BACKGROUND: Antibiotics in current use target a surprisingly small number of cellular functions: cell wall, DNA, RNA, and protein biosynthesis. Targeting of novel essential pathways is expected to play an important role in the discovery of new antibacterial agents against bacterial pathogens, such as Pseudomonas aeruginosa, that are difficult to control because of their ability to develop resistance, often multiple, to all current classes of clinical antibiotics. RESULTS: We aimed to identify novel essential genes in P. aeruginosa by shotgun antisense screening. This technique was developed in Staphylococcus aureus and, following a period of limited success in Gram-negative bacteria, has recently been used effectively in Escherichia coli. To also target low expressed essential genes, we included some variant steps that were expected to overcome the non-stringent regulation of the promoter carried by the expression vector used for the shotgun antisense libraries. Our antisense screenings identified 33 growth-impairing single-locus genomic inserts that allowed us to generate a list of 28 "essential-for-growth" genes: five were "classical" essential genes involved in DNA replication, transcription, translation, and cell division; seven were already reported as essential in other bacteria; and 16 were "novel" essential genes with no homologs reported to have an essential role in other bacterial species. Interestingly, the essential genes in our panel were suggested to take part in a broader range of cellular functions than those currently targeted by extant antibiotics, namely protein secretion, biosynthesis of cofactors, prosthetic groups and carriers, energy metabolism, central intermediary metabolism, transport of small molecules, translation, post-translational modification, non-ribosomal peptide synthesis, lipopolysaccharide synthesis/modification, and transcription regulation. This study also identified 43 growth-impairing inserts carrying multiple loci targeting 105 genes, of which 25 have homologs reported as essential in other bacteria. Finally, four multigenic growth-impairing inserts belonged to operons that have never been reported to play an essential role. CONCLUSIONS: For the first time in P. aeruginosa, we applied regulated antisense RNA expression and showed the feasibility of this technology for the identification of novel essential genes.
BACKGROUND: Antibiotics in current use target a surprisingly small number of cellular functions: cell wall, DNA, RNA, and protein biosynthesis. Targeting of novel essential pathways is expected to play an important role in the discovery of new antibacterial agents against bacterial pathogens, such as Pseudomonas aeruginosa, that are difficult to control because of their ability to develop resistance, often multiple, to all current classes of clinical antibiotics. RESULTS: We aimed to identify novel essential genes in P. aeruginosa by shotgun antisense screening. This technique was developed in Staphylococcus aureus and, following a period of limited success in Gram-negative bacteria, has recently been used effectively in Escherichia coli. To also target low expressed essential genes, we included some variant steps that were expected to overcome the non-stringent regulation of the promoter carried by the expression vector used for the shotgun antisense libraries. Our antisense screenings identified 33 growth-impairing single-locus genomic inserts that allowed us to generate a list of 28 "essential-for-growth" genes: five were "classical" essential genes involved in DNA replication, transcription, translation, and cell division; seven were already reported as essential in other bacteria; and 16 were "novel" essential genes with no homologs reported to have an essential role in other bacterial species. Interestingly, the essential genes in our panel were suggested to take part in a broader range of cellular functions than those currently targeted by extant antibiotics, namely protein secretion, biosynthesis of cofactors, prosthetic groups and carriers, energy metabolism, central intermediary metabolism, transport of small molecules, translation, post-translational modification, non-ribosomal peptide synthesis, lipopolysaccharide synthesis/modification, and transcription regulation. This study also identified 43 growth-impairing inserts carrying multiple loci targeting 105 genes, of which 25 have homologs reported as essential in other bacteria. Finally, four multigenic growth-impairing inserts belonged to operons that have never been reported to play an essential role. CONCLUSIONS: For the first time in P. aeruginosa, we applied regulated antisense RNA expression and showed the feasibility of this technology for the identification of novel essential genes.
Authors: Michael A Jacobs; Ashley Alwood; Iyarit Thaipisuttikul; David Spencer; Eric Haugen; Stephen Ernst; Oliver Will; Rajinder Kaul; Christopher Raymond; Ruth Levy; Liu Chun-Rong; Donald Guenthner; Donald Bovee; Maynard V Olson; Colin Manoil Journal: Proc Natl Acad Sci U S A Date: 2003-11-14 Impact factor: 11.205
Authors: R Allyn Forsyth; Robert J Haselbeck; Kari L Ohlsen; Robert T Yamamoto; Howard Xu; John D Trawick; Daniel Wall; Liangsu Wang; Vickie Brown-Driver; Jamie M Froelich; Kedar G C; Paula King; Melissa McCarthy; Cheryl Malone; Brian Misiner; David Robbins; Zehui Tan; Zhan-yang Zhu Zy; Grant Carr; Deborah A Mosca; Carlos Zamudio; J Gordon Foulkes; Judith W Zyskind Journal: Mol Microbiol Date: 2002-03 Impact factor: 3.501
Authors: S Y Gerdes; M D Scholle; J W Campbell; G Balázsi; E Ravasz; M D Daugherty; A L Somera; N C Kyrpides; I Anderson; M S Gelfand; A Bhattacharya; V Kapatral; M D'Souza; M V Baev; Y Grechkin; F Mseeh; M Y Fonstein; R Overbeek; A-L Barabási; Z N Oltvai; A L Osterman Journal: J Bacteriol Date: 2003-10 Impact factor: 3.490
Authors: Geoffrey L Winsor; David K W Lam; Leanne Fleming; Raymond Lo; Matthew D Whiteside; Nancy Y Yu; Robert E W Hancock; Fiona S L Brinkman Journal: Nucleic Acids Res Date: 2010-10-06 Impact factor: 16.971