Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi‐enzyme complexes in Escherichia coli
2020; Springer Nature; Volume: 39; Issue: 5 Linguagem: Inglês
10.15252/embj.2019102246
ISSN1460-2075
AutoresManuel Banzhaf, Hamish C. L. Yau, Jolanda Verheul, Adam Lodge, George Kritikos, André Mateus, Baptiste Cordier, Ann Kristin Hov, Frank Stein, Morgane Wartel, Manuel Pazos, Alexandra S. Solovyova, Eefjan Breukink, Sven van Teeffelen, Mikhail M. Savitski, Tanneke den Blaauwen, Athanasios Typas, Waldemar Vollmer,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle3 February 2020Open Access Outer membrane lipoprotein NlpI scaffolds peptidoglycan hydrolases within multi-enzyme complexes in Escherichia coli Manuel Banzhaf Manuel Banzhaf European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author Hamish CL Yau Hamish CL Yau Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Jolanda Verheul Jolanda Verheul Bacterial Cell Biology & Physiology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Adam Lodge Adam Lodge Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author George Kritikos George Kritikos European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author André Mateus André Mateus orcid.org/0000-0001-6870-0677 European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author Baptiste Cordier Baptiste Cordier orcid.org/0000-0002-6042-9787 Microbial Morphogenesis and Growth Lab, Institut Pasteur, Paris, France Search for more papers by this author Ann Kristin Hov Ann Kristin Hov European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author Frank Stein Frank Stein European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author Morgane Wartel Morgane Wartel European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author Manuel Pazos Manuel Pazos Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Alexandra S Solovyova Alexandra S Solovyova Newcastle University Protein and Proteome Analysis, Newcastle Upon Tyne, UK Search for more papers by this author Eefjan Breukink Eefjan Breukink Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Sven van Teeffelen Sven van Teeffelen Microbial Morphogenesis and Growth Lab, Institut Pasteur, Paris, France Search for more papers by this author Mikhail M Savitski Mikhail M Savitski orcid.org/0000-0003-2011-9247 European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany European Molecular Biology Laboratory, Structural & Computational Unit, Heidelberg, Germany Search for more papers by this author Tanneke den Blaauwen Corresponding Author Tanneke den Blaauwen [email protected] orcid.org/0000-0002-5403-5597 Bacterial Cell Biology & Physiology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Athanasios Typas Corresponding Author Athanasios Typas [email protected] orcid.org/0000-0002-0797-9018 European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany European Molecular Biology Laboratory, Structural & Computational Unit, Heidelberg, Germany Search for more papers by this author Waldemar Vollmer Corresponding Author Waldemar Vollmer [email protected] orcid.org/0000-0003-0408-8567 Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Manuel Banzhaf Manuel Banzhaf European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author Hamish CL Yau Hamish CL Yau Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Jolanda Verheul Jolanda Verheul Bacterial Cell Biology & Physiology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Adam Lodge Adam Lodge Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author George Kritikos George Kritikos European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author André Mateus André Mateus orcid.org/0000-0001-6870-0677 European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author Baptiste Cordier Baptiste Cordier orcid.org/0000-0002-6042-9787 Microbial Morphogenesis and Growth Lab, Institut Pasteur, Paris, France Search for more papers by this author Ann Kristin Hov Ann Kristin Hov European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author Frank Stein Frank Stein European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author Morgane Wartel Morgane Wartel European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany Search for more papers by this author Manuel Pazos Manuel Pazos Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Alexandra S Solovyova Alexandra S Solovyova Newcastle University Protein and Proteome Analysis, Newcastle Upon Tyne, UK Search for more papers by this author Eefjan Breukink Eefjan Breukink Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Sven van Teeffelen Sven van Teeffelen Microbial Morphogenesis and Growth Lab, Institut Pasteur, Paris, France Search for more papers by this author Mikhail M Savitski Mikhail M Savitski orcid.org/0000-0003-2011-9247 European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany European Molecular Biology Laboratory, Structural & Computational Unit, Heidelberg, Germany Search for more papers by this author Tanneke den Blaauwen Corresponding Author Tanneke den Blaauwen [email protected] orcid.org/0000-0002-5403-5597 Bacterial Cell Biology & Physiology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Athanasios Typas Corresponding Author Athanasios Typas [email protected] orcid.org/0000-0002-0797-9018 European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany European Molecular Biology Laboratory, Structural & Computational Unit, Heidelberg, Germany Search for more papers by this author Waldemar Vollmer Corresponding Author Waldemar Vollmer [email protected] orcid.org/0000-0003-0408-8567 Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK Search for more papers by this author Author Information Manuel Banzhaf1,8,‡, Hamish CL Yau2,9,‡, Jolanda Verheul3,‡, Adam Lodge2,10, George Kritikos1, André Mateus1, Baptiste Cordier4, Ann Kristin Hov1,11, Frank Stein1, Morgane Wartel1, Manuel Pazos2, Alexandra S Solovyova5, Eefjan Breukink6, Sven van Teeffelen4, Mikhail M Savitski1,7, Tanneke den Blaauwen *,3, Athanasios Typas *,1,7 and Waldemar Vollmer *,2 1European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany 2Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK 3Bacterial Cell Biology & Physiology, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, The Netherlands 4Microbial Morphogenesis and Growth Lab, Institut Pasteur, Paris, France 5Newcastle University Protein and Proteome Analysis, Newcastle Upon Tyne, UK 6Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands 7European Molecular Biology Laboratory, Structural & Computational Unit, Heidelberg, Germany 8Present address: Institute of Microbiology & Infection and School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK 9Present address: Faculty of Science, Agriculture and Engineering, Newcastle University, Newcastle Upon Tyne, UK 10Present address: Iksuda Therapeutics, The Biosphere, Newcastle Upon Tyne, UK 11Present address: École polytechnique fédérale de Lausanne SV IBI-SV UPDALPE, AAB 013, Lausanne, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +31 20 525 3852; Email: [email protected] *Corresponding author. Tel: +49 6221 3878156; Email: [email protected] *Corresponding author. Tel: +44 191 208 3216; Email: [email protected] The EMBO Journal (2020)39:e102246https://doi.org/10.15252/embj.2019102246 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus requires the combined activity of peptidoglycan synthases and hydrolases. In Escherichia coli, the activity of two PG synthases is driven by lipoproteins anchored in the outer membrane (OM). However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the OM lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and division biosynthetic complexes based on its localization and genetic interactions. Consistent with such a role, we reconstitute PG multi-enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that peptidoglycan regulators and adaptors are part of PG biosynthetic multi-enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases. Synopsis In bacteria, enzyme activities regulating peptidoglycan biosynthesis and degradation have to be adjusted during cell wall growth. Here, the outer membrane-anchored lipoprotein NlpI is shown to facilitate formation of peptidoglycan synthase and hydrolase multi-enzyme complexes to coordinate correct enlargement of the cell wall peptidoglycan layer in E. coli. NlpI binds to different classes of peptidoglycan hydrolases. NlpI can specifically form multimeric complexes with various endopeptidases. NlpI contributes to peptidoglycan biosynthetic complexes active in cell elongation and cell division based on its cellular localization and genetic interactions. NlpI forms multi-enzyme complexes containing peptidoglycan synthases and hydrolases in vitro. Introduction Peptidoglycan (PG) provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stresses. The PG layer or sacculus is a mesh-like structure composed of glycan chains connected by peptides and surrounds the cytoplasmic membrane (CM; Vollmer et al, 2008a; Silhavy et al, 2010). Given the internal turgor of the cells, PG layer growth requires the coordinated action of synthases and hydrolases to enlarge the sacculus without rupture. This important task is executed by large protein complexes, the elongasome and the divisome, which recruit PG enzymes together with regulators, cytoskeletal, morphogenesis and other structural proteins (Typas et al, 2012; Typas & Sourjik, 2015; den Blaauwen et al, 2017). It has been previously hypothesized that the formation of these complexes enables the cell to coordinate and regulate the activities of various synthetic and hydrolytic PG enzymes in a spatiotemporal manner (Höltje, 1993). Within these complexes, the key bifunctional penicillin-binding protein (PBP) PG synthases are activated by cognate outer membrane (OM)-anchored lipoproteins (Paradis-Bleau et al, 2010; Typas et al, 2010, 2012; Dorr et al, 2014; Egan et al, 2014, 2018; Greene et al, 2018; Moré et al, 2019) and coordinate their action with another, cell constriction-related protein complex (Gray et al, 2015). However, with the exception of the amidases (Uehara et al, 2010; Yang et al, 2012; Peters et al, 2013; Tsang et al, 2017), it is less clear how Gram-negative bacteria control the activities of their repertoire of hydrolases, i.e. the endopeptidases (EPases), carboxypeptidases (CPases) and lytic transglycosylases. NlpI is an OM-anchored lipoprotein predicted to be involved in cell division and responsible for targeting the PG EPase MepS for proteolytic degradation (Ohara et al, 1999; Singh et al, 2015). Deletion of nlpI causes cell filamentation at elevated temperature (42°C) or low osmolarity, whilst overexpressing NlpI results in the formation of prolate spheroids (Ohara et al, 1999). Deletion of nlpI has further implications on the stability of the OM as it increases membrane vesicle formation, in a manner that depends on the activity of two EPases; PBP4 in stationary phase and MepS in exponential phase. This vesicle formation phenotype is suppressed by a deletion of mepS (Schwechheimer et al, 2015). Many of its pleiotropic effects may be due to the ability of NlpI to target the EPase MepS for proteolytic degradation by forming a complex with the tail-specific protease Prc (Su et al, 2017). NlpI and MepS both interact with Prc, but whilst MepS is digested, only 12 C-terminal amino acids of NlpI are removed (Singh et al, 2015). In the absence of NlpI, the half-life of MepS increases from ~2 min to ~45 min. Further, in the ΔnlpI mutant, uncontrolled levels of MepS have been shown to impair cell growth on low osmolarity medium and lead to the formation of long filaments (Singh et al, 2012, 2015). NlpI forms a homodimer (Wilson et al, 2005) with the 33 kDa monomers having their OM-binding N-termini in close proximity. Each monomer consists of 14 α-helices forming 4 canonical but distinct tetratricopeptide helix-turn-helix repeats (TPR) and 2 non-TPR helix motifs. TPR are found in many protein-interacting modules (Zeytuni & Zarivach, 2012). A putative binding cleft is formed from the curvature of the helices on each monomer, which would be available for protein–protein interactions (Das et al, 1998; Wilson et al, 2005). It is hence possible that NlpI acts as a scaffold for the formation of protein complexes. In this study, we provide evidence that in addition to targeting MepS for degradation, NlpI scaffolds hydrolases within PG multi-enzyme complexes in E. coli. Results Deletion of NlpI alters abundance and thermostability of envelope biogenesis proteins Deletion of nlpI causes several pleiotropic phenotypes and morphological changes. To link the observed phenotypes to changes in protein abundance and activity, we compared an nlpI knockout strain (∆nlpI) to wild-type E. coli using two-dimensional thermal proteome profiling (2D-TPP; Savitski et al, 2014; Mateus et al, 2018). In TPP, both protein abundance and thermostability can be measured. The latter depends on the intrinsic physical properties of the protein and on external factors that stabilize its fold, such as protein–protein and protein–ligand interactions. Numerous proteins changed abundance and thermostability in the ΔnlpI cells (Tables EV1 and EV2). In agreement with its periplasmic location and links to envelope integrity (Schwechheimer et al, 2015), deletion of nlpI resulted in changes in abundance and thermostability of major envelope components, including outer membrane proteins (OMPs), the β-barrel assembly machinery (BAM; Noinaj et al, 2017) and the Tol-Pal complex (Egan, 2018; Fig 1A and B). As expected, both MepS abundance and thermostability were dramatically elevated in ΔnlpI cells, since in the absence of NlpI, MepS is not targeted for degradation by Prc (Singh et al, 2015; Fig 1A and B). We also observed that other PG biogenesis proteins showed mild increases in abundance and these included several PG hydrolases (PBP5, PBP6a, MltA, MltG), LdtB, LdtF and PG synthases (PBP1A, PBP1B; Fig 1A). A number of these also decreased in thermostability, with lytic transglycosylases (MltA, MltC, MltE), the LD-transpeptidase LdtF and the PG synthases and their regulators (PBP1B, LpoA, LpoB) showing the strongest effects (Fig 1B). In contrast, all amidases (AmiA, AmiB and AmiC) decreased in abundance (Fig 1A). Moreover, the amidase regulator NlpD (which binds to AmiC and controls its activity; Uehara et al, 2010) and the YraP protein, which was recently implicated in the activation of NlpD, were strongly destabilized (Fig 1B; Tsang et al, 2017). Figure 1. In vivo and in vitro proteomics-based assays link NlpI to several classes of PG hydrolases A, B. Wild-type and ΔnlpI cells were heated at a range of temperatures, and the soluble components were labelled by TMT, combined and quantified by LC-MS, using the published 2D-TPP protocol (Mateus et al, 2018). Shown are volcano plots of two replicates depicting changes in protein abundance (A) and thermostability (B). A local FDR (false discovery rate) < 0.01 was set as a threshold for significance. Highlighted proteins: outer membrane proteins (OMPs, light green), β-barrel assembly machinery (BAMs, red), PG synthases/regulators (green), PG hydrolases and regulators (blue) and the Tol-Pal complex (violet). All other proteins were coloured grey and not labelled to increase the plot clarity. Full results can be found in Tables EV1 and EV2. C, D. Affinity chromatography with immobilized NlpI. Membrane extracts from E. coli were incubated in low and high salt binding conditions (50 and 400 mM NaCl, respectively), and then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label-free LC-MS analysis. The plot shows the log2 fold enriched proteins when compared to those eluted from a parallel empty column control, versus the log10 P-value, in low (4 replicates) (C) and high (2 replicates) (D) salt. Highlighted points are all interactions with PG enzymes and their regulators, as well as members of the divisome. All other proteins were coloured grey and not labelled to increase the plot clarity; many were non-physiological interactions with abundant cytoplasmic proteins. Full results can be found in Tables EV3 and EV4. GO enrichments can be found in Tables EV9 and EV10. Download figure Download PowerPoint To ensure that pleiotropic changes are not due to polar gene expression caused by inactivation of NlpI, we complemented the ΔnlpI mutant by expressing endogenous NlpI from an arabinose inducible, medium copy number plasmid (pBAD30). The complemented strain restored cell length and partially cell width to wild-type values (Appendix Figs S5F, and S12A and B). The lack of full complementation of cell widths could be due to our inability to precisely restore the level and regulation of NlpI and, consequently, the level of MepS (Ohara et al, 1999), Overall, our results indicate that almost all effects in the ΔnlpI mutant are due to cells lacking NlpI. To test whether the observed changes are due to higher abundance of MepS in the ΔnlpI mutant, we repeated the 2D-TPP with an ΔnlpIΔmepS mutant (Appendix Fig S1A and B). Several of the changes observed in the ΔnlpI cells remained in the ΔnlpIΔmepS background (Appendix Fig S1A and B), including the destabilization of many cell wall enzymes and regulators. We also directly compared the 2D-TPP profiles of ΔnlpI and ΔnlpIΔmepS mutants (Appendix Fig S1C and D), with the major difference between both proteomes being that some OMPs were more stable in ΔnlpI cells. Importantly, the stability changes occurring for PG enzymes were not observed in this comparison, indicating that they occur independently of MepS levels. Altogether, these results provide the first evidence that NlpI affects PG biogenesis beyond the known interaction with the EPase MepS. NlpI pulls down several classes of PG hydrolases and multiple divisome proteins The decrease in thermostability of several PG biogenesis proteins in ΔnlpI cells raised the possibility that NlpI may interact with these proteins. To investigate this further, we applied detergent-solubilized E. coli membrane proteins to immobilized NlpI to identify potential interaction partners. Affinity chromatography was performed both in low salt binding conditions (50 mM) to pull down larger PG multi-enzyme complexes, and in high salt binding conditions (400 mM) to identify stronger, salt-resistant and possibly direct binding partners. As a control, we used a column containing Tris-coupled sepharose beads and compared elution fractions with label-free mass spectrometry (Tables EV3 and EV4). To investigate relevant NlpI interaction partners, we first performed gene ontology (GO) enrichment analysis and confirmed that proteins pulled down are enriched in several relevant GO terms, such as "cell wall organization" and "peptidoglycan metabolic processes" (Tables EV9 and EV10). Next, we focused on proteins located in the periplasmic space and highlighted known PG biogenesis proteins (Fig 1C and D). For both affinity chromatography experiments, we were unable to detect the known NlpI binding partner MepS in the applied extract, likely due to its low cellular levels in wild-type cells (Fig 3D). In low salt binding conditions, NlpI retained several envelope biogenesis proteins, such as the PG synthases PBP1A, PBP1B, PBP1C, the divisome proteins EnvC, PBP3, FtsK, FtsQ and FtsX, the lytic transglycosylases MltA and MltC, the amidase AmiC and the EPases PBP4 and PBP7, amongst others (Fig 1C). This shows that NlpI is able to pull-down full or partial PG-synthase complexes. When challenged in high salt binding conditions, many of the aforementioned interactions were lost. However, immobilized NlpI still retained the divisome proteins PBP3, FtsK, FtsQ and FtsX, the amidase AmiA and its regulator EnvC, and the lytic transglycosylases MltA at 400 mM NaCl, suggesting strong, salt-resistant interactions (Fig 1D). The in vivo proteomics of ΔnlpI and the subsequent affinity chromatography revealed strong links of NlpI to several classes of PG hydrolases, PG synthases and divisome proteins. To investigate whether NlpI has a broader role in regulating EPases beyond MepS (Singh et al, 2015), we next focused on characterizing the interactions of NlpI with EPases and PG synthases in more detail. NlpI dimerizes and interacts with several EPases To confirm the observed interactions between NlpI and EPases, we performed various biochemical assays. A soluble version of NlpI lacking its membrane anchor was used for all these assays. Firstly, we determined that NlpI is predominantly a homodimer using analytical ultracentrifugation (AUC). The experimentally determined sedimentation coefficient was 4.16 S, which is close to the calculated sedimentation coefficient of 4.52 S, based on the crystal structure of the NlpI dimer (1XNF.pdb; Wilson et al, 2005) (Appendix Fig S2A). We measured the apparent dissociation constant (KD) for the NlpI dimer as 126 ± 9 nM by microscale thermophoresis (MST): titrating a fluorescently labelled NlpI (fl-NlpI) against a serial dilution of unlabelled NlpI (Fig 2A and Appendix Fig S2B). Binding of the unlabelled NlpI to fl-NlpI resulted in changes to the thermophoretic mobility of fl-NlpI, which is expressed as a change in fluorescence and plotted against ligand concentration to derive the binding affinity. The formation of a dimer by NlpI in solution is consistent with previous work (Su et al, 2017). We next tested the specificity of a previously reported interaction between NlpI and the EPase MepS, using MST (Singh et al, 2015). We found that NlpI and MepS interacted directly, with an apparent KD of 145 ± 52 nM (Fig 2A and B). NlpI also interacted with MepM and PBP4 with similar apparent KD's of 152 ± 42 nM and 177 ± 49 nM, respectively (Fig 2A and Appendix Fig S2B). Assaying for an interaction between NlpI and PBP7 by MST revealed a more complex binding curve, which could only be fit assuming a Hill coefficient of ~ 3 (Appendix Fig S2B). This resulted in an apparent EC50 value of 422 ± 25 nM and suggested an element of positive cooperativity in the NlpI-PBP7 binding. Figure 2. NlpI interacts with several EPases and is able to form trimeric complexes with them A. Dissociation constants for interactions between NlpI with MepM, MepS, PBP4, PBP7 as determined by microscale thermophoresis (MST). The values are mean ± SD of three independent experiments. The corresponding MST binding curves are shown in Appendix Fig S2B. B. MepS-NlpI interaction by MST as an example plot for Fig 2A. The same plot is also shown in Appendix Fig S2B. MST curve plotted is the mean data ± SD of three independent experiments. Fl, fluorescently labelled; FNorm, normalized fluorescence. C, D. NlpI has different binding sites for MepS and PBP4, and MepS and PBP7 as shown by the ability of labelled MepS to bind pre-formed NlpI-PBP4 (C) and NlpI-PBP7 (D) complexes by a fixed concentration MST assay. Values are mean ± SD of 3–6 independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in P-values corrected for multiple testing. Relevant P-values are highlighted directly in the figure (*< 0.05; **< 0.01, ***< 0.001), and all P-values can be found in Table EV7. Download figure Download PowerPoint We also tested the interactions between NlpI and EPases (MepM, MepS, PBP4 and PBP7) by Ni2+-NTA pull-down assays and confirmed the interactions found by MST (Appendix Fig S3A). We could not detect an interaction between NlpI and the carboxypeptidase PBP5 or the lytic transglycosylase Slt, suggesting that NlpI does not interact with all hydrolases in general (Appendix Fig S3A). Using a combination of MST and Ni2+-NTA pull-down assays, we also tested for interactions between the EPases. Of the four EPases, which we studied and all possible combinations tested, the only interactions we found were between MepS-MepM and PBP4-PBP7 (Appendix Figs S2C and S3B). NlpI scaffolds trimeric complexes between different EPases Since NlpI bound multiple EPases, we tested whether NlpI could also form trimeric complexes with them. As a starting point, we tested whether NlpI could scaffold MepS and PBP4 in a fixed concentration MST assay. In the presence of 3 μM NlpI, the normalized fluorescence (FNorm) of fl-MepS increased, confirming the interaction between NlpI and MepS (Fig 2C). In contrast, fl-MepS did not interact with PBP4, even when that was used in excess (30 μM; Fig 2C). Interestingly, fl-MepS was able to bind to a saturated NlpI-PBP4 complex indicating the formation of a trimeric complex between NlpI, PBP4 and MepS (Fig 2C). NlpI pre-incubated with excess BSA did not give the same increase in fl-MepS signal, indicating that the FNorm increase was specific to the binding of NlpI-PBP4 (Fig 2C). We also tested whether NlpI was able to scaffold MepS and PBP7. Fl-MepS could bind pre-incubated NlpI-PBP7 complexes indicating that NlpI can scaffold both EPases and likely has different binding sites for MepS and PBP7 (Fig 2D). Using a three-component Ni2+-NTA pull-down assay, we were also able to resolve an NlpI-mediated complex containing PBP7 and MepS (Appendix Fig S3C). The trimeric complexes were not due to direct interactions between the EPases (Appendix Fig S2C and S3B), but rather due to NlpI scaffolding both EPases simultaneously. Thus, NlpI can scaffold at least two different trimeric EPase complexes, with MepS-PBP4 and MepS-PBP7. NlpI affects EPase activity of MepM and MepS in vitro Although NlpI interacted with and complexed several EPases, the cellular role of such complexes remained unclear. Hence, we investigated whether NlpI increased or decreased the activity of these EPases using in vitro PG digestion assays with purified sacculi or pre-digested muropeptides. EPases cleave the peptide bond between neighbouring peptides, resulting in a decrease in TetraTetra (bis-disaccharide tetrapeptide) muropeptides. Therefore, we quantified the remaining cross-linked PG substrate following incubation with the respective EPase and used the decrease in TetraTetra as an indication of EPase activity (Fig 3A and Appendix Fig S4A). Our results show that NlpI reduced the activity of MepM, which was more active by itself against sacculi. In contrast, MepS was inactive against sacculi and pre-digested muropeptides, but the addition of NlpI slightly activated MepS against muropeptides (Fig 3A; see also methods). We did not observe significant differences in the activity of PBP4 or PBP7 in the presence of NlpI (Fig 3A). These results suggest that NlpI is able to modulate the activity in vitro of certain (e.g. MepM and slightly MepS), but not all, EPases. Figure 3. NlpI genetically interacts with MepS and affects the enzyme activity of MepS and MepM HPLC-based PG digestion assay representing EPase activity. The graph shows the relative percentage of the muropeptide TetraTetra present at the end of the incubation period for each protein as described in Materials and Methods. MepM and PBP4 were incubated with sacculi, whilst MepS and PBP7 were incubated with soluble muropeptides, both from E. coli MC1061, respectively. Values are mean ± SD of three independent experiments
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