FlhE functions as a chaperone to prevent formation of periplasmic flagella in Gram-negative bacteria.

The bacterial flagellum, which facilitates motility, is composed of ~20 structural proteins organized into a long extracellular filament connected to a cytoplasmic rotor-stator complex via a periplasmic rod. Flagellum assembly is regulated by multiple checkpoints that ensure an ordered gene expression pattern coupled to the assembly of the various building blocks. Here, we use epifluorescence, super-resolution, and transmission electron microscopy to show that the absence of a periplasmic protein (FlhE) prevents proper flagellar morphogenesis and results in the formation of periplasmic flagella in Salmonella enterica. The periplasmic flagella disrupt cell wall synthesis, leading to a loss of normal cell morphology resulting in cell lysis. We propose that FlhE functions as a periplasmic chaperone to control assembly of the periplasmic rod, thus preventing formation of periplasmic flagella.

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 µM) include PBPs (PBP1a, KD = 0.07 µM; PBP5 = 0.4 µM); other lytic transglycosylases (SltB2, KD = 0.09 µM; RlpA, KD = 0.4 µM); a type VI secretion system effector (Tse5, KD = 0.3 µM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 µM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.

Control of biofilm formation by an Agrobacterium tumefaciens pterin-binding periplasmic protein conserved among diverse Proteobacteria.

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages.

Biosensor that Detects Stress Caused by Periplasmic Proteins.

Escherichia coli is often used as a factory to produce recombinant proteins. In many cases, the recombinant protein needs disulfide bonds to fold and function correctly. These proteins are genetically fused to a signal peptide so that they are secreted to the oxidizing environment of the periplasm (where the enzymes required for disulfide bond formation exist). Currently, it is difficult to determine in vivo whether a recombinant protein is efficiently secreted from the cytoplasm and folded in the periplasm or if there is a bottleneck in one of these steps because cellular capacity has been exceeded. To address this problem, we have developed a biosensor that detects cellular stress caused by (1) inefficient secretion of proteins from the cytoplasm and (2) aggregation of proteins in the periplasm. We demonstrate how the fluorescence fingerprint obtained from the biosensor can be used to identify induction conditions that do not exceed the capacity of the cell and therefore do not cause cellular stress. These induction conditions result in more effective biomass and in some cases higher titers of soluble recombinant proteins.

A critical role of the periplasm in copper homeostasis in Gram-negative bacteria.

Copper is essential for life, but is toxic in excess. Copper homeostasis is achieved in the cytoplasm and the periplasm as a unique feature of Gram-negative bacteria. Especially, it has become clear the role of the periplasm and periplasmic proteins regarding whole-cell copper homeostasis. Here, we addressed the role of the periplasm and periplasmic proteins in copper homeostasis using a Systems Biology approach integrating experiments with models. Our analysis shows that most of the copper-bound molecules localize in the periplasm but not cytoplasm, suggesting that Escherichia coli utilizes the periplasm to sense the copper concentration in the medium and sequester copper ions. In particular, a periplasmic multi-copper oxidase CueO and copper-responsive transcriptional factor CusS contribute both to protection against Cu(I) toxicity and to incorporating copper into the periplasmic components/proteins. We propose that Gram-negative bacteria have evolved mechanisms to sense and store copper in the periplasm to expand their living niches.

A genetically encoded fluorescent biosensor for extracellular L-lactate.

L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

Interplay between DsbA1, DsbA2 and C8J_1298 Periplasmic Oxidoreductases of Campylobacter jejuni and Their Impact on Bacterial Physiology and Pathogenesis.

The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges between cysteine residues that stabilize protein structures and ensure their proper functioning. Here, we report the detailed analysis of the Dsb pathway of Campylobacter jejuni. The oxidizing Dsb system of this pathogen is unique because it consists of two monomeric DsbAs (DsbA1 and DsbA2) and one dimeric bifunctional protein (C8J_1298). Previously, we showed that DsbA1 and C8J_1298 are redundant. Here, we unraveled the interaction between the two monomeric DsbAs by in vitro and in vivo experiments and by solving their structures and found that both monomeric DsbAs are dispensable proteins. Their structures confirmed that they are homologs of EcDsbL. The slight differences seen in the surface charge of the proteins do not affect the interaction with their redox partner. Comparative proteomics showed that several respiratory proteins, as well as periplasmic transport proteins, are targets of the Dsb system. Some of these, both donors and electron acceptors, are essential elements of the C. jejuni respiratory process under oxygen-limiting conditions in the host intestine. The data presented provide detailed information on the function of the C. jejuni Dsb system, identifying it as a potential target for novel antibacterial molecules.

Chaperone Spy Protects Outer Membrane Proteins from Folding Stress via Dynamic Complex Formation.

Gram-negative bacteria have a multicomponent and constitutively active periplasmic chaperone system to ensure the quality control of their outer membrane proteins (OMPs). Recently, OMPs have been identified as a new class of vulnerable targets for antibiotic development, and therefore a comprehensive understanding of OMP quality control network components will be critical for discovering antimicrobials. Here, we demonstrate that the periplasmic chaperone Spy protects certain OMPs against protein-unfolding stress and can functionally compensate for other periplasmic chaperones, namely Skp and FkpA, in the Escherichia coli K-12 MG1655 strain. After extensive in vivo genetic experiments for functional characterization of Spy, we use nuclear magnetic resonance and circular dichroism spectroscopy to elucidate the mechanism by which Spy binds and folds two different OMPs. Along with holding OMP substrates in a dynamic conformational ensemble, Spy binding enables OmpX to form a partially folded ß-strand secondary structure. The bound OMP experiences temperature-dependent conformational exchange within the chaperone, pointing to a multitude of local dynamics. Our findings thus deepen the understanding of functional compensation among periplasmic chaperones during OMP biogenesis and will promote the development of innovative antimicrobials against pathogenic Gram-negative bacteria. IMPORTANCE Outer membrane proteins (OMPs) play critical roles in bacterial pathogenicity and provide a new niche for antibiotic development. A comprehensive understanding of the OMP quality control network will strongly impact antimicrobial discovery. Here, we systematically demonstrate that the periplasmic chaperone Spy has a role in maintaining the homeostasis of certain OMPs. Remarkably, Spy utilizes a unique chaperone mechanism to bind OmpX and allows it to form a partially folded ß-strand secondary structure in a dynamic exchange of conformations. This mechanism differs from that of other E. coli periplasmic chaperones such as Skp and SurA, both of which maintain OMPs in disordered conformations. Our study thus deepens the understanding of the complex OMP quality control system and highlights the differences in the mechanisms of ATP-independent chaperones.

Porin threading drives receptor disengagement and establishes active colicin transport through Escherichia coli OmpF.

Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane.

Competing stress-dependent oligomerization pathways regulate self-assembly of the periplasmic protease-chaperone DegP.

DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a "resting" hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP's substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults.

DegP Initiates Regulated Processing of Filamentous Hemagglutinin in Bordetella bronchiseptica.

Filamentous hemagglutinin (FhaB) is a critical virulence factor for both Bordetella pertussis, the causal agent of whooping cough, and the closely related species Bordetella bronchiseptica. FhaB is an adhesin, suppresses inflammatory cytokine production, and protects against phagocytic cell clearance during infection. Regulated degradation of the FhaB C-terminal prodomain is required to establish a persistent infection in mice. Two proteases, CtpA in the periplasm and SphB1 on the bacterial surface, are known to mediate FhaB processing, and we recently determined that CtpA functions before, and controls the FhaB cleavage site of, SphB1. However, the data indicate that another periplasmic protease must initiate degradation of the prodomain by removing a portion of the FhaB C terminus that inhibits CtpA-mediated degradation. Using a candidate approach, we identified DegP as the initiating protease. Deletion of degP or substitution of its predicted catalytic residue resulted in reduced creation of FHA' (the main product of FhaB processing) and an accumulation of full-length FhaB in whole-cell lysates. Also, FHA' was no longer released into culture supernatants in degP mutants. Alterations of the FhaB C terminus that relieve inhibition of CtpA abrogate the need for DegP, consistent with DegP functioning prior to CtpA in the processing pathway. DegP is not required for secretion of FhaB through FhaC or for adherence of the bacteria to host cells, indicating that DegP acts primarily as a protease and not a chaperone for FhaB in B. bronchiseptica. Our results highlight a role for HtrA family proteases in activation of virulence factors in pathogenic bacteria. IMPORTANCE Two-partner secretion (TPS) systems are broadly distributed among Gram-negative bacteria and play important roles in bacterial pathogenesis. FhaB-FhaC is the prototypical member of the TPS family and we here identified the protease that initiates a processing cascade that controls FhaB function. Our results are significant because they provide insight into the molecular mechanism underlying the ability of Bordetella species to prevent clearance by phagocytic cells, which is critical for bacterial persistence in the lower respiratory tract. Our findings also highlight an underappreciated role for HtrA family proteases in processing specific bacterial virulence factors.

Ecotin and LamB in Escherichia coli influence the susceptibility to Type VI secretion-mediated interbacterial competition and killing by Vibrio cholerae.

BACKGROUND: A prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet. METHODS: Escherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses. RESULTS: The T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition. CONCLUSIONS: Our study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae. GENERAL SIGNIFICANCE: We envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.

Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly.

Antibodies are essential tools in research and diagnostics. Although antibody fragments typically obtained from in vitro selection can be rapidly produced in bacteria, the generation of full-length antibodies or the modification of antibodies with probes is time and labor intensive. Protein ligation such as SpyTag technology could covalently attach domains and labels to antibody fragments equipped with a SpyTag. However, we found that the established periplasmic expression of antibody fragments in E. coli led to quantitative cleavage of the SpyTag by the proteases Tsp and OmpT. Here we report successful periplasmic expression of SpyTagged Fab fragments and demonstrate the coupling to separately prepared SpyCatcher modules. We used this modular toolbox of SpyCatcher proteins to generate reagents for a variety of immunoassays and measured their performance in comparison with traditional reagents. Furthermore, we demonstrate surface immobilization, high-throughput screening of antibody libraries, and rapid prototyping of antibodies based on modular antibody assembly.

Endogenous membrane stress induces T6SS activity in Pseudomonas aeruginosa.

The type 6 secretion system (T6SS) is a dynamic organelle encoded by many gram-negative bacteria that can be used to kill competing bacterial prey species in densely occupied niches. Some predatory species, such as Vibrio cholerae, use their T6SS in an untargeted fashion while in contrast, Pseudomonas aeruginosa assembles and fires its T6SS apparatus only after detecting initial attacks by other bacterial prey cells; this targeted attack strategy has been termed the T6SS tit-for-tat response. Molecules that interact with the P. aeruginosa outer membrane such as polymyxin B can also trigger assembly of T6SS organelles via a signal transduction pathway that involves protein phosphorylation. Recent work suggests that a phospholipase T6SS effector (TseL) of V. cholerae can induce T6SS dynamic activity in P. aeruginosa when delivered to or expressed in the periplasmic space of this organism. Here, we report that inhibiting expression of essential genes involved in outer membrane biogenesis can also trigger T6SS activation in P. aeruginosa Specifically, we developed a CRISPR interference (CRISPRi) system to knock down expression of bamA, tolB, and lptD and found that these knockdowns activated T6SS activity. This increase in T6SS activity was dependent on the same signal transduction pathway that was previously shown to be required for the tit-for-tat response. We conclude that outer membrane perturbation can be sensed by P. aeruginosa to activate the T6SS even when the disruption is generated by aberrant cell envelope biogenesis.

Gene-specific mutagenesis enables rapid continuous evolution of enzymes in vivo.

Various in vivo mutagenesis methods have been developed to facilitate fast and efficient continuous evolution of proteins in cells. However, they either modify the DNA region that does not match the target gene, or suffer from low mutation rates. Here, we report a mutator, eMutaT7 (enhanced MutaT7), with very fast in vivo mutation rate and high gene-specificity in Escherichia coli. eMutaT7, a cytidine deaminase fused to an orthogonal RNA polymerase, can introduce up to ∼4 mutations per 1 kb per day, rivalling the rate in typical in vitro mutagenesis for directed evolution of proteins, and promotes rapid continuous evolution of model proteins for antibiotic resistance and allosteric activation. eMutaT7 provides a very simple and tunable method for continuous directed evolution of proteins, and suggests that the fusion of new DNA-modifying enzymes to the orthogonal RNA polymerase is a promising strategy to explore the expanded sequence space without compromising gene specificity.

Chromosome Segregation and Peptidoglycan Remodeling Are Coordinated at a Highly Stabilized Septal Pore to Maintain Bacterial Spore Development.

Asymmetric division, a hallmark of endospore development, generates two cells, a larger mother cell and a smaller forespore. Approximately 75% of the forespore chromosome must be translocated across the division septum into the forespore by the DNA translocase SpoIIIE. Asymmetric division also triggers cell-specific transcription, which initiates septal peptidoglycan remodeling involving synthetic and hydrolytic enzymes. How these processes are coordinated has remained a mystery. Using Bacillus subtilis, we identified factors that revealed the link between chromosome translocation and peptidoglycan remodeling. In cells lacking these factors, the asymmetric septum retracts, resulting in forespore cytoplasmic leakage and loss of DNA translocation. Importantly, these phenotypes depend on septal peptidoglycan hydrolysis. Our data support a model in which SpoIIIE is anchored at the edge of a septal pore, stabilized by newly synthesized peptidoglycan and protein-protein interactions across the septum. Together, these factors ensure coordination between chromosome translocation and septal peptidoglycan remodeling to maintain spore development.

DegP protease is essential for tolerance to salt stress in the plant growth-promoting bacterium Gluconacetobacter diazotrophicus PAL5.

The use of plant growth-promoting bacteria represents an alternative to the massive use of mineral fertilizers in agriculture. However, some abiotic stresses commonly found in the environment, like salinity, can affect the efficiency of this approach. Here, we investigated the key mechanisms involved in the response of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus to salt stress by using morphological and cell viability analyses, comparative proteomics, and reverse genetics. Our results revealed that the bacteria produce filamentous cells in response to salt at 100 mM and 150 mM NaCl. However, such a response was not observed at higher concentrations, where cell viability was severely affected. Proteomic analysis showed that salt stress modulates proteins involved in several pathways, including iron uptake, outer membrane efflux, osmotic adjustment, cell division and elongation, and protein transport and quality control. Proteomic data also revealed the repression of several extracytoplasmic proteins, especially those located at periplasm and outer membrane. The role of such pathways in the tolerance to salt stress was analyzed by the use of mutant defectives for Δtbdr (iron uptake), ΔmtlK and ΔotsA (compatible solutes synthesis), and ΔdegP (quality control of nascent extracytoplasmic proteins). ΔdegP presented the highest sensitivity to salt stress, Δtbdr, andΔmtlK also showed increased sensitivity, but ΔotsA was not affected. This is the first demonstration that DegP protein, a protease with minor chaperone activity, is essential for tolerance to salt stress in G. diazotrophicus. Our data contribute to a better understanding of the molecular bases that control the bacterial response/tolerance to salt stress, shedding light on quality control of nascent extracytoplasmic proteins.

Characterization of ecotin homologs from Campylobacter rectus and Campylobacter showae.

Ecotin, first described in Escherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in various Campylobacter species, including Campylobacter rectus and Campylobacter showae residing in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotins in vitro, the orthologs from C. rectus and C. showae were recombinantly expressed and purified from E. coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins from C. rectus and C. showae inhibit NE, factor Xa and trypsin, but not the Campylobacter jejuni serine protease HtrA or its ortholog in E. coli, DegP. To further evaluate ecotin function in vivo, an E. coli ecotin-deficient mutant was complemented with the C. rectus and C. showae homologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of the E. coli ecotin-deficient mutant can be rescued upon expression of ecotins from C. rectus and C. showae. In addition, the C. rectus and C. showae ecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen, Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.

The hypothetical periplasmic protein PA1624 from Pseudomonas aeruginosa folds into a unique two-domain structure.

The crystal structure of the 268-residue periplasmic protein PA1624 from the opportunistic pathogen Pseudomonas aeruginosa PAO1 was determined to high resolution using the Se-SAD method for initial phasing. The protein was found to be monomeric and the structure consists of two domains, domains 1 and 2, comprising residues 24-184 and 185-268, respectively. The fold of these domains could not be predicted even using state-of-the-art prediction methods, and similarity searches revealed only a very distant homology to known structures, namely to Mog1p/PsbP-like and OmpA-like proteins for the N- and C-terminal domains, respectively. Since PA1624 is only present in an important human pathogen, its unique structure and periplasmic location render it a potential drug target. Consequently, the results presented here may open new avenues for the discovery and design of antibacterial drugs.

Roles of the Tol-Pal system in the Type III secretion system and flagella-mediated virulence in enterohemorrhagic Escherichia coli.

The Tol-Pal system is a protein complex that is highly conserved in many gram-negative bacteria. We show here that the Tol-Pal system is associated with the enteric pathogenesis of enterohemorrhagic E. coli (EHEC). Deletion of tolB, which is required for the Tol-Pal system decreased motility, secretion of the Type III secretion system proteins EspA/B, and the ability of bacteria to adhere to and to form attaching and effacing (A/E) lesions in host cells, but the expression level of LEE genes, including espA/B that encode Type III secretion system proteins were not affected. The Citrobacter rodentium, tolB mutant, that is traditionally used to estimate Type III secretion system associated virulence in mice did not cause lethality in mice while it induced anti-bacterial immunity. We also found that the pal mutant, which lacks activity of the Tol-Pal system, exhibited lower motility and EspA/B secretion than the wild-type parent. These combined results indicate that the Tol-Pal system contributes to the virulence of EHEC associated with the Type III secretion system and flagellar activity for infection at enteric sites. This finding provides evidence that the Tol-Pal system may be an effective target for the treatment of infectious diseases caused by pathogenic E. coli.