Sofía Fraile

SEVA 2.0: an update of the Standard European Vector Architecture for de-/re-construction of bacterial functionalities

The Standard European Vector Architecture 2.0 database (SEVA-DB 2.0, http://seva.cnb.csic.es) is an improved and expanded version of the platform released in 2013 (doi: 10.1093/nar/gks1119) aimed at assisting the choice of optimal genetic tools for de-constructing and re-constructing complex prokaryotic phenotypes. By adopting simple compositional rules, the SEVA standard facilitates combinations of functional DNA segments that ease both the analysis and the engineering of diverse Gram-negative bacteria for fundamental or biotechnological purposes. The large number of users of the SEVA-DB during its first two years of existence has resulted in a valuable feedback that we have exploited for fixing DNA sequence errors, improving the nomenclature of the SEVA plasmids, expanding the vector collection, adding new features to the web interface and encouraging contributions of materials from the community of users. The SEVA platform is also adopting the Synthetic Biology Open Language (SBOL) for electronic-like description of the constructs available in the collection and their interfacing with genetic devices developed by other Synthetic Biology communities. We advocate the SEVA format as one interim asset for the ongoing transition of genetic design of microorganisms from being a trial-and-error endeavor to become an authentic engineering discipline.

Monitoring Intracellular Levels of XylR in Pseudomonas putida with a Single-Chain Antibody Specific for Aromatic-Responsive Enhancer-Binding Proteins

We have isolated a recombinant phage antibody (Phab) that binds a distinct epitope of the subclass of the sigma(54)-dependent prokaryotic enhancer-binding proteins that respond directly to aromatic effectors, e.g., those that activate biodegradative operons of Pseudomonas spp. The DNA segments encoding the variable (V) domains of the immunoglobulins expressed by mice immunized with the C-terminal half of TouR (TouRDeltaA) of Pseudomonas stutzeri OX1 were amplified and rearranged in vitro as single-chain Fv (scFv) genes. An scFv library was thereby constructed, expressed in an M13 display system, and subjected to a panning procedure with TouR. One clone (named B7) was selected with high affinity for TouR and XylR (the regulator of the upper TOL operon of the pWW0 plasmid). The epitope recognized by this Phab was mapped to the peptide TPRAQATLLRVL, which seems to be characteristic of the group of enhancer-binding proteins to which TouR and XylR belong and which is located adjacent to the Walker B motif of the proteins. The Phab B7 was instrumental in measuring directly the intracellular levels of XylR expressed from its natural promoter in monocopy gene dosage in Pseudomonas putida under various conditions. Growth stage, the physical form of the protein produced (XylR or XylRDeltaA), and the presence or absence of aromatic inducers in the medium influenced the intracellular pool of these molecules. XylR oscillated from a minimum of approximately 30 molecules (monomers) per cell during exponential phase to approximately140 molecules per cell at stationary phase. Activation of XylR by aromatic inducers decreased the intracellular concentration of the regulator. The levels of the constitutively active variant of XylR named XylRDeltaA were higher, fluctuating between approximately 90 and approximately 570 molecules per cell, depending on the growth stage. These results are compatible with the present model of transcriptional autoregulation of XylR and suggest the existence of mechanisms controlling the stability of XylR protein in vivo.

Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies

Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirMEHEC). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirMEHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirMEHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions.

Secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coli.

The tolerance of the haemolysin transport system (Hly) for exporting dimeric protein substrates to the supernatants of Escherichia coli cultures was examined. A strong dimerization domain (i.e. an amphipathic α‐helix capable of forming a leucine zipper in the yeast transcription factor GCN4) was inserted into an epitope‐tagged version of the 23 kDa C‐terminal secretion signal of haemolysin (EHlyA). The zipper‐containing polypeptide (ZEHlyA) was effectively secreted by E. coli cells carrying the HlyBD transporter and accumulated in the culture media as a stable dimer as determined by gel filtration chromatography. In vivo protein cross‐linking experiments and coexpression with a secretion‐deficient derivative of ZEHlyA indicated that leucine zipper‐dependent dimerization occurs following secretion. To test whether dimerization allows the correct folding of the secreted polypeptide, immunoglobulin VHH‐domains obtained from camel antibodies were fused to EHlyA and ZEHlyA. Functional dimerization of the ZEHlyA hybrid was anticipated to increase the apparent binding affinity (i.e. avidity) of the VHH moiety, thus becoming an excellent reporter of correct protein folding and dimerization. Both VHH‐EHlyA and VHH‐ZEHlyA hybrids were quantitatively secreted and found in the extracellular medium as active monomers and dimers respectively. When compared with their monomeric counterparts, the dimeric VHH‐ZEHlyA molecules showed superior binding properties to their cognate antigen, with a 10‐fold increase in their avidity. These data reveal a non‐anticipated permissiveness of the Hly type I transport machinery for the secretion of substrates with dimerization capacity.

Increasing Signal Specificity of the TOL Network of Pseudomonas putida mt-2 by Rewiring the Connectivity of the Master Regulator XylR

Prokaryotic transcription factors (TFs) that bind small xenobiotic molecules (e.g., TFs that drive genes that respond to environmental pollutants) often display a promiscuous effector profile for analogs of the bona fide chemical signals. XylR, the master TF for expression of the m-xylene biodegradation operons encoded in the TOL plasmid pWW0 of Pseudomonas putida, responds not only to the aromatic compound but also, albeit to a lesser extent, to many other aromatic compounds, such as 3-methylbenzylalcohol (3MBA). We have examined whether such a relaxed regulatory scenario can be reshaped into a high-capacity/high-specificity regime by changing the connectivity of this effector-sensing TF within the rest of the circuit rather than modifying XylR structure itself. To this end, the natural negative feedback loop that operates on xylR transcription was modified with a translational attenuator that brings down the response to 3MBA while maintaining the transcriptional output induced by m-xylene (as measured with a luxCDABE reporter system). XylR expression was then subject to a positive feedback loop in which the TF was transcribed from its own target promoters, each known to hold different input/output transfer functions. In the first case (xylR under the strong promoter of the upper TOL operon, Pu), the reporter system displayed an increased transcriptional capacity in the resulting network for both the optimal and the suboptimal XylR effectors. In contrast, when xylR was expressed under the weaker Ps promoter, the resulting circuit unmistakably discriminated m-xylene from 3MBA. The non-natural connectivity engineered in the network resulted both in a higher promoter activity and also in a much-increased signal-to-background ratio. These results indicate that the working regimes of given genetic circuits can be dramatically altered through simple changes in the way upstream transcription factors are self-regulated by positive or negative feedback loops.

Monitoring biodegradative enzymes with nanobodies raised in Camelus dromedarius with mixtures of catabolic proteins.

Functional studies of biodegradative activities in environmental microorganisms require molecular tools for monitoring catabolic enzymes in the members of the native microbiota. To this end, we have generated repertories of single-domain V(HH) fragments of camel immunoglobulins (nanobodies) able to interact with multiple proteins that are descriptors of environmentally relevant processes. For this, we immunized Camelus dromedarius with a cocktail of up to 12 purified enzymes that are representative of major types of detoxifying activities found in aerobic and anaerobic microorganisms. Following the capture of the antigen-binding modules from the mRNA of the camel lymphocytes and the selection of sub-libraries for each of the enzymes in a phage display system we found a large number of V(HH) modules that interacted with each of the antigens. Those associated to the enzyme 2,3 dihydroxybiphenyl dioxygenase of Burkholderia xenovorans LB400 (BphC) and the arsenate reductase of Staphylococcus aureus (ArsC) were examined in detail and found to hold different qualities that were optimal for distinct protein recognition procedures. The repertory of anti-BphC V(HH) s included variants with a strong affinity and specificity for linear epitopes of the enzyme. When the anti-BphC V(HH) library was recloned in a prokaryotic intracellular expression system, some nanobodies were found to inhibit the dioxygenase activity in vivo. Furthermore, anti-ArsC V(HH) s were able to discriminate between proteins stemming from different enzyme families. The easiness of generating large collections of binders with different properties widens considerably the molecular toolbox for analysis of biodegradative bacteria and opens fresh possibilities of monitoring protein markers and activities in the environment.

Characterisation of the putative effector interaction site of the regulatory HbpR protein from Pseudomonas azelaica by site-directed mutagenesis.

Bacterial transcription activators of the XylR/DmpR subfamily exert their expression control via σ54-dependent RNA polymerase upon stimulation by a chemical effector, typically an aromatic compound. Where the chemical effector interacts with the transcription regulator protein to achieve activation is still largely unknown. Here we focus on the HbpR protein from Pseudomonas azelaica, which is a member of the XylR/DmpR subfamily and responds to biaromatic effectors such as 2-hydroxybiphenyl. We use protein structure modeling to predict folding of the effector recognition domain of HbpR and molecular docking to identify the region where 2-hydroxybiphenyl may interact with HbpR. A large number of site-directed HbpR mutants of residues in- and outside the predicted interaction area was created and their potential to induce reporter gene expression in Escherichia coli from the cognate PC promoter upon activation with 2-hydroxybiphenyl was studied. Mutant proteins were purified to study their conformation. Critical residues for effector stimulation indeed grouped near the predicted area, some of which are conserved among XylR/DmpR subfamily members in spite of displaying different effector specificities. This suggests that they are important for the process of effector activation, but not necessarily for effector specificity recognition.

NanoPad: An integrated platform for bacterial production of camel nanobodies aimed at detecting environmental biomarkers

The presence of given antigens in environmental samples (e.g. biodegradative enzymes) reports the quality and catalytic vigor of particular soils or aquatic ecosystems. In this context, we have developed the NanoPad system consisting of a complete platform for isolation, amplification, and extracellular production of specific antibodies against antigens that diagnose the occurrence of protein markers in crude environmental samples. The workflow starts with the inoculation of camels (Camelus dromedarius) with various proteins (e.g. catabolic enzymes) for generating a phage display library of variable heavy‐chain antibody H fragment (VHH) domains that bind the different antigens. Instead of being subjected to a conventional panning, such a library is then probed with a Western‐panning technique that allows direct isolation of specific binders of proteins blotted on membranes from polyacrylamide gels. Finally, VHHs are fused to the C‐domain of hemolysin for secretion to the culture media as virtually pure dimeric proteins that can be used as a primary antibody without further processing. The value of NanoPad is shown with the selection of nanobodies for detection of biphenyl 2,3‐dioxygenase, a key enzyme for biodegradation of polychlorinated biphenyls. The thereby generated anti‐biphenyl 2,3‐dioxygenase VHHs revealed the presence of this enzyme in the metaproteome of an oil refinery waste treatment plant.

Phenotypic knockouts of selected metabolic pathways by targeting enzymes with camel-derived nanobodies (VHHs)

Surveying the dynamics of metabolic networks of Gram-negative bacteria often requires the conditional shutdown of enzymatic activities once the corresponding proteins have been produced. We show that given biochemical functions can be entirely suppressed in vivo with camel antibodies (VHHs, nanobodies) that target active sites of cognate enzymes expressed in the cytoplasm. As a proof of principle, we raised VHHs against 2,5-dihydroxypyridine dioxygenase (NicX) of Pseudomonas putida, involved in nicotinic acid metabolism. Once fused to a thioredoxin domain, the corresponding nanobodies inhibited the enzyme both in Escherichia coli and in P. putida cells, which then accumulated the metabolic substrate of NicX. VHHs were further engineered to track the antigen in vivo by C-terminal fusion to a fluorescent protein. Conditional expression of the resulting VHHs allows simultaneously to track and target proteins of interest and enables the design of transient phenotypes without mutating the genetic complement of the bacteria under study.