Dual-fluorescent bacterial two-hybrid system for quantitative Protein-Protein interaction measurement via flow cytometry.

Characterization of protein-protein interactions (PPIs) is essential for understanding cellular signal transduction pathways. However, quantitative measurement of the binding strength remains challenging. Building upon the classical bacterial adenylate cyclase two-hybrid (BACTH) system, we previously demonstrated that the relative reporter protein expression (RRPE), defined as the level of reporter expression normalized to that of the interacting protein, is an intrinsic characteristic associated with the binding strength between the two interacting proteins. In this study, we inserted fluorescent protein tdTomato in the chromosome as the reporter protein by CRISPR/Cas9 technology and employed a 12-amino acid tetracysteine (TC) to tag one of the interacting proteins, which can be further labeled by a membrane-permeable biarsenical dye. The combined use of tdTomato and TC-tag offers rapid and high-throughput analysis of the expression levels of both the reporter protein and one of the interacting proteins at the single-cell level by multicolor flow cytometry, which simplifies the quantitative measurement of PPI. The use of the as-developed RRPE-tdTomato-TC-BACTH approach was demonstrated in three demanding applications. First, binding affinities could be correctly ranked for discriminating interaction strengths with a tenfold difference or of the same order of magnitude. We demonstrate that the method is sensitive enough to discriminate affinities with a small difference of 1.4-fold. Moreover, residues involved in PPI can be easily mapped and ranked. Lastly, protein interaction inhibitors can be rapidly screened.

Deciphering the Antitoxin-Regulated Bacterial Stress Response via Single-Cell Analysis.

Bacterial toxin-antitoxin (TA) systems, which are diverse and widespread among prokaryotes, are responsible for tolerance to drugs and environmental stresses. However, the low abundance of toxin and antitoxin proteins renders their quantitative measurement in single bacteria challenging. Employing a laboratory-built nano-flow cytometer (nFCM) to monitor a tetracysteine (TC)-tagged TA system labeled with the biarsenical dye FlAsH, we here report the development of a sensitive method that enables the detection of basal-level expression of antitoxin. Using the Escherichia coli MqsR/MqsA as a model TA system, we reveal for the first time that under its native promoter and in the absence of environmental stress, there exist two populations of bacteria with high or low levels of antitoxin MqsA. Under environmental stress, such as bile acid stress, heat shock, and amino acid starvation, the two populations of bacteria responded differently in terms of MqsA degradation and production. Subsequently, resumed production of MqsA after amino acid stress was observed for the first time. Taking advantage of the multiparameter capability of nFCM, bacterial growth rate and MqsA production were analyzed simultaneously. We found that under environmental stress, the response of bacterial growth was consistent with MqsA production but with an approximate 60 min lag. Overall, the results of the present study indicate that stochastic elevation of MqsA level facilitates bacterial survival, and the two populations with distinct phenotypes empower bacteria to deal with fluctuating environments. This analytical method will help researchers gain deeper insight into the heterogeneity and fundamental role of TA systems.

Specific detection of live Escherichia coli O157:H7 using tetracysteine-tagged PP01 bacteriophage.

Sensitive and rapid detection of Escherichia coli O157:H7, one of the most notorious bacterial pathogens, is urgently needed for public health protection. Yet, the existing methods are either lack of speed or limited in discriminating viable and dead cells. Using a recombinant bacteriophage, here we report the development of a rapid and sensitive method for live E. coli O157:H7 detection. First, the wild-type PP01 phage was engineered with a tetracysteine (TC)-tag fused with the small outer capsid (SOC) protein. Then, this PP01-TC phage was used to inoculate bacterial sample for 30min. Specific infection and rapid replication of PP01-TC phage in viable E. coli O157:H7 host cell yields a large number of progeny phages with capsids displaying TC tags that can be fluorescently labeled by a membrane permeable biarsenical dye (FlAsH). The bright green fluorescence of single E. coli O157:H7 cells can be readily detected by flow cytometry (FCM) and fluorescence microscopy. High specificity of the assay was verified with seven other bacterial strains. Practical application in E. coli O157:H7 detection in drinks was successfully demonstrated with artificially contaminated 100% apple juice. In less than three hours (including sample preconcentration) and with 40mL of sample volume, as low as 1cfu/mL E. coli O157:H7 can be detected in the presence of large excess of other nontarget bacteria via fluorescence microscopic measurement. The as-developed TC-PP01-FlAsH approach shows a great potential in the safeguard of liquid food products by providing rapid, sensitive, and specific detection of live E. coli O157:H7.

[Study on the relationship between structure and spectroscopy of two compounds containing 2,2'-bipy and [MoO3] cluster skeletons].

Two compounds of molybdate with 2, 2'-bipy and [MoO3]: [(2, 2'-bipy)2 (MoO3)3]n (I) and [(2, 2'-bipy) (MoO3)]n (II) were successfully synthesized by hydrothermal synthesis method with programmable temperature control. In order to clarify the relationship between the structure and spectroscopy of these two compounds, both of them were characterized by means of X-ray powder diffraction (XRD), Fourier transform infrared spectra(FTIR), thermal perturbation 2D-IR correlation spectrum (2D-IR COS), thermogravimetric analysis(TGA), scanning electron microscopy(SEM), High temperature infrared analysis, UV-Vis DRS adsorption spectra and solid fluorescence spectrum to investigate the relationship between structure and properties of the title compounds. The powder XRD patterns of the complexes are well matched with the simulation based on single-crystal analysis, which indicate that compound I and II are in a pure phase. The characteristics of vibration frequency of FTIR and thermal perturbation relative spectral response of 2D-IR peak is consistent with thecompound I and II structure analysis. The synchronous and asynchronous correlation 2D-IR spectra of compounds also identified the compounds I and II molybdenum-oxygen cluster skeletons sequencing of vibration intensity change with temperature consistent with the high temperature infrared analysis. Through the TGA and high temperature infrared analysis it was found that the decomposition temperature was more than 300 degrees C and maximum weight losses rates above 800 degrees C, which suggest that they have good thermal stability. According to the UV-Vis DRS spectrum of the compound I and II there exists a wide ultraviolet absorption band in a range of 225 to 350 nm. The compound I and II steady-state fluorescence spectrum under the excitation of 277 and 295 nm respectively revealed compound I and II have the strongest emission peak at 460 and 480 nm respectively. This paper illustrates the coordination situation of these two compounds, and reveals the inherent law of valence electrons in molecule energy level transition. In the meantime it was verified that the weak interaction not only plays a role of stability in the frame of the structure of the complexes, but also plays an important role in heat resistance.