Regulation of the First Committed Step in Lipopolysaccharide Biosynthesis Catalyzed by LpxC Requires the Essential Protein LapC (YejM) and HslVU Protease

Abstract

We previously showed that lipopolysaccharide (LPS) assembly requires the essential LapB protein to regulate FtsH-mediated proteolysis of LpxC protein that catalyzes the first committed step in the LPS synthesis. To further understand the essential function of LapB and its role in LpxC turnover, multicopy suppressors of ΔlapB revealed that overproduction of HslV protease subunit prevents its lethality by proteolytic degradation of LpxC, providing the first alternative pathway of LpxC degradation. Isolation and characterization of an extragenic suppressor mutation that prevents lethality of ΔlapB by restoration of normal LPS synthesis identified a frame-shift mutation after 377 aa in the essential gene designated lapC, suggesting LapB and LapC act antagonistically. The same lapC gene was identified during selection for mutations that induce transcription from LPS defects-responsive rpoEP3 promoter, confer sensitivity to LpxC inhibitor CHIR090 and a temperature-sensitive phenotype. Suppressors of lapC mutants that restored growth at elevated temperatures mapped to lapA/lapB, lpxC and ftsH genes. Such suppressor mutations restored normal levels of LPS and prevented proteolysis of LpxC in lapC mutants. Interestingly, a lapC deletion could be constructed in strains either overproducing LpxC or in the absence of LapB, revealing that FtsH, LapB and LapC together regulate LPS synthesis by controlling LpxC amounts.

Keywords: FabZ; HslV/U protease; LapB; LapC; LpxC; RpoE; YejM; lipopolysaccharide.

Figure 1

Model of regulation of balanced LPS and phospholipid biosynthesis: R-3-hydroxymyristoyl-ACP serves as a common metabolic precursor for the LpxC–dependent LPS biosynthesis and the FabZ-mediated phospholipid biosynthesis. LpxC stability is regulated by previously established FtsH/LapB and, from this study, via the negative regulation by LapC counteracting the LapB/FtsH pathway and also at high temperature by HslVU proteolysis of LpxC.

Figure 2

Schematic drawing illustrating three major approaches that identified additional players in the regulation of LpxC proteolysis. The multicopy suppressor approach led to discovery of the HslVU-dependent proteolysis of LpxC in the absence of LapB (A). In search of suppressors that bypass the lethality of a lapB deletion, the lapC377fs mutation was isolated that restored the normal LPS synthesis and the viability in the absence of LapB (B). In the third approach, based on mutagenesis, CHIR090- and temperature-sensitive mutants with defects in LPS identified the lapC gene. Suppressors of lapC mutants revealed that LapC works together with LapB/FtsH to regulate LpxC proteolysis and the essential lapC gene is dispensable in the absence of LapB (C).

Figure 3

Overexpression of either the hslV gene alone or hslVU genes results in enhanced proteolysis of LpxC. (A) Cultures of ΔlapA/B strain carrying the inducible hslV gene on a plasmid were grown in M9 medium, adjusted to an OD₅₉₅ of 0.2 in LB medium. Expression of the hslV gene was induced by the addition of 75 μM IPTG at 42 °C. An equivalent amount of total proteins from indicated time points were resolved by SDS-PAGE and LpxC levels were determined by immunoblot analysis using LpxC-specific antibodies, revealing a gradual decrease of LpxC. (B) Expression of hslVU genes from the pET24b vector in BL21 strain was induced by the addition of 75 μM IPTG at 30 °C for 15 min at an OD₅₉₅ of 0.1. Cultures were washed and shifted to 42 °C and further host transcription stopped by the addition of rifampicin. LpxC levels were determined using the equivalent amount of total proteins by immunoblot analysis using LpxC-specific antibodies.

Figure 4

The absence of hslV and hslVU genes confers the sensitivity to the LpxC inhibitor CHIR090. Exponentially grown cultures of the wild type and its isogenic derivatives lacking genes encoding HslVU protease subunits were adjusted to an OD₅₉₅ of 0.1 and serially spot diluted at 30 °C on LA agar supplemented with or without varying concentrations of CHIR090 as indicated. As a control, the isogenic CHIR090-sensitive strain with the lapC190 mutation was included. Data presented are from one of the representative experiments.

Figure 5

The lapC377fs mutation restores the wild-type LPS content in ΔlapB. Equivalent amounts of whole cell lysate treated with Proteinase K were resolved on a 14% SDS-Tricine gel and LPS revealed by silver staining. The arrow indicates the position of LPS and the relevant genotype of strains is depicted.

Figure 6

The lapC377fs mutation suppresses the accumulation of LPS precursors and restores the normal LPS synthesis in ΔlapB. Charge-deconvoluted mass spectra in the negative ion mode of LPS from the wild type (A), its ΔlapB (B) and ΔlapB lapC377fs (C) derivatives. Cultures were grown in phosphate-limiting medium at 30 °C. Mass numbers refer to monoisotopic peaks. Mass peaks with rectangular boxes correspond to the glycoform containing the third Kdo. Ovals—derivatives with two Kdo residues with either complete or incomplete core.

Figure 7

Truncation of the C-terminal periplasmic domain of LapC confers the sensitivity to the LpxC inhibitor CHIR090. Exponentially grown cultures of the wild type and its isogenic derivatives with point mutations in the lapC gene were adjusted to an OD₅₉₅ of 0.1 and serially spot diluted at 30 °C on LA agar supplemented with or without varying concentrations of CHIR090 as indicated.

Figure 8

Mutations that cause truncation of periplasmic domain of LapC induce transcription from the rpoEP3 promoter, which specifically responds to severe defects in LPS. Exponentially grown isogenic strains of the wild type and its derivatives with various lapC mutations as indicated, carrying the single-copy chromosomal rpoEP3-lacZ fusion, were analyzed for the β-galactosidase activity. Cultures were adjusted to an OD₅₉₅ of 0.05 and allowed to grow in LB medium at 30 °C. Aliquots of samples were drawn after different time intervals and used to measure the β-galactosidase activity. Error bars represent a S.E. of three independent measurements.

Figure 9

lapC190 mutant bacteria exhibit a reduction in LPS amounts and the temperature-sensitivity (Ts) phenotype. (A) Exponentially grown cultures with indicated genotypes were adjusted to an OD₅₉₅ of 0.1 and spot diluted on LA and MacConkey agar at different temperatures. (B) Exponentially grown cultures of the wild type, its lapC190 derivative and strains with various suppressor mutations mapping to the lpxC gene were grown under permissive growth conditions. Equivalent amounts of total cellular proteins were resolved by SDS-PAGE and proteins transferred by Western blotting and subjected to immunoblotting, using LpxC-specific antibodies. (C) The position of amino acids residues in the structure of LpxC (PDB 4MQY, [49]), whose specific alterations suppress the Ts phenotype of a lapC190 mutation and cause elevation of LpxC levels.

Figure 10

LPS levels are highly reduced in lapC mutants lacking the C-terminal periplasmic domain, which are restored by various suppressor mutations mapping to either lpxC, lapA or lapB genes. Isogenic bacterial cultures of the wild type and its derivatives with the indicated genotype were grown up to an OD₅₉₅ of 0.5 at 30 °C. An equivalent portion of whole cell lysates were applied to a 14% SDS-Tricine gel and transferred by Western blotting. Relative amounts of LPS were revealed by immunoblotting with the LPS-specific monoclonal antibody WN1 222-5 using chemiluminescence detection kit.

Figure 11

Suppressors of lapC190 that restore growth at high temperature mapping either to the promoter region of the lapA/B operon or in the lapA gene reduce the LapB abundance. (A) Isogenic cultures of wild type, its lapC190 derivative and lapC190 with a specific suppressor mutation in the lapA gene or its promoter region were adjusted to an OD₅₉₅ of 0.1, serially diluted, 5 μL aliquots spotted on either LA agar or MacConkey agar at various indicated temperatures and plates incubated for 24 h. (B) Immunoblots of whole cell lysates obtained from isogenic strains with indicated genotypes using LapB-specific antibodies. An equivalent amount of total proteins was loaded. As controls, extracts from ΔlapA/B and purified LapB (lanes 1&10) were applied. (C) The position of various suppressor mutations in the lapA gene is indicated. The presence of IS element in either the coding region or the promoter region is indicated by a triangle at the specific nt position. Other mutations causing frame shifts or introducing stop codons, resulting into either truncation of LapA or alterations in the amino acid sequence, are indicated and underlined with the indicated stop codon as * symbol.

Figure 12

Suppressors mapping to the lapB gene that restore growth of lapC mutant bacteria reduce the LapB abundance. (A) Growth of isogenic cultures of strains with lapC190 and with suppressor mutations in the lapB gene was quantified by spot dilution on LA and MacConkey agar at various temperatures. The genotype and incubation temperature are indicated. (B) Immunoblot of total cellular proteins from various strains, used in growth measurement, with LapB-specific antibodies. Purified LapB (lane 10) serves as a control to validate the cross-reactivity of LapB and its position on immunoblot. Equivalent amount of proteins were resolved by SDS-PAGE prior to immunoblotting. (C) The position of various mutations in LapB with the indicated relevant TPR motif are shown in the structure of LapB (PDB 4ZLH, [25]).

Figure 12

Suppressors mapping to the lapB gene that restore growth of lapC mutant bacteria reduce the LapB abundance. (A) Growth of isogenic cultures of strains with lapC190 and with suppressor mutations in the lapB gene was quantified by spot dilution on LA and MacConkey agar at various temperatures. The genotype and incubation temperature are indicated. (B) Immunoblot of total cellular proteins from various strains, used in growth measurement, with LapB-specific antibodies. Purified LapB (lane 10) serves as a control to validate the cross-reactivity of LapB and its position on immunoblot. Equivalent amount of proteins were resolved by SDS-PAGE prior to immunoblotting. (C) The position of various mutations in LapB with the indicated relevant TPR motif are shown in the structure of LapB (PDB 4ZLH, [25]).

Figure 13

FtsH A296V suppressor mutation causes the reduction in FtsH levels. (A) Immunoblot analysis of total cellular extracts obtained from the wild type, its derivatives with lapC190, lapC190 ftsH A296V and as the negative control isogenic strain with ΔftsH sfhC21 mutation. An equivalent amount of proteins was resolved by SDS-PAGE and immunoblots were treated with a FtsH-specific antibody. Arrow indicates the position of FtsH. (B) The position of FtsH amino acid residue 296 on its crystal structure (PDB 1LV7 [54]) is shown.

Figure 14

LapC and LapB show physical interaction. Purification profile of proteins from IM fractions after 100 μM and 500 μM IPTG addition to induce lapA and lapB transcription. Lanes 1 and 3 indicate co-purification of His-tagged LapA with LapB and LapC from IM fractions of total proteins. Lane 2 depicts elution profile of His₆-LapC and its co-elution with LapB. Proteins were resolved on a 12% SDS-PAGE. The identity of LapC, LapB, LapA and FtsH are shown by arrows.

Figure 15

Transcription of the lapC gene is induced upon a shift to high temperature. qRT-PCR analysis of mRNA isolated from wild-type bacteria grown up to an OD₅₉₅ of 0.2 in M9 minimal medium either at 30 °C or after a 15-min shift to 42 °C. Data presented are from RNA isolated from three biological replicates and error bars are indicated.