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Proc Natl Acad Sci U S A. Jul 5, 2006; 103(27): 10420–10425.
Published online Jun 27, 2006. doi:  10.1073/pnas.0604343103
PMCID: PMC1482837

The QseC sensor kinase: A bacterial adrenergic receptor


Quorum sensing is a cell-to-cell signaling mechanism in which bacteria respond to hormone-like molecules called autoinducers (AIs). The AI-3 quorum-sensing system is also involved in interkingdom signaling with the eukaryotic hormones epinephrine/norepinephrine. This signaling activates transcription of virulence genes in enterohemorrhagic Escherichia coli O157:H7. However, this signaling system has never been shown to be involved in virulence in vivo, and the bacterial receptor for these signals had not been identified. Here, we show that the QseC sensor kinase is a bacterial receptor for the host epinephrine/norepinephrine and the AI-3 produced by the gastrointestinal microbial flora. We also found that an α-adrenergic antagonist can specifically block the QseC response to these signals. Furthermore, we demonstrated that a qseC mutant is attenuated for virulence in a rabbit animal model, underscoring the importance of this signaling system in virulence in vivo. Finally, an in silico search found that the periplasmic sensing domain of QseC is conserved among several bacterial species. Thus, QseC is a bacterial adrenergic receptor that activates virulence genes in response to interkingdom cross-signaling. We anticipate that these studies will be a starting point in understanding bacterial–host hormone signaling at the biochemical level. Given the role that this system plays in bacterial virulence, further characterization of this unique signaling mechanism may be important for developing novel classes of antimicrobials.

Keywords: AI-3, enterohemorrhagic Escherichia coli, epinephrine, quorum sensing, two-component systems

The gastrointestinal (GI) tract is the site of the most complex heterogeneous environment in the mammalian host, where the majority of the microbial flora reside in the large intestine (1). It is estimated that the microbial population within the human GI tract exceeds the total number of mammalian cells by at least an order of magnitude (1). The bacterial flora is beneficial to the host by facilitating nutrient assimilation and immune competence (2). Conversely, adverse interactions between pathogenic or opportunistic bacteria and the host can lead to disease. The high density and diversity of bacteria within the GI tract suggests that the members of this community communicate among themselves as well as with the host to coordinate any number of advantageous processes. Little is currently known about the communicative relationship(s) among the normal flora, bacterial pathogens, and the host. It has been widely held that bacteria must sense and recognize the host environment to express genes essential for colonization and/or virulence expression. Along those lines, quorum sensing is a cell-to-cell signaling mechanism wherein bacteria respond to chemical hormone-like molecules called autoinducers (AIs). When an AI reaches a critical concentration threshold, the bacteria recognize and respond to this signal by altering their patterns of gene expression. A signaling system has been identified in which a bacterial chemical signal (AI-3) is produced and used for interkingdom signaling between prokaryotic and eukaryotic cells. Namely, AI-3 cross-signals with the eukaryotic hormones epinephrine (epi) and/or norepinephrine (NE) (3).

Enterohemorrhagic Escherichia coli (EHEC) 0157:H7 colonizes the human colon, resulting in the development of often fatal hemorrhagic colitis and hemolytic uremic syndrome (4). EHEC exploits the AI-3/epi/NE signaling system to activate its virulence genes (3). These signals may be sensed by histidine sensor kinases (HKs) in the membrane of EHEC that relay this information to a complex regulatory cascade (3). HKs are, arguably, among the most widely used sensors of all of the signal transduction enzymes in nature, being present in bacteria, archaea, and eukarya (5, 6). Although there are no known HKs present in animals, eukaryotes such as yeast, fungi, plants, and protozoa use HKs to regulate hormone-dependent developmental processes (6). Thus, it has been suggested that HKs originated in bacteria and were later transferred into eukaryotes and archaea (7). Of relevance to EHEC, QseB/QseC comprise a two-component system, in which QseC is the predicted HK and QseB the predicted response regulator. QseB/QseC activate transcription of the flagella regulon responsible for swimming motility in EHEC (8). An EHEC mutant unable to produce AI-3 activates transcription of the flagella/motility genes and, consequently, swimming motility in response to both AI-3 and epi given exogenously. However, a qseC (sensor mutant) is unable to activate expression of these genes in response to both these signals (3).

In this study, we demonstrate that QseC specifically senses the bacterial AI-3 signal and the host epi/NE hormones. QseC directly binds to these signals, and this binding can be blocked by the α-adrenergic antagonist phentolamine (PE). The role of QseC in pathogenesis has also been defined by using a rabbit infection animal model, demonstrating that a qseC mutant is attenuated for virulence. Taken together, these results suggest that QseC is a bacterial adrenergic receptor that is crucial for interkingdom signaling.


QseC Senses the Host Hormones Epi and/or NE.

We have previously reported that a qseC mutant did not activate expression of the flagella and motility genes in response to AI-3, epi, and/or NE (3). These results led us to hypothesize that QseC could be the sensor for these signals and may act as a bacterial adrenergic receptor for these compounds. We tested this hypothesis at the molecular level by expressing and purifying MycHis-tagged QseC under native conditions, and performing in vitro autophosphorylation assays. Because most HKs, including QseC, are membrane-bound, we reconstituted QseC into liposomes. This system can be used to study signal transduction and transmembrane signaling, which depends on the membrane-intrinsic portions of the protein linking the periplasmic sensory and cytoplasmic kinase domains (9, 10). As depicted in Fig. 1A, QseC adopts an inside-out orientation, in which the periplasmic signal-recognition domain is inside the liposomes, whereas the kinase domain is outside. Orientation of proteins in liposomes has been established (10). In the case of QseC as well as on previous studies (9, 10), the inside-out orientation can be concluded from the accessibility of ATP to the kinase site without disruption of the liposomes. To verify QseC protein incorporation into the liposomes, Western blot analysis using anti-Myc antibody was performed. The recognition of the Myc-tag by the anti-Myc antibody without disruption of the liposomes further confirms the inside-out orientation of QseC (data not shown). QseC autophosphorylation was weak in the liposomes alone (Fig. 1B).

Fig. 1.
QseC increases autophosphorylation in response to epi. (A) Graphic depiction of the inside-out orientation of QseC in the liposome. QseC has two transmembrane domains and a HK domain, indicating that its membrane location may allow autophosphorylation ...

To test whether QseC could sense epi, QseC-loaded liposomes were treated with the physiologically intestinal-relevant concentration of 5–50 μM epi (11). Addition of epi to QseC-loaded liposomes during the freeze–thaw step enabled this signal to gain access to the internal space of these liposomes (containing the periplasmic sensing domain of QseC). QseC then increased its level of autophosphorylation dramatically (compared with liposomes to which no epi was added) (Fig. 1B). The addition of 5 μM NE also increased the autophosphorylation of QseC (Fig. 2D and F). Thus, QseC directly responds to the presence of epi/NE. Of note, DcuS (E. coli fumarate and succinate sensor) loaded into liposomes increased its autophosphorylation in response to 20 mM fumarate, as expected, but not to 50 μM epi (Fig. 2A), confirming that QseC and DcuS each sense and respond to different signaling compounds.

Fig. 2.
QseC specifically senses epi/NE/AI-3. Each graph represents results from three separate experiments. Student’s t test was performed to determine whether the results were statistically significant as compared with the control (no signal added). ...

We did not observe autophosphorylation of full-length, soluble QseC in free solution under various buffer conditions (data not shown). This is not inordinate; however, given that autophosphorylation studies of most other HKs have been performed by using truncated proteins containing only the cytoplasmic kinase domain. An important caveat is that these studies preclude signal-recognition assessment by HKs, because the recombinant proteins do not contain the periplasmic domain necessary for signal recognition (1218). In order for QseC to respond to epi/NE, these signals had to be loaded within the liposomes, where the periplasmic domain is located; loading these signals from outside yielded no response (data not shown). Thus, QseC is one of the rare HKs whose signals have been biochemically determined.

To further assess the signal specificity of QseC, we tested whether QseC could sense and respond to other intestinal hormones. QseC autophosphorylation did not increase in response to other intestinal hormones, such as gastrin, secretin, or galanin (Fig. 2B). In fact, the basal level of QseC autophosphorylation in response to any of these hormones was not significantly different from the negative control, where no signal was added, indicating that these other gastric hormones, unlike epi/NE do not play any role in signaling through QseC.

The neuronal response to epi/NE in the intestinal tract can be blocked by β- and α-adrenergic receptor antagonists, such as propranolol (PO) or PE, respectively (19). Because QseC autophosphorylation increases in response to epi/NE, we investigated whether we could block this response using PO or PE. The addition of PO or PE alone to the liposomes did not have a significant effect on QseC autophosphorylation, whereas the addition of 5 μM epi again induced QseC autophosphorylation (Fig. 2C). To address whether PO and PE could act as antagonists of epi for QseC autophosphorylation, liposomes were loaded with 5 μM epi and an excess concentration (50 μM) of PO or PE. Autophosphorylation of QseC was increased by epi in the presence of PO (Fig. 2C), suggesting that PO cannot antagonize the recognition of epi by QseC. However, in the presence of PE, QseC did not respond to epi (Fig. 2C), suggesting that this α-adrenergic antagonist can block the recognition of epi by QseC.

QseC Binds to NE.

Because QseC autophosphorylation increases in response to epi/NE, we reasoned that these compounds may be directly interacting with the membrane-bound QseC to stimulate kinase activity. To study binding, 5 or 10 μM of [3H]NE, alone or in conjunction with 50 μM PE, were loaded into QseC liposomes. In the presence of [γ32P]dATP, QseC autophosphorylation increased with the addition of [3H]NE, as expected (Fig. 2 D and F). The addition of 5 μM [3H]NE plus 50 μM PE did not show a significant increase in QseC autophosphorylation when compared with liposomes without signal added. These results confirm that PE antagonizes NE recognition. To determine whether the [3H]NE was binding to QseC, we excised the bands that contained phosphorylated QseC protein, suspended them in scintillation fluid, and counted, which allows the differential counting of [3H]NE (to assess binding) and γ[32]ATP (to assess autophosphorylation). (Fig. 2 E and F). The addition of 5 μM [3H]NE to the QseC liposome resulted in a 2.25-fold increase in the amount of [3H] bound to QseC, which correlated with the increase in QseC autophosphorylation (Fig. 2 D and E). The addition of 10 μM [3H]NE to these liposomes resulted in a 4-fold increase in the amount of [3H]NE bound to QseC and also an increase in QseC autophosphorylation. No further increase in autophosphorylation was observed by addition of increasing amounts of [3H]NE (Fig. 2F). This finding could be due to the fact that QseC is very sensitive, and even a small amount of signal activates full autophosphorylation for a quick response. Finally, when 5 μM [3H]NE and 50 μM PE were both loaded into the liposomes, a dramatic, 6-fold decrease in the amount of [3H]NE bound to QseC was observed (Fig. 2E). QseC autophosphorylation did not increase significantly in the presence of excess PE (Fig. 2 D and F). These data support our hypothesis that PE antagonizes the recognition of NE by QseC.

QseC Senses the Epi/NE Bacterial Cross-Signal AI-3.

Epi/NE cross-signals with AI-3 (3). To test whether QseC could also respond to AI-3, we loaded the QseC liposomes with purified AI-3 (100 nM). The addition of purified AI-3 significantly increased QseC autophosphorylation to a level similar to that of epi (Fig. 2G). As a negative control, we loaded the QseC liposomes with another bacterial AI, AI-2 (100 μM), which does not activate EHEC virulence genes (3). As predicted, the addition of synthesized AI-2 did not induce a significant difference in QseC autophosphorylation as compared with liposomes to which no signal was added (Fig. 2G).

QseB/QseC Is a Cognate Two-Component System.

QseB is predicted to be the cognate response regulator for QseC (Fig. 3A). To determine whether QseB receives a phosphate from the QseC HK, we again used the QseC liposomes. After loading the liposomes with 50 μM epi, 5 μg of QseB, and 250 μCi (1 Ci = 37 GBq)of [γ32P]dATP were added to the reaction. The QseC HK initiated autophosphorylation after 10 min in the presence of epi and transferred its phosphate to its cognate response regulator, QseB, after 30 min (Fig. 3B). After 120 min, QseC transferred a large proportion of its phosphate to QseB. These data confirm that QseB and QseC is a functional two-component system.

Fig. 3.
QseB/QseC is a functional two-component system. (A) Model of signaling begins with QseC responding to epi/NE and AI-3. QseC increases its autophosphorylation and transfers its phosphate to QseB. Phosphorylated QseB then activates both its own transcription ...

Phosphorylated QseB binds to and activates the transcription of the flhDC promoter, which encodes the FlhDC master regulators of the flagella regulon, and also binds to its own promoter (8, 20). To investigate the effect of epi and PE on downstream gene activation through QseB/QseC, we performed transcriptional analysis of the flhDC promoter in WT, qseC, and luxS mutants (which do not produce AI-3) (3) and complemented strains of EHEC. In agreement with previous results (8), transcription of flhDC in the qseC and luxS mutants was decreased 4- and 2.5-fold compared with WT and complemented strains (Fig. 3C). The addition of 5 μM epi to WT did not significantly change the transcription of flhDC, most likely because of the fact that AI-3, which cross-signals with epi, is already produced by EHEC during growth in culture (3). In support of this hypothesis, addition of 5 μM of epi or 100 nM of AI-3 to a luxS mutant (unable to produce AI-3) (3) increased transcription of flhDC to WT levels, confirming that both signals activate flhDC transcription. Addition of either signal to a qseC mutant did not yield an increase in flhDC transcription, further suggesting that the qseC mutant cannot sense these signals. The addition of 50 μM PE to the luxS mutant (in the presence of just 5 μM epi) or the WT strain (in the presence of both AI-3 produced by EHEC and 5 μM epi) resulted in decreased transcription of flhDC in both strains to the same low level of transcription observed in the qseC mutant (Fig. 3C). These data suggest that PE is blocking QseC autophosphorylation in response to both AI-3 and epi/NE, thereby affecting QseBC-dependent transcription of flhDC. Consequently, when the autophosphorylation of QseC is blocked by PE, QseC cannot transfer a phosphate to QseB, and unphosphorylated QseB cannot interact with the flhDC promoter to activate transcription (8). A negative bla::lacZ control showed no transcriptional difference in any conditions or strains tested (Fig. 3C).

QseC Is Involved in Bacterial Pathogenesis.

The AI-3/epi/NE signaling system has been shown to regulate several virulence genes in EHEC (3). However, the role of this signaling system in pathogenesis in vivo has not been addressed. There is no small-animal model for EHEC; therefore, we used rabbits infected with rabbit enteropathogenic E. coli (REPEC). REPEC is a natural pathogen of rabbits, colonizing the large bowel and causing diarrhea between days 4 and 7 in these animals. The onset of diarrhea in these rabbits resolves by 10–12 days after infection (21). REPEC causes the same lesions in the intestinal cells and possesses the same virulence factors as EHEC. Additionally, a REPEC qseC mutant has the same phenotypes as the EHEC mutant (Fig. 5, which is published as supporting information on the PNAS web site). Using this infection model, we showed that a qseC mutant is attenuated for virulence in rabbits (Fig. 4), thereby establishing that QseB/QseC are involved in pathogenesis. Rabbits inoculated with the WT strain did not gain any weight because of diarrhea (Fig. 4 A and C). Rabbits inoculated with the qseC mutant gained weight as did PBS controls (WT vs. qseC, P < 0.03; PBS vs. qseC, P < 0.18) (Fig. 4A). The WT strain caused diarrhea in six of seven rabbits by day 6 after inoculation (Fig. 4C). None of the PBS control animals presented diarrhea (Fig. 4B). The qseC mutant was attenuated in rabbits in comparison with WT, with two of eight rabbits presenting diarrhea (WT vs. qseC, P < 0.0005) (Fig. 4D).

Fig. 4.
A qseC mutant is attenuated for virulence in rabbits. (A) Cumulative weight gain of rabbits inoculated with WT, qseC, or PBS by day 6. Development of diarrhea by rabbits inoculated with PBS (B), WT (C), and qseC (D). The WT caused diarrhea in six of seven ...


Very few signals or inhibitors of two-component systems are currently known. Roychoudhury (22) was the first to identify an inhibitory compound of the AlgR21 two-component system of Pseudomonas aeruginosa. AlgR21 regulates the transcription of alginate, a complex carbohydrate that inhibits access of antimicrobials to the site of infection in cystic fibrosis patients (23). Of the 25,000 synthetic and natural compounds screened, only 15 inhibitors were identified, many of which contained aromatic rings (22). Additionally, extensive screening assays have identified several inhibitors, including several for the Bacillus subtilis KinA/SpoOF two-component system (24, 25). Although a few compounds have been reported as inhibitors of two-component systems, their mechanisms of action and true stimulatory signals are largely unknown (26). Even less is known about the signaling compounds that specifically activate HKs. One of the few systems for which a defined signal has been identified is the DcuSR two-component system of E. coli, which controls the expression of genes of C (4)-dicarboxylate metabolism (27). Janausch et al. (9) have shown that the autophosphorylation of liposome-reconstituted DcuS is stimulated by the signals fumarate and succinate. Here, we show that both the AI-3 signal produced by the microbial flora and the epi/NE signals produced by the host are sensed by the QseC HK. Upon sensing these signals, QseC autophosphorylates and then transfers its phosphate to QseB, which then regulates both is own transcription and the transcription of the flagella and motility genes (8, 20). EHEC likely is then able to exploit its motility apparatus to swim to the intestinal epithelium and initiate infection. The observation that a qseC mutant is attenuated for virulence in a rabbit infection model (Fig. 4), further underscores the role of this signaling system in bacterial pathogenesis.

QseC acts a bacterial adrenergic receptor and links cross-kingdom signaling by sensing a bacterial hormone-like compound (AI-3) and the host hormones epi and/or NE. It is noteworthy that QseC does not share primary sequence homology with adrenergic receptors; hence, it may serve as a functional analog, not homolog, of these G protein-coupled receptors. An in silico search using the periplasmic (signal-sensing) domain of QseC reveals a high degree of conservation among different bacterial species (Fig. 6, which is published as supporting information on the PNAS web site). The QseC sensor is found in Shigella sp., Salmonella sp., Erwinia carotovora, Haemophilus influenzae, Pasteurella multocida, Actinobacillus pleuropneumoniae, Chromobacterium violaceium, Rubrivivax gelatinosus, Thiobacillus denitrificans, Ralstonia eutropa, Ralstonia metallidurans, and Psychrobacter sp. This search also revealed homology to a fungal protein of unknown function from Aspergillus nidulans. Taken together, these findings suggest that QseC may have an ancient evolutionary history.

One of the daunting medical challenges at present surrounds the issue of microbial antibiotic resistance. It once was believed that modern medicine conquered infectious diseases through the discovery of both first- and second-generation antimicrobials. However, very few novel antibiotics have been discovered in the past 30 years, and, during this period, profound selective pressure through both justified and indiscriminant use of treatment has allowed bacteria to quickly evolve mechanisms of resistance to virtually all known antibiotics. Hence, diseases such as tuberculosis are again developing into significant public health problems and, thus, are considered to be reemerging infectious diseases. Many other infections, like the hemorrhagic colitis and hemolytic uremic syndrome caused by EHEC, comprise newly emerging infectious diseases. These combined health threats constitute a compelling argument for the development of novel classes of antimicrobial compounds. Our combined data, reported herein, in addition to published data (3), indicate that adrenergic antagonists can inhibit the AI-3/epi/NE signaling cascade in EHEC and render it unable to induce its virulence genes (in response to these signals), suggesting that antagonists targeting this signaling cascade might constitute a novel class of antimicrobials (3). Furthermore, these antimicrobials will possibly be useful against other human pathogens, including enteropathogenic E. coli, Salmonella, Shigella, and Yersinia, etc., all of which ostensibly harbor this signaling cascade. A broad understanding of the QseB/QseC signaling cascade will be instrumental for investigating further the intriguing concept of interkingdom sensory signaling.

Materials and Methods

Strains and Plasmids.

E. coli strains were grown in LB at 37°C. The EHEC WT strain, 86-24, was isolated from an outbreak in the United States (28). The EHEC isogenic qseC (VS138), luxS (VS94), and complemented strains (VS179 and VS95, respectively) have been described (29, 30). The REPEC qseC mutant (VS243) was generated by allelic exchange using vector pVS132 as described (29). The qseC mutant (VS243) was complemented with plasmid pVS178 (29), generating strain VS247.

Inoculation of Rabbits.

Bacterial strains were inoculated in 2-month-old New Zealand White rabbits. Three groups of animals (seven to eight in each group) were inoculated with the WT, the qseC mutant (VS243) strain, or PBS (negative control). Bacteria were cultured in LB broth, washed once, suspended in sterile PBS, and adjusted to OD600 = 1. Subsequent dilution of this bacterial suspension at 1:100 was made to obtain ≈1 × 107 colony-forming units per ml for inoculation. Bacterial viable counts were determined by plating. These experiments were repeated twice with two different groups of animals to ensure their reproducibility.

Before inoculation, rabbits were starved overnight. Rabbits were inoculated intragastrically via a pediatric feeding tube with 10 ml of 10% bicarbonate solution, followed by a 3-ml inoculum. Rabbits were weighed daily and observed for stool characteristics and clinical signs of illness for 13 days. Stools were graded as normal (hard pellets no diarrhea) or diarrhea (completely liquid). Rabbits were killed 13 days after inoculation. Differences in weight gain between experimental groups were analyzed by the Student t test. Differences in diarrheal disease between the experimental groups were analyzed by using a χ2 test.

β-Galactosidase Assays.

The lacZ fusions (flhDC::lacZ from pVS182 and bla::lacZ from pVSAP) were constructed as described, and assayed for β-galactosidase activity by using o-nitrophenyl-β-d-galactopyranoside as a substrate (29).

Reconstitution of QseC-His into Liposomes.

As described in ref. 8, E. coli strains containing either pVS154 (QseB-MycHis) or pVS155 (QseC-MycHis) were induced with 0.2% arabinose, and purified through nickel columns according to the manufacturer’s instructions (Qiagen).

Liposomes were reconstituted as described by Janausch et al. (31). Briefly, 50 mg of E. coli phospholipids (20 mg/ml in chloroform; Avanti Polar Lipids) were evaporated and then dissolved into 5 ml of potassium phosphate buffer containing 80 mg of N-octyl-β-d-glucopyranoside. The solution was dialyzed overnight against potassium phosphate buffer. The resulting liposome suspension was subjected to freeze–thaw in liquid N2. Liposomes were then destabilized by the addition of 26.1 mg of dodecylmaltoside, and 2.5 mg of QseC-MycHis was added, followed by stirring at room temperature for 10 min. Two hundred-sixty milligrams of Biobeads were then added to remove the detergent, and the resulting solution was allowed to incubate at 4°C overnight. The supernatant was then incubated with fresh Biobeads for 1 h in the morning. The resulting liposomes containing reconstituted QseC-MycHis were frozen in liquid N2 and stored at −80°C until used. Orientation of HKs in the liposome system has been established by other groups (12) and can be concluded from the accessibility of ATP to the kinase site and anti-Myc antisera to the C-terminal QseC-MycTag without disruption of the liposomes.

Phosphorylation of QseC-MycHis in Liposomes.

Twenty microliters of the liposomes containing QseC-MycHis were adjusted to 10 mM MgCl2 and 1 mM DTT, and various concentrations of agonist or antagonist, frozen and thawed rapidly in liquid N2, and kept at room temperature for 1 h (this allows for the signals to be loaded within the liposomes). [γ32P]dATP (0.625 μl) (110 TBq/mmol) was added to each reaction. To some reactions, 10 μg of QseB-MycHis was added. At each time point (0, 10, 30, 60, or 120 min), 20 μl of SDS loading buffer was added. For all experiments involving QseC alone, a time point of 10 min was used. The samples were run on SDS/PAGE without boiling and visualized via PhosphorImager. The bands were quantitated by using imagequant version 5.0 software (Amersham Pharmacia).

Agonists and Antagonists.

Various concentrations of agonist or antagonist (Sigma) were added to each of the liposome experiments, resulting in final concentrations as follows: 5 μM or 50 μM epi, 5 μM or 10 μM NE, 50 μM PE, 50 μM PO, 50 μM gastrin, 50 μM galanin, and 50 μM secretin. Synthetic AI-2 (≈100 μM) and purified AI-3 (≈100 nM) were obtained as described (3). AI-3 was a gift from B. Sangras and J. R. Falck (University of Texas Southwestern Medical Center) and was purified from EHEC strain 86-24 as described (3). Tritiated NE was obtained from Amersham Pharmacia Biosciences and used at a final concentration of 5 or 10 μM.

Determination of Tritiated Ligand Binding.

To determine the concentration of tritiated NE that was bound to QseC-MycHis in the liposomes, 20 μl of the liposome containing QseC-MycHis were adjusted to 10 mM MgCl2, 1 mM DTT, and 5 μM tritiated NE, 10 μM tritiated NE, or 5 μM tritiated NE plus 50 μM PE. The liposomes were frozen and thawed rapidly in liquid N2 and kept at room temperature for 1 h. [γ32P] (0.625 μl) dATP (110 TBq/mmol) was added to each reaction. After 10 min, SDS loading dye was added, and the samples were run on SDS/PAGE and visualized by PhosphorImager. The bands containing phosphorylated QseC-His were excised and counted in a scintillation counter.

Supplementary Material

Supporting Figures:


We thank G. Unden (Johannes Gutenberg-Universität, Mainz, Germany) for the DcuS protein; J. R. Falck for the AI-3; Hernan Rios and Katherine Davis for assistance with the rabbits’ infections; and Michael Gale, Jr., Lora Hooper, James B. Kaper, Michael Norgard, Kim Orth, Melissa Kendall, and David Rasko for critical reading of the manuscript. This work was supported by National Institutes of Health (NIH) Grant AI053067 and an Ellison Foundation award. M.B.C. was supported through NIH Training Grant 5-T32-AI007520-07.


enterohemorrhagic E. coli
histidine sensor kinase
rabbit enteropathogenic E. coli.


Conflict of interest statement: No conflicts declared.


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