Autoproteolysis and Intramolecular Dissociation of Yersinia YscU Precedes Secretion of Its C-Terminal Polypeptide YscU_CC

Abstract

Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscU_CC. Here we show that depletion of calcium induces intramolecular dissociation of YscU_CC from YscU followed by secretion of the YscU_CC polypeptide. Thus, YscU_CC behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscU_CC in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscU_CC dissociation for Yop secretion. We propose that YscU_CC orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.

Introduction

In 1952, Hills and Spurr showed that virulent strains of Yersinia pestis (Pasturella pestis) were unable to grow and divide when incubated at 37°C; instead, they required incubation at 27°C [1]. This phenotype was surprising, because Y. pestis causes lethal infections in rodents and humans, which have a body temperature close to 37°C. Moreover, no typical nutritional requirements could explain this phenotype. Later, Kupferberg and Smith demonstrated that addition of 2.5 mM calcium to the growth medium supported growth of Y. pestis at 37°C [2]. This unusual requirement for calcium was later shown to be correlated to the massive synthesis and secretion of a number of proteins, called Yersinia outer proteins (Yops). This was based on the observation that 2.5 mM calcium in the growth medium blocked Yop secretion, while depletion of calcium induced massive Yop secretion that also results in stop of bacteria proliferation [3], [4], [5]. Synthesis and secretion of Yops are dependent on a virulence plasmid [6], a common feature of all human pathogenic Yersinia (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis). Yops are synthesized during infection, which indicates their importance in virulence [3]. Yop secretion involves the type III secretion system (T3SS) of Yersinia, which is encoded by the same virulence plasmid that carries the yop genes. The T3SS is a dedicated secretion system that forms a multi-protein complex of around 25 proteins spanning the inner and outer bacterial membranes [7]. It is built up by a basal body located in the membrane showing high homology with a corresponding structure of the bacterial flagellum. A needle is anchored to the basal body forming hollow tube measuring around 60 to 80 nm in length and 8 nm in external width with an inner diameter of 3 nm [8], [9]. It has been postulated that Yops are transferred to the target cell through the needle structure [8]. This model has however been challenged in recent work from our laboratory [10] where we show that bacterial surface localized Yop-effectors can be translocated into the target cell. Hence, translocation can occur via a mechanism that is distinct from the postulated micro-injection model. Yersinia employs the T3SS to secrete Yops into the external environment and to translocate Yops into the cytoplasm of eukaryotic target cells [11]. These processes are highly regulated. It has been shown that Y. pseudotuberculosis up-regulates yop expression after contact with eukaryotic cells, and this requires a functional T3SS [12], [13]. Importantly, target cell contact can be mimicked by depleting calcium in the growth medium and simultaneously shifting the temperature from 26°C to 37°C [11]. Modulation of calcium levels in the growth medium has been an invaluable tool for increasing our understanding of T3SSs in Yersinia virulence. Several seminal and general discoveries have been made based on the calcium effect, including T3SS mediated secretion, translocation, and target cell induced expression of effector proteins [12], [13], [14].

The YscU protein of Yersinia is an integral inner-membrane protein with four membrane spanning segments (Figure 1A) and is required for T3SS function. It belongs to a family of proteins (YscU/FlhB class) that is characterized by auto-cleavage at a highly conserved N↑PTH motif (amino acids 263–266) [15]. Autoproteolysis of YscU is required for proper regulation of Yops synthesis and secretion. Furthermore, the Yop synthesis and secretion is lost when the full yscU gene or the N↑PTH coding sequence are deleted, indicating the importance of YscU for T3SS function. Similar phenotypes are observed when point mutations affect cleavage at the N↑PTH motif; this illustrates the importance of cleavage for calcium regulation [16], [17], [18], [19]. Full length YscU (denoted YscU) contains two domains, the transmembrane domain (TM) and a soluble cytoplasmic domain, denoted YscU_C (Figure 1A and Figure 1B). Autoproteolysis of YscU occurs between asparagine 263 and proline 264 at the N↑PTH motif and results in a 10 kDa C-terminal polypeptide fragment, denoted YscU_CC that is attached to the remainder of the protein through protein-protein interactions. In context of the cytoplasmic domain, YscU_C (which is used extensively in this article), cleavage generates two fragments; the YscU_CC fragment and a 6 kDa N-terminal fragment denoted YscU_CN [16], [18] (Figure 1A).

Figure 1. Domain structure of YscU and crystallographic structure of YscU_C.

(A) Schematic domain structure of the integral membrane protein, YscU of Y. pseudotuberculosis. The full-length protein contains 354 amino acid residues. The N-terminal 210 residues constitute four transmembrane helices (TM). The cytosolic domain of YscU (YscU_C) undergoes autoproteolytic cleavage at the N↑PTH-motif (amino acids 263–266), which leaves an N-terminal cytoplasmic polypeptide, denoted YscU_CN, and a C-terminal polypeptide, denoted YscU_CC. (B) Ribbon drawing of the cleaved cytosolic domain YscU_C (2JLI.PDB) from Y. pestis [39]. YscU_CN and YscU_CC resulting from cleavage at the N↑PTH motif are colored in orange and grey, respectively.

Both YscP (FliK) and YscU (FlhB) have been linked to the “substrate specificity switch”, first identified by MacNab and coworkers in the flagellum T3SS [15], [20]. This switching machinery changes secretion specificity from early hook substrates to late filament substrates as one step in the assembly of the flagellum [21]. It has been suggested that the C-terminal domain of FliK (FliK_C) binds to the C-terminal cytosolic domain of FlhB (FlhB_C), causing a conformational change in FlhB_C that is required for the switch [20]. An yscP mutant was impaired in switching from the early secretion of needle subunits (YscF) to the late export of Yops [16], [22]. This led to a phenotype with unusually long needles unable to secrete Yop proteins, thus YscP is an essential protein for T3SS mediated secretion [16], [22], [23]. A similar phenotype was observed for the yscU mutant, N263A, which highlighted the importance of YscU autoproteolysis in the substrate specificity switch [16]. Interestingly, an yscP null mutant was suppressed by single amino acid substitutions in YscU_C, and these suppressor mutants partially restored Yop secretion [17]. This suggested that YscU and YscP interact, and that this interaction was essential for proper control of needle formation and Yop secretion [16]. A direct interaction between the YscU and YscP orthologs, Flik and FlhB has been shown with surface plasmon resonance experiments [24]. In analogy, mutations in the corresponding yscP gene in Shigella flexneri (spa32) and Salmonella thypimurium (invJ) [25] also caused defective substrate switching. It has been shown that Spa32 (YscP) and Spa40 (YscU) interact [26], [27]. Given the high functional similarity between the T3SSs of different species, it is likely that the substrate specificity switch is regulated by a similar mechanism in different pathogens.

Here, we studied the functional role of YscU in Yop secretion by exploiting the calcium regulation of substrate switching in Y. pseudotuberculosis. We combined in vivo and in vitro methods to examine the steps of YscU autoproteolysis, subsequent dissociation, and secretion of YscU_CC and how they affect Yop secretion in Y. pseudotuberculosis during growth in calcium depleted media.

Materials and Methods

Bacterial Strains, Plasmids, and Growth Conditions

Bacterial strains and plasmids used in this study are listed in the supporting material (“Table S1”). Escherichia coli strains were grown in Luria-Bertani broth (LB) or on Luria agar plates at 37°C. Y. pseudotuberculosis was grown at either 26°C or 37°C in Hepes buffered LB or on Luria agar plates (unless specified in the text). Antibiotics were used for selection according to the resistance markers carried by the strains at the following concentrations: kanamycin, 50 µg/ml; chloramphenicol, 25 µg/ml; and carbenicillin, 100 µg/ml. EGTA was added to the media at a final concentration of 5 mM and 20 mM MgCl₂ to create calcium depleted conditions.

Yop Secretion Assay

Cultures were started at an absorbance of OD₆₀₀ = 0.1 in Hepes buffered LB with the appropriate antibiotics. Bacteria were grown at 26°C for 2 h and shifted to 37°C for 3 h in calcium-supplemented or calcium-depleted conditions (except where specified in the text). Cultures were harvested and centrifuged for 10 min at 4 000×g. Aliquots (4.5 ml) of filtrated supernatant were combined with 10% (v/v) trichloroacetic acid (TCA) for protein precipitation. Precipitated proteins were solubilized in SDS-PAGE loading buffer. The pelleted cells were resuspended in an equal volume of LB and lysed with SDS-PAGE loading buffer. Cells and supernatants were loaded at equivalent protein concentrations (according to OD₆₀₀) and separated by SDS-PAGE. Proteins were either stained with Coomassie R250 or, alternatively, transferred onto a PVDF membrane (GE Healthcare) for immunoblotting. Anti-Yop antibodies were diluted at 1∶5 000 and horseradish peroxidase-conjugated anti-rabbit IgG was diluted at 1∶10 000 (GE Healthcare). Proteins were detected with a chemiluminescence detection kit (GE Healthcare).

YscU_CC Overexpression and Secretion Assay

We grew YPIII/pIB102 bacterial strains, which contained the pBADmycHis B plasmid (Invitrogen) with the yscU_CC expression sequence (see supporting “Material and methods S1”), and control strains contained an empty vector. The growth conditions were as described above, except 0.2% (v/v) of L-arabinose was added after 1 h at 26°C to induce biosynthesis of YscU_CC. After separation by SDS-PAGE, proteins were stained with Coomassie R250 (Yop secretion) or transferred onto a PVDF membrane (GE Healthcare) for immunoblotting. Anti-YscU_CC peptide antibodies were diluted at 1∶5 000 [16] and horseradish peroxidase-conjugated anti-rabbit IgG was diluted at 1∶10 000 (GE Healthcare). Proteins were detected with a chemiluminescence detection kit (GE Healthcare).

GST-pulldown Assay

We purified GST-YscU_C-His₆ and GST-A268F-His₆ proteins in 2 steps, by combining GST- and Ni-NTA affinity chromatography. Purified proteins (80 µM) were incubated in 25 mM Tris, pH 7.4, 1 mM EDTA, and 150 mM NaCl at 37°C and 30°C. At different time points, 250 µl samples were taken, centrifuged to remove aggregates (15 min, 16 000×g at 4°C), and loaded on GST-SpinTrap™ columns (GE Healthcare). Columns were washed twice with 25 mM Tris, pH 7.4, 150 mM NaCl buffer, and eluted twice by adding 20 mM GSH solution, pH 8.0. Eluted samples were mixed with SDS-sample buffer and boiled. Proteins were subsequently separated and visualized with 4–12% Bis-Tris Gel SDS-PAGE (Invitrogen).

Protein Purification of YscU_C Variants

All YscU_C constructs were cloned as GST fusion with a cleavage site for PreScission Protease between the GST domain and YscU_C (see supporting “Material and methods S2”). After transformation into E. coli BL21 (DE3) pLysS the protein synthesis was induced with IPTG and performed overnight at 30°C in LB medium containing carbenicillin and chloramphenicol. The bacterial cells were harvested by centrifugation at 5 000 rpm at 4°C and stored at −80°C until use. The protein purification of YscU_C variants was performed with an ÄKTA purifier system (GE Healthcare). The bacterial pellet was resuspended in 50 mM Tris pH 7.4 and 2 mM DTT, and cells were disrupted by sonication. The lysate was clarified by centrifugation at 15 000 rpm and 4°C, and the supernatant passed through a 0.45 µm syringe filter (Corning). The lysate, containing the soluble GST fusion protein, was loaded on a 5 mL GSTrap FF column (GE Healthcare) and eluted with 20 mM GSH solution at pH 8.0. Fractions with the fusion protein were pooled, dialyzed at 4°C against cleavage buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT). The GST protein was cleaved from the target protein by adding PreScission Protease (GE Healthcare). To remove GST and non-cleaved GST-fusion protein the solution was passed through a Glutathione Sepharose 4B column. The flowthrough with YscU_C, was subjected to cation exchange chromatography (5 ml SP Sepharose, GE Healthcare). All eluted fractions with YscU_C were pooled, concentrated with Amicon Ultra-15 Centrifugal Filter Units (Millipore, Billerica, MA), and polished with size-exclusion chromatography (HiPrep 26/60 Sephacryl S-100HR, GE Healthcare) in phosphate buffered saline at pH 7.4. Fractions with YscU_C were pooled stored as 100 µM stock at 20°C until use.

Analytical Size Exclusion Chromatography

Protein samples (YscU_CC) were applied to a Superose 6 10/300 GL column (GE Healthcare) and size-exclusion chromatography (SEC) was performed with a flow rate of 0.5 ml/min in phosphate buffered saline at pH 7.4. Protein elution was followed by monitoring the UV absorption at 260 nm, 280 nm, and 220 nm. All samples with signal peaks at 280 nm were analyzed by SDS-PAGE. Prior to analytical SEC, the column was calibrated with a gel filtration protein standard (Bio-Rad).

NMR Spectroscopy

NMR experiments were performed on a Bruker DRX 600 MHz spectrometer equipped with a 5-mm triple resonance z-gradient cryoprobe. Temperature calibration was conducted with a home-made probe, inserted into the sample compartment of the cryoprobe. The NMR samples contained unlabeled, ¹⁵N-labeled, or ¹⁵N/¹³C enriched protein in a buffer consisting of 10% ²H₂0 (v/v), 50 mM NaCl, and 30 mM phosphate buffer at pH 7.4. Backbone YscU_C resonance assignments were accomplished with triple resonance experiments, HNCA [28], HNCOCA, HNCACB [29], and CBCACONH [28], supplemented with a ¹⁵N NOESY-HSQC experiment. Chemical shift perturbations were calculated according to: Δω = 0.2 · |Δ¹⁵N|+|Δ¹H| (ppm).

The time series of one-dimensional ¹H NMR spectra to probe dissociation were acquired with a pulse program from the Bruker library, which incorporated excitation sculpting for water suppression. For each spectrum, 64 scans were accumulated with a relaxation recovery delay of 2 s between scans. For each protein, time series were acquired at 30°C and 37°C. To quantify dissociation kinetics, we integrated the methyl group resonances in the 0.2 to 0.4 ppm spectral region. Each time course of the NMR signal was fit with a single exponential decay function of the form: I = I₀ exp^{(−t/τdiss)}+A, where τ_diss was the lifetime of the decay, and A was a baseline offset. NMR data was processed with NMRPipe [30] and visualized in ANSIG for Windows [31].

Circular Dichroism

Circular dichroism (CD) spectra were recorded on a Jasco J-810 spectropolarimeter, equipped with a Peltier element for temperature control and a 0.1 cm quartz cuvette. The proteins were measured at 10 µM in a buffer of 10-fold diluted phosphate buffered saline at pH 7.4. For all experiments with calcium, different buffers were used (phosphate, MOPS, and Pipes) to exclude possible calcium precipitation effects. Thermal dissociation experiments were performed by monitoring the CD signal at 220 nm as a function of temperature. All thermal profiles were acquired in the interval of 20°C to 90°C. The thermal scan rate was varied from 0.5 to 2°C/min, without any significant change in protein behavior; we selected 1°C/min as the standard condition in this study for CD spectroscopy-based temperature perturbation experiments. The inflection point for dissociation, T _diss, was quantified by fitting thermal curves with a two-state equation [32]. Calcium binding affinity was quantified by fitting a one-site binding model to the CD data.

Results

Dissociation of YscU_CC from YscU_CN

We recently published results showing that specific yscU mutants (N263A and P264A), defective in autoproteolysis, were impaired in their ability to secrete Yops into the culture supernatant at wild-type levels [16]. These mutations strongly reduced the autoproteolytic activity of the YscU protein. Especially the yscU mutant P264A was severely suppressed in autoproteolysis leading to an almost complete inhibition of Yop secretion [16]. These results and earlier findings showing that deletion of the autoproteolytic cleavage motif NPTH leads to a complete loss of Yop secretion indicated that the cleavage of YscU is required for Yop secretion [18]. Here, we decided to study YscU in more detail. Because YscU is an integral inner-membrane protein, we produced a polypeptide that comprised the cytosolic segment of YscU including the motif for autoproteolytic cleavage (YscU_C; Figure 1A and Figure 1B) for in vitro studies. For detailed investigation of conformational changes in YscU_C we have used the spectroscopic methods nuclear magnetic resonance (NMR) and circular dichroism (CD).

It was previously proposed that the YscU_CC polypeptide dissociates from the remaining, membrane-anchored segment of YscU to allow Yop secretion [16]. To test this hypothesis, we developed biophysical NMR and CD based protocols for the quantification of YscU_CC dissociation from YscU_CN in vitro. The high quality, ¹H-¹⁵N HSQC spectrum of YscU_C at 20°C (Figure 2A, blue contours) and the chemical shift dispersion showed that YscU_C was a folded protein under the experimental conditions. We assigned 91% of the non-proline backbone resonances in our protein construct that contained residues 211–354. In the YscU_C crystal structure (2JLI.PDB) the N-terminal residues 241–255 are in a helical conformation. The helix protrudes into solution and the first residue that makes contact with the remainder of the protein is residue number 250. In solution the first helical residue that we could identify based on NOE contacts was residue 251, and the assigned preceding residues are adopting an unstructured and flexible conformation as inferred from high signal intensities and narrow chemical shift dispersion. After incubation at 60°C for 10 min, the NMR spectrum of YscU_C at 20°C showed a dramatic perturbation (Figure 2A, red contours); only resonances that corresponded to the YscU_CN polypeptide were visible. The narrow chemical shift dispersion and high peak intensities of the YscU_CN resonances showed that the polypeptide adopted an unfolded conformation also after dissociation from YscU_CC. The absence of signals that corresponded to the YscU_CC polypeptide was due to the formation of aggregated particles too large for detection with NMR spectroscopy (see supporting material, “Results S1”).

Figure 2. In vitro dissociation of YscU_C.

(A) ¹H-¹⁵N HSQC spectra of YscU_C at 20°C, pH 7.4, before (blue contours) and after (red contours) incubation at 60°C for 10 min. Only resonances that corresponded to YscU_CN were visible after the thermal treatment. (B) Thermal up- and down-scans of YscU_C at pH 7.4 monitored with CD spectroscopy at 220 nm in the absence of calcium. (C) Thermal signatures of P264A, a non-cleavable mutant, at pH 7.4 in the absence of calcium. Up- and down scans of YscU_C and P264A are shown in red and blue circles, respectively.

The NMR experiments showed that YscU_CC dissociation from YscU_CN was triggered by subjecting YscU_C to thermal perturbation, and that dissociation was an irreversible process. The irreversibility provided a tool for quantifying dissociation kinetics (discussed below). To obtain an accurate value of the dissociation temperature (T _diss), we observed YscU_C dissociation by subjecting the protein to a thermal cycle, and we followed this event with CD at 220 nm. The CD signal at 220 nm contains contributions from both alpha helical and β-strand secondary structures [33]. Thus, because YscU_C contains, both alpha helices and β-strands, 220 nm was a suitable wavelength for following changes in the YscU_C structure (Figure 1B). The thermogram of YscU_C (Figure 2B) was composed of two distinct transitions (55°C and 77°C), and the overall thermal response was not reversible, as the CD signal did not reach its initial value after a complete thermal cycle (see supporting material, “Results S2”). The transition at 55°C corresponded to the dissociation of YscU_C; this was in good agreement with the NMR results that showed an upper limit of T _diss equal to 60°C. The high temperature transition (77°C) was reversible, but with distinct signs of hysteresis (Figure S1). Because this transition was not relevant in the context of dissociation, we did not study it further. Of note, because dissociation is an irreversible process, T _diss is scan-rate dependent. Therefore, all CD spectroscopy-based temperature perturbation experiments were conducted at a fixed scan rate of 1°C/min.

To address the questions whether the dissociation of YscU_C might have biological relevance we subjected the non-cleavable YscU_C mutant P264A to a thermal denaturation. The thermal signature of P264A was dramatically perturbed compared to the wild-type, and the thermogram displayed one irreversible transition at 55°C (Figure 2C). To investigate the biological relevance further, we asked whether calcium might affect the YscU_C dissociation in vitro. The thermogram in the presence of calcium was remarkably similar to that of the non-cleavable variant P264A in the absence of calcium (Figure 3A). Hence, calcium mimicked the effect of a mutation that suppressed the YscU_C auto-processing activity. P264A contained one polypeptide chain that could not dissociate; thus, the data suggested that calcium prevented dissociation of YscU_CC from the YscU_CN polypeptide.

Figure 3. Calcium effects on YscU_C stability and Yop secretion.

(A) Thermal up- and down-scans of YscU_C at pH 7.4 monitored with CD spectroscopy at 220 nm in the presence of 2.5 mM calcium. Up- and down scans of YscU_C are shown in red and blue circles, respectively. (B) Titration of calcium to YscU_C monitored with CD spectroscopy at 220 nm. The calcium binding isotherm to YscU_C was fit to a one-site binding model (red line). The resulting K_d was 800 µM. (C) Western Blot analysis of YopB, YopD, and YopE in wild-type Y. pseudotuberculosis. The bacteria were grown for 2 h at 26°C and shifted to 37°C for 3 h (temperature shift for induction of Yop secretion) with varying concentrations of free calcium. “Pellet” indicates intracellular proteins; “supernatant” denotes secreted proteins. The LB growth medium was initially supplemented with 1 mM EGTA to complex residual calcium content (approximately 500 µM); thereafter, calcium was added to set the indicated concentrations of free calcium.

Next we investigated the ability of YscU_C to bind calcium in vitro and compared it to the calcium concentration needed to block the Yop secretion in vivo. The CD signal at 220 nm indicated the YscU_C binds calcium with a dissociation constant (K_d) of 800 µM, assuming a one-site binding model (Figure 3B). To benchmark the K_d value of calcium against the calcium concentration required for inhibition of the Yersinia T3SS in vivo, we analyzed Yop secretion and expression at different calcium levels in the growth medium. The T3SS was down regulated at calcium concentrations between 0.5 to 1 mM (Figure 3C). Hence, the in vitro K_d value for calcium interaction with YscU_C (800 µM) was well in the concentration interval that inhibited Yop secretion in vivo. Comparative analysis with different divalent cations revealed no exclusive specificity of YscU_C towards calcium. Different alkaline earth metals, Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺ showed comparable effects in vitro on YscU_C interaction and dissociation (Table 1). It was not surprising that Ba²⁺ and Sr²⁺ showed a similar effect as Ca²⁺ since these ions have been shown to effect Yop secretion similarly to Ca²⁺ [34]. On the other hand Mg²⁺ has no effect on Yop secretion suggesting that the in vivo regulation of calcium controlled secretion is dependent on additional factors. This promiscuous metal binding property of YscU_C is consistent with the absence of any known calcium binding motif in YscU_C. In accordance we observed with NMR spectroscopy that calcium binding is mediated through a large set of residues confined to the YscU_CC polypeptide (Figure S2).

Table 1. Dissociation temperatures of YscU_C in presence of different divalent cations and in vitro binding affinities.

	CaCl2		BaCl2	SrCl2		MgCl2
Dissociationtemperature, Tdiss
Tdiss (°C)	58.5±0.8		57.1±0.3	55.8±0.1		57.9±0.1
Dissociation constant, Kd
Kd (µM)	800±40		900±140	840±100		130±10

CD spectroscopy at 220 nm was used to monitor thermal up- and down-scans of YscU_C in presence of different divalent cations at a scan rate of 1°C/min to determine dissociation temperatures (T _diss, compare Figure 3A). To measure the binding isotherms (K_d) of different divalent cations towards YscU_C (compare Figure 3B) titrations monitored with CD spectroscopy at 220 nm were performed.

Nevertheless the in vitro data clearly showed that YscU_C was poised for dissociation into YscU_CN and YscU_CC fragments, and that this event can be inhibited by calcium and other divalent cations. Thus we wondered whether the dissociation of YscU_C could be monitored directly in Yersinia, and whether it can be linked to T3SS regulated Yop secretion. To test this idea, we first probed for the presence of YscU_CC in the culture supernatants after incubating the wild-type Y. pseudotuberculosis strain at 37°C in the absence or presence of 2.5 mM calcium. Remarkably, YscU_CC was found in the culture supernatant from the calcium depleted cultures, and no YscU_CC was found in cultures with 2.5 mM calcium (Figure 4). To explore this finding further, the gene for the YscU_CC polypeptide (amino acids 264 to 354 of YscU) was cloned into the pBAD vector under the control of an inducible araC promoter. This allowed the in trans overexpression of yscU_CC in Y. pseudotuberculosis. After promoter induction, the levels of secreted YscU_CC were analyzed in cultures grown with or without calcium. We found that induction of yscU_CC expression caused increased secretion of YscU_CC into the culture supernatant when compared to the wild-type levels secreted by a strain that contained the control vector. Further, secretion of YscU_CC was blocked in the presence of 2.5 mM calcium in the medium. To investigate the requirement of a functional T3SS for YscU_CC secretion we analyzed the secretion behavior of a Y. pseudotuberculosis ΔyscC null mutant. In absence of YscC no secretion of either Yops or YscU_CC was observed (Figure 4) showing that secretion of YscU_CC is dependent on a functional T3SS. To link our in vitro findings of YscU_C and calcium directly to in vivo events we analyzed the secretion behavior of a ΔyopN mutant that has lost its calcium regulation and secretes Yops in presence and absence of calcium in similar amounts [35]. It was found that the ΔyopN mutant secreted YscU_CC independently of the calcium concentration. Thus, YscU_CC was secreted from the ΔyopN mutant in the presence of calcium showing a similar secretion profile as the Yop substrates (Figure 4).

Figure 4. In vivo dissociation and secretion of YscU_CC in different Yersinia strains.

Calcium dependent regulation of Yop and YscU_CC secretion in wild-type Y. pseudotuberculosis, in a ΔyscC mutant and ΔyscN mutant strain without and with in trans complementation of YscU_CC. Bacteria transformed with empty pBADmycHis B (pBAD), or pBAD with one additional yscU_CC copy (pBAD(YscU_CC)), were grown for 2 h at 26°C and 3 h at 37°C in calcium depleted (−) or calcium supplemented (+) medium. The expression of yscU_CC was induced by addition of arabinose. Yop secretion is coupled to the secretion of YscU_CC in all analysed Yersinia strains and required a functional T3SS. Secreted Yops visualized on Coomassie stained PAGE gels; YscU_CC visualized on immunoblots with anti-YscU_CC peptide antibodies. “pellet” indicates intracellular proteins; “supernatant” denotes secreted proteins. The YopJ protein (black box) was subjected to densitometric analysis for quantification of secretion levels (see Table 2).

In conclusion, YscU_CC showed a secretion pattern similar to that of Yops. This suggested that YscU_CC constituted a novel substrate of the T3SS in Yersinia. Furthermore, the fact that YscU_CC was secreted in the wild-type strain demonstrated that YscU_CC was able to dissociate from the remaining membrane bound part of YscU in vivo.

pH-dependencies of YscU_CC Dissociation/secretion and Yop Secretion

Unpublished observations from our laboratory indicated that Yop secretion, but not bacterial growth, was influenced by the pH of the growth medium. Further it has been shown that autoproteolysis of YscU_C was pH dependent [15]. Here, we addressed the question of whether dissociation of YscU_C was also pH dependent. First, we subjected cleaved YscU_C to a thermal cycle at pH 6.0 (instead of pH 7.4) by monitoring the thermal signature with CD spectroscopy at 220 nm (Figure 5A). When the resulting thermogram was super-imposed on the thermograms of the wild-type YscU_C at pH 7.4 in presence of 2.5 mM calcium and the non-cleavable mutant P264A in the absence of calcium, they were virtually identical. From this observation, we concluded that YscU_C dissociation was prevented by low pH. YscU_C contains two histidine residues (positions 266 and 324) in the YscU_CC fragment that may explain the observed pH-dependency. By monitoring the CD signal at 220 nm as a function of pH, we identified one ionization event in the pH interval of 6.0 to 7.4, with a pKa value of around 6.3 (Figure 5B), and one ionization event around pH 8.0. NMR analyses revealed that both histidines were protonated in response to a pH drop from 7.4 to 6.0 (Figure 5C, 5D). This indicated that the protonation of histidines 266 and 324 was responsible for the observed differences in thermally induced dissociation of YscU_C at pH 6.0 and 7.4. This observation was interesting, because all known YscU orthologs in other T3SSs harbor a conserved histidine residue at the position that corresponds to amino acid 266 in the N↑PTH motif. To further dissect the relevance of the two histidines we replaced histidine 324 with alanine (H324A) and studied the in vitro response of this mutant to both thermal- and pH perturbations. Since it has been shown that mutation of histidine 266 leads to an yscU mutant affected in autoproteolysis this position is not suitable for an alanine replacement [36]. The H324A variant showed similar dissociation behavior in vitro but with reduced thermal stability at pH 7.4 and pH 6.0 compared to wild-type (Figure S3A and S3B). The pH-dependency of the CD-signal at 220 nm of the histidine mutant H324A showed a distinct difference compared to the wild-type protein (Figure S3C). Whereas the wild-type protein displayed two ionization events (at pH 6.3 and 8.0) the mutant only displayed the ionization event at pH 6.0. Hence, the pKa of the N↑PTH histidine is around 6, and protonation/deprotonation of this histidine is likely responsible for the difference in thermally induced dissociation at pH 6.0 and 7.4. Next, we wondered whether these biophysical observations reflected biological effects in vivo. To investigate the pH-dependency of Yop secretion, we cultivated wild-type Y. pseudotuberculosis in Hepes buffered media at pH values between 6.0 and 7.5 in calcium depleted media and monitored the secretion of YscU_CC and Yops (Figure 5E and Table 2). Both Yop and YscU_CC secretion was maximal at pH levels between 7.0 and 7.5. Secretion gradually decreased when pH was lowered, and at pH 6.0, we observed a pronounced inhibitory effect on the secretion of YscU_CC as well as the Yops (albeit not as strong as the inhibition by calcium). Importantly, bacterial growth was not affected by changing the pH of the growth medium; thus, perturbations of external pH values did not cause any general effects on bacterial proliferation (Figure S4). These in vivo and in vitro results suggested that the pH-dependent secretion of the secretion of Yops and YscU_CC was a consequence of the molecular behavior of YscU; i.e., the dissociation and secretion of YscU_CC was a prerequisite for maximal secretion of Yop substrates into the culture medium.

Figure 5. pH-dependencies of YscU_C dissociation in vitro and Yop/YscU_CC secretion in vivo.

(A) Thermal up- and down-scans of YscU_C at pH 6.0, monitored with CD spectroscopy at 220 nm in the absence of calcium. The thermal signature of YscU_C displayed one large-amplitude transition at 55°C. Up- and down scans of YscU_C are shown in red and blue circles, respectively. (B) The pH-dependency of the YscU_C monitored with CD spectroscopy at 220 nm. (C) Chemical shift perturbations of YscU_C quantified from ¹H-¹⁵N HSQC spectra, in response to a pH-shift from 7.4 to 6.0, displayed against the primary sequence. The blue line indicates the threshold value (0.05 ppm) used in Figure 5D. The chemical shift perturbation of the two histidines at positions 266 and 324 are shown in red. (D) Structural distributions of residues that show significant chemical shift perturbations in response to a pH-shift from 7.4 to 6.0 are shown in red on the YscU_C structure (2JLI.PDB). The YscU_CN and YscU_CC fragments are colored orange and gray, respectively. The two histidine residues (266 and 324) in the folded part of YscU_C are indicated. (E) Coomassie stained gels show Yop secretion under different pH conditions. (top panel) “pellet” indicates intracellular proteins; (middle panel) “supernatant” denotes secreted proteins. The YopJ protein (black box) was subjected to densitometric analysis for quantification of secretion levels (see Table 2). (bottom panel) The pH-dependency of YscU_CC secretion was visualized on immunoblots with anti-YscU_CC peptide antibodies.

Table 2. Comparative densitometric analysis of pH and calcium-dependent Yops secretion in Y. pseudotuberculosis.

condition	observed growth attenuation	observed Yop secretion	secretion efficiency (%)
pBAD, +Ca2+	no	no	0
pBAD/YscUCC), +Ca2+	no	no	0
pBAD, −Ca2+	yes (+++)	yes (+++)	99
pBAD/YscUCC), −Ca2+a	yes (+++)	yes (+++)	100
pH 6.0	yes (+)	yes (+)	1
pH 6.5	yes (++)	yes (++)	32
pH 7.0	yes (+++)	yes (+++)	75
pH 7.5b	yes (+++)	yes (+++)	100

To compare and quantify the Yop secretion efficiency in wild-type Y. pseudotuberculosis under different conditions, Coomassie stained Yop secretion profiles were subjected to densitometric analysis with Multi Gauge software (Fuji Film). The protein YopJ (boxed in Figure 4 and Figure 5E) was selected for quantitative analysis. Growth kinetics in media with different pH’s (Figure S4B) showed attenuation of bacterial growth directly linked to the observed Yop secretion efficiency.

^asecretion efficiency was set to 100%;

^bsecretion efficiency at pH 7.5 was set to 100%.

Yops are Secreted at Lower Temperatures in Destabilized YscU_CC Variants Compared to Wild-type

The results described above suggested that dissociation of YscU_CC from the remainder of YscU, followed by secretion of the YscU_CC polypeptide, was important for Yop secretion. We and other groups previously showed that single amino acid substitutions in YscU_C could suppress the Yop secretion-deficient phenotype of the Y. pseudotuberculosis ΔyscP mutant [17], [21], [37]. Identification of such suppressor mutations is generally considered as strong genetic evidence for protein-protein interactions. Thus, second-site suppressor mutations are expected to be localized at protein surfaces that are prime positions for protein-protein interactions. In sharp contrast, all amino acid substitutions on the YscU_CC polypeptide were either fully or partially buried in the protein structure (Figure 6). Consequently, the underlying mechanism of these mutations must be more complex than a direct protein-protein interaction with YscP. Mutations at buried positions generally act to destabilize proteins; therefore, we reasoned that the altered secretion behavior of the suppressor mutants might be attributed to perturbed stabilities (T _diss) and dissociation rates (k _diss = 1/τ_diss) compared to wild-type YscU_C.

Figure 6. YscU_C suppressor mutations are buried in the structure.

The spatial locations of single mutations in YscU_C that suppressed the non-secreting ΔyscP phenotype in vivo are shown on the YscU_C structure of Y. pestis (2JLI.PDB). All positions are either fully or partially buried in the protein structure. YscU_CN and YscU_CC polypeptides are colored orange and gray, respectively.

To test this notion, we monitored the dissociation kinetics of YscU_C suppressor mutants A268F, Y287G, V292T and wild-type YscU_C with CD and NMR spectroscopy in vitro. The resulting dissociation kinetics are reported as dissociation lifetimes (τ_diss), or the reciprocal of the dissociation rate ( = 1/k _diss). Because Yop secretion is triggered by a temperature shift from 26°C to 37°C [3], [4], [6], we initially performed the assays at 37°C. We found that all YscU_C mutants were folded (Figure S5A) and displayed decreased thermal stabilities compared to wild-type YscU_C, evident from the reduced dissociation temperatures (Figure 7A and Table 3). We quantified the dissociation kinetics with both CD and NMR spectroscopy for the YscU_C variant V292T; with CD, we detected changes in ellipticity at 220 nm that accompanied dissociation (Figure S5B); with NMR, we detected the loss of resonance intensities for residues in the YscU_CC fragment (Figure S6). The time-dependent signals were well described by first order processes; accordingly, all kinetic traces could be fit accurately with single exponential decay functions. Both CD and NMR results indicated that wild-type YscU_C displayed very slow dissociation kinetics at 37°C in the observed time frame; in comparison, the YscU_C variant V292T displayed a significantly enhanced rate of dissociation (Figure 7B, Figure S6A, S6B). It should be noted that the observed variations in the dissociation lifetimes (τ _diss) for the suppressor mutants are dependent on the method used for quantification (Table 3). For instance, τ _diss for the V292T variant was 61 min and 140 min, based on CD and NMR, respectively. This discrepancy could be attributed to differences in sensitivity; the CD signal was directly sensitive to the dissociation process, but NMR required both dissociation and aggregation for a reduction in signal intensity. Hence, both dissociation and aggregation were slow processes that occurred at similar time scales in vitro. After one hour at 37°C, a significant fraction of the V292T variant displayed dissociated species (65% and 38% from CD and NMR, respectively), but wild-type YscU_C remained intact under the same conditions.

Figure 7. YscU_C suppressor mutant stabilities and dissociation kinetics.

(A) Thermal induced unfolding of YscU_C and suppressor mutants. Dissociation temperatures (T _diss) of single suppressor mutants at pH 7.4 were quantified with the CD signal at 220 nm and a scan rate of 1°C/min; YscU_C (black), A268F (blue), Y287G (green), and V292T (red). All suppressor mutants are destabilized compared to wild-type YscU_C. T _diss values are summarized in Table 3. (B), (C) Dissociation kinetics quantified as dissociation life-times (τ_diss) of YscU_C (black) and V292T (red) at pH 7.4 followed with NMR-spectroscopy at (B) 37°C and (C) 30°C, respectively. Primary NMR data for (B) is shown in Figure S6. Solid lines correspond to fits of the experimental data to single exponential decays. (D) Time dependent GST-pulldown experiments show the dissociation of wild-type YscU_C and the suppressor mutant A268F at 30°C and 37°C after varying incubation times. The suppressor mutant A268F displayed pronounced dissociation of YscU_CC-His₆ at 37°C and moderate dissociation at 30°C; wild-type YscU_C displayed no dissociation of YscU_CC-His₆ at 37°C or at 30°C over the observed time period. Note! Dissociation of YscU_CC is manifested as disappearance of YscU_CC-His₆ over time since the dissociation is irreversible and YscU_CC-His₆ cannot bind itself to the used resin.

Table 3. Dissociation temperatures and kinetics of wild-type YscU_C and the suppressor mutants V292T, Y287G, and A268F probed with NMR and CD spectroscopy.

Circular dichroisma	T diss (°C)b	τ diss at 37°C (min)
YscUC, wild-type	55.2±1.4	stablec
V292T	49.5±0.5	60.8±3.2
Y287G	48.9±0.3	138.4±10.5
A268F	44.8±0.5	70.1±4.8
NMR spectroscopyd	τ diss at 30°C (min)	τ diss at 37°C (min)
YscUC, wild-type	29914±3526	827±71
V292T	714±47	140±3

CD spectroscopy at 220 nm was performed at a scan rate of 1°C/min to determine the dissociation temperature (T _diss) of YscU_C and suppressor mutants (Figure 7A). The kinetics of the dissociation process (τ _diss) was monitored with CD spectroscopy at 220 nm and NMR spectroscopy at 37°C and 30°C. See also Figure 7B and 7C; Figure S5B.

^ameasured by following the CD signal at 220 nm;

^bmeasured with a scan-rate of 1°C/min;

^cdissociation was too slow to fit with a single exponential decay function;

^dmeasured by following methyl group intensities in one dimensional ¹H spectra.

V292T displayed a significantly reduced thermal stability compared to wild-type YscU_C; therefore, we also performed NMR-based kinetic experiments at 30°C (Figure 7C). At this temperature, wild-type YscU_C was stable over the entire experiment (860 min), but the V292T variant dissociated at a rate equal to the rate for wild-type YscU_C at 37°C, within experimental error (Table 3). We confirmed these results with the A268F suppressor mutant. Further we used a GST pull down assay, and the dissociation of GST-YscU_C-His₆ respectively GST-A268F-His₆ was monitored at 30°C and 37°C. Indeed, GST-YscU_C-His₆ was completely stable at both temperatures, but GST-A268F-His₆ dissociated to a significant extent over time, visible in the decrease of the YscU_CC-His₆ content at both 30°C and 37°C (Figure 7D).

Because the dissociation of YscU_C is required for Yop secretion in vivo, we expected suppressor mutant strains to secrete Yops at lower temperatures compared to the wild-type strain. To test this hypothesis, we probed for the presence of secreted Yops and YscU_CC in the culture supernatant in strains that carried either yscU or yscU suppressor mutants A268F or V292T after a temperature shift from 26°C to 30°C. Both the A268F and V292T mutant strain secreted Yops and YscU_CC already at 30°C; in contrast, the wild-type strain showed almost no secretion at this temperature (Figure 8). Probing for LcrV and YscI as early substrates in T3SS confirmed the direct link between secretion of YscU_CC and T3SS substrates (Figure S7B). Despite the increased secretion of Yops at reduced temperatures, the pH regulation was still active in these Yersinia mutants. A268F and V292T revealed the same secretion pattern like the wild-type when grown in media with different pH (Figure 5E and Figure S7A). Importantly, all strains secreted Yops in a calcium-regulated manner; this showed that the suppressor mutants retained calcium sensing capability (data not shown).

Figure 8. Yersinia strains with destabilized yscU suppressor mutants secrete YscU_CC and Yops at lower temperatures (30°C) than wild-type.

Coomassie stained analysis of Yop secretion in Y. pseudotuberculosis incubated at 30°C. Bacteria expressing either wild-type yscU or one of the suppressor mutants, A268F or V292T. “pellet” indicates intracellular proteins; “supernatant” denotes secreted proteins. Secretion of YscU_CC was analyzed on immunoblots with anti-YscU_CC peptide antibodies. Yersinia harboring yscU suppressor mutants showed strongly elevated secretion of Yops after cultivation at 30°C.

Our results showed a strong link between in vivo and in vitro results for dissociation of the YscU_CC polypeptide from the remainder of the protein. This demonstrated that not only autoproteolytic cleavage but also dissociation and secretion of YscU_CC is a key step in the regulation of Yop secretion.

Discussion

The YscU protein of Yersinia pseudotuberculosis and orthologs in other bacteria display autoproteolytic activity, with cleavage at a conserved N↑PTH motif. It is reasonable to assume that a strictly conserved autoproteolytic activity in a protein is linked to a specific function in the organism. Auto-processing has been observed in other proteins; e.g., in the SEA domain of the membrane-bound MUC1 protein, the processing occurs at a conserved GD↑PH site. It has been suggested that this cleavage introduces a molecular-mechanical fracture that protects epithelial cells from rupture [38]. It was previously postulated that autoproteolysis of YscU exposes a new binding surface to other T3SS proteins by changing the charge distribution at the cleavage site [39]. Here, we propose an alternative model, where dissociation of YscU_CC from the membrane anchored segment of YscU, followed by YscU_CC secretion via the T3SS, plays a central role in the substrate specificity switch [21].

We showed that T3SS mediated Yop secretion correlated with the secretion of YscU_CC and presumed a fully functional T3SS. Secretion of YscU_CC required autocatalytic cleavage of the cytosolic domain (YscU_C) of the inner membrane protein YscU that was linked in earlier studies to be one key regulator in the substrate specificity switch of T3SS mediated Yop secretion [17]. In vitro analysis with recombinantly produced YscU_C confirmed the dissociation capacity of the protein and revealed potential regulating factors, like divalent ions (i.e. calcium), pH and temperature. We showed that T3SS mediated Yop secretion was strongly affected by 0.5 to 1 mM calcium and pH values below 6.5. The calcium concentration had an “all or nothing” effect on Yop secretion. In contrast, the inhibitory effect of low pH values was less pronounced. Surprisingly Ca²⁺-ions bound to YscU_C with a K_d of 800 µM in vitro; notably, this value was consistent with the threshold value for calcium dependent down-regulation of Yop secretion in vivo. Additional in vitro analysis including different divalent cations Mg²⁺, Sr²⁺ and Ba²⁺ indicated that the alkaline earth metal ions interacted and stabilized YscU_CC binding to about the same extent. These results indicated that the calcium regulation of the Yersinia T3SS in vivo is complex and cannot be explained only on basis of the YscU_C calcium interaction. The N↑PTH histidine, is unfortunately not suitable for mutations to other amino acids since it affects the autoproteolytic activity [36]. However experiments where histidine 324 was replaced with alanine indicated that, overall, the N↑PTH histidine is responsible for the pH-dependency of YscU_C dissociation. Thus these findings suggest that pH is also an extracellular queue regulating the Yersinia T3SS.

It is known that the YscP protein plays an important role in the regulation of the substrate specificity switch [17]. We recently proposed that YscP activity may stimulate displacement of YscU_CC from the remaining YscU part, and that this activity was triggered by target cell contact or calcium depletion. We further suggested that, in yscU suppressor mutants, the point mutations in YscU_CC induced a perturbation in the YscU structure, which destabilized the interaction between YscU_CC and the remaining membrane anchored part of YscU [16]. This was suggested because the whole yscP gene was deleted; thus, it was likely that the YscU_CC mutations caused a gain of function. In the present study, we confirmed this hypothesis by showing that destabilization of the suppressor mutants led to premature YscU_CC dissociation, which then induced Yop secretion. Strains that carried the suppressor mutations secreted Yops at 30°C in calcium-depleted medium. This finding contrasted with findings in the wild-type strain, which showed almost no Yop secretion at 30°C. Nevertheless, all suppressor mutants retained the wild-type calcium regulation of Yop secretion indicating that calcium exhibits a stabilizing effect on the interaction between YscU_CC and the remainder of YscU.

Surprisingly, we found that YscU_CC was also secreted via the T3SS; this was remarkable, given that YscU is an inner membrane protein [40]. Our results favor a model where YscU orchestrates obstruction of the T3SS secretion channel. This block is thus, relieved through YscU_CC secretion in calcium depleted and in pH ≥6.5 conditions. In support for this model is the observation that yscU mutants impeded for autoproteolysis and subsequent secretion of YscU_CC (mutations within the NPTH motif) are unable to secrete the Yop-proteins [18]. Further, these mutants secrete elevated amounts of the early substrate YscF showing that the T3SS is active in these mutants and allows secretion of early but not late substrates [16]. The model explains why these mutants are also defective in Yop secretion. Further support for this model was our finding that the “Ca²⁺-blind” yopN mutant, unable to respond to extracellular calcium, secreted YscU_CC as well as Yops in Ca²⁺ containing conditions indicating that YscU_CC secretion is tightly coupled to Yop secretion. Thus, YopN is essential for the calcium response in Yersinia and in addition our results suggest that YopN has a role in the Ca²⁺ response in vivo leading to stabilization of YscU. Moreover, Mg²⁺ blocks YscU_CC displacement in vitro similarly to Ca²⁺ but in contrast the pathogen secretes Yops in vivo at high concentrations of Mg²⁺, indicating that Mg²⁺ is not interacting with YscU_C during in vivo conditions. Thus it is possible that YopN discriminates between the ions, allowing transport/uptake of Ca²⁺ but not Mg²⁺ during growth at 37°C in vivo. YopN has also been shown to be surface located when Yersinia is grown at 37°C in presence of calcium which agrees with its putative role as calcium scavenger [35].

This study has provided a new handle for investigating the function of YscU/FlhB proteins in other bacteria. Given the conservation within this family of proteins, it is likely that they also exhibit regulatory roles that involve secretion of the processed C-terminus of the protein. However, depending on life style, the actual triggering signal may differ among different pathogens. For example, Salmonella invasion is controlled by environmental pH, low oxygen, and acetate [41]. Although different intracellular regulatory pathways have been linked to these signals, virtually nothing is known about signal reception and interpretation by the pathogen. Based on the results presented here, it would not be surprising to find that some signals stimulated secretion of the Salmonella YscU_CC homolog, SpaS_CC.

Supporting Information

Figure S1

Reversibility of the high temperature (77°C) transition of YscU_C. Thermal up- and down-scans of 10 µM YscU_C were monitored with CD spectroscopy at 220 nm. YscU_C was subjected to two sequential thermal cycles (one cycle: 20°C to 95°C and then back to 20°C). The color coding indicates: first up-scan (black), first down-scan (open gray), second up-scan (open blue) and second down-scan (open red). After initial loss of secondary structure, the high temperature transition is reversible.

(TIF)

Figure S2

Calcium induced chemical shift perturbations in YscU_C. (A) Chemical shift differences were monitored with two-dimensional ¹H-¹⁵N HSQC NMR spectra of YscU_C before and after saturation with calcium. Chemical shift differences are plotted against the primary sequence. The blue line indicates the threshold value (0.06 ppm) used in (B) to highlight amino acid residues affected by addition of calcium. Residues responding to calcium are confined to the YscU_CC fragment. (B) Structural distributions of residues that show significant chemical shift perturbations in response to calcium binding are shown in red on the YscU_C structure (2JLI.PDB). YscU_CN and YscU_CC fragments are colored orange and gray, respectively.

(TIF)

Figure S3

Biophysical analysis of the YscU_C variant H324A. (A) Thermal up- and down-scans of H324A at pH 7.4 monitored with CD spectroscopy at 220 nm in the absence of calcium. (B) Thermal up- and down-scans of H324A at pH 6.0 monitored with CD spectroscopy at 220 nm in the absence of calcium. Up- and down scans of H324A are shown in red and blue circles, respectively. (C) The pH-dependency of the CD-signal at 220 nm for H324A.

(TIF)

Figure S4

Y. pseudotuberculosis growth kinetics were pH independent between pH 6.0 and pH 7.5. Bacterial growth of wild-type Y. pseudotuberculosis was analyzed by monitoring the optical density (OD₆₀₀) under (A) calcium-supplemented and (B) calcium-depleted conditions. Bacteria were cultivated 2 h at 26°C, then 3 h at 37°C. Samples were taken every hour to monitor the growth based on the measured OD₆₀₀. No significant differences in growth kinetics were observed within the monitored pH-interval of 6.0 to 7.5.

(TIF)

Figure S5

Secondary structure analysis and dissociation kinetics of YscU_C suppressor mutants monitored with CD spectroscopy. (A) Far-UV CD spectra of 10 µM wild-type YscU_C (black) and the suppressor mutants A268F (blue), Y287G (green), V292T (red). The similarity in the shape of the CD signals indicates that all YscU_C variants have similar secondary structure. (B) Dissociation kinetics of YscU_C (black) and suppressor mutants A268F (blue), Y287G (green), and V292T (red) were monitored with CD spectroscopy at 220 nm and 37°C. The solid lines represent the best fit of a single exponential decay function to determine τ _diss. Dissociation kinetics are summarized in Table 3. Note that the data points at time = zero have been normalized for clarity.

(TIF)

Figure S6

Primary NMR data used to quantify dissociation kinetics of wild-type YscU_C and V292T at 37°C. Shown are expansions of methyl resonances from one-dimensional ¹H spectra at various time points for (A) wild-type YscU_C and (B) the V292T mutant (see Figure 7B and 7C).

(TIF)

Figure S7

Secretion analysis of ysc U suppressor mutants A268F and V292T . (A) Secretion analysis of A268F and V292T after cultivation in Hepes-buffered LB at different pH values. Yop secretion was induced by calcium depletion and a temperature shift from 26°C to 37°C. A268F and V292T showed elevated Yop secretion at pH≥6.5 and strong inhibition at pH 6.0 (B) Secretion analysis of A268F and V292T at pH 7.5 and 30°C compared to wild-type. The T3SS was induced by calcium depletion and a temperature shift to 30°C. A268F and V292T showed a strongly elevation of Yop secretion. Coomassie stained gels demonstrate secreted Yops. “pellet” indicates intracellular proteins; “supernatant” denotes secreted proteins. The secretion of YscI and LcrV was visualized on immunoblots with anti-YscI and anti-LcrV antibodies.

(TIF)

Figure S8

Size exclusion chromatography-based estimation of the YscU_CC aggregate size. Chromatogram of analytical size exclusion chromatography of purified YscU_CC. YscU_CC eluted in the void volume of the SEC column.

(TIF)

Figure S9

CD-based analysis of YscU_CC produced by thermal stimulation or by recombinant protein production. Comparison of YscU_C CD spectra at 20°C after one completed thermal cycle to 95°C. Filled circles show the YscU_CC produced by thermal stimulation and open circles show the purified YscU_CC fragment, produced in vitro by recombinant protein production. The similarity of the spectra shows that the residual CD signal of YscU_C after one thermal cycle is dominated by the YscU_CC fragment.

(TIF)

Table S1

Bacterial strains and plasmids used in this study.

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Table S2

Primers used in this study.

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Materials and Methods S1

Procedure for cloning yscU _CC into pBADmyc His B.

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Materials and Methods S2

Cloning procedure for GST fusion proteins.

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Results S1

YscU_CC aggregated after dissociation from YscU_CN. Analytical size exclusion chromatography was used to show that YscU_CC aggregates after dissociation from the YscU_CN polypeptide.

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Results S2

Persistent secondary structure in aggregated YscU_CC from CD spectroscopy. CD spectroscopy on thermally and recombinantly produced YscU_CC aggregates was used to show that YscU_CC contains elements of secondary structure in the aggregated state.

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Funding Statement

This research was financially supported by the Swedish Research Council (HWW and MWW), the UCMR Linnaeus Postdoctoral Program (to HWW), the Laboratory of Molecular Infection Medicine Sweden (MIMS, to HWW), and an Umeå University Young Researcher Award (to MWW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.