SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes.

The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB) and as a physical repository of plasmid vectors for genetic analysis and engineering of Gram-negative bacteria with a structure and nomenclature that follows a strict, fixed architecture of functional DNA segments. While the current update keeps the basic features of earlier versions, the platform has been upgraded not only with many more ready-to-use plasmids but also with features that expand the range of target species, harmonize DNA assembly methods and enable new applications. In particular, SEVA 4.0 includes (i) a sub-collection of plasmids for easing the composition of multiple DNA segments with MoClo/Golden Gate technology, (ii) vectors for Gram-positive bacteria and yeast and [iii] off-the-shelf constructs with built-in functionalities. A growing collection of plasmids that capture part of the standard-but not its entirety-has been compiled also into the DB and repository as a separate corpus (SEVAsib) because of its value as a resource for constructing and deploying phenotypes of interest. Maintenance and curation of the DB were accompanied by dedicated diffusion and communication channels that make the SEVA platform a popular resource for genetic analyses, genome editing and bioengineering of a large number of microorganisms.

Engineering Tropism of Pseudomonas putida toward Target Surfaces through Ectopic Display of Recombinant Nanobodies.

Gram-negative bacteria are endowed with complex outer membrane (OM) structures that allow them to both interact with other organisms and attach to different physical structures. However, the design of reliable bacterial coatings of solid surfaces is still a considerable challenge. In this work, we report that ectopic expression of a fibrinogen-specific nanobody on the envelope of Pseudomonas putida cells enables controllable formation of a bacterial monolayer strongly bound to an antigen-coated support. To this end, either the wild type or a surface-naked derivative of P. putida was engineered to express a hybrid between the ß-barrel of an intimin-type autotransporter inserted in the outer membrane and a nanobody (VHH) moiety that targets fibrinogen as its cognate interaction partner. The functionality of the thereby presented VHH and the strength of the resulting cell attachment to a solid surface covered with the cognate antigen were tested and parametrized with Quartz Crystal Microbalance technology. The results not only demonstrated the value of using bacteria with reduced OM complexity for efficient display of artificial adhesins, but also the potential of this approach to engineer specific bacterial coverings of predetermined target surfaces.

Naked Bacterium: Emerging Properties of a Surfome-Streamlined Pseudomonas putida Strain.

Environmental bacteria are most often endowed with native surface-attachment programs that frequently conflict with efforts to engineer biofilms and synthetic communities with given tridimensional architectures. In this work, we report the editing of the genome of Pseudomonas putida KT2440 for stripping the cells of most outer-facing structures of the bacterial envelope that mediate motion, binding to surfaces, and biofilm formation. To this end, 23 segments of the P. putida chromosome encoding a suite of such functions were deleted, resulting in the surface-naked strain EM371, the physical properties of which changed dramatically in respect to the wild type counterpart. As a consequence, surface-edited P. putida cells were unable to form biofilms on solid supports and, because of the swimming deficiency and other alterations, showed a much faster sedimentation in liquid media. Surface-naked bacteria were then used as carriers of interacting partners (e.g., Jun-Fos domains) ectopically expressed by means of an autotransporter display system on the now easily accessible cell envelope. Abstraction of individual bacteria as adhesin-coated spherocylinders enabled rigorous quantitative description of the multicell interplay brought about by thereby engineered physical interactions. The model was then applied to parametrize the data extracted from automated analysis of confocal microscopy images of the experimentally assembled bacterial flocks for analyzing their structure and distribution. The resulting data not only corroborated the value of P. putida EM371 over the parental strain as a platform for display artificial adhesins but also provided a strategy for rational engineering of catalytic communities.

SEVA 3.0: an update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts.

The Standard European Vector Architecture 3.0 database (SEVA-DB 3.0, http://seva.cnb.csic.es) is the update of the platform launched in 2013 both as a web-based resource and as a material repository of formatted genetic tools (mostly plasmids) for analysis, construction and deployment of complex bacterial phenotypes. The period between the first version of SEVA-DB and the present time has witnessed several technical, computational and conceptual advances in genetic/genomic engineering of prokaryotes that have enabled upgrading of the utilities of the updated database. Novelties include not only a more user-friendly web interface and many more plasmid vectors, but also new links of the plasmids to advanced bioinformatic tools. These provide an intuitive visualization of the constructs at stake and a range of virtual manipulations of DNA segments that were not possible before. Finally, the list of canonical SEVA plasmids is available in machine-readable SBOL (Synthetic Biology Open Language) format. This ensures interoperability with other platforms and affords simulations of their behaviour under different in vivo conditions. We argue that the SEVA-DB will remain a useful resource for extending Synthetic Biology approaches towards non-standard bacterial species as well as genetically programming new prokaryotic chassis for a suite of fundamental and biotechnological endeavours.

Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system.

BACKGROUND: The hemolysin (Hly) secretion system of E. coli allows the one-step translocation of hemolysin A (HlyA) from the bacterial cytoplasm to the extracellular medium, without a periplasmic intermediate. In this work, we investigate whether the Hly secretion system of E. coli is competent to secrete a repertoire of functional single-domain VHH antibodies (nanobodies, Nbs), facilitating direct screening of VHH libraries and the purification of selected Nb from the extracellular medium. RESULTS: We employed a phagemid library of VHHs obtained by immunization of a dromedary with three protein antigens from enterohemorrhagic E. coli (EHEC), namely, the extracellular secreted protein A (EspA), the extracellular C-terminal region of Intimin (Int280), and the translocated intimin receptor middle domain (TirM). VHH clones binding each antigen were enriched and amplified by biopanning, and subsequently fused to the C-terminal secretion signal of HlyA to be expressed and secreted in a E. coli strain carrying the Hly export machinery (HlyB, HlyD and TolC). Individual E. coli clones were grown and induced in 96-well microtiter plates, and the supernatants of the producing cultures directly used in ELISA for detection of Nbs binding EspA, Int280 and TirM. A set of Nb sequences specifically binding each of these antigens were identified, indicating that the Hly system is able to secrete a diversity of functional Nbs. We performed thiol alkylation assays demonstrating that Nbs are correctly oxidized upon secretion, forming disulphide bonds between cysteine pairs despite the absence of a periplasmic intermediate. In addition, we show that the secreted Nb-HlyA fusions can be directly purified from the supernatant of E. coli cultures, avoiding cell lysis and in a single affinity chromatography step. CONCLUSIONS: Our data demonstrate the Hly secretion system of E. coli can be used as an expression platform for screening and purification of Nb binders from VHH repertories.

Rationally rewiring the connectivity of the XylR/Pu regulatory node of the m-xylene degradation pathway in Pseudomonas putida.

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 σ(54)-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 σ(54) 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 σ(54) 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 σ(54) 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.

Phenotypic knockouts of selected metabolic pathways by targeting enzymes with camel-derived nanobodies (V(HH)s).

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.

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.

Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two ß-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 ß-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 ß-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirM(EHEC)). 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 ß-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirM(EHEC) binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin ß-domain. The specificity of the selected clones against TirM(EHEC) 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.

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, VHH s 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 VHH s revealed the presence of this enzyme in the metaproteome of an oil refinery waste treatment plant.

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.

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 σ(54)-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 P(C) 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.

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.

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 alpha-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 V(HH)-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 V(HH) moiety, thus becoming an excellent reporter of correct protein folding and dimerization. Both V(HH)-EHlyA and V(HH)-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 V(HH)-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.