Aitor de las Heras

The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes

The Standard European Vector Architecture database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.

Regulation of Listeria virulence: PrfA master and commander

Listeria monocytogenes is the causative agent of listeriosis, a severe foodborne infection. These bacteria live as soil saprotrophs on decaying plant matter but also as intracellular parasites, using the cell cytosol as a replication niche. PrfA, a regulatory protein, integrates a number of environmental cues that signal the transition between these two contrasting lifestyles, activating a set of key virulence factors during host infection. While a number of details concerning the general mode of action of this virulence master switch have been elucidated, others remain unsolved. Recent work has revealed additional mechanisms that contribute to L. monocytogenes virulence modulation, often via cross-talk with PrfA, or by regulating new genes involved in host colonization.

Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes

Although different biological approaches for detection of anti‐personnel mines and other unexploded ordnance (UXO) have been entertained, none of them has been rigorously documented thus far in the scientific literature. The industrial 2,4,6 trinitrotoluene (TNT) habitually employed in the manufacturing of mines is at all times tainted with a small but significant proportion of the more volatile 2,4 dinitrotoluene (2,4 DNT) and other nitroaromatic compounds. By using mutation‐prone PCR and DNA sequence shuffling we have evolved in vitro and selected in vivo variants of the effector recognition domain of the toluene‐responsive XylR regulator of the soil bacterium Pseudomonas putida that responds to mono‐, bi‐ and trinitro substituted toluenes. Re‐introduction of such variants in P. putida settled the transcriptional activity of the cognate promoters (Po and Pu) as a function of the presence of nitrotoluenes in the medium. When strains bearing transcriptional fusions to reporters with an optical output (luxAB, GFP) were spread on soil spotted with nitrotoluenes, the signal triggered by promoter activation allowed localization of the target compounds on the soil surface. Our data provide a proof of concept that non‐natural transcription factors evolved to respond to nitroaromatics can be engineered in soil bacteria and inoculated on a target site to pinpoint the presence of explosives. This approach thus opens new ways to tackle this gigantic humanitarian problem.

Stable implantation of orthogonal sensor circuits in Gram‐negative bacteria for environmental release

A broad host range, orthogonal genetic platform has been developed to format sensor circuits in the chromosome of Gram-negative microorganisms destined for environmental release as bioindicators of toxic or perilous compounds (e.g. explosives) in soil. The genetic scheme includes the generation of a genomic landing pad for the sensor module with a Tn5-mini-transposon bearing an optimal attTn7 sequence and a choice of reporter systems with optical and enzymatic outputs. The array of functional elements thereby inserted in the chromosome match that of a cognate plasmid vector which delivers the transcription factors and the promoters to a frame that places the regulatory parts in front of the reporters. Site-specific recombination sites allow the deletion of antibiotic resistances and enables reporter output prior to deliberate release. The system thus allows the production and maintenance of cells in a pre-release state and its intentional conversion in deliverable strains that fulfil all safety, stability and performance criteria. The combination of such a genetic platform with a variant of the transcriptional regulator XylR of Pseudomonas putida that responds to 2,4-dinitrotoluene has been the basis for the production of strains that emit light upon exposure to residues of explosives in a soil microcosm.

Engineering input/output nodes in prokaryotic regulatory circuits

A large number of prokaryotic regulatory elements have been interfaced artificially with biological circuits that execute specific expression programs. Engineering such circuits involves the association of input/output components that perform discrete signal-transfer steps in an autonomous fashion while connected to the rest of the network with a defined topology. Each of these nodes includes a signal-recognition component for the detection of the relevant physicochemical or biological stimulus, a molecular device able to translate the signal-sensing event into a defined output and a genetic module capable of understanding such an output as an input for the next component of the circuit. The final outcome of the process can be recorded by means of a reporter product. This review addresses three such aspects of forward engineering of signal-responding genetic parts. We first recap natural and non-natural regulatory assets for designing gene expression in response to predetermined signals - chemical or otherwise. These include transcriptional regulators developed by in vitro evolution (or designed from scratch), and synthetic riboswitches derived from in vitro selection of aptamers. Then we examine recent progress on reporter genes, whose expression allows the quantification and parametrization of signal-responding circuits in their entirety. Finally, we critically examine recent work on other reporters that confer bacteria with gross organoleptic properties (e.g. distinct odour) and the interfacing of signal-sensing devices with determinants of community behaviour.

PrfA regulation offsets the cost of Listeria virulence outside the host.

Summary Virulence traits are essential for pathogen fitness, but whether they affect microbial performance in the environment, where they are not needed, remains experimentally unconfirmed. We investigated this question with the facultative pathogen L isteria monocytogenes and its PrfA virulence regulon. PrfA‐regulated genes are activated intracellularly (PrfA ‘ON’) but shut down outside the host (PrfA ‘OFF’). Using a mutant PrfA regulator locked ON (PrfA*) and thus causing PrfA‐controlled genes to be constitutively activated, we show that virulence gene expression significantly impairs the listerial growth rate (μ) and maximum growth (A) in rich medium. Deletion analysis of the PrfA regulon and complementation of a L. monocytogenes mutant lacking all PrfA‐regulated genes with PrfA* indicated that the growth reduction was specifically due to the unneeded virulence determinants and not to pleiotropic regulatory effects of PrfA ON. No PrfA*‐associated fitness disadvantage was observed in infected eukaryotic cells, where PrfA‐regulated virulence gene expression is critical for survival. Microcosm experiments demonstrated that the constitutively virulent state strongly impaired L .u2009monocytogenes performance in soil, the natural habitat of these bacteria. Our findings provide empirical proof that virulence carries a significant cost to the pathogen. They also experimentally substantiate the assumed, although not proven, key role of virulence gene regulation systems in suppressing the cost of bacterial virulence outside the host.

Association of dnt genes of Burkholderia sp. DNT with the substrate-blind regulator DntR draws the evolutionary itinerary of 2,4-dinitrotoluene biodegradation

The regulation of the DNT pathway for biodegradation of 2,4‐dinitrotoluene of Burkholderia sp. DNT has been examined by exporting each of its components to Pseudomonas putida KT2440. The cognate regulator DntR does not respond to the pathway substrate, but to the non‐substrate salicylate. In order to examine whether such a response to an unrelated inducer was specific or rather a vestige of a previous evolutionary stage, the complete dnt complement or parts of it were expressed functionally for accumulation of various metabolic intermediates. Their effect on expression of dnt genes was then followed both biochemically and by means of a luminescent reporter engineered in the surrogate host. DntR was not only unresponsive to DNT biodegradation products, but it also failed to influence expression of dnt genes at all. Comparison of the dntR/dntA divergent promoter region with similar ones found in various catabolic systems indicated that the leading segment of the DNT biodegradation pathway evolved from a matching portion of naphthalene biodegradation routes existing in other bacteria. That a useless but still active transcriptional factor occurs along enzymes that have already evolved a new substrate specificity suggests that emergence of novel catalytic abilities precedes their submission to cognate regulatory devices, not vice versa.

Cooperative amino acid changes shift the response of the σ54-dependent regulator XylR from natural m-xylene towards xenobiotic 2,4-dinitrotoluene

XylR is a σ54‐dependent transcriptional factor of Pseudomonas putida that activates the Pu promoter of the TOL plasmid upon binding its natural effector, m‐xylene. The search for mutants of the signal‐sensing module of XylR that respond to the xenobiotic compound 2,4‐dinitrotoluene recurrently yields protein variants with a broad effector range. These mutants had amino acid changes not only in the effector recognition moiety (A module), but also in the inter‐domain B linker of the protein. A random mutagenesis and selection/counterselection setup was adopted to optimize the 2,4‐DNT reaction of XylRv17, one of the best 2,4‐DNT responders and thus recreate how this regulator can adjust its specificity to novel effectors by individual changes on the evolving protein. Site‐specific mutagenesis was then used to decipher the contribution of individual mutations in XylRv17 and in one of the mutants evolved from it (XylR28) to the 2,4‐DNT response. This approach allowed us to capture a new XylR version with novel mutations that fixed the protein in an intermediate stage of the progress from an effector‐promiscuous, pluri‐potent protein type to a more specific form where the natural response to m‐xylene was decreased and the non‐native acquired response to 2,4‐DNT was increased.

In situ detection of aromatic compounds with biosensor Pseudomonas putida cells preserved and delivered to soil in water-soluble gelatin capsules

While many types of bacteria have been engineered to produce an optical output in response to given analytes in a culture, their use for extensive, in situ monitoring of distinct chemical species in soil is hampered by a dearth of practicable spreading schemes. In this work, we report and validate a comprehensive system for the long-term preservation of Pseudomonas putida cells genetically designed for biosensing benzene, toluene, ethylbenzene, and xylenes (BTEX) in soil, along with a procedure to formulate, spread, and vigorously activate such bacteria at the desired site and occasion. To this end, various known lyoprotectants were tested for promoting the long-term maintenance of biosensor cells with quite variable outcomes. While a formulation of inositol and maltodextrines was optimal for preservation of freeze-dried BTEX-sensing bacteria, adsorption of P. putida cells to corncob powder (an abundant residue of the corn industry) endowed the resulting material with a lasting viability at ambient conditions. In any case, the thereby preserved bacterial biomass acquired physical and mechanical properties adequate for formulating the biosensor agent in water-soluble but otherwise hard dry gelatine capsules with a long shelf life. When such capsules were spread in a soil microcosm and subsequently liquefied with water or high humidity, the released microorganisms formed spots that gave an intense luminiscent signal upon exposure to effectors of the sensor circuit implanted in the chromosome of the P. putida strain. We argue that the procedures described here can facilitate implementation of wide-area biological detection strategies for revealing the location of toxic or perilous chemicals.

Engineering Whole-Cell Biosensors with No Antibiotic Markers for Monitoring Aromatic Compounds in the Environment

A cornerstone of Synthetic Biology is the engineering of gene regulatory networks. Construction of such biological circuits has been used not only to elucidate the dynamics of gene expression but also for designing whole-cell biosensors that translate environmental signals into quantifiable outputs. To this end, distinct components of given regulatory systems are rationally rewired in a way that translates an external stimulus (for instance, the presence of one chemical species) into a measurable readout typically fluorescence or luminescence. Various biosensors for BTEX (a mixture of benzene, toluene, ethylbenzene and xylenes) are based on XylR, the main transcriptional regulator of the TOL pathway of Pseudomonas putida mt-2. In the presence of its natural effectors (e.g., m-xylene, toluene or 3-methylbenzylalcohol), XylR triggers expression of the upper pathway genes by means of the Pu promoter. Available biosensors combine the xylR gene and a direct fusion between the cognate Pu promoter and the luxCDABE operon, all components stably integrated in the chromosome of P. putida. A versatile development of the same biosensing concept is described, aimed at increasing the sensitivity of the genetic circuit toward XylR inducers. The new platform utilizes mini-transposon vectors tailored for engineering an artificial expression cascade that operates as an amplifier of the signal/response ratio of the biosensor. This strategy was applied to the construction of a strain that carries a transcriptional fusion between the Pu promoter and T7 RNA polymerase (which becomes under the control of XylR and its effectors), along with a T7 promoter controlling expression of the luxCDABE operon. This simple regulatory architecture produced a dramatic increase of bioluminescence emission in respect to the strain that carries only the direct fusion between the Pu promoter and the luxCDABE reporter.A cornerstone of Synthetic Biology is the engineering of gene regulatory networks. Construction of such biological circuits has been used not only to elucidate the dynamics of gene expression but also for designing whole-cell biosensors that translate environmental signals into quantifiable outputs. To this end, distinct components of given regulatory systems are rationally rewired in a way that translates an external stimulus (for instance, the presence of one chemical species) into a measurable readout typically fluorescence or luminescence. Various biosensors for BTEX (a mixture of benzene, toluene, ethylbenzene and xylenes) are based on XylR, the main transcriptional regulator of the TOL pathway of Pseudomonas putida mt-2. In the presence of its natural effectors (e.g., m-xylene, toluene or 3-methylbenzylalcohol), XylR triggers expression of the upper pathway genes by means of the Pu promoter. Available biosensors combine the xylR gene and a direct fusion between the cognate Pu promoter and the luxCDABE operon, all components stably integrated in the chromosome of P. putida. A versatile development of the same biosensing concept is described, aimed at increasing the sensitivity of the genetic circuit toward XylR inducers. The new platform utilizes mini-transposon vectors tailored for engineering an artificial expression cascade that operates as an amplifier of the signal/response ratio of the biosensor. This strategy was applied to the construction of a strain that carries a transcriptional fusion between the Pu promoter and T7 RNA polymerase (which becomes under the control of XylR and its effectors), along with a T7 promoter controlling expression of the luxCDABE operon. This simple regulatory architecture produced a dramatic increase of bioluminescence emission in respect to the strain that carries only the direct fusion between the Pu promoter and the luxCDABE reporter.