Altered lipid A structures and polymyxin hypersensitivity of Rhizobium etli mutants lacking the LpxE and LpxF phosphatases

Abstract

The lipid A of Rhizobium etli, a nitrogen-fixing plant endosymbiont, displays significant structural differences when compared to that of Escherichia coli. An especially striking feature of R. etli lipid A is that it lacks both the 1- and 4'-phosphate groups. The 4'-phosphate moiety of the distal glucosamine unit is replaced with a galacturonic acid residue. The dephosphorylated proximal unit is present as a mixture of the glucosamine hemiacetal and an oxidized 2-aminogluconate derivative. Distinct lipid A phosphatases directed to the 1 or the 4'-positions have been identified previously in extracts of R. etli and Rhizobium leguminosarum. The corresponding structural genes, lpxE and lpxF, respectively, have also been identified. Here, we describe the isolation and characterization of R. etli deletion mutants in each of these phosphatase genes and the construction of a double phosphatase mutant. Mass spectrometry confirmed that the mutant strains completely lacked the wild-type lipid A species and accumulated the expected phosphate-containing derivatives. Moreover, radiochemical analysis revealed that phosphatase activity was absent in membranes prepared from the mutants. Our results indicate that LpxE and LpxF are solely responsible for selectively dephosphorylating the lipid A molecules of R. etli. All the mutant strains showed an increased sensitivity to polymyxin relative to the wild-type. However, despite the presence of altered lipid A species containing one or both phosphate groups, all the phosphatase mutants formed nitrogen-fixing nodules on Phaseolus vulgaris. Therefore, the dephosphorylation of lipid A molecules in R. etli is not required for nodulation but may instead play a role in protecting the bacteria from cationic antimicrobial peptides or other immune responses of plants.

Figure 1. Structures of the major lipid A species present in E. coli and R. etli

Panel A. The predominant lipid A moiety of E. coli LPS consists of a hexa-acylated disaccharide of glucosamine, substituted with monophosphate groups at the 1- and 4′-positions. There is very little acyl chain heterogeneity [8]. Panels B and C. The major lipid A species in R. etli and R. leguminosarum lack phosphate substituents and are more heterogeneous in with respect to fatty acyl chain length [14, 15]. Components B and C are constructed around the typical glucosamine disaccharide found in the lipid A of other Gram-negative organisms. Components D and E feature an aminogluconate unit in place of the proximal glucosamine residue, derived by LpxQ-catalyzed oxidation of B and C respectively [28, 29]. All R. etli lipid A species contain a galacturonic acid moiety at position-4′ in place of the more typical monophosphate group, and they also contain an unusual C28 secondary acyl chain that may be further derivatized at the 27-OH moiety with a β-hydroxybutyrate group [13]. Components C and E differ from B and D by the absence of a hydroxyacyl chain at position 3, which is removed by the deacylase PagL [30]. Dashed bonds highlight the most prominent micro-heterogeneity of R. etli lipid A with respect to its acyl chains and the presence of the β-hydroxybutyrate substituent. Micro-heterogeneity arising from minor branched-chain or even chain fatty acids is not indicated, but is apparent in the mass spectra shown below.

Figure 2. Topography of LPS assembly and lipid A modification in R. etli

With the exception of the UDP-diacylglucosamine hydrolase LpxH, which is replaced by LpxI (L. E. Metzler and C. R. H. Raetz, in preparation), the constitutive enzymes that generate the phosphorylated intermediate Kdo₂-lipid IV_A in E. coli are also present in R. etli [17]. The assembly of R. etli lipid A diverges after Kdo₂-lipid IV_A formation, starting with the addition of the 27-hydroxyoctacosanoate chain (27OHC28:0), catalyzed by LpxXL [20]. After completion of core glycosylation and transport to the outer surface of the inner membrane by MsbA, LpxF and LpxE remove the phosphate moieties at the 4′- and 1-positions, respectively [22, 23, 25]. Next, RgtD is thought to utilize dodecaprenyl phosphate-galacturonic acid to incorporate the 4′-galacturonic acid residue, and RgtA/RbtB similarly modify the outer Kdo unit [26, 27]. After completion of O-antigen assembly (not shown) and transport to the outer membrane by the Lpt proteins [68], the ester-linked hydroxyacyl chain at position 3 may be removed by the deacylase PagL [30, 31], and the proximal glucosamine may be converted to the aminogluconate unit by the oxidase LpxQ [28, 29]. The orientation of the LpxQ active site within the outer membrane is not established unequivocally.

Figure 3. Predicted structures of the major lipid A species in lpxF, lpxE, and lpxE/lpxF deletion mutants of R. etli

Panel A. The proposed major lipid A species (B’, C’, D’ and E’) of the R. etli LpxF mutant should resemble that of the wild-type, except that the 4′-galacturonic acid unit would be replaced with a monophosphate group. Panel B. The proposed major lipid A species (F and G) of the R. etli LpxE mutant should be less heterogeneous than wild-type, because the proximal glucosamine unit cannot be oxidized by LpxQ. Panel C. The proposed major lipid A species (H and I) of the double mutant should retain phosphate groups at both the 1- and 4′-positions, resembling the lipid A of Sinorhizobium meliloti [49]. The exact masses predicted for these predominant lipid A molecular species are shown in Table II.

Figure 4. TLC analysis of lipid A species released by acetic acid hydrolysis from wild-type and phosphatase mutants

Approximately 5 μg of the lipid A molecules released from wild-type R. etli or the single phosphatase mutants by hydrolysis in 1% acetic acid were spotted onto a silica gel 60 TLC plate, which was developed in the solvent system CHCl₃/MeOH/H₂O/NH₄OH (40:25:4:2 v/v). The lipids were detected after chromatography by spraying with 10% sulfuric acid in ethanol and charring on a hot plate. The tentative assignments of the lipid species are based on their relative migrations in this experiment, which were previously validated for the wild-type [14,15].

Figure 5. ESI/MS analysis of lipid A species released by acetic acid hydrolysis from wild-type and single phosphatase mutants

The lipid A released from the wild-type or the phosphatase deletion mutants by 1% acetic acid hydrolysis was analyzed by negative ion ESI/MS. Panel A. The peaks in the singly charged region corresponding to the four major components of wild-type R. etli lipid A (B, C, D, and E), shown in Figs. 1B and 1C, are labeled and color coded. Component A (Supporting Fig. 3) is an elimination artifact derived from D during hydrolysis [14, 15]. There is additional micro-heterogeneity (see below) with regard to acyl chain length or the presence of the β-hydroxybutyrate substituent for each component, in agreement with previous studies [13]. Panel B. The spectrum in the singly charged region of the lipid A from the LpxF 4′-phosphatase mutant shows the same relative pattern of peaks as the wild-type, but each component (B’, C’, D’, and E’) is shifted by 96 amu because of the replacement of the 4′-galacturonic acid moiety with a phosphate group (Fig. 3A). Panel C. The spectrum in the singly charged region of the lipid A species from the LpxE 1-phosphatase mutant (Fig. 3B) show peaks at increased m/z values because of the retention of the phosphate group at the 1-position. There are also fewer lipid A components in this mutant because oxidation of the proximal glucosamine unit by LpxQ cannot occur. The hydrolysis artifacts (A and A’), seen in wild-type R. etli and mutant CS506 respectively, are therefore not present in this strain. Additional micro-heterogeneity in fatty acid chain length (14 or 28 atomic mass units) and partial substitution with β-hydroxybutyrate (86 amu) is observed in the lipid A species of all these strains. Each major lipid A species and some of its corresponding acyl chain variants are grouped by color. Symbols indicate the following amu shifts: for Panels A and B: + = (−28) * = (−86) • = (−86 and −28); for Panel C: + = (−28) * = (−86) • = (−86 and −28) = + 28 28) = +14 α = (−86 and + 28) ¢ = −14 π = (−86 and + 14) ∂ = (−86 and + 14 and + 28) λ = (−86 and −14).

Figure 6. ESI/MS analysis of lipid A species released by acetic acid hydrolysis from the double phosphatase mutant

The spectrum in the negative ion mode was the accumulation of 60 scans from 200 to 2400 atomic mass units. Only the doubly-charged ion of the spectrum are shown, because the peaks of the singly charged are very small. The two major lipid A components of this mutant (H and I) lack the galacturonic modification at the 4′-position, which is replaced by a phosphate group, and do not contain the aminogluconate unit (Fig. 3C). Heterogeneity in fatty acid chain length (14 or 28 amu) and partial substitution with β-hydroxybutyrate (86 amu) is observed. Symbols indicate the following amu shifts: + = (−28) * = (−86) • = (−86 and −28) 28) = + 28 28) = +14 β = (+28 and +14) ¢ = −14 π = (−86 and + 14) α = (−86 and + 28) ∂ = (−86 and + 14 and + 28) λ = (−86 and −14).

Figure 7. Absence of 4′-phosphatase activity in membranes R. etli lpxF deletion mutant CS506

Membranes of the indicated strains (0.25 mg/mL) were assayed for 4′-phosphatase activity at 30 °C with 5 μM [4′-³²P]Kdo₂-lipid IV_A. The products were separated by TLC using the solvent chloroform:pyridine:88% formic acid:water, (30:70:16:10, v/v), followed by detection with a PhosphorImager. No enzyme; parent, R. etli CE3 wild-type; CS501, R. etli ΔlpxE; CS506, R. etli ΔlpxF.

Figure 8. Nitrogen fixation in nodules of wild-type and mutant R. etli

Nitrogen fixation activity was determined using an acetylene reduction assay. Every strain was analyzed in four independent experiments, each comprised of 8 plants, as highlighted by the differential shading of the four columns. Columns show the relative nitrogen fixation activity and error bars indicate the standard deviations within each of the four independent experiments. Nitrogen fixation activity is expressed as the amount of ethylene formed per weight of fresh nodule, normalized to the nitrogen fixation activity of the wild-type CE3. Parent, R. etli CE3 wild-type; CS501, R. etli lipid A 1-phosphatase ΔlpxE mutant; CS506, R. etli lipid A 4′-phosphatase ΔlpxF mutant; CS513, R. etli mutant deficient in ΔlpxF and ΔlpxE.