An infrequent molecular ruler controls flagellar hook length inSalmonella enterica

Article7 June 2011free access An infrequent molecular ruler controls flagellar hook length in Salmonella enterica Marc Erhardt Corresponding Author Marc Erhardt Département de Médecine, Université de Fribourg, Fribourg, Switzerland Department of Biology, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Hanna M Singer Hanna M Singer Département de Médecine, Université de Fribourg, Fribourg, Switzerland Department of Biology, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Daniel H Wee Daniel H Wee Département de Médecine, Université de Fribourg, Fribourg, Switzerland Department of Biology, University of Utah, Salt Lake City, UT, USA Search for more papers by this author James P Keener Corresponding Author James P Keener Department of Mathematics, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Kelly T Hughes Corresponding Author Kelly T Hughes Département de Médecine, Université de Fribourg, Fribourg, Switzerland Department of Biology, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Marc Erhardt Corresponding Author Marc Erhardt Département de Médecine, Université de Fribourg, Fribourg, Switzerland Department of Biology, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Hanna M Singer Hanna M Singer Département de Médecine, Université de Fribourg, Fribourg, Switzerland Department of Biology, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Daniel H Wee Daniel H Wee Département de Médecine, Université de Fribourg, Fribourg, Switzerland Department of Biology, University of Utah, Salt Lake City, UT, USA Search for more papers by this author James P Keener Corresponding Author James P Keener Department of Mathematics, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Kelly T Hughes Corresponding Author Kelly T Hughes Département de Médecine, Université de Fribourg, Fribourg, Switzerland Department of Biology, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Author Information Marc Erhardt 1,2, Hanna M Singer1,2,‡, Daniel H Wee1,2,‡, James P Keener 3 and Kelly T Hughes 1,2 1Département de Médecine, Université de Fribourg, Fribourg, Switzerland 2Department of Biology, University of Utah, Salt Lake City, UT, USA 3Department of Mathematics, University of Utah, Salt Lake City, UT, USA ‡These authors contributed equally to this work *Corresponding authors: Département de Biologie, Université de Fribourg, Chemin du Musée 10, Fribourg CH-1700, Switzerland. Tel.: +41 26 300 9435; Fax: +41 26 300 9741; E-mail: [email protected] or Tel.: +41 26 300 9436; Fax: +41 26 300 9741; E-mail: [email protected] of Mathematics, University of Utah, Salt Lake City, UT 84112, USA. Tel.: +1 801 581 6089; Fax: +1 801 581 4148; E-mail: [email protected] The EMBO Journal (2011)30:2948-2961https://doi.org/10.1038/emboj.2011.185 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 The bacterial flagellum consists of a long external filament connected to a membrane-embedded basal body at the cell surface by a short curved structure called the hook. In Salmonella enterica, the hook extends 55 nm from the cell surface. FliK, a secreted molecular ruler, controls hook length. Upon hook completion, FliK induces a secretion-specificity switch to filament-type substrate secretion. Here, we demonstrate that an infrequent ruler mechanism determines hook length. FliK is intermittently secreted during hook polymerization. The probability of the specificity switch is an increasing function of hook length. By uncoupling hook polymerization from FliK expression, we illustrate that FliK secretion immediately triggers the specificity switch in hooks greater than the physiological length. The experimental data display excellent agreement with a mathematical model of the infrequent ruler hypothesis. Merodiploid bacteria expressing simultaneously short and long ruler variants displayed hook-length control by the short ruler, further supporting the infrequent ruler model. Finally, the velocity of FliK secretion determines the probability of a productive FliK interaction with the secretion apparatus to change secretion substrate specificity. Introduction Bacteria propel themselves through liquid environments by rotating helical flagellar filaments (Figure 1A) (Berg and Anderson, 1973). The bacterial flagellum is a motor organelle that is composed of three main structural parts: (i) a basal body that includes rotor and stator structures embedded in the cytoplasmic membrane, a rod traversing the periplasmic space and a flagellar-specific protein export system; (ii) the hook, a flexible coupling structure that functions as a universal joint between the basal body and (iii) the rigid filament serving as a propeller that extends several microns from the cell (Macnab, 2003; Chevance and Hughes, 2008). This sophisticated nanomachine is evolutionarily and structurally related to the virulence-associated injectisome or needle complex of pathogenic bacteria (Hueck, 1998; Cornelis, 2006). Common features of both the flagellum and the injectisome systems are a type III protein export machine at the base of the structures (Blocker et al, 2003; Cornelis, 2006) and an intrinsic control mechanism for length control of the flagellar hook or injectisome needle, respectively (Journet et al, 2003; Shibata et al, 2007). For the flagellum, rod-hook-type substrates are exported via the flagellar type III protein export system until the hook is of appropriate length (55±6 nm) (Hirano et al, 1994) and then the type III secretion system switches substrate specificity and starts exporting filament-type substrates (Williams et al, 1996). For the injectisome needle system of Yersinia enterocolitica, the needle polymerizes to a length of 58±10 nm (Journet et al, 2003) before substrate specificity is switched towards export of effector proteins (Sorg et al, 2007). Figure 1.(A) Schematic of axial components of the bacterial flagellum. The structure of the bacterial flagellum can be divided into three parts: (i) the basal-body structure that harbours the flagellar-specific type III secretion apparatus at the base; (ii) the hook that functions as a flexible coupling structure between the basal body and (iii) the rigid filament. Stator elements (Mot proteins) that span the inner membrane and apply torque to the C-ring in response to transmembrane proton flow are not shown. Rod-hook length is determined by a molecular ruler, FliK, that in turn induces a switch in secretion specificity from rod-hook-type to filament-type substrates upon hook completion, presumably by interaction with FlhB, a component of the type III secretion apparatus at the base of the structure. (B) Schematic of experimental outline. An overnight culture of a strain expressing the flagellar master operon flhDC from a Tc-inducible PtetA promoter is diluted into fresh LB and grown for 3 h. After 3 h growth, flagellar gene expression is induced by addition of Tc and 30 min after induction transcription of Class 3 promoters is observed, which indicates HBB completion (Karlinsey et al, 2000). Here, we uncoupled FliK expression from flagellar genes expression to analyse the effects of late FliK induction on switching from HBB-type secretion to filament-type secretion in a strain deleted for its native fliK gene and expressing fliK from the inducible ParaBAD promoter (PtetA-flhD+C+ ParaBAD-fliK+ ΔfliK). In the first sample (‘wild-type’), flagellar gene expression and fliK expression are induced simultaneously by addition of Tc to induce the flagellar master regulator flhDC (PtetA-flhD+C+) and Ara to induce fliK expression (ParaBAD-fliK+) resulting in hooks of wild-type length. In the second sample (‘polyhook’), only Tc is added to induce flagellar gene expression, giving rise to polyhooks because FliK is not induced. In the third sample (‘late fliK induction’), flagellar gene expression is induced for 45 min without FliK expression. This allows for hook-length growth beyond the physiological length. Afterwards, fliK is induced by addition of Ara and the culture grown for an additional 30 min to allow for induction of the secretion-specificity switch and filament assembly. Download figure Download PowerPoint In Salmonella enterica, the secretion-specificity switch is thought to occur by an interaction between secreted FliK and the substrate-specificity-determining component of the flagellar secretion apparatus, FlhB (Minamino et al, 2006). Null mutants of fliK and dominant-negative alleles of flhB fail to switch the secretion specificity to filament-type substrates and continue uncontrolled hook polymerization (Patterson-Delafield et al, 1973; Hirano et al, 1994; Kutsukake et al, 1994; Minamino et al, 1999; Fraser et al, 2003). Homologous proteins of FliK and FlhB in the Yersinia ssp. injectisome system are YscP and YscU (Magdalena et al, 2002; Journet et al, 2003). The C-terminal domains of FliK and YscP are thought to be responsible for induction of the specificity switch within the type III secretion apparatus, presumably by interaction with FlhB or YscU, respectively (Hirano et al, 1994; Minamino et al, 2006; Sorg et al, 2007). Export of FliK and YscP is required for hook- and needle-length control, respectively (Minamino et al, 1999; Agrain et al, 2005). Deletions and insertions in FliK (YscP) revealed a linear correlation between length of the hook (needle) structure and the length of FliK (YscP), illustrating that these proteins determine hook (needle) length as a molecular ruler that directly measures the length of the structure (Journet et al, 2003; Shibata et al, 2007). The fundamental problem is how, during the process of being secreted, the ruler molecule is able to transmit hook (needle)-length information beyond the cell surface back to the type III export apparatus in the inner membrane in order to flip the switch. Several models for the mechanism of how FliK (YscP) regulates hook (needle) length have been proposed. Initially, a molecular ruler model was not considered for the flagellar system because all fliK mutants isolated resulted in longer, not shorter, hooks (Kawagishi et al, 1996). However, mutants in the C-ring components fliG, fliM or fliN were identified that resulted in short hooks (Makishima et al, 2001). Thus, it was proposed that the cytoplasmic rotor of the flagellum functions as a measuring cup. This C-ring cup would fill up with hook subunits that would correspond to the required number of hook molecules for the assembly of a hook of appropriate length. Upon emptying of the cup, FliK would be able to access and interact with FlhB (Makishima et al, 2001). Recent results show, however, that controlled hook lengths are observed in mutants missing parts or all of the C-ring (Konishi et al, 2009; Erhardt et al, 2010). Later, a static-ruler model has been proposed, where a single ruler molecule resides in the secretion channel and is attached to the growing tip of the needle (hook) structure (Journet et al, 2003). In this model, needle (hook) subunits must be able to pass by the retained ruler inside a secretion channel of around 2 nm in diameter (Shaikh et al, 2005). Finally, an alternative model was proposed, where FliK is intermittently secreted throughout hook growth and the length signal is determined via a stochastic process, where the probability of hook growth termination is an increasing function of hook length (Erhardt et al, 2010; Keener, 2010). In this work, we present experimental evidence in favour of this infrequent molecular ruler model and provide for the first time a mechanism for flagellar hook-length determination by FliK in which the velocity of FliK secretion dictates the probability of a productive interaction with the secretion apparatus for the specificity switch to occur. Results Experimental approach and motility of the model strains The infrequent ruler model for flagellar hook-length regulation predicts that the FliK ruler can be intermittently secreted with hook (FlgE) subunits at any time during hook polymerization. During secretion, FliK takes temporal measurements of hook length. The probability of a productive interaction of the FliK C-terminus with the type III secretion apparatus, which is a prerequisite for the switch in secretion specificity, increases with hook length. Termination of hook polymerization would be unlikely for short hooks, but highly probable at longer hook lengths. A possible mechanism is that the speed of FliK secretion is facilitated, while the hook is shorter than its physiological length. This would prevent a productive interaction of the FliK C-terminus with the FlhB component of the secretion system within the cytoplasmic membrane. In hooks of the physiological length or greater, the rate of FliK secretion is slow enough to allow ample time for the C-terminus of FliK to interact with FlhB and flip the specificity switch to late substrates. One prediction of the infrequent ruler hypothesis is that FliK triggers the secretion-specificity switch every time FliK is secreted through a hook of physiological length or greater. To this end, we envisaged an S. enterica model strain in which fliK induction (and FliK protein secretion) is uncoupled and independently controlled from hook-basal-body (HBB) assembly. In this strain, induction of flagellar gene expression is controlled by a tetracycline (Tc)-inducible promoter (Karlinsey et al, 2000), thereby enabling us to control and synchronize expression of the flagellar master regulator flhDC (PtetA-flhD+C+) and accordingly HBB gene expression and assembly. It has been previously reported that approximately 30 min after induction of the flhDC operon, the secretion-specificity switch has occurred, which corresponds to HBB completion (Karlinsey et al, 2000). Approximately 60 min after induction of flagellar genes, external filaments of about 2 μm length are observed (Karlinsey et al, 2000). In a strain that is deleted for its chromosomal fliK gene, induction of the flagellar master regulator will result in HBB assembly. However, in the absence of FliK, the secretion apparatus will fail to flip the secretion-specificity switch and hook growth will continue beyond physiological lengths, resulting in a polyhook phenotype. To control FliK expression in the cell, the fliK gene was placed under arabinose (Ara) induction (ParaBAD-fliK+). This model strain (PtetA-flhD+C+ ParaBAD-fliK+ ΔfliK) allows for induction of FliK at times after hook length has reached its physiological length. As shown in Supplementary Figure S1, the model strain displayed motility comparable to wild type on soft agar plates containing Tc and Ara as inducers of both FliK and flagellar gene expression. To test the infrequent ruler model for hook-length determination, the model strain was grown under three different conditions (Figure 1B). For the ‘wild-type’ control, flagellar genes (PtetA-flhD+C+) and fliK expression (ParaBAD-fliK) were induced simultaneously by addition of both inducers for 75 min. In case of the polyhook control, flagellar gene expression in the absence of fliK expression was induced by addition of only Tc for 75 min. No FliK or FliC secretion was detected under those conditions (Supplementary Figure S2). To assess the effects of late FliK secretion in a population, where the majority of the hooks have polymerized beyond the physiological length, expression of the flagellar master regulator was induced with Tc for 45 min followed by late induction of fliK expression by addition of Ara for an additional 30 min to allow for the switch to late-substrate secretion mode. Switch to late-substrate secretion occurred immediately after FliK induction in hooks greater than the physiological length In the absence of FliK, addition of Tc for 45 min ensures that hooks polymerize beyond their physiological length. It takes 30 min to grow the completed HBB structure after induction of the flagellar master operon (flhDC) (Karlinsey et al, 2000). We induced fliK 45 min after addition of Tc followed by an additional 10 or 30 min growth to determine if FliK could induce the secretion-specificity switch in HBBs with elongated hooks. Figure 2A shows an in vivo analysis of this specificity switch using detection of flagellar filaments by immunostaining as a marker for the switch to late-substrate secretion. Flagellar filaments will only form if the secretion apparatus flipped to late-secretion mode by interaction of the ruler molecule FliK with the FlhB component of the secretion system. For a quantitative analysis of the number of HBBs in a synchronized culture that flipped to late-substrate secretion, HBB complexes and filaments were immunostained using hook- and filament-specific antibodies (Figure 2A). As displayed, almost every HBB switched to late, filament-type secretion, if FliK was induced late after the hooks had polymerized beyond their physiological length. Importantly, after only 10 min of fliK induction, short filaments were attached to nearly every HBB, where observed (Figure 2A, ‘late FliK 10 min’). This suggested that the first ruler molecule secreted into HBBs with elongated hooks immediately flipped the specificity switch, as predicted by the infrequent ruler hypothesis. The switch allowed for the activation of late-substrate gene transcription by the secretion of an inhibitor of late gene transcription. Transcription of the filament gene fliC started approximately 5 min after induction of fliK, as shown in Figure 2B, and we could detect short filaments by immunofluorescence 10 min after fliK induction (Figure 2A). We observed an average filament length of 1.1 μm 10 min after late fliK induction (Figure 2C), corresponding to an approximate average growth rate of 0.22 μm/min, if filament gene transcription initiated 5 min after fliK induction (see above). We observed filaments of up to 7 μm in length 30 min after late fliK induction (or approximately 25 min after induction of filament gene transcription) and this translated to a maximal growth rate of up to 0.28 μm/min. Reported filament growth rates in vivo ranged from 0.10 to 0.55 μm/min (Stocker and Campbell, 1959; Iino, 1974). Figure 2.FliK induces secretion-specificity switch in hooks >wt length. (A) Immunostaining of assembled HBB complexes and filaments. ‘WT’: simultaneous induction of fliK and flagellar genes expression; ‘polyhook’: flagellar genes were induced without induction of fliK; ‘late FliK’: late fliK induction after 45 min of flagellar genes expression for 10 and 30 min, respectively. Tc was not removed prior to addition of Ara. Representative fluorescent microscopy images of strain TH16941 (PtetA-flhD+C+ ParaBAD-fliK+ ΔfliK flgE∷3xHA) are shown. DNA (blue), hooks (white) and filaments (green). Scale bar=2 μm. (B) Time lapse of fliC transcription after fliK induction. FliK expression in strain TH17502 harbouring a fliC-lac reporter was induced late after 45 min of flagellar genes expression. β-galactosidase activity was assayed according to Materials and methods. The inducer of flagellar gene expression, Tc, was not removed prior to addition of Ara. Data are presented as mean±s.d. of three independent, biological replicates. (C) Filament-length distribution after 10 and 30 min of late fliK induction compared with the simultaneous induction of fliK and flagellar genes expression (‘WT’). Number of filaments measured: ‘WT’=34, ‘10 min’=36 and ‘30 min’=47. Download figure Download PowerPoint In the control sample, where fliK expression was never induced, rarely a filament was observed (about 5–10% of detected HBBs) (Figure 2A, ‘polyhook’). The low frequency of filaments can be explained by a combination of spontaneous switching of the type III secretion apparatus to late secretion and basal expression of the ParaBAD-fliK allele that would result in some FliK expression and secretion. Next, we obtained the hook-length distribution of the model strain under different FliK induction conditions (Figure 3C). In the first sample, both flagellar genes and fliK were induced simultaneously (labelled ‘WT’ in the figure). In the second sample, only flagellar genes were induced (labelled ‘polyhook’ in the figure) and in the third sample, fliK expression was induced only after 45 min of flagellar gene expression (labelled ‘late FliK’ in the figure). In case of simultaneous expression of HBB genes and fliK405, an average hook length of 43±6 nm was observed (Figure 3C, left panel). This corresponds to the prediction of nine FliK molecules secreted per 42 nm hook under conditions, where FliK was over-expressed from the Ara promoter. The average hook length is approximately 12 nm shorter as previously observed under wild-type conditions (Hirano et al, 1994). However, this result can be explained by simultaneous, non-hierarchical expression of HBB genes and fliK, contrary to what is the case under wild-type conditions, and overproduction of fliK expressed from the strong ParaBAD promoter. In fact, it has been previously reported that over-expression of fliK produced shorter hooks (45±6 or 46±7 nm) (Muramoto et al, 1998; Minamino et al, 2009). The slight differences in hook length under FliK over-expression conditions can be explained by the fact that in previous over-expression experiments, a population of wild-type hooks was already present prior to the start of the FliK over-expression. However, we started expression of FliK and flagellar HBB genes simultaneously, which would result in the presence of over-produced FliK already before the construction of any hooks. Figure 3.Late FliK secretion induces secretion-specificity switch in elongated hooks. Left panels (WT): simultaneous induction of fliK and flagellar genes expression. Middle panels (polyhook): flagellar genes were induced without induction of fliK. Right panels (late FliK): late fliK induction after 45 min of flagellar genes expression. (A) Representative fluorescent microscopy images of strain TH16941 (PtetA-flhD+C+ ParaBAD-fliK+ ΔfliK flgE∷3xHA). Tc was removed prior to addition of Ara to prevent formation of nascent HBBs. Number of cells counted for the presence/absence of HBB–filament complexes: ‘WT’=400, ‘polyhook’=642, ‘late FliK’=321. Fraction of HBBs with attached filaments is given in the upper left corner. DNA (blue), hooks (white) and filaments (green). Scale bar=2 μm. (B) Representative electron micrograph images of hooks isolated from strain TH16791 (PtetA-flhD+C+ ParaBAD-fliK+ ΔfliK). Scale bar=50 nm. (C) Histogram of measured hooks of strain TH16791. Number of measured hooks: ‘WT’=66, ‘polyhook’=229, ‘late FliK’=228. (D) CDF of hooks measured for TH16791. Measured hook lengths shown as asterisks and Pi(L) (solid curve) computed from equation 8 using L*=470 nm. Download figure Download PowerPoint When fliK was not expressed in the polyhook sample, hook-length control was completely abolished with hooks up to 1.2 μm length (Figure 3C, middle panel). This is consistent with the hook-length distribution observed in a fliK deletion strain (Patterson-Delafield et al, 1973). Importantly, in the sample, where fliK expression was induced late after physiological HBB completion, hook length appears to be partially controlled (Figure 3C, right panel). Hooks longer than 500 nm were not observed contrary to the polyhook sample and the histogram revealed a prominent population of 42±5 nm. In this sample, fliK was induced for 30 min, while the inducer for HBB genes was still present due to experimental limitation that did not allow us to remove the inducer of HBB genes. Accordingly, production of nascent ‘wild-type’ HBBs during the 30-min time frame, where both HBB inducer and FliK were present, accounted for this prominent peak. Induction of HBB genes for 30 min is sufficient to allow formation of wild-type HBB as shown previously (Karlinsey et al, 2000). This explains the prominent ‘wild-type’ peak at 42 nm. It is important to stress, however, that the isolation procedure for HBBs requires either very long hooks (e.g. as found in a polyhook phenotype) or attached filaments and this suggested that the longer hooks up to 500 nm length indeed switched to late-substrate secretion and had a filament attached during the HBB preparation. Wild-type HBBs were re-purified from strain TH16791 (ParaBAD-fliK405) and independently imaged on a different electron microscope to confirm our length measurements. We observed an average hook length of 43±5 nm, the same as described above (Supplementary Figure S6). In order to exclude that only nascent HBBs switched to filament-type secretion after late FliK induction (e.g. as seen in Figure 2), we repeated the late fliK induction experiment under conditions, where Tc, the inducer of flagellar genes, was removed after 45 min before the induction of fliK by the addition of Ara (Figure 3A). This ensured that during the following 30 min of FliK expression, no nascent HBBs were produced and FliK was secreted through old HBBs with elongated hooks. Under these conditions, approximately 95% of detected HBBs indeed switched to filament-type secretion (Figure 3A, right panel). In the Materials and methods, we present a mathematical model of the infrequent ruler mechanism that allowed us to use wild-type hook-length data and polyhook data to predict the length distribution of hooks produced by late fliK induction. First, we used the experimentally obtained hook-length data from the wild-type sample (Figure 3D, left panel) to estimate the function Pc(L), the probability of FliK interaction with FlhB at hook-length L. The length of the wild-type ruler was captured by the parameter = 39 nm. The cumulative distribution function (CDF) P(L) for this data is shown in Figure 3D, with data points shown as asterisks. An estimate of Pc(L) is shown in Supplementary Figure S3B. The second data set is polyhook data, determined from a culture in which there was no fliK induction. The culture was grown for 75 min. The histogram of lengths is shown in Figure 3C (middle panel) and the CDF Pp(L) for this collection of polyhooks is shown in Figure 3D (middle panel). The third type of data is from a culture grown for 75 min, with induction of fliK at time T0=45 min (Figure 3, right panel). Figure 3D (right panel) shows the CDF of the data (shown as asterisks) and the predicted CDF Pi(L) determined from equation (8), using the functions Pc(L) and Pp(L). The agreement between the curve Pi(L) and the data is striking. There is some difference, however, which is possibly explained by the fact that in the derivation of equation (8), the velocity of hook growth is assumed to be constant, independent of length. A better estimate of the length distribution at the time of induction would require more detailed knowledge of the velocity of hook growth as a function of length. In spite of this caveat, however, the excellent agreement between the late fliK induction data and the prediction based on information from sample 1 (WT) and sample 2 (polyhook) gives strong evidence in favour of the hypothesis that hook-length determination is by an infrequent ruler mechanism with a switching probability function Pc(L). This analysis was further applied to several data sets with varying induction times T0. The results with the same agreement are shown in Figure 4 (T0=45, 55 and 65 min). Figure 4.Late induction of fliK at varying times T0. Strain TH16791 (PtetA-flhD+C+ ParaBAD-fliK+ ΔfliK) was grown in the presence of Tc (inducer of flagellar genes) for (A) 45 min, (B) 55 min and (C) 65 min. Afterwards, fliK expression was induced by addition of Ara for a total sample time of 80 min. Left panels: histogram of measured hooks. Number of measured hooks: ‘T0=45 min’=286, ‘T0=55 min’=461, ‘T0=65 min’=134. Right panels: CDF of measured hooks, data shown as asterisks and Pi(L) (solid curve) computed from equation 8 using L*=600 nm (T0=45 min), 440 nm (T0=55 min) and 260 nm (T0=65 min). Download figure Download PowerPoint Secretion of FliK deletion and insertion alleles in elongated hooks immediately induced the secretion-specificity switch To further assess the ability of late FliK secretion in triggering the specificity switch in hooks greater than the physiological length, we engineered FliK deletion and insertion variants and tested their ability to control hook length after late fliK induction. First, a long FliK variant was generated by inserting a 164-amino-acid fragment of YscP between amino acids 140 and 141 of FliK, resulting in FliK570. A short FliK variant was constructed by deleting amino acids 161 through 202 of FliK, resulting in FliK363. FliK570 (reported hook length 81.6±9.5 nm) and FliK363 (reported hook length 43.5±8.0 nm) retain hook-length control if expressed from the native PfliF promoter (Shibata et al, 2007). In order to allow for inducible expression, both FliK variants were expressed from the chromosomal ParaBAD promoter. The ability of the FliK variants to flip the specificity switch to late-substrate secretion was first analysed by filament immunostaining (Figure 5A; Supplementary Figure S4A). Under conditions where nascent HBBs were no longer produced after fliK induction, late FliK secretion switched 91% (FliK570) and 96% (FliK363) of the detected HBBs to filament-type secretion (Figure 5A). Next, th