Strategies for identifying virulence genes using microarrays.

Microarray-based methods for the study of pathogenic bacteria. (a) Comparative genomic hybridization compares the chromosomal DNA content of two related strains. Genomic DNA is isolated from two strains and labeled with different fluorophores (red or green). The samples are then mixed and hybridized to a microarray, which generally contains fragments that are complementary to each gene in the genome. Chromosomal regions that differ between the strains are identified by comparing the intensities of the two fluorophores for each locus represented on the microarray. Yellow represents the binding of both red- and green-labeled DNA. (b) Expression analysis measures the relative abundance of RNA transcripts in bacteria subjected to different stimuli. RNA is isolated from two pools of bacteria that were grown under different conditions and the RNA is labeled with different fluorophores. Stimulus-specific gene expression patterns are determined by simultaneous hybridization of the RNA samples to a microarray. (c) Transposon site hybridization identifies conditionally essential genes. Each mutant strain carries a single transposon insertion (triangle). The figure depicts multiple insertions present in a pool of mutants. Two mutant pools are subjected to different growth conditions. Strains harboring insertions in genes that are essential for growth under a particular condition are lost from the pool. Labeled RNA that is complementary to the chromosomal DNA adjacent to each insertion site is generated from each pool. Microarray hybridization detects genes that have been lost from the pool that are essential for growth in the specific condition.