Comprehensive identification of conditionally essential genes in mycobacteria - PubMed (original) (raw)

Comprehensive identification of conditionally essential genes in mycobacteria

C M Sassetti et al. Proc Natl Acad Sci U S A. 2001.

Abstract

An increasing number of microbial genomes have been completely sequenced, and the identified genes are categorized based on their homology to genes of known function. However, the function of a large number of genes cannot be determined on this basis alone. Here, we describe a technique, transposon site hybridization (TraSH), which allows rapid functional characterization by identifying the complete set of genes required for growth under different conditions. TraSH combines high-density insertional mutagenesis with microarray mapping of pools of mutants. We have made large pools of independent transposon mutants in mycobacteria by using a mariner-based transposon and efficient phage transduction. By using TraSH, we have defined the set of genes required for growth of Mycobacterium bovis bacillus Calmette-Guérin on minimal but not rich medium. Genes of both known and unknown functions were identified. Of the genes with known functions, nearly all were involved in amino acid biosynthesis. TraSH is a powerful method for categorizing gene function that should be applicable to a variety of microorganisms.

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Figures

Figure 1

Efficient transduction of a mariner transposon to generate diverse mutant libraries. (A) Cultures of the indicated mycobacterial species were infected with the transducing phage and the resulting kanamycin-resistant colonies were counted. (B) M. bovis BCG transposon mutants (105) or wild-type BCG (106 cfu) were plated on the indicated antibiotics and resistant colonies were counted. “Fold increase” represents the frequency of antibiotic-resistant strains in the library divided by the frequency of spontaneous resistance. (C) Insertions throughout the katG gene are found in INH-resistant mutants. PCR was performed on 11 isoniazid-resistant clones by using one primer that hybridizes to a region flanking the katG gene and one that hybridizes to the inverted repeat region of the transposon. PCR products between 168 and 2,390 bp represent insertions in the katG orf.

Figure 2

Schematic representation of TraSH procedure. (A) Chromosomal region encompassing genes A–C from six different mutant strains (rectangles) is shown. Each mutant carries a single transposon insertion (triangles) that disrupts the function of a gene. Pools of mutants are grown under two different selective conditions. Genes A and C are nonessential for growth. Gene B is essential only under growth condition 2, and mutants harboring insertions in this gene are lost from this pool (represented by light shading). TraSH target that is complementary to the chromosomal DNA flanking each transposon insertion is generated from the two pools, labeled with different fluorophores, and hybridized to a microarray. The DNA probes representing genes A and C on the microarray will hybridize to the target generated from both pools. However, the target representing gene B will only be present in the pool from growth condition 1. By measuring the ratio of the two fluorophores for each probe, differential gene requirements are detected. (B) Method for generating TraSH target. The chromosomal DNA containing a transposon insertion (green) is digested with frequent cutting restriction enzymes, and adapters (blue) are ligated to the ends of the resulting DNA. PCR is then performed with primers (red) that hybridize to transposon and adapter sequences. To avoid the amplification of fragments that do not contain transposon sequence, the adapter contains the adapter–primer sequence but not the complementary strand, which serves as a binding site for this primer. Thus, the binding site must be generated by extension from the transposon-specific primer. To allow for the linear extension of these products, the transposon primer was designed with a higher melting temperature than the adapter primer, and higher annealing temperatures were used in the initial cycles of PCR (see Materials and Methods). Extension of the adapter itself, which would generate a binding site for the adapter primer, is prevented by a 3′-amino modification. The resulting PCR product is used as a template for the in vitro transcription of RNA target. This RNA is labeled and used for microarray hybridization.

Figure 3

TraSH identifies mutations that result in auxotrophy. The bar graphs display the fluorescence intensity observed for each gene across the genomic region surrounding auxotrophic mutations (in bold). Gray bars and white bars represent fluorescence observed from pools grown on minimal or rich medium, respectively. Data are the average of duplicate measurements from a single microarray and are represented on a log scale. Error bars representing standard deviations are shown for all features. The absence of error bars indicates that the value was too small to be plotted. Genes for which the intensity from the rich medium pool did not exceed two-times the background were omitted.

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Comprehensive identification of conditionally essential genes in mycobacteria - PubMed (original) (raw)