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: Genomics research has enabled crucial insights into the adaptive evolution of Mycobacterium tuberculosis as an obligate human pathogen. Here, we highlight major recent advances and evaluate the potential for genomics approaches to inform tuberculosis control efforts in high-burden settings.Genome Biology 11/2014; 15(11):514. · 10.47 Impact Factor
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ABSTRACT: Mycobacteriophages have provided numerous essential tools for mycobacterial genetics, including delivery systems for transposons, reporter genes, and allelic exchange substrates, and components for plasmid vectors and mutagenesis. Their genetically diverse genomes also reveal insights into the broader nature of the phage population and the evolutionary mechanisms that give rise to it. The substantial advances in our understanding of the biology of mycobacteriophages including a large collection of completely sequenced genomes indicates a rich potential for further contributions in tuberculosis genetics and beyond.Microbiology spectrum. 03/2014; 2(2):1-36.
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ABSTRACT: To persist and cause disease in the host, Mycobacterium tuberculosis must adapt to its environment during infection. These adaptations include changes in nutrient utilization and alterations in growth rate. M tuberculosis Rv1422 is a conserved gene of unknown function that was found in a genetic screen to interact with the mce4 cholesterol uptake locus. Rv1422 protein is phosphorylated by the M. tuberculosis Ser/Thr kinases PknA and PknB, which regulate cell growth and cell wall synthesis. Bacillus subtilis strains lacking the Rv1422 homologue yvcK grow poorly on several carbon sources, and yvcK is required for proper localization of peptidoglycan synthesis. Here, we show that M. smegmatis and M. tuberculosis strains lacking Rv1422 have growth defects in minimal medium containing limiting amounts of several different carbon sources. These strains also have morphologic abnormalities, including shortened and bulging cells, findings that suggest a cell wall defect. In both mycobacterial species, Rv1422 localizes uniquely to the growing cell pole, the site of peptidoglycan synthesis in mycobacteria. M. tuberculosis ΔRv1422 is markedly attenuated for virulence in a mouse infection model, where it elicits decreased inflammation in the lungs and shows impaired bacterial persistence. These findings lead us to name this gene cuvA (carbon utilization and virulence protein A) and suggest a model in which deletion of cuvA leads to changes in nutrient uptake and/or metabolism that affect cell wall structure, morphology and virulence. Its role in virulence suggests that CuvA may be a useful target for novel inhibitors of M. tuberculosis during infection.Infection and Immunity 07/2014; · 4.16 Impact Factor