Identification of essential genes in Streptococcus pneumoniae by allelic replacement mutagenesis.

To find potential targets of novel antimicrobial agents, we identified essential genes of Streptococcus pneumoniae using comparative genomics and allelic replacement mutagenesis. We compared the genome of S. pneumoniae R6 with those of Bacillus subtilis, Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus, and selected 693 candidate target genes with > 40% amino acid sequence identity to the corresponding genes in at least two of the other species. The 693 genes were disrupted and 133 were found to be essential for growth. Of these, 32 encoded proteins of unknown function, and we were able to identify orthologues of 22 of these genes by genomic comparisons. The experimental method used in this study is easy to perform, rapid and efficient for identifying essential genes of bacterial pathogens.

[1]  Hong-Yu Ou,et al.  EG: a database of essential genes , 2004, Nucleic Acids Res..

[2]  James R. Brown,et al.  A Global Approach to Identify Novel Broad-Spectrum Antibacterial Targets among Proteins of Unknown Function , 2004, Journal of Molecular Microbiology and Biotechnology.

[3]  Cheorl-Ho Kim.  A Salmonella typhimurium rfaE mutant recovers invasiveness for human epithelial cells when complemented by wild type rfaE (controlling biosynthesis of ADP-L-glycero-D-mannoheptose-containing lipopolysaccharide). , 2003, Molecules and cells.

[4]  S. Ehrlich,et al.  Essential Bacillus subtilis genes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[5]  S. D. Mills.  The role of genomics in antimicrobial discovery. , 2003, The Journal of antimicrobial chemotherapy.

[6]  M. Lonetto,et al.  Characterization of a Novel Fucose-Regulated Promoter (PfcsK) Suitable for Gene Essentiality and Antibacterial Mode-of-Action Studies in Streptococcus pneumoniae , 2003, Journal of bacteriology.

[7]  R. Levesque,et al.  Structure and function of the Mur enzymes: development of novel inhibitors , 2002, Molecular microbiology.

[8]  J. Clements,et al.  Novel approaches to antimicrobial therapy: peptide deformylase. , 2002, Current opinion in drug discovery & development.

[9]  B. Dougherty,et al.  Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae. , 2002, Nucleic acids research.

[10]  D. Adam.  Global antibiotic resistance in Streptococcus pneumoniae. , 2002, The Journal of antimicrobial chemotherapy.

[11]  E. Koonin,et al.  Essential genes are more evolutionarily conserved than are nonessential genes in bacteria. , 2002, Genome research.

[12]  G. Pozzi,et al.  Construction of new unencapsulated (rough) strains of Streptococcus pneumoniae. , 2002, Research in microbiology.

[13]  Howard Xu,et al.  A genome‐wide strategy for the identification of essential genes in Staphylococcus aureus , 2002, Molecular microbiology.

[14]  J. Mekalanos,et al.  A genome-scale analysis for identification of genes required for growth or survival of Haemophilus influenzae , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[15]  E. Rubin,et al.  Comprehensive identification of conditionally essential genes in mycobacteria , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Elliot J. Lefkowitz,et al.  Genome of the Bacterium Streptococcus pneumoniae Strain R6 , 2001, Journal of bacteriology.

[17]  Martin Rosenberg,et al.  Identification of Critical Staphylococcal Genes Using Conditional Phenotypes Generated by Antisense RNA , 2001, Science.

[18]  S. Salzberg,et al.  Complete Genome Sequence of a Virulent Isolate of Streptococcus pneumoniae , 2001, Science.

[19]  M. Page,et al.  Peptide Deformylase as an Antibacterial Drug Target: Target Validation and Resistance Development , 2001, Antimicrobial Agents and Chemotherapy.

[20]  Paul S. Hoffman,et al.  Systematic Identification of Selective Essential Genes in Helicobacter pylori by Genome Prioritization and Allelic Replacement Mutagenesis , 2001, Journal of bacteriology.

[21]  O. White,et al.  Global transposon mutagenesis and a minimal Mycoplasma genome. , 1999, Science.

[22]  T J Dougherty,et al.  Concordance analysis of microbial genomes. , 1998, Nucleic acids research.

[23]  Manuel Peitsch,et al.  A genome-based approach for the identification of essential bacterial genes , 1998, Nature Biotechnology.

[24]  D. Lipman,et al.  A genomic perspective on protein families. , 1997, Science.

[25]  D. Morrison,et al.  An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[26]  P. Manning,et al.  Cloning and expression in Escherichia coli of the Streptococcus pneumoniae gene encoding pneumolysin , 1986, Infection and immunity.

[27]  P. Trieu-Cuot,et al.  Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III. , 1983, Gene.