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Construction and use of an stx1 transcriptional fusion to gfp

Abram Aertsen, Rob Van Houdt, Chris W. Michiels
DOI: http://dx.doi.org/10.1016/j.femsle.2005.02.024 73-77 First published online: 1 April 2005


Shiga toxins (Stxs), also termed Vero toxins, are cytotoxic ribosome inactivating proteins that are produced by a number of gastrointestinal pathogens and that contribute to the severity of the associated diseases. In this work, we constructed and validated a transcriptional fusion of the stx1AB promoter to the gfp reporter gene. The cloned promoter region encompasses both the proximal and the distal promoter regions of stx1AB, mediating control by the host's iron-responsive Fur repressor and the Stx prophage's Q antiterminator protein, respectively. The probe was validated by demonstrating its responsiveness towards mitomycin C and EDTA, and the contribution of host and phage encoded factors could be separated by studying stx1 expression in either wild-type or isogenic lysogenic cells. Moreover, stx1AB expressing populations could be visualized by flow cytometry. The potential use of such a probe for non-destructive online detection of stx1AB expression and visualization of stx1AB expressing populations is further discussed.

  • Escherichia coli
  • Shiga toxin
  • Flow cytometry
  • GFP promoter fusion

1 Introduction

Shiga toxins (Stxs) constitute a family of heteromultimeric proteins belonging to the AB family of toxins and form a branch of the potent class 2 ribosome inhibiting proteins [1]. The cytotoxicity of Stxs was first demonstrated on Vero cells, lending them the name of Vero toxins, and production of Stx was originally identified in the enteropathogen Shigella dysenteriae type 1. Later, several Stx producing Escherichia coli (STEC) strains have also been isolated. In pathogenic STEC strains Stx production can significantly contribute to virulence by increasing the severity of gastrointestinal illnesses that vary from uncomplicated diarrhea to hemorrhagic colitis and a severe complication called hemolytic uremic syndrome [2].

With regard to public health, both the dissemination of stx genes and the regulation of their expression are of tremendous interest. While in S. dysenteriae type 1 the stxAB operon is embedded in remnants of a defective bacteriophage genome, it often resides on still functional lambdoid prophages in the case of STEC strains [3]. As a result, the capacity to produce Stx can be easily transmitted horizontally in a process termed lysogenic conversion [4]. Moreover, induction of the SOS response, often as a result of DNA damaging treatments, is known to trigger the lytic cycle of lambdoid prophages [5] and results in a burst of Stx phage particles, in turn enhancing the probability of infection and lysogenization of naive E. coli or other susceptible bacterial hosts [6,7]. Interestingly, owing to their localization in the late region of the phage genome, expression of stx genes was found to be coregulated with transcription of late phage genes associated with the lytic cycle [8,9]. Additional regulation in the case of stx1 allows for its expression under low iron conditions [10], a phenomenon also observed for several other virulence genes and thought of as a tactic to turn on virulence traits upon entering the host organism where iron is scarce.

The techniques that are currently employed to monitor Stx production or expression involve either immunological methods for direct toxin detection [11], or specific stx mRNA quantification and promoter fusions to reporter proteins, such as alkaline phosphatase [10] and β-galactosidase [12]. Notwithstanding the importance of revealing the patterns of stx expression under different conditions, none of these techniques allow rapid and online detection and quantification of stx expression. In this study, we therefore constructed and validated an stx1 gfp transcriptional fusion, allowing online and sensitive measurement of stx1AB expression, and further discuss its use in a variety of applications.

2 Materials and methods

2.1 Strains and growth conditions

E. coli strain MG1655 [13] was used in this study. MG1655 H-19B was constructed earlier [7] by lysogenizing MG1655 with Stx phage H-19B, encoding stx1AB. MG1655 Δfur::Kn was kindly provided by Touati et al. [14]. MG1655 PsulA was constructed earlier [15] and contains a transcriptional fusion of the sulA promoter to gfp in pFPV25 [16]. Stationary phase cultures were obtained by growth in Luria Bertani broth (LB) [17] overnight at 37 °C under well-aerated conditions. Exponential phase cultures were obtained by diluting overnight cultures 1/100 in fresh prewarmed LB and incubating further at 37 °C until late exponential phase (OD600= 0.6). Ampicillin (100 μg/ml, Ap100) and kanamycin (50 μg/ml, Kn50) were added when necessary.

2.2 Induction and measurement of gfp expression

Exponential phase cultures of strains carrying promoter fusions were collected by centrifugation (5 min at 6000g), and resuspended in fresh LB. To measure GFP production after induction by mitomycin C (Applichem, Darmstadt, Germany) or EDTA, 300 μl samples were transferred to microplate wells and placed in a fluorescence reader (Fluoroscan Ascent FL, Thermolabsystems, Brussels, Belgium). Fluorescence at 520 nm was then measured at 30 min intervals with intermittent shaking (every 5 min) at 37 °C, using an excitation wavelength of 480 nm. At the same time OD600 was measured and fluorescence was expressed per unit of OD600. Fold induction was then expressed as the ratio of the fluorescence of induced versus control cultures. Alternatively, GFP expressing populations were analyzed by flow cytometry, essentially as described before [15].

3 Results and discussion

3.1 Construction of an stx1-gfp transcriptional fusion

stx1AB is carried by stx1 coding Stx phages, such as H-19B, and is located in the late lytic region. The promoter region directly upstream of stx1AB contains a Fur box, making it responsive to the cellular iron status through the action of the Fur regulatory protein encoded by the host MG1655 [10]. Aside from this proximal promoter element, however, stx1 transcription is also driven by a more distal promoter element, Embedded Image in the presence of the phage encoded antiterminator protein Q which interacts with the qut site (Fig. 1) [9]. The latter is produced at the end of a regulatory cascade that is initiated when the prophage enters the lytic cycle, and extends transcription from Embedded Image towards the late region. To study the regulation of both promoter elements in the stx1 gfp transcriptional fusion, a 793 bp promoter fragment directly upstream of stx1AB, Pstx1, was obtained by PCR from MG1655 H-19B, using primers 5′-CAGTGGATCCTGGCACGGAAACATGGGT-3′ and 5′-TCAGTCTAGATTACGTCTTTGCAGTCGAGAAGTC-3′. Pstx1 was subsequently digested by BamHI and XbaI and cloned upstream of gfp in BamHI and XbaI digested pFPV25 [16]. The resulting plasmid, pAA310, is depicted in Fig. 1.

Figure 1

Map of the pAA310 plasmid showing the Stx1 phage fragment cloned upstream of gfp. Black boxes indicate regulatory sites: the qut site interacts with the Q antiterminator protein, while the Fur box indicates the Fur binding region. Black arrows represent genes. BamHI and XbaI cloning sites are shown. It should be noted that the 20 bp between the XbaI site and the gfp start codon harbor a ribosome binding site (5′-AAGAAG-3′) (16).

3.2 Validation of the stx1-gfp transcriptional fusion

Under certain stress conditions stx1 expression is governed both by host as well as by prophage encoded functions, and by comparing stress-induced expression of Pstx1 in a wild-type and in an isogenic H-19B lysogen background, the contribution of host and prophage encoded factors can be separated. Indeed, in the presence of millimolar concentrations of EDTA, a chelating agent sequestering soluble iron and derepressing the Fur regulon, expression of Pstx1 rapidly increased >2-fold both in wild-type as well as in H-19B lysogenized cells (Fig. 2(a), compare diamonds and triangles, respectively), indicating no involvement of prophage encoded factors. On the other hand, when the same strains were exposed to mitomycin C, a DNA damaging agent and a potent SOS inducer, expression of Pstx1 was induced only in the lysogenic strain (Fig. 2 (b), triangles). This is in agreement with the fact that the SOS response triggers the lytic cycle of the H-19B prophage and thus Q mediated expression of the late region, including stx1AB. In the absence of prophage, this cycle cannot be initiated and no Q antiterminator protein can be produced. When comparing the expression of stx1 (triangles) and sulA (squares) in Fig. 2 (b), a well-known gene from the SOS regulon, the former shows a lag of ca. 3 h, indicative of the supplementary time needed to (i) initiate the prophage's lytic cycle by the RecA mediated cleavage of its CI repressor [5], and (ii) initiate the late stage of the lytic cycle with concomitant Q mediated expression of stx1AB (measured by Pstx1) [9]. For comparison, upon induction with EDTA, no SOS response was triggered (Fig. 2(a), squares), and prophage latency was retained thus excluding Q mediated stx expression, and allowing for immediate Fur mediated derepression of stx1. It can be concluded that the Pstx1 fragment in pAA310 integrates both host and phage attributed regulation, and that these effects can be separated in a wild-type or lysogenic background.

Figure 2

Induction of the Pstx1 promoter, measured as GFP production in MG1655 wild-type (♦) and MG1655 H-19B (▲) cells during exposure to (a) 8 mM EDTA or (b) 2 μg/ml mitomycin C. Induction of PsulA (♦) in MG1655 serves as a measure of induction of the SOS response. Fold induction was calculated as the ratio of expression between induced and uninduced cultures of the same strain. Expression was determined as fluorescence divided by the OD600 of the culture. Means ± standard deviations of four independent experiments are shown.

3.3 Use of the stx1-gfp transcription fusion to study stx1 expression in E. coli populations

Opposed to alkaline phosphatase or β-galactosidase reporter proteins [10,12], the rapid and online non-destructive detection of GFP allows for the visualization of stx1AB expression throughout a population by epifluorescence microscopy or flow cytometry. Monitoring stx1AB expression using the latter technique also offers high sensitivity, since changes in expression can in principle be detected in populations of no more than a few hundred cells. We applied flow cytometry analysis and observed that under our standard growth conditions in LB medium Pstx1 is constitutively expressed, and this expression seems independent from prophage encoded factors, since the presence or absence of H-19B prophage had little or no influence on Pstx1 expression (compare MG1655 wild-type in Fig. 3(a) with its isogenic H-19B lysogen in Fig. 3 (b)). This is in agreement with a recent study by Livny and Friedman [18], who by a RIVET based reporter approach calculated that only ca. 0.005% of H-19B lysogens are spontaneously induced per generation during growth in LB.

Figure 3

Flow cytometry analysis of Pstx1 expression, measured as GFP production in late exponential cultures of (a) MG1655 wild-type, (b) MG1655 H-19B, and (c) MG1655 Δfur:Kn. The curves each represent populations of 105 cells. Representative results of four independent experiments were shown. Cells containing pFPV25, and thus harboring a promoterless gfp gene (negative control) do not express GFP and have a mean fluorescence of ca. 2 (data not shown).

These results indicate, that in the absence of its lytic development, very little regulation is imposed by the prophage, and stx1AB expression is predominantly governed by the host's Fur protein. To examine the impact of the Fur repressor, pAA310 was transformed to MG1655 Δfur::Kn, and the resulting population was analyzed by flow cytometry (Fig. 3 (c)). As expected, in the absence of Fur, the Pstx1 expression levels of cells increased significantly (ca. 6-fold; compare Fig. 3 (c) with (a)). Interestingly, during the preparation of this manuscript, it was shown that production of Stx2 from 933W lysogens is not detectable when its lytic cycle was impaired by replacing the CI repressor with an uncleavable variant [19]. This is in agreement with our observations with stx1AB expression, since the stx2AB regulon encoded by phage 933W contains no Fur box and is therefore not regulated by the host's iron homeostasis.

Since Shiga toxins comprise an important virulence trait, understanding the regulation of their expression is of increasing interest and the availability of versatile monitoring methods is crucial. Besides for rapid online monitoring of stx1AB expression in pure cultures as demonstrated in this work, the described stx1 gfp transcriptional fusion can also be used in more complex settings, in which the quantification of Stx toxin produced by a population by immunological or mRNA dependent methods may be biased either by difficult sampling or by the presence of other Stx producing populations. In these cases, a population of interest can be transformed with pAA310 and its stx1AB expression as influenced by various environmental conditions can be highlighted from the total population. Moreover, in mixed populations, non-lysogenic strains carrying the probe can be used to report infection by Stx phage, since upon infection and initiation of the lytic cycle in these strains, the Q protein of the invading phage will induce expression from Pstx1.


We acknowledge financial support by research grants OT/01/35 from the K.U. Leuven Research Fund and G.0195.02 from F.W.O. Vlaanderen.


  1. [1].
  2. [2].
  3. [3].
  4. [4].
  5. [5].
  6. [6].
  7. [7].
  8. [8].
  9. [9].
  10. [10].
  11. [11].
  12. [12].
  13. [13].
  14. [14].
  15. [15].
  16. [16].
  17. [17].
  18. [18].
  19. [19].
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