Bioluminescence resonance energy transfer system for measuring dynamic protein-protein interactions in bacteria.

Bioluminescence Resonance Energy Transfer System for Measuring Dynamic Protein-Protein Interactions in Bacteria Boyu Cui,a Yao Wang,a Yunhong Song,a Tietao Wang,a Changfu Li,a Yahong Wei,a Zhao-Qing Luo,b Xihui Shena State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, Chinaa; Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USAb B.C. and Y.W. contributed equally to this article.

ABSTRACT Protein-protein interactions are important for virtually every biological process, and a number of elegant approaches have been designed to detect and evaluate such interactions. However, few of these methods allow the detection of dynamic and real-time protein-protein interactions in bacteria. Here we describe a bioluminescence resonance energy transfer (BRET) system based on the bacterial luciferase LuxAB. We found that enhanced yellow fluorescent protein (eYFP) accepts the emission from LuxAB and emits yellow fluorescence. Importantly, BRET occurred when LuxAB and eYFP were fused, respectively, to the interacting protein pair FlgM and FliA. Furthermore, we observed sirolimus (i.e., rapamycin)-inducible interactions between FRB and FKBP12 and a dose-dependent abolishment of such interactions by FK506, the ligand of FKBP12. Using this system, we showed that osmotic stress or low pH efficiently induced multimerization of the regulatory protein OmpR and that the multimerization induced by low pH can be reversed by a neutralizing agent, further indicating the usefulness of this system in the measurement of dynamic interactions. This method can be adapted to analyze dynamic protein-protein interactions and the importance of such interactions in bacterial processes such as development and pathogenicity. IMPORTANCE Real-time measurement of protein-protein interactions in prokaryotes is highly desirable for determining the roles

of protein complex in the development or virulence of bacteria, but methods that allow such measurement are not available. Here we describe the development of a bioluminescence resonance energy transfer (BRET) technology that meets this need. The use of endogenous excitation light in this strategy circumvents the requirement for the sophisticated instrument demanded by standard fluorescence resonance energy transfer (FRET). Furthermore, because the LuxAB substrate decanal is membrane permeable, the assay can be performed without lysing the bacterial cells, thus allowing the detection of protein-protein interactions in live bacterial cells. This BRET system added another useful tool to address important questions in microbiological studies. Received 9 March 2014 Accepted 10 April 2014 Published 20 May 2014 Citation Cui B, Wang Y, Song Y, Wang T, Li C, Wei Y, Luo Z-Q, Shen X. 2014. Bioluminescence resonance energy transfer system for measuring dynamic protein-protein interactions in bacteria. mBio 5(3):e01050-14. doi:10.1128/mBio.01050-14. Invited Editor Ilya Manukhov, State Research Institute of Genetics and Selection of Industrial Microorganisms (GosNIIGenetika) Editor Stephen Winans, Cornell University Copyright © 2014 Cui et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Xihui Shen, [email protected], or Zhao-Qing Luo, [email protected].

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rotein complexes of multiple components govern essentially all biological processes, and a number of elegantly designed methods have been developed to identify interacting proteins and to evaluate such interactions (1–3). Successful utilization of these methods has led to the identification of numerous interacting proteins and the elucidation of many important biological processes (4, 5). Two-hybrid systems represent one class of such methods. These systems, depending upon the activity of the reporter proteins used as the readout, can be divided into two categories. In the first category, exemplified by the yeast two-hybrid system, a transcriptional factor was split into two halves; each has lost the original activity in the activation of transcription. Fusion of interacting proteins to each of the fragments restores such activity by bringing these two fragments into physical proximity (1). The second category involves in the use of specific enzymes whose enzymatic activity can be restored when the split fragments are brought together by interacting proteins (6). For example, because of its simplicity in experimental manipulation, the cyclic

May/June 2014 Volume 5 Issue 3 e01050-14

AMP synthase (Cya)-based system has been widely used in the study of protein-protein interaction in bacteria (7). Whereas two-hybrid systems offer the advantage of screening through numerous candidate proteins from complex libraries potentially representing every protein of a given organism, they are not feasible for measuring real-time dynamic binding. This limitation was overcome by several protein fragment complementation assays (PCA) (8) and by fluorescence and bioluminescence resonance energy transfer (FRET and BRET, respectively) (9, 10). In PCA, inactive fragments of reporter proteins, such as the green fluorescent protein (GFP) and light-emitting luciferase, were fused to the proteins of interest and the binding of these proteins restores their activity (11, 12). In FRET and BRET, energy generated by either a fluorescent protein or by a light-emitting luciferase is transferred to an acceptor fluorophore (13, 14). Such transfer occurs when the molecules are in close proximity, generally within 10 to 100 Å, depending on the extent of spectral overlap (15), leading to the emission of a different fluorescence signal (13,

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RESEARCH ARTICLE

16, 17). These methods allow direct visualization of the interactions in the native organism and thus can reveal the cellular compartment within which the interactions occur (18). BRET assays have been widely used to analyze protein-protein interactions in eukaryotic cells, particularly in experiments that mandate real-time dynamic measurement of the bindings (9, 18– 20). For example, by using this method, Perroy et al. successfully measured the kinetics of ubiquitination of ␤-arrestin 2 (21). However, similar applications are limited in bacterial cells, probably due to factors such as lower sensitivity caused by suboptimal expression of the luciferase of eukaryotic origin. Indeed, although the use of firefly Hotaria parvula luciferase (HpL) and Gaussia princeps luciferase (GLuc) has been reported (22–24), the 35-kDa Renilla luciferase (Rluc) is the most commonly used lightemitting luciferase in BRET (25). We hypothesized that a BRET assay based on a luciferase of bacterial origin may have the potential to overcome the limitations associated with the existing systems and allow the development of methods more suitable for measuring interactions in prokaryotic cells. To this end, we established a BRET system using the LuxAB from Photorhabdus luminescens (26) and enhanced yellow fluorescent protein (eYFP) (27). We found that the spectrum of LuxAB emission is sufficiently broad to excite fluorescence of eYFP but not that of enhanced GFP (eGFP) or mOrange, a fluorescent protein from Discosoma sp. (28). Using this system, we observed interactions between FlgM and FliA, two proteins in the flagellar regulon; we also examined sirolimus (i.e., rapamycin)dependent, FK506-titratable dynamic interactions between FRB, the sirolimus-binding domain of mTOR (29, 30), and the FK506binding protein FKBP12 (31). This system was further used to evaluate the impact of distinct environmental stimuli on the multimerization of OmpR, the transcriptional regulatory protein involved in sensing osmolarity/pH changes (32–35). Our results established LuxAB-based BRET as a potentially useful tool in dissecting dynamic protein-protein interactions in bacterial cells. RESULTS

Excitation of eYFP by LuxAB. To establish a BRET system based on the bacterial luciferase LuxAB, we first examined the ability of its emission to excite several receptor fluorophores. To this end, we chose to test GFP, the monomeric fluorophore mOrange, derived from the coral red fluorescent protein DsRed, and eYFP, the GFP mutant that emits yellow fluorescence. Based on their photophysical properties, a significant spectral overlap only exists between LuxAB and eYFP (Fig. 1A), which potentially can be exploited for a BRET assay in which LuxAB and eYFP will be brought into proximity by interacting proteins, respectively, fused to the fluorophores (Fig. 1B and C). On the other hand, due to the lack of significant spectral overlap between LuxAB and GFP or mOrange, BRET is not expected when either of these two proteins is used as the receptor fluorophore. We thus made constructs that allow lac promoter (Plac)dependent expression of each of these three fluorophores fused to the carboxyl end of LuxAB (Fig. 2A). As expected, the light produced by Escherichia coli cells expressing LuxAB became detectable around 430 nm and peaked at 490 nm (Fig. 2B to D). Cells expressing GFP, mOrange, or eYFP also emitted fluorescence signals with their respective spectra (Fig. 2B to D). Furthermore, the emission spectrum of cells expressing LuxAB-GFP or LuxABmOrange strain was indistinguishable from that of LuxAB itself

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(Fig. 2B and C), indicating that resonance energy transfer did not occur. Importantly, in cells expressing LuxAB-eYFP, in addition to the peak at 490 nm, another peak was detected at 527 nm, a wavelength that is identical to the emission of eYFP itself (Fig. 2D). This excitation is consistent with the significant spectral overlap between LuxAB and eYFP (Fig. 1A). Furthermore, the signal intensity of this peak was comparable to that emitted by eYFP, suggesting that the excitation signal from LuxAB is able to trigger the fluorescence of eYFP almost at the maximal level. The inability of LuxAB to excite GFP or mOrange is consistent with the fact that no spectral overlap exists between LuxAB and each of these fluorophores, which have major excitation peaks at 395 nm (36) and 548 nm (28), respectively (Fig. 2B and C). Because the luciferase of Photorhabdus luminescens is not related to eYFP from Aequorea, intrinsic interactions between LuxAB and eYFP are very unlikely. Thus, we chose these two fluorophores to establish a BRET assay for analyzing protein-protein interactions. The sensitivity of the LuxAB-based BRET depends upon the orientation of protein fusion. To provide the proof-of-concept evidence for this method, we examined the interactions between the alternative sigma factor FliA specific for the flagellar regulon (37) and the anti-sigma factor FlgM (38, 39). FliA and FlgM were fused to the carboxyl or amino terminus of LuxAB and eYFP, respectively (Fig. 3A). BRET signals indicative of interactions between FlgM and FliA were detected when FliA was fused to the carboxyl end of LuxAB irrespective of the orientation of the eYFPFlgM fusion (Fig. 3B and C). Although the eYFP-FlgM fusion was expressed at a high level (see Fig. S1 in the supplemental material), its BRET signal was considerably lower than that of the FlgMeYFP fusion (Fig. 3C). Similar results were obtained when the ratio of emission at 527 nm to that at 490 nm (Y:B ratio) was measured (Fig. 3D). On the other hand, luminescence signals could not be detected when FliA was fused to the N-terminal end of LuxAB, and no further experiments were performed with this construct. Thus, the orientation of the fusion is important for the sensitivity or sometimes even for the functionality of LuxABbased BRET to work. Dynamic interactions between FRB and FKBP12. To determine the usefulness of the LuxAB-based BRET system, we first set out to analyze the interactions of two proteins known to regulatably interact with each other. To this end, we chose FRB, the sirolimus-binding domain of mTOR and its binding partner, FKBP12, a 12-kDa protein that also binds the immunosuppressant FK506 with high affinity (30). This interacting protein pair offers at least two advantages in the evaluation of the LuxAB-based BRET. First, their interaction depends on sirolimus (40) (Fig. 4A), a property that allows us to examine whether intrinsic association between the LuxAB and eYFP fusions occurs. Second, the FRBFKBP12 complex can be abolished by FK506 (31, 41), which potentially will enable us to determine the reversibility of the interactions in real time. To ensure that these proteins are properly expressed in bacterial cells, we first codon optimized the mouse cDNA of both FKBP12 and FRB in accordance with the codon preference of eubacteria, such as E. coli (see Fig. S2 in the supplemental material). To determine the interactions between these two proteins, we used the ratio of emission at 527 nm to that at 490 nm (Y:B ratio) to measure the intensity of BRET or the strength of the interaction (19). E. coli cells expressing both LuxAB-FKBP12 and FRB-eYFP were measured for BRET under different conditions.



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FIG 1 Emission spectra of LuxAB and eYFP and schematics of fusion constructs for the LuxAB-based BRET assay. (A) The bioluminescence spectra illustrate the emission spectra of LuxAB and the enhanced yellow fluorescent acceptor protein. The luciferase substrate used in this case was decanal. Spectral resolution of the system is marked on the x axis. (B) When testing proteins X and Y do not interact, eYFP cannot be excited by the emission from LuxAB, and thus no BRET occurs. (C) The binding between X= and Y= brings LuxAB and eYFP to close proximity, which allows the transfer of the energy to the receptor fluorophore and the emission of fluorescence signals.

In samples receiving the solvent dimethyl sulfoxide (DMSO), no BRET signal was detected, and the Y:B ratio was constantly at approximately 0.55 (Fig. 4B) over the experimental duration. In samples receiving 100 nM sirolimus, BRET signals became detectable within 2 min and plateaued 10 min after addition of the inducer (Fig. 4B). Interestingly, when FK506, the inhibitory competitor for FKBP12 was added together with sirolimus to the bacterial cultures, the emission of BRET signal was arrested, which is consistent with the fact that FK506 competes for the binding site of sirolimus, leading to reduced interactions between FKBP12 and FRB (Fig. 4B). The sirolimus-dependent BRET signals between this protein pair indicate that no intrinsic association occurs between LuxAB and eYFP. To further explore the usefulness of this method, we sought to determine the inhibitory constant of FK506 against sirolimus by measuring its effects on the Y:B ratio at different concentrations. The inhibitory effect of FK506 became detectable at 0.001 nM and reached the maximal at 10 ␮M (Fig. 4C). Calculations based on these results revealed that in our system, the 50% inhibitory constant (IC50) of FK506 was 0.37 nM (Fig. 4C). These results indicate that the LuxAB-based BRET method allows the measurement of dynamic protein-protein interactions.


Multimerization of OmpR measured by the LuxAB-based BRET. Many important bacterial regulatory proteins function by binding to promoters of their target genes as multimers upon receiving proper environmental stimuli. Because of the lag caused by transcription, translation, and the final maturation of the protein produced by conventional reporter genes such as lacZ and gfp, it has been difficult to determine the kinetics of the response of the regulatory proteins. To test the usefulness of the LuxAB-based BRET in analyzing protein multimerization in response to environmental cues, we chose OmpR, the widely distributed bacterial regulatory protein that responds to signals such as changes in osmolarity and pH (34, 35, 42). The interaction between OmpR molecules became detectable within 5 min after the bacterial cultures were treated with either osmolarity shock or low pH (Fig. 5A and B). Importantly, such multimerization did not occur in a mutant in which Asp 55, the recipient residue for EnvZ-mediated phosphorylation, was mutated (Fig. 5A and B). Moreover, no interaction was detected between wild-type OmpR and the D55A mutant (Fig. 5B), indicating that phosphorylation of both monomers is required for the formation of the dimer. Taken together, these results indicate that the formation of active OmpR dimer

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Real-Time Dynamic Protein Interactions in Bacteria


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FIG 2 BRET in E. coli cells expressing LuxAB and different receptor fluorophores. (A) Diagrams of the expression cassettes of Plac-LuxAB-eYFP. Plac, the lac promoter region; Ter, transcription terminator region. The nucleotide sequence of the 15-amino-acid fusion linker between luxAB and eYFP is shown below the Plac-LuxAB-eYFP diagram. (B to D) Luminescence or fluorescence emission spectra of strains expressing LuxAB, mOrange, or eYFP or the fusion proteins LuxAB-GFP (B), LuxAB-mOrange (C), and LuxAB-eYFP (D), respectively. Luminescence reactions were initiated by the addition of decanal to 1% in the cultures. All spectra were normalized by using the signals of suspensions of overnight cultures.

depends on the proper signaling cascade that leads to its posttranslational modification. We next examined the kinetics of OmpR multimerization in response to changes in environmental cues by neutralizing the acidic medium at different time points. A culture in which OmpR multimerization had been established by low pH was split into several subcultures, and NaOH was added to one of the subcultures at different times. When NaOH was added 5 min after acidmediated induction, a time point when the multimerization had just started to occur, little dimerization was detected (Fig. 5C), which was consistent with the fact that activation of OmpR did not occur in a neutral-pH medium. After the dimer has formed (10 or 15 min after low-pH induction), neutralization of the medium brought down the strength of BRET signals within 10 to 15 min (Fig. 5C). Because the half-life of P-OmpR is 1 to 2 h (43) and the upstream regulator EnvZ can function as a kinase or phosphatase upon sensing distinct environmental cues (44), the reduction in BRET signals likely was attributed to the disassembly of established dimers under unfavorable conditions. To correlate the kinetics of OmpR multimerization with the transcription of genes under its control, we measured the expression of a bioluminescence reporter fused to PompC, an OmpRregulated promoter (33). Bioluminescence did not become de-

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tectable 20 min after the osmolarity of the medium was disrupted (Fig. 5D). The time required for transcription, translation, and other events necessary for the production of the measurable product (light) apparently accounted for the longer time needed to detect gene expression. These results further validate the usefulness of the LuxAB-based BRET system in the study of dynamic protein-protein interactions. DISCUSSION

Many elegantly designed genetic methods, such as two-hybrid systems (1), PCA (8), and bimolecular fluorescence complementation (BiFC) (45), have been developed to measure protein-protein interactions. However, in prokaryotes, most, if not all of these methods only offer the detection of steady-state interactions (46, 47). Here we described the development of a bioluminescencebased BRET system that for the first time allows real-time measurement of protein-protein interactions in live bacterial cells. In this system, the excitation light source is the bacterial LuxAB, and the receptor fluorophore is eYFP. This strategy has several advantages. First, the endogenous excitation light circumvents the problems associated with the need for an exogenous light source, which often requires a sophisticated instrument. Second, because the LuxAB substrate decanal is membrane permeable, the assay can be


FIG 3 Interactions between FlgM and FliA measured by the LuxAB/eYFP BRET system. (A) Schematics of constructs expressing relevant LuxAB or eYFP fusions. Proteins were fused both N terminal and C terminal of LuxAB and eYFP, respectively. With the exception of pLac::FliA-LuxAB, all fusions were expressed. (B and C) Luminescence spectra of strains harboring pLac::luxAB-fliA/pTac::eYFP-flgM and pLac::luxAB-fliA/pTac::eYFP-flgM. A strain harboring pLac::luxAB-FliA and pTac::eYFP was used as the negative control. (D) Ratios of 527 nm to 490 nm (Y:B ratios) from strains expressing indicated fusions. In all cases, luminescence production was initiated by the addition of decanal to 1% in the cultures.

performed without lysing the bacterial cells. This feature makes experiments aiming at addressing potentially reversible interactions possible. Third, with further modifications, such as having the protein fusions be expressed from the bacterial chromosome by the cognate promoter, this system will allow the detection of a protein interaction close to the natural condition. The analyses of interactions of two protein pairs with distinct features indicate that the BRET signals were generated by specific binding of the testing proteins. First, the binding between FRB and FKBP12 occurred only when sirolimus, the molecule that directly links these two proteins, was added to the bacterial cultures. Second, no multimerization of OmpR was detected in the absence of the environmental cues important for its activation. Importantly, the use of FK506 to reverse sirolimus-mediated binding between FRB and FKBP allowed us to obtain an IC50 of 0.37 nM for the competitive inhibitor, which is comparable to values obtained from other methods (30), further indicating the reliability of this method. Our study of the multimerization of OmpR also revealed some novel features associated with this important regulatory protein in response to environmental stimuli. First, in our system, multimerization of OmpR became apparent within 5 min of changing the


environment to favor its activation. However, the expression of the target gene as measured by a bioluminescence reporter did not become detectable until 20 to 30 min after induction (Fig. 5D). Such delay may even be longer in the response of the cell to the environmental challenge because the proteins directly responsible for dealing with the stress will need to target to the right cellular location to fulfill their functions. Second, apo-OmpR is believed to mainly exist as a monomer (34, 35, 48): our results indicate that phosphorylated OmpR cannot form multimers with apo-OmpR. Phosphorylation may allow OmpR to assume a conformation distinctly different from that of apo-OmpR, which prevents the formation of heterodimers. Our results also suggested that changes in environmental cues could potentially lead to disassembly of the multimer. However, the mechanism of such disassembly remains to be determined; it is possible that dephosphorylation of p-OmpR by the phosphatase activity of EnvZ (44), the upstream regulator of EnvZ, is attributed to the disassembly of the dimer. Alternatively, the reduction of the BRET signal upon the removal of the stimulus may be caused at least in part by the decay of existing proteins and the inability of the newly synthesized proteins to form multimers. In addition to providing the proof-of-principle evidence for

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FIG 4 Interactions between FRB and FKBP12 measured by the LuxAB-based BRET. (A) Molecule model of sirolimus-induced FRB-FKBP12 association detected by BRET. (B) Sirolimus-induced FRB-FKBP12 association and its dissociation triggered by FK506. A 100 nM concentration of sirolimus (noted on the figure by the common name “rapamycin”), 10 ␮M FK506, or the same volume of DMSO (negative control) was added together with 1% decanal to bacterial cells, and the BRET signals were recorded for 60 min. (C) Competitive inhibition coefficient of FK506 against sirolimus (IC50). Samples expressing FRB-eYFP and LuxAB-FKBP containing 100 nM sirolimus were treated with the indicated concentrations of FK506, and the BRET signals were measured to calculate the Y:B ratios. Similar results were obtained in at least three independent experiments done in triplicate, and results from one representative experiment are shown.

the method, the analysis of binding between FliA and FlgM revealed that protein fusions of different orientations could affect the sensitivity and potentially even the usefulness of the method. Reasons such as the conformation, abundance, and stability of the fusion proteins can account for such differences. This phenomenon should be of note in the use of this method to analyze interactions of novel protein pairs. As indicated by the abolishment of the sirolimus-induced FRB-FKBP interaction by FK506, this method can be adapted to establish screenings aiming at identifying molecules capable of inhibiting the binding of proteins involved in important biological processes. Clearly, modifications such as engineering the bacterium of interest to produce the substrate for LuxAB may provide more accurate measurements for some experimental systems. Similarly, construction of bacterial strains expressing the relevant protein fusions from the chromosome by their cognate promoters will allow the study of the proteins of interest under conditions more closely resembling the natural conditions. Nevertheless, the development of a novel BRET system that allows real-time measurement of protein-protein interactions in live bacterial cells has added another useful tool to address important questions in the field.

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MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table S1 in the supplemental material. Strains were grown as described previously (39). Briefly, Escherichia coli strains were cultured in Luria-Bertani (LB) medium containing appropriate antibiotics aerobically on a rotary shaker (180 rpm) or on agar plates at 37°C. Yersinia pseudotuberculosis strains were cultured in Yersinia Luria-Bertani (YLB) broth at 26°C with appropriate antibiotics when necessary. Antibiotics were added at the following concentrations: ampicillin, 100 ␮g/ml; kanamycin, 50 ␮g/ml; tetracycline, 10 ␮g/ml; chloramphenicol, 30 ␮g/ml. DNA manipulation. General DNA manipulations and transformations were performed using standard genetic and molecular techniques (49). Restriction enzyme digestion, ligation, and plasmid purification were done in accordance with the manufacturer’s instructions (TaKaRa, Dalian, China). PCR was performed with TaKaRa Taq or Pyrobest DNA polymerase (TaKaRa, Dalian, China). Plasmid DNA was isolated with the Tiangen Biotech plasmid DNA Miniprep spin columns, and chromosomal DNA was purified with the Tiangen Biotech bacterial genomic DNA purification kit (Tiangen Biotech, Beijing, China). DNA fragments were purified from agarose gels by using the EasyPure quick gel extraction kit (Transgen, Beijing, China). DNA sequencing and primer synthesis were carried out by Sangon Biotech (Shanghai, China).


FIG 5 Kinetics of OmpR dimerization under different conditions. (A) High-salt-concentration-induced dimerization of OmpR requires the phosphorylation site D55. Salt was added to 1.5 M to cultures expressing the indicated testing protein fusions, and the BRET signals were recorded for an experimental duration of 60 min (B) Wild-type OmpR does not interact with its D55A mutant. The pH of cultures expressing the indicated protein fusion pairs was lowered to 4, and the BRET signals were recorded for 60 min. Note that no BRET occurred in cultures expressing OmpR and its D55A mutant fused to the two BRET components, respectively. (C) Abolishment of the inducing condition led to reduction of dimerization. NaOH was added to cultures that had been induced by low pH for 5, 10, or 15 min, and the BRET signals were measured at the indicated time intervals for 60 min. A culture that received an equal volume of fresh medium was used as a control. (D) Kinetics of the PompC promoter activity in response to high salt. Salt was added to cells containing the PompC::LuxAB reporter, and the expression of bioluminescence was monitored for 90 min. Note that it takes about 30 min to detect significant LuxAB activity.

Plasmid construction. The plasmids and primers used in this study are listed in Tables S1 and S2, respectively, in the supplemental material. To construct LuxAB-eYFP, LuxAB-GFP, and LuxAB-mOrange fusion expression cassettes, the coding region of luxAB was amplified with primers LuxA-XbaI-F and LuxB-L15-BglII-R and cloned into XbaI/BamHIdigested pBluescript II KS⫹ to give pBluescript-luxAB. A 15-amino-acid linker (GGGSG)3 was fused in frame to the luxB open reading frame (ORF) by deleting the stop codon TAA and adding codons for the linker after the luxB coding region in primer LuxB-L15-BglII-R. The lac promoter was amplified with primers pLac-SacI-F and pLac-XbaI-R, digested with SacI/XbaI, and then ligated into pBluescript-luxAB, resulting in plasmid pBluescript-pLac::luxAB (see Fig. S3 in the supplemental material). The eYFP coding region was amplified with primers eYFP-BamHI-F and eYFP-SalI-R, digested with BamHI/SalI, and inserted into similarly digested pBluescript-pLac::luxAB to produce pBluescript-pLac::luxABeYFP, in which the eYFP ORF is fused in frame with the luxB gene, spaced by the (GGGSG)3 linker. The plasmids pBluescript-pLac::luxAB-mOrange and pBluescript-pLac::luxAB-gfp were constructed similarly, with the ORFs of GFP and mOrange amplified with primer pairs GFP-BamHI-F/ GFP-SalI-R and mOrange-BamHI-F/mOrange-SalI-R, by using the plasmids pEGFP-N1 and pmOrange, respectively, as the template. The tac promoter was amplified with primers pTac-SacI-F and pTac-


XbaI-R, digested with SacI/XbaI, and then ligated into pBluescript II KS⫹, resulting in pBluescript-pTac. An eYFP coding sequence with or without the stop codon was PCR amplified using primers eYFP-EcoRI-F/eYFPSalI-R and eYFP-SpeI-F/eYFP-SalI-R=, respectively, and then cloned into the corresponding sites of plasmid pBluescript-pTac, to give pBluescriptpTac::⍀-eYFP and pBluescript-pTac::eYFP-⍀. The lac promoter was amplified with primer pair pLac-SphI-F/pLacSpeI-R, digested with SphI/SpeI, and ligated into similarly digested pKT100 (see Fig. S3 in the supplemental material), to produce pKT100pLac. The luxAB coding region was amplified with primer pair LuxASpeI-F/LuxB-L15-BamHI-R and LuxA-L15-XbaI-F/LuxB-KpnI-R and cloned into the SpeI/BamHI and XbaI/KpnI sites of the vector pKT100pLac, respectively, resulting in plasmids pKT100-pLac::luxAB-⍀ and pKT100-pLac::⍀-luxAB. For the plasmid pKT100-pLac::luxAB-⍀, a 15amino-acid linker (GGGSG)3 was fused in frame to the luxB ORF by removing the stop codon TAA. Finally, for plasmid pKT100-pLac::⍀luxAB, the 15-amino-acid linker (GGGSG)3 was fused in-frame to the ORF of luxA directly upstream of the start codon. The coding regions of flgM and fliA were amplified from the genome of Y. pseudotuberculosis strain YpIII with primer pairs FliA-BamHI-F/FliASalI-R, FliA-BamHI-F/FliA-SalI-R-TAA, FlgM-BamHI-F/FlgM-SalI-R, and FlgM-BamHI-F/FlgM-SalI-R-TAA, respectively. PCR products of

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flgM and fliA were digested with BamHI/SalI and inserted into pBluescript-pTac::⍀-eYFP, pBluescript-pTac::eYFP-⍀, pKT100-pLac:: ⍀-luxAB, and pKT100-pLac::luxAB-⍀, respectively, to produce plasmids pLac::luxAB-fliA, pLac::fliA-luxAB, pTac::eYFP-flgM, and pTac::flgMeYFP. In the resulting plasmids, the fusion proteins FlgM and FliA were fused in frame to the upstream and downstream sequences of eYFP and LuxAB and were all transcribed from the lac promoter. The cDNA encoding the mouse FK506 binding protein (FKBP) and the 11-kDa FKBP12-rapamycin binding domain within FRAP (FRB) were codon optimized according to Neurospora codon usage data from the Kazusa DNA Research Institute (http://www.kazusa.or.jp/codon) and synthesized by GenScript Co., Ltd. (Nanjing, China). For each residue, the codon with the highest usage frequency in the bacterium was chosen. The entire coding regions of FRB and FKBP were cloned into pBluescriptpTac::⍀-eYFP and pKT100-pLac::luxAB-⍀, respectively, to produce the plasmids pBluescript-pTac::FRB-eYFP and pKT100-pLac::luxAB-FKBP. The ompR gene fragment was amplified with primers OmpR-FBamHI and OmpR-R-SalI, using E. coli genomic DNA as the template. The PCR product was digested with BamHI and SalI and inserted into similarly digested pBluescript-pTac::⍀-eYFP and pKT100-pLac:: luxAB-⍀, to give pBluescript-pTac::OmpR-eYFP and pKT100-pLac:: luxAB-OmpR. Site-directed mutagenesis was performed according to the QuikChange protocol (Stratagene, La Jolla, CA) as described by the supplier with primers OmpR*-F and OmpR*-R, to construct pBluescriptpTac::OmpR(D55A)-eYFP and pKT100-pLac::luxAB-OmpR(D55A). To construct the reporter plasmid pBluescript-POmpC::luxAB, the ompC promoter was amplified with primer pair pOmpC-SacI-F/pOmpCXbaI-R from E. coli genomic DNA. The PCR product was cloned into SacI/XbaI-digested pBluescript-luxAB, which harbors an ampicillin resistance gene and a promoterless luxAB reporter gene. The integrity of the insert in all constructs was confirmed by DNA sequencing. Bioluminescence and fluorescence assay. E. coli strains were grown overnight in LB medium containing appropriate antibiotics at 37°C. To eliminate the background fluorescence in LB medium, the E. coli cultures were washed and resuspended in M9 minimal salts medium before assay. To detect eYFP fluorescence, 3 ml of E. coli cells resuspended in M9 medium (optical density at 600 nm [OD600] of 1.0) was transferred to a 4-ml cuvette. Fluorescence excitation and emission measurements were performed on an F-7000 fluorescence spectrophotometer with a 150-W xenon lamp (Hitachi). eYFP fluorescence was excited at 510 nm, and its emission was scanned from 515 nm to 600 nm. For the luminescence assay, the lamp was shut off, and the cuvette was gently bubbled with air to supply sufficient oxygen for the luciferase reaction after the addition of 1% decanal (Aladdin). Bioluminescence emission spectra were collected between the wavelengths 400 nm and 600 nm as described previously (14). Detection of dynamic protein interactions by the LuxAB-based BRET system. The plasmids pBluescript-pTac::OmpR-eYFP and pKT100pLac::luxAB-OmpR were cotransformed into E. coli strain DH5␣, and the transformant was used to examine OmpR dimerization under different conditions by growing the cells at 37°C in LB medium to an OD600 of 1.0. To detect the induced OmpR dimerization by high-osmolarity stress or acid stress, 3 ml E. coli cells resuspended in M9 medium (OD600 of 1.0) was transferred to a 4-ml cuvette, and protein dimerization was initiated by the addition of hydrochloric acid to pH 4.0 or NaCl to 1.5 M in the presence of 1% decanal. Similarly, the sirolimus-induced FKBP-FRB interaction was detected by using E. coli DH5␣ cotransformed with pBluescript-pTac::FRB-eYFP and pKT100-pLac::luxAB-FKBP, and the interaction was initiated by adding 100 nM sirolimus (dissolved in DMSO) and 1% decanal. To obtain a competitive inhibitor coefficient of FK506 to sirolimus, eight groups of E. coli cells expressing FRB-eYFP and LuxABFKBP were treated with 100 nM sirolimus and incubated with different concentrations of FK506 for bioluminescence analysis. The BRET sensor fragments do not affect the dissociation constant for the FRB-sirolimusFKBP complex and the inhibition constant for FK506 (41, 50). The BRET

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assay was measured for indicated time durations (0 to 60 min) in live cells after being mixed and bubbled during the experiments (41). BRET imaging of E. coli cultures. Cells of 1.5-ml overnight E. coli cultures grown at 37°C in LB medium with appropriate antibiotics were washed twice with M9 medium and resuspended in 1.5 ml M9 medium containing 1% decanal. For imaging of the induced FKBP-FRB BRET signal, 100 nM sirolimus was added into the suspension. Two hundred microliters of each suspension was immediately transferred to wells of a dark microwell plate (Costar). The small volume of the sample allowed good gas exchange, and so bubbling was not required. Six duplicates were made for each strain, which were then divided into two groups. One group was visualized through a 480-nm (⫾5-nm) interference filter and the other through a 530-nm (⫾5-nm) filter. Images were captured with a cooled charge-coupled device (CCD) camera of the ChemiDocXRS⫹ gel imaging system (Bio-Rad). LuxAB reporter fusion and bioluminescence assay. To determine the dynamics of OmpC transcription induced by high-osmolarity stress or acid stress, the reporter plasmid pBluescript-POmpC::luxAB was transformed into E. coli DH5␣ as a reporter strain. One hundred microliters of a log-phase cell culture of E. coli reporter strain was transferred into a 1.5-ml microcentrifuge tube. Luminescence reactions were initiated by the addition of NaCl to a final concentration of 1.5 M or hydrochloric acid (stock concentration, 0.1 mol/liter) to a pH of 4.0 in the presence of 1% decanal. Luminescence activity was detected on a GloMax 20/20 luminometer (Promega) as described previously (51). All experiments were performed independently at least three times, and the data shown are from one representative experiment done in triplicate. Western blot analysis. E. coli cells expressing the BRET vectors, eYFP fusion proteins, were harvested and lysed with passive lysis buffer (Promega) for 30 min on ice. Equal amounts of total protein were resolved by 12% SDS-polyacrylamide gel and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 4% milk for 2 h at room temperature, membranes were probed with anti-YFP mouse monoclonal antibody (1:2,000; Millipore, Billerica, MA) diluted in Trisbuffered saline–Tween (TBST) at 4°C overnight. The membrane was washed five times with TBST and subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Shanghai Genomics) in TBST buffer for 1 h. The proteins were visualized by using the ECL Plus kit (GE Healthcare, United Kingdom) following the manufacturer’s instructions.

SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at http://mbio.asm.org/ lookup/suppl/doi:10.1128/mBio.01050-14/-/DCSupplemental. Figure S1, PDF file, 0.1 MB. Figure S2, PDF file, 0.2 MB. Figure S3, PDF file, 0.3 MB. Table S1, PDF file, 0.2 MB. Table S2, PDF file, 0.1 MB.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China grant 31170121 to X.S. and grants 31170100 and 31370150 to Y.W., Specialized Research Fund for the Doctoral Program of Higher Education of China grant 20110204120023 to X.S., and NIH-NIAID grant K02AI085403 to Z.-Q.L.

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Corrigendum: Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM).