The Lipid A 1-Phosphatase, LpxE, Functionally Connects Multiple Layers of Bacterial Envelope Biogenesis

Dephosphorylation of the lipid A 1-phosphate by LpxE in Gram-negative bacteria plays important roles in antibiotic resistance, bacterial virulence, and modulation of the host immune system. Our results demonstrate that in addition to removing the 1-phosphate from lipid A, LpxEs also dephosphorylate undecaprenyl pyrophosphate, an important metabolite for the synthesis of the essential envelope components, peptidoglycan and O-antigen. Therefore, LpxEs participate in multiple layers of biogenesis of the Gram-negative bacterial envelope and increase antibiotic resistance. This discovery marks an important step toward understanding the regulation and biogenesis of the Gram-negative bacterial envelope.

ABSTRACT

Although distinct lipid phosphatases are thought to be required for processing lipid A (component of the outer leaflet of the outer membrane), glycerophospholipid (component of the inner membrane and the inner leaflet of the outer membrane), and undecaprenyl pyrophosphate (C₅₅-PP; precursors of peptidoglycan and O antigens of lipopolysaccharide) in Gram-negative bacteria, we report that the lipid A 1-phosphatases, LpxEs, functionally connect multiple layers of cell envelope biogenesis in Gram-negative bacteria. We found that Aquifex aeolicus LpxE structurally resembles YodM in Bacillus subtilis, a phosphatase for phosphatidylglycerol phosphate (PGP) with a weak in vitro activity on C₅₅-PP, and rescues Escherichia coli deficient in PGP and C₅₅-PP phosphatase activities; deletion of lpxE in Francisella novicida reduces the MIC value of bacitracin, indicating a significant contribution of LpxE to the native bacterial C₅₅-PP phosphatase activity. Suppression of plasmid-borne lpxE in F. novicida deficient in chromosomally encoded C₅₅-PP phosphatase activities results in cell enlargement, loss of O-antigen repeats of lipopolysaccharide, and ultimately cell death. These discoveries implicate LpxE as the first example of a multifunctional regulatory enzyme that orchestrates lipid A modification, O-antigen production, and peptidoglycan biogenesis to remodel multiple layers of the Gram-negative bacterial envelope.

INTRODUCTION

The Gram-negative bacterial cell envelope consists of three essential molecular architectures—the inner membrane, the peptidoglycan layer, and the outer membrane—that together protect bacteria against mechanical stress, maintain cell shape, and shield these microorganisms from the damage of detergents and antibiotics. These architectures are formed by distinct molecules, with phospholipids constituting the inner membrane and inner leaflet of the outer membrane, peptide-conjugated carbohydrates constituting the peptidoglycan layer, and lipopolysaccharides (LPS) anchoring at the outer leaflet of the outer membrane through the hydrophobic lipid A moiety. As peptidoglycan, phospholipids, and LPS are synthesized through distinct pathways, how Gram-negative bacteria orchestrate the biogenesis and remodeling across three layers of the cell envelope for optimal bacterial growth and virulence remains incompletely understood.

As the major lipid species coating the outer surface of Gram-negative bacteria, lipid A is the predominant signaling molecule that is detected by the mammalian Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) innate immune receptor (1) and caspase-4/-5/-11 (2) to trigger the host innate immune response to bacterial infection. With few exceptions, Gram-negative bacteria constitutively synthesize the 1,4′-bisphosphorylated tetra-acyl-lipid A intermediate, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-linked lipid IV_A (Kdo₂-lipid IV_A), via the action of seven conserved enzymes in the Raetz pathway (3) (see Fig. S1A in the supplemental material), which are essential to nearly all Gram-negative bacteria and are attractive targets for novel antibiotics (4–6). Gram-negative bacteria additionally harbor modification enzymes that further process the Kdo₂-lipid IV_A intermediate to generate unique lipid A molecules in each bacterial species to adapt to environmental changes and evade the host immune response (7). For example, the lipid A 1-phosphate is a key determinant for lipid A recognition by the mammalian TLR4/MD-2 innate immune receptor (8). Removal of the lipid A 1-phosphate by the membrane-embedded phosphatase LpxE strongly protects bacteria against host cationic peptides and the last-resort antibiotic colistin (9), significantly dampens the host innate immune response, and dramatically increases colonization and survival of Helicobacter pylori in the gastric mucosa (10).

FIG S1

Lipid A biosynthesis and modification. (A) Schematic illustration of Kdo₂-lipid A biosynthesis and modification. The lipid A 1-phosphatase LpxE is in pink. (B) Lipid A structure of A. pyrophilus. Download FIG S1, TIF file, 2.8 MB ^{(2.9MB, tif)} .

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In order to gain molecular insights into the structure and function of the lipid A 1-phosphatase LpxE, we identified the previously uncharacterized gene aq_1706 from Aquifex aeolicus as the gene for the thermophilic LpxE enzyme (LpxE_AA). Our structural analysis of LpxE_AA shows distinct features between LpxE_AA and Escherichia coli PgpB (PgpB_EC) enzymes but reveals a surprising structural similarity to YodM, a phosphatase of phosphatidylglycerol phosphate (PGP) in the Gram-positive bacterium Bacillus subtilis with a weak in vitro activity on undecaprenyl pyrophosphate (C₅₅-PP). Consistent with our structural analysis, we found that LpxE_AA possesses substantial in vitro activities toward Kdo₂-lipid A/lipid IV_A, C₅₅-PP, and PGP and complements E. coli strains deficient in C₅₅-PP phosphatase and PGP phosphatase activities. In addition to the LpxE enzyme from A. aeolicus, distant LpxE orthologs from Francisella, Helicobacter, and Rhizobium also complement E. coli strains deficient in the C₅₅-PP phosphatase activity, supporting the notion that the multifunctional lipid phosphatase activity is a general feature of LpxE enzymes. Significantly, deletion of the native lpxE gene sensitizes Francisella novicida to bacitracin, an antibiotic that sequesters C₅₅-PP to disrupt peptidoglycan synthesis; furthermore, suppression of plasmid-encoded lpxE in the F. novicida strain deficient in the endogenous C₅₅-PP phosphatase activity results in noticeable changes in cell morphology, profound reduction of O-antigen repeats in LPS, and loss of cell viability. Taken together, these observations reveal a previously unappreciated contribution of LpxE to peptidoglycan biogenesis and LPS O-antigen modification beyond its well-recognized role as the lipid A 1-phosphatase to orchestrate the remodeling of multiple layers of the Gram-negative bacterial envelope to respond to environmental changes, evade host immune surveillance, and promote bacterial viability and virulence.

RESULTS

A distant ortholog of LpxE_FN in A. aeolicus.

LpxE is a member of the lipid phosphatase/phosphotransferase (LPT) family, a well-distributed family of lipid-processing enzymes also known as the integral transmembrane branch of the type II phosphatidic acid phosphatase (PAP2) superfamily (11, 12). This family is characterized by a conserved tripartite active site motif of KX₆RP---PSGH---SRX₅HX₃D and activity independent of Mg²⁺ or other cations (13). The LPT family includes enzymes responsible for processing several types of lipids in Gram-negative bacteria, including the membrane-embedded PgpB, which dephosphorylates PGP and C₅₅-PP (14) (Fig. S2). Even though PgpB and LpxE are both members of the LPT family, they have been reported to have distinct substrate specificities: PgpB is unable to utilize lipid A as a substrate (15), whereas purified LpxE from Rhizobium leguminosarum (LpxE_RL) utilizes PGP ∼1,000 times less efficiently than lipid A species as a substrate in vitro (16).

FIG S2

Lipid phosphatases. Reactions of PGP and C₅₅-PP phosphatases are shown in panels A and B, respectively. Download FIG S2, TIF file, 2.8 MB ^{(2.9MB, tif)} .

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In order to gain a molecular understanding of the LpxE structure and function, we searched for a thermophilic LpxE enzyme from Aquificae to facilitate structural analysis. The lipid A of Aquifex pyrophilus LPS contains d-galacturonic acid in place of phosphates at the 1- and 4′-positions (17) (Fig. S1B). As the 1,4′-bisphosphorylated lipid IV_A is a common lipid A intermediate before further modification (18, 19) and as Aquificae has the conserved biosynthetic enzymes to make 1,4′-bisphosphorylated lipid IV_A, incorporation of the d-galacturonic acid moiety requires the removal of 1-phosphate from lipid A, indicating the presence of the lipid A 1-phosphatase activity in Aquificae. Such a rationale led us to search for the gene responsible for the lipid A 1-phosphatase activity in A. aeolicus VF5, as no lipid A 1-phosphatase has been reported in any Aquifex species.

A Position-Specific Iterated Basic Local Alignment Search Tool (PSI-BLAST) (20) search revealed a distant ortholog of F. novicida LpxE (LpxE_FN) (15), Aq_1706 (E value, 0.81; sequence identity, 13.84%), in the genome of A. aeolicus VF5. Aq_1706 shares little sequence identity with other LpxE enzymes (sequence identities of Aq_1706 with LpxE of Helicobacter and Rhizobium are 16.58% and 14.57%, respectively), except for the well-conserved tripartite active-site motif of KX₆RP---PSGH---SRX₅HX₃D (Fig. S3). In order to determine if aq_1706 encodes the lipid A 1-phosphatase activity in vivo, we overexpressed Aq_1706 in the heptose transferase-deficient E. coli strain WBB06, which produces Kdo₂-lipid A instead of full-length LPS, to facilitate mass spectrometry analysis of lipid A modifications (21). Since E. coli does not encode LpxE activity, mass spectrometry analysis of the extracted lipids showed normal lipid A containing 1-phosphate with an m/z of 1,117.633 for the [M-2H]²⁻ ion species (calculated m/z, 1,117.661 for the exact mass of 2,237.336 of Kdo₂-lipid A) from E. coli cells expressing a control vector; in contrast, overexpression of Aq_1706 in E. coli led to the disappearance of the intact lipid A species and significant accumulation of lipid A molecules lacking the 1-phosphate group, with an m/z of 1,077.647 for the [M-2H]²⁻ ion species (calculated m/z, 1,077.678 for the exact mass of 2,157.370 of 1-dephospho Kdo₂-lipid A), consistent with the anticipated lipid A 1-phosphatase activity (Fig. 1A). In order to verify that the loss of phosphate occurred at the 1-position, but not at the 4′-position, we further tested the ability of LpxE to dephosphorylate 4′-³²P-labeled Kdo₂-lipid IV_A, which was previously shown to be an efficient substrate for LpxE enzymes with specific activity comparable to that for the substrate Kdo₂-lipid A (16). We found that treatment of Kdo₂-[4′-³²P] lipid IV_A with membrane extracts from E. coli overexpressing Aq_1706, but not those carrying a control vector, resulted in time-dependent reduction of the Kdo₂-lipid IV_A band and accumulation of an upper-shifted band on the thin-layer chromatography (TLC) plate (Fig. 1B), reflecting the removal of 1-phosphate but retention of the ³²P-labeled 4′-phosphate group. Taken together, these observations verify aq_1706 in A. aeolicus as the gene that encodes the thermophilic lipid A 1-phosphatase LpxE (LpxE_AA).

FIG 1.

Characterization of Aq_1706 from A. aeolicus as the lipid A 1-phosphatase LpxE_AA. (A) Mass spectrometry analysis of lipid A species in the heptose-deficient E. coli strain WBB06 (left) and the WBB06 strain overexpressing LpxE_AA (right). (B) ³²P-autoradiographic TLC-based Kdo₂-lipid IV_A 1-dephosphorylation assay of the membrane extract of the C41(DE3) strain overexpressing LpxE_AA.

FIG S3

Sequence alignment of LpxE orthologs. Alignment was achieved by Clustal Omega (F. Sievers, A. Wilm, D. Dineen, T. J. Gibson, et al., Mol Syst Biol 7:539, 2011) with manual adjustment. Conserved residues in the signature motifs of the PAP2 family of enzymes are labeled. Download FIG S3, TIF file, 2.9 MB ^{(3MB, tif)} .

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Structural analysis of LpxE_AA reveals a striking similarity to YodM in B. subtilis.

After verifying the lipid A 1-phosphatase activity of LpxE_AA, we cloned and purified LpxE_AA. Consistent with the TMHMM analysis (http://www.cbs.dtu.dk/services/TMHMM/), high-yield expression of LpxE_AA was achieved in a maltose-binding protein (MBP) fusion construct containing an N-terminal PelB secretion signal (22), suggesting that the N terminus of LpxE_AA is located at the periplasmic side of the inner membrane. The crystal structure of LpxE_AA containing an I63M mutation was determined at 2.38 Å (Fig. 2A; statistics shown in Table S1). The selenomethionine substitution of the nonconserved I63 residue (I63M) was designed to enhance the selenium single anomalous dispersion (Se-SAD) signal for de novo phasing. The overall structure of LpxE_AA contains seven α-helices, including an N-terminal amphiphilic helix lying at the periplasmic surface of the inner membrane and five tightly packed transmembrane helices (α3 to α7). Apart from α2, which originates from the periplasmic surface and penetrates halfway across the inner membrane at an ∼45° angle and immediately connects to transmembrane helix α3, the remaining helices are oriented largely in parallel or antiparallel with each other and perpendicularly to the membrane plane. Looking from the periplasmic surface, helix 5 (α5) is located at the center, which is surrounded by α2, α3, α4, α7, and α6 in a counterclockwise fashion (Fig. 2B).

FIG 2.

Crystal structure of LpxE_AA. (A) Ribbon representation of LpxE_AA, with blue to red colors corresponding to the N to C termini. The sulfate molecule is shown in the space-filling model. Individual helices and membrane locations are labeled. (B) Top view of LpxE_AA. (C) The active site of LpxE_AA. The sulfate molecule is shown in the stick model. Side chains of H143 and D147 from the RX₅HX₃D motif and conserved residues coordinating the sulfate molecule, including K73 and R80 from the KX₆RP motif and R137 of the RX₅HX₃D motif, are shown in the stick model. Hydrogen bonds are shown by dashed lines. The sulfate group is additionally stabilized by the interaction with the electrical dipole of helix α5 (indicated by gray hydrogen bonds). The conserved PSG motif is colored in coral, with Cα atoms shown in spheres. (D) Superimposition of LpxE_AA (cyan) with YodM_BS (PDB code 5JKI; pink), revealing striking structural similarities. The major differences between the two structures are highlighted, with dashed circles indicating missing structural features (helix or loop) and arrows indicating conformational discrepancy.

TABLE S1

Data collection and refinement statistics of LpxE_AA. Download Table S1, DOCX file, 0.01 MB ^{(14.3KB, docx)} .

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The active site of LpxE is located at the periplasmic surface of the inner membrane and is defined by conserved motifs specific to the PAP2 enzymes (K⁷³X₆ R⁸⁰P---R¹³⁷X₅ H¹⁴³X₃ D¹⁴⁷) located at the C-terminal end of α4, the α4-α5 loop, α6, the α6-α7 loop, and the N terminus of α7 (Fig. 2C). Fortuitously, a sulfate molecule is found in the active site, which is a structural analog of the 1-phosphate group of lipid A. The sulfate group is extensively recognized by K73 and R80 of the K⁷³X₆R⁸⁰P motif and R137 of the R¹³⁷X₅H¹⁴³X₃D¹⁴⁷ motif. The catalytically important H143 is located 3.3 Å away from the sulfur atom of the sulfate group, ready to carry out inline attack to remove the phosphate group of the lipid substrate. D147, the last residue of the R¹³⁷X₅H¹⁴³X₃D¹⁴⁷ motif, forms a hydrogen bond with H143. Although the corresponding aspartate residue is found in most LpxE enzymes (Fig. S3), it is absent in the LpxE ortholog from H. pylori (LpxE_HP), suggesting that it is not absolutely required for catalysis. The first three residues of the PSGH motif are conserved in LpxE_AA, with the central serine residue (S101) serving as a helix cap to stabilize helix α5, but the histidine residue is replaced with an aspartate residue in LpxE_AA (Fig. 2C).

The LpxE_AA structure shows noticeable conformational discrepancy with the previously reported structures of PgpB_EC (PDB codes 4PX7 and 5JWY) (23, 24), another PAP2 family enzyme, with overall backbone root mean square deviations (RMSDs) of ∼4.5 Å (Fig. S4); surprisingly, LpxE_AA is structurally similar to the recently reported YodM in B. subtilis (PDB code 5JKI) (25), a PGP phosphatase with a weak in vitro activity on C₅₅-PP, with an overall backbone RMSD of 1.2 Å (Fig. 2D). The major differences of these two enzymes are the absence of an N-terminal transmembrane helix in LpxE_AA in comparison with YodM, a longer α4-α5 loop in LpxE_AA, and a significant conformational variation of the α4-α5 loop surrounding the active site.

FIG S4

Superimposition of LpxE_AA with PgpB_EC structures_. RMSDs of ∼4.5 Å were observed between the coordinates of LpxE_AA and PgpB_EC (PDB codes 4PX7 [J. Fan, D. Jiang, Y. Zhao, J. Liu, et al., Proc Natl Acad Sci U S A 111:7636–7640, 2014] and 5JWY [S. L. Tong, Y. B. Lin, S. Lu, M. T. Wang, et al., J Biol Chem 291:18342–18352, 2016]). LpxE_AA and PgpB_EC are shown in Cα traces, with LpxE_AA colored in cyan and PgpB_EC in gray. Download FIG S4, TIF file, 2.8 MB ^{(2.9MB, tif)} .

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LpxE_AA is a trifunctional lipid phosphatase in vitro and functionally complements E. coli mutants deficient in C₅₅-PP or PGP phosphatase activities.

Surprised by the structural similarity between LpxE_AA and YodM_BS, we asked whether LpxE_AA could function as a C₅₅-PP and PGP phosphatase. To address this question, we compared the specific activities of purified LpxE_AA toward Kdo₂-lipid A, PGP, and C₅₅-PP using the malachite green assay to detect the release of inorganic phosphate. As expected, LpxE_AA efficiently catalyzed the hydrolysis of 1-phosphate from Kdo₂-lipid A, with a specific activity of 2.04 ± 0.46 μmol/mg/min. Moreover, LpxE_AA catalyzed C₅₅-PP more efficiently than it catalyzed Kdo₂-lipid A, with a specific activity of 3.58 ± 0.47 μmol/mg/min—a value that is ∼1.8-fold higher than that toward Kdo₂-lipid A. Finally, LpxE_AA also displayed significant activity toward PGP, with a specific activity of 0.75 ± 0.11 μmol/mg/min, ∼40% of its activity toward Kdo₂-lipid A (Table 1). Taken together, our biochemical assays validate LpxE_AA as a trifunctional LPT enzyme that efficiently dephosphorylates chemically diverse Kdo₂-lipid A (glycolipids), PGP (phosphoglycerol lipid), and C₅₅-PP (isoprenyl lipid) in vitro.

TABLE 1.

Specific activities of enzymes in this study

Enzyme	Specific activity (μmol/mg/min)
	Kdo2-lipid A	C55-PP	PGP
LpxEAA	2.04 ± 0.46	3.58 ± 0.47	0.75 ± 0.11
LpxEFN	3.25 ± 0.21	2.99 ± 0.45	0.038 ± 0.009
UppPFN	0.010 ± 0.005	22.71 ± 2.62	0.031 ± 0.007

In order to obtain further evidence of the trifunctional role of LpxE_AA in cells, we examined whether LpxE_AA could functionally rescue lethal E. coli mutants lacking C₅₅-PP phosphatase or PGP phosphatase activities. E. coli contains four C₅₅-PP phosphatases, BacA, PgpB, YbjG, and LpxT. A deletion mutant, ΔybjG ΔbacA ΔpgpB::kan, in E. coli is lethal unless rescued by a plasmid expressing BacA, PgpB, or YbjG (26). To examine if LpxE_AA could function as a C₅₅-PP phosphatase in cells, we set up complementation of the lethal ΔybjG ΔpgpB ΔbacA::kan E. coli mutant carrying lpxE_AA on a low-copy-number, temperature-sensitive pMAK705 vector (pMAK-lpxE_AA). The E. coli bacA gene, encoding the C₅₅-PP phosphatase, was used as the positive control (pMAK-bacA_EC). We found that overexpression of LpxE_AA and BacA_EC from pMAK705-derived plasmids complemented the lethal phenotype of the ΔybjG ΔpgpB ΔbacA::kan triple knockout in E. coli on an LB agar plate at 30°C; such a complementation effect was lost when cells were grown at 42°C, consistent with the loss of the temperature-sensitive pMAK705 plasmid encoding LpxE_AA or BacA_EC and confirming that LpxE_AA functionally complements the loss of C₅₅-PP phosphatase activity in E. coli (Fig. 3A).

FIG 3.

LpxE_AA complements E. coli strains deficient in C₅₅-PP phosphatase or PGP phosphatase activities. (A) Complementation of the C₅₅-PP phosphatase-deficient E. coli strain (BW25113 ΔybjG ΔpgpB ΔbacA::kan) by the temperature-sensitive pMAK705 plasmid harboring bacA_EC (positive control, pMAK-bacA_EC) or lpxE_AA (pMAK-lpxE_AA). WT E. coli cells carrying pMAK705 or C₅₅-PP phosphatase-deficient E. coli cells carrying pMAK-bacA_EC or pMAK-lpxE_AA were grown at 30°C or 42°C. From left to right are spots of 10-fold serial dilutions from 10² to 10⁵. (B) Complementation of the PGP phosphatase-deficient E. coli strain (W3110 ΔpgpA ΔpgpB ΔpgpC::kan) by the temperature-sensitive pMAK705 plasmid harboring pgpA_EC (positive control, pMAK-pgpA_EC) or lpxE_AA (pMAK-lpxE_AA). WT E. coli cells carrying pMAK705 or PGP phosphatase-deficient E. coli cells carrying pMAK-pgpA_EC or pMAK-lpxE_AA were grown at 30°C or 42°C. From left to right are spots of 10-fold serial dilutions from 10² to 10⁵. (C) Chromosomal complementation of C₅₅-PP phosphatase activity-deficient E. coli with lpxE_AA. The left, middle, and right images show the construction of different E. coli C₅₅-PP phosphatase gene deletion mutants, the growth of WT E. coli cells and C₅₅-PP phosphatase-deficient cells complemented by a chromosomal copy of lpxE_AA, and the PCR verification of ybjG, bacA, and pgpB knockouts of the target mutant strain, respectively. (D) Chromosomal complementation of PGP phosphatase activity-deficient E. coli with lpxE_AA. The left, middle, and right images show the construction of different E. coli PGP phosphatase gene deletion mutants, the growth of WT E. coli cells and PGP phosphatase-deficient cells complemented by a chromosomal copy of lpxE_AA, and PCR verification of pgpA, pgpC, and pgpB knockouts of the target mutant strain, respectively. Since the expected sizes of pgpB (1,106 bp) and pgpB::P_L-lpxE_AA (1,093 bp) are similar using primers flanking pgpB in the final strain, the knockout of pgpB was established by also verifying the PCR result of the mother strain (strain 5: W3110 ΔpgpA ΔpgpB::P_L-lpxE_AA-frt-kan-frt).

We similarly tested whether LpxE_AA functionally complements the loss of PGP phosphatase activity in E. coli. E. coli has three PGP phosphatases, PgpA, PgpB, and PgpC (27). A ΔpgpA ΔpgpB ΔpgpC::kan triple-knockout mutant is lethal unless it is rescued by a plasmid harboring an active PGP phosphatase (27). Overexpression of LpxE_AA or the positive control PgpA_EC from the temperature-sensitive pMAK705 plasmid supported the growth of the ΔpgpA ΔpgpB ΔpgpC::kan triple-knockout mutant strains at 30°C but not at 42°C. In contrast, the control strain (W3110/pMAK705) grew well at both temperatures (Fig. 3B). These observations confirm that LpxE_AA is a functional PGP phosphatase in E. coli.

While the pMAK705 vector-encoded LpxE_AA complemented E. coli triple knockouts lacking C₅₅-PP phosphatase or PGP phosphatase activities, pMAK705 has a higher copy number (pSC101 origin, ∼5 copies/cell) than that of the chromosome in E. coli (single copy/cell). In order to mitigate the concern that the observed genetic complementation was caused by multiple copies of the lpxE_AA gene, we replaced the pgpB gene in the chromosome of E. coli (BW25113) ΔybjG ΔbacA with a gene cassette (P_L-lpxE_AA-FRT-kan-FRT) containing lpxE_AA and a kanamycin resistance gene under the control of the P_L promoter (28). The resulting E. coli strain (E. coli BW25113 ΔybjG ΔbacA ΔpgpB::P_L-lpxE_AA-FRT-kan-FRT) grew on an LB agar plate, and the proper knockouts of bacA, ybjG, and pgpB were verified by PCR (Fig. 3C), confirming that the chromosomal copy of lpxE_AA complemented the loss of C₅₅-PP phosphatase activity. Using a similar approach, we also replaced the pgpB gene of E. coli (W3110) ΔpgpA with P_L-lpxE_AA-FRT-kan-FRT, removed the kanamycin resistance cassette (29), and then knocked out pgpC. The resulting strain (E. coli W3110 ΔpgpA ΔpgpB::P_L-lpxE_AA ΔpgpC::kan) also grew on an LB agar plate, and knockouts of pgpA, pgpB, and pgpC were verified by PCR (Fig. 3D), confirming that the chromosomal copy of lpxE_AA similarly complemented the loss of PGP phosphatase activity.

Altogether, the substantial phosphatase activities of LpxE_AA toward Kdo₂-lipid A, C₅₅-PP, and PGP in vitro and its ability to complement the loss of C₅₅-PP and PGP phosphatase activities in E. coli—both via the plasmid-borne gene and via chromosomal knock-in—strongly support the multifunctionality of LpxE_AA in Gram-negative bacterial envelope biogenesis.

LpxE_FN is a bifunctional lipid phosphatase in vitro and functionally complements an E. coli mutant deficient in the C₅₅-PP phosphatase activity.

Despite the intriguing observation of the multifunctionality of LpxE_AA, it is challenging to establish the biological consequence in its native host due to the difficulty of culturing and genetic manipulation of A. aeolicus. Therefore, we asked if other LpxE enzymes from genetically trackable bacteria similarly display multifunctional lipid phosphatase activities. In order to answer this question, we chose LpxE_FN, a distant ortholog of LpxE_AA, for further characterization. The ability of LpxE_FN to dephosphorylate lipid A at the 1-position was previously reported (15), but its activity toward other lipid substrates has not been thoroughly investigated. We first conducted similar complementation experiments using E. coli strains deficient in either the C₅₅-PP phosphatase activity or PGP phosphatase activity carrying the temperature-sensitive pMAK-lpxE_FN. We found that LpxE_FN complemented the loss of C₅₅-PP phosphatase activity of E. coli (ΔybjG ΔpgpB ΔbacA::kan) at 30°C but not at 42°C, indicating that LpxE_FN is a functional C₅₅-PP phosphatase in E. coli (Fig. 4A). However, we were unable to complement E. coli deficient in the PGP activity (ΔpgpA ΔpgpB ΔpgpC::kan) with a plasmid encoding LpxE_FN (pMAK-lpxE_FN). Consistently, we found that purified LpxE_FN displayed significant phosphatase activity toward both Kdo₂-lipid A and C₅₅-PP and processed these two substrates with similar efficiencies (specific activities of 3.25 ± 0.21 μmol/mg/min for Kdo₂-lipid A and 2.99 ± 0.45 μmol/mg/min for C₅₅-PP), but its activity toward PGP was ∼100-fold lower (specific activity of 0.038 ± 0.009 μmol/mg/min) (Table 1), confirming that LpxE_FN is a bifunctional lipid phosphatase.

FIG 4.

F. novicida harbors two C₅₅-PP phosphatases, LpxE_FN and UppP_FN. (A) Complementation of the C₅₅-PP phosphatase-deficient E. coli strain (BW25113 ΔybjG ΔbacA ΔpgpB::kan) by the temperature-sensitive pMAK705 plasmid harboring bacA_EC (positive control, pMAK-bacA_EC), lpxE_FN (pMAK-lpxE_FN), or uppP_FN (ftn_1552, pMAK-uppP_FN). WT E. coli cells carrying pMAK705 or C₅₅-PP phosphatase-deficient E. coli cells carrying pMAK-bacA_EC, pMAK-lpxE_FN, or pMAK-uppP_FN were grown at 30°C or 42°C. From left to right are spots of 10-fold serial dilutions from 10² to 10⁵. (B) Schematic illustration of the construction of different F. novicida gene deletion strains. Viable and lethal strains are in black and red, respectively. The presence of the proper gene deletion was verified by PCR using primers at ∼0.25-kb or 0.6-kb positions flanking lpxE or uppP, respectively. (C) Viability of F. novicida mutants. While F. novicida mutants containing ΔlpxE_FN::kan, ΔuppP_FN::kan, or ΔuppP_FN ΔlpxE_FN::lpxE_AA were viable, no colonies could be isolated for F. novicida mutants containing ΔuppP_FN ΔlpxE_FN::kan. (D) Construction of the conditional lethal F. novicida strain containing ΔlpxE_FN ΔuppP_FN complemented by aTc-inducible pEDL17-lpxE_FN. The sequence of strain construction is shown at the top. The presence of the desired gene deletion was verified by PCR (left). Prolonged withdrawal of aTc from the growth medium resulted in a slow bactericidal phenotype (right).

F. novicida harbors two C₅₅-PP phosphatases: LpxE_FN and FTN_1552.

It is important to note that the lipid A 1-phosphatase activity is not essential in bacteria but the C₅₅-PP phosphatase activity is. Prior to this study, no enzyme encoding the C₅₅-PP phosphatase activity had been identified in F. novicida. As the transposon mutant of lpxE_FN is not lethal in F. novicida (30), we reasoned that there must exist another enzyme encoding the C₅₅-PP phosphatase activity in F. novicida. By searching for F. novicida proteins homologous to E. coli enzymes containing C₅₅-PP phosphatase activity (i.e., BacA_EC, YbjG_EC, PgpB_EC, and LpxT_EC) using PSI-BLAST (20), we have identified a PAP2 family protein of unknown function, FTN_1552, as a potential candidate of the C₅₅-PP phosphatase (PSI-BLAST of PgpB_EC: E value of 0.003 and sequence identity of 16.47%). We found that the temperature-sensitive pMAK705 vector harboring ftn_1552 complemented the E. coli strain deficient in C₅₅-PP phosphatase activity (ΔybjG ΔpgpB ΔbacA::kan), confirming ftn_1552 as the gene encoding the C₅₅-PP phosphatase activity (Fig. 4A). FTN_1552 was subsequently renamed UppP_FN. Purified UppP_FN appears to be a specific enzyme for C₅₅-PP, with a specific activity of 22.71 ± 2.62 μmol/mg/min, and displays little activity toward Kdo₂-lipid A and PGP (specific activities of 0.010 ± 0.005 μmol/mg/min and 0.031 ± 0.007 μmol/mg/min, respectively [Table 1]). Importantly, while F. novicida strains containing a chromosomal deletion of either lpxE_FN or uppP_FN were viable, we were unable to generate F. novicida strains containing both deletions (ΔlpxE_FN ΔuppP_FN) in the chromosome (Fig. 4B and C). However, F. novicida cells were viable in the ΔuppP_FN background when lpxE_FN was replaced with lpxE_AA (Fig. 4B and C). Furthermore, when F. novicida was first transformed with a plasmid (pEDL17) bearing lpxE_FN under the control of an anhydrotetracycline (aTc) promoter, we were also able to obtain viable F. novicida colonies containing chromosomal deletions of both lpxE_FN and uppP_FN (U112 ΔlpxE_FN ΔuppP_FN::kan/pEDL17-lpxE_FN). The presence of proper chromosomal deletions of the lpxE_FN and uppP_FN genes was verified by PCR (Fig. 4D). As expected, the viability of such a strain depends on the expression of plasmid-encoded LpxE_FN: prolonged withdrawal of aTc suppressed the bacterial growth and slowly resulted in cell lysis in culture (Fig. 4D), reinforcing the notion that UppP_FN and LpxE_FN share redundant C₅₅-PP phosphatase activities in Francisella.

LpxE_FN functionally connects multiple layers of envelope biogenesis in F. novicida.

After establishing that LpxE_FN shares C₅₅-PP phosphatase activity with UppP_FN, we further examined the biological implication of the multifunctional enzymatic activity of LpxE_FN in its native host, F. novicida. We first verified the role of LpxE_FN as a lipid A 1-phosphatase. Wild-type (WT) F. novicida cells contain both LPS (i.e., core oligosaccharide and O-antigen-modified Kdo-lipid A3 without 1- and 4′-phosphates) and free lipid A species A1 and A2, which do not contain core oligosaccharides/Kdo or O-antigen (lipid A2 differs from lipid A1 in that it has an additional α-linked glucose moiety attached to its 6′-position; also see the schematic lipid A structures of WT F. novicida in Fig. 5A) (31). Both lipid A1 and lipid A2 are further modified by FlmK, which transfers galactosamine from C₅₅-P-galactosamine to the 1-phosphate of lipid A (32, 33). As the core oligosaccharide and O-antigen-modified lipid A are inefficiently extracted by the Bligh-Dyer method for mass spectrometry analysis, we examined the effect of ΔlpxE_FN in the F. novicida strain deficient in the glycosyltransferase activity (ΔlpcC), which produces Kdo-lipid A3 (instead of LPS), in addition to lipids A1 and A2 found in the wild-type cells (Fig. 5A). Accumulations of Kdo-(1-phospho)-lipid A3 and Kdo-(galactosamine-1-phospho)-lipid A3, as well as the disappearance of Kdo-lipid A3, were observed in F. novicida when lpxE_FN was deleted (ΔlpxE_FN), confirming the lipid A 1-phosphatase activity of LpxE_FN in cells (Fig. 5A and Fig. S5).

FIG 5.

LpxE_FN functionally contributes to multiple layers of the F. novicida bacterial envelope biogenesis. (A) Deletion of lpxE_FN results in accumulation of 1-phosphorylated Kdo-lipid A3 species. The profiles of total lipid extracts from F. novicida U112 WT, ΔlpcC, and ΔlpcC ΔlpxE strains were analyzed by TLC. Lipid A species are labeled. Abbreviations: GalN, galactosamine; A1, lipid A1; A2, lipid A2; Kdo-A3, Kdo-lipid A3; Kdo-(GalN-1-P)-A3, Kdo-(galactosamine-1-phospho)-lipid A3; Kdo-(1-P)-A3, Kdo-(1-phospho)-lipid A3. (B) Deletion of lpxE_FN sensitizes F. novicida to bacitracin as reflected by reduced MIC. Error bars represent standard deviations from quadruplet measurements. (C) Suppression of the plasmid-encoded LpxE_FN expression in the ΔlpxE_FN ΔuppP_FN::kan mutant of F. novicida causes cell deformation. Images of wild-type cells and ΔlpxE_FN ΔuppP_FN::kan F. novicida mutant cells without and with LpxE_FN expression are shown in the left, middle, and right images, respectively. (D) Lack of LPS phenotypes in F. novicida cells containing the single deletion of lpxE_FN or uppP_FN. (E) Suppression of the plasmid-encoded LpxE_FN expression in the ΔlpxE_FN ΔuppP_FN::kan mutant of F. novicida causes the loss of O-antigen repeats in LPS. LPS profiles from wild-type F. novicida cells carrying the pDEL17 vector (VC) or the C₅₅-PP phosphatase-deficient (ΔlpxE_FN ΔuppP_FN::kan) cells carrying the pDEL17-lpxE_FN vector without or with aTc were analyzed on SDS-PAGE gels using the Pro-Q Emerald LPS staining kit. O-antigen-containing LPS, including both high- and low-repeat species, and free lipid A/core species are labeled.

FIG S5

Mass spectrometry analysis of purified lipid A species of F. novicida mutants from preparative TLC. Accumulated new lipid A species from F. novicida mutants (ΔlpcC::tet and ΔlpxE::kan ΔlpcC::tet) isolated from TLC are identified using mass spectrometry as Kdo-lipid A3 (A), Kdo-(galactosamine-1-phospho)-lipid A3 (B), and Kdo-(1-phospho)-lipid A3 (C). Download FIG S5, TIF file, 2.8 MB ^{(2.9MB, tif)} .

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As the C₅₅-PP phosphatase activity of LpxE_FN (2.99 ± 0.45 μmol/mg/min) is only ∼7-fold smaller than that of UppP_FN (22.71 ± 2.62 μmol/mg/min), we asked whether LpxE_FN could functionally contribute to the bacterial envelope biogenesis beyond lipid A modification at the 1-phosphate position. We first compared the sensitivities of the wild-type F. novicida strain (U112) and mutant strains containing either the lpxE or uppP deletion to bacitracin, an antibiotic sequestering C₅₅-PP. We found that while the loss of uppP in F. novicida generated an 8.5-fold drop of MIC in comparison with that of the WT strain (0.5 μM versus 4.25 μM), as expected, the loss of lpxE also resulted in ∼1.7-fold drop of the MIC of bacitracin (2.5 μM for F. novicida ΔlpxE) (Fig. 5B), implicating a functional role of LpxE_FN in the recycling of C₅₅-PP.

In order to isolate the biological effect of LpxE_FN, we utilized the F. novicida strain containing chromosomal deletions of both uppP_FN and lpxE_FN, which is complemented by a plasmid carrying lpxE_FN under the control of an aTc promoter. We found that the loss of plasmid-mediated expression of LpxE_FN due to withdrawal of aTc in the growth medium resulted in cell enlargement, reflecting defective peptidoglycan biosynthesis (Fig. 5C). Strikingly, while no change of O-antigen repeats was observed in F. novicida cells containing the chromosomal deletion of either lpxE or uppP in comparison with WT cells (Fig. 5D), transient suppression of LpxE led to a dramatic reduction of the LPS O-antigen repeats, including both high- and low-repeat species (Fig. 5E) (34), suggesting a contribution of LpxE to the O-antigen biogenesis. These observations are consistent with the notion that the biosynthesis and transport of peptidoglycan and O-antigen depend on C₅₅-P, the product of LpxE_FN (and UppP_FN) activity, and reveal a previously unappreciated function of LpxE in the biogenesis and remodeling of multiple components across the bacterial envelope: peptidoglycan, free lipid A, and the O-antigen repeat of LPS.

DISCUSSION

LpxE enzymes are important virulence factors that promote bacterial survival, fitness, and pathogenicity. In H. pylori and Rhizobium etli CE3, the chromosomal knockout of lpxE resulted in increased susceptibility to positively charged antimicrobial peptides such as polymyxin B and colistin (9, 35), presumably due to the retention of 1-phosphate of lipid A. Previous studies showed that Rhizobium LpxE displays over a 1,000-fold preference of Kdo₂-lipid A/lipid IV_A over PGP; therefore, LpxE has been regarded as a highly specific monofunctional enzyme whose sole activity is to remove the 1-phosphate from lipid A. In this study, based on the striking structural similarity between LpxE_AA and YodM_BS, a PGP phosphatase with a weak in vitro activity on C₅₅-PP phosphatase, we discovered that LpxE is a multifunctional lipid phosphatase. The LpxE enzyme from A. aeolicus displays significant activities toward Kdo₂-lipid A/lipid IV_A, C₅₅-PP and PGP and functionally complements E. coli strains deficient in C₅₅-PP or PGP phosphatase activities. Likewise, the LpxE enzyme from F. novicida is a dual-function enzyme that processes Kdo₂-lipid A and C₅₅-PP with similar efficiencies. Strikingly, deletion of lpxE_FN in its native host F. novicida resulted in accumulation of phosphorylated lipid A species and increased sensitivity to bacitracin; in the C₅₅-PP phosphatase-deficient (ΔuppP and ΔlpxE double-knockout mutant) F. novicida, suppression of LpxE_FN expression from the plasmid resulted in cell deformation due to defective peptidoglycan biosynthesis and the loss of O-antigen repeats in LPS associated with reduced O-antigen transport, both of which are critically dependent on the recycling of C₅₅-PP to C₅₅-P. Taken together, these results show that LpxE enzymes from A. aeolicus and F. novicida functionally connect multiple layers of bacterial envelope biogenesis and remodeling. Such multiple functional roles are not unique to LpxE enzymes from A. aeolicus and F. novicida: we found that LpxE enzymes from H. pylori and R. leguminosarum also complemented E. coli deficient in C₅₅-PP phosphatase activities (Fig. S6), suggesting that these LpxE enzymes can similarly process Kdo₂-lipid A and C₅₅-PP to synchronize lipid A modification with peptidoglycan biosynthesis and O-antigen modification of LPS.

FIG S6

Complementation of the C₅₅-PP phosphatase-deficient E. coli strain by LpxE enzymes from R. leguminosarum (LpxE_RL) and H. pylori (LpxE_HP). Wild-type E. coli cells carrying an empty vector (BW25113/pMAK705) or C₅₅-PP phosphatase-deficient E. coli cells (BW25113 ΔybjG ΔpgpB ΔbacA::kan) carrying pMAK-lpxE_RL or lpxE_HP were grown at 30°C or 42°C, respectively. From left to right are spots of 10-fold serial dilutions from 10² to 10⁵. Download FIG S6, TIF file, 2.2 MB ^{(2.3MB, tif)} .

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It is appropriate to ask why LpxE has evolved into a multifunctional enzyme. There are several potential explanations. First, it is conceivable that the peptidoglycan biosynthesis is such an essential process that multiple enzymes, including LpxE, are employed as the backup enzymes for the C₅₅-PP phosphatase-mediated recycling reaction for peptidoglycan charging and biosynthesis. Second, it is possible that LpxE from Aquifex species represents an ancestral lipid phosphatase, which, although primitive, is sufficient to conduct all lipid phosphatase activities to support the bacterial envelope biogenesis and remodeling, while other LPT family of lipid phosphatases, such as the PGP phosphatase, evolved later as specialized, highly efficient enzymes. Third, it is also likely that LpxE evolved as a multifunctional enzyme to coordinate lipid A modification and the biogenesis of other layers of bacterial envelope. As 1,4′-bisphosphorylated lipid A chelates metal ions to form a fortified layer for bacterial protection, removal of the 1-phosphate could weaken the lipid A layer and increase membrane permeability. It is conceivable that the weakened lipid A layer is compensated by the elevated peptidoglycan biosynthesis and enhanced O-antigen decoration of LPS. Thus, bestowing LpxE with the multifunctionality toward Kdo₂-lipid A and C₅₅-PP (and, in the case of A. aeolicus, PGP) enables LpxE to orchestrate lipid A modification with bacterial envelope remodeling at multiple layers (Fig. 6) in order to promote the optimal bacterial growth and enhance bacterial survival in nature and the human host.

FIG 6.

Multiple roles of LpxE in the biogenesis of the Gram-negative bacterial envelope. Shown is a schematic illustration of the multifunctional roles of LpxE in the biogenesis of the cell wall polymer (peptidoglycan), outer membrane (LPS), and inner membrane (PGP). Shared phosphatase activities of LpxEs from diverse bacteria, including Aquifex (Aquificales), Francisella (Gammaproteobacteria), Rhizobium (Alphaproteobacteria), and Helicobacter (Epsilonproteobacteria), toward lipid A and C₅₅-PP are indicated by solid arrows, whereas the unique PGP phosphatase activity of LpxE_AA is indicated by a dashed red line.

The Gram-negative bacterial envelope contains three layers. How Gram-negative bacteria coordinate the biogenesis and remodeling of different layers of the bacterial envelope has remained an area of active investigation. Our study has revealed the first biological evidence of a multifunctional enzyme, LpxE in F. novicida, that natively couples lipid A 1-dephosphorylation with C₅₅-PP recycling to enhance peptidoglycan biogenesis and O-antigen decoration of LPS, promote cell viability against antimicrobial peptides, evade host immune surveillance, and ultimately support bacterial pathogenesis. We suggest that such a multifunctional role represents a common but previously unappreciated mechanism for Gram-negative bacteria to coordinate bacterial envelop biogenesis across different layers.

MATERIALS AND METHODS

Data collection and refinement statistics of LpxE_AA are listed in Table S1. All strains and plasmids used in this work are listed in Tables S2 and S3, respectively.

TABLE S2

Plasmids used in this work. Download Table S2, DOCX file, 0.02 MB ^{(20.5KB, docx)} .

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TABLE S3

Strains used in this work. Download Table S3, DOCX file, 0.02 MB ^{(25.6KB, docx)} .

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Plasmid and strain constructions and growth conditions are described in the supplemental material in detail.

Extraction of lipid A species, TLC and mass spectrometry analyses of lipid A species, and assay conditions are described in the Supplementary Methods section of the supplemental material.

Characterizations of F. novicida U112 mutants are described in the Supplementary Methods section of the supplemental material.

TEXT S1

Supplemental methods. Download Text S1, DOCX file, 0.1 MB ^{(78.3KB, docx)} .

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