Assembly of lipopolysaccharide in Escherichia coli requires the essential LapB heat shock protein

Abstract

Here, we describe two new heat shock proteins involved in the assembly of LPS in Escherichia coli, LapA and LapB (lipopolysaccharide assembly protein A and B). lapB mutants were identified based on an increased envelope stress response. Envelope stress-responsive pathways control key steps in LPS biogenesis and respond to defects in the LPS assembly. Accordingly, the LPS content in ΔlapB or Δ(lapA lapB) mutants was elevated, with an enrichment of LPS derivatives with truncations in the core region, some of which were pentaacylated and exhibited carbon chain polymorphism. Further, the levels of LpxC, the enzyme that catalyzes the first committed step of lipid A synthesis, were highly elevated in the Δ(lapA lapB) mutant. Δ(lapA lapB) mutant accumulated extragenic suppressors that mapped either to lpxC, waaC, and gmhA, or to the waaQ operon (LPS biosynthesis) and lpp (Braun's lipoprotein). Increased synthesis of either FabZ (3-R-hydroxymyristoyl acyl carrier protein dehydratase), slrA (novel RpoE-regulated non-coding sRNA), lipoprotein YceK, toxin HicA, or MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) suppressed some of the Δ(lapA lapB) defects. LapB contains six tetratricopeptide repeats and, at the C-terminal end, a rubredoxin-like domain that was found to be essential for its activity. In pull-down experiments, LapA and LapB co-purified with LPS, Lpt proteins, FtsH (protease), DnaK, and DnaJ (chaperones). A specific interaction was also observed between WaaC and LapB. Our data suggest that LapB coordinates assembly of proteins involved in LPS synthesis at the plasma membrane and regulates turnover of LpxC, thereby ensuring balanced biosynthesis of LPS and phospholipids consistent with its essentiality.

Keywords: Endotoxin; Glycobiology; Glycolipid Structure; Glycosyltransferases; Heptosyltransferase; Lipid A; Lipopolysaccharide (LPS); Myristoyltransferase; RpoH; Tetratricopeptide Repeat (TPR).

FIGURE 1.

Proposed LPS structures of major glycoforms observed in E. coli K12. Shown is a schematic drawing of LPS glycoform I (A), II (B), and VII (C) with various nonstoichiometric substitutions. D–F, chemical structures of different lipid A variants: pentaacylated (D), hexaacylated with two lauroyl chains (E), and typical hexaacylated lipid A (F). Mass numbers of these lipid A species are indicated. R1 and R2, modification by Ara4N and P-EtN, respectively, observed in lipid A-modifying conditions.

FIGURE 2.

LapB is essential for the bacterial growth in rich medium. Isogenic bacterial cultures of the wild type and ΔlapB and Δ(lapA lapB) mutants were grown overnight in M9 medium at 30 °C. Cultures were adjusted to A₆₀₀ of 0.05 in 12 ml of prewarmed M9 medium at 30 and 42 °C (A). Aliquots of samples were drawn at different intervals, and the bacterial growth was monitored by measuring A₆₀₀. In parallel, growth of the wild type and its Δ(lapA lapB) derivative was monitored after shift at A₆₀₀ of 0.05 in prewarmed LB medium (B). An equivalent amount of bacterial cells obtained from the wild type and its ΔlapB derivative grown on M9 agar at 30 °C were used to prepare whole cell lysates. Samples were digested with proteinase K and applied on a 16.5% SDS-Tricine gel, and LPS was revealed by silver staining (C). The asterisk in the lane corresponding to the LPS from ΔlapB mutant indicates faster migrating species.

FIGURE 3.

Transcriptional regulation of lapA and lapB genes. A, nucleotide sequence of the promoter region of the lap operon. Transcriptional start sites were identified from RNA obtained from the wild-type bacteria grown in M9 medium at 30 °C and after a 15-min shift to 42 °C. The arrows indicate the position of transcription start sites. The site marked as P2_hs corresponds to the heat shock promoter. The corresponding −10 and −35 elements are shown. The P1 and P3 start sites represent initiation sites under non-heat shock conditions. The P1 start is located upstream of the pgpB gene as indicated. The highly conserved T and G nucleotides present in the putative −35 region of the P1 promoter are indicated. The TG dinucleotide upstream of the −10 region of the P3 promoter, corresponding to the presence of putative extended −10 promoter, is shown in orange. B, alignment of −10 and −35 regions of the lapABP2_hs promoter with well characterized heat shock promoters. C, the activity of lapABP2_hs and groESL promoters was measured using strains carrying single-copy chromosomal promoter fusions. Cultures were grown as described above; heat-shocked at 42 °C for 5, 10, 15, 20, and 30 min; and analyzed for β-galactosidase activity. Shown is Western blot analysis of whole cell extracts from strains expressing LapA-FLAG (D) and LapB-FLAG (E). Cultures were grown in M9 medium at 30 °C up to an A₆₀₀ of 0.2. One-ml aliquots were shifted to prewarmed tubes held at 42 °C and incubated for 5, 10, 15, or 20 min. Proteins were precipitated by TCA (10%) and analyzed on 12% SDS-PAGE, followed by immunoblotting with anti-FLAG antibody.

FIGURE 4.

ΔlapB mutant accumulates precursor forms of LPS and restoration of normal LPS composition upon complementation. Shown are charge-deconvoluted ESI FT-ICR mass spectra in the negative ion mode of native LPS obtained from the wild-type strain (A) and its ΔlapB derivative (B) grown in M9 medium at 30 °C. C–G, spectra of LPS obtained from cultures grown in 121 medium at 30 °C. The relevant genotype is indicated. Mass numbers refer to monoisotopic peaks. In B, mass peaks, mostly corresponding to the substitution by phosphate or sodium adducts or due to carbon change length polymorphism, are not labeled. Rectangular boxes, mass peaks corresponding to the glycoform containing the third Kdo. Ovals, derivatives with two Kdo residues with either complete core or incomplete core.

FIGURE 5.

Defects in the lipid A biogenesis of ΔlapB derivatives. Shown are charge-deconvoluted ESI FT-ICR mass spectra in the negative ion mode depicting the lipid A composition and its modifications from the LPS obtained under permissive growth conditions. The relevant genotype corresponding to panels A–D is indicated. Part of the negative ion mass spectra of the native LPS after nonspecific fragmentation, leading to the cleavage of the labile lipid A-Kdo linkage, is presented. The mass peaks corresponding to the penta- and hexaacylated lipid A part and substitutions with P-EtN and/or Ara4N are indicated. Insets, predicted chemical composition of the hexa- and pentaacylated lipid A part.

FIGURE 6.

Co-purification of LapA and LapB with Lpt proteins. Purification of LapA and LapB and co-purification with Lpt proteins upon co-overexpression were performed as described under “Experimental Procedures.” Samples after the induction of LapA (A) and LapB (B), after washing columns and after elution with indicated concentrations of imidazole, were resolved on 12.5% SDS-PAGE. The identity of co-purifying proteins is indicated by numbers in A and B. Lanes 5 (A and B), purified samples of LapA and LapB, respectively, obtained after blocking host protein synthesis by the addition of rifampicin. Lanes 6 (A and B), complex after co-expression of LapB with LptC. Purified samples corresponding to lanes 5 and 7 in the A representing LapA and LapB, respectively, were digested with proteinase K and applied onto a 16.5% SDS-Tricine gel, followed by silver staining to reveal LPS (C). D, as a negative control, 300 μg of purified LPS from the wild-type bacteria was applied over nickel-nitrilotriacetic acid beads, pre-equilibrated with the same buffer used in the purification of LapA. One μg of purified LPS, a portion of the flow-through (FT), and LPS after elution were digested with proteinase K and analyzed as described above. E and F, WaaC heptosyltransferase I co-purifies with LapB. His-tagged WaaC was overproduced in the strain expressing LapB-FLAG from the chromosome. WaaC purification profile was examined by the analysis of WaaC elutions on 12% SDS-PAGE (E). The arrows indicate the position of LapB and WaaC. The same samples as used in E were analyzed on 12% SDS-PAGE and tested for the presence of LapB-FLAG, using FLAG antibody after immunoblotting (F).

FIGURE 7.

The accumulation of LpxC in Δ(lapA lapB) mutant and suppression of its growth defects by the overexpression of the fabZ gene. A, cultures were grown at 30 °C in M9 medium up to an A₆₀₀ of 0.2, and aliquots were shifted to 37 and 42 °C for 30 min in LB medium. An equivalent amount of total protein was analyzed on 12% SDS-PAGE, followed by immunoblotting using LpxC antibodies. Note the elevated amounts of LpxC in Δ(lapA lapB) mutant and its reduction in lpxC186 background and upon overexpression of the slrA sRNA. B, cultures of Δ(lapA lapB) mutant carrying the vector alone or expressing the fabZ gene from plasmid under the control of the ptac promoter were grown in M9 medium at 30 °C, adjusted to an A₆₀₀ of 0.1, and serially diluted. Five-μl aliquots were spotted on M9 plate supplemented with 25 μm IPTG and incubated at 42 °C.

FIGURE 8.

A single point mutation in the waaC gene suppresses ΔlapB. Charge-deconvoluted ESI FT-ICR mass spectra in the negative ion mode of LPS isolated from strains with ΔwaaC deletion (A) and ΔlapB with waaCT187K suppressor mutation (B). Cultures were grown in permissive conditions at 30 °C in M9 medium. Mass peaks corresponding to pentaacylated lipid A species lacking the myristoyl chain, and mass peaks representing carbon chain polymorphism are drawn schematically. Mass numbers refer to monoisotopic peaks, and the predicted compositions of mass peaks are indicated. C, equivalent amount of bacterial cells obtained from isogenic strains carrying the ΔwaaC deletion (lane 1) and ΔlapB waaCT187K derivative (lane 2) grown on LA agar at 30 °C and processed to obtain whole cell lysates. Samples were digested with proteinase K and applied on a 16.5% SDS-Tricine gel, and LPS was revealed by silver staining. Isogenic bacterial cultures were grown at 30 °C in M9 medium up to an A₆₀₀ of 0.2 and harvested by centrifugation. An equivalent amount of total protein was analyzed on 12% SDS-PAGE, followed by immunoblotting using LpxC antibodies (D), and as a loading control, the same samples were tested by immunoblotting using antibodies to the α subunit of RNA polymerase, in the bottom panel (D). The relevant genotype is indicated.

FIGURE 9.

RpoE-regulated novel sRNA slrA as a multicopy suppressor of Δ(lapA lapB) mutation and its role in modulating RpoE activity. A, nucleotide sequence of the gene encoding slrA RNA and mapping of 5′ and 3′ ends by RACE. The arrows indicate the beginning of the primary transcript or processing sites. Nucleotides in red correspond to the region encoding the mature form of slrA sRNA, which is modeled in B. The −10 and −35 promoter elements are underlined and aligned with conserved RpoE-regulated promoters, and consensus is shown (A and C). Shown are elevated levels of the slrA promoter activity monitored by single-copy slrA-lacZ promoter fusion in a ΔrseA derivative as compared with that in the wild type (D). Cultures of Δ(lapA lapB) derivative with vector alone or expressing the slrA gene from a plasmid were grown at 30 °C in the M9 medium up to an A₆₀₀ of 0.2 and analyzed by serial spot dilution for the restoration of growth on MacConkey agar at 42 °C (E). Exponentially grown cultures of the wild type (wt), its slrA^C derivative with constitutive expression of slrA, ΔdegP, and ΔdegP slrA^C derivatives were grown in LB medium at 30 °C, adjusted to an A₆₀₀ of 0.1, and serially spot-diluted on LA plates. Plates were incubated at 30 and 42 °C (F). Isogenic cultures were grown as described in the legend to Fig. 7A. A portion of the cultures were shifted to 37 and 42 °C for 30 min in LB medium. An equivalent amount of proteins were resolved on 12% SDS-PAGE and immunoblotted with DegP antibodies (G).

FIGURE 10.

Effect in the accumulation of LPS precursors of Δ(lapA lapB) derivatives lacking DnaK/J or YceK. Shown are charge-deconvoluted ESI FT-ICR mass spectra in the negative ion mode of LPS isolated from strains carrying different mutations: Δ(lapA lapB) (A), Δ(dnaK dnaJ) (B), Δ(lapA lapB dnaK dnaJ) (C), ΔyceK (D), Δ(lapA lapB yceK) (E), and Δ(lapA lapB) + pdnaK⁺dnaJ⁺ (F). Cultures were grown under permissive growth conditions at 30 °C in 121 medium. Mass numbers are the monoisotopic masses of major peaks, and the predicted compositions of mass peaks are indicated.

FIGURE 11.

Defects in the accumulation of myristoyltransferase LpxM, early glycosyltransferases, and LptD in Δ(lapA lapB) mutant. Cultures of the wild type and its Δ(lapA lapB) derivatives carrying chromosomal single-copy C-terminal 3× FLAG epitope fused to various enzymes were grown at 30 °C in M9 medium up to an A₆₀₀ of 0.2, shifted to 42 °C for 90 min in LB medium. An equivalent portion of total proteins was fractionated to obtain IM proteins and total protein aggregates. Samples obtained after fractionation into aggregates, IM, and samples without fractionation (total fractions) from 30 or 42 °C were analyzed on 12% SDS-PAGE and immunoblotted with FLAG antibody. Shown are Western blots of samples from LpxM-FLAG derivatives (A) and Western blots from samples with FLAG tag on different glycosyltransferases (B). B (lanes 9 and 10), samples from aggregate fractions obtained from a strain with the Δ(lapA lapB) mutation in WaaC-3×FLAG background carrying plasmid-borne slrA RNA and subjected to immunoblotting with FLAG antibody. The relevant genotype, temperature, and correspondence to samples without fractionation (total) and after fractionation are marked in each case. C, cultures of the wild type with LptD-3×FLAG and its Δ(lapA lapB) derivative were grown in M9 medium at 30 °C up to an A₆₀₀ of 0.2. A portion of the culture was shifted for 90 min to 42 °C and fractionated to obtain OM fractions. Proteins were resolved on 12% SDS-PAGE and subjected to immunoblotting with FLAG antibody. Lanes 1–4, samples without fractionation, representing the total amount of LptD. Lanes 5–9, samples from OM fractions. In lane 9, a 5× volume of sample is applied from the OM fraction obtained from Δ(lapA lapB) mutant after the shift to 42 °C.

FIGURE 12.

Induction of stress response pathways in Δ(lapA lapB) derivatives. Isogenic cultures of the wild type and its Δ(lapA lapB) derivative, carrying a specific single-copy chromosomal promoter fusion, were grown to early log phase at 30 °C in M9 medium and adjusted to an A₆₀₀ of 0.02 and allowed to grow further. Samples were analyzed for β-galactosidase activity after different intervals. Data are shown after a 90-min incubation (A). B and C, cultures of the wild type and its Δ(lapA lapB) derivative were grown in M9 medium up to A₆₀₀ of 0.2 at 30 °C. Aliquots were shifted to 37 and 42 °C and allowed to grow for 30 min in prewarmed flasks. Cultures were harvested by centrifugation, and an equivalent amount of total protein was applied on 12% SDS-PAGE. The relative abundance of DegP (B) and DnaK (C) was revealed by immunoblotting using DegP and DnaK antibodies, respectively. The relevant genotype and temperature are indicated.

FIGURE 13.

Modeling of LapB from E. coli. The primary amino acid sequence was analyzed for the best fitting model on the Phyre 2 server. Mutations identified in this work are indicated.

FIGURE 14.

Proposed model of LapA and LapB function in the assembly of LPS. LapB functions at key steps, including the presentation of LpxC to FtsH protease, serving as a docking site for the LPS assembly by various IM-associated or IM-anchored enzymes, ensuring that only the completely synthesized LPS is translocated. In this process, LapB could couple LPS synthesis with its translocation. This mature LPS would be flipped by MsbA, and in subsequent steps LapA and LapB could function together with transenvelope Lpt complex components because such proteins were found to co-purify. In the absence of LapB, LpxC accumulates, causing toxicity due to the imbalance between LPS and phospholipids. Overexpression of the fabZ gene product can restore this balance between phospholipids and LPS.