Francisella tularensis is a highly infectious pathogen that causes tularemia. Francisella lipid A contains an unusual galactosamine (GalN) unit, attached to its 1-phosphate moiety. Two genes, flmF2 and flmK, are required for the addition of GalN to Francisella lipid A, but the relevant enzymes and the GalN donor substrate have not been characterized. We now report the purification and identification of a novel minor lipid from Francisella novicida that functions as the GalN donor. On the basis of electrospray ionization mass spectrometry (ESI/MS) and NMR spectroscopy, we propose that this compound is undecaprenyl phosphate-β-D-GalN. Approximately 0.5 mg of pure lipid was obtained from 10 g of F. novicida by chloroform/methanol extraction, followed by DEAE-cellulose chromatography, mild alkaline hydrolysis, and thin-layer chromatography. ESI/MS in the negative mode revealed a molecular ion [M - H](-) at m/z 1006.699, consistent with undecaprenyl phosphate-GalN. (31)P NMR spectroscopy showed a single phosphorus atom in the phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the sugar. (1)H NMR studies showed the presence of a polyisoprene chain and a sugar consistent with a β-D-GalN unit. Heteronuclear multiple-quantum coherence (HMQC) analysis confirmed that nitrogen is attached to C-2 of the sugar. Purified undecaprenyl phosphate-β-D-GalN supports the in vitro modification of lipid IV(A) by membranes of Escherichia coli cells expressing FlmK, an orthologue of E. coli ArnT, the enzyme that transfers 4-amino-4-deoxy-l-arabinose to lipid A in polymyxin-resistant strains. The discovery of undecaprenyl phosphate-β-D-GalN suggests Francisella modifies lipid A with GalN on the periplasmic surface of the inner membrane.

lipid A, modification, Francisella tularensis, Undecaprenyl phosphate, tularemia

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
0	bDGalpN	96.12	55.26	70.46	68.66	77.08	61.98
	P


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
0	bDGalpN	5.065	3.217	3.77	3.907	3.671	3.725-3.789
	P


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
0	bDGalpN	96.12/5.065	55.26/3.217	70.46/3.77	68.66/3.907	77.08/3.671	61.98/3.725-3.789
	P

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

N[C@H]1[C@H](OP(=O)(O)O)O[C@H](CO)[C@H](O)[C@@H]1O

259.151 g/mol (C₆H₁₄NO₈P)