Molecular ruler determines needle length for the Salmonella Spi-1 injectisome

Daniel H. Wee; Kelly T. Hughes

doi:10.1073/pnas.1423492112

Edited by Philippe J. Sansonetti, Institut Pasteur, Paris, France, and approved February 11, 2015 (received for review December 8, 2014)

Significance

Type-III secretion systems facilitate the virulence capability of Gram-negative bacterial pathogens. They are part of complex nanomachines that include the bacterial flagellum, which confers motility, and the injectisome, which secretes proteins from the infecting bacterial cytoplasm into eukaryotic host cells. Here we examine the mechanisms of length control for the main component of the injectisome and flagellar basal structures, the needle and the hook, respectively. The defined lengths of the hook and needle are important for their function. We find that both systems use a secreted, molecular ruler to effect length determination at the nanometer-scale. Understanding the mechanisms of length control in these systems provides insight into bacterial pathogens and a biological model for the controlled construction of nanomaterials.

Abstract

The type-III secretion (T3S) systems of bacteria are part of self-assembling nanomachines: the bacterial flagellum that enables cells to propel themselves through liquid and across hydrated surfaces, and the injectisome that delivers pathogenic effector proteins into eukaryotic host cells. Although the flagellum and injectisome serve different purposes, they are evolutionarily related and share many structural similarities. Core features to these T3S systems are intrinsic length control mechanisms for external cellular projections: the hook of the flagellum and the injectisome needle. We present evidence that the Spi-1 injectisome, like the Salmonella flagellar hook, uses a secreted molecular ruler, InvJ, to determine needle length. This result supports a universal length control mechanism using molecular rulers for T3S systems.

Length control mechanisms of macromolecular structures are used throughout biology for maintaining cellular functions (1 ⇓⇓⇓⇓–6). Mechanisms of length control have evolved independently and range from the kinetically controlled length of the metaphase spindle involved in eukaryotic cell-division, to the scaffolds controlling bacteriophage tail-lengths (2, 5). One of the best-studied and controversial mechanisms of length control is that of bacterial Type III secretion (T3S) system, which includes the virulence-associated injectisome of Salmonella pathogenicity island 1 (Spi-1), and the bacterial motility organelle, the flagellum.

The injectisome is a hypodermic needle-like organelle used by pathogenic Gram-negative bacteria to inject effector proteins into eukaryotic host cells (7). This complex organelle is embedded in the membranes and cell wall of Gram-negative bacteria. The basal structure of the injectisome includes a rod-like component that spans the periplasmic space between the inner and outer membranes, and an injectisome-specific T3S apparatus within the cytoplasmic membrane (7 ⇓–9). The T3S system associated with the injectisome secretes proteins required for its structure and assembly, as well as effector proteins into host cells to facilitate pathogenesis. Extending from the injectisome basal-body at the cell surface is a rigid needle-like structure, which grows to defined lengths for different bacterial species, and serves as a conduit between the bacterium and the host cell (10 ⇓–12). At the tip of the needle is the translocon, which allows the needle to dock to the host membrane (13 ⇓–15) (Fig. 1A).

Fig. 1.

(A) Schematic of axial components in a cross-section of the Spi-1 injectisome. (B) Schematic of axial components of the bacterial flagellum. (C) Model of hook-length determination by the infrequent ruler mechanism: (i) The secreted N terminus of FliK (FliK_N), shown in red halts hook polymerization and is slow to diffuse. (ii) The lack of interactions in short hooks or the folding of the secreted FliK_N as it exits the secretion channel rapidly pulls the FliK molecule past FlhB (shown in orange) through the channel without induction of the secretion-specificity switch. (iii) FliK is secreted outside of the cell and hook polymerization continues. (iv) The hook has grown to the physiological length and a new FliK molecule is secreted, again halting hook growth. (v) The C terminus of FliK (FliK_C) is now closely aligned to FlhB, the slower rate of FliK secretion provides sufficient time for a productive FliK–FlhB interaction that induces the secretion-specificity switch. (D) The static ruler model for length control of the Yersinia injectisome: (i) A single YscP (shown in red) molecule enters the secretion channel and remains there during needle polymerization. The N terminus of YscP interacts with the needle or tip-complex and is pulled through as the needle continues to polymerize, bringing the C terminus of YscP (YscP_C) near the secretion apparatus (YscU – shown in orange). (ii) When YscP_C and YscU are close enough to interact, a secretion specificity switch occurs, halting needle polymerization. (E) The inner-rod model of length control of the Spi-1 injectisome: Here, both the inner-rod (shown in green) and needle (shown in blue) are assembled simultaneously. (i) Assembly of the inner-rod is facilitated by InvJ (shown in red). (ii) The completion of the inner-rod leads to substrate switching and the interruption of secretion of the inner-rod and needle proteins, thus determining the length of the needle substructure.

Like the injectisome, the flagellum contains a membrane and cell wall spanning basal-body, which also functions as a rotary motor in motility, and includes an integral membrane-associated, flagellar-specific T3S apparatus (16, 17) (Fig. 1B). From the basal-body at the cell surface extends a flexible tube, called the hook, which also grows to defined lengths and serves as a universal-joint to transmit torque generated by the basal-body to a long (up to 20 µm) helical propeller-like structure called the flagellar filament (18, 19). A defining characteristic of T3S systems is an intrinsic length control mechanism for hook and needle length that is associated with a switch in the type of substrates that are recognized and exported by the secretion apparatus (3, 4, 20, 21). For the flagellum of Salmonella Typhimurium, assembly of the basal-body is followed by the secretion of ∼130 FlgE protein subunits that polymerize to form the hook (19). When the hook reaches a terminal length of ≥ 40 nm, the T3S apparatus switches secretion specificity to filament or late-type secretion substrates to complete flagellum assembly (20, 22). Here, the length of the hook and induction of the secretion specificity switch is determined by a secreted, molecular ruler called FliK (23 ⇓–25).

Assembly of the injectisome proceeds in a similar fashion to the flagellum; however, the ring structures that are in the outer membrane form independent to the inner membrane-associated components of the basal-body (10, 26, 27). Subunits that make up an inner-rod structure that spans the cell wall and periplasmic space and subunits that assembly into the external needle are part of the early class of secreted substrates (8, 12). The injectisome is completed when the needle reaches an optimal length after which, the T3S system switches specificity for late secreted substrates including the translocon and effector proteins (10, 21, 28).

Many mechanisms have been proposed for length determination for the hook of the flagellar system and the needle of the injectisome system. Length control of the flagellar hook and injectisome needle by a molecular ruler was first proposed based on work on the Yersinia spp. injectisome system (3). For the injectisome system of Yersinia enterocolitica, the needles grow to a length of 58 ± 10 nm, whereas Yersinia pestis needles grow to a length of about 41 nm (3). It was shown that the N-terminal length of YscP, a nonstructural, early secretion substrate and functional homolog of FliK, determined needle lengths of the Yersinia injectisomes (10, 29, 30). An important difference between the YscP molecules from Y. enterocolitica and Y. pestis is that Y. pestis YscP is 60 amino acid residues shorter in length, which corresponded to shorter needles. Increasing or decreasing the lengths of the N-terminal domain of YscP or FliK resulted in a linear correlation between the length of the needle and the length of YscP, or the length of the hook and FliK (3). Additionally, the C-terminal domain of YscP and FliK is believed to interact with the component of the T3S apparatus that governs secretion specificity, known as YscU in Yersinia spp. and FlhB in the flagellar system. Loss of YscP/FliK, or dominant mutant alleles in the T3S switch component yscU/flhB, will give rise to a poly-needle and poly-hook phenotype, respectively, where the needles or hooks polymerize indefinitely and do not switch secretion specificity modes (21, 31).

Although both the flagellum of S. Typhimurium and injectisome of Yersinia spp. use molecular rulers for controlling length, the mechanisms for how measurements are achieved are proposed to work in different ways (22, 28, 32). In Yersina injectisomes, a static-ruler model has been proposed (32). Here, the ruler molecule remains anchored by its N terminus to the tip of the needle in the secretion channel as the needle substrates are secreted and polymerize into the growing needle. The anchored YscP is pulled along with the growing needle until its C terminus is in proximity to the secretion apparatus to induce the secretion specificity switch (Fig. 1D). Alternatively, in the flagellar system, FliK has been shown to act as an infrequent ruler, with multiple FliK molecules secreted through the growing structure until a minimal hook-length is achieved (22, 33). In this model, the probability of inducing the secretion specificity switch is a function of the velocity of FliK secretion, which is inversely correlated to hook length. FliK enters the secretion channel and proceeds to the hook-tip and exits the cell from its N terminus (FliK_N) followed by the C terminus (FliK_C). Once FliK_N exits the cell, FliK_C continues through the channel at an increased rate of secretion that is too fast to allow FliK_C to interact with FlhB (22). It is only when the hook has reached a sufficient length where FliK_C can interact with FlhB prior to the exit of FliK_N from the cell does the secretion specificity switch at FlhB occur (Fig. 1C). Interestingly, injectisomes in S. enterica were proposed to measure needle lengths by a mechanism that is different from those proposed for FliK and YscP.

S. enterica encodes two injectisomes: the Salmonella pathogenicity island 1 (Spi-1) and Salmonella pathogenicity island 2 (Spi-2) injectisomes, which are required to cause gasteroenteritis in humans (34). The Spi-1 needle is composed of PrgI subunits that polymerize to a length of ∼25 nm before undergoing the secretion specificity switch. An early secretion substrate, InvJ, was believed to be the molecular ruler analog in Spi-1 injectisomes, because its absence results in a poly-needle phenotype; however, these injectisomes were reported to be missing their inner-rod structure (10, 28). Additionally, overexpression of the inner-rod subunit protein PrgJ was found to result in slightly shorter needle lengths, giving rise to an inner-rod length-control model for the Spi-1 needle (28). In this model, needle length is determined by the rate of inner-rod assembly, where the complete inner-rod structure presumably interacts with the T3S apparatus to induce the secretion specificity switch, thus terminating needle assembly (Fig. 1E). A recent study supporting the inner-rod model reports the isolation of mutant PrgJ-alleles that give rise to abnormally long needles that still undergo the secretion specificity switch and assemble the inner-rod (35). It is reasoned that these mutant alleles are defective in PrgJ–PrgJ interactions causing slower inner-rod assembly, and therefore longer needles. Still, it remained unclear as to what role InvJ plays in Spi-1 injectisome needle length control. In this study, we address the hypothesis that InvJ, like its functional homologs FliK and YscP, acts as a molecular ruler in determining needle length for the Spi-1 injectisome. Our work demonstrates that InvJ acts as a molecular ruler and supports a universal molecular ruler model for length control in T3S systems.

Results

InvJ Acts as a Molecular Ruler to Determine Needle Length.

Hydrophobic cluster analysis of T3S system molecular rulers, including InvJ, FliK, and YscP, has revealed a conserved C-terminal domain that interacts with the T3S apparatus causing the secretion specificity switch and subsequent termination of needle or hook polymerization (29). Previous studies provide evidence to suggest that the N terminus of InvJ (InvJ_N) is the ruler domain for determining needle length, and its C terminus (InvJ_C) is responsible for inducing the secretion specificity switch by interacting with the FlhB/YscU homolog in the Spi-1 T3S apparatus, SpaS, to induce the secretion specificity switch, as is the case for FliK and YscP (29, 36, 37). Increasing or decreasing the length of the ruler molecule in either the flagellar system (FliK), or the injectisome system of Yersinia spp. (YscP) results in a linear correlation in hook or needle length, respectively (3, 4).

Because Spi-1 injectisome needle-length is reportedly controlled by PrgJ copolymerization with PrgJ inner-rod subunits, we expected that, unlike FliK and YscP, varying InvJ lengths should have no effect on needle length. Conversely, if InvJ acted like FliK and YscP as a molecular ruler, then amino acid insertions that lengthen the N-terminal region of InvJ should result in needles with length increases corresponding to the number of amino acids inserted.

Segments of varying lengths from the ruler domain of YscP were used to extend the length of InvJ. These fragments were inserted into various positions before amino acid 220 in InvJ to avoid possible disruption of the C-terminal region (InvJ_C). The Spi-1 master-regulatory gene, hilD, was expressed from an arabinose promoter (P_araBAD-hilD⁺⁺) to control Spi-1 induction. In addition, cells were cultured in Spi-1 inducing media (38). Cells were harvested, and intact injectisomes were isolated, negatively stained, and imaged by transmission electron microscopy. Needle lengths were measured and distributed along a histogram as shown in Fig. 2A.

Fig. 2.

(A) Negatively stained electron-micrographs of intact injectisomes isolated from strains containing varying InvJ lengths (Lower Right). Histogram of measured needle lengths from corresponding micrographs from the right (Left). Number of measured injectisomes (n), mean length of injectisomes, and number of additional amino acids in InvJ are indicated above the electron micrographs (Upper Left). The red-line on the histogram denotes the Gaussian distribution. (B) Average needle lengths plotted against the number of amino acid residues in InvJ.

The wild-type needle length of the Spi-1 injectisome was 20.8 ± 4 nm. This length is similar to the wild-type peak lengths reported in Marlovits et al. (28). We next measured needle lengths for the InvJ length-variants. All InvJ length-variants tested produced needles of controlled lengths that increased with increased InvJ lengths. This conclusion was supported by the Gaussian distribution for needle lengths plotted on a histogram (Fig. 3A). When the mean needle-lengths of each InvJ length-variant were plotted against their corresponding InvJ lengths, a linear correlation of ∼0.2 nm per inserted amino acid residue was observed (Fig. 2B). This result is similar to the 0.17 and 0.19 nm per amino acid residue correlations that were reported for Hook–FliK and Needle–YscP lengths, respectively (3, 4). The spacing between amino acids is 0.17 nm in an alpha-helix and 0.4 nm in an extended peptide structure (39). Thus, the change in needle length we observed with amino acid additions in InvJ closely associates with the distance between amino acid residues in an alpha-helix, which is the predicted secondary structure of the inserted fragments of YscP (30). We also verified that all InvJ length variants were secreted (Fig. S1). This finding supports the model that InvJ acts as a secreted molecular ruler. There was no effect of the inserted YscP sequences on the amount of InvJ that was produced or secreted, further supporting that the observed needle length variation was due solely to InvJ length variation.

Fig. 3.

(A) Western blot analysis of cultured supernatant (S), periplasmic (P), and cellular (C) fractions for the presence of InvJ (Upper) and DnaK (Lower) in wild-type, ∆invJ and ∆prgJ backgrounds. (B) Western blot analysis of cultured supernatants (Upper) and cellular (Lower) fractions for the presence of InvJ in wild-type (WT), invJ (InvJ++), prgJ (PrgJ++), and prgI (PrgI++) overexpression backgrounds. (C) Relative levels of InvJ secretion from blots show in B to wild-type (WT = 1).

Strains deleted for the C terminus of InvJ (InvJ_∆C) produced the poly-needle phenotype of an invJ null mutant (Fig. 4). These mutants did not induce the secretion specificity switch, and secreted InvJ_∆C at wild-type levels (Figs. S2 and S3). This finding further demonstrates similarities between InvJ and the well characterized YscP and FliK molecular ruler systems. This result also supports the requirement of InvJ_C to induce the secretion specificity switch and suggests that length control is universal in all T3S systems.

Fig. 4.

Electron micrographs of negatively stained whole cells from chromosomal-prgJM83A (Top Left), chromosomal-prgJS75A (Top Right), invJ_∆C (Bottom Left). PrgJ S75A. Arrows indicate needle structures. Electron micrograph of purified injectisome from chromosomal-prgJV81A (Middle Left) and prgI–prgJ overexpression strain (Right) clearly display the short and elongated needles produced from these strains, respectively.

Secretion Competition Between Early T3S Substrates Affects Needle Length.

The hypothesis that injectisome needle-lengths were controlled by copolymerization with the inner-rod structure was in part an interpretation of the effect of prgJ overexpression on needle length. Marlovits et al. (28) reported that wild-type S. enterica produced injectisomes with needle-lengths varying between 7 and 57 nm and a peak length around 25 nm. When the inner-rod protein, PrgJ, was overproduced, they observed injectisomes with needle-lengths varying between 7 and 47 nm and a peak length of about 20 nm. Furthermore, cryo-EM analysis of poly-needles from a ∆invJ mutant revealed that they lacked the inner-rod structure. The 10% reduction in average needle length by PrgJ overexpression and lack of an inner-rod structure in the ∆invJ mutant led Marlovits et al. to propose the needle length control model based on the rate of PrgJ polymerization. This model purports that InvJ facilitates inner-rod assembly and copolymerizes with the needle in the periplasm. Completion of the inner-rod structure induces the secretion specificity switch and terminates needle assembly. Therefore, overexpression of PrgJ resulted in a faster completion of the inner-rod, and thereby shorter needle structures.

Our findings suggest that the simplest interpretation of their data would be that needle length is shortened due to overexpression of a secretion competitor. Excess PrgJ could reduce the secretion rate of PrgI resulting in an increase in the frequency of InvJ measurements during needle elongation. Alternatively, overproduction of PrgJ could somehow facilitate InvJ secretion. Either possibility could affect the frequency of InvJ ruler measurements and needle length and predict that overproduction of PrgJ would lower InvJ concentrations outside of the cell. Furthermore, PrgI overexpression, which produces elongated needles, should also show reduced InvJ secretion.

InvJ was proposed to interact with PrgJ for inner-rod assembly. We tested whether InvJ is secreted in the absence of PrgJ. If InvJ secretion was independent of PrgJ, we expected to detect InvJ secreted into the periplasm. The periplasmic fraction from the ΔprgJ strain was isolated following osmotic shock and examined for the presence of InvJ by Western blot analysis. As expected, in the wild-type background InvJ was present in the cellular, periplasmic, and supernatant fractions, whereas, in the prgJ mutant, InvJ was not secreted to the supernatant, but was secreted to the periplasm (Fig. 3A). This finding indicates that PrgJ is not necessary for InvJ secretion.

We then investigated how excess levels of PrgJ or PrgI affected InvJ secretion, and thereby needle length. A copy of prgI, prgJ, and invJ coding regions were cloned into plasmid pTrc99A containing an IPTG-inducible promoter (P_trc), to produce an excess of PrgJ, PrgI or InvJ secretion substrates, respectively (40). The amount of InvJ secreted in the overproduction strains along with a wild-type control was determined by Western blot analysis of the extracellular fractions.

Overexpression of PrgJ resulted in ∼40% reduction in InvJ secreted levels compared with the control with PrgJ expressed only from its native Spi-1 locus (Fig. 3C). Both strains had comparable levels of cellular InvJ to wild type (Fig. 3B). We also observed the same reduction in the InvJ secreted level when PrgI was overproduced. Overproduction of either PrgJ or PrgI resulted in a reduction of the InvJ secreted level by about one third. Thus, the overexpression of one Spi-1 secretion substrate results in reduced secreted levels of another Spi-1 secretion substrate.

Mutant Alleles of prgJ Reported to Affect Needle Length Control Show Wild-Type Needle Lengths When Expressed from the Chromosome.

A recent study by Lefebre and Galán (35) identified mutations in the inner-rod protein, PrgJ, which gave rise to aberrantly long needles that were competent in inducing the secretion specificity switch. Lefebre and Galán argued that these results suggested a defect in PrgJ–PrgJ interactions that caused inner-rods to assembly at a slower rate, allowing the needle to polymerize to longer lengths before the inner-rod was able to induce the secretion specificity switch. However, the Lefebre and Galán study cloned all PrgJ mutants together with a wild-type copy of the needle subunit, PrgI, into a low-copy plasmid expression system (pWSK29) to maintain a wild-type PrgJ:PrgI ratio (35). We reasoned that the abnormally long needles produced by mutant PrgJ alleles was simply an artifact of PrgI overexpression, because previous studies have shown aberrantly long needle lengths greater than 92 nm were obtained when PrgI was expressed from the same low-copy plasmid (28). Length control by a molecular ruler mechanism predicts that overexpression of PrgI would produce longer needles because the frequency of InvJ secretion relative to the rate of needle polymerization would be reduced, allowing needles to polymerize beyond their wild-type lengths before encountering a ruler molecule.

We investigated whether the prgJ needle-length control mutations would produce the extended needle structures when expressed at wild-type levels from the chromosome and not subject to potential overexpression artifacts. We chose three mutations in prgJ (S75A, V81A, and M83A) that were reported to give rise to long needles and introduced these mutations into the chromosome prgJ locus (35). None of the three chromosomal prgJ mutants gave rise poly-needles; the needles were similar to those observed with wild-type (prgJ⁺) allele (Fig. 4).

We also examined whether overexpression of prgI and prgJ at wild-type ratios would result in elongated needles. We cloned the coding region of prgI–prgJ into the plasmid pTrc99a to produce excess amounts of PrgI and PrgJ subunits at wild-type ratios. Consistent with our data, we found that needles grow beyond wild-type lengths (40). It is possible that the elongated needles from the prgJ mutant strains reported by Lefebre and Galán (35) were due to the overexpression of prgI (Fig. 4). Although the pWSK29 plasmid used in their study is a lower copy number plasmid than the pTrc99a backbone used in this study. Plasmid copy number differences could account for the differences observed between these two studies. We would not expect plasmid copy number to affect needle length according to the inner-rod model, provided that prgI and prgJ coexpression does not change. Our findings provide further evidence that needle length is not determined by the rate of inner rod versus needle polymerization, but rather support a molecular ruler model where the frequency of measurements relative to the rate of needle polymerization determines needle length.

Discussion

In 1972, Silverman and Simon reported hook-length control mutants for the flagellum of Escherichia coli, and the following year hook-length control mutants were reported for Salmonella Typhimurium (41). Loss-of-function alleles of a gene, later named fliK, resulted in the uncontrolled hook-lengths in these two species. More than two decades later, the laboratories of Shin-Ichi Aizawa, Kazuhiro Kutsukake, and Robert Macnab began the study of FliK-dependent hook-length control in the Salmonella flagellar system (23, 42, 43). Hook growth is terminated through an interaction between the C terminus of FliK (FliK_C) and an integral cytoplasmic membrane component of the flagellar T3S (fT3S) system, FlhB, which is conserved in all T3S systems (25, 44). The conformation of FlhB determines whether the fT3S system is specific for the rod-hook-type or early class of secretion substrates or for the filament-type of late class of secretion substrates (45 ⇓–47). Before the N terminus of FliK exiting the secretion channel, the secretion rate is slow, but in structures with short or no hooks, the C terminus of FliK is not in the vicinity of FlhB when the N terminus is exiting the channel to catalyze the secretion-specificity switch. Once hooks reach a length where the entire molecule of FliK remains within the extended hook channel, FliK is passing through the channel at a slow rate when FliK_C is in the vicinity of FlhB to induce the secretion-specificity switch (22).

Similarly, for the virulence-associated type III secretion (vT3S) system of the Salmonella pathogenicity island 1 (Spi-1) injectisome, loss of the FliK functional homolog, InvJ, resulted in injectisome structures with needles of uncontrolled lengths (10). An interaction between the C terminus of InvJ and the Spi-1 FliK homolog, SpaS, is thought to produce a conformational change in SpaS that results in the cessation of needle secretion and the initiation of late substrate secretion, which includes virulence effector proteins (36).

The idea that both FliK and InvJ could act as molecular rulers came from work in the laboratory of Guy Cornelis (3). Like Salmonella Typhimurium, Yersinia pestis and Y. enterocolitica possess vT3S systems with a FliK functional homolog, YscP, and an FlhB homolog, YcsU (31). Loss-of-function mutants in the yscP gene of either species resulted in needles of uncontrolled lengths, whereas lengthening or shortening YscP produced injectisomes with longer or shorter needles corresponding to 0.19 nm per amino acid inserted or deleted.

Following the work of Cornelis and his colleagues, it was shown that the FliK protein also acted as a molecular ruler. Lengthening or shortening FliK produced flagellar structures with longer or shorter hooks corresponding to 0.17 nm per amino acid inserted or deleted (4). Further studies revealed that FliK acted to measure a minimal length of about 40 nm (22). Additionally, the YscP/FliK homolog of Shigella flexerni’s injectisome, Spa32, has also been demonstrated to increase needle length when additional amino acids are introduced (48). We decided to test whether InvJ could act as a molecular ruler in the Spi-1 system, regardless of the presence of additional length control by inner-rod completion. Just like YscP and FliK, increasing the length of InvJ corresponded to increased needle lengths of ∼0.2 nm per amino acid inserted, confirming that InvJ acts as a molecular ruler to control Spi-1 needle length. Furthermore, like the molecular rulers YscP and FliK, the C terminus of InvJ proved to be essential for inducing the Spi-1 secretion specificity switch.

The effect of secretion substrate competition on Spi-1 needle-length control explains the shorter needle lengths when PrgJ was overproduced in Marlovits et al. (28). The reduction in needle length by prgJ overexpression was a key factor leading to the inner-rod model of length control. As was previously shown with FliK in the flagellar system, increasing the frequency a ruler molecule enters the secretion channel during assembly of the structure being measured will lead to a more accurate measurement of the needle and produce shorter needle lengths on average (49 ⇓–51). Alternatively, increasing the rate of hook or needle polymerization would have the added effect of decreasing the frequency a molecular ruler enters the secretion channel to make a measurement and further contribute to the longer hook or needle lengths observed. In Lefebre and Galán (35), they report mutants in prgJ that correspond to increased needle lengths. However, in that study, prgI was coexpressed with the prgJ mutants under an inducible promoter in a plasmid-based expression system, and not from the chromosome (35). Based on previous reports, we believe that having prgI coexpressed with prgJ from a plasmid led to the overproduction of needle subunits and an increase in the rate of needle assembly (28). Our own findings show normal needle length when prgJ length control mutants were expressed from their native locus. Our study induces Spi-1 by expression of hilD from the arabinose promoter, which could also account for some of the differences between the two studies. However, the data in Fig. 2 demonstrating a direct correlation between InvJ and needle lengths is consistent with a molecular ruler model and not with an inner-rod model.

The question for us is not how PrgJ measures needle length, but how PrgJ affects InvJ and PrgI secretion. The experimental results we report here show experimental evidence in support for a conserved molecular ruler mechanism of length control across all T3S systems. Additionally, we show that secretion competition between early Spi-1 substrates can affect needle lengths by altering frequency of measurements with respect to the rate of needle polymerization.

Materials and Methods

Bacterial Strains and Culture Conditions.

Cells were cultured in lysogeny broth (LB) at 0.3 M NaCl. Arabinose (0.2% wt/vol) was supplemented to cultures as needed. The generalized transducing phage of S. Typhimurium P22 HT105/1 int-201 was used in all transduction crosses (52). InvJ variants were constructed using the Lambda-RED method (53).

Injectisome Isolation, Electron Microscopy, and Needle Measurements.

Needle purification was carried out by the methods described in Marlovits et al. (28). Purified injectisomes were negatively stained with 2% (wt/vol) uranyl acetate or 2% (wt/vol) PTA on carbon-formvar coated copper grids. Images were captured user a Hitachi H-7100 electron microscope at an acceleration voltage of 125 kV. Needle lengths from intact injectisomes were measured using NIH ImageJ 1.42q software, and the average needle length was determined by nonlinear regression analysis of the Gaussian distribution using GraphPad Prism 5.0c software.

Protein Secretion Assay, Osmotic Shock, SDS/PAGE, and Western Blotting.

Protein secretion was analyzed by Western blot. Overnight cultures were diluted 1:100 in 0.3 M NaCl LB media supplemented with 0.2% arabinose and grown until the cultures reached an optical density at 600 nm of ∼0.8–1.0. Cell cultures were then centrifuged for 10 min, and the supernatant was collected and filtered through a low-protein binding 0.45-µm cellulose acetate filter (Tisch Scientific syringe filter) to remove all cells from the supernatant fraction. Aliquots (2 mL) of the filtered supernatant were then mixed with 10% TCA (final concentration) to precipitate the secreted proteins, and washed with ice-cold acetone before further analysis.

Isolation of the periplasmic fraction was performed by osmotic shock. Harvested cells were resuspended in 20% Sucrose, 1 mM EDTA, 30 mM Tris⋅HCl solution at pH 8.0. Cultures were incubated for 10 min at room temperature, then centrifuged at 13,000 × g for 10 min at 4 °C. The resulting supernatant was removed and the pelleted cells were rapidly resuspened in ice-cold water. Cultures were incubated at 4 °C for 10 min, then centrifuged at 13,000 × g for 10 min at 4 °C. The resulting supernatant, which contains the periplasmic fraction, was collected and mixed with 10% TCA (final concentration).

Precipitated proteins from the supernatant and periplasmic fractions were dissolved in Laemmli sample buffer and adjusted to the sample with the lowest OD unit (∼200 OD units per µL). Fifteen microliters of each sample (equivalents of ∼3,000 OD units) were loaded onto a 12% SDS/PAGE gel. Proteins were analyzed by immunoblotting using anti-InvJ antibodies (rabbit) and visualized using secondary anti-rabbit-700 antibodies (LI-COR). Protein density was obtained using NIH ImageJ 1.42q software.

Acknowledgments

We thank Samuel I. Miller for providing InvJ antibody, and the K.T.H. laboratory, Marc Erhardt, and James P. Keener for helpful discussions and critical readings of the manuscript. This work was supported by PHS (Public Health Service) Grant GM056141 from the National Institutes of Health.

Footnotes

↵¹To whom correspondence should be addressed. Email: daniel.wee{at}utah.edu.

Author contributions: D.H.W. and K.T.H. designed research; D.H.W. performed research; D.H.W. and K.T.H. analyzed data; and D.H.W. and K.T.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1423492112/-/DCSupplemental.

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(2007) FliK regulates flagellar hook length as an internal ruler. Mol Microbiol 64(5):1404–1415
.
OpenUrl CrossRef PubMed
↵
1. Katsura I,
2. Hendrix RW
(1984) Length determination in bacteriophage lambda tails. Cell 39(3 Pt 2):691–698
.
OpenUrl CrossRef PubMed
↵
1. Oda T,
2. Yanagisawa H,
3. Kamiya R,
4. Kikkawa M
(2014) A molecular ruler determines the repeat length in eukaryotic cilia and flagella. Science 346(6211):857–860
.
OpenUrl Abstract/FREE Full Text
↵
1. Kubori T, et al.
(1998) Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280(5363):602–605
.
OpenUrl Abstract/FREE Full Text
↵
1. Marlovits TC, et al.
(2004) Structural insights into the assembly of the type III secretion needle complex. Science 306(5698):1040–1042
.
OpenUrl Abstract/FREE Full Text
↵
1. Bergeron JRC, et al.
(2013) A refined model of the prototypical Salmonella SPI-1 T3SS basal body reveals the molecular basis for its assembly. PLoS Pathog 9(4):e1003307
.
OpenUrl CrossRef PubMed
↵
1. Kubori T,
2. Sukhan A,
3. Aizawa SI,
4. Galán JE
(2000) Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system. Proc Natl Acad Sci USA 97(18):10225–10230
.
OpenUrl Abstract/FREE Full Text
↵
1. Tamano K,
2. Katayama E,
3. Toyotome T,
4. Sasakawa C
(2002) Shigella Spa32 is an essential secretory protein for functional type III secretion machinery and uniformity of its needle length. J Bacteriol 184(5):1244–1252
.
OpenUrl Abstract/FREE Full Text
↵
1. Sukhan A,
2. Kubori T,
3. Galán JE
(2003) Synthesis and localization of the Salmonella SPI-1 type III secretion needle complex proteins PrgI and PrgJ. J Bacteriol 185(11):3480–3483
.
OpenUrl Abstract/FREE Full Text
↵
1. Broz P, et al.
(2007) Function and molecular architecture of the Yersinia injectisome tip complex. Mol Microbiol 65(5):1311–1320
.
OpenUrl CrossRef PubMed
↵
1. Blocker AJ, et al.
(2008) What’s the point of the type III secretion system needle? Proc Natl Acad Sci USA 105(18):6507–6513
.
OpenUrl Abstract/FREE Full Text
↵
1. Lunelli M,
2. Hurwitz R,
3. Lambers J,
4. Kolbe M
(2011) Crystal structure of PrgI-SipD: Insight into a secretion competent state of the type three secretion system needle tip and its interaction with host ligands. PLoS Pathog 7(8):e1002163
.
OpenUrl CrossRef PubMed
↵
1. Hueck CJ
(1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62(2):379–433
.
OpenUrl Abstract/FREE Full Text
↵
1. Macnab RM
(2004) Type III flagellar protein export and flagellar assembly. Biochim Biophys Acta Mol Cell Res 1694(1-3):207–217
.
OpenUrl CrossRef PubMed
↵
1. Macnab RM
(2003) How bacteria assemble flagella. Annu Rev Microbiol 57:77–100
.
OpenUrl CrossRef PubMed
↵
1. Samatey FA, et al.
(2004) Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism. Nature 431(7012):1062–1068
.
OpenUrl CrossRef PubMed
↵
1. Williams AW, et al.
(1996) Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium. J Bacteriol 178(10):2960–2970
.
OpenUrl Abstract/FREE Full Text
↵
1. Sorg I, et al.
(2007) YscU recognizes translocators as export substrates of the Yersinia injectisome. EMBO J 26(12):3015–3024
.
OpenUrl Abstract/FREE Full Text
↵
1. Erhardt M,
2. Singer HM,
3. Wee DH,
4. Keener JP,
5. Hughes KT
(2011) An infrequent molecular ruler controls flagellar hook length in Salmonella enterica. EMBO J 30(14):2948–2961
.
OpenUrl Abstract/FREE Full Text
↵
1. Hirano T,
2. Yamaguchi S,
3. Oosawa K,
4. Aizawa S
(1994) Roles of FliK and FlhB in determination of flagellar hook length in Salmonella typhimurium. J Bacteriol 176(17):5439–5449
.
OpenUrl Abstract/FREE Full Text
↵
1. Minamino T,
2. Doi H,
3. Kutsukake K
(1999) Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium. Biosci Biotechnol Biochem 63(7):1301–1303
.
OpenUrl CrossRef PubMed
↵
1. Minamino T,
2. Ferris HU,
3. Moriya N,
4. Kihara M,
5. Namba K
(2006) Two parts of the T3S4 domain of the hook-length control protein FliK are essential for the substrate specificity switching of the flagellar type III export apparatus. J Mol Biol 362(5):1148–1158
.
OpenUrl CrossRef PubMed
↵
1. Spreter T, et al.
(2009) A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system. Nat Struct Mol Biol 16(5):468–476
.
OpenUrl CrossRef PubMed
↵
1. Schraidt O,
2. Marlovits TC
(2011) Three-dimensional model of Salmonella’s needle complex at subnanometer resolution. Science 331(6021):1192–1195
.
OpenUrl Abstract/FREE Full Text
↵
1. Marlovits TC, et al.
(2006) Assembly of the inner rod determines needle length in the type III secretion injectisome. Nature 441(7093):637–640
.
OpenUrl CrossRef PubMed
↵
1. Agrain C, et al.
(2005) Characterization of a Type III secretion substrate specificity switch (T3S4) domain in YscP from Yersinia enterocolitica. Mol Microbiol 56(1):54–67
.
OpenUrl CrossRef PubMed
↵
1. Wagner S, et al.
(2009) The helical content of the YscP molecular ruler determines the length of the Yersinia injectisome. Mol Microbiol 71(3):692–701
.
OpenUrl CrossRef PubMed
↵
1. Edqvist PJ, et al.
(2003) YscP and YscU regulate substrate specificity of the Yersinia type III secretion system. J Bacteriol 185(7):2259–2266
.
OpenUrl Abstract/FREE Full Text
↵
1. Wagner S,
2. Stenta M,
3. Metzger LC,
4. Dal Peraro M,
5. Cornelis GR
(2010) Length control of the injectisome needle requires only one molecule of Yop secretion protein P (YscP). Proc Natl Acad Sci USA 107(31):13860–13865
.
OpenUrl Abstract/FREE Full Text
↵
1. Keener JP
(2010) A molecular ruler mechanism for length control of extended protein structures in bacteria. J Theor Biol 263(4):481–489
.
OpenUrl CrossRef PubMed
↵
1. Galán JE
(2001) Salmonella interactions with host cells: Type III secretion at work. Annu Rev Cell Dev Biol 17:53–86
.
OpenUrl CrossRef PubMed
↵
1. Lefebre MD,
2. Galán JE
(2014) The inner rod protein controls substrate switching and needle length in a Salmonella type III secretion system. Proc Natl Acad Sci USA 111(2):817–822
.
OpenUrl Abstract/FREE Full Text
↵
1. Zarivach R, et al.
(2008) Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS. Nature 453(7191):124–127
.
OpenUrl CrossRef PubMed
↵
1. Collazo CM,
2. Zierler MK,
3. Galán JE
(1995) Functional analysis of the Salmonella typhimurium invasion genes invl and invJ and identification of a target of the protein secretion apparatus encoded in the inv locus. Mol Microbiol 15(1):25–38
.
OpenUrl CrossRef PubMed
↵
1. Bajaj V,
2. Lucas RL,
3. Hwang C,
4. Lee CA
(1996) Co-ordinate regulation of Salmonella typhimurium invasion genes by environmental and regulatory factors is mediated by control of hilA expression. Mol Microbiol 22(4):703–714
.
OpenUrl CrossRef PubMed
↵
1. Pauling L,
2. Corey RB,
3. Branson HR
(1951) The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. Proc Natl Acad Sci USA 37(4):205–211
.
OpenUrl FREE Full Text
↵
1. Amann E,
2. Ochs B,
3. Abel KJ
(1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69(2):301–315
.
OpenUrl CrossRef PubMed
↵
1. Silverman MR,
2. Simon MI
(1972) Flagellar assembly mutants in Escherichia coli. J Bacteriol 112(2):986–993
.
OpenUrl Abstract/FREE Full Text
↵
1. Kutsukake K,
2. Minamino T,
3. Yokoseki T
(1994) Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium. J Bacteriol 176(24):7625–7629
.
OpenUrl Abstract/FREE Full Text
↵
1. Minamino T,
2. González-Pedrajo B,
3. Yamaguchi K,
4. Aizawa SI,
5. Macnab RM
(1999) FliK, the protein responsible for flagellar hook length control in Salmonella, is exported during hook assembly. Mol Microbiol 34(2):295–304
.
OpenUrl CrossRef PubMed
↵
1. Morris DP, et al.
(2010) Kinetic characterization of Salmonella FliK-FlhB interactions demonstrates complexity of the Type III secretion substrate-specificity switch. Biochemistry 49(30):6386–6393
.
OpenUrl CrossRef PubMed
↵
1. Minamino T,
2. Macnab RM
(2000) Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching. J Bacteriol 182(17):4906–4914
.
OpenUrl Abstract/FREE Full Text
↵
1. Minamino T, et al.
(2004) Domain organization and function of Salmonella FliK, a flagellar hook-length control protein. J Mol Biol 341(2):491–502
.
OpenUrl CrossRef PubMed
↵
1. Ferris HU,
2. Minamino T
(2006) Flipping the switch: Bringing order to flagellar assembly. Trends Microbiol 14(12):519–526
.
OpenUrl CrossRef PubMed
↵
1. Botteaux A,
2. Sani M,
3. Kayath CA,
4. Boekema EJ,
5. Allaoui A
(2008) Spa32 interaction with the inner-membrane Spa40 component of the type III secretion system of Shigella flexneri is required for the control of the needle length by a molecular tape measure mechanism. Mol Microbiol 70(6):1515–1528
.
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1. Muramoto K,
2. Makishima S,
3. Aizawa SI,
4. Macnab RM
(1998) Effect of cellular level of FliK on flagellar hook and filament assembly in Salmonella typhimurium. J Mol Biol 277(4):871–882
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2. Makishima S,
3. Aizawa S,
4. Macnab RM
(1999) Effect of hook subunit concentration on assembly and control of length of the flagellar hook of Salmonella. J Bacteriol 181(18):5808–5813
.
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1. Moriya N,
2. Minamino T,
3. Hughes KT,
4. Macnab RM,
5. Namba K
(2006) The type III flagellar export specificity switch is dependent on FliK ruler and a molecular clock. J Mol Biol 359(2):466–477
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2. Roth JR
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Proceedings of the National Academy of Sciences: 112 (13)

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[1] ↵
Marshall WF,
Rosenbaum JL
(2001) Intraflagellar transport balances continuous turnover of outer doublet microtubules: Implications for flagellar length control. J Cell Biol 155(3):405–414
.
OpenUrl Abstract/FREE Full Text

[2] Marshall WF,

[3] Rosenbaum JL

[4] ↵
Goshima G,
Wollman R,
Stuurman N,
Scholey JM,
Vale RD
(2005) Length control of the metaphase spindle. Curr Biol 15(22):1979–1988
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OpenUrl CrossRef PubMed

[5] Goshima G,

[6] Wollman R,

[7] Stuurman N,

[8] Scholey JM,

[9] Vale RD

[10] ↵
Journet L,
Agrain C,
Broz P,
Cornelis GR
(2003) The needle length of bacterial injectisomes is determined by a molecular ruler. Science 302(5651):1757–1760
.
OpenUrl Abstract/FREE Full Text

[11] Journet L,

[12] Agrain C,

[13] Broz P,

[14] Cornelis GR

[15] ↵
Shibata S, et al.
(2007) FliK regulates flagellar hook length as an internal ruler. Mol Microbiol 64(5):1404–1415
.
OpenUrl CrossRef PubMed

[16] Shibata S, et al.

[17] ↵
Katsura I,
Hendrix RW
(1984) Length determination in bacteriophage lambda tails. Cell 39(3 Pt 2):691–698
.
OpenUrl CrossRef PubMed

[18] Katsura I,

[19] Hendrix RW

[20] ↵
Oda T,
Yanagisawa H,
Kamiya R,
Kikkawa M
(2014) A molecular ruler determines the repeat length in eukaryotic cilia and flagella. Science 346(6211):857–860
.
OpenUrl Abstract/FREE Full Text

[21] Oda T,

[22] Yanagisawa H,

[23] Kamiya R,

[24] Kikkawa M

[25] ↵
Kubori T, et al.
(1998) Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280(5363):602–605
.
OpenUrl Abstract/FREE Full Text

[26] Kubori T, et al.

[27] ↵
Marlovits TC, et al.
(2004) Structural insights into the assembly of the type III secretion needle complex. Science 306(5698):1040–1042
.
OpenUrl Abstract/FREE Full Text

[28] Marlovits TC, et al.

[29] ↵
Bergeron JRC, et al.
(2013) A refined model of the prototypical Salmonella SPI-1 T3SS basal body reveals the molecular basis for its assembly. PLoS Pathog 9(4):e1003307
.
OpenUrl CrossRef PubMed

[30] Bergeron JRC, et al.

[31] ↵
Kubori T,
Sukhan A,
Aizawa SI,
Galán JE
(2000) Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system. Proc Natl Acad Sci USA 97(18):10225–10230
.
OpenUrl Abstract/FREE Full Text

[32] Kubori T,

[33] Sukhan A,

[34] Aizawa SI,

[35] Galán JE

[36] ↵
Tamano K,
Katayama E,
Toyotome T,
Sasakawa C
(2002) Shigella Spa32 is an essential secretory protein for functional type III secretion machinery and uniformity of its needle length. J Bacteriol 184(5):1244–1252
.
OpenUrl Abstract/FREE Full Text

[37] Tamano K,

[38] Katayama E,

[39] Toyotome T,

[40] Sasakawa C

[41] ↵
Sukhan A,
Kubori T,
Galán JE
(2003) Synthesis and localization of the Salmonella SPI-1 type III secretion needle complex proteins PrgI and PrgJ. J Bacteriol 185(11):3480–3483
.
OpenUrl Abstract/FREE Full Text

[42] Sukhan A,

[43] Kubori T,

[44] Galán JE

[45] ↵
Broz P, et al.
(2007) Function and molecular architecture of the Yersinia injectisome tip complex. Mol Microbiol 65(5):1311–1320
.
OpenUrl CrossRef PubMed

[46] Broz P, et al.

[47] ↵
Blocker AJ, et al.
(2008) What’s the point of the type III secretion system needle? Proc Natl Acad Sci USA 105(18):6507–6513
.
OpenUrl Abstract/FREE Full Text

[48] Blocker AJ, et al.

[49] ↵
Lunelli M,
Hurwitz R,
Lambers J,
Kolbe M
(2011) Crystal structure of PrgI-SipD: Insight into a secretion competent state of the type three secretion system needle tip and its interaction with host ligands. PLoS Pathog 7(8):e1002163
.
OpenUrl CrossRef PubMed

[50] Lunelli M,

[51] Hurwitz R,

[52] Lambers J,

[53] Kolbe M

[54] ↵
Hueck CJ
(1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62(2):379–433
.
OpenUrl Abstract/FREE Full Text

[55] Hueck CJ

[56] ↵
Macnab RM
(2004) Type III flagellar protein export and flagellar assembly. Biochim Biophys Acta Mol Cell Res 1694(1-3):207–217
.
OpenUrl CrossRef PubMed

[57] Macnab RM

[58] ↵
Macnab RM
(2003) How bacteria assemble flagella. Annu Rev Microbiol 57:77–100
.
OpenUrl CrossRef PubMed

[59] Macnab RM

[60] ↵
Samatey FA, et al.
(2004) Structure of the bacterial flagellar hook and implication for the molecular universal joint mechanism. Nature 431(7012):1062–1068
.
OpenUrl CrossRef PubMed

[61] Samatey FA, et al.

[62] ↵
Williams AW, et al.
(1996) Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium. J Bacteriol 178(10):2960–2970
.
OpenUrl Abstract/FREE Full Text

[63] Williams AW, et al.

[64] ↵
Sorg I, et al.
(2007) YscU recognizes translocators as export substrates of the Yersinia injectisome. EMBO J 26(12):3015–3024
.
OpenUrl Abstract/FREE Full Text

[65] Sorg I, et al.

[66] ↵
Erhardt M,
Singer HM,
Wee DH,
Keener JP,
Hughes KT
(2011) An infrequent molecular ruler controls flagellar hook length in Salmonella enterica. EMBO J 30(14):2948–2961
.
OpenUrl Abstract/FREE Full Text

[67] Erhardt M,

[68] Singer HM,

[69] Wee DH,

[70] Keener JP,

[71] Hughes KT

[72] ↵
Hirano T,
Yamaguchi S,
Oosawa K,
Aizawa S
(1994) Roles of FliK and FlhB in determination of flagellar hook length in Salmonella typhimurium. J Bacteriol 176(17):5439–5449
.
OpenUrl Abstract/FREE Full Text

[73] Hirano T,

[74] Yamaguchi S,

[75] Oosawa K,

[76] Aizawa S

[77] ↵
Minamino T,
Doi H,
Kutsukake K
(1999) Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium. Biosci Biotechnol Biochem 63(7):1301–1303
.
OpenUrl CrossRef PubMed

[78] Minamino T,

[79] Doi H,

[80] Kutsukake K

[81] ↵
Minamino T,
Ferris HU,
Moriya N,
Kihara M,
Namba K
(2006) Two parts of the T3S4 domain of the hook-length control protein FliK are essential for the substrate specificity switching of the flagellar type III export apparatus. J Mol Biol 362(5):1148–1158
.
OpenUrl CrossRef PubMed

[82] Minamino T,

[83] Ferris HU,

[84] Moriya N,

[85] Kihara M,

[86] Namba K

[87] ↵
Spreter T, et al.
(2009) A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system. Nat Struct Mol Biol 16(5):468–476
.
OpenUrl CrossRef PubMed

[88] Spreter T, et al.

[89] ↵
Schraidt O,
Marlovits TC
(2011) Three-dimensional model of Salmonella’s needle complex at subnanometer resolution. Science 331(6021):1192–1195
.
OpenUrl Abstract/FREE Full Text

[90] Schraidt O,

[91] Marlovits TC

[92] ↵
Marlovits TC, et al.
(2006) Assembly of the inner rod determines needle length in the type III secretion injectisome. Nature 441(7093):637–640
.
OpenUrl CrossRef PubMed

[93] Marlovits TC, et al.

[94] ↵
Agrain C, et al.
(2005) Characterization of a Type III secretion substrate specificity switch (T3S4) domain in YscP from Yersinia enterocolitica. Mol Microbiol 56(1):54–67
.
OpenUrl CrossRef PubMed

[95] Agrain C, et al.

[96] ↵
Wagner S, et al.
(2009) The helical content of the YscP molecular ruler determines the length of the Yersinia injectisome. Mol Microbiol 71(3):692–701
.
OpenUrl CrossRef PubMed

[97] Wagner S, et al.

[98] ↵
Edqvist PJ, et al.
(2003) YscP and YscU regulate substrate specificity of the Yersinia type III secretion system. J Bacteriol 185(7):2259–2266
.
OpenUrl Abstract/FREE Full Text

[99] Edqvist PJ, et al.

[100] ↵
Wagner S,
Stenta M,
Metzger LC,
Dal Peraro M,
Cornelis GR
(2010) Length control of the injectisome needle requires only one molecule of Yop secretion protein P (YscP). Proc Natl Acad Sci USA 107(31):13860–13865
.
OpenUrl Abstract/FREE Full Text

[101] Wagner S,

[102] Stenta M,

[103] Metzger LC,

[104] Dal Peraro M,

[105] Cornelis GR

[106] ↵
Keener JP
(2010) A molecular ruler mechanism for length control of extended protein structures in bacteria. J Theor Biol 263(4):481–489
.
OpenUrl CrossRef PubMed

[107] Keener JP

[108] ↵
Galán JE
(2001) Salmonella interactions with host cells: Type III secretion at work. Annu Rev Cell Dev Biol 17:53–86
.
OpenUrl CrossRef PubMed

[109] Galán JE

[110] ↵
Lefebre MD,
Galán JE
(2014) The inner rod protein controls substrate switching and needle length in a Salmonella type III secretion system. Proc Natl Acad Sci USA 111(2):817–822
.
OpenUrl Abstract/FREE Full Text

[111] Lefebre MD,

[112] Galán JE

[113] ↵
Zarivach R, et al.
(2008) Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS. Nature 453(7191):124–127
.
OpenUrl CrossRef PubMed

[114] Zarivach R, et al.

[115] ↵
Collazo CM,
Zierler MK,
Galán JE
(1995) Functional analysis of the Salmonella typhimurium invasion genes invl and invJ and identification of a target of the protein secretion apparatus encoded in the inv locus. Mol Microbiol 15(1):25–38
.
OpenUrl CrossRef PubMed

[116] Collazo CM,

[117] Zierler MK,

[118] Galán JE

[119] ↵
Bajaj V,
Lucas RL,
Hwang C,
Lee CA
(1996) Co-ordinate regulation of Salmonella typhimurium invasion genes by environmental and regulatory factors is mediated by control of hilA expression. Mol Microbiol 22(4):703–714
.
OpenUrl CrossRef PubMed

[120] Bajaj V,

[121] Lucas RL,

[122] Hwang C,

[123] Lee CA

[124] ↵
Pauling L,
Corey RB,
Branson HR
(1951) The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. Proc Natl Acad Sci USA 37(4):205–211
.
OpenUrl FREE Full Text

[125] Pauling L,

[126] Corey RB,

[127] Branson HR

[128] ↵
Amann E,
Ochs B,
Abel KJ
(1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69(2):301–315
.
OpenUrl CrossRef PubMed

[129] Amann E,

[130] Ochs B,

[131] Abel KJ

[132] ↵
Silverman MR,
Simon MI
(1972) Flagellar assembly mutants in Escherichia coli. J Bacteriol 112(2):986–993
.
OpenUrl Abstract/FREE Full Text

[133] Silverman MR,

[134] Simon MI

[135] ↵
Kutsukake K,
Minamino T,
Yokoseki T
(1994) Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium. J Bacteriol 176(24):7625–7629
.
OpenUrl Abstract/FREE Full Text

[136] Kutsukake K,

[137] Minamino T,

[138] Yokoseki T

[139] ↵
Minamino T,
González-Pedrajo B,
Yamaguchi K,
Aizawa SI,
Macnab RM
(1999) FliK, the protein responsible for flagellar hook length control in Salmonella, is exported during hook assembly. Mol Microbiol 34(2):295–304
.
OpenUrl CrossRef PubMed

[140] Minamino T,

[141] González-Pedrajo B,

[142] Yamaguchi K,

[143] Aizawa SI,

[144] Macnab RM

[145] ↵
Morris DP, et al.
(2010) Kinetic characterization of Salmonella FliK-FlhB interactions demonstrates complexity of the Type III secretion substrate-specificity switch. Biochemistry 49(30):6386–6393
.
OpenUrl CrossRef PubMed

[146] Morris DP, et al.

[147] ↵
Minamino T,
Macnab RM
(2000) Domain structure of Salmonella FlhB, a flagellar export component responsible for substrate specificity switching. J Bacteriol 182(17):4906–4914
.
OpenUrl Abstract/FREE Full Text

[148] Minamino T,

[149] Macnab RM

[150] ↵
Minamino T, et al.
(2004) Domain organization and function of Salmonella FliK, a flagellar hook-length control protein. J Mol Biol 341(2):491–502
.
OpenUrl CrossRef PubMed

[151] Minamino T, et al.

[152] ↵
Ferris HU,
Minamino T
(2006) Flipping the switch: Bringing order to flagellar assembly. Trends Microbiol 14(12):519–526
.
OpenUrl CrossRef PubMed

[153] Ferris HU,

[154] Minamino T

[155] ↵
Botteaux A,
Sani M,
Kayath CA,
Boekema EJ,
Allaoui A
(2008) Spa32 interaction with the inner-membrane Spa40 component of the type III secretion system of Shigella flexneri is required for the control of the needle length by a molecular tape measure mechanism. Mol Microbiol 70(6):1515–1528
.
OpenUrl CrossRef PubMed

[156] Botteaux A,

[157] Sani M,

[158] Kayath CA,

[159] Boekema EJ,

[160] Allaoui A

[161] ↵
Muramoto K,
Makishima S,
Aizawa SI,
Macnab RM
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Molecular ruler determines needle length for the Salmonella Spi-1 injectisome

Significance

Abstract

Results

InvJ Acts as a Molecular Ruler to Determine Needle Length.

Secretion Competition Between Early T3S Substrates Affects Needle Length.

Mutant Alleles of prgJ Reported to Affect Needle Length Control Show Wild-Type Needle Lengths When Expressed from the Chromosome.

Discussion

Materials and Methods

Bacterial Strains and Culture Conditions.

Injectisome Isolation, Electron Microscopy, and Needle Measurements.

Protein Secretion Assay, Osmotic Shock, SDS/PAGE, and Western Blotting.

Acknowledgments

Footnotes

References

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Search

Significance

Abstract

Results

InvJ Acts as a Molecular Ruler to Determine Needle Length.

Secretion Competition Between Early T3S Substrates Affects Needle Length.

Mutant Alleles of prgJ Reported to Affect Needle Length Control Show Wild-Type Needle Lengths When Expressed from the Chromosome.

Discussion

Materials and Methods

Bacterial Strains and Culture Conditions.

Injectisome Isolation, Electron Microscopy, and Needle Measurements.

Protein Secretion Assay, Osmotic Shock, SDS/PAGE, and Western Blotting.

Acknowledgments

Footnotes

References

Citation Manager Formats

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