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

Allosteric mutants show that PrfA activation is dispensable for vacuole escape but required for efficient spread and <em>Listeria</em> survival <em>in vivo</em>

The transcriptional regulator PrfA controls key virulence determinants of the facultative intracellular pathogen Listeria monocytogenes. PrfA-dependent gene expression is strongly induced within host cells. While the basis of this activation is unknown, the structural homology of PrfA with the cAMP receptor protein (Crp) and the finding of constitutively activated PrfA* mutants suggests it may involve ligand-induced allostery. Here, we report the identification of a solvent-accessible cavity within the PrfA N-terminal domain that may accommodate an activating ligand. The pocket occupies a similar position to the cAMP binding site in Crp but lacks the cyclic nucleotide-anchoring motif and has its entrance on the opposite side of the β-barrel. Site-directed mutations in this pocket impaired intracellular PrfA-dependent gene activation without causing extensive structural/functional alterations to PrfA. Two substitutions, L48F and Y63W, almost completely abolished intracellular virulence gene induction and thus displayed the expected phenotype for allosteric activation-deficient PrfA mutations. Neither PrfA(allo) substitution affected vacuole escape and initial intracellular growth of L. monocytogenes in epithelial cells and macrophages but caused defective cell-to-cell spread and strong attenuation in mice. Our data support the hypothesis that PrfA is allosterically activated during intracellular infection and identify the probable binding site for the effector ligand. They also indicate that PrfA allosteric activation is not required for early intracellular survival but is essential for full Listeria virulence and colonization of host tissues

Comparison of Listeria monocytogenes Exoproteomes from biofilm and planktonic state:Lmo2504, a protein associated with biofilms

The food-borne pathogen Listeria monocytogenes is the causative agent of the severe human and animal disease listeriosis. The persistence of this bacterium in food processing environments is mainly attributed to its ability to form biofilms. The search for proteins associated with biofilm formation is an issue of great interest, with most studies targeting the whole bacterial proteome. Nevertheless, exoproteins constitute an important class of molecules participating in various physiological processes, such as cell signaling, pathogenesis, and matrix remodeling. The aim of this work was to quantify differences in protein abundance between exoproteomes from a biofilm and from the planktonic state. For this, two field strains previously evaluated to be good biofilm producers (3119 and J311) were used, and a procedure for the recovery of biofilm exoproteins was optimized. Proteins were resolved by two-dimensional difference gel electrophoresis and identified by electrospray ionization-tandem mass spectrometry. One of the proteins identified in higher abundance in the biofilm exoproteomes of both strains was the putative cell wall binding protein Lmo2504. A mutant strain with deletion of the gene for Lmo2504 was produced (3119Δlmo2504), and its biofilm-forming ability was compared to that of the wild type using the crystal violet and the ruthenium red assays as well as scanning electron microscopy. The results confirmed the involvement of Lmo2504 in biofilm formation, as strain 3119Δlmo2504 showed a significantly (P < 0.05) lower biofilm-forming ability than the wild type. The identification of additional exoproteins associated with biofilm formation may lead to new strategies for controlling this pathogen in food processing facilities

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

Caracterización de genes de poligalacturonasas de "Fusarium oxysporum" f.sp. "Radicis lycopersici" y su análisis en sistemas heterólogos

El género Fusarium agrupa a numerosas especies de hongos filamentosos siendo, la mayoría, importantes patógenos de plantas. Una de las más importantes es Fusarium oxysporum, especie cosmopolita que parásita alrededor de 100 especies de plantas, muchas de ellas de interés comercial. La primera barrera que tienen que superar los fitopatógenos para completar con éxito el proceso de patogénesis es la pared celular vegetal, estructura compleja de la cual un componente fundamental es la pectina. Los fitopatógenos producen numerosas enzimas que les permiten degradar la pared celular. Entre ellas las enzimas pécticas y en especial las poligalacturonasas (PGs) ha sido las más estudiadas debido a que son las primeras en actuar. El objetivo fundamental de esta tesis es el estudio de genes de PGs del patógeno del tomate Fusarium oxysporum f.sp radicis lycopersici (FORL). Se obtuvieron las secuencias completas de cuatro genes codificadores de PGs, dos con modo de acción exo y dos endo, mediante el escrutinio de una genoteca geonómica y se analizó su estructura. Asimismo, se obtuvieron las secuencias de aminoácidos y las estructuras terciarias teóricas con las cuales se realizó un análisis comparativo. Se establecieron las relaciones filogenéticas de las PGs de FORL con respecto a otras descritas en hongos filamentosos. Se analizó el patrón de expresión de estos genes en cultivos in vitro pudiendo establecer que están sometidos a una regulación transcripcional compleja y que presentan un patrón de regulación diferencial. Posteriormente, realizamos un análisis in silico de las regiones 5́ reguladoras para poder determinar que factores estaban relacionados con la regulación de estos genes, así como, un estudio funcional de las regiones promotoras de los cuatro genes en Saccharomyces cerevisiae. Por último se utilizó Pichia pastoris para expresar las EXOPGs de FORL consiguiendo la expresión heteróloga de una de ellas y su posterior caracterización bioquímica

Edwin: an integrated high-throughput platform for the automated analysis of microbial gene expression at protein and RNA level 

de las Heras, Aitor. (2015). Edwin: an integrated high-throughput platform for the automated analysis of microbial gene expression at protein and RNA level, [dataset]. University of Edinburgh. http://dx.doi.org/10.7488/ds/29

Breaking effector discrimination with a semi-constitutive variant of XylR.

<p>(a) Specific bioluminescence produced by cultures of <i>P. putida</i> BX (encoding wild-type XylR) and <i>P. putida</i> BX17 (encoding XylRv17) after 6 h of incubation in the absence of effectors. (b) Specific bioluminescence produced by cultures of <i>P. putida</i> BX17, <i>P. putida</i> Ps·RBX and <i>P. putida</i> Ps·RBX17 cultures over time without inducers. (c) Same, following addition of 1.0 mM of 3MBA. Note that the insensitivity of <i>P. putida</i> Ps·RBX to the suboptimal inducer is lost in the equivalent construct expressing XylRv17, which recovers a level of <i>Pu</i> output comparable to that of the wild-type.</p

The master control loop (MCL).

<p>The sketch to the left shows a common arrangement of regulatory elements in devices that control expression of pathways for biodegradation and detoxification of environmental pollutants. The motif involves an upstream signal (the effector X) that influences expression of a cognate regulator Y that, in turn, binds the inducer X for acting on a target promoter Z. The motif has 4 transfer functions (a, b, c, d) that can be combined to produce a large number of regulatory possibilities. In this work, we documented that changing the sign of the auto-regulation loop that governs <i>xylR</i> expression from its native negative architecture (middle) to a positive interaction (right) causes the system to discriminate between an optimal and a suboptimal effector of the system.</p

Effector sensitivity of strains expressing <i>xylR</i> through a negative or positive feedback loop.

<p>(a) Specific bioluminescence emitted by <i>P. putida</i> BX and <i>P. putida</i> Pu·RBX following addition of 1.0 mM 3MBA. (b) As in (a), but with <i>P. putida</i> BX and <i>P. putida</i> Pu·RBX cultures 6 hours after adding different concentrations of 3MBA as indicated.</p

Structure of the TOL network of <i>P. putida</i> mt-2.

<p>The TOL pathway encompasses two different operons, the <i>upper</i> operon, (<i>xylUWCMABN</i>), the products of which transform <i>m</i>-xylene into 3-methylbenzoate, and the <i>lower</i> operon (<i>xylXYZLTEGFJQKIH</i>) that produces enzymes for further metabolism of this compound into TCA cycle intermediates. XylR and XylS are the transcriptional regulators that control the expression of either operon. The master regulatory gene <i>xylR</i> is encoded in a location adjacent to the end of the <i>lower</i> operon and is expressed from the <i>Pr</i> promoter. XylR is produced in an inactive form (R) that, in the presence of the pathway substrate (<i>m</i>-xylene) or pathway intermediates, such as 3-methylbenzyl alcohol (3MBA), changes to an active form (Ra). XylRa then activates both <i>Pu</i> and <i>Ps</i>, triggering expression of the <i>upper</i> pathway and XylS, respectively. At the same time, XylRa acts as repressor of its own transcription, thereby decreasing its own expression. In the absence of <i>m</i>-xylene, XylS is produced at low levels and changes from the inactive form (S) to an active state (Sa) by binding 3-methylbenzoate. In turn, XylSa is able to induce expression of the <i>meta</i> pathway by activating the <i>Pm</i> promoter (note that operons and regulatory elements not to scale).</p