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Lipoprotein cofactors located in the outer membrane activate bacterial cell wall polymerases - PubMed

  • ️Fri Jan 01 2010

Lipoprotein cofactors located in the outer membrane activate bacterial cell wall polymerases

Catherine Paradis-Bleau et al. Cell. 2010.

Abstract

Most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by polysaccharide polymerases called penicillin-binding proteins (PBPs). Because they are the targets of penicillin and related antibiotics, the structure and biochemical functions of the PBPs have been extensively studied. Despite this, we still know surprisingly little about how these enzymes build the PG layer in vivo. Here, we identify the Escherichia coli outer-membrane lipoproteins LpoA and LpoB as essential PBP cofactors. We show that LpoA and LpoB form specific trans-envelope complexes with their cognate PBP and are critical for PBP function in vivo. We further show that LpoB promotes PG synthesis by its partner PBP in vitro and that it likely does so by stimulating glycan chain polymerization. Overall, our results indicate that PBP accessory proteins play a central role in PG biogenesis, and like the PBPs they work with, these factors are attractive targets for antibiotic development.

Copyright © 2010 Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1. Cell wall structure and assembly

A. Schematic of bacterial cells with the cell wall (PG layer) in green. Gram-negative cells have a relatively thin PG layer surrounded by an additional (outer) membrane. Above the cells is a schematic detailing the structure of PG, which continues in all directions to envelop the cell (green arrows). M, N-acetylmuramic acid; G, N-acetylglucosamine. Dots represent the attached peptides. B. Overview of PG assembly. A generic multi-protein complex (grey) containing a class A PBP (purple) is shown. For simplicity, the other PG assembly factors thought to participate in the final stages of PG construction are not specifically labeled. See text for details. PGT, peptidoglycan glycosyltransferase domain; TP, transpeptidase domain.

Figure 2
Figure 2. Synthetic lethal screens and terminal phenotypes

A-B. Colonies of transposon mutants used for the slb mutant screen grown on an indicator plate (LB-IPTG-Xgal) for 2 days at 30°C. The arrow points to a rare solid-blue colony that retained the unstable plasmid. The boxed region in (A) is enlarged in (B). C. Schematics indicating the approximate locations of the transposon insertions in the lpo genes. Triangles represent transposon insertion points (green: transcription of the KanR cassette is in the same direction as the target gene; red: transcription is in the opposite direction). D. Plating defects of representative slb and sla mutants isolated in the screens. Cells of TU122/pTU110 [ΔponB/Plac∷ponB lacZ] (top) or TU121/pCB1 [ΔponA/Plac∷gfp-ponA lacZ] (bottom) and their derivatives with the indicated transposon insertions were grown overnight at 30°C. Culture densities were normalized, 10-fold serial dilutions were prepared for each, and 5 μl of each dilution was spotted onto LB with or without IPTG as indicated. E. Cells of TU121(attλTB309) [ΔponA(Para∷ponA)] (top) or MM11 [Para∷ponB] (bottom) and their derivatives were grown overnight at 37°C. Serial dilutions were prepared as in (D) and dilutions were spotted onto the indicated media. F-G. Strains used in (E) were grown in LB-arabinose at 37°C to an OD600 = 0.6-1.1. They were then pelleted, washed three times with LB, and resuspended in LB-glucose at an OD600 = 0.08 or 0.02 for TU121(attλTB309) (F) or MM11 derivatives (G), respectively. Cell growth following subculture (t = 0) was then monitored by regular OD600 measurements. Please also see data in Figure S2 and S3.

Figure 3
Figure 3. Lpo factors are essential for growth and PBP1 function

A. Cells of MM22(attHKMM10) [ΔlpoA(Plac∷lpoA-gfp)] and its derivatives were grown overnight in LB-IPTG (50μM) at 37°C. Serial dilutions were prepared as in Figure 2, and spotted onto the indicated solid media. Identical results were obtained when LpoB was depleted in the absence of LpoA (data not shown). B. Cultures of cells from (A) were grown to an OD600 of 0.3-0.4 in LB-IPTG (50μM) at 37°C. The cells were then pelleted, washed three times with LB, and resuspended in LB-glucose at an OD600 = 0.02. Cell growth following subculture (t = 0) was then monitored by regular OD600 measurements. C-D. Cells of MM13 [Para∷ponB ΔlpoA] (C) or CB4(attλTB309) [ΔponA ΔlpoB(Para∷ponA)] (D) containing the low-copy plasmids pTB284 [Plac-con∷gfp], pCB62 [Plac-con∷ponA], or pCB72 [Plac-con∷ponB] were grown to an OD600 of 0.5-0.7 in LB-Ara-Spc (C) or M9-Ara-Spc (D) at 37°C. They were then washed, diluted into LB-IPTG (1mM), and growth at 37°C was followed as in (B). E-F. To determine the extent of PBP1a or PBP1b overproduction in the cultures from (C) and (D), respectively, extracts were prepared from cells harvested at times indicated by the arrows above the growth curves (C-D). Immunoblot analysis was then performed to determine the levels of PBP1a (E) or PBP1b (F) in strains MM13/pCB62 or CB4(attλTB309)/pCB72, respectively, relative to corresponding control strains harboring pTB284. Numbers above lanes indicate the amount of total protein loaded. G. Cultures of TB28 [WT], MM13, or CB4(attλTB309) containing the aforementioned plasmids were diluted, plated on the indicated media containing Spc, and incubated overnight at 37°C. Plasmids pCB62 [Plac-con∷ponA] and pCB72 [Plac-con∷ponB] possess native ribosome binding sites and 5′UTRs for ponA and ponB, respectively. pCB72 likely has a higher basal level of expression than pCB62 since it can correct the PBP1B- LpoA- phenotype of MM13 without IPTG induction. Plac-con is a synthetic lac promoter with consensus -35 and -10 elements. Please also see data in Figure S1 and S4.

Figure 4
Figure 4. LpoA and LpoB are outer membrane lipoproteins

A-F. Cytological assay of membrane localization. Cells of MM13 [Para∷ponB ΔlpoA] (A-C) or CB4(attλTB309) [ΔponA ΔlpoB(Para∷ponA)] (D-F) harboring the integrated expression constructs (A) attHKMM10 [Plac∷lpoA-gfp], (B) attHKCB42 [Plac∷ssdsbA-lpoA-gfp], (C) attHKMM50 [Plac∷lpoA(D+2D+3)-gfp], (D) attHKCB28 [Plac∷lpoB-gfp], (E) attHKCB41 [Plac∷ssdsbA-lpoB-gfp], or (F) attHKMM51 [Plac∷lpoB(D+2E+3)-gfp] were grown at 30°C to mid-log in M9-arabinose supplemented with 100 μM IPTG. The cells were then plasmolyzed and visualized using GFP (panels 1) and phase contrast (panels 2) optics. Arrows highlight clear examples of protein localization (outer membrane, A and D; periplasm B and E; inner membrane, C and F). Bar equals 2 microns. G-H. Functionality of signal sequence mutants. Cultures of cells from (A-F) were diluted and plated on the indicated media as described in Figure 2. Please also see data in Figure S5 and S6

Figure 5
Figure 5. LpoA and LpoB specifically interact with their cognate PBP

A-D. H-LpoA (A-B) or H-LpoB (C-D) was incubated with PBP1a (A and C) or PBP1b (B and D) for 60 min at room temperature in binding buffer [20mM Tris (pH 7.4), 0.1% Triton-X-100, and either 300mM or 150mM NaCl for A-B or C-D, respectively]. Ni-NTA resin (Qiagen) was then added to each reaction and they were further incubated for 2 hr at 4°C with rotation. The resin was pelleted by centrifugation, washed twice with binding buffer containing 20mM imadazole, and the proteins retained on the resin were eluted with sample buffer containing EDTA (100 mM). Proteins in the initial reaction (input), initial supernatant (UB), wash supernatants (W1 and W2), and eluate were separated on a 12% SDS polyacrylamide gel and stained with Coomassie Brilliant Blue. FtsZ was included in each reaction as a non-specific control. All proteins were present in the initial binding reaction at a concentration of 4 μM. Positions of molecular weight markers (numbers in kDa) are given to the left of each gel.

Figure 6
Figure 6. LpoB activates PBP1b PGT activity and affects polymer length

A. PG synthesis in EP cells. EP cells from the indicated strains were incubated with or without LpoB (0.5-4 μM, as indicated). Reactions were initiated with the addition of UDP-M-pentapeptide (4 nmol) and UDP-[14C]G. After 60 min they were boiled in 4% SDS and filtered. Labeled PG retained on the filter was quantified by liquid scintillation counting. B-C. PBP PGT activity was measured by the incorporation of lipid-II into peptidoglycan in the presence of penicillin G. [14C]G-labeled lipid-II (4-8 μM) was incubated with or without LpoA or LpoB (50 nM) prior to the addition of PBP1a or PBP1b (50 nM), which initiated the reaction. At the indicated time points, reactions were quenched and analyzed for remaining substrate and PG product by paper chromatography. Results of single experiments are shown. They are representative of multiple trials (see Supplementary Information). D. Glycan chains generated in reactions similar to those in (B-C) were separated on an acrylamide gel (9%) and visualized using a phosphorimager. Lipid-II substrate was present at 4 μM in each reaction and protein amounts are indicated above the gel lanes.

Figure 7
Figure 7. Model for Lpo protein function

Shown are schematic diagrams of putative PBP-containing complexes in E. coli drawn as in Figure 1. The partial redundancy of PBP1a (lavender) and PBP1b (brown) suggest that they form part of independent PG synthesizing (sub)complexes that can substitute for one another. LpoA is an essential component of the PBP1a complex (left) that potentially stimulates the transpeptidase activity of this PBP (see text for details). LpoB is an essential component of the PBP1b complex and activates its PGT activity. Please also see model in Figure S7.

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