Development of a Fur-dependent and tightly regulated expression system in Escherichia colifor toxic protein synthesis
© Guan et al.; licensee BioMed Central Ltd. 2013
Received: 8 September 2012
Accepted: 8 March 2013
Published: 19 March 2013
There is a continuous demanding for tightly regulated prokaryotic expression systems, which allow functional synthesis of toxic proteins in Escherichia coli for bioscience or biotechnology application. However, most of the current promoter options either are tightly repressed only with low protein production levels, or produce substantial protein but lacking of the necessary repression to avoid mutations initiated by leaky expression in the absence of inducer. The aim of this study was to develop a tightly regulated, relatively high-efficient expression vector in E. coli based on the principle of iron uptake system.
By using GFP as reporter, PfhuA with the highest relative fluorescence units, but leaky expression, was screened from 23 iron-regulated promoter candidates. PfhuA was repressed by ferric uptake regulator (Fur)-Fe2+ complex binding to Fur box locating at the promoter sequence. Otherwise, PfhuA was activated without Fur-Fe2+ binding in the absence of iron. In order to improve the tightness of PfhuA regulation for toxic gene expression, Fur box in promoter sequence and fur expression were refined through five different approaches. Eventually, through substituting E. coli consensus Fur box for original one of PfhuA, the induction ratio of modified PfhuA (named PfhuA1) was improved from 3 to 101. Under the control of PfhuA1, strong toxic gene E was successfully expressed in high, middle, low copy-number vectors, and other two toxic proteins, Gef and MazF were functionally synthesized without E. coli death before induction.
The features of easy control, tight regulation and relatively high efficiency were combined in the newly engineered PfhuA1. Under this promoter, the toxic genes E, gef and mazF were functionally expressed in E. coli induced by iron chelator in a tightly controllable way. This study provides a tightly regulated expression system that might enable the stable cloning, and functional synthesis of toxic proteins for their function study, bacterial programmed cell death in biological containment system and bacterial vector vaccine development.
With the advent of the post-genomic era coming, the need is boosting to express a growing number of genes originating from different organisms . Unfortunately, many of these foreign genes severely interfere with the survival of Escherichia coli cells, which could lead to bacteria death or cause significant defects in bacteria growth. What’s more, following the development of biological technology, many genetically engineered E. coli have been constructed and developed for different purposes, such as bioremediation, biomedicine and bioenergy . However, their practical applications in the field are still restricted because engineered bacteria may cause new environmental contaminations. To minimize the potential risks, biological containment system was designed to monitor and restrict the distribution of engineered bacteria. Generally, containment systems are based on a ‘killer gene’ and a tight ‘regulatory circuit’ that controls expression of the killer gene in response to the presence or absence of environmental signals [3, 4]. In order to activate the killer gene expression only at expected condition, usually a specific environment, the promoter that controls bacteria programmed cell death needs to be easily controllable, tightly regulated and environment-responsive.
In general, there are five solutions to manage killer or highly toxic genes expression: manipulation of transcriptional and translational control elements of highly toxic genes, manipulation of the coding sequence, manipulation of the copy number, addition of stabilizing sequences, and empirical selection of E. coli strains . Among these solutions, manipulation of transcriptional control elements aims at blocking the leaky expression from the common used promoters, such as Plac, Ptrc, Ptac, PT7, PpL, PtetA or PlacUV5, which is critical in toxic gene expression [6, 7]. As a result, the manipulation strategies of transcriptional control elements have been described to reduce the possibility of a unexpected toxic event. The strategies include suppression of basal expression from leaky inducible promoters, suppression of read-through transcription from cryptic promoters, tight control of plasmid copy numbers and proteins production as inactive (but reversible) forms [5, 8–10].
Since the incomplete repression of promoter presents a major problem when cloning genes that encode lethal product to the bacterial host , it is obviously critical to tighten the control of gene expression by utilizing a tightly controllable promoter that permits E. coli normal growth until the very moment of highly toxic gene induction. Starting from this point, several tightly regulated expression circuits have been developed, such as Prha, hybrid Plac/ara-1, and PBAD-based vectors . The widely used PBAD derivative expression systems have been verified as useful solutions for their stringentness in toxic protein production in E. coli. Expression is induced to high levels on media containing L-arabinose, and tightly shuts off on media with glucose but without L-arabinose, which shows more stringent regulation of target gene expression than other expression systems. However, some problems still exist in this system, such as few vectors available and catabolism repression by glucose .
Usually, environment-responsive promoters are commonly engineered in inducible expression system, such as promoters sensing pH, temperature, oxygen concentration or iron availability . From iron-uptake systems that are evolved by bacteria growing in iron-limiting environment, many iron-related promoters have been reported [13–15]. The promoter from iron-uptake regulon is strongly repressed in iron rich conditions by Fur, but fully derepressed in absence of iron . Usually, Fur protein complexing with ferrous irons binds with high affinity to the 19-bp inverted repeat consensus sequence known as the Fur box (GATAATGAT [A/T] ATCATTATC) in the relevant promoter area, which controls transcription of iron-responsive genes in microorganisms . Fur inhibits transcription initiation by blocking the entry of RNA polymerase (RNAP) to the promoter. This ability is tightly dependent on the relative affinities of RNAP and Fur to binding sites in the DNA .
In this study, a novel tightly inducible expression system was developed as an alternative of the current ones. This system, designated pYPfhuA1, was capable of extremely tight regulation and allowed cloning of genes encoding highly toxic products within various copy-number plasmids. In detail, using E. coli Top10 as bacterial host, the strong iron-regulated promoter PfhuA was selected as the primary candidate. By modifying Fur box in promoter sequences and Fur repressor synthesis, the tightness of PfhuA was decreased to varying degrees. Thereinto, PfhuA1, which could be strict repressed by excess iron and efficiently induced by iron chelators, was the ideal promoter candidate for toxic gene expression in E. coli host.
Preliminary screening for iron-regulated promoters
To evaluate the regulation performance of PfhuA, the GFP synthesis was detected when Top10/ptPfhuAG growing in LB medium supplemented with repressor (40 μM FeSO4) or inducer (200 μM 2,2’-dipyridyl). As shown in Figure 1B, even with the repressor addition, an obvious leaky expression was detected under PfhuA transcription. Based on the data, the induction ratio that showed the tightness of promoter was calculated as 3.4, indicating that the tightness of PfhuA had to be improved for the toxic gene expression.
Modifications of PfhuAto improve its tightness
Comparison of different promoter modified strategies
Modified strategies and descriptions
Relative induction fold
E. coli Top10
200 ± 10
200 ± 9
260 ± 11
7812 ± 31
Original fhuA promoter
3292 ± 20
10116 ± 37
Changed the Fur box in −10 region into a conserved Fur box from E. coli
250 ± 12
5466 ± 26
Modified the Fur box in −10 region into an enhanced Fur box 
196 ± 5
1885 ± 17
Integrated a designed Fur box in −35 region
200 ± 9
662 ± 19
Inserted the SD-fur-TT circuit in the vector
230 ± 9
3464 ± 24
Inserted the Pfur-SDfur-TT circuit in the vector
195 ± 7
2389 ± 26
Performance evaluation of pPfhuA1as an inducible tightly regulated expression system
In order to see the time response of ptPfhuA1 by which it is effectively turned on, GFP yields along with E. coli growth were detected. GFP expression in the ptPfhuA1-bearing culture was continuously increased until 9 h post induction (Figure 3B). During the first 1 h induction, the relative fluorescence value increased by 3.4 folds, which meant the expression circuit had been switched on. Afterwards, the GFP expression increased very slowly from 1 to 5 h, and underwent a jump from 5 to 9 h. At last, the GFP expression achieved its peak at 9 h post induction, corresponding to the beginning of stationary phase in E. coli growth. Thus, the whole expression circle for PfhuA1 lasted for 9 h. In other side, the GFP synthesis always lagged behind the E. coli growth. This is desirable in toxic gene expression to reduce the damage of protein products to cells.
Toxic protein expression under the control of modified PfhuA1
With the successful application of pYPfhuA1 in the regulation of E gene expression, its broad availability was the next concern. Then, other two toxic proteins were synthesized using pYPfhuA1 in E. coli host. The gef encoding Gef of E. coli belongs to the hok killer gene family in Gram-negative bacteria and its expression kills the cell from the inside by interfering with a vital function in the cell membrane . The mazF from a toxin-antitoxin module mazEF specifies a stable toxin that cleaves mRNA at a specific site(s) which is responsible for programmed cell death in E. coli. Here patPfhuA1 was used, which is a middle-low-copy number vector with ~30 copies per cell . Colony forming units (CFUs) of the transformants were detected in the presence of inducer (Figure 4B). Comparing with the negative control Top10, all the recombinant strains were grown similarly in the presence of iron (data are not shown), and no leaky expression phenotype was appeared. However, the growth of Top10/patPfhuA1gef and Top10/patPfhuA1mazF was repressed when cultured with 2,2’-dipyridyl as the result of gef and mazF expression. At 0 h, 2,2’-dipyridyl was added. During the first 2 h after induction, all three strains performed to adapt the iron-limiting condition and maintained in a similar density of 109 CFU/ml. However, after 2 h, their growth behaviors were obviously different. Top10 strain grew fast to 2.4×109 CFU/ml in exponential way until 9 h post induction. However, the strains Top10/patPfhuA1gef and Top10/patPfhuA1mazF grew exponentially until 7 h post induction and then kept at 6×108 CFU/ml and 1×109 CFU/ml, respectively. Therefore, the growth repression shown by recombinant strains revealed that toxic genes gef and mazF were functionally expressed after induction under the control of PfhuA1. Furthermore, pYPfhuA1 could be applied in different toxic gene expressions.
One of the most notable merits for this system is its tight regulation by iron-limitation signal. Escolar et al. once pointed out that the actual sequences recognized by Fur consisted of a minimal array of three conserved 6 bp (GATAAT) units which could be extended laterally by discrete additions of repeats of the same unit, thereby giving rise to new sites to which the repressor binds with a range of affinities . It was assumed that the affinity would vary depending on both the number of repeats present on each operator and the conservation of their sequences, which would allow an entire range and hierarchy of transcription responses depending on small changes in the iron status in the cell . Based on this hypothesis, we designed three types of Fur boxes differing in sequence conservation and 6-bp unit repeat number. It was found that the more repeats of conserved 6-bp units or the more conserved Fur box, the stronger affinity between Fur and the promoter, which was following by the tighter promoter. This verified the hypothesis mentioned above. In another side, this strategy also provides a new way for modifying other inducible promoters. However, a problem in this strategy is that the transcription efficiency decreases along with the tightness improvement. This is in common, because most of the regulatory systems have an intrinsic limitation in the range of induction, and attempts to mutate promoters to reduce basal expression usually result in concomitant reduction of induced levels. To overcome this drawback, Royo et al. used the nasF attenuator and the NasR-dependent anti-termination system from Klebsiella oxytoca to construct a novel expression circuit which could conditionally prevent undesired transcription from any transcriptional initiation signal, while keeping induced levels intact [10, 25]. This point of view gives us a new reference for further optimization of expression vector constructed here.
Although iron is one of the most abundant elements on Earth, in aerobic environments it is predominantly found as ferric (hydro)oxides that are relatively insoluble at neutral pH, and thus, ionic ferric (Fe3+) concentrations are exceedingly low . Therefore, as an iron limitation responsive vector, pYPfhuA1 has great potential in developing ‘suicidal’ containment system for environmentally relevant application. Furthermore, in our previous research, iron-limiting condition was verified as an in vivo stimulating signal, and promoters derived from iron uptake system could be induced in vivo. Therefore, this means PfhuA1 could be used in the construction of in vivo inducible antigen synthesis system, which could alleviate the toxicity or metabolic burden of the host strain and improve the immunogenicity for bacterial vector vaccine [27–32]. Moreover, in vivo regulated and delayed attenuation strategy has been developed for maintaining the invasive abilities of bacterial vector to the greatest extent, such that the recombinant vaccine has the ability as virulent wild-type strain to reach effector lymphoid tissues before display of attenuation to preclude onset of any disease symptoms . pYPfhuA1 could also be applied in construction of in vivo delayed attenuation vaccine. In all, pYPfhuA1 could be induced by iron chelators in medium, in vitro aerobic environments and in vivo animal host, and has extensive application in biotechnology area.
In all, as the expression vector particularly developed for toxic protein synthesis, pYPfhuA1 owns its notable advantages, which include easy control, tight regulation, relatively high efficiency and multi-environments response potential. Consequently, pYPfhuA1 has great potential in functional study of toxic genes, construction of biological containment system, or bacterial vector vaccine development.
Bacterial strains and growth conditions
Plasmids used in this study
pUC18 derivative with rrnBT1T2 terminator, Apr
pUT derivative, with a reporter gene gfp, Apr
pUC18 derivative with rrnBT1T2 terminator, pBBR1 ori., Apr
pUC18 derivative with rrnBT1T2 terminator, p15A ori., Apr
pUC18 derivative with rrnBT1T2 terminator, pAT153 ori., Apr
pUT derivative containing PfhuAgfp TT, Apr
pUT derivative containing PfhuA1gfp TT, Apr
pUT derivative containing PfhuA2gfp TT, Apr
pUT derivative containing PfhuA3gfp TT, Apr
pUT derivative containing PfhuAgfp TT and SD-fur TT from E. coli, Apr
pUT derivative containing PfhuAgfp TT and Pfurfur TT from E. coli, Apr
pUT derivative containing PfhuA1E TT, Apr
pUTb derivative containing PfhuA1E TT, Apr
pUTa derivative containing PfhuA1E TT, Apr
pUTat derivative containing PfhuA1E TT, Apr
pUTat derivative containing PfhuA1gef TT, Apr
pUTat derivative containing PfhuA1mazF TT, Apr
A reporter plasmid pUTtG constructed previously was used as the promoter screening vector . The primers from the former work were applied to amplify the candidate promoters from bacterium chromosomes or plasmids . The amplified promoter products, possessing the native start codon, Shine-Dalgarno sequence, and −35 and −10 promoter elements plus additional upstream bases, were inserted into pUTtG, and the resultant plasmids were transformed into E. coli Top10 named Top10/ptPXG (X mean different promoters) for iron-regulated promoter screening.
PCR primers used in this study
General DNA procedure and online analysis tools
General DNA operations were carried out following the standard protocols. Automated DNA sequencing and primer synthesis were completed by Life Technologies (Shanghai, China). PfhuA characteristic regions were predicted by online tools: BPROM-bacterial promoter prediction program from Softberry (http://www.linux1.softberry.com/berry.phtml?topic=bprom%26group=programs%26subgroup=gfindb), BDGP-Promoter (http://www.fruitfly.org/seq_tools/promoter.html) and SCOPE-Suite for Computational identification Of Promoter Elements .
GFP synthesis detection
Overnight cell cultures were inoculated (1:100, v/v) into fresh LB medium containing appropriate antibiotics and cultured in a shaker at 200 rpm and 37°C. At middle log phase typically with an optical density at OD600 = 0.8-1.0, 2, 2’-dipyridyl was added to induce the expression of GFP. After 20 hours or defined time points of iron-limiting induction, 1 ml cell culture sample was taken, centrifuged at 12,000 g for 3 min, washed and resuspended in PBS (pH 7.2) to the same OD600 value (OD600 = 1.0). For each sample, 100 μl of cell suspension was added into a 96-well flat-bottom polystyrene plate (Costar, USA) and measured with a fluorescence plate reader (TECAN, GENios Pro, Austria). Excitation wavelength was set at 485 nm and emission was detected at 535 nm.
Toxic gene expression detection
The E. coli strains Top10/ptPfhuA1E, Top10/pbPfhuA1E, Top10/paPfhuA1E, Top10/patPfhuA1gef and Top10/patPfhuA1mazF were overnight grown at 37°C in LB medium supplemented with 40 μM FeSO4 to ensure tight repression of toxic gene. To induce toxic gene expression, the culture was diluted to an OD600 of 0.1 and cultured in a shaker at 200 rpm and 37°C. At early log phase (OD600 = 0.3-0.4), 2,2’-dipyridyl was added into culture to create iron-limiting condition. Cell samples were taken at different time points after induction to measure both the OD600 and the colony formation units (CFUs) to determine the bacteria growth in iron-limiting medium.
This work was supported by the joint project, National Natural Science Foundation of China- Austrian Science Fund (30811130545).
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