The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets.

Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale ''negative selection'' technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. Genes required in the lung included those with characterized functions in other models of H. influenzae pathogenesis and genes not previously implicated in infection. Genes implicated in vivo have reported or potential roles in survival during nutrient limitation, oxidative stress, and exposure to antimicrobial membrane perturbations, suggesting that these conditions are encountered by H. influenzae during pulmonary infection. The results demonstrate an efficient means to identify genes required for bacterial survival in experimental models of pathogenesis, and this approach should function similarly well in selections conducted in vitro and in vivo with any organism amenable to insertional mutagenesis.Illumina ͉ mariner ͉ mutagenesis ͉ pathogenesis ͉ transposon W hole-genome analytic techniques have been developed to identify bacterial genes essential for growth or survival in vitro or during infection of model hosts. The most direct of these approaches can be classified as ''negative selection'' strategies, in which large pools of diverse mutants are analyzed to identify mutations that reduce fitness under a particular condition. ''Signature-tagged mutagenesis'' utilizes DNA arrays representing unique hybridization tags that are introduced into each mutant within a library of strains to be evaluated (1). The ''transposon-site hybridization'' and ''microarray tracking of transposon mutants'' methods use microarrays displaying each gene of the target organism to monitor the relative abundance of transposon insertions in these genes under varied selection conditions (2-4). Each of these methods has been effectively used to identify virulence genes in diverse bacteria. For many pathogens, however, generation of large banks of uniquely tagged mutants is impractical and whole-genome microarrays may be unavailable, particularly for newly recognized organisms or genetically diverse species. In both microarray-based methods, hybridization is used to detect the abundance of a given mutation within the library of mutants. Therefore, quantification is limited by background hybridization levels and the dynamic range of signal detection. A method that generates an output that allows precise noise filtering and a broad dynamic range would represent a significant advancement ...

ABSTRACTmariner family transposons are widespread among eukaryotic organisms. These transposons are apparently horizontally transmitted among diverse eukaryotes and can also transpose in vitro in the absence of added cofactors. Here we show that transposons derived from the mariner element Himar1 can efficiently transpose in bacteria in vivo. We have developed simple transposition systems by using minitransposons, made up of short inverted repeats f lanking antibiotic resistance markers. These elements can efficiently transpose after expression of transposase from an appropriate bacterial promoter. We found that transposition of mariner-based elements in Escherichia coli produces diverse insertion mutations in either a targeted plasmid or a chromosomal gene. With Himar1-derived transposons we were able to isolate phage-resistant mutants of both E. coli and Mycobacterium smegmatis. mariner-based transposons will provide valuable tools for mutagenesis and genetic manipulation of bacteria that currently lack well developed genetic systems.

Signal transduction molecules within the two-component family represent a conserved adaptation for the control of genes involved in pathogenesis. The Bordetella virulence control locus, bvgAS, activates and represses gene expression in response to environmental signals. While infection requires virulence gene activation, the role of gene repression during infection is not understood. By altering regulatory genes and reversing regulatory connections, we found evidence that the BvgAS-repressed genes responsible for motility are neither required nor expressed during colonization of the host. Expression of this Bvg- phase-specific phenotype in the Bvg+ growth phase resulted in a defect in tracheal colonization. Therefore, BvgAS promotes virulence both by activating genes required for colonization and by repressing genes that inhibit the development of infection.