TY - JOUR
AU - , de Lorenzo, Víctor
AB - Abstract The XylR/Pu regulatory node of the m-xylene biodegradation pathway of Pseudomonas putida mt-2 is one of the most intricate cases of processing internal and external cues into a single controlling element. Despite this complexity, the performance of the regulatory system is determined in vivo only by the occupation of Pu by m-xylene-activated XylR and σ54-RNAP. The stoichiometry between these three elements defines natural system boundaries that outline a specific functional space. This space can be expanded artificially following different strategies that involve either the increase of XylR or σ54 or both elements at the same time (each using a different inducer). In this work we have designed a new regulatory architecture that drives the system to reach a maximum performance in response to one single input. To this end, we first explored using a simple mathematical model whether the output of the XylR/Pu node could be amended by simultaneously increasing σ54 and XylR in response to only natural inducers. The exacerbation of Pu activity in vivo was tested in strains bearing synthetic transposons encoding xylR and rpoN (the σ54 coding gene) controlled also by Pu, thereby generating a P. putida strain with the XylR/Pu output controlled by two intertwined feed forward loops (FFLs). The lack of a negative feedback loop in the expression node enables Pu activity to reach its physiological maximum in response to a single input. Only competition for cell resources might ultimately check the upper activity limit of such a rewired m-xylene sensing device. Insight, innovation, integration Environmental bacteria with a history of exposure to chemical pollutants often carry metabolic operons that become transcribed when cells meet unusual recalcitrant and/or xenobiotic compounds. Prokaryotic transcriptional factors that recognize such molecules as effectors for activating cognate promoters become the starting point for the development of biosensors useful for environmental monitoring or components of synthetic biology circuits. Yet, the parameters that control naturally occurring regulatory nodes are frequently inadequate to meet the specifications that are needed for the given genetic constructs. Predictions made by a simple model elaborated with available data guided us to experimentally exacerbate the activity of the m-xylene responsive and sigma-54 dependent promoter Pu of the soil bacterium Pseudomonas putida by genetic rewiring of the 3 key constituents of the control system. This opens the way to engineering better whole-cell sensors for small molecules. Introduction Regulatory networks have been revealed as complex webs of interacting molecular components, which can adopt different conformations.1 In fact, the shape and the strength of the network components contribute to define and fine-tune the response.2,3 Bacterial promoters are key elements of these regulatory networks. They integrate physiological and environmental signals triggering gene transcription demarcated by a specific functional space. This space is usually constrained by a number of parameters defined during the evolution of the regulatory system.4 A goal of Synthetic Biology is to reprogram signal processing pathways by rearranging the regulatory nodes, and generate predictable and beneficial applications.5 Sometimes this entails strategies to amplify the output of a specific regulatory system by expanding its natural boundaries.6,7 In general, the approaches employed to optimize the output rely on the introduction of exogenous modules in the cell that act as amplifiers.8,9 Nevertheless, our previous work revealed that it is possible to amplify the signal of a specific system without introducing non-native elements, just by rewiring the host regulatory network.6 Although amplification devices can increase the response of a specific regulatory system, only by simultaneous removal of extant physiological constraints it is possible to reach its maximum potential.4 We have examined these questions using the Pu promoter of the environmental bacterium Pseudomonas putida mt-2 as the preferred experimental system. This promoter is one of the most intricate cases of processing internal and external cues into a single regulatory element.10–12,Pu and the various factors it interacts with belong to a complex metabolic and regulatory network that determines a pathway for biodegradation of m-xylene borne by the TOL plasmid pWW0 of this bacterium.4,13 This pathway encompasses two catabolic operons, which are subject to a complex regulatory circuit that involves the interplay between various transcription factors.14,15 XylR is the main transcriptional regulator that controls the system.15 This regulator, in the presence of its natural inducers (m-xylene, 3-methylbenzyl alcohol) triggers the Pu and Ps promoters driving the expression of both the catabolic genes of the upper TOL operon and xylS respectively.16 The activation of the Ps promoter not only produces XylS, the second regulator of the system, but also leads to the repression of xylR expression due to the divergent disposition of Ps and the xylR promoter (PR).17 XylR acts in concert with the RNA polymerase (RNAP) containing the alternative sigma factor σ54 (ref. 15 and 18) both positioned at distant places of the DNA sequence Pu promoter. With the assistance of the DNA-bending factor IHF (integration host factor19,20) they form a tridimensional transcription initiation complex. Yet, the one sufficient condition for full promoter performance in vivo is the complete occupation of Pu by m-xylene-activated XylR and σ54-RNAP.4 Based on this fact we have demonstrated that it is possible to increase the output of the system by enhancing the levels of both XylR and σ54-RNAP individually, or in combination.4,6 Nevertheless, the approach that we reported involved two input signals: one to increase the XylR amount and another one to trigger the heterologous system responsible for the overexpression of the σ54 RNA subunit. As this strategy was of considerable interest for designing e.g. whole cell biosensors, and other heterologous expression devices, we wondered if it was possible to re-design it and achieve the same optimized output in response to one single signal. To this end, we first explored using a simple mathematical model whether the output of the XylR/Pu device could be increased further by simultaneously increasing σ54 and XylR levels in response to single TOL pathway inducers. For testing the predicted outcomes in vivo, we constructed transposon vectors encoding xylR and rpoN (the σ54 coding gene) controlled by Pu and we used them to generate a strain with the XylR/Pu node output controlled by two (positive) feed-forward loops (FFLs). The results show that it is possible to magnify Pu output by implementing two intertwined FFLs with xylR and rpoN which, by changing the stoichiometry between the key regulatory elements, expand the extant functional boundaries of the system. Results and discussion Rationale for expanding the functional boundaries of the XylR/Pu regulatory node Our previous results proved that it is possible to expand the natural functional space of the XylR/Pu system by changing the boundaries imposed by the two elements that control Pu: σ54 and XylR.4 The native XylR regulatory scenario defines the limits of the Pu output by adjusting the XylR molecule number with a negative feedback loop (NFL17). Also this output is limited by the defined number of σ54-containing species in the whole RNAP pool available for Pu binding (Fig. 1a). In previous studies we followed different strategies to increase the response of the system. Our first approach (Fig. 1b) focused on an induction-dependent augmentation of XylR molecules by reshaping the xylR architecture and replacing the natural NFL mediated by the PR promoter by a positive FFL generated by placing xylR under the control of Pu.6 On the other hand, we engineered the system to increase the number of σ54 RNA polymerase subunit molecules by using a heterologous expression system dependent on an external inducer (Fig. 1c). Both approaches led to a similar increase of the output of the system. Only by combining both strategies (Fig. 1d) it was possible to fill completely the potential functional space defined by these two elements. However, in these experiments4 we used a salicylate-dependent heterologous expression system to overproduce the σ54 subunit of RNA polymerase, thereby making necessary the use of both external inputs (natural XylR inducers and salicylate) to enable the system to give its optimal response. Besides, the requirement of another inducer for increasing σ54 levels could interfere with the expression of other genes of the regulon.21 On this basis, we set out to redesign the connectivity of the components shown in Fig. 1d to eliminate the need of an extra inducer – other than the specific effectors of XylR. Fig. 1 Open in new tabDownload slide Relational scheme of the key components of the natural and synthetic regulatory architectures of the XylR/Pu regulatory node. (a) Natural regulatory architecture of XylR/Pu node: in the presence of m-xylene XylR (R) activates Pu (output) and inhibits its own expression via PR. In this natural configuration, σ54 is a necessary factor for expression of Pu but its input comes separately from the rest of the components. (b) Synthetic amplifier of Pu performance based on changes in the XylR amount: in the presence of m-xylene, XylR (R) both turns Pu on (output) and self-activates its expression through the Pu promoter also. In this configuration, the natural amount of σ54 also contributes to the final output. (c) Synthetic amplifier of Pu performance (output) based on changes in the σ54 amount: in the same native regulatory architecture described before, it is possible to modify the output of the system by increasing the amount of σ54 with an external inducer. (d) Finally, rearrangement of the XylR/Pu node combining both synthetic amplifiers. Fig. 1 Open in new tabDownload slide Relational scheme of the key components of the natural and synthetic regulatory architectures of the XylR/Pu regulatory node. (a) Natural regulatory architecture of XylR/Pu node: in the presence of m-xylene XylR (R) activates Pu (output) and inhibits its own expression via PR. In this natural configuration, σ54 is a necessary factor for expression of Pu but its input comes separately from the rest of the components. (b) Synthetic amplifier of Pu performance based on changes in the XylR amount: in the presence of m-xylene, XylR (R) both turns Pu on (output) and self-activates its expression through the Pu promoter also. In this configuration, the natural amount of σ54 also contributes to the final output. (c) Synthetic amplifier of Pu performance (output) based on changes in the σ54 amount: in the same native regulatory architecture described before, it is possible to modify the output of the system by increasing the amount of σ54 with an external inducer. (d) Finally, rearrangement of the XylR/Pu node combining both synthetic amplifiers. Optimized XylR/Pu performance in response to a single input can be achieved with two intertwined positive FFLs To explore the scenario mentioned before, we designed a circuit where the Pu promoter was controlling not only the expression of xylR but also the overexpression of σ54. In this situation two FFLs cooperate to increase the amount of both XylR and σ54 upon induction of the system with e.g. m-xylene (Fig. 2c). In order to formalize this regulatory scheme we first simulated the performance of Pu after the induction of the system in two scenarios: [i] Pu controls the expression of xylR but σ54 levels are left constant (native levels, Fig. 2a and b) and [ii] the same xylR regulatory architecture but added with an extra copy of rpoN (encoding σ54 factor) controlled also by Pu (Fig. 2c and d). The readout of either architecture is Pu promoter activity (a parameter that can be measured, see below). As shown before, the first scenario predicts that addition of the aromatic inducer raises the XylR levels and therefore the output of the system (Fig. 2b6). The situation changes when an extra copy of rpoN controlled by Pu is introduced into the simulation (Fig. 2c). The model then predicts that the system output (i.e. transcriptional Pu activity) will be amplified because of two convergent effects: [i] the augmentation of the sigma factor after induction enlarges the share of σ54-containing RNAP for Pu binding, thereby increasing its own expression and, [ii] there will be a further increase of XylR levels due to the strengthening of the Pu promoter. This arrangement generates two autonomous but linked positive feedback loops: one controlling the expression of xylR and another one enhancing the expression of σ54, both triggered and sustained by exposure to a single aromatic inducer. As shown below, these predictions were examined in detail by following emission of bioluminescence by a Pu–luxCDABE reporter system as well as by monitoring XylR and σ54 levels in vivo with specific antibodies for each protein. Fig. 2 Open in new tabDownload slide Modeling the reshaped XylR/Pu regulatory node with alternative configurations of σ54 expression where two intertwined positive feedback loops influence the output of the system. (a) Relational map of reference: in the presence of m-xylene, XylR self-activates its expression but the levels of σ54 are kept constitutive (and thus not represented as a variable in the system). (b) Dynamic model. Arrows signal the moment of induction by m-xylene. (c) Alternative regulatory configuration in which m-xylene causes Pu to activate xylR and σ54 expression. Augmentation of σ54 intensifies its own expression and therefore the amount of XylR and Pu activity in a positive merge of two FFLs. (d) Dynamic model. Fig. 2 Open in new tabDownload slide Modeling the reshaped XylR/Pu regulatory node with alternative configurations of σ54 expression where two intertwined positive feedback loops influence the output of the system. (a) Relational map of reference: in the presence of m-xylene, XylR self-activates its expression but the levels of σ54 are kept constitutive (and thus not represented as a variable in the system). (b) Dynamic model. Arrows signal the moment of induction by m-xylene. (c) Alternative regulatory configuration in which m-xylene causes Pu to activate xylR and σ54 expression. Augmentation of σ54 intensifies its own expression and therefore the amount of XylR and Pu activity in a positive merge of two FFLs. (d) Dynamic model. Simultaneous increase of σ54 and XylR levels in response to Pu inducer m-xylene In order to test our model we engineered a mini-Tn5 transposon determining transcription of the rpoN gene under the control of Pu (Fig. 3a). The transposon Tn5 [Pu·RpoN] (Fig. 3a, module #3) was then delivered to the chromosome of the previously described P. putida Pu·RBX strain6 bearing in its chromosome transcriptional fusions Pu → luxCDABE (Fig. 3a, module #1) and Pu → xylR (Fig. 3a, module #2). The resulting strain (P. putida Pu·RpoN·Pu·RBX) thus bears an extra copy of rpoN transcribed from Pu (and thus sensitive to XylR-mediated induction with m-xylene) besides the native rpoN gene present in the extant genomic location. In order to test whether this new regulatory architecture increased the intracellular σ54 and XylR concentrations we grew both strains in LB and we exposed them or not to saturating vapour of m-xylene. After induction, protein extracts from each strain were prepared at different time points and levels of the σ54 factor and XylR were examined in Western blot assays (Fig. 3b) with recombinant antibodies22 against either σ54 or XylR.23 The results of Fig. 3b showed an increase of the σ54 molecules 3 hours after the induction of strain P. putida Pu·RpoN·Pu·RBX with m-xylene with respect to the one lacking the Pu → rpoN module (Fig. 3b, upper panel). Concerning XylR contents, both strains showed an increase after m-xylene (Fig. 3b lower panel) induction in accordance with the results predicted using the model (Fig. 2b and d) regarding the presence of both strains of the module Pu → xylR. Nevertheless, the augmentation of XylR in P. putida Pu·RpoN·Pu·RBX was higher than the one observed in the P. putida Pu·RBX strain due to the effect of the overexpression of intracellular σ54. These data confirmed that it was possible to obtain increased levels of both XylR and σ54 by implementing the regulatory architecture of Fig. 2c in which the expression of both proteins is magnified in response to a single input. But how does this translate in actual performance of Pu promoter activity? Fig. 3 Open in new tabDownload slide Augmentation of XylR and σ54 in the XylR/Pu regulatory system. (a) Genetic constructs. The figure shows a sketch (not to scale) of the genetic modules borne by the P. putida strains used in the experiment: The Pu–luxCDABE reporter (module #1) has a promoterless luminescence-determining operon controlled by the Pu promoter. Module #2 determines xylR transcription engineered in an auto-activation loop in which the gene is transcribed through the Pu promoter. Module #3 is a specialized device in which expression of the rpoN gene (encoding σ54) has been placed under the control of Pu. The P. putida strains used in this experiment are P. putida Pu·RBX and P. putida Pu·RpoN·Pu·RBX. Both bear the chromosome modules #1 and #2 and P. putida Pu·RpoN·Pu·RBX also carries module #3. (b) Western blot of P. putida Pu·RBX and P. putida Pu·RpoN·Pu·RBX extracts prepared from cells collected at different time points after exposing cultures to the saturating vapour of m-xylene and probed with an anti-σ54 antibody (upper panel) and anti-XylR (lower panel). Fig. 3 Open in new tabDownload slide Augmentation of XylR and σ54 in the XylR/Pu regulatory system. (a) Genetic constructs. The figure shows a sketch (not to scale) of the genetic modules borne by the P. putida strains used in the experiment: The Pu–luxCDABE reporter (module #1) has a promoterless luminescence-determining operon controlled by the Pu promoter. Module #2 determines xylR transcription engineered in an auto-activation loop in which the gene is transcribed through the Pu promoter. Module #3 is a specialized device in which expression of the rpoN gene (encoding σ54) has been placed under the control of Pu. The P. putida strains used in this experiment are P. putida Pu·RBX and P. putida Pu·RpoN·Pu·RBX. Both bear the chromosome modules #1 and #2 and P. putida Pu·RpoN·Pu·RBX also carries module #3. (b) Western blot of P. putida Pu·RBX and P. putida Pu·RpoN·Pu·RBX extracts prepared from cells collected at different time points after exposing cultures to the saturating vapour of m-xylene and probed with an anti-σ54 antibody (upper panel) and anti-XylR (lower panel). Effect of concurrent increase of XylR and σ54 in transcription in the Pu readout P. putida strains Pu·RBX and Pu·RpoN·Pu·RBX were tested in parallel for Pu activity using light emission stemming from their Pu → luxCDABE insert as a proxy of transcription initiation. The data shown in Fig. 4a revealed that Pu output in the P. putida strain with m-xylene inducible expression of rpoN increased after 5 h by about twofold when compared to the reference strain P. putida Pu·RBX. These experimental figures were consistent with the predictions of Fig. 2, in which (within a certain parameter set), increasing σ54 on top of the already exacerbated XylR was expected to augment Pu output by a factor of ∼2. To examine whether this improved responsiveness was dependent on having a prime effector of the XylR protein (i.e. m-xylene) or could be maintained also with a suboptimal inducer we recorded light emission of P. putida strains Pu·RBX and Pu·RpoN·Pu·RBX along time but using 3-methylbenzyl alcohol (3MBA) instead of m-xylene as the aromatic inducer. The results are shown in Fig. 4. For a more rigorous comparison of the two conditions fold-induction with respect to non-inducing conditions (rather than specific luminescence) was plotted vs. time. While P. putida Pu·RBX displayed an inducibility of 60–80 fold, the strain carrying the Pu → rpoN module (P. putida PuRpoN·Pu·RBX) reached ∼120-fold at the peak of its activity. Note, however that light emission caused by 3MBA did not start taking off until 6 h after inducer addition (in contrast to the much earlier response to m-xylene, Fig. 4a). Since the regulatory architecture of Fig. 2c sets Pu activation by XylR to happen earlier than σ54 overproduction, it is possible that a less efficient inducer delay the accumulation of both factors until they reach a critical level. But once this happens (by 5–6 h after induction in Fig. 4b), the same architecture causes a faster induction rate. Fig. 4 Open in new tabDownload slide Dynamics of Pu response to XylR inducers in P. putida Pu·RBX and P. putida Pu·RpoN·Pu·RBX. (a) Pu–luxCDABE activity upon induction of cells with the optimal effector m-xylene. The strains indicated were grown in LB medium and exposed to saturating vapours of m-xylene at t = 0 and then for the period of time indicated. (b) Fold-induction with 3-methylbenzylacohol (suboptimal effector) with respect to non-induced conditions (baseline at t = 0 was 0.66). The same bacteria were grown in the presence of 1 mM 3MBA for the time indicated and luminescence emissions recorded as described in Experimental procedures. Fig. 4 Open in new tabDownload slide Dynamics of Pu response to XylR inducers in P. putida Pu·RBX and P. putida Pu·RpoN·Pu·RBX. (a) Pu–luxCDABE activity upon induction of cells with the optimal effector m-xylene. The strains indicated were grown in LB medium and exposed to saturating vapours of m-xylene at t = 0 and then for the period of time indicated. (b) Fold-induction with 3-methylbenzylacohol (suboptimal effector) with respect to non-induced conditions (baseline at t = 0 was 0.66). The same bacteria were grown in the presence of 1 mM 3MBA for the time indicated and luminescence emissions recorded as described in Experimental procedures. Outlook The work above shows that one can amplify dramatically the net transcriptional activity of the Pu promoter of the TOL plasmid in response to m-xylene by rationally rewiring the connectivity of its key components: the Pu promoter proper, XylR and σ54. This is in contrast to habitual approaches with the same purpose, which typically rely on either generation of mutants in the promoter DNA or in the amino acid sequence of the cognate transcriptional factors. In our case we have re-connected the constituents by means of two intertwined positive FFLs that deliver high amounts of the two limiting proteins in a self-activation fashion. Simultaneous escalation of both the signal-specific (XylR) factor and one or more global regulatory components (the σ54 in our case) are likely to take this promoter to its maximum possible transcriptional activity in vivo. In reality, as the circuit lacks any restraining feedback loop, once the forward cascade of Fig. 2c gets started upon m-xylene induction the engineered regulatory node cannot but amplify itself over time. But eventually, the hyper-activity of this σ54-dependent system is likely to reach its ceiling by competing for the host's gene expression machinery. This may occur by [i] displacing other sigma factors out of the RNAP pool and/or [ii] draining the metabolic currency that fuels the functioning of the synthetic implant. Current efforts try to tackle this problem (named retroactivity) with additional genetic isolation devices24,25 so that the functioning of the genetic constructs has a minimal influence on the physiology and viability of the host. Experimental procedures Strains, culture conditions, and general procedures P. putida strains used in study are derivatives of the reference strain KT2440 inserted with various combinations of the genetic cassettes indicated in each case (Table 1). E. coli CC118λpir was used as the host for propagating plasmids based on a R6K origin of replication.26 Bacteria were grown in Luria-Bertani (LB) medium. When required, the medium was amended with specified concentrations of 3-methylbenzyl alcohol (3MBA) or m-xylene vapour. Antibiotics were used at the following concentrations: piperacillin (Pip) 40 μg ml−1, chloramphenicol (Cm) 30 μg ml−1, gentamycin (Gm) 10 μg ml−1, streptomycin (Sm) 50 μg ml−1, and potassium tellurite (Tel) at 80 μg ml−1. For PCR reactions, 50–100 ng of the DNA template indicated in each case was mixed in a 50 μl mixture with 0.2 μM of each of the primers specified and 2.5 units of Pfu DNA polymerase (Stratagene). Samples were then subjected to 30 cycles of 1 min at 95 °C, 30 s at 58 °C and 1 min at 72 °C. The clones were first checked by colony PCR27 using 1.25 units of Taq DNA polymerase (Roche) and later confirmed by DNA sequencing. Other gene cloning techniques and molecular biology procedures were carried out according to standard methods.27 Table 1 Strains and plasmids Strain or plasmid(s) Relevant characteristics Ref. Strains E. coli CC118 λpir CC118 lysogenized with λpir phage 29 E. coli DH5a Routine cloning host strain 27 P. putida Pu·LUX P. putida strain carrying in the chromosome a Pu–luxCDABE fusion 6 P. putida Pu·RBX P. putida strain carrying in the chromosome a Pu–luxCDABE fusion and xylR under the control of Pu (positive feedback loop) 6 P. putida Pu·RpoN·Pu·RBX P. putida Pu·RBX carrying rpoN under the control of Pu promoter This study Plasmids RK600 CmR; ColE1oriV RK2mob+ tra+ 30 pUT/mini-Tn5 Sm/Sp Mini-Tn5 Sm/Sp delivery plasmid 31 pUC18Not pUC18 with NotI sites flanking the polylinker 29 pPu2 pUC18Not containing the Pu promoter This study pPuMRpoN pUC18Not containing a fusion Pu-rpoN This study pTn5 [Pu·RpoN] Mini-Tn5 delivery vector carrying the Pu promoter controlling rpoN expression This study pPu·RBX pUC18NotI carrying a Pu-xylR fusion 6 pTn7-PuRBX Mini-Tn7 delivery vector carrying a Pu–xylR fusion 6 Strain or plasmid(s) Relevant characteristics Ref. Strains E. coli CC118 λpir CC118 lysogenized with λpir phage 29 E. coli DH5a Routine cloning host strain 27 P. putida Pu·LUX P. putida strain carrying in the chromosome a Pu–luxCDABE fusion 6 P. putida Pu·RBX P. putida strain carrying in the chromosome a Pu–luxCDABE fusion and xylR under the control of Pu (positive feedback loop) 6 P. putida Pu·RpoN·Pu·RBX P. putida Pu·RBX carrying rpoN under the control of Pu promoter This study Plasmids RK600 CmR; ColE1oriV RK2mob+ tra+ 30 pUT/mini-Tn5 Sm/Sp Mini-Tn5 Sm/Sp delivery plasmid 31 pUC18Not pUC18 with NotI sites flanking the polylinker 29 pPu2 pUC18Not containing the Pu promoter This study pPuMRpoN pUC18Not containing a fusion Pu-rpoN This study pTn5 [Pu·RpoN] Mini-Tn5 delivery vector carrying the Pu promoter controlling rpoN expression This study pPu·RBX pUC18NotI carrying a Pu-xylR fusion 6 pTn7-PuRBX Mini-Tn7 delivery vector carrying a Pu–xylR fusion 6 Open in new tab Table 1 Strains and plasmids Strain or plasmid(s) Relevant characteristics Ref. Strains E. coli CC118 λpir CC118 lysogenized with λpir phage 29 E. coli DH5a Routine cloning host strain 27 P. putida Pu·LUX P. putida strain carrying in the chromosome a Pu–luxCDABE fusion 6 P. putida Pu·RBX P. putida strain carrying in the chromosome a Pu–luxCDABE fusion and xylR under the control of Pu (positive feedback loop) 6 P. putida Pu·RpoN·Pu·RBX P. putida Pu·RBX carrying rpoN under the control of Pu promoter This study Plasmids RK600 CmR; ColE1oriV RK2mob+ tra+ 30 pUT/mini-Tn5 Sm/Sp Mini-Tn5 Sm/Sp delivery plasmid 31 pUC18Not pUC18 with NotI sites flanking the polylinker 29 pPu2 pUC18Not containing the Pu promoter This study pPuMRpoN pUC18Not containing a fusion Pu-rpoN This study pTn5 [Pu·RpoN] Mini-Tn5 delivery vector carrying the Pu promoter controlling rpoN expression This study pPu·RBX pUC18NotI carrying a Pu-xylR fusion 6 pTn7-PuRBX Mini-Tn7 delivery vector carrying a Pu–xylR fusion 6 Strain or plasmid(s) Relevant characteristics Ref. Strains E. coli CC118 λpir CC118 lysogenized with λpir phage 29 E. coli DH5a Routine cloning host strain 27 P. putida Pu·LUX P. putida strain carrying in the chromosome a Pu–luxCDABE fusion 6 P. putida Pu·RBX P. putida strain carrying in the chromosome a Pu–luxCDABE fusion and xylR under the control of Pu (positive feedback loop) 6 P. putida Pu·RpoN·Pu·RBX P. putida Pu·RBX carrying rpoN under the control of Pu promoter This study Plasmids RK600 CmR; ColE1oriV RK2mob+ tra+ 30 pUT/mini-Tn5 Sm/Sp Mini-Tn5 Sm/Sp delivery plasmid 31 pUC18Not pUC18 with NotI sites flanking the polylinker 29 pPu2 pUC18Not containing the Pu promoter This study pPuMRpoN pUC18Not containing a fusion Pu-rpoN This study pTn5 [Pu·RpoN] Mini-Tn5 delivery vector carrying the Pu promoter controlling rpoN expression This study pPu·RBX pUC18NotI carrying a Pu-xylR fusion 6 pTn7-PuRBX Mini-Tn7 delivery vector carrying a Pu–xylR fusion 6 Open in new tab Bioluminescence assays P. putida strains were first pre-grown in test tubes overnight in LB medium at 30 °C. Then they were diluted to an OD600 of 0.05 in 100 ml flasks and cultured to an OD600 = 1.0. At that point they were exposed, wherever indicated, to diverse amounts of m-xylene vapour or 1 mM 3MBA. Then, at indicated time points, 200 μl aliquots of the cultures were placed in 96-well plates (NUNC) and light emission and OD600 were measured on a Victor II 1420 Multilabel Counter (Perkin Elmer). The specific bioluminescence values were calculated by dividing the obtained values of total light emission (in arbitrary units) by the ones that reflect the optical density of the culture (OD600). The specific bioluminescence values shown represent the average of at least three biological replicates. Protein techniques Whole-cell protein extracts were prepared by pelleting the cells (10 000 × g, 5 min) from 1 ml of LB culture and re-suspending them in 50 μl of 10 mM Tris HCl, pH 7.5 and then in 50 μl of 2× SDS-sample buffer (120 mM Tris-HCl pH 6.8, 2% w/v SDS, 10% v/v glycerol, 0.01% w/v bromophenol blue, and 2% v/v 2-mercapto-ethanol). After resuspension, samples were boiled for 10 min, sonicated briefly (∼5 s) and centrifuged (14 000 × g, 10 min). Samples with thereby prepared extracts equivalent to ∼108 cells per lane were loaded on SDS-PAGE gels (Miniprotean system, Bio-Rad). Following electrophoresis, gels were transferred to polyvinylidene difluoride membranes using semi-dry electrophoresis transfer apparatus (Bio-Rad). Membranes were next blocked for 2 h at room temperature with MBT buffer (0.1% Tween and 5% skimmed milk in phosphate-buffered saline, PBS). For inmmunodetection of XylR, phage-based antibodies (Phab23) were used following the method described before.6 For immunodetection of σ54, we used the recombinant antibody scFv C222 according to a previously described protocol.4 Mathematical methods The simple models (toy models) presented in this work were made by setting a number of ordinary differential equations describing the TOL control network. Simulations and other calculations were done using MATLAB®. (See ESI† for further details.) Genetic constructs The transposon bearing a Pu–luxCDABE reporter system (which is present in all the strains used in this study) used to engineer P. putida PuLUX has been described before.6 Also the mini-Tn7 derivative bearing a cassette expressing the xylR wild type version under the control of its native Pu promoter and the P. putida strain engineered with it P. putida Pu·RBX (i.e., subject to a XylR self-amplifying loop6) is described in a previous work. The pTn5 [Pu·RpoN] construct for Pu dependent overexpression of the σ54 sigma factor was engineered using pUT mini-Tn5 Sm/Sp26 as the assembly vector as follows: a 238 bp fragment containing the Pu promoter was amplified with the primers Pu8F (XbaI) (gcTCTAGACCCGGGAAAGCGCGATGA) and Pu9R (BamHI) (cgcGGATCCTGAAGGGTCACCACTATTTTT) using the pMAD plasmid as a template.12 This XbaI/BamHI fragment was then cloned into pUC18Not26 rendering pPu2. Then a 1551 bp fragment containing the rpoN gene was obtained by PCR using RpoN 7F (gcGGATCCTTACACTTAGTTAAATTGCTAAC) and RpoN 5R (GgGGTACCCTACATCAGTCGCTTGCGTT) primers and pTn5 [Psal·RpoN] as the template4 and is inserted into pPu2 as the BamHI/KpnI fragment generating pPuRpoN. Finally to construct pTn5 [Pu·RpoN] a NotI fragment containing the Pu-rpoN fusion was excised from pPuRpoN and cloned into pUTmini-Tn5 Sm/Sp. This pTn5 [Pu·RpoN] was then mobilized into the P. putida Pu·RBX, generating P. putida Pu·RpoN·Pu·RBX. Plasmid transfer and mini-transposon delivery into P. putida Plasmids and transposons were conjugally passed from the donor E. coli strain indicated in each case into different P. putida recipients using a filter mating technique.26 To this end, a mixture of the donor, the recipient and the helper strain E. coli HB101 (pRK600) was placed on 0.45 μm filters in a 1 : 1 : 3 ratio and incubated for 8 h at 30 °C on the surface of LB-agar plates. Mini-Tn7 derivatives were co-mobilized along with the transposase-encoding gene tnsABCD into the recipient strains by including E. coli CC118 λpir (pTNS128) in the mating mixture. After incubation, cells were resuspended in 10 mM MgSO4 in either case, and appropriate dilutions plated on M9/succinate amended with suitable antibiotics for counter-selection of the donor and helper strains and growth of the P. putida clones that had acquired the desired plasmids or insertions. Bona fide transposition was verified in each case by checking the sensitivity of individual exconjugants to the delivery vector marker, piperacillin. Acknowledgements This work was partially funded by MINECO grant MTM2014-56948-C2, the ARYSIS and EMPOWERPUTIDA (6336636) contracts of the EU, the ERANET-IB program and the PROMT project of the CAM. The authors declare no conflict of interest. References 1 M. E. Wall , W. S. Hlavacek and M. A. Savageau, Nat. Rev. Genet. , 2004 , 5 , 34 – 42 . Crossref Search ADS PubMed 2 R. Silva-Rocha and V. de Lorenzo, Annu. Rev. Microbiol. , 2010 , 64 , 257 – 275 . Crossref Search ADS PubMed 3 S. A. F. T. Van Hijum , M. H. Medema and O. P. Kuipers, Microbiol. Mol. Biol. Rev. , 2009 , 73 , 481 – 509 . Crossref Search ADS PubMed 4 A. de las Heras , E. Martinez-Garcia, M. R. Domingo-Sananes and V. de Lorenzo, Mol. BioSyst. , 2015 , 11 , 734 – 742 . Crossref Search ADS PubMed 5 D. E. Chang , S. Leung, M. R. Atkinson, A. Reifler, D. Forger and A. J. 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TI - Rationally rewiring the connectivity of the XylR/Pu regulatory node of the m-xylene degradation pathway in Pseudomonas putida
JO - Integrative Biology
DO - 10.1039/c5ib00310e
DA - 2016-04-18
UR - https://www.deepdyve.com/lp/oxford-university-press/rationally-rewiring-the-connectivity-of-the-xylr-pu-regulatory-node-of-cpePzs94Ds
SP - 571
VL - 8
IS - 4
DP - DeepDyve
ER -