Systematic identification of essential genes by in vitro mariner mutagenesis

Abstract

Although the complete DNA sequences of several microbial genomes are now available, nearly 40% of the putative genes lack identifiable functions. Comprehensive screens and selections for identifying functional classes of genes are needed to convert sequence data into meaningful biological information. One particularly significant group of bacterial genes consists of those that are essential for growth or viability. Here, we describe a simple system for performing transposon mutagenesis on naturally transformable organisms along with a technique to rapidly identify essential or conditionally essential DNA segments. We show the general utility of this approach by applying it to two human pathogens, Haemophilus influenzae and Streptococcus pneumoniae, in which we detected known essential genes and assigned essentiality to several ORFs of unknown function.

Figure 1

Schematic diagram of the two steps required for GAMBIT (a) Strategy for producing chromosomal mutations by using in vitro transposon mutagenesis. (b) Genetic footprinting for detection of essential genes. Target DNA mutagenized in vitro with the Himar1 transposon was introduced into bacteria by transformation and homologous recombination. Recombinants were selected for drug resistance encoded by the transposon, and insertions in essential genes were lost from the pool during growth. PCR with primers that hybridize to the transposon and to specific chromosomal sites yielded a product corresponding to each mutation in the pool. DNA regions containing no insertions yielded a blank region on electrophoresis gels.

Figure 2

Southern blot analysis of H. influenzae transposon mutants. Genomic DNA was isolated from 16 individual mutants and was digested with AseI, which cleaves once within magellan1. Digested DNA was subjected to agarose gel electrophoresis, was transferred to nitrocellulose, and was hybridized with a probe composed solely of magellan1 minitransposon-derived DNA.

Figure 3

Genetic footprinting of H. influenzae mutant pools. Genetic footprinting was carried out by using a Himar1-specific primer and a chromosomal primer. In a, the positions of molecular weight standards are indicated; other panels are labeled with locus names by HI number. In c and d, cells were selected on BXV, MIc, or BXV containing trimethoprim (“Tri”). In f, in vitro mutagenesis of a chromosomal fragment that included the secA gene was performed, and the mutagenized DNA was transformed into both wild-type H. influenzae and an H. influenzae strain containing pSecA.

Figure 4

Essential ORFs of H. influenzae. Five chromosomal segments are shown. ORFs with essential functions are shown in black, ORFs that are nonessential are shown in white, and ORFs in which mutations produce growth attenuation are shown in gray. The direction of transcription for each ORF is shown along with the TIGR designation below the ORF and the closest homologue above the ORF. Conserved hypothetical ORFs of unknown function are designated CH. [*, Essential ORFs that can sustain a highly limited number of discrete insertions (<2/kbp).]