Elucidation of a novel lipid A α-(1,1)-GalA transferase gene (rgtF) from Mesorhizobium loti: Heterologous expression of rgtF causes Rhizobium etli to synthesize lipid A with α-(1,1)-GalA

Abstract

An unusual α-(1,1)-galacturonic acid (GalA) lipid A modification has been reported in the lipopolysaccharide of a number of interesting Gram-negative bacteria, including the nitrogen-fixing bacteria Azospirillum lipoferum, Mesorhizobium huakuii and M. loti, the stalk-forming bacterium Caulobacter crescentus and the hyperthermophilic bacterium Aquifex aeolicus. However, the α-(1,1)-GalA transferase (GalAT) gene, which we have named RgtF, was not identified. Species of the Rhizobium genera produce lipid A with α-(1,4')-GalA but not α-(1,1)-GalA. The Rhizobium GalAT, RgtD, is the lipid A α-(1-4')-GalAT which utilizes the lipid donor dodecaprenyl-phosphate GalA (Dod-P-GalA) for GalA transfer. An additional Rhizobium GalAT, RgtE, is required for the biosynthesis of Dod-P-GalA. We predicted candidate rgtF genes in bacterial species known to produce lipid A with α-(1,1)-GalA. In order to determine the predicted rgtF gene function, we cloned the M. loti rgtF gene into an expression plasmid and introduced that plasmid into Rhizobium etli strains that do not contain the rgtF gene nor produce lipid A α-(1,1)-GalA. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis combined with NMR studies revealed that the lipid As from these rgtF-complemented strains were modified with an additional α-(1,1)-GalA attached to the proximal glucosamine.

Occurrence of an Unusual Hopanoid-containing Lipid A Among Lipopolysaccharides from Bradyrhizobium Species

“…The presence of D-GlcpN3N in the lipid A backbone of the LPS of nitrogen-fixing bacteria is rather common. This amino sugar was reported for lipid A of the LPS from Mesorhizobium loti (18,43), M. huakuii (20), A. caulinodans (24), and other symbiotic bacteria belonging to the genera Ochrobactrum and Phyllobacterium.…”

“…This unique substitution of the lipid A backbone had been noticed 3 A. Choma, personal communication. earlier in Mesorhizobium huakuii (20) and Mesorhizobium loti strains (43), which contained a non-reducing trisaccharide backbone comprising a ␤-(136)-D-GlcpN3N disaccharide partly substituted with phosphate at C-4Ј and an ␣-(131)-linked D-GalpA in stoichiometric amounts. Other microorganisms that produce lipid A substituted with ␣-(131)-D-GalpA include A. pyrophilus (48), the stalk-forming bacterium Caulobacter crescentus (51), and the plant-growth promoting bacterium Azospirillum lipoferum (52).…”

“…A gene cluster that encoded for D-GalA transferase (rgtF, ArnT-like glycosyl transferase), lipid A 1-phosphatase (lpxE), and glycosyl transferase (rgtE), in the relative order: rgtE, duf, rgtF, and lpxE (where duf is a domain with unknown function), was identified in M. loti MAFF303099 genome (43). Similar gene clusters were found also in other mesorhizobial strains (Mesorhizobium ciceri WSM1271 and Mesorhizobium opportunistum WSM2075), as well as in C. crescentus CB15 and A. lipoferum B510 (43).…”

“…A gene cluster that encoded for D-GalA transferase (rgtF, ArnT-like glycosyl transferase), lipid A 1-phosphatase (lpxE), and glycosyl transferase (rgtE), in the relative order: rgtE, duf, rgtF, and lpxE (where duf is a domain with unknown function), was identified in M. loti MAFF303099 genome (43). Similar gene clusters were found also in other mesorhizobial strains (Mesorhizobium ciceri WSM1271 and Mesorhizobium opportunistum WSM2075), as well as in C. crescentus CB15 and A. lipoferum B510 (43). BLAST similarity searches of B. japonicum USDA 110 genome revealed the presence of a similar gene cluster, which contained putative rgtF, lpxE, and rgtE genes, encoding proteins with high sequence identity (37-55%) with respective proteins of M. loti (data not shown), and thus proving the presence of genes necessary for the synthesis of ␣-(131)-D-GalpA substituted lipid A in this strain.…”

“…BLAST similarity searches of B. japonicum USDA 110 genome revealed the presence of a similar gene cluster, which contained putative rgtF, lpxE, and rgtE genes, encoding proteins with high sequence identity (37-55%) with respective proteins of M. loti (data not shown), and thus proving the presence of genes necessary for the synthesis of ␣-(131)-D-GalpA substituted lipid A in this strain. Brown and co-workers showed that the R. leguminosarum rgtE Ϫ mutant, which produces LPS completely devoid of D-GalpA, was severely compromised in membrane stability (43). Thus, we assume that the synthesis of D-GalpAcontaining lipid A by Bradyrhizobium bacteria, apart from the presence of the hopanoid substituent, may cause an additional stabilization of the outer membrane, most probably by the presence of divalent cationic bridging, which is similar to mammalian pathogens that produce phosphate-containing LPS (lipid A) (10, 43).…”