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Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease

2020; Springer Nature; Volume: 39; Issue: 10 Linguagem: Inglês

10.15252/embj.2019102935

1460-2075

Jakub Began, Baptiste Cordier, Jana Březinová, Jordan Delisle, R. Hexnerova, Pavel Srb, Petra Rampírová, Milan Kožíšek, Mathieu Baudet, Yohann Couté, Anne Galinier, Václav Veverka, Thierry Doan, Kvido Střı́šovský,

Endoplasmic Reticulum Stress and Disease

Article13 January 2020Open Access Source DataTransparent process Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease Jakub Began Jakub Began Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czech Republic Search for more papers by this author Baptiste Cordier Baptiste Cordier orcid.org/0000-0002-6042-9787 Laboratoire de Chimie Bactérienne (LCB), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7283, Aix Marseille Univ, Marseille Cedex 20, France Search for more papers by this author Jana Březinová Jana Březinová orcid.org/0000-0002-2076-9891 Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic Search for more papers by this author Jordan Delisle Jordan Delisle orcid.org/0000-0003-3275-132X Laboratoire de Chimie Bactérienne (LCB), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7283, Aix Marseille Univ, Marseille Cedex 20, France Search for more papers by this author Rozálie Hexnerová Rozálie Hexnerová Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Search for more papers by this author Pavel Srb Pavel Srb Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Search for more papers by this author Petra Rampírová Petra Rampírová Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Search for more papers by this author Milan Kožíšek Milan Kožíšek Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Search for more papers by this author Mathieu Baudet Mathieu Baudet CEA, Inserm, IRIG-BGE, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Yohann Couté Yohann Couté orcid.org/0000-0003-3896-6196 CEA, Inserm, IRIG-BGE, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Anne Galinier Anne Galinier orcid.org/0000-0001-5988-016X Laboratoire de Chimie Bactérienne (LCB), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7283, Aix Marseille Univ, Marseille Cedex 20, France Search for more papers by this author Václav Veverka Václav Veverka orcid.org/0000-0003-3782-5279 Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic Search for more papers by this author Thierry Doan Corresponding Author Thierry Doan [email protected] orcid.org/0000-0002-5909-4289 Laboratoire de Chimie Bactérienne (LCB), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7283, Aix Marseille Univ, Marseille Cedex 20, France Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7255, Aix Marseille Univ, Marseille Cedex 20, France Search for more papers by this author Kvido Strisovsky Corresponding Author Kvido Strisovsky [email protected] orcid.org/0000-0003-3677-0907 Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Search for more papers by this author Jakub Began Jakub Began Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czech Republic Search for more papers by this author Baptiste Cordier Baptiste Cordier orcid.org/0000-0002-6042-9787 Laboratoire de Chimie Bactérienne (LCB), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7283, Aix Marseille Univ, Marseille Cedex 20, France Search for more papers by this author Jana Březinová Jana Březinová orcid.org/0000-0002-2076-9891 Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic Search for more papers by this author Jordan Delisle Jordan Delisle orcid.org/0000-0003-3275-132X Laboratoire de Chimie Bactérienne (LCB), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7283, Aix Marseille Univ, Marseille Cedex 20, France Search for more papers by this author Rozálie Hexnerová Rozálie Hexnerová Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Search for more papers by this author Pavel Srb Pavel Srb Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Search for more papers by this author Petra Rampírová Petra Rampírová Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Search for more papers by this author Milan Kožíšek Milan Kožíšek Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Search for more papers by this author Mathieu Baudet Mathieu Baudet CEA, Inserm, IRIG-BGE, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Yohann Couté Yohann Couté orcid.org/0000-0003-3896-6196 CEA, Inserm, IRIG-BGE, Univ. Grenoble Alpes, Grenoble, France Search for more papers by this author Anne Galinier Anne Galinier orcid.org/0000-0001-5988-016X Laboratoire de Chimie Bactérienne (LCB), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7283, Aix Marseille Univ, Marseille Cedex 20, France Search for more papers by this author Václav Veverka Václav Veverka orcid.org/0000-0003-3782-5279 Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic Search for more papers by this author Thierry Doan Corresponding Author Thierry Doan [email protected] orcid.org/0000-0002-5909-4289 Laboratoire de Chimie Bactérienne (LCB), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7283, Aix Marseille Univ, Marseille Cedex 20, France Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7255, Aix Marseille Univ, Marseille Cedex 20, France Search for more papers by this author Kvido Strisovsky Corresponding Author Kvido Strisovsky [email protected] orcid.org/0000-0003-3677-0907 Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic Search for more papers by this author Author Information Jakub Began1,2,‡, Baptiste Cordier3,‡, Jana Březinová1,4, Jordan Delisle3, Rozálie Hexnerová1, Pavel Srb1, Petra Rampírová1, Milan Kožíšek1, Mathieu Baudet5, Yohann Couté5, Anne Galinier3, Václav Veverka1,6, Thierry Doan *,3,7 and Kvido Strisovsky *,1 1Institute of Organic Chemistry and Biochemistry, Czech Academy of Science, Prague, Czech Republic 2Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czech Republic 3Laboratoire de Chimie Bactérienne (LCB), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7283, Aix Marseille Univ, Marseille Cedex 20, France 4Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic 5CEA, Inserm, IRIG-BGE, Univ. Grenoble Alpes, Grenoble, France 6Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic 7Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM), Institut de Microbiologie de la Méditerranée (IMM), CNRS, UMR 7255, Aix Marseille Univ, Marseille Cedex 20, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 4 91 16 44 85; E-mail: [email protected] *Corresponding author. Tel: +420 2201 83468; E-mail: [email protected] The EMBO Journal (2020)39:e102935https://doi.org/10.15252/embj.2019102935 See also: G Liu et al (May 2020) and JD Knopf & MK Lemberg (May 2020) 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 Magnesium homeostasis is essential for life and depends on magnesium transporters, whose activity and ion selectivity need to be tightly controlled. Rhomboid intramembrane proteases pervade the prokaryotic kingdom, but their functions are largely elusive. Using proteomics, we find that Bacillus subtilis rhomboid protease YqgP interacts with the membrane-bound ATP-dependent processive metalloprotease FtsH and cleaves MgtE, the major high-affinity magnesium transporter in B. subtilis. MgtE cleavage by YqgP is potentiated in conditions of low magnesium and high manganese or zinc, thereby protecting B. subtilis from Mn2+/Zn2+ toxicity. The N-terminal cytosolic domain of YqgP binds Mn2+ and Zn2+ ions and facilitates MgtE cleavage. Independently of its intrinsic protease activity, YqgP acts as a substrate adaptor for FtsH, a function that is necessary for degradation of MgtE. YqgP thus unites protease and pseudoprotease function, hinting at the evolutionary origin of rhomboid pseudoproteases such as Derlins that are intimately involved in eukaryotic ER-associated degradation (ERAD). Conceptually, the YqgP-FtsH system we describe here is analogous to a primordial form of "ERAD" in bacteria and exemplifies an ancestral function of rhomboid-superfamily proteins. Synopsis Functions and substrates of prokaryotic members of the rhomboid intramembrane protease family remain poorly understood. Here, characterization of a bacterial rhomboid role in membrane transporter regulation exemplifies an ancestral pseudoprotease function analogous to rhomboid-family client adaptors in eukaryotic ERAD. Bacillus subtilis rhomboid protease YqgP cleaves the high-affinity magnesium transporter MgtE. MgtE cleavage by YqgP is enhanced in conditions of low environmental magnesium and high manganese or zinc. Manganese binding to the cytosolic extramembrane domain of YqgP mediates metal-dependent stimulation of MgtE cleavage. Metal-stimulated MgtE degradation protects B. subtilis from Mn2+/Zn2+ toxicity. YqgP acts independently as substrate adaptor of the AAA+ metalloprotease/dislocase FtsH to facilitate full degradation of MgtE. Introduction Rhomboids are intramembrane serine proteases widespread across the tree of life. In eukaryotes, rhomboid proteases regulate signalling via the epidermal growth factor receptor, mitochondrial quality control or invasion of malaria parasites into the host cells (reviewed in Urban, 2016). Rhomboid proteases also pervade the prokaryotic kingdom (Koonin et al, 2003; Kinch & Grishin, 2013), suggesting sufficient biological significance for evolutionary conservation and expansion. However, their functions in bacteria are much less well-understood than in eukaryotes. Notably, in eukaryotes, the rhomboid superfamily includes a number of pseudoproteases of important biological roles, such as iRhoms and Derlins, but the mechanistic and evolutionary aspects of rhomboid pseudoprotease functions have not been clarified (Ticha et al, 2018). In the Gram-negative eubacterium Providencia stuartii, the rhomboid protease AarA is required to process a pro-form of TatA, a component of the twin-arginine translocase secretion apparatus (Stevenson et al, 2007), but this function does not seem to be widely conserved because most bacteria encode a mature form of TatA. In the archaebacterium Haloferax volcanii, a rhomboid protease is involved in protein glycosylation of the S-layer (Parente et al, 2014; Costa et al, 2018), but the molecular mechanism has not been uncovered yet. Similarly, the main model rhomboid protease GlpG of Escherichia coli, widely distributed in Gram-negative bacteria, is extremely well-characterised structurally and mechanistically (Strisovsky et al, 2009; Baker & Urban, 2012; Zoll et al, 2014), yet its biological function is unknown. A recent report proposed a role of GlpG in the survival of a pathogenic strain of E. coli in the mouse gut (Russell et al, 2017), but the mechanism has not been demonstrated and it is not clear how specific the phenotype is, because rescue experiment has not been reported. Current knowledge of functions of rhomboid proteases in Gram-positives is similarly sketchy. An isolated report indicated that YqgP may play a role in cell division and may be required for glucose export (hence the therein coined alternative name GluP) (Mesak et al, 2004), but rescue experiment was not reported and the involved substrates were not investigated, leaving the molecular mechanisms of these functions unknown. Therefore, to shed light on one of the significantly populated subclasses of bacterial rhomboid proteases in a model Gram-positive organism, we focused on Bacillus subtilis YqgP and used quantitative proteomics to identify its substrates and interactors. We find that YqgP cleaves the high-affinity magnesium transporter MgtE and that YqgP interacts with the membrane-anchored metalloprotease FtsH. At low extracellular concentration of magnesium cations and high concentration of manganese or zinc cations, cleavage of MgtE by YqgP is potentiated, and the globular N-terminal cytosolic domain of YqgP represents the manganese/zinc-sensing unit. The second molecular role of YqgP is presenting MgtE or its cleavage products as substrates to FtsH, for which the proteolytic activity of YqgP is dispensable but its unoccupied active site is essential. YqgP thus fulfils both protease and pseudoprotease functions in tandem with FtsH, representing an ancestral proteolytic platform dedicated to the regulated degradation of polytopic membrane proteins, functionally equivalent to regulatory ERAD in eukaryotes. Our results shed light on the evolution of membrane proteostasis control in response to environmental stimuli, and on the arising of pseudoproteases, which are surprisingly common in the rhomboid superfamily (Adrain & Freeman, 2012; Freeman, 2014). Results Quantitative proteomics reveals candidate interactors and substrates of Bacillus subtilis rhomboid protease YqgP Bacillus subtilis genome encodes two rhomboid protease genes, ydcA and yqgP [also known as gluP (Mesak et al, 2004)]. No proteolytic activity has been detected for YdcA so far (Urban et al, 2002b; Lemberg et al, 2005), while YqgP is a commonly used model rhomboid protease cleaving a number of synthetic substrates (Lemberg et al, 2005; Urban & Wolfe, 2005; Ticha et al, 2017a,b). YqgP has homologs in a number of Gram-positive bacteria, including Bacilli, but also Staphylococci, Listeriae and others (http://www.ebi.ac.uk/interpro/protein/P54493/similar-proteins), and thus represents an attractive system to explore the cell biology and functions of bacterial rhomboid proteases. To reveal the repertoire of YqgP substrates, we generated a strain deficient in yqgP and re-expressed yqgP or its catalytically dead mutant S288A (Lemberg et al, 2005) in this background from an ectopic chromosomal locus (strains BS50 and BS51, respectively, see Table EV2). We then conducted quantitative proteomic analysis of membrane fractions of both of these strains employing SILAC labelling and SDS–PAGE pre-fractionation (GeLC experiment), where we were seeking peptides belonging to proteins migrating at a lower-than-expected apparent molecular weight selectively in the YqgP-expressing strain relative to the S288A mutant expressing one. This analysis yielded the high-affinity magnesium transporter MgtE as the top candidate substrate of YqgP (Fig 1A–C, Dataset EV1, PRIDE dataset PXD014578). Figure 1. Quantitative proteomics reveals candidate interactors and substrates of rhomboid protease YqgP from Bacillus subtilis Schematic representation of the SILAC-based quantitative proteomic experiment in B. subtilis. Cells overexpressing active YqgP (BS50, Table EV2) or its catalytically dead mutant YqgP.S288A (BS51), both auxotrophic for lysine, were grown in parallel in "heavy" (containing 13C615N2-lysine isotope) or "light" (containing stable 12C614N2-lysine) M9 minimal medium, respectively. After mixing the cell cultures in the 1:1 ratio (based on OD600), cell suspension was lysed and the fraction enriched for transmembrane proteome was analysed in a GeLC-MS/MS experiment. Using bioinformatic analysis of the MS data, highest-scoring candidate substrates were further evaluated. Diagrammatic representation of a result of GeLC-MS/MS analysis of excised gel regions described in (A). Each coloured block depicts a quantified peptide at its amino acid position according to the horizontal axis. Possible ongoing proteolysis was identified by high abundance ratio of YqgP/YqgP.S288A for a given protein and lower apparent molecular weight than expected for the corresponding full-length protein, as estimated from the position of the respective gel slice relative to the molecular weight marker. Table of best substrate candidates of two GeLC experiments. In order to assess possible cleavage sites in combination with topology information, the QARIP software (Ivankov et al, 2013) was used to summarise results from Experiments 1 and 2. Abundance ratios (YqgP/YqgP.S288A) for the intracellular parts of proteins were calculated for each gel slice separately by QARIP from peptide ratios computed by MaxQuant (Cox & Mann, 2008). Substrate candidates highlighted in red were identified in both experiments. Schematic representation of the affinity co-purification experiment in B. subtilis to identify YqgP interactors. Results of MS analyses of affinity co-purification experiments in wild-type B. subtilis control (strain BTM2, Table EV2) and B. subtilis deficient in endogenous YqgP expressing the wild-type YqgP-sfGFP bait (strain BTM84, Table EV2) or the proteolytically inactive YqgP.S288A-sfGFP bait (strain BBM1, Table EV2). Proteins were considered as potential interactors of YqgP if they were identified only in both positive co-purifications with a minimum of three weighted spectral counts or enriched at least five times in positive bait samples compared with control ones based on weighted spectral counts. The protein highlighted in red was the only overlapping hit between the two proteomic approaches. Download figure Download PowerPoint In a complementary approach, we used affinity co-immunopurification and label-free quantitative proteomics to identify proteins associating with YqgP (Fig 1D and E). We ectopically expressed either a functional YqgP-sfGFP fusion or catalytically dead YqgP.S288A-sfGFP fusion as the sole copy of YqgP in the cell. We solubilised the isolated membranes using the NP-40 detergent, isolated YqgP-sfGFP by anti-GFP affinity pull-down and analysed the co-isolated proteins by MS-based proteomics. Using this approach, we identified several high-confidence interactor candidates including the membrane-anchored protease FtsH, and ATPase subunits A, D, F and G (Fig 1E, Dataset EV2, PRIDE dataset PXD014566). The only high-confidence overlap between the two proteomic datasets was the high-affinity magnesium transporter MgtE (Fig 1C and E), promoting it to the highest likelihood candidate substrate. MgtE is the main magnesium transporter in B. subtilis, it is essential for magnesium homeostasis (Wakeman et al, 2014), and we thus examined the possible functional relationship between YqgP and MgtE. YqgP cleaves the high-affinity magnesium transporter MgtE between its first and second transmembrane helices To validate the results of quantitative proteomics and gain more insight into the role of YqgP in the physiology of B. subtilis, we first examined the proteolytic status of endogenous MgtE by immunoblotting using an in-house generated antibody recognising its N-terminal cytosolic domain (MgtE 1–275). Both endogenous and ectopically expressed YqgP cleaved endogenous MgtE, yielding distinct cleavage products (Fig 2A). The cleavage was abrogated by a rhomboid-specific peptidyl ketoamide inhibitor (Ticha et al, 2017b) at low nanomolar levels, confirming that it was a rhomboid-specific event (Fig 2B). Judging by the apparent molecular size of the N-terminal cleavage fragment compared with the in vitro-translated reference fragments (Lemberg & Martoglio, 2003) of MgtE, we concluded that the cleavage by YqgP occurred within the extracytoplasmic loop between TMH1 and TMH2 of MgtE. This region is close to or within the periplasmic end of TMH2 (Fig 2C and D), which is consistent with the topology and mechanism of a rhomboid protease (Strisovsky, 2013, 2016). MgtE transporters function as homodimers with a cytosolic amino-terminal cystathionine-beta-synthase (CBS) domain that senses intracellular Mg2+ and a carboxy-terminal five-transmembrane helical pore for Mg2+ import (Hattori et al, 2007; Takeda et al, 2014). Cleavage of MgtE between TMH1 and TMH2 by YqgP would thus likely inactivate the transporter. Figure 2. YqgP cleaves the high-affinity magnesium transporter MgtE between its first and second transmembrane helices Steady-state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living Bacillus subtilis (BTM2 and BTM501, respectively, Table EV2), in minimal medium at low magnesium concentration (10 μM). Strain lacking YqgP (ΔyqgP, BTM78, Table EV2) was used as a control. Proteins were detected by immunoblotting with chemiluminescence detection. The cleavage is efficiently inhibited by 1 μM STS736, a specific peptidyl ketoamide rhomboid inhibitor (Ticha et al, 2017b). To map the cleavage site region within endogenous MgtE (second lane from the left, from strain BTM501), MgtE-derived reference fragments encoding first 300, 315 and 330 amino acids, as well as full-length MgtE (black arrow), were in vitro-transcribed and in vitro-translated. Mobility of the N-terminal cleavage product (red arrow) of MgtE on SDS–PAGE was compared to the mobilities of the translated reference fragments. Diagrammatic display of the mapping shows that YqgP cleaves MgtE in a periplasmic region between transmembrane helices 1 and 2 (red arrow). Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2–275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies. Download figure Download PowerPoint Manganese excess activates cleavage of MgtE by YqgP under conditions of magnesium starvation It is known that MgtE homologs can also transport Mn2+ (Takeda et al, 2014) under low Mg2+ concentration (Mg starvation) and that high concentrations of Mn2+ are toxic for bacteria, mainly because Mn2+ can mis-metalate Mg2+-binding sites (Hohle & O'Brian, 2014; Chandrangsu et al, 2017). We therefore hypothesised that YqgP could be involved in the regulation of MgtE under Mn2+ stress, and we tested the influence of manganese on the in vivo activity of YqgP. While at high extracellular magnesium concentration (1 mM) the effect of 100 μM MnCl2 was not detectable, at low extracellular magnesium concentration (0.01 mM), when endogenous MgtE is upregulated, a shift from 1 to 100 μM MnCl2 activated the YqgP-dependent cleavage of MgtE fourfold (Fig 3A). Figure 3. Magnesium starvation and manganese excess activate cleavage of MgtE by YqgP, which is beneficial in manganese-stress conditions Detection and quantification of the cleavage of endogenous MgtE by YqgP in living Bacillus subtilis cells (BS72, Table EV2) depending on the concentrations of magnesium and manganese ions. Cells were cultivated in glucose M9 minimal medium with limiting (0.01 mM) or high (1 mM) concentration of MgSO4, in the presence or absence of 100 μM MnCl2, and analysed by Western blotting with near-infrared detection (upper panel). Black arrow denotes full-length MgtE, and red arrow denotes its N-terminal cleavage product formed by YqgP. The corresponding fluorescence signals were quantified by densitometry, and are displayed as relative specific activity, which is substrate conversion normalised to enzyme expression level (lower panel). Growth curves of wild-type (BTM843, Table EV2), yqgP-deficient (BTM844, Table EV2) and rescue (BTM845, Table EV2) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese stress elicited by adding 75 μM MnSO4 in mid-exponential phase (stress phase denoted by blueish background). All strains further contain a deletion in the putative manganese efflux pump MntP (ΔywlD, Table EV2). Bottom panel shows that manganese is more toxic in the yqgP-deficient strain than in the wild-type strain and that reintroduction of YqgP rescues fitness during manganese stress to above wild-type level. Top panel shows no difference between the strains in the absence of manganese stress. Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time point, which illustrates the reproducibility of the assay. Top panel: growth curves of wild-type (BTM843), yqgP-deficient (BTM844) and rescue (BTM845) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese shock elicited by adding 75 μM MnSO4 in mid-exponential phase. Bottom panel: manganese toxicity is prevented by further adding 5 mM magnesium (MgSO4) in otherwise identical conditions. Inhibition of YqgP by a rhomboid-specific peptidyl ketoamide inhibitor (STS736, i.e. compound 9 from Ticha et al, 2017b) abolishes the YqgP-induced fitness of B. subtilis under manganese stress, in a dose-dependent manner, both with endogenous YqgP (top panel) and overexpressed YqgP (bottom panel). Media and growth conditions were identical to those used in panel (B). Overexpression of heterologous MgtE inhibits growth of yqgP-deficient strain (BTM610, Table EV2) in rich LB medium supplemented with 75 μM MnSO4. Cell fitness is improved by overexpression of YqgP (BTM611, Table EV2) or its catalytically dead mutant YqgP.S288A (BTM612, Table EV2). For clarity, for panels (C–E), representative experiments of 2–3 independent biological replicates are shown. Download figure Download PowerPoint We next tested the importance of YqgP for growth in manganese-stress conditions. During growth in minimal medium at low magnesium concentration (10 μM), adding 75 μM manganese at mid-log phase caused growth arrest and lysis of wild-type B. subtilis, which was more severe upon deletion of yqgP, and fully rescued by ectopic expression of YqgP (Fig 3B). This effect was highly reproducible. Interestingly, all strains resumed growth at later time points, indicating the possible existence of an adaptive mechanism. This did not appear to involve the second B. subtilis rhomboid YdcA, because deletion of ydcA did not modify the phenotypic behaviour of B. subtilis regardless of the presence or absence of yqgP (Fig EV1). Regardless, the yqgP-deficient strains showed a marked growth disadvantage even in the latter phase of the growth curve, indicating that the role of YqgP for the fitness of B. subtilis in these conditions is significant. The toxic effect of 50 μM manganese sulphate was fully prevented by growth in high (5 mM) magnesium sulphate (Fig 3C), which was consistent with a similar effect on MgtE cleavage by YqgP (Fig 3A). In addition, increasing concentrations of YqgP inhibitor had a dose-dependent effect on the protective role of YqgP both when it was overexpressed or present at endogenous levels (Fig 3D), indicating that the pronounced protective effect of ectopically expressed YqgP was a result of its overexpression. Finally, the phenotype manifested also during steady-state growth in rich LB medium supplemented with 75 μM manganese sulphate, where yqgP-deficient B. subtilis ectopically expressing MgtE was delayed in growth compared with the same strain ectopically expressing YqgP (Fig 3E). Intriguingly, ectopic expression of a proteolytically inactive S288A mutant of YqgP also rescued the phenotype of the parent strain, which was initially surprising and suggested that YqgP may have a role independent of its protease activity. Together, these results reveal that a physiological function of YqgP is the protection from manganese stress by contributing to the degradation of the main magnesium transporter MgtE in B. subtilis (Wakeman et al, 2014). Click here to expand this figure. Figure EV1. Analysis of the role of ydcA in the phenotypic behaviour of Bacillus subtilis during Mn stressGrowth curves of wild-type (BTM843, Table EV2), yqgP-deficient (BTM844, Table EV2), ydcA-deficient (BTM1001, Table EV2), yqgP ydcA-deficient (BTM1003, Table EV2) and YqgP rescue (BTM845 and BTM1005, Table EV2) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese stress elicited by adding 75 μM MnSO4 in mid-exponential phase (stress phase denoted by blueish background, in bottom panel). All strains further contain a deletion in the putative manganese efflux pump MntP (ΔywlD, Table EV2). Bottom panel shows that manganese is more toxic in both yqgP-deficient strains (black squares or open black circles) than in the wild type or ydcA rhomboid-deficient strains. The overexpression of YqgP in both ΔyqgP and ΔyqgPΔydcA strains rescues fitness during manganese stress to above wild-type level. Top panel shows no difference between the strains in the absence of manganese stress.