Development of genomic array footprinting for identification of conditionally essential genes in Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput, genome-wide technique, genomic array footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in nonreproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1 showed that both have an equal degree of saturation but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for surviving zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high-zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae.

FIG. 1.

Schematic representation of the GAF procedure. A large S. pneumoniae transposon library is grown under nonselective and selective conditions. Subsequently, chromosomal DNA containing transposons (gray rectangle) with outward-facing T7 RNA polymerase promoters (arrow with T7) is isolated from each population. The DNA is digested, and the DNA adjacent to the transposon insertion site is amplified using in vitro transcription with T7 RNA polymerase. The RNA is used in standard microarray procedures. Hybridization of these probes to a microarray will reveal which genes were disrupted in the mutants that disappeared during selection: those spots to which only material derived from the nonselective condition will hybridize (gray spots).

FIG. 2.

Detection of transposon insertion sites using PCR-based methods. (A) Scatterplot of the signal intensities of two parallel reactions on the chromosomal DNA of a genome-wide library using MATT. There is no correlation between the signal intensities for each gene (r², 0.036), indicating that the procedure is nonreproducible. (B) Scatterplot of the signal intensities of two parallel reactions on the chromosomal DNA of a genome-wide library using a TOPO-isomerase adapter combined with a nested PCR. There is no correlation between the signal intensities in each gene (r², 0.010), indicating that the procedure is nonreproducible. (C) Scatterplot of the signal intensities of two parallel reactions on the chromosomal DNA of a genome-wide library using TraSH. Correlation between the signal intensities for each gene indicates that the procedure is fairly reproducible (r², 0.825).

FIG. 3.

In vitro transcription on purified chromosomal DNA restriction fragments leads to specific and sensitive detection of transposon insertion sites. (A) Scatterplot of the comparison of reactions on the chromosomal DNA of a mutant library consisting of 90 unique marinerT7 mutants and the DNA of the same mutants to which the chromosomal DNA of 9 defined mutants has been added. Straight lines denote a twofold difference in signal intensities between the Cy5 and Cy3 channels. Genes identified by sequencing as added to the library are indicated as black squares. (B to D) Scatterplots of the signal intensities of two parallel reactions on the chromosomal DNA of a genome-wide library digested with TacI (B), DdeI (C), and AluI (D). Correlation between the signal intensities in each plot is >0.96, indicating that the procedure is highly reproducible.

FIG. 4.

czcD is a conditionally essential gene when S. pneumoniae is grown under high-Zn²⁺ conditions. (A) Growth of the wild type (filled triangles) and a czcD deletion mutant (open triangles) in GM17. (B) Growth of the wild type (filled triangles) and a czcD deletion mutant (open triangles) in GM17 to which 0.5 mM ZnSO₄ was added. Results are representative of at least three replicate experiments; the error bars indicate the standard deviations. OD₅₉₅, optical density at 595 nm. (C) Schematic representation of in silico digestion of the R6 chromosomal region containing czcD and its neighboring genes with TaqI. Arrows indicate the locations of genes, and gray rectangles indicate the genomic regions that are present as amplicons on the microarray.