The deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine (UDP-3-O-acyl-GlcNAc) by LpxC is the committed reaction of lipid A biosynthesis. CHIR-090, a novel N-aroyl-l-threonine hydroxamic acid, is a potent, slow, tight-binding inhibitor of the LpxC deacetylase from the hyperthermophile Aquifex aeolicus, and it has excellent antibiotic activity against Pseudomonas aeruginosa and Escherichia coli, as judged by disk diffusion assays. We now report that CHIR-090 is also a two-step slow, tight-binding inhibitor of E. coli LpxC with Ki = 4.0 nM, Ki* = 0.5 nM, k5 = 1.9 min-1, and k6 = 0.18 min-1. CHIR-090 at low nanomolar levels inhibits LpxC orthologues from diverse Gram-negative pathogens, including P. aeruginosa, Neisseria meningitidis, and Helicobacter pylori. In contrast, CHIR-090 is a relatively weak competitive and conventional inhibitor (lacking slow, tight-binding kinetics) of LpxC from Rhizobium leguminosarum (Ki = 340 nM), a Gram-negative plant endosymbiont that is resistant to this compound. The KM (4.8 microM) and the kcat (1.7 s-1) of R. leguminosarum LpxC with UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine as the substrate are similar to values reported for E. coli LpxC. R. leguminosarum LpxC therefore provides a useful control for validating LpxC as the primary target of CHIR-090 in vivo. An E. coli construct in which the chromosomal lpxC gene is replaced by R. leguminosarum lpxC is resistant to CHIR-090 up to 100 microg/mL, or 400 times above the minimal inhibitory concentration for wild-type E. coli. Given its relatively broad spectrum and potency against diverse Gram-negative pathogens, CHIR-090 is an excellent lead for the further development of new antibiotics targeting the lipid A pathway.