Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Research Article J Mol Microbiol Biotechnol 2010;19:169–179 DOI: 10.1159/000322157 Cloning and Targeted Disruption of Two Lipopolysaccharide Biosynthesis Genes, kdsA and waaG, of Pseudomonas aeruginosa PAO1 by Site-Directed Mutagenesis Deepak Perumal   a, b Kishore R. Sakharkar   b, d Thean Hock Tang   d Vincent T.K. Chow   c Chu Sing Lim   b Areejit Samal   e, f Norio Sugiura   g Meena K. Sakharkar   a, b, g a  Advanced Design and Modeling Laboratory, Nanyang Technological University, b  Pharmacogenomics Group, BioMedical Engineering Research Centre, Research Technoplaza, Nanyang Technological University, and c  Human Genome Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; d  Infectious Disease Cluster, Advanced Medical & Dental Institute, Universiti Sains Malaysia, Penang, Malaysia; e  Max Planck Institute for Mathematics in the Sciences, Leipzig, Germany; f  Laboratoire de Génétique Végétale du Moulon, Université Paris-Sud, Ferme du Moulon, Gif-sur-Yvette, France; g  School of Life and Environmental Science, University of Tsukuba, Tsukuba, Japan ed as putative target candidates for the gene disruption ex- periments using plasmid insertion mutagenesis to deter- mine essentiality. The introduction of a selectable ampicillin and kanamycin resistance marker into the chromosome re- sulted in lack of recovery of antibiotic-resistant colonies sug- gesting the essentiality of these genes for the survival of P. aeruginosa. Several molecular analyses were carried out in order to confirm the essentiality of these genes. We propose that the above two validated drug targets are essential and can be screened for functional inhibitors for the discovery of novel therapeutic compounds against antibiotic-resistant opportunistic pathogen P. aeruginosa. Copyright © 2010 S. Karger AG, Basel Introduction Antibiotics target genes that are required for bacterial growth and survival. Hence, knowledge on essential genes is of importance both for understanding of the Key Words Essential genes  Plasmid insertion mutagenesis  Lipopolysaccharide biosynthesis  Antibacterial drugs Abstract The emergence of antibiotic resistance in bacterial patho- gens poses a great challenge to public health and empha- sizes the need for new antimicrobial targets. The recent de- velopment of microbial genomics and the availability of ge- nome sequences allows for the identification of essential genes which could be novel and potential targets for anti- bacterial drugs. However, these predicted targets need ex- perimental validation to confirm essentiality. Here, we report on experimental validation of a two potential targets in the lipopolysaccharide (LPS) biosynthesis pathway of the patho- gen Pseudomonas aeruginosa PAO1 using insertion duplica- tion. Two genes, kdsA and waaG, from LPS encoding proteins 2-dehydro-3-deoxyphosphooctonate aldolase and UDP- glucose (heptosyl) LPS -1,3-glucosyltransferase were select- Published online: October 29, 2010 Prof. Meena K. Sakharkar, PhD School of Life and Environmental Science, University of Tsukuba Tsukuba, Ibaraki (Japan) Tel. +81 29 853 8834 E-Mail meena.sak.gn  @  u.tsukuba.ac.jp © 2010 S. Karger AG, Basel 1464–1801/10/0194–0169$26.00/0 Accessible online at: www.karger.com/mmb Downloaded by: Universiti Sains Malaysia 198.143.39.65 - 9/9/2015 6:05:37 AM