Abstract
BackgroundFrancisella 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.ResultsLpxA 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.ConclusionThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0336-x) contains supplementary material, which is available to authorized users.
Highlights
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
Extragenic suppressor mutation enrichment in the ΔripA background To enrich for extragenic suppressor mutations in the ΔripA strain we performed several rounds of infections lasting 36 hours in J774A.1 cells whereby the bacteria isolated from the first round of infections were used to inoculate a new flask of J774A.1 cells
Using whole genome sequencing with the Genome Analyzer IIx (Illumina) we mapped the suppressor mutation in S102 by conducting comparative analysis aligning the sequencing reads from the suppressor mutant strain to the annotated genome on NCBI (NC_007880) (Table 1)
Summary
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. The ΔripA strain infects host cells and escapes the phagosome entering the cytoplasm similar to wild type F. tularensis, but fails to replicate within the cytosol [1]. Both transcription and translation of ripA are elevated at neutral pH coinciding with F. tularensis leaving the acidified vacuole and entering the neutral cytoplasm [3]. Given the scant information obtained from bioinformatics analysis of RipA, and the eclectic
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