title: Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA.
abstract: BACKGROUND
  Francisella tularensis is a Gram-negative bacterium that infects hundreds of species including humans, and has evolved to grow efficiently within a plethora of cell types. RipA is a conserved membrane protein of F. tularensis, which is required for growth inside host cells. As a means to determine RipA function we isolated and mapped independent extragenic suppressor mutants in ∆ripA that restored growth in host cells. Each suppressor mutation mapped to one of two essential genes, lpxA or glmU, which are involved in lipid A synthesis. We repaired the suppressor mutation in lpxA (S102, LpxA T36N) and the mutation in glmU (S103, GlmU E57D), and demonstrated that each mutation was responsible for the suppressor phenotype in their respective strains. We hypothesize that the mutation in S102 altered the stability of LpxA, which can provide a clue to RipA function. LpxA is an UDP-N-acetylglucosamine acyltransferase that catalyzes the transfer of an acyl chain from acyl carrier protein (ACP) to UDP-N-acetylglucosamine (UDP-GlcNAc) to begin lipid A synthesis.
  
  
  RESULTS
  LpxA was more abundant in the presence of RipA. Induced expression of lpxA in the ΔripA strain stopped bacterial division. The LpxA T36N S102 protein was less stable and therefore less abundant than wild type LpxA protein.
  
  
  CONCLUSIONS
  These data suggest RipA functions to modulate lipid A synthesis in F. tularensis as a way to adapt to the host cell environment by interacting with LpxA.
authors.forename: Cheryl N
authors.initials: CN
authors.lastname: Miller
authors.forename: Shaun P
authors.initials: SP
authors.lastname: Steele
authors.forename: Jason C
authors.initials: JC
authors.lastname: Brunton
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Jenkins
authors.forename: Eric D
authors.initials: ED
authors.lastname: LoVullo
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Taft-Benz
authors.forename: Artur
authors.initials: A
authors.lastname: Romanchuk
authors.forename: Corbin D
authors.initials: CD
authors.lastname: Jones
authors.forename: Garry D
authors.initials: GD
authors.lastname: Dotson
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Collins
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Kawula
mesh_terms: D000217:Acyltransferases / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D005604:Francisella tularensis / Q000235:genetics; D008050:Lipid A / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D013489:Suppression, Genetic / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D008050:Lipid A; D000217:Acyltransferases; C050304:acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine acyltransferase