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J Bacteriol. 2017 April 15; 199(8): e00861-16.
Published online 2017 March 28. Prepublished online 2017 January 30. doi:  10.1128/JB.00861-16
PMCID: PMC5370426

Genetic and Mechanistic Analyses of the Periplasmic Domain of the Enterohemorrhagic Escherichia coli QseC Histidine Sensor Kinase

Thomas J. Silhavy, Editor
Thomas J. Silhavy, Princeton University;


The histidine sensor kinase (HK) QseC senses autoinducer 3 (AI-3) and the adrenergic hormones epinephrine and norepinephrine. Upon sensing these signals, QseC acts through three response regulators (RRs) to regulate the expression of virulence genes in enterohemorrhagic Escherichia coli (EHEC). The QseB, QseF, and KdpE RRs that are phosphorylated by QseC constitute a tripartite signaling cascade having different and overlapping targets, including flagella and motility, the type three secretion system encoded by the locus of enterocyte effacement (LEE), and Shiga toxin. We modeled the tertiary structure of QseC's periplasmic sensing domain and aligned the sequences from 12 different species to identify the most conserved amino acids. We selected eight amino acids conserved in all of these QseC homologues. The corresponding QseC site-directed mutants were expressed and still able to autophosphorylate; however, four mutants demonstrated an increased basal level of phosphorylation. These mutants have differential flagellar, motility, LEE, and Shiga toxin expression phenotypes. We selected four mutants for more in-depth analyses and found that they differed in their ability to phosphorylate QseB, KdpE, and QseF. This suggests that these mutations in the periplasmic sensing domain affected the region downstream of the QseC signaling cascade and therefore can influence which pathway QseC regulates.

IMPORTANCE In the foodborne pathogen EHEC, QseC senses AI-3, epinephrine, and norepinephrine, increases its autophosphorylation, and then transfers its phosphate to three RRs: QseB, QseF, and KdpE. QseB controls expression of flagella and motility, KdpE controls expression of the LEE region, and QseF controls the expression of Shiga toxin. This tripartite signaling pathway must be tightly controlled, given that flagella and the type three secretion system (T3SS) are energetically expensive appendages and Shiga toxin expression leads to bacterial cell lysis. Our data suggest that mutations in the periplasmic sensing loop of QseC differentially affect the expression of the three arms of this signaling cascade. This suggests that these point mutations may change QseC's phosphotransfer preferences for its RRs.

KEYWORDS: enterohemorrhagic E. coli, EHEC, LEE, QseC, two-component system


Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes outbreaks of bloody diarrhea and hemolytic-uremic syndrome (HUS) worldwide. EHEC colonizes the colon, where it forms attaching and effacing (A/E) lesions on enterocytes. The hallmark of these lesions is the effacement of the microvilli and the rearrangement of the cytoskeleton to form a pedestal-like structure that cups the bacterium (1,3). A/E lesion formation requires the expression of a type three secretion system (T3SS) encoded within the locus of enterocyte effacement (LEE) (4, 5). Several LEE-encoded proteins are also necessary for A/E lesion formation, such as the adhesin intimin (6) and its receptor Tir (7). The LEE-encoded T3SS translocates effector proteins encoded both within and outside the LEE region. One such effector is EspFu/TccP, which is essential for A/E lesion formation (8,15). Additionally, EHEC also produces Shiga toxins (Stx) that are responsible for HUS (16). This toxin is encoded by an integrated lambdoid phage in the EHEC chromosome. The genes encoding Stx are located within the late-phage genes and expressed along with the phage's late genes when the phage enters the lytic cycle due to activation of the cell's SOS response (17,19).

Among many signals, there are three chemical signals sensed by EHEC that have been extensively reported to activate transcription of its virulence genes: a bacterial autoinducer (AI-3) produced by the normal gastrointestinal flora and two hormones (epinephrine/norepinephrine) produced by the host (20). These signals are detected by two membrane-bound histidine sensor kinases, QseC and QseE, which subsequently relay this information to a complex regulatory cascade to activate the transcription of key virulence genes (21, 22). When in the presence of these signals, QseC first autophosphorylates and then transfers the phosphate to QseB, its cognate response regulator (RR). The qseBC genes are cotranscribed in an operon that is also autoregulated (23). QseB is involved in regulation of the flagellar and motility genes (24, 25) (Fig. 1). This RR directly binds to the promoters of qseBC and flhDC (the master transcription regulator of the flagellar regulon), thereby allowing expression of these genes to be influenced by exogenous signals from the host or host flora. In the case of flhDC, the operon is activated by phosphorylated QseB but is repressed by unphosphorylated QseB (24, 25).

QseC signaling cascade. QseC senses AI-3 epinephrine (E) and norepinephrine (NE) to increase its autophosphorylation. QseE senses E and NE and sulfate (SO4) and phosphate (PO4). QseC is at the top of this signaling cascade, because QseC activates qseE ...

In addition to phosphorylating its cognate RR, QseC also phosphorylates the noncognate RRs QseF and KdpE (25). Through these RRs, QseC regulates expression of the LEE and Stx genes at the transcriptional and posttranscriptional levels (25,27). There are 41 genes within the LEE, and the majority of them are organized within five major operons (LEE1 to -5) (28,32). The first gene within LEE1 is ler, which encodes the master transcriptional activator of all LEE genes (29, 32, 33). KdpE in conjunction with Cra (a member of the LacI family that uses fluctuations in sugar concentrations to activate or inhibit the expression of its target genes [34]) directly binds to the regulatory region of ler (LEE1) to activate expression of all the LEE genes in a cascade fashion (25, 26, 35). Cra is active during gluconeogenesis (34); consequently, the growth of EHEC under glycolytic conditions inhibits LEE expression while growth under gluconeogenic conditions activates expression of these genes, and this sugar-dependent regulation is achieved through KdpE and Cra (26).

The QseF RR is phosphorylated by both QseC and QseE (25). While QseC senses AI-3, epinephrine, and norepinephrine, QseE senses epinephrine/norepinephrine, phosphates, and sulfates (21, 22). QseF is involved in the regulation of the stxAB genes encoding Shiga toxin (25), and QseF and QseB directly activate expression of the glmY small RNA (sRNA) that is encoded upstream of the qseEGF operon (36). The glmY sRNA posttranscriptionally controls expression of the LEE and espFu genes, which are necessary for A/E lesion formation (37). The GlmY sRNA is known to stabilize the GlmZ sRNA (constitutively encoded elsewhere in the E. coli chromosome [38]), and GlmZ is the sRNA that base pairs with its target mRNAs in an Hfq-dependent manner (38). GlmY/GlmZ destabilizes the LEE4 and LEE5 mRNA and promotes translation of EspFu, modulating the levels of A/E lesions produced by EHEC (38).

Although the phosphorylation cascade and resulting genetic regulation controlled by QseC have been studied extensively, the structural requirements for kinase activity are still unclear. The purpose of this study was to identify amino acids in the periplasmic domain of QseC that affect its ability to function as a kinase. Despite the fact that the periplasmic domain is predicted to be the site of signal sensing, the amino acids described here appear to play a role in controlling the interaction between QseC and the three RRs, QseB, KdpE, and QseF.


Eight residues within the QseC periplasmic domain are conserved among homologues.

QseC is present in several species of bacteria. We aligned the periplasmic domain of several QseC proteins to identify the most conserved amino acids in this domain to target for mutagenesis (39). When the periplasmic domain (amino acid [aa] residues 37 through 155) of EHEC QseC was aligned with the same region of homologous proteins, at least eight strictly conserved residues were identified (Fig. 2A). To better understand the position of these residues within the periplasmic domain, a model of this domain was predicted using the Modeller software. When the positions of the residues were visualized on the predicted model (Fig. 2B), the conserved residues appeared to be located in one of two structural regions. Residues D101, F117, R130, V145, Q147, and W129 were predicted to be located in the vicinity of a series of β-sheets, forming a potential binding pocket. In both CitA and DcuS, two histidine kinases (HKs) that resemble the topology and secondary structure of QseC, this binding pocket is the site of ligand binding (40, 41). For this reason, these residues were chosen for site-directed mutagenesis. Three of these residues (R130, V145, and Q147), plus two more residues (D45 and R152), were also strictly conserved. D45 and R152 were both located within or near α-helices proximal to the potential binding pocket. Due to their location and conservation within the predicted structure, all eight of these residues were chosen for site-directed mutagenesis.

Alignment of QseC homologues' periplasmic domain and model structure of this domain. (A) Sequence alignment of the periplasmic domain of EHEC QseC to the same region of QseC homologues representing a range of bacterial families. The conserved residues ...

QseC autophosphorylation.

Using liposomes, we have previously reported that QseC increases its autophosphorylation in response to epinephrine, norepinephrine, and AI-3 (42, 43). Here, we again confirmed that QseC increases its autophosphorylation in liposomes in the presence of epinephrine. In liposomes, QseC's response to epinephrine can be observed between 2 and 10 min (Fig. 3A). Because it has been previously reported that QseC autophosphorylation could be monitored using membrane vesicles (44), we also assessed the conditions in these assays under which QseC would still respond to signals, given that membrane preparation for several site-directed mutants would be a more straightforward protocol than that for liposomes. We observed that in the membrane autophosphorylation of QseC was faster and that high levels of NaCl enhanced QseC autophosphorylation, decreasing its response to epinephrine. These results suggest that the conditions previously used to assess QseC autophosphorylation using Tris-buffered saline (TBS) buffer (containing 150 mM NaCl) (44) are not conducive to probing epinephrine sensing. QseC already autophosphorylated to high levels at 1 min, and increased concentrations of NaCl abolished its response to epinephrine, with 100 mM NaCl completely bypassing the response to this signal (Fig. 3B).

QseC autophosphorylation in response to epinephrine. (A) Autophosphorylation of QseC reconstituted in liposomes with and without 50 μM epinephrine (Epi). (B) Autophosphorylation of QseC in membrane vesicles with and without 100 μM Epi ...

Characterization of QseC mutants.

We changed the eight conserved residues in QseC's periplasmic domain (D101, F117, W129 D45, R130, V145, Q147, and R152) into alanines. These mutations were introduced into a cloned copy of qseC in a pBADMycHisA plasmid under the control of an arabinose-inducible promoter. The altered plasmids, as well as the plasmid carrying the wild-type (WT) gene, were used to complement strain VS138, a qseC-null mutant of EHEC. All of the mutant proteins were expressed (Fig. 4A) and were capable of autophosphorylation in membrane vesicles (Fig. 4B), suggesting that all of them maintained their kinase activity. The D101A, F117A, W129A, and V145A mutants had basal autophosphorylation activity comparable to that of WT QseC (Fig. 4B and andC).C). Notably, the D45A, R130A, Q147A, and R152A mutants demonstrated increased basal levels of autophosphorylation compared to WT QseC (Fig. 4B and andCC).

Expression and autophosphorylation of WT QseC and point mutants. (A) Membrane proteins from VS138 (qseC-null mutant) expressing wild-type QseC or one of the point mutants cloned in pBADMycHisA were resolved by SDS-PAGE. QseC was detected using anti-His ...

One of the QseC-regulated phenotypes in EHEC is motility. The qseC-null mutant (VS138) has reduced motility compared to the WT, and this phenotype can be complemented with qseC cloned in a plasmid (pVS155) (24, 45). We assayed VS138 (qseC-null mutant) complemented with WT QseC (pVS155) or the eight amino acid mutants for motility. When these strains were assayed for motility (Fig. 5A), only three strains were significantly hindered in motility: the D45A, R130A, and R152A mutants. The remaining strains were able to swim through soft agar at a rate similar to that of the strain complemented with WT qseC. Quantification of transcription of fliC, the gene encoding the major flagellin protein, was also used to determine the effect of these mutations on the flagellar biosynthesis pathway. Quantification of the fliC transcript by reverse transcription-quantitative PCR (qRT-PCR) identified two additional mutations, V145A and Q147A, that adversely affected the complemented strains' ability to produce flagellin (Fig. 5B). A potential explanation for the fact that these two strains were still motile is that even with lowered levels of fliC transcript, these strains still produced enough flagellin to assemble functional flagella. Interestingly, the D45A mutant appears to have fliC transcripts at levels equivalent to the WT, despite being less motile. This suggests that this mutation might be affecting some other aspect of flagellar biosynthesis or motility, such as motor assembly/rotation or flagellin secretion.

QseC-dependent motility and fliC expression. (A) Motility of VS138 (qseC-null mutant) complemented with either wild-type QseC or one of the eight point mutants. Representative images of each of the strains are shown in insets above the graph. The graph ...

Next, we tested LEE and Stx expression in these mutants compared to that with WT QseC by qRT-PCR. Expression of ler (the master regulator of the LEE genes [29]) was decreased in six of the mutants but not in the Q147A mutant, where it was unchanged, and the R152A mutant, where it was increased (Fig. 6A). Expression of tir, which is required for A/E lesion formation (7), is also decreased in all mutants except for the Q147A mutant, in which its expression is highly increased. Expression of stx2 is significantly decreased in the W129A mutant and increased in the R152A mutant, being largely unchanged in the others (Fig. 6A). To delve deeper into QseC-regulated phenotypes, we streamlined further assays to four mutations that belonged to different phenotypic categories: D45A (increased basal level of autophosphorylation, decreased motility but unchanged fliC expression, decreased ler and tir expression, and unchanged stx2 expression), V145A (normal levels of basal autophosphorylation, unchanged motility but decreased fliC expression, decreased ler and tir expression, and unchanged stx2 expression), Q147A (increased autophosphorylation, unchanged motility but decreased fliC expression, unchanged ler but increased tir expression, and unchanged stx2 expression), and R152A (increased autophosphorylation, decreased motility and fliC expression, increased ler and decreased tir expression, and increased stx2 expression). Western blots for the LEE-secreted effector EspA are mostly congruent with the LEE transcriptional profiling, with decreased levels of secreted EspA in the D45A and V145A mutants and increased levels of secreted EspA in the Q147A and R152A mutants (Fig. 6B). It is worth noting that both the LEE5 (which contains tir) and LEE4 (which contains espA) operons are highly posttranscriptionally regulated (27, 46,48), which can account for the differences in ler and tir transcription and EspA expression in the Q147A and R152A mutants. We also quantified A/E lesion formation in these mutants, and the D45A, VS145A, and R152A mutants formed A/E lesions at levels similar to those with WT QseC, but the Q147A mutant demonstrated enhanced A/E lesion formation (Fig. 6C and andDD).

QseC-dependent LEE expression and A/E lesion formation. (A) qRT-PCR analysis of the LEE genes ler and tir and stx2 from VS138 (qseC-null mutant) and VS138 complemented with either wild-type QseC (Comp) or one of the point mutants. RNA was extracted from ...

QseC phosphorylates three RRs: QseB, KdpE, and QseF. These RRs regulate different and overlapping targets of this signaling cascade, including phenotypes such as flagella, motility, and LEE and Stx expression (25) (Fig. 1). Because expression of flagella, LEE, and Stx probably occurs at different times and under diverse environmental conditions, we investigated the conditions under which these RRs are expressed. QseB is highly expressed in mid- and late-logarithmic growth by EHEC in LB medium (Fig. 7), which is the environmental condition conducive to flagellar expression and motility (25). KdpE is expressed only in late-mid-logarithmic growth in low-glucose Dulbecco's modified Eagle's medium (DMEM) (gluconeogenic conditions) (Fig. 7), which is the environmental condition under which the LEE genes are expressed at optimal levels (26). QseF is expressed throughout growth under all conditions tested, LB, low-glucose DMEM (gluconeogenic), and high-glucose DMEM (glycolytic) (Fig. 7), which could reflect the fact that QseF's regulation of the LEE and Stx occurs indirectly and, in the case of the LEE, posttranscriptionally (25, 27).

Expression of RRs phosphorylated by QseC under different environmental conditions. qseB, qseF, and kdpE RNAs from WT EHEC grown in LB, low-glucose DMEM, and high-glucose DMEM to different OD600 values at 37°C were extracted and analyzed by qRT-PCR. ...

Because these RRs regulate different arms of the QseC signaling cascade, we reconstituted QseC in liposomes and assessed phosphotransfer of the QseC mutants to these three RRs. Phosphotransfer from QseC to QseB and KdpE could be quantified because these proteins differ in size, but phosphotransfer from QseC to QseF could not be quantified because they have the same molecular weight (Fig. 8B and andC).C). The D45A and R152A mutants demonstrated decreased phosphotransfer to both QseB and KdpE. The V145A mutant had unaltered phosphotransfer to QseB but decreased phosphotransfer to KdpE. The Q147A mutant had increased phosphotransfer to QseB and decreased phosphotransfer to KdpE (Fig. 8B and andCC).

Phosphotransfer from WT QseC and point mutants to QseB, QseF, and KdpE. (A) β-Galactosidase activity of the qseBC promoter-lacZ fusion in the absence or presence of epinephrine (50 μM), measured by the Miller assay. (B) Representative ...

To further probe the different interactions between QseC and these RRs, we assessed ler and stx2 transcription in the ΔqseC ΔkdpE mutant and the ΔqseBC strain complemented with WT QseC or the four site-directed mutant genes. In the ΔqseC ΔkdpE mutant, complementation with WT QseC had a discrete impact in increasing ler and stx2 transcription (Fig. 8D), which suggests that the QseC-KdpE arm of this signaling cascade plays an important role in the expression of these genes. Transcription of ler was highly increased (15-fold) by complementation with WT QseC in the ΔqseBC mutant (Fig. 8E), again suggesting an important role for KdpE but not QseB in QseC-dependent activation of ler (25). The levels of ler transcription in the D45A and VS145A mutants were comparable to that in the ΔqseC ΔkdpE mutant with WT QseC (Fig. 8D), suggesting that these point mutants act through KdpE in regard to ler transcriptional activation. ler expression was decreased in the ΔqseBC mutant with the D45A and V145A mutations (Fig. 8E), which may be reflective of the dependence on KdpE for this activation in these mutants, given that they demonstrate lower levels of KdpE phosphorylation than strains with WT QseC (Fig. 8B and andC).C). Transcription of ler was unchanged for the R152 mutant compared to that with WT QseC in the ΔqseC ΔkdpE and ΔqseBC strains (Fig. 8D and andE),E), again suggesting that posttranscriptional regulation (27, 46,48) may play a role in the regulation of the LEE by this mutant. Transcription of ler was increased in the ΔqseC ΔkdpE mutant with Q147A, while it was decreased in the ΔqseBC mutant compared to the strain with WT QseC (Fig. 8D and andE).E). These data suggest that there is an unknown QseB-dependent regulation of ler transcription triggered by this mutation, given that QseB is overphosphorylated by the Q147A mutant (Fig. 8B) and ler transcription increases in the absence of KdpE, which in theory could increase the pools of phospho-QseB within the cell, and decreases in the absence of QseB.

As in the ΔqseC ΔkdpE mutant, transcription levels of stx2 were also discretely impacted in the ΔqseBC mutant (Fig. 8D and andE),E), suggesting that neither KdpE nor QseB plays an important role in QseC-dependent stx2 activation, which has been previously shown to occur mostly through QseF (25). However, while levels of transcription of stx2 are not different between the strain with WT QseC and any of the ΔqseBC mutants (Fig. 8E), its transcription is increased in the D45A, Q147A and R152A ΔqseC ΔkdpE mutants (Fig. 8D). It is also possible that several of these phenotypes are affected by dysregulation of QseF, but unfortunately, we could not measure QseF's phosphorylation by QseC.

To investigate whether any of these mutations impacted the ability of QseC to sense and respond to epinephrine, we assessed the expression of a qseBC-lacZ transcriptional fusion (qseBC autoregulates its own expression in response to epinephrine [23]) in the absence and presence of epinephrine in the qseC strain complemented with WT QseC or the point mutants. Expression of qseBC was enhanced by epinephrine in the strain with WT QseC and all four mutants, suggesting that all mutants can still sense this signal (Fig. 8A). However, the effect of epinephrine on qseBC transcription was enhanced in the V145A, Q147A, and R152A mutants, suggesting that they are hyperresponders to this signal (Fig. 8A).


From a structural standpoint, QseC is typical of most sensor kinases; however, it is one of the only kinases that cannot autophosphorylate constitutively when only its cytoplasmic domain is expressed in vitro, requiring proper insertion of the full protein into the lipid membrane for autophosphorylation to occur (49). This suggests that the structure of the periplasmic and transmembrane regions is integral to its ability to autophosphorylate and to transfer that phosphoryl group to an RR. The point mutations described in this study are all located in the periplasmic domain (Fig. 2), but the mutations that displayed the largest effect on QseC function (D45A, V145A, Q147A, and R152A) were located near the periplasmic face of the inner membrane. In addition, three of these mutations (V145A, Q147A, and R152A) are located adjacent to a predicted HAMP domain (residues 164 to 234) (Fig. 2) that is theorized to play a role in kinase multimerization (50). These mutations had a strong effect on QseC's ability to phosphorylate its RRs and may have differentially affected the arms of this tripartite signaling cascade (Fig. 5 to to8).8). These data suggest that modifications in the periplasmic sensing domain of a sensor kinase alter its abilities for phosphotransfer to RRs.

Interestingly, the effect seen on RR phosphorylation varied among regulators. This suggests that QseC interacts with the RRs in different ways. One explanation is that QseC has a different affinity for each of the three RRs in this study (QseB, KdpE, and QseF). The fact that KdpE was phosphorylated poorly after any mutation in the periplasmic domain might indicate that QseC has a lower affinity for this RR than it does for the other two (Fig. 8B). Any alteration to the periplasmic domain, particularly in the vicinity of the HAMP domain, may have an adverse effect on QseC's ability to form multimers and efficiently phosphorylate RRs. If this is the case, we would expect the RR with the lowest affinity with QseC to be affected the most.

It the case of QseB, the different mutations in QseC had a range of effects on RR phosphorylation. Some mutations (D45A and R152A) resulted in lower QseB phosphorylation, while the Q147A mutation resulted in higher QseB phosphorylation, and the V145A mutation did not appear to have any effect on phosphorylation. Motility is decreased in both D45A and R152A mutants, congruent with the lower level of phosphorylation of QseB by these mutants (Fig. 5 and and8).8). However, flagellin expression, although decreased in R152A, is not altered in V45A, suggesting that the motility defect in V45A does not occur at the level of flagellin expression and may be due to effects on flagellar assembly and/or motor assembly/rotation. The V145A mutant has decreased ability to phosphorylate QseB, congruent with the lower expression of fliC; however, it does not demonstrate any motility defects (Fig. 5 and and8),8), suggesting that other aspects of flagellar assembly and rotation can compensate for lower flagellin expression in this mutant. The Q147A mutant overphosphorylates QseB but has decreased fliC expression and nonaltered motility (Fig. 5 and and8),8), suggesting again that other aspects of flagellar assembly and rotation are altered by the mutation.

Because QseC and QseF have similar molecular weights, making it difficult to quantify QseF autophosphorylation, we refrain from discussing the potential effects of these QseC mutations on QseC-QseF interactions.

Another structural difference is that the D45A mutation is located near the first transmembrane helix while the other three mutations are adjacent to the second transmembrane helix and the HAMP domain. Since the second transmembrane region and the HAMP domain are theoretically involved in multimerization, the presence of V145A, Q147A, and R152A mutations near this location might result in an alteration in structure or multimerization different from that caused by the D45A mutation and therefore have different effects on the phosphorylation of QseB and QseF. Moreover, one has to take into account that the RRs are differentially expressed under different environmental conditions (Fig. 7), which may change the pool of available RRs within the bacterial cell to be phosphorylated by QseC.

The notion that HKs are exquisitely faithful to their cognate RRs has been proposed using truncated HKs where only the cytoplasmic domain was utilized (51). Here, we show that mutations in the periplasmic sensing loop of QseC change phenotypes associated with the different RRs phosphorylated by this HK. Taken as a whole, this study indicates that the interactions between an HK and various RRs may be unique to each kinase/regulator pair and that it is possible to influence these interactions through specific mutations within the periplasmic domain of the kinase. By better understanding these effects, we may be able to better understand how kinases have evolved to regulate multiple two-component systems and how we can better target these kinases to selectively disrupt one kinase/regulator pair while leaving another kinase/regulator pair unaffected.


Plasmids and strains.

The plasmids and strains used in this study are listed in Table 1. Unless otherwise noted, all strains were grown in either Luria-Bertani (LB) medium or low-glucose Dulbecco's modified Eagle's medium (DMEM) at 37°C and 250 rpm.

Strains and plasmids used in this study

In silico analysis.

Known QseC homologues were identified by BLAST search using EHEC QseC as the target sequence. Several pathogenic or commensal homologue examples (total of 12) were chosen, and the periplasmic domains (equivalent to residues 37 through 155 of the EHEC protein) of these homologues were aligned using CLC Sequence Viewer 6 (CLC bio). The structure of the periplasmic domain of EHEC QseC was predicted with Modeler version 9.10 (52, 53), using the known structures of E. coli CitA (40) and DcuS (41) as the template.

Generation of mutants.

Nonpolar mutants were constructed using the λ red protocol to all be in the same genetic background (54). The ΔqseC ΔkdpE mutant was constructed by knocking out kdpE on VS138 using kdpEλRed-F and kdpEλRed-R primers (55). The ΔqseBC mutant was constructed by knocking out qseB on VS138 using qseBλRed-F and qseBλRed-R primers (25).

Generation of QseC point mutants.

The QuikChange II site-directed mutagenesis kit (Agilent Technologies) was used to introduce alanine substitution mutations in a plasmid carrying a C-terminally His-tagged copy of the qseC gene under the control of the araBAD promoter (pVS155) (45). The primers (Table 2) were used to amplify pVS155, and the resulting PCR product was treated with DpnI and transformed into XL1-Blue cells. The mutations were confirmed by sequencing plasmid DNA.

Oligonucleotides used in this study

Motility assays.

Either pVS155 or derivatives containing point mutations were introduced into VS138, a qseC-null mutant of WT EHEC 86-24 (45). These strains were subsequently used in motility assays performed using the soft agar method, as previously described (24). Briefly, strains were stabbed in triplicate into soft agar motility plates (1% tryptone, 0.25% NaCl, 0.3% agar) containing 0.2% arabinose and incubated at 37°C. The diameter of the resulting halos was measured after 8 h.

RNA extraction and qRT-PCR.

Cultures were grown in low-glucose DMEM to an optical density at 600 nm (OD600) of 1.0. RNA from 3 biological replicates was extracted using the RiboPure bacterial isolation kit, according to the manufacturer's protocols (Ambion). qRT-PCR was performed as described previously (25). Briefly, diluted extracted RNA was mixed with validated primers (Table 2), RNase inhibitor, and reverse transcriptase (Applied Biosystems). The mixture was used in a one-step reaction utilizing an ABI 7500 sequence detection system. Data were collected using ABI Sequence Detection 1.2 software, normalized to endogenous rpoA levels, and analyzed using the comparative critical threshold cycle (CT) method. Analyzed data were presented as fold changes over WT levels. Student's unpaired t test was used to determine statistical significance. A P value of ≤0.05 was considered significant.

Detection of membrane proteins.

Complemented VS138 strains with pVS155 (WT QseC) or the site-directed mutants were grown in LB supplemented with 0.2% arabinose to an OD600 of 0.6, and cells were pelleted and lysed by five passages through an EmulsiFlex C3 emulsifier. The resulting lysate was cleared by centrifugation at 26,000 × g. Membranes were isolated from these lysates by ultracentrifugation at 179,000 × g. The pelleted membranes were solubilized in SDS-PAGE sample buffer, and proteins were separated on a 10% acrylamide gel. These proteins were transferred by Western blotting, probed with an anti-His antiserum primary antibody, and then incubated with a secondary antibody conjugated to streptavidin-horseradish peroxidase (HRP). ECL reagent (GE) was added and the membranes were exposed to film.

Detection of secreted proteins.

Secreted proteins were extracted and detected as previously described (4). Complemented VS138 strains with WT QseC (pVS155) or the point mutants were grown in low-glucose DMEM to an OD600 of 1.0. Cells were pelleted by centrifugation and suspended in phosphate-buffered saline. Supernatants were passed through a 0.22-μm-pore-size filter and treated with EDTA (5 mM final concentration), phenylmethylsulfonyl fluoride (PMSF) (50 μg/ml final concentration), and aprotinin (0.5 μg/ml final concentration). One hundred micrograms of bovine serum albumin (BSA) was added to each sample as a control. The volume of the supernatants was reduced to 50 μl using centrifugal filters with a molecular weight cutoff (MWCO) of 10,000. Ten micrograms of protein from each sample was separated on 4 to 15% SDS gradient gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The blot was stained with 0.1% Ponceau S in 5% acetic acid to visualize the BSA loading control. The presence of secreted EspA was detected with polyclonal anti-EspA antibodies.

Fluorescein actin staining assays.

Assays were performed as described by Knutton et al. (56). Briefly, HeLa cells were grown on coverslips in wells containing DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-glutamine (PSG) antibiotic mix at 37°C and 5% CO2 overnight to about 80% confluence. The wells were then thoroughly washed with phosphate-buffered saline (PBS) and replaced with fresh medium supplemented with arabinose (0.2% final concentration) and lacking antibiotics. Overnight static cultures of bacteria were then used to infect at a dilution of 100:1 (bacteria to DMEM). After a 6-h infection at 37°C and 5% CO2, the coverslips were washed, fixed, and permeabilized. The samples were then treated with fluorescein isothiocyanate (FITC)-labeled phalloidin and propidium iodide (PI) to visualize actin accumulation and bacteria, respectively. PI also stained HeLa nuclei red. The coverslips were then mounted on slides and visualized with a Zeiss Axiovert microscope. Pedestal formation was quantified as the percentage of pedestals formed per attached bacterium. Replicate coverslips from multiple experiments were quantified, and statistical analysis was performed using the Student t test. Serially diluted samples of the original bacterial cultures were also plated to confirm that similar CFU ratios were used for infection.

Autophosphorylation and phosphotransfer assays using proteoliposomes.

His-tagged QseC proteins and response regulators (QseB, KdpE, and QseF) were isolated from cultures grown to mid-log phase in LB and induced with either 0.2% arabinose (kinases) or 400 mM isopropyl-β-d-thiogalactopyranoside (IPTG) (response regulators) at 30°C overnight. The cells were pelleted, resuspended in 50 mM phosphate buffer containing 150 mM NaCl, and lysed by passage through an EmulsiFlex C3 emulsifier. The resulting lysate was cleared by centrifugation, and proteins were isolated using the standard nickel-nitrilotriacetic acid (Ni-NTA) technique, as described by the manufacturer (Qiagen).

QseC autophosphorylation experiments were performed as described by Clarke et al. (21). Briefly, as described previously (24), the E. coli strain containing pVS155 (qseC::Myc-His) was grown at 37°C in LB to an OD600 of 0.7, at which point arabinose was added to a final volume of 0.2% and allowed to induce for 3 h (21). Nickel columns were utilized, according to the manufacturer's instructions (Qiagen). Autophosphorylation experiments were performed with QseC embedded in liposomes. Liposomes were reconstituted as described by Janausch et al. (57). Briefly, 50 mg of E. coli phospholipids (20 mg/ml in chloroform; Avanti Polar Lipids) was 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/thawing in liquid N2. Liposomes were then destabilized by the addition of 26.1 mg of dodecyl-maltoside, and 2.5 mg of QseC-Myc-His was added, followed by stirring at room temperature for 10 min. Two hundred sixty-one milligrams of Bio-Beads was 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 Bio-Beads for 1 h in the morning. The resulting liposomes containing reconstituted QseC-Myc-His were frozen in liquid N2 and stored at −80°C until used. Orientation of HKs in the liposome system has been established by previous groups (58) and can be concluded from the accessibility of ATP to the kinase site and anti-Myc antisera to the C-terminal QseC Myc tag without disruption of the liposomes (21). Twenty microliters of the liposomes containing QseC-Myc-His was adjusted to 10 mM MgCl2 and 1 mM dithiothreitol (DTT), no signal, or 50 μM epinephrine, frozen and thawed rapidly in liquid N2, and kept at room temperature for 1 h. A volume of 0.625 μl of [γ32P]dATP (110 terabecquerels [TBq]/mmol) was added to each reaction mixture. At the 1-, 2-, 5-, and 10-min time points, 20 μl of SDS loading buffer was added (21). The samples were run on SDS-PAGE gels without boiling, according to standard procedures (59), and visualized via a phosphorimager.

Phosphotransfer assays were performed by first adding DTT (1 mM final concentration), MgCl2 (0.5 mM final concentration), and epinephrine-bitartrate (50 μM final concentration) to an aliquot of liposome. These were then subjected to three freeze/thaw cycles using liquid nitrogen and held at room temperature for 1 h. In a 20-μl reaction mixture, response regulator was added to 10 μl of loaded liposome at a 1:1 molar ratio to the kinase, and the reaction was started by adding 0.3 μl of [γ32P]dATP (3 μCi). Reactions were stopped by adding 5 μl of SDS-PAGE sample buffer supplemented with additional SDS to a concentration of 18% (wt/vol). Proteins were resolved, without boiling, on 10% SDS-PAGE gels. Radiolabeled proteins were visualized by exposing the gel to a phosphorimaging screen. The bands were quantitated using the ImageQuant version 5.0 software (Amersham).

Autophosphorylation in membranes.

Membranes were prepared from VS138 (qseC mutant) with or without pVS155 (QseC in pBADMycHisA) grown in LB and induced with 0.02% arabinose. Total membranes were isolated by ultracentrifugation, suspended in 50 mM Tris buffer (pH 7.5) with 100 mM NaCl, and stored at −80°C. For the autophosphorylation assays, the membranes were washed with Tris-EDTA (TE) and assayed in 10 mM Tris (pH 7.5)–2 mM MgCl2–0 to 100 mM NaCl and 2.5 μM [γ32P]dATP (110 TBq/mmol) in the absence or presence of 100 μM epinephrine on ice for 0 to 3 min. Assays were stopped with sample buffer, and assay mixtures were loaded on a 10% SDS gel. Radiolabeled proteins were visualized by exposing the gel to a phosphorimaging screen. We also performed Western blotting with anti-His antisera on blots from these genes to ensure the identity of QseC.


We thank members of the Sperandio lab for collegial discussions of this work.

This work was supported by National Institutes of Health (NIH) grants AI053067, AI05135, AI077613, and AI114511. C.T.P. was supported through NIH training grant 5 T32 AI7520-14.

The contents of this article are solely the responsibility of the authors and do not represent the official views of the NIH NIAID.


1. Moon HW, Whipp SC, Argenzio RA, Levine MM, Giannella RA 1983. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun 41:1340–1351. [PMC free article] [PubMed]
2. Knutton S, Baldini MM, Kaper JB, McNeish AS 1987. Role of plasmid-encoded adherence factors in adhesion of enteropathogenic Escherichia coli to HEp-2 cells. Infect Immun 55:78–85. [PMC free article] [PubMed]
3. Tzipori S, Wachsmuth IK, Chapman C, Birden R, Brittingham J, Jackson C, Hogg J 1986. The pathogenesis of hemorrhagic colitis caused by Escherichia coli O157:H7 in gnotobiotic piglets. J Infect Dis 154:712–716. doi:.10.1093/infdis/154.4.712 [PubMed] [Cross Ref]
4. Jarvis KG, Giron JA, Jerse AE, McDaniel TK, Donnenberg MS, Kaper JB 1995. Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. Proc Natl Acad Sci U S A 92:7996–8000. doi:.10.1073/pnas.92.17.7996 [PubMed] [Cross Ref]
5. McDaniel TK, Jarvis KG, Donnenberg MS, Kaper JB 1995. A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci U S A 92:1664–1668. doi:.10.1073/pnas.92.5.1664 [PubMed] [Cross Ref]
6. Jerse AE, Yu J, Tall BD, Kaper JB 1990. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci U S A 87:7839–7843. doi:.10.1073/pnas.87.20.7839 [PubMed] [Cross Ref]
7. Kenny B, DeVinney R, Stein M, Reinscheid DJ, Frey EA, Finlay BB 1997. Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell 91:511–520. doi:.10.1016/S0092-8674(00)80437-7 [PubMed] [Cross Ref]
8. Deng W, Puente JL, Gruenheid S, Li Y, Vallance BA, Vazquez A, Barba J, Ibarra JA, O'Donnell P, Metalnikov P, Ashman K, Lee S, Goode D, Pawson T, Finlay BB 2004. Dissecting virulence: systematic and functional analyses of a pathogenicity island. Proc Natl Acad Sci U S A 101:3597–3602. doi:.10.1073/pnas.0400326101 [PubMed] [Cross Ref]
9. Campellone KG, Robbins D, Leong JM 2004. EspFU is a translocated EHEC effector that interacts with Tir and N-WASP and promotes Nck-independent actin assembly. Dev Cell 7:217–228. doi:.10.1016/j.devcel.2004.07.004 [PubMed] [Cross Ref]
10. Gruenheid S, Sekirov I, Thomas NA, Deng W, O'Donnell P, Goode D, Li Y, Frey EA, Brown NF, Metalnikov P, Pawson T, Ashman K, Finlay BB 2004. Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol 51:1233–1249. doi:.10.1046/j.1365-2958.2003.03911.x [PubMed] [Cross Ref]
11. Mundy R, Jenkins C, Yu J, Smith H, Frankel G 2004. Distribution of espI among clinical enterohaemorrhagic and enteropathogenic Escherichia coli isolates. J Med Microbiol 53:1145–1149. doi:.10.1099/jmm.0.45684-0 [PubMed] [Cross Ref]
12. Dahan S, Wiles S, La Ragione RM, Best A, Woodward MJ, Stevens MP, Shaw RK, Chong Y, Knutton S, Phillips A, Frankel G 2005. EspJ is a prophage-carried type III effector protein of attaching and effacing pathogens that modulates infection dynamics. Infect Immun 73:679–686. doi:.10.1128/IAI.73.2.679-686.2005 [PMC free article] [PubMed] [Cross Ref]
13. Garmendia J, Frankel G 2005. Operon structure and gene expression of the espJ–tccP locus of enterohaemorrhagic Escherichia coli O157:H7. FEMS Microbiol Lett 247:137–145. doi:.10.1016/j.femsle.2005.04.035 [PubMed] [Cross Ref]
14. Shaw RK, Smollett K, Cleary J, Garmendia J, Straatman-Iwanowska A, Frankel G, Knutton S 2005. Enteropathogenic Escherichia coli type III effectors EspG and EspG2 disrupt the microtubule network of intestinal epithelial cells. Infect Immun 73:4385–4390. doi:.10.1128/IAI.73.7.4385-4390.2005 [PMC free article] [PubMed] [Cross Ref]
15. Tobe T, Beatson SA, Taniguchi H, Abe H, Bailey CM, Fivian A, Younis R, Matthews S, Marches O, Frankel G, Hayashi T, Pallen MJ 2006. An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination. Proc Natl Acad Sci U S A 103:14941–14946. doi:.10.1073/pnas.0604891103 [PubMed] [Cross Ref]
16. Karmali MA, Petric M, Lim C, Fleming PC, Arbus GS, Lior H 1985. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J Infect Dis 151:775–782. doi:.10.1093/infdis/151.5.775 [PubMed] [Cross Ref]
17. Neely MN, Friedman DI 1998. Functional and genetic analysis of regulatory regions of coliphage H-19B: location of Shiga-like toxin and lysis genes suggest a role for phage functions in toxin release. Mol Microbiol 28:1255–1267. doi:.10.1046/j.1365-2958.1998.00890.x [PubMed] [Cross Ref]
18. Neely MN, Friedman DI 2000. N-mediated transcription antitermination in lambdoid phage H-19B is characterized by alternative NUT RNA structures and a reduced requirement for host factors. Mol Microbiol 38:1074–1085. [PubMed]
19. Wagner PL, Neely MN, Zhang X, Acheson DW, Waldor MK, Friedman DI 2001. Role for a phage promoter in Shiga toxin 2 expression from a pathogenic Escherichia coli strain. J Bacteriol 183:2081–2085. doi:.10.1128/JB.183.6.2081-2085.2001 [PMC free article] [PubMed] [Cross Ref]
20. Sperandio V, Torres AG, Jarvis B, Nataro JP, Kaper JB 2003. Bacteria-host communication: the language of hormones. Proc Natl Acad Sci U S A 100:8951–8956. doi:.10.1073/pnas.1537100100 [PubMed] [Cross Ref]
21. Clarke MB, Hughes DT, Zhu C, Boedeker EC, Sperandio V 2006. The QseC sensor kinase: a bacterial adrenergic receptor. Proc Natl Acad Sci U S A 103:10420–10425. doi:.10.1073/pnas.0604343103 [PubMed] [Cross Ref]
22. Reading NC, Rasko DA, Torres AG, Sperandio V 2009. The two-component system QseEF and the membrane protein QseG link adrenergic and stress sensing to bacterial pathogenesis. Proc Natl Acad Sci U S A 106:5889–5894. doi:.10.1073/pnas.0811409106 [PubMed] [Cross Ref]
23. Clarke MB, Sperandio V 2005. Transcriptional autoregulation by quorum sensing E. coli regulators B and C (QseBC) in enterohemorrhagic E. coli (EHEC). Mol Microbiol 58:441–455. doi:.10.1111/j.1365-2958.2005.04819.x [PubMed] [Cross Ref]
24. Clarke MB, Sperandio V 2005. Transcriptional regulation of flhDC by QseBC and sigma (FliA) in enterohaemorrhagic Escherichia coli. Mol Microbiol 57:1734–1749. doi:.10.1111/j.1365-2958.2005.04792.x [PubMed] [Cross Ref]
25. Hughes DT, Clarke MB, Yamamoto K, Rasko DA, Sperandio V 2009. The QseC adrenergic signaling cascade in enterohemorrhagic E. coli (EHEC). PLoS Pathog 5:e1000553. doi:.10.1371/journal.ppat.1000553 [PMC free article] [PubMed] [Cross Ref]
26. Njoroge JW, Nguyen Y, Curtis MM, Moreira CG, Sperandio V 2012. Virulence meets metabolism: Cra and KdpE gene regulation in enterohemorrhagic Escherichia coli. mBio 3(5):e00280-12. doi:.10.1128/mBio.00280-12 [PMC free article] [PubMed] [Cross Ref]
27. Gruber CC, Sperandio V 2014. Posttranscriptional control of microbe-induced rearrangement of host cell actin. mBio 5(1):e01025-13. doi:.10.1128/mBio.01025-13 [PMC free article] [PubMed] [Cross Ref]
28. Elliott SJ, Hutcheson SW, Dubois MS, Mellies JL, Wainwright LA, Batchelor M, Frankel G, Knutton S, Kaper JB 1999. Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli. Mol Microbiol 33:1176–1189. [PubMed]
29. Mellies JL, Elliott SJ, Sperandio V, Donnenberg MS, Kaper JB 1999. The Per regulon of enteropathogenic Escherichia coli: identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler). Mol Microbiol 33:296–306. doi:.10.1046/j.1365-2958.1999.01473.x [PubMed] [Cross Ref]
30. Elliott SJ, Wainwright LA, McDaniel TK, Jarvis KG, Deng YK, Lai LC, McNamara BP, Donnenberg MS, Kaper JB 1998. The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol Microbiol 28:1–4. [PubMed]
31. Abe A, de Grado M, Pfuetzner RA, Sanchez-Sanmartin C, Devinney R, Puente JL, Strynadka NC, Finlay BB 1999. Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion. Mol Microbiol 33:1162–1175. [PubMed]
32. Sánchez-SanMartín C, Bustamante VH, Calva E, Puente JL 2001. Transcriptional regulation of the orf19 gene and the tir-cesT-eae operon of enteropathogenic Escherichia coli. J Bacteriol 183:2823–2833. doi:.10.1128/JB.183.9.2823-2833.2001 [PMC free article] [PubMed] [Cross Ref]
33. Elliott SJ, Sperandio V, Giron JA, Shin S, Mellies JL, Wainwright L, Hutcheson SW, McDaniel TK, Kaper JB 2000. The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun 68:6115–6126. doi:.10.1128/IAI.68.11.6115-6126.2000 [PMC free article] [PubMed] [Cross Ref]
34. Ramseier TM, Negre D, Cortay JC, Scarabel M, Cozzone AJ, Saier MH Jr 1993. In vitro binding of the pleiotropic transcriptional regulatory protein, FruR, to the fru, pps, ace, pts and icd operons of Escherichia coli and Salmonella Typhimurium. J Mol Biol 234:28–44. doi:.10.1006/jmbi.1993.1561 [PubMed] [Cross Ref]
35. Njoroge J, Sperandio V 2012. Enterohemorrhagic Escherichia coli virulence regulation by two bacterial adrenergic kinases, QseC and QseE. Infect Immun 80:688–703. doi:.10.1128/IAI.05921-11 [PMC free article] [PubMed] [Cross Ref]
36. Reichenbach B, Gopel Y, Gorke B 2009. Dual control by perfectly overlapping sigma 54- and sigma 70-promoters adjusts small RNA GlmY expression to different environmental signals. Mol Microbiol 74:1054–1070. doi:.10.1111/j.1365-2958.2009.06918.x [PubMed] [Cross Ref]
37. Gruber CC, Sperandio V 2014. Posttranscriptional control of microbe-induced rearrangement of host cell actin. mBio 5(1):e01025-13. doi:.10.1128/mBio.01025-13 [PMC free article] [PubMed] [Cross Ref]
38. Urban JH, Vogel J 2008. Two seemingly homologous noncoding RNAs act hierarchically to activate glmS mRNA translation. PLoS Biol 6:e64. doi:.10.1371/journal.pbio.0060064 [PubMed] [Cross Ref]
39. Kendall MM, Sperandio V 2016. What a dinner party! Mechanisms and functions of interkingdom signaling in host-pathogen associations. mBio 7(2):e01748-15. doi:.10.1128/mBio.01748-15 [PMC free article] [PubMed] [Cross Ref]
40. Reinelt S, Hofmann E, Gerharz T, Bott M, Madden DR 2003. The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain. J Biol Chem 278:39189–39196. doi:.10.1074/jbc.M305864200 [PubMed] [Cross Ref]
41. Pappalardo L, Janausch IG, Vijayan V, Zientz E, Junker J, Peti W, Zweckstetter M, Unden G, Griesinger C 2003. The NMR structure of the sensory domain of the membranous two-component fumarate sensor (histidine protein kinase) DcuS of Escherichia coli. J Biol Chem 278:39185–39188. doi:.10.1074/jbc.C300344200 [PubMed] [Cross Ref]
42. Clarke MB, Hughes DT, Zhu C, Boedeker EC, Sperandio V 2006. The QseC sensor kinase: a bacterial adrenergic receptor. Proc Natl Acad Sci U S A 103:10420–10425. doi:.10.1073/pnas.0604343103 [PubMed] [Cross Ref]
43. Rasko DA, Moreira CG, Li de R, Reading NC, Ritchie JM, Waldor MK, Williams N, Taussig R, Wei S, Roth M, Hughes DT, Huntley JF, Fina MW, Falck JR, Sperandio V 2008. Targeting QseC signaling and virulence for antibiotic development. Science 321:1078–1080. doi:.10.1126/science.1160354 [PMC free article] [PubMed] [Cross Ref]
44. Kostakioti M, Hadjifrangiskou M, Pinkner JS, Hultgren SJ 2009. QseC-mediated dephosphorylation of QseB is required for expression of genes associated with virulence in uropathogenic Escherichia coli. Mol Microbiol 73:1020–1031. doi:.10.1111/j.1365-2958.2009.06826.x [PMC free article] [PubMed] [Cross Ref]
45. Sperandio V, Torres AG, Kaper JB 2002. Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli. Mol Microbiol 43:809–821. doi:.10.1046/j.1365-2958.2002.02803.x [PubMed] [Cross Ref]
46. Lodato PB, Hsieh PK, Belasco JG, Kaper JB 2012. The ribosome binding site of a mini-ORF protects a T3SS mRNA from degradation by RNase E. Mol Microbiol 86:1167–1182. [PMC free article] [PubMed]
47. Shakhnovich EA, Davis BM, Waldor MK 2009. Hfq negatively regulates type III secretion in EHEC and several other pathogens. Mol Microbiol 74:347–363. doi:.10.1111/j.1365-2958.2009.06856.x [PMC free article] [PubMed] [Cross Ref]
48. Bhatt S, Edwards AN, Nguyen HT, Merlin D, Romeo T, Kalman D 2009. The RNA binding protein CsrA is a pleiotropic regulator of the locus of enterocyte effacement pathogenicity island of enteropathogenic Escherichia coli. Infect Immun 77:3552–3568. doi:.10.1128/IAI.00418-09 [PMC free article] [PubMed] [Cross Ref]
49. Yamamoto K, Hirao K, Oshima T, Aiba H, Utsumi R, Ishihama A 2005. Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli. J Biol Chem 280:1448–1456. doi:.10.1074/jbc.M410104200 [PubMed] [Cross Ref]
50. Aravind L, Ponting CP 1999. The cytoplasmic helical linker domain of receptor histidine kinase and methyl-accepting proteins is common to many prokaryotic signalling proteins. FEMS Microbiol Lett 176:111–116. doi:.10.1111/j.1574-6968.1999.tb13650.x [PubMed] [Cross Ref]
51. Laub MT, Goulian M 2007. Specificity in two-component signal transduction pathways. Annu Rev Genet 41:121–145. doi:.10.1146/annurev.genet.41.042007.170548 [PubMed] [Cross Ref]
52. Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Eramian D, Shen MY, Pieper U, Sali A 2006 Comparative protein structure modeling using Modeller. Curr Protoc Bioinformatics Chapter 5:Unit 5.6. doi:.10.1002/0471250953.bi0506s15 [PMC free article] [PubMed] [Cross Ref]
53. Martí-Renom MA, Stuart AC, Fiser A, Sanchez R, Melo F, Sali A 2000. Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct 29:291–325. doi:.10.1146/annurev.biophys.29.1.291 [PubMed] [Cross Ref]
54. Datsenko KA, Wanner BL 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645. doi:.10.1073/pnas.120163297 [PubMed] [Cross Ref]
55. Carlson-Banning KM, Sperandio V 2016. Catabolite and oxygen regulation of enterohemorrhagic Escherichia coli virulence. mBio 7(6):e01852-16. doi:.10.1128/mBio.01852-16 [PMC free article] [PubMed] [Cross Ref]
56. Knutton S, Baldwin T, Williams PH, McNeish AS 1989. Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun 57:1290–1298. [PMC free article] [PubMed]
57. Janausch IG, Garcia-Moreno I, Lehnen D, Zeuner Y, Unden G 2004. Phosphorylation and DNA binding of the regulator DcuR of the fumarate-responsive two-component system DcuSR of Escherichia coli. Microbiology 150:877–883. doi:.10.1099/mic.0.26900-0 [PubMed] [Cross Ref]
58. Dioum EM, Rutter J, Tuckerman JR, Gonzalez G, Gilles-Gonzalez MA, McKnight SL 2002. NPAS2: a gas-responsive transcription factor. Science 298:2385–2387. doi:.10.1126/science.1078456 [PubMed] [Cross Ref]
59. Sambrook J, Fritsch EF, Maniatis T 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
60. Griffin PM, Ostroff SM, Tauxe RV, Greene KD, Wells JG, Lewis JH, Blake PA 1988. Illnesses associated with Escherichia coli O157:H7 infections. A broad clinical spectrum. Ann Intern Med 109:705–712. [PubMed]

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