Use of the riboflavin synthase gene (ribC) as a model for development of an essential gene disruption and complementation system for Haemophilus influenzae

Applied and Environmental Microbiology
Amna Saeed-KotheScott D Mills

Abstract

We have developed a system for rapid and reliable assessment of gene essentiality in Haemophilus influenzae Rd strain KW20. We constructed two "suicide" complementation vectors (pASK5 and pASK6) containing 5' and 3' regions of the nonessential ompP1 gene flanking a multiple cloning site and a selectable marker (a chloramphenicol resistance gene or a tetracycline resistance cassette). Transformation of H. influenzae with the complementation constructs directs chromosomal integration of a gene of interest into the ompP1 locus, where the strong, constitutive ompP1 promoter drives its expression. This single-copy, chromosome-based complementation system is useful for confirming the essentiality of disrupted genes of interest. It allows genetic analysis in a background free of interference from any upstream or downstream genetic elements and enables conclusive assignment of essentiality. We validated this system by using the riboflavin synthase gene (ribC), a component of the riboflavin biosynthetic pathway. Our results confirmed the essentiality of ribC for survival of H. influenzae Rd strain KW20 and demonstrated that a complementing copy of ribC placed under control of the ompP1 promoter reverses the lethal phenotype of a strain ...Continue Reading

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Citations

Oct 11, 2011·The Journal of Biological Chemistry·Ed T BuurmanAdam B Shapiro
Dec 30, 2010·Antimicrobial Agents and Chemotherapy·Scott D MillsSuzanne S Stokes
Jun 19, 2013·BMC Microbiology·Randy J HempelTerrence L Stull
Jan 16, 2016·European Journal of Clinical Microbiology & Infectious Diseases : Official Publication of the European Society of Clinical Microbiology·F S BitarafS L Mousavi Gargari
Jan 16, 2010·Microbiology·Paul W WhitbyTerrence L Stull
Nov 18, 2005·FEMS Microbiology Letters·Daniel J MortonTerrence L Stull
Mar 16, 2016·PloS One·Jennifer M Kress-BennettBenjamin A Janto
Nov 16, 2016·American Journal of Respiratory and Critical Care Medicine·Kristian Riesbeck
Jan 3, 2006·Biochemical Pharmacology·Scott D Mills
Nov 18, 2018·PLoS Computational Biology·Joana C XavierIsabel Rocha
Jan 30, 2020·Frontiers in Cellular and Infection Microbiology·Müberra Fatma CesurTunahan Çakır

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