Abstract
Until now, the gene responsible for the 3-O-deacylation of lipid A among nitrogen-fixing endosymbionts has not been characterized. Several Gram-negative animal pathogens such as Salmonella enterica, Pseudomonas aeruginosa, and Bordetella bronchiseptica contain an outer membrane 3-O-deacylase (PagL) that has been implicated in host immune evasion. The role of 3-O-deacylated lipid A among nitrogen-fixing endosymbionts, plant endophytes, and plant pathogens has not been studied. However, D'Haeze et al. (D'Haeze, W., Leoff, C., Freshour, G., Noel, K. D., and Carlson, R. W. (2007) J. Biol. Chem. 282, 17101-17113) reported that the lipopolysaccharide from Rhizobium etli CE3 bacteroids isolated from host bean root nodules contained exclusively tetraacylated lipid A that lacked a lipid A β-hydroxymyristyl residue, an observation that is consistent with the possibility of PagL activity being important in symbiosis. A putative pagL gene was identified in the R. etli genome sequence. With this information, we created a pagL(-) mutant strain derived from R. etli CE3. Using mass spectrometry, we demonstrated that the mutant lacks 3-O-deacylated lipid A. The parent and mutant LPS were very similar as determined by gel electrophoresis and glycosyl composition analysis using gas chromatography/mass spectrometry. However, fatty acid analysis showed that the mutant lipid A contained larger amounts of β-hydroxypentadecanoic acid than that of the parent. Furthermore, the mutant was adversely affected in establishing symbiosis with its host, Phaseolus vulgaris.
Highlights
R. etli lipid-A de-O-acylation occurs during symbiosis
Because the expected product resulting from PagL activity is structure D, these results strongly suggest that the R. etli CE3 pagL gene product is responsible for the removal of a primary -hydroxy fatty acid from the lipid A
Mass spectrometric evidence suggests that the endosymbionts R. etli CE3 [9] and S. fredii NGR 234 [33], when grown under laboratory conditions, produce 3-O-deacylated lipid A molecules
Summary
R. etli lipid-A de-O-acylation occurs during symbiosis. Results: R. etli produces PagL, which de-O-acylates lipid A, and is active on E. coli lipid A, and a pagL mutant has a symbiotic defect. 282, 17101–17113) reported that the lipopolysaccharide from Rhizobium etli CE3 bacteroids isolated from host bean root nodules contained exclusively tetraacylated lipid A that lacked a lipid A -hydroxymyristyl residue, an observation that is consistent with the possibility of PagL activity being important in symbiosis. Partial disruption of LPS biosynthesis (e.g., O-chain biosynthesis and lipid A fatty acid acylation) leads to disruption of proper symbiosis in many nitrogen-fixing symbiotic systems (4 – 8). The outer membrane 3-O-deacylase enzyme PagL has been reported in a number of Gram-negative pathogens including Salmonella typhimurium, Salmonella enterica, Pseudomonas aeruginosa, and Bordetella bronchiseptica [13] and has been implicated in the evasion of the host immune response [14, 15]. We further characterized the effect of the mutation on LPS synthesis and symbiosis with the host plant P. vulgaris
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