Transposon site hybridization in Mycobacterium tuberculosis.
ABSTRACT Microarray mapping of transposon insertions can be used to quantify the relative abundance of different transposon mutants within a complex pool after exposure to selective pressure. The transposon site hybridization (TraSH) method applies this strategy to the study of Mycobacterium tuberculosis and can be adapted to the study of other microorganisms. This chapter describes the methods used to mutagenize mycobacteria with transposons, extract genomic DNA, amplify genomic DNA adjacent to transposon ends using polymerase chain reaction and T7 transcription, and synthesize labeled cDNA. It also describes methods used to construct an appropriate microarray, hybridize labeled cDNA, and analyze the microarray data. Important considerations involved in the experimental design of the selective pressure, the design of the microarray, and the statistical analysis of collected data are discussed.
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ABSTRACT: Salmonella spp. are major cause of human morbidity and mortality worldwide. Upon entry into the human host, Salmonella spp. must overcome the resistance to colonization mediated by the gut microbiota and the innate immune system. They successfully accomplish this by inducing inflammation and mechanisms of innate immune defense. Many models have been developed to study Salmonella spp. interaction with the microbiota that have helped to identify factors necessary to overcome colonization resistance and to mediate disease. Here we review the current state of studies into this important pathogen/microbiota/host interaction in the mammalian gastrointestinal tract.Frontiers in Microbiology 05/2011; 2:101. DOI:10.3389/fmicb.2011.00101 · 3.94 Impact Factor
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ABSTRACT: We present a conceptual review of transposition-based strategies for determining gene essentiality on a one-by-one basis in bacteria. Many of the techniques are described in greater detail in individual chapters of this volume. The second section of this chapter deals with transposition-deletion-based strategies for determining the essentiality of blocks of genes. This latter approach has the potential to experimentally define the minimal required genome for a given organism.Methods in Molecular Biology 02/2008; 416:13-26. DOI:10.1007/978-1-59745-321-9_2 · 1.29 Impact Factor
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ABSTRACT: Nitric oxide (NO), which is an important component of immunity to Mycobacterium tuberculosis, has both cytotoxic and immune regulatory functions. We examined the way that this molecule interacts with M. tuberculosis in vivo by screening for bacterial mutations that alter growth in mice that are unable to produce inducible NO synthase (iNOS), the dominant source of NO during infection. We found that very few bacterial genes appeared to be specifically required for resistance to NO in vivo. Instead, mutations in several virulence factors caused greater attenuation in the absence of iNOS. Among these were mutants incapable of transporting the lipid phthiocerol dimycocerosate (PDIM). Although PDIM has been implicated in NO defense, this result indicates that PDIM has other roles during infection. We additionally found that PDIM transport is required for virulence in mice lacking interferon-gamma . Thus, PDIM is important for resisting an interferon-gamma-independent mechanism of immunity.The Journal of Infectious Diseases 10/2009; 200(5):774-82. DOI:10.1086/605128 · 5.78 Impact Factor