The structural analysis of the periplasmic domain of Sinorhizobium meliloti chemoreceptor McpZ reveals a novel fold and suggests a complex mechanism of transmembrane signaling.

Chemotaxis is a fundamental process whereby bacteria seek out nutrient sources and avoid harmful chemicals. For the symbiotic soil bacterium Sinorhizobium meliloti, the chemotaxis system also plays an essential role in the interaction with its legume host. The chemotactic signaling cascade is initiated through interactions of an attractant or repellent compound with chemoreceptors or methyl-accepting chemotaxis proteins (MCPs). S. meliloti possesses eight chemoreceptors to mediate chemotaxis. Six of these receptors are transmembrane proteins with periplasmic ligand-binding domains (LBDs). The specific functions of McpW and McpZ are still unknown. Here, we report the crystal structure of the periplasmic domain of McpZ (McpZPD) at 2.7 Å resolution. McpZPD assumes a novel fold consisting of three concatenated four-helix bundle modules. Through phylogenetic analyses, we discovered that this helical tri-modular domain fold arose within the Rhizobiaceae family and is still evolving rapidly. The structure, offering a rare view of a ligand-free dimeric MCP-LBD, reveals a novel dimerization interface. Molecular dynamics calculations suggest ligand binding will induce conformational changes that result in large horizontal helix movements within the membrane-proximal domains of the McpZPD dimer that are accompanied by a 5 Å vertical shift of the terminal helix toward the inner cell membrane. These results suggest a mechanism of transmembrane signaling for this family of MCPs that entails both piston-type and scissoring movements. The predicted movements terminate in a conformation that closely mirrors those observed in related ligand-bound MCP-LBDs.

Biochemical analysis of protein-protein interfaces underlying the regulation of bacterial secretion systems.

Bacterial pathogens such as Pseudomonas aeruginosa use complex regulatory networks to tailor gene expression patterns to meet complex environmental challenges. P. aeruginosa is capable of causing both acute and chronic persistent infections, each type being characterized by distinct symptoms brought about by distinct sets of virulence mechanisms. The GacS/GacA phosphorelay system sits at the heart of a complex regulatory network that reciprocally governs the expression of virulence factors associated with either acute or chronic infections. A second non-enzymatic signaling cascade involving four proteins, ExsA, ExsC, ExsD, and ExsE is a key player in regulating the expression of the type three secretion system, an essential facilitator of acute infections. Both signaling pathways involve a remarkable array of non-canonical interactions that we sought to characterize. In the following section, we will outline several strategies, we adapted to map protein-protein interfaces and quantify the strength of biomolecular interactions by pairing complex mutational analyses with FRET binding assays and Bacterial-Two-Hybrid assays with appropriate functional assays. In the process, protocols were developed for disrupting large hydrophobic interfaces, deleting entire domains within a protein, and for mapping protein-protein interfaces formed primarily through backbone interactions.

RetS inhibits Pseudomonas aeruginosa biofilm formation by disrupting the canonical histidine kinase dimerization interface of GacS.

Bacterial signaling histidine kinases (HKs) have long been postulated to function exclusively through linear signal transduction chains. However, several HKs have recently been shown to form complex multikinase networks (MKNs). The most prominent MKN, involving the enzymes RetS and GacS, controls the switch between the motile and biofilm lifestyles in the pathogenic bacterium Pseudomonas aeruginosa. While GacS promotes biofilm formation, RetS counteracts GacS using three distinct mechanisms. Two are dephosphorylating mechanisms. The third, a direct binding between the RetS and GacS HK regions, blocks GacS autophosphorylation. Focusing on the third mechanism, we determined the crystal structure of a cocomplex between the HK region of RetS and the dimerization and histidine phosphotransfer (DHp) domain of GacS. This is the first reported structure of a complex between two distinct bacterial signaling HKs. In the complex, the canonical HK homodimerization interface is replaced by a strikingly similar heterodimeric interface between RetS and GacS. We further demonstrate that GacS autophosphorylates in trans, thus explaining why the formation of a RetS-GacS complex inhibits GacS autophosphorylation. Using mutational analysis in conjunction with bacterial two-hybrid and biofilm assays, we not only corroborate the biological role of the observed RetS-GacS interactions, but also identify a residue critical for the equilibrium between the RetS-GacS complex and the respective RetS and GacS homodimers. Collectively, our findings suggest that RetS and GacS form a domain-swapped hetero-oligomer during the planktonic growth phase of P. aeruginosa before unknown signals cause its dissociation and a relief of GacS inhibition to promote biofilm formation.

Backbone Interactions Between Transcriptional Activator ExsA and Anti-Activator ExsD Facilitate Regulation of the Type III Secretion System in Pseudomonas aeruginosa.

The type III secretion system (T3SS) is a pivotal virulence mechanism of many Gram-negative bacteria. During infection, the syringe-like T3SS injects cytotoxic proteins directly into the eukaryotic host cell cytoplasm. In Pseudomonas aeruginosa, expression of the T3SS is regulated by a signaling cascade involving the proteins ExsA, ExsC, ExsD, and ExsE. The AraC-type transcription factor ExsA activates transcription of all T3SS-associated genes. Prior to host cell contact, ExsA is inhibited through direct binding of the anti-activator protein ExsD. Host cell contact triggers secretion of ExsE and sequestration of ExsD by ExsC to cause the release of ExsA. ExsA does not bind ExsD through the canonical ligand binding pocket of AraC-type proteins. Using site-directed mutagenesis and a specific in vitro transcription assay, we have now discovered that backbone interactions between the amino terminus of ExsD and the ExsA beta barrel constitute a pivotal part of the ExsD-ExsA interface. Follow-up bacterial two-hybrid experiments suggest additional contacts create an even larger protein-protein interface. The discovered role of the amino terminus of ExsD in ExsA binding explains how ExsC might relieve the ExsD-mediated inhibition of T3SS gene expression, because the same region of ExsD interacts with ExsC following host cell contact.

Helix Cracking Regulates the Critical Interaction between RetS and GacS in Pseudomonas aeruginosa.

Recent paradigm shifting discoveries have demonstrated that bacterial signaling kinases engage in unexpected regulatory crosstalk, yet the underlying molecular mechanisms remain largely uncharacterized. The Pseudomonas aeruginosa RetS/GacS system constitutes an ideal model for studying these mechanisms. The in-depth analysis of the kinase region of RetS and RetS/GacS interactions presented here refutes a longstanding model, which posited the formation of a catalytically inactive RetS/GacS heterodimer. Crystallographic studies uncovered structurally dynamic features within the RetS kinase region, suggesting that RetS uses the reversible unfolding of a helix, or helix cracking, to control interactions with GacS. The pivotal importance of this helical region for regulating GacS and, by extension, Pseudomonas aeruginosa virulence, was corroborated via in vivo assays. The implications of this work extend beyond the RetS/GacS system because the helix cracking occurs right next to a highly conserved catalytic residue histidine-424, suggesting this model could represent an emergent archetype for histidine kinase regulation.

Structure of the sensory domain of McpX from Sinorhizobium meliloti, the first known bacterial chemotactic sensor for quaternary ammonium compounds.

The α-proteobacterium Sinorhizobium meliloti can live freely in the soil or engage in a symbiosis with its legume host. S. meliloti facilitates nitrogen fixation in root nodules, thus providing pivotal, utilizable nitrogen to the host. The organism has eight chemoreceptors, namely McpT to McpZ and IcpA that facilitate chemotaxis. McpX is the first known bacterial sensor of quaternary ammonium compounds (QACs) such as choline and betaines. Because QACs are exuded at chemotaxis-relevant concentrations by germinating alfalfa seeds, McpX has been proposed to contribute to host-specific chemotaxis. We have determined the crystal structure of the McpX periplasmic region (McpXPR) in complex with the proline betaine at 2.7 Å resolution. In the crystal, the protein forms a symmetric dimer with one proline betaine molecule bound to each monomer of McpXPR within membrane-distal CACHE module. The ligand is bound through cation-πinteractions with four aromatic amino acid residues. Mutational analysis in conjunction with binding studies revealed that a conserved aspartate residue is pivotal for ligand binding. We discovered that, in a striking example of convergent evolution, the ligand-binding site of McpXPR resembles that of a group of structurally unrelated betaine-binding proteins including ProX and OpuAC. Through this comparison and docking studies, we rationalized the specificity of McpXPR for this specific group of ligands. Collectively, our structural, biochemical, and molecular docking data have revealed the molecular determinants in McpX that are crucial for its rare ligand specificity for QACs.

The type IV pilus assembly ATPase PilB functions as a signaling protein to regulate exopolysaccharide production in Myxococcus xanthus.

Myxococcus xanthus possesses a form of surface motility powered by the retraction of the type IV pilus (T4P). Additionally, exopolysaccharide (EPS), the major constituent of bacterial biofilms, is required for this T4P-mediated motility in M. xanthus as the putative trigger of T4P retraction. The results here demonstrate that the T4P assembly ATPase PilB functions as an intermediary in the EPS regulatory pathway composed of the T4P upstream of the Dif signaling proteins in M. xanthus. A suppressor screen isolated a pilB mutation that restored EPS production to a T4P- mutant. An additional PilB mutant variant, which is deficient in ATP hydrolysis and T4P assembly, supports EPS production without the T4P, indicating PilB can regulate EPS production independently of its function in T4P assembly. Further analysis confirms that PilB functions downstream of the T4P filament but upstream of the Dif proteins. In vitro studies suggest that the nucleotide-free form of PilB assumes the active signaling conformation in EPS regulation. Since M. xanthus PilB possesses conserved motifs with high affinity for c-di-GMP binding, the findings here suggest that c-di-GMP can regulate both motility and biofilm formation through a single effector in this surface-motile bacterium.

Using a Concept Inventory to Reveal Student Thinking Associated with Common Misconceptions about Antibiotic Resistance.

Misconceptions, also known as alternate conceptions, about key concepts often hinder the ability of students to learn new knowledge. Concept inventories (CIs) are designed to assess students' understanding of key concepts, especially those prone to misconceptions. Two-tiered CIs include prompts that ask students to explain the logic behind their answer choice. Such two-tiered CIs afford an opportunity for faculty to explore the student thinking behind the common misconceptions represented by their choice of a distractor. In this study, we specifically sought to probe the misconceptions that students hold prior to beginning an introductory microbiology course (i.e., preconceptions). Faculty-learning communities at two research-intensive universities used the validated Host-Pathogen Interaction Concept Inventory (HPI-CI) to reveal student preconceptions. Our method of deep analysis involved communal review and discussion of students' explanations for their CI answer choice. This approach provided insight valuable for curriculum development. Here the process is illustrated using one question from the HPI-CI related to the important topic of antibiotic resistance. The frequencies with which students chose particular multiple-choice responses for this question were highly correlated between institutions, implying common underlying misconceptions. Examination of student explanations using our analysis approach, coupled with group discussions within and between institutions, revealed patterns in student thinking to the participating faculty. Similar application of a two-tiered concept inventory by general microbiology instructors, either individually or in groups, at other institutions will allow them to better understand student thinking related to key concepts in their curriculum.

Crystal Structure of a Type IV Pilus Assembly ATPase: Insights into the Molecular Mechanism of PilB from Thermus thermophilus.

Type IV pili (T4P) mediate bacterial motility and virulence. The PilB/GspE family ATPases power the assembly of T4P and type 2 secretion systems. We determined the structure of the ATPase region of PilB (PilBATP) in complex with ATPγS to provide a model of a T4P assembly ATPase and a view of a PilB/GspE family hexamer at better than 3-Šresolution. Spatial positioning and conformations of the protomers suggest a mechanism of force generation. All six PilBATP protomers contain bound ATPγS. Two protomers form a closed conformation poised for ATP hydrolysis. The other four molecules assume an open conformation but separate into two pairs with distinct active-site accessibilities. We propose that one pair represents the post-hydrolysis phase while the other pair appears poised for ADP/ATP exchange. Collectively, the data suggest that T4P assembly is powered by coordinating concurrent substrate binding with ATP hydrolysis across the PilB hexamer.

Structural Analysis of the Regulatory Domain of ExsA, a Key Transcriptional Regulator of the Type Three Secretion System in Pseudomonas aeruginosa.

Pseudomonas aeruginosa employs a type three secretion system to facilitate infections in mammalian hosts. The operons encoding genes of structural components of the secretion machinery and associated virulence factors are all under the control of the AraC-type transcriptional activator protein, ExsA. ExsA belongs to a unique subfamily of AraC-proteins that is regulated through protein-protein contacts rather than small molecule ligands. Prior to infection, ExsA is inhibited through a direct interaction with the anti-activator ExsD. To activate ExsA upon host cell contact this interaction is disrupted by the anti-antiactivator protein ExsC. Here we report the crystal structure of the regulatory domain of ExsA, which is known to mediate ExsA dimerization as well as ExsD binding. The crystal structure suggests two models for the ExsA dimer. Both models confirmed the previously shown involvement of helix α-3 in ExsA dimerization but one also suggest a role for helix α-2. These structural data are supported by the observation that a mutation in α-2 greatly diminished the ability of ExsA to activate transcription in vitro. Additional in vitro transcription studies revealed that a conserved pocket, used by AraC and the related ToxT protein for the binding of small molecule regulators, although present in ExsA is not involved in binding of ExsD.

Self-association is required for occupation of adjacent binding sites in Pseudomonas aeruginosa type III secretion system promoters.

ExsA is a member of the AraC/XylS family of transcriptional regulators and is required for expression of the Pseudomonas aeruginosa type III secretion system (T3SS). All P. aeruginosa T3SS promoters contain two adjacent binding sites for monomeric ExsA. The amino-terminal domain of ExsA (NTD) is thought to mediate interactions between the ExsA monomers bound to each site. Threading the NTD onto the AraC backbone revealed an α-helix that likely serves as the primary determinant for dimerization. In this study, we performed alanine scanning mutagenesis of the ExsA α-helix (residues 136 to 152) to identify determinants required for self-association. Residues L137, C139, L140, K141, and L148 exhibited self-association defects and were required for maximal activation by ExsA. Disruption of self-association resulted in decreased binding to T3SS promoters, particularly loss of binding by the second ExsA monomer. Removing the NTD or increasing the space between the ExsA-binding sites restored the ability of the second ExsA monomer to bind the PexsC promoter. This finding indicated that, in the absence of self-association, the NTD prevents binding by a second monomer. Similar findings were seen with the PexoT promoter; however, binding of the second ExsA monomer in the absence of self-association also required the presence of a high-affinity site 2. Based on these data, ExsA self-association is necessary to overcome inhibition by the NTD and to compensate for low-affinity binding sites, thereby allowing for full occupation and activation of ExsA-dependent promoters. Therefore, ExsA self-association is indispensable and provides an attractive target for antivirulence therapies.

A naturally variable residue in the S1 subsite of M1 family aminopeptidases modulates catalytic properties and promotes functional specialization.

M1 family metallo-aminopeptidases fulfill a wide range of critical and in some cases medically relevant roles in humans and human pathogens. The specificity of M1-aminopeptidases is dominated by the interaction of the well defined S1 subsite with the side chain of the first (P1) residue of the substrate and can vary widely. Extensive natural variation occurs at one of the residues that contributes to formation of the cylindrical S1 subsite. We investigated whether this natural variation contributes to diversity in S1 subsite specificity. Effects of 11 substitutions of the S1 subsite residue valine 459 in the Plasmodium falciparum aminopeptidase PfA-M1 and of three substitutions of the homologous residue methionine 260 in Escherichia coli aminopeptidase N were characterized. Many of these substitutions altered steady-state kinetic parameters for dipeptide hydrolysis and remodeled S1 subsite specificity. The most dramatic change in specificity resulted from substitution with proline, which collapsed S1 subsite specificity such that only substrates with P1-Arg, -Lys, or -Met were appreciably hydrolyzed. The structure of PfA-M1 V459P revealed that the proline substitution induced a local conformational change in the polypeptide backbone that resulted in a narrowed S1 subsite. The restricted specificity and active site backbone conformation of PfA-M1 V459P mirrored those of endoplasmic reticulum aminopeptidase 2, a human enzyme with proline in the variable S1 subsite position. Our results provide compelling evidence that changes in the variable residue in the S1 subsite of M1-aminopeptidases have facilitated the evolution of new specificities and ultimately novel functions for this important class of enzymes.

Self-trimerization of ExsD limits inhibition of the Pseudomonas aeruginosa transcriptional activator ExsA in vitro.

The opportunistic pathogen Pseudomonas aeruginosa ranks among the leading causes of nosocomial infection. The type III secretion system (T3SS) aids acute Pseudomonas aeruginosa infection by injecting potent cytotoxins into host cells to suppress the host's innate immune response. Expression of all T3SS-related genes is strictly dependent on the transcription factor ExsA. Consequently, ExsA and the biological processes that regulate ExsA function are of great biomedical interest. The present study focused on the ExsA-ExsC-ExsD-ExsE signaling cascade, which ties host cell contact to the upregulation of T3SS gene expression. Prior to T3SS induction, the antiactivator protein ExsD binds to ExsA and blocks ExsA-dependent transcription by interfering with ExsA dimerization and promoter interactions. Upon host cell contact, ExsD is sequestered by the T3SS chaperone ExsC, resulting in the release of ExsA and upregulation of the T3SS. Previous studies have shown that the ExsD-ExsA interactions are not freely reversible. Because independently folded ExsD and ExsA were not found to interact, it has been hypothesized that folding intermediates of the two proteins form the complex. Here, we demonstrate, for the first time, that ExsD alone is sufficient to inhibit ExsA-dependent transcription in vitro and that no other cellular factors are required. More significantly, we show that independently folded ExsD and ExsA are capable of interacting, but only at 37 °C and not at 30 °C. Guided by the crystal structure of ExsD, we designed a monomeric variant of the protein, and demonstrated that ExsD trimerization prevents ExsD from inhibiting ExsA-dependent transcription at 30 °C. We propose that this unique mechanism plays an important role in T3SS regulation.

The catalytic domain of the germination-specific lytic transglycosylase SleB from Bacillus anthracis displays a unique active site topology.

Bacillus anthracis produces metabolically inactive spores. Germination of these spores requires germination-specific lytic enzymes (GSLEs) that degrade the unique cortex peptidoglycan to permit resumption of metabolic activity and outgrowth. We report the first crystal structure of the catalytic domain of a GSLE, SleB. The structure revealed a transglycosylase fold with unique active site topology and permitted identification of the catalytic glutamate residue. Moreover, the structure provided insights into the molecular basis for the specificity of the enzyme for muramic-δ-lactam-containing cortex peptidoglycan. The protein also contains a metal-binding site that is positioned directly at the entrance of the substrate-binding cleft.

Orientation of Pseudomonas aeruginosa ExsA monomers bound to promoter DNA and base-specific contacts with the P(exoT) promoter.

ExsA is a transcriptional activator of the Pseudomonas aeruginosa type III secretion system (T3SS) and a member of the AraC/XylS protein family. Each of the 10 ExsA-dependent promoter regions that define the T3SS regulon has two adjacent binding sites for monomeric ExsA. Whereas the promoter-proximal sites (binding site 1) contain highly conserved GnC and TGnnA sequences that are separated by ∼10 bp, the promoter-distal sites (binding site 2) share no obvious sequence similarity to each other or to the binding site 1 consensus. In the present study, we used footprinting with Fe-BABE (a protein-labeling reagent that can be conjugated to cysteine residues) to demonstrate that the two ExsA monomers bind to the P(exsC), P(exsD), P(exoT), and P(pcrG) promoters in a head-to-tail orientation. The footprinting data further indicate that the conserved GnC and TGnnA sequences constitute binding site 1. When bound to site 1, the first helix-turn-helix (HTH) motif of ExsA interacts with the conserved GnC sequence, and the second HTH interacts at or near the TGnnA sequences. Genetic data using the P(exoT) promoter indicate that residues L198 and T199 in the first HTH motif of ExsA contact the guanine in the GnC sequence and that residue K202, also in the first HTH motif, contacts the cytosine. Likewise, evidence is presented that residues Q248, Y250, T252, and R257 located in the second HTH motif contribute to the recognition of the TGnnA sequence. These combined data define interactions of ExsA with site 1 on the P(exoT) promoter and provide insight into the nature of the interactions involved in recognition of binding site 2.

Phosphorylation and dephosphorylation among Dif chemosensory proteins essential for exopolysaccharide regulation in Myxococcus xanthus.

Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as a difD difE double mutant phenocopied the difE single mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifE approximately P), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifD approximately P). Here we report in vitro phosphorylation studies with all the Dif chemosensory proteins that were expressed and purified from Escherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifD approximately P has an unusually long half-life for dephosphorylation in vitro, DifG efficiently dephosphorylated DifD approximately P as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifE approximately P.

Structural evidence suggests that antiactivator ExsD from Pseudomonas aeruginosa is a DNA binding protein.

The opportunistic pathogen P. aeruginosa utilizes a type III secretion system (T3SS) to support acute infections in predisposed individuals. In this bacterium, expression of all T3SS-related genes is dependent on the AraC-type transcriptional activator ExsA. Before host contact, the T3SS is inactive and ExsA is repressed by the antiactivator protein ExsD. The repression, thought to occur through direct interactions between the two proteins, is relieved upon opening of the type III secretion (T3S) channel when secretion chaperone ExsC sequesters ExsD. We have solved the crystal structure of Delta20ExsD, a protease-resistant fragment of ExsD that lacks only the 20 amino terminal residues of the wild-type protein at 2.6 A. Surprisingly the structure revealed similarities between ExsD and the DNA binding domain of transcriptional repressor KorB. A model of an ExsD-DNA complex constructed on the basis of this homology produced a realistic complex that is supported by the prevalence of conserved residues in the putative DNA binding site and the results of differential scanning fluorimetry studies. Our findings challenge the currently held model that ExsD solely acts through interactions with ExsA and raise new questions with respect to the underlying mechanism of ExsA regulation.

New protein fold revealed by a 1.65 A resolution crystal structure of Francisella tularensis pathogenicity island protein IglC.

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes the fulminating disease tularemia and is considered to be a potential bioweapon. F. tularensis pathogenicity island proteins play a key role in modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm of macrophages. The 23 kDa pathogenicity island protein IglC is essential for the survival and proliferation of F. tularensis in macrophages. Seeking to gain some insight into its function, we determined the crystal structure of IglC at 1.65 A resolution. IglC adopts a beta-sandwich conformation that exhibits no similarity with any known protein structure.

Structure of the POZ domain of human LRF, a master regulator of oncogenesis.

The proto-oncogenic properties of the POK family of transcriptional repressors BCL6, PLZF, and LRF have been well established. These proteins utilize their amino-terminal POZ domains for multimerization and the recruitment of co-repressors. Because LRF represses the production of the tumor suppressor p19(Arf) (ARF), it is regarded as an attractive therapeutic target for the treatment of many types of cancer. The crystal structure of the LRF POZ domain reveals a high degree of structural conservation with the corresponding domains of BCL6 and PLZF. However, striking differences between the electrostatic properties of the BCL6 and LRF POZ domains suggest that if, like BCL6, LRF interacts with the co-repressor SMRT, it almost certainly uses a different mechanism to do so. These differences may also explain why LRF interacts with BCL6 but not with PLZF. Finally, the conservation of crystal packing contacts suggests the probable location of the interface that mediates LRF/BCL6 complex formation.

Selection and characterization of Yersinia pestis YopN mutants that constitutively block Yop secretion.

Secretion of Yop effector proteins by the Yersinia pestis plasmid pCD1-encoded type III secretion system (T3SS) is regulated in response to specific environmental signals. Yop secretion is activated by contact with a eukaryotic cell or by growth at 37 degrees C in the absence of calcium. The secreted YopN protein, the SycN/YscB chaperone and TyeA form a cytosolic YopN/SycN/YscB/TyeA complex that is required to prevent Yop secretion in the presence of calcium and prior to contact with a eukaryotic cell. The mechanism by which these proteins prevent secretion and the subcellular location where the block in secretion occurs are not known. To further investigate both the mechanism and location of the YopN-dependent block, we isolated and characterized several YopN mutants that constitutively block Yop secretion. All the identified amino-acid substitutions that resulted in a constitutive block in Yop secretion mapped to a central domain of YopN that is not directly involved in the interaction with the SycN/YscB chaperone or TyeA. The YopN mutants required an intact TyeA-binding domain and TyeA to block secretion, but did not require an N-terminal secretion signal, an intact chaperone-binding domain or the SycN/YscB chaperone. These results suggest that a C-terminal domain of YopN complexed with TyeA blocks Yop secretion from a cytosolic, not an extracellular, location. A hypothetical model for how the YopN/SycN/YscB/TyeA complex regulates Yop secretion is presented.