LppB shares a high degree of homology with Lpp and LppA but features a cysteine residue in the penultimate position of the C-terminal lysine.

A. Amino acids sequence alignment of Lpp proteins of E. coli and S. Typhimurium LT2. The six residues preceding the C-terminal lysine (Lys58 in the matured peptide sequence of LppB starting from the tri-acylated cysteine, Cys21) are indicated within a pink box. Numbers above the first sequence line indicate amino acid positions relative to the start of each protein sequence. Conserved residues are shown as black letters on a grey background, and similar residues are shown as white letters in black boxes. The secondary structure of E. coli Lpp is depicted above the alignment. B. Schematic comparison of the genetic neighborhood of lpp in E. coli, S. Typhi, and S. Typhimurium. The lpp and ldtE genes face each other in both E. coli and S. enterica species and are positioned between the suf (iron-sulfur cluster) and pykF operons. LdtE catalyzes the formation of 3-3 crosslinks in the peptidoglycan 48. Phylogeny is midpoint rooted. Genes and intergenic regions are drawn to scale. Arrow tags represent genes, with arrow orientation indicating transcriptional direction. Colors depict homologous relationships. Illustration created with ESPript and BioRender.com/0swneug.

LppB crosslinks to the peptidoglycan via K58.

LppB crosslinking to peptidoglycan (PG). Lpp and LppB variants were expressed from pSC284 and pSC283 derivatives, respectively, in the Δlpp strain (lanes 2-5; 7-10). The left panel depicts untreated cell lysate (lanes 1-5) while the right panel depicts cell lysates treated with 25 μg/μL of lysozyme (lanes 6-10). Bands corresponding to the monomeric form of Lpp and LppB are observed at ∼7.5 kDa. Bands corresponding to the dimeric form of LppB are observed at ∼ 15 kDa. Bands of higher molecular weight corresponding to Lpp or LppB bound to various muropeptides are observed except in strains expressing the K58R variant of Lpp or LppB (lanes 8 and 10 respectively). Cell lysates were TCA-precipitated and Western blot analysis was performed using anti-Lpp antibody. Representative images from experiments conducted in biological triplicates are shown.

LdtB is the primary L,D-transpeptidase for efficient peptidoglycan crosslinking of LppB.

LppB crosslinking to peptidoglycan (PG) in the presence or absence of the L,D-transpeptidases. A. LdtB is required for efficient LppB crosslinking. LppB was expressed from pSC283 (pLppB) in the Δlpp mutant or in double mutants Δlpp/ΔldtA, Δlpp/ΔldtB, Δlpp/ΔldtC strains (lanes 3-6). Deletion of ldtB (lane 5) caused the strongest reduction of LppB-peptidoglycan crosslinking, whereas loss of ldtA or ldtC had only minor effects. Wild-type and Δlpp controls are shown in lanes 1 and 2, respectively. A schematic (right) illustrates the LdtB-mediated crosslink: the C-terminal lysine residue of LppB (K58) is covalently attached to the diaminopimelic acid (mDAP) moiety of the peptidoglycan. B. Functional complementation of LppB crosslinking in the absence of LdtB. LdtA (pEP25; lanes 11 and 18), LdtB (pAA186; lanes 12 and 19), and LdtC (pEP26; lanes 13 and 20) were ectopically expressed alongside LppB (pLppB) in the Δlpp/ΔldtB strain (lanes 10 and 17). Wild-type lanes (7 and 14), Δlpp lanes (8 and 15) and Δlpp complemented with pLppB lanes (9 and 16) served as controls. LdtB restoration (lanes 12 and 19) fully rescued crosslinking, while LdtA provided partial activity (lanes 11 and 18) and LdtC had minimal effect (lanes 13 and 20), confirming LdtB as the predominant transpeptidase. For both figures, sample were treated with 25 μg/μL of lysozyme, cell lysates were TCA-precipitated and treated with 30 mM DTT for reduction when indicated. Western blot analysis was performed using anti-Lpp antibody. Representative images from experiments conducted in biological triplicates are shown.

LppB predominantly forms disulfide-linked homodimers in vivo.

LppB mostly exists in an oxidized state in vivo, forming mixed disulfide homodimers. Lpp and LppB variants were expressed from pSC284 and pSC283 derivatives, respectively, in the Δlpp strain (lanes 4-5; 9-10; 12-16; 18-22). The wild type is shown in lanes 1, 6, 11 and 17 as control. A. Oxidized state of LppB in vivo. The left panel depicts untreated cell lysate while the right panel depicts cell lysate treated with 30 mM DTT to reduce disulfide bonds. Monomeric forms of Lpp and LppB migrate at ∼ 7.5 kDa, whereas bands corresponding to the dimeric form of LppB are observed at ∼ 15 kDa. DTT treatment reduced homodimer abundance with a corresponding increase of monomer signal (compare lane 5 and 10), confirming disulfide bond-mediated dimerization. B. Cysteine-dependent homodimerization of LppB. The left panel depicts cell lysates treated with 25 μg/μL of lysozyme (lanes 11-16). The right panel depicts samples additionally treated with 30 mM DTT to reduce disulfide bonds (lanes 17-22). High–molecular weight bands corresponding to Lpp bound to peptidoglycan fragments were observed, except in lanes carrying the lysine substitution mutants (K58R; lanes 13,15,19,21). Substitution of the cysteine (C57R) abolished LppB homodimeric species (LppB-S-S-LppB; compare lane 14 and 16). DTT treatment similarly reduced the LppB homodimer signal and increased monomer abundance (compare lane 14 and 20). Western blot analysis was performed using anti-Lpp antibody, and representative images from experiments conducted in biological triplicates are shown.

LppB drives disulfide-linked dimerization of Lpp/LppB heterotrimers in vivo.

LppB was expressed from pSC283 (pLppB) in the wild-type and Δlpp strains using varying isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentrations for induction. A. LppB forms a homodimeric mixed disulfide in the presence or absence of Lpp B. -C. Chemical crosslinking with Dithiobis-succinimidyl-propionate (DSP) or with Bis-sulfosuccinimidyl-suberate (BS3) reveals higher order oligomers. Distinct bands corresponding to Lpp homodimers and to Lpp-LppB heterotetramers (Lpp-LppB-S-S-LppB-Lpp) are observed. D. Reducing conditions disrupt disulfide-linked oligomers. Treatment of samples from A. with 30 mM DTT decreased homodimer abundance and increased monomer signal. E. Reducing conditions on DSP-crosslinked complexes disrupts both disulfide-linked and chemically crosslinked homodimers and heterotetramers. Treatment of samples from B. with 30 mM DTT results in monomer enrichment and decrease of higher order oligomers. F. Reducing conditions on BS3-crosslinked complexes only disrupts disulfide-linked homodimers and heterotetramers. Treatment of samples from C. with 30 mM DTT results in monomers and dimers (LppB homodimers and Lpp-LppB heterodimers) enrichment and disulfide-linked heterotetramers (Lpp-LppB-S-S-LppB-Lpp) and homodimers (LppB-S-S-LppB) decrease. G. Schematic representation of oligomeric states of Lpp (represented as A) and LppB (represented as B) observed in panels A-F. Dimeric and tetrameric crosslinked species are depicted. Created with BioRender.com/aersfld. H. Structural model of an Lpp-LppB heterohexamer. Two LppA-LppB mixed disulfide trimers [LppA2LppB] are linked via a disulfide bond to form a hexameric complex [LppA2LppB]-S-S-[LppBLppA2] embedded in the OM. The two LppB helices appear in cyan, with disulfide-bonded Cys57s and Lys58s displayed as spheric atoms (sulfur atoms in yellow; nitrogen atoms in the amino group in blue). N-terminal acyl chains (green) are embedded in the OM (shown as stick).

LppB negatively modulates Lpp-PG crosslinking.

LppB was expressed from pSC283 (pLppB) in wild-type cells using the indicated isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentrations for induction. Cells were harvested and treated with lysozyme for peptidoglycan digestion. A. Dose-dependent inhibition of Lpp-PG crosslinking by LdtB. Progressive induction of LppB reduced the abundance of Lpp–PG crosslinked species. B. Effect of reducing conditions on Lpp-PG crosslinking. Samples from panel A were additionally treated with 30 mM DTT to reduce disulfide bonds. The inhibitory effect of LppB on Lpp–PG crosslinking persisted after reduction, demonstrating that this modulating activity is independent of the disulfide bond in LppB. C. Comparison of LppB and Lpp expression effect on Lpp-PG crosslinking. LppB expression decreases Lpp–PG crosslinking, whereas Lpp expression does not. Left panel: LppB expressed from pSC283 (pLppB) at 0 or 40 μM IPTG. Right panel: Lpp expressed from pSC284 (pLpp) under the same conditions. Cells were harvested and treated with lysozyme for PG digestion. Western blot analysis was performed using anti-Lpp antibody. Representative images from experiments conducted in biological triplicates are shown. (see Fig. S6).

Formation of LppA and LppB heterotrimers.

The plot shows the probability of AAB heterotrimer formation (PAAB) as a function of the fraction of LppB (b). PAAB increases up ∼ b=0.33, then declines as the relative fraction of LppA decreases. Since LppB is expected to be present at low levels in Salmonella, the region where b is small (highlighted by the dotted square) is particularly informative. Within this range, PAAB increases approximately linearly, with a ∼ 2.4-3-fold rise as the fraction of LppB increases

Acidic pH increases LppB expression levels.

Lpp and LppB variants were expressed from pSC284 and pSC283 derivatives respectively, in the Δlpp strain. A. Lpp and LppB oligomeric states in neutral pH conditions. Untreated cell lysates from cells grown at pH 7 show monomeric Lpp and LppB at ∼7.5 kDa and LppB homodimers at ∼15 kDa. B. Lpp and LppB oligomeric states in acidic pH conditions. Untreated cell lysate from cells grown at pH 5.5 reveal higher LppB expression compared with neutral conditions (compare lanes 5 and 10). Monomeric and dimeric species migrate as in panel A. C. - D. Effect of reducing conditions. Lysates from panels A and B were treated with 30 mM DTT to reduce disulfide bonds. DTT treatment decreased LppB dimer abundance and increased monomer signal at both pH conditions, confirming disulfide bond–dependent dimerization. Western blot analysis was performed using anti-Lpp antibody. Representative images from experiments conducted in biological triplicates are shown.

Acidic pH enhances LppB-mediated protection against SDS-EDTA sensitivity.

The contribution of LppB to envelope integrity increases under acidic conditions. Wild-type and Δlpp mutant complemented with pSC284 and pSC283 derivatives were grown in LB at the indicated pH. Cells were harvested, serially diluted, and 5 μL of each dilution was spotted on indicated plates and incubated overnight at 37 °C. A. At pH 7, LppB expression provides weak protection against SDS-EDTA-induced growth defects compared to Lpp. B. At pH 5.5, LppB expression restores resistance to SDS-EDTA-induced growth defects to levels comparable to those conferred by Lpp. Representative images from experiments conducted in biological triplicates are shown.

E. coli LdtA/B/C share strong sequence similarity with their S. Typhimurium homologs ErfK/Ybis/YcfS.

Amino acid sequence alignments of the L,D-transpeptidases from E. coli K12 and S. Typhimurium LT2. Numbers above the first sequence line indicate amino acid positions relative to the start of each protein sequence. Conserved residues are shown as black letters on a grey background, and similar residues are shown as white letters in black boxes. A. Amino acids sequence alignment of E. coli LdtA and S. Typhimurium ErfK. The secondary structure of LdtA is depicted above the alignment. B. Amino acids sequence alignment of E. coli LdtB and S. Typhimurium YbiS. The secondary structure of LdtB is depicted above the alignment. C. Amino acids sequence alignment of E. coli LdtC and S. Typhimurium YcfS. The secondary structure of LdtA is depicted above the alignment. D. Pairwise percent identity matrix. Percent identity values were calculated from global pairwise alignments performed with EMBOSS Needle and represent the proportion of identical residues across the full alignment length, including gaps.

LppB forms homodimeric mixed disulfide independently of expression levels or Lpp presence.

LppB was expressed from pSC283 (pLppB) in the wild-type and Δlpp strains using varying concentrations of isopropyl ß-D-1-thiogalactopyranoside (IPTG) for induction. Bands corresponding to the homodimeric mixed-disulfide form of LppB (∼15 kDa) were detected under all tested conditions indicating that LppB homodimerization occurs independently of its expression level or the presence of Lpp. Western blot analysis was performed using anti-Lpp antibody. Representative images from experiments conducted in biological triplicates are shown.

LppB forms homodimeric mixed disulfide and heterotrimers with Lpp in vivo.

LppB was expressed from pSC283 (pLppB) in the wild-type and Δlpp strains using varying concentrations of isopropyl ß-D-1-thiogalactopyranoside (IPTG) for induction. A. Chemical crosslinking with Bis-sulfosuccinimidyl-suberate (BS3) reveals distinct oligomeric states of Lpp and LppB. Bands corresponding to Lpp or LppB monomers and homodimers and to Lpp-LppB heterotetramers (Lpp-LppB-S-S-LppB-Lpp) were detected. The samples analyzed here are identical to those used in Fig. 5C. B. Effect of reducing conditions on BS3-crosslinked Lpp and LppB oligomeric states. Samples from panel A were treated with 30 mM DTT to reduce disulfide bonds formed through native cysteine only. Disulfide bond formed through BS3 are irreversible. The samples analyzed here are identical to those used in Fig. 5F. C. Effect of alkylating conditions on reduced BS3-crosslinked Lpp and LppB oligomeric states. Reduced samples from panel B were treated with 4-Acetamido-4’-Maleimidylstilbene-2,2’-Disulfonic Acid (AMS), which adds ∼ 500 Da per free thiol. AMS treatment caused mobility shifts for LppB dimers and Lpp-LppB heterodimers, but not for Lpp homodimers, confirming the presence of reactive cysteines in LppB. Western blot analysis was performed using anti-Lpp antibody.

LppB negatively modulates Lpp-peptidoglycan crosslinking.

Comparative effects of LppB and Lpp on Lpp-peptidoglycan crosslinking. LppB was expressed from pSC283 (pLppB) and Lpp from pSC284 (pLpp) in wild-type cells using the indicated concentrations of isopropyl β-D-1-thiogalactopyranoside (IPTG) for induction. The left panel shows LppB expressed at 0 or 40 μM IPTG; the right panel shows Lpp expressed under the same conditions. Increasing LppB expression reduced Lpp-peptidoglycan crosslinking, whereas Lpp overexpression had no detectable effect. Cells were harvested and treated with lysozyme for PG digestion. Western blot analysis was performed using anti-Lpp antibody, and representative images from experiments conducted in biological triplicates are shown.

E. coli Lpp paralog YqhH shares sequence similarity with S. Typhimurium LppA and LppB, and a conserved C-terminal cysteine is found in YqhH and LppB.

Amino acid sequence alignment of E. coli YqhH with LppA and LppB from S. Typhimurium LT2. YqhH contains a conserved cysteine residue at its C-terminus that aligns with Cys57 of LppB (highlighted within an orange box and marked by a star). The predicted secondary structure of E. coli YqhH is depicted above the alignment. Illustration created with ESPript and BioRender.com/99cogwn.

Bacterial strains used in this study.

Plasmids used in this study.

Primers used in this study.