Systematic identification of essential genes by in vitro mariner mutagenesis.
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.
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
B J
BJ
Akerley
E J
EJ
Rubin
A
A
Camilli
D J
DJ
Lampe
H M
HM
Robertson
J J
JJ
Mekalanos
D001483:Base Sequence; D017931:DNA Primers; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D005798:Genes, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics*; D016296:Mutagenesis; D016366:Open Reading Frames; D013296:Streptococcus pneumoniae / Q000235:genetics*; D019895:Transposases
D017931:DNA Primers; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C111652:mariner transposases; D019895:Transposases