Francisella tularensis during their respective intracellular cycles inside murine bone marrow-derived macrophages. Standard immunofluorescence techniques are used to define the intracellular localization of the pathogens based on their co-localization with specifically labeled macrophage organelles. In addition, we describe an assay... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538666 Fri, 01 Feb 2008 05:00:00 +0100 1538666 http://www.medworm.com/index.php?rid=1538665&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_19 Staphylococcus aureus nasal colonization is a well-known risk factor for development of S. aureus infections in humans, but despite this established association, we are only beginning to understand the factors, both host and pathogen, that play a role in the colonization of the nares by S. aureus. The cotton rat is a model for many human respiratory pathogens and has proved its utility as a robust model for S. aureus nasal colonization. In this animal model, S. aureus is instilled in the nostrils of adult cotton rats, the bacteria rapidly colonize, and 7 days later S. aureus nasal colonization is enumerated by surgical removal of the nose and recovery of the colonizing S. aureus. This model is an excellent animal model to allow for the evaluation of the efficacy of various therapies, inclu... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538665 Fri, 01 Feb 2008 05:00:00 +0100 1538665 http://www.medworm.com/index.php?rid=1538664&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_7 Borrelia burgdorferi, the causative agent of Lyme disease, is an obligate parasite that cycles between vertebrate hosts and tick vectors. Attempts to understand the genetic factors that allow B. burgdorferi to sense, adapt to, and survive in different environments have been limited by a relatively low transformation rate. Here, we describe a mariner-based transposon system that achieves saturating levels of random mutagenesis in B. burgdorferi. In comparison with allelic exchange, which targets a single locus, transposon mutagenesis can create libraries of mutants encompassing disruptions of all genes. Suitably designed screens or selections of such a library permit the recovery of mutants exhibiting a desired phenotype. The system described here allows rapid identification of the genetic ... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538664 Fri, 01 Feb 2008 05:00:00 +0100 1538664 http://www.medworm.com/index.php?rid=1538663&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_17 The response of Borrelia burgdorferi to the challenge of reactive oxygen species (ROS) is a direct result of its limited biosynthetic capabilities and lack of biologically significant levels of intracellular Fe. In other bacteria, the major target for oxidative damage is DNA as a consequence of the reaction of “free” intracellular with ROS through the Fenton reaction. Therefore, cellular defenses in these bacteria are focused on protecting this essential cellular component. This does not seem to be the case for B. burgdorferi. In this chapter, we describe methods that were used to analyze the potential targets for ROS in B. burgdorferi. Surprisingly, membrane lipids (e.g., linoleic and linolenic acids) derived from host are the major target of ROS in the Lyme disease spirochete... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538663 Fri, 01 Feb 2008 05:00:00 +0100 1538663 http://www.medworm.com/index.php?rid=1538662&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-032-8_14 Electron microscopy of bacterial pathogens and interactions between bacteria and host cells and tissues provides valuable insights into structural and molecular properties and processes involved in pathogenesis. Applications for electron microscopy in bacterial pathogenesis range from discovering etiologic agents and following chronological events during infections by conventional examination of clinical samples to assessing molecular host–cell responses to infection and in situ interactions between receptors and ligands using specific immune-labeling techniques. This chapter focuses on techniques for preparing samples of bacteria and host cells for conventional transmission (TEM) and scanning electron microscopy (SEM) and use of luminescent nanocrystals or “quantum dots”... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538662 Fri, 01 Feb 2008 05:00:00 +0100 1538662 http://www.medworm.com/index.php?rid=1538686&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-457-5_19 Baculovirus-based insecticides are currently being used worldwide, and new products are in development in many countries. The most dramatic examples of successful baculovirus insecticides are found in soybean in Brazil and cotton in China. Production of baculoviruses is generally done in larvae of a convenient host species, and the level of sophistication varies tremendously between field-collection of infected insects at the one extreme and automated mass manufacturing at the other. Currently, only products with wild type baculoviruses as active ingredients are commercially available. Baculoviruses encoding insecticidal proteins are considered attractive, especially for crops with little tolerance to feeding damage, where speed-of-kill is an important characteristic. Successful field test... Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538686 Fri, 06 Jul 2007 04:00:00 +0100 1538686 http://www.medworm.com/index.php?rid=1538685&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-457-5_10 Improved methods of baculovirus cloning and insect cell culture and their commercialization have made the use of the baculovirus expression vector system (BEVS) a routine tool for the production of preparative quantities of recombinant protein. This chapter outlines basic techniques for small-scale protein production using the BEVS, including protocols for expression from adherent and suspension insect cell cultures, titer estimation, and expression optimization. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538685 Fri, 06 Jul 2007 04:00:00 +0100 1538685 http://www.medworm.com/index.php?rid=1538684&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-457-5_22 Probing the baculovirus infection process is essential in optimizing the recombinant protein production. Typically, researchers monitor the infection process in the stirred tank reactor, which, however, contains a population of cells infected at different times after virus inoculation. This chapter describes a two-stage reactor system consisting of an upstream continuous stirred tank reactor and a downstream tubular reactor with segmented plug flow for probing baculovirus infection and production. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology info http://www.medworm.com/rss/comments.php?id=1538684 Fri, 06 Jul 2007 04:00:00 +0100 1538684