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UDP-3-O-(R-3-hydroxyacyl)GlcN N-acyltransferase (LpxD) has been shown to be essential to survival of lipid A producing Gram-negative bacteria. In this study, LpxD-binding peptides 12 amino acids in length were identified from a phage-bound random peptide library screen. Three peptides displayed antibacterial activity when expressed intracellularly, one of which (RJPXD33) represented 15% of the total hits. RJPXD33 binds to E. coli LpxD with a Kd of 6 μM and is competitive with R-3-hydroxymyristoyl-ACP binding. RJPXD33 can be C-terminally fused in vivo with thioredoxin or N-terminally modified in vitro with beta-alanyl-fluorescein and maintain LpxD binding. The latter was used to develop an LpxD fluorescent binding assay used to evaluate unlabeled ligands, and is amenable to small molecule library screening. Furthermore, RJPXD33 also binds to and inhibits E. coli UDP-N-acetylglucosamine acyltransferase (LpxA) with a Kd of 20 μM, unearthing the possibility for the development of small molecule, dual-binding LpxA/LpxD inhibitors as novel antimicrobials.

This work demonstrates coupling of the newly described electrophoretic enrichment technique of gradient elution isotachophoresis (GEITP) to a low-cost, conventional ultraviolet absorbance detector to realize sensitive measurements with a universal detector, eliminating the need for fluorescent analytes or derivatization. The effects of various parameters on enrichment were studied, including current density varied by leading electrolyte concentration, current density varied by applied electric field, and counter-flow acceleration across varying capillary inner diameters. Optimized parameters were applied to the enrichment and separation of the amino acids tryptophan (Trp) and tyrosine (Tyr). Limits of detection for Trp and Tyr were 51 and 215 nM, respectively, reflecting sensitivity enhancements of 860- and 1900-fold. Analysis times were less than 6 min, and peak height RSDs were less than 4%. A demonstration of enrichment and separation of these amino acids from artificial cerebrospinal fluid is additionally shown as a first step to realizing biochemical monitoring by GEITP.

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. Conclusion 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. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0336-x) contains supplementary material, which is available to authorized users.

UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(R-3-hydroxyacyl)-glucosamine acyltransferase (LpxD) catalyze the first and third steps of Lipid A biosynthesis, respectively. Both enzymes have been found to be essential for survival among Gram-negative bacteria which synthesize lipopolysaccharide, and are viable targets for antimicrobial development. Catalytically, both acyltransferases catalyze an acyl-acyl carrier protein (ACP) dependent transfer of a fatty acyl moiety to a UDP-glucosamine core ring. Herein, we exploit the single free-thiol unveiled on holo-ACP after transfer of the fatty acyl group to the glucosamine ring using the thiol specific labeling reagent, ThioGlo. The assay is continuously monitored as a change in fluorescence at λex = 379 nm and λem = 513 nm using a microtiter plate reader. This assay marks the first continuous and non-radioactive assay for either acyltransferase.