Rocío Lopez-Igual, Joaquín Bernal-Bayard, Alfonso Rodríguez-Patón, Jean-Marc Ghigo and Didier Mazel, Engineered toxin-intein antimicrobials can selectively target and kill antibiotic-resistant bacteria in mixed populations, Nature Biotechnology, April 2019.
https://doi.org/10.1038/s41587-019-0105-3
https://doi.org/10.1038/s41587-019-0105-3
Marcos Rodríguez Regueira, Jesús Daza García & Alfonso Rodríguez-Patón. The multicellular incoherent feedforward loop motif generates spatial patterns. BioRxiv, 2019: 579342.
Paula Gregorio-Godoy, Guillermo Perez del Pulgar, Marcos Rodriguez-Regueira, Alfonso Rodríguez-Patón. Deriving general conditions and mechanisms for division of labor using the cell-based simulator gro. bioRxiv, 2018: 363093.
Martín Eduardo Gutiérrez, Paula Gregorio-Godoy, Guillermo Pérez del Pulgar, Luis Enrique Muñoz, Sandra Sáez, Alfonso Rodríguez-Patón.
A new improved and extended version of the multicell bacterial simulator gro. Biorxiv (Preprint). 2017 January 2 DOI: 10.1101/097444
Abstract
gro is a cell programming language developed in Klavins Lab for simulating colony growth and cell-cell communication. It is used as a synthetic biology prototyping tool for simulating multicellular biocircuits. In this work, we present several extensions made to gro that improve the performance of the simulator, make it easier to use and provide new functionalities. The new version of gro is between one and two orders of magnitude faster than the original version. It is able to grow microbial colonies with up to 105 cells in less than 20 minutes. A new library, CellEngine, accelerates the resolution of spatial physical interactions between growing and dividing cells by implementing a new shoving algorithm. A genetic library, CellPro, based on Probabilistic Timed Automata, simulates gene expression dynamics using simplified and easy to compute digital proteins. We also propose a more convenient language specification layer, ProSpec, based on the idea that proteins drive cell behavior. CellNutrient, another library, implements Monod-based growth and nutrient uptake functionalities. The intercellular signaling management was improved and extended in a library called CellSignals. Finally, bacterial conjugation, another local cell-cell communication process, was added to the simulator. To show the versatility and potential outreach of this version of gro, we provide studies and novel examples ranging from synthetic biology to evolutionary microbiology. We believe that the upgrades implemented for gro have made it into a powerful and fast prototyping tool capable of simulating a large variety of systems and synthetic biology designs.
A new improved and extended version of the multicell bacterial simulator gro. Biorxiv (Preprint). 2017 January 2 DOI: 10.1101/097444
Abstract
gro is a cell programming language developed in Klavins Lab for simulating colony growth and cell-cell communication. It is used as a synthetic biology prototyping tool for simulating multicellular biocircuits. In this work, we present several extensions made to gro that improve the performance of the simulator, make it easier to use and provide new functionalities. The new version of gro is between one and two orders of magnitude faster than the original version. It is able to grow microbial colonies with up to 105 cells in less than 20 minutes. A new library, CellEngine, accelerates the resolution of spatial physical interactions between growing and dividing cells by implementing a new shoving algorithm. A genetic library, CellPro, based on Probabilistic Timed Automata, simulates gene expression dynamics using simplified and easy to compute digital proteins. We also propose a more convenient language specification layer, ProSpec, based on the idea that proteins drive cell behavior. CellNutrient, another library, implements Monod-based growth and nutrient uptake functionalities. The intercellular signaling management was improved and extended in a library called CellSignals. Finally, bacterial conjugation, another local cell-cell communication process, was added to the simulator. To show the versatility and potential outreach of this version of gro, we provide studies and novel examples ranging from synthetic biology to evolutionary microbiology. We believe that the upgrades implemented for gro have made it into a powerful and fast prototyping tool capable of simulating a large variety of systems and synthetic biology designs.
Andrei Paun, Alfonso Rodríguez-Patón. P Systems Simulating Bacterial Conjugation: Universality and Properties. Fundamenta Informaticae 7 June 2017 DOI: 10.3233/FI-2017-1532.
Abstract
We refine the modeling in the P systems area of the way bacteria transmit genetic information in bacterial colonies, specifically the conjugation process. We study this new model from the computational power perspective using methods and ideas in the area; we are able to prove the universality of these systems. We show that systems working in a homogeneous manner and using only 75 species of objects in the regions and 13 species of “on-membrane” objects are enough for reaching universality. The system starts in a initial state with only few (nine) bacteria needed and the “bacteria” from this system are homogeneous, all have the same rules.
Abstract
We refine the modeling in the P systems area of the way bacteria transmit genetic information in bacterial colonies, specifically the conjugation process. We study this new model from the computational power perspective using methods and ideas in the area; we are able to prove the universality of these systems. We show that systems working in a homogeneous manner and using only 75 species of objects in the regions and 13 species of “on-membrane” objects are enough for reaching universality. The system starts in a initial state with only few (nine) bacteria needed and the “bacteria” from this system are homogeneous, all have the same rules.
Artificial symmetry-breaking for morphogenetic engineering bacterial colonies
ACS Synthetic Biology, 6:256–265 (2017) DOI: 10.1021/acssynbio.6b00149
Isaac N. Nuñez, Tamara F. Matute, Ilenne Del Valle, Anton Kan, Atri Choksi, Drew Endy, Jim Haseloff, Timothy Rudge, and Fernan Federici
Abstract
Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies. The former represents an artificial patterning mechanism based on plasmid segregation while the latter plays the role of artificial cell differentiation by spatial co-localization of ubiquitous and segregated components. This separation of patterning from actuation facilitates the design-build-test-improve engineering cycle. We created computational modules for CellModeller representing these basic functions and used it to guide the design process and explore the design space in silico. We applied these tools to encode spatially structured functions such as metabolic complementation, RNAPT7 gene expression and CRISPRi/Cas9 regulation. Finally, as a proof of concept, we used CRISPRi/Cas technology to regulate cell growth by controlling methionine synthesis. These mechanisms start from single cells enabling the study of morphogenetic principles and the engineering of novel population scale structures from the bottom up.
ACS Synthetic Biology, 6:256–265 (2017) DOI: 10.1021/acssynbio.6b00149
Isaac N. Nuñez, Tamara F. Matute, Ilenne Del Valle, Anton Kan, Atri Choksi, Drew Endy, Jim Haseloff, Timothy Rudge, and Fernan Federici
Abstract
Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems. Here, we describe the engineering of two mechanisms, symmetry-breaking and domain-specific cell regulation, as elementary functions for the prototyping of morphogenetic instructions in bacterial colonies. The former represents an artificial patterning mechanism based on plasmid segregation while the latter plays the role of artificial cell differentiation by spatial co-localization of ubiquitous and segregated components. This separation of patterning from actuation facilitates the design-build-test-improve engineering cycle. We created computational modules for CellModeller representing these basic functions and used it to guide the design process and explore the design space in silico. We applied these tools to encode spatially structured functions such as metabolic complementation, RNAPT7 gene expression and CRISPRi/Cas9 regulation. Finally, as a proof of concept, we used CRISPRi/Cas technology to regulate cell growth by controlling methionine synthesis. These mechanisms start from single cells enabling the study of morphogenetic principles and the engineering of novel population scale structures from the bottom up.
Concerted action of NIC relaxase and auxiliary protein MobC in RA3 plasmid conjugation
Molecular Microbiology, 2 June 2016, DOI: 10.1111/mmi.13401
Jolanta Godziszewska, Gabriel Moncalián, Matilde Cabezas, Aneta A. Bartosik, Fernando de la Cruz, Grazyna Jagura-Burdzy
Abstract
Conjugative transfer of the broad-host-range RA3 plasmid, the archetype of the IncU group, relies on the relaxase NIC that belongs to the as yet uncharacterized MOBP4 subfamily. NIC contains the signature motifs of HUH relaxases involved in Tyr nucleophilic attack. However, it differs in the residue involved in His activation for cation coordination and was shown here to have altered divalent cation requirements. NIC is encoded in the mobC-nic operon preceded directly by oriT, where mobC encodes an auxiliary transfer protein with a dual function: autorepressor and stimulator of conjugative transfer. Here an interplay between MobC and NIC was demonstrated. MobC is required for efficient NIC cleavage of oriT in supercoiled DNA whereas NIC assists MobC in repression of the mobC-nic operon. A 7-bp arm of IR3 (IR3a) was identified as the binding site for NIC and the crucial nucleotides in IR3a for NIC recognition were defined. Fully active oriTRA3was delineated to a 47-bp DNA segment encompassing a conserved cleavage site sequence, the NIC binding site IR3a and the MobC binding site OM. This highly efficient RA3 conjugative system with defined requirements for minimal oriT could find ample applications in biotechnology and computational biology where simple conjugative systems are needed.
Molecular Microbiology, 2 June 2016, DOI: 10.1111/mmi.13401
Jolanta Godziszewska, Gabriel Moncalián, Matilde Cabezas, Aneta A. Bartosik, Fernando de la Cruz, Grazyna Jagura-Burdzy
Abstract
Conjugative transfer of the broad-host-range RA3 plasmid, the archetype of the IncU group, relies on the relaxase NIC that belongs to the as yet uncharacterized MOBP4 subfamily. NIC contains the signature motifs of HUH relaxases involved in Tyr nucleophilic attack. However, it differs in the residue involved in His activation for cation coordination and was shown here to have altered divalent cation requirements. NIC is encoded in the mobC-nic operon preceded directly by oriT, where mobC encodes an auxiliary transfer protein with a dual function: autorepressor and stimulator of conjugative transfer. Here an interplay between MobC and NIC was demonstrated. MobC is required for efficient NIC cleavage of oriT in supercoiled DNA whereas NIC assists MobC in repression of the mobC-nic operon. A 7-bp arm of IR3 (IR3a) was identified as the binding site for NIC and the crucial nucleotides in IR3a for NIC recognition were defined. Fully active oriTRA3was delineated to a 47-bp DNA segment encompassing a conserved cleavage site sequence, the NIC binding site IR3a and the MobC binding site OM. This highly efficient RA3 conjugative system with defined requirements for minimal oriT could find ample applications in biotechnology and computational biology where simple conjugative systems are needed.
Orthogonal intercellular signaling for programmed spatial behavior.
Molecular Systems Biology 12:849-861, (2016). DOI: 10.15252/msb.20156590
Grant PK, Dalchau N, Brown JR, Federici F, Rudge TJ, Yordanov B, Patange O, Phillips A, Haseloff J.
Abstract
Bidirectional intercellular signaling is an essential feature of multicellular organisms, and the engineering of complex biological systems will require multiple pathways for intercellular signaling with minimal crosstalk. Natural quorum-sensing systems provide components for cell communication, but their use is often constrained by signal crosstalk. We have established new orthogonal systems for cell-cell communication using acyl homoserine lactone signaling systems. Quantitative measurements in contexts of differing receiver protein expression allowed us to separate different types of crosstalk between 3-oxo-C6- and 3-oxo-C12-homoserine lactones, cognate receiver proteins, and DNA promoters. Mutating promoter sequences minimized interactions with heterologous receiver proteins. We used experimental data to parameterize a computational model for signal crosstalk and to estimate the effect of receiver protein levels on signal crosstalk. We used this model to predict optimal expression levels for receiver proteins, to create an effective two-channel cell communication device. Establishment of a novel spatial assay allowed measurement of interactions between geometrically constrained cell populations via these diffusible signals. We built relay devices capable of long-range signal propagation mediated by cycles of signal induction, communication and response by discrete cell populations. This work demonstrates the ability to systematically reduce crosstalk within intercellular signaling systems and to use these systems to engineer complex spatiotemporal patterning in cell populations.
Molecular Systems Biology 12:849-861, (2016). DOI: 10.15252/msb.20156590
Grant PK, Dalchau N, Brown JR, Federici F, Rudge TJ, Yordanov B, Patange O, Phillips A, Haseloff J.
Abstract
Bidirectional intercellular signaling is an essential feature of multicellular organisms, and the engineering of complex biological systems will require multiple pathways for intercellular signaling with minimal crosstalk. Natural quorum-sensing systems provide components for cell communication, but their use is often constrained by signal crosstalk. We have established new orthogonal systems for cell-cell communication using acyl homoserine lactone signaling systems. Quantitative measurements in contexts of differing receiver protein expression allowed us to separate different types of crosstalk between 3-oxo-C6- and 3-oxo-C12-homoserine lactones, cognate receiver proteins, and DNA promoters. Mutating promoter sequences minimized interactions with heterologous receiver proteins. We used experimental data to parameterize a computational model for signal crosstalk and to estimate the effect of receiver protein levels on signal crosstalk. We used this model to predict optimal expression levels for receiver proteins, to create an effective two-channel cell communication device. Establishment of a novel spatial assay allowed measurement of interactions between geometrically constrained cell populations via these diffusible signals. We built relay devices capable of long-range signal propagation mediated by cycles of signal induction, communication and response by discrete cell populations. This work demonstrates the ability to systematically reduce crosstalk within intercellular signaling systems and to use these systems to engineer complex spatiotemporal patterning in cell populations.
Analyzing Repast Symphony models in R with RRepast package
Biorxiv (Preprint). 2016 May 2 DOI: 10.1101/047985
Antonio Prestes García, Alfonso Rodríguez-Patón.
Abstract
In order to produce dependable results, the output of models must be carefully evaluated and compared to the experimental data. One of the main goals of analyzing a model is the understanding the effect of input factors on the model output. This task is carried out using a methodology known as sensitivity analysis. The analysis of Individual-based Models is hindered by the lack of simple tools allowing a complete and throughout evaluation without much effort. This kind of model tends to have a high level of complexity and the manual execution of a large experimental setup is generally not a feasible choice. Thus, it is required that model evaluation should ideally be simple and robust without demanding a high level of knowledge from modelers. In this work we present the RRepast, an open source GNU R package for executing, calibrating and analyzing Repast Symphony models directly from the R environment.
Biorxiv (Preprint). 2016 May 2 DOI: 10.1101/047985
Antonio Prestes García, Alfonso Rodríguez-Patón.
Abstract
In order to produce dependable results, the output of models must be carefully evaluated and compared to the experimental data. One of the main goals of analyzing a model is the understanding the effect of input factors on the model output. This task is carried out using a methodology known as sensitivity analysis. The analysis of Individual-based Models is hindered by the lack of simple tools allowing a complete and throughout evaluation without much effort. This kind of model tends to have a high level of complexity and the manual execution of a large experimental setup is generally not a feasible choice. Thus, it is required that model evaluation should ideally be simple and robust without demanding a high level of knowledge from modelers. In this work we present the RRepast, an open source GNU R package for executing, calibrating and analyzing Repast Symphony models directly from the R environment.
Sensitivity analysis of Repast Computational Ecology models with R/Repast.
Ecology and Evolution Journal, 2016 Oct 15 DOI: 10.5281/zenodo.160954
Antonio Prestes García, Alfonso Rodríguez-Patón.
Abstract
Computational ecology is an emerging interdisciplinary discipline founded mainly on modeling and simulation methods for studying ecological systems. Among the existing modeling formalisms, the individual-based modeling is particularly well suited for capturing the complex temporal and spatial dynamics as well as the nonlinearities arising in ecosystems, communities or populations due to individual variability. In addition, being a bottom up approach, it is useful for providing new insights on the local mechanisms which are generating some observed global dynamics. Of course no conclusions about model results could be taken seriously if they are based on a single model execution and they are not analyzed carefully. Therefore, a sound methodology should always be used for underpinning the interpretation of model results. The sensitivity analysis is a methodology for quantitatively assessing the effect of input uncertainty in the simulation output which should be incorporated compulsorily to every work based on in silico experimental setup. In this paper we present R/Repast a GNU R package for running and analyzing Repast Simphony models accompanied by two worked examples on how to perform global sensitivity analysis and how to interpret the results.
Ecology and Evolution Journal, 2016 Oct 15 DOI: 10.5281/zenodo.160954
Antonio Prestes García, Alfonso Rodríguez-Patón.
Abstract
Computational ecology is an emerging interdisciplinary discipline founded mainly on modeling and simulation methods for studying ecological systems. Among the existing modeling formalisms, the individual-based modeling is particularly well suited for capturing the complex temporal and spatial dynamics as well as the nonlinearities arising in ecosystems, communities or populations due to individual variability. In addition, being a bottom up approach, it is useful for providing new insights on the local mechanisms which are generating some observed global dynamics. Of course no conclusions about model results could be taken seriously if they are based on a single model execution and they are not analyzed carefully. Therefore, a sound methodology should always be used for underpinning the interpretation of model results. The sensitivity analysis is a methodology for quantitatively assessing the effect of input uncertainty in the simulation output which should be incorporated compulsorily to every work based on in silico experimental setup. In this paper we present R/Repast a GNU R package for running and analyzing Repast Simphony models accompanied by two worked examples on how to perform global sensitivity analysis and how to interpret the results.
BactoSim - An Individual-Based Simulation Environment for Bacterial Conjugation.
PAAMS 2015: 275-279 DOI: 10.1007/978-3-319-18944-4_26
Antonio Prestes García, Alfonso Rodríguez-Patón.
Abstract
BactoSim is an agent-based platform for simulating the conjugation in spatially structured bacterial populations, which are the conditions typically found on naturally occurring colonies such as biofilms or in agar-based laboratory cultures. The model provides a set of key indicators which can be visualized in real time as the simulation evolves and saved as for further analysis.analysis and how to interpret the results.
PAAMS 2015: 275-279 DOI: 10.1007/978-3-319-18944-4_26
Antonio Prestes García, Alfonso Rodríguez-Patón.
Abstract
BactoSim is an agent-based platform for simulating the conjugation in spatially structured bacterial populations, which are the conditions typically found on naturally occurring colonies such as biofilms or in agar-based laboratory cultures. The model provides a set of key indicators which can be visualized in real time as the simulation evolves and saved as for further analysis.analysis and how to interpret the results.
A Preliminary Assessment of Three Strategies for the Agent-Based Modeling of Bacterial Conjugation
PACBB 2015: 1-9 DOI: 10.1007/978-3-319-19776-0_1
Antonio Prestes García, Alfonso Rodríguez-Patón.
Abstract
Bacterial conjugation is a cell-cell communication by which neighbor cells transmit circular DNA strands called plasmids. The transmission of these plasmids has been traditionally modeled using differential equations. Recently agent-based systems with spatial resolution have emerged as a promising tool that we use in this work to assess three different schemes for modeling the bacterial conjugation. The three schemes differ basically in which point of cell cycle the conjugation is most prone to happen. One alternative is to allow a conjugative event occurs as soon a suitable recipient is found, the second alternative is to make conjugation equally like to happen throughout the cell cycle and finally, the third one technique to assume that conjugation is more likely to occur in a specific point late in the cell cycle.
PACBB 2015: 1-9 DOI: 10.1007/978-3-319-19776-0_1
Antonio Prestes García, Alfonso Rodríguez-Patón.
Abstract
Bacterial conjugation is a cell-cell communication by which neighbor cells transmit circular DNA strands called plasmids. The transmission of these plasmids has been traditionally modeled using differential equations. Recently agent-based systems with spatial resolution have emerged as a promising tool that we use in this work to assess three different schemes for modeling the bacterial conjugation. The three schemes differ basically in which point of cell cycle the conjugation is most prone to happen. One alternative is to allow a conjugative event occurs as soon a suitable recipient is found, the second alternative is to make conjugation equally like to happen throughout the cell cycle and finally, the third one technique to assume that conjugation is more likely to occur in a specific point late in the cell cycle.
Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures
PLOS ONE. 2016 March; vol 11, no 3. DOI: 10.1371/journal.pone.0152666
Sandra Sagredo, Fernando de la Cruz , Gabriel Moncalián
Abstract
During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.
PLOS ONE. 2016 March; vol 11, no 3. DOI: 10.1371/journal.pone.0152666
Sandra Sagredo, Fernando de la Cruz , Gabriel Moncalián
Abstract
During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.
Tanzawaic Acids, a Chemically Novel Set of Bacterial Conjugation Inhibitors
PLOS ONE, 2016 Jan 26, vol. 11, no 1. DOI: 10.1371/ journal.pone.0148098
María Getino , Raúl Fernández-López , Carolina Palencia-Gándara , Javier CamposGómez , Jose M. Sánchez-López , Marta Martínez , Antonio Fernández , Fernando de la Cruz
Abstract
Bacterial conjugation is the main mechanism for the dissemination of multiple antibiotic resistance in human pathogens. This dissemination could be controlled by molecules that interfere with the conjugation process. A search for conjugation inhibitors among a collection of 1,632 natural compounds, identified tanzawaic acids A and B as best hits. They specially inhibited IncW and IncFII conjugative systems, including plasmids mobilized by them. Plasmids belonging to IncFI, IncI, IncL/M, IncX and IncH incompatibility groups were targeted to a lesser extent, whereas IncN and IncP plasmids were unaffected. Tanzawaic acids showed reduced toxicity in bacterial, fungal or human cells, when compared to synthetic conjugation inhibitors, opening the possibility of their deployment in complex environments, including natural settings relevant for antibiotic resistance dissemination.
PLOS ONE, 2016 Jan 26, vol. 11, no 1. DOI: 10.1371/ journal.pone.0148098
María Getino , Raúl Fernández-López , Carolina Palencia-Gándara , Javier CamposGómez , Jose M. Sánchez-López , Marta Martínez , Antonio Fernández , Fernando de la Cruz
Abstract
Bacterial conjugation is the main mechanism for the dissemination of multiple antibiotic resistance in human pathogens. This dissemination could be controlled by molecules that interfere with the conjugation process. A search for conjugation inhibitors among a collection of 1,632 natural compounds, identified tanzawaic acids A and B as best hits. They specially inhibited IncW and IncFII conjugative systems, including plasmids mobilized by them. Plasmids belonging to IncFI, IncI, IncL/M, IncX and IncH incompatibility groups were targeted to a lesser extent, whereas IncN and IncP plasmids were unaffected. Tanzawaic acids showed reduced toxicity in bacterial, fungal or human cells, when compared to synthetic conjugation inhibitors, opening the possibility of their deployment in complex environments, including natural settings relevant for antibiotic resistance dissemination.
Characterization of intrinsic properties of promoters.
ACS Synthetic Biology. 2016 Jan 15;5(1):89-98. DOI: 10.1021/acssynbio.5b00116
Rudge TJ, Brown JR, Federici F, Dalchau N, Phillips A, Ajioka JW, Haseloff J.
Abstract
Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators. However, transcription rates observed from promoters can vary significantly depending on the growth rate of host cells and the experimental and genetic context of measurement. Further, in vivo measurement methods must accommodate variation in translation, protein folding and maturation rates of reporter proteins, as well as metabolic load. The external factors affecting transcription activity may be considered extrinsic, and the goal of characterization should be to obtain quantitative measures of the intrinsic characteristics of promoters. We have developed a promoter characterization method that is based on a mathematical model for cell growth and reporter gene expression and exploits multiple in vivo measurements to compensate for variation due to extrinsic factors. First, we used optical density and fluorescent reporter gene measurements to account for the effect of differing cell growth rates. Second, we compared the output of reporter genes to that of a control promoter using concurrent dual-channel fluorescence measurements. This allowed us to derive a quantitative promoter characteristic (ρ) that provides a robust measure of the intrinsic properties of a promoter, relative to the control. We imposed different extrinsic factors on growing cells, altering carbon source and adding bacteriostatic agents and demonstrated that the use of ρ values reduced the fraction of variance due to extrinsic factors from 78% to less than 4%. This is a simple and reliable method for quantitative description of promoter properties.
c aquí para editar.
ACS Synthetic Biology. 2016 Jan 15;5(1):89-98. DOI: 10.1021/acssynbio.5b00116
Rudge TJ, Brown JR, Federici F, Dalchau N, Phillips A, Ajioka JW, Haseloff J.
Abstract
Accurate characterization of promoter behavior is essential for the rational design of functional synthetic transcription networks such as logic gates and oscillators. However, transcription rates observed from promoters can vary significantly depending on the growth rate of host cells and the experimental and genetic context of measurement. Further, in vivo measurement methods must accommodate variation in translation, protein folding and maturation rates of reporter proteins, as well as metabolic load. The external factors affecting transcription activity may be considered extrinsic, and the goal of characterization should be to obtain quantitative measures of the intrinsic characteristics of promoters. We have developed a promoter characterization method that is based on a mathematical model for cell growth and reporter gene expression and exploits multiple in vivo measurements to compensate for variation due to extrinsic factors. First, we used optical density and fluorescent reporter gene measurements to account for the effect of differing cell growth rates. Second, we compared the output of reporter genes to that of a control promoter using concurrent dual-channel fluorescence measurements. This allowed us to derive a quantitative promoter characteristic (ρ) that provides a robust measure of the intrinsic properties of a promoter, relative to the control. We imposed different extrinsic factors on growing cells, altering carbon source and adding bacteriostatic agents and demonstrated that the use of ρ values reduced the fraction of variance due to extrinsic factors from 78% to less than 4%. This is a simple and reliable method for quantitative description of promoter properties.
c aquí para editar.
Orthogonal intercellular signaling for programmed spatial behavior.
Molecular Systems Biology. 2016 Jan 25;12(1):849. DOI: 10.15252/msb.20156590
Grant PK, Dalchau N, Brown JR, Federici F, Rudge TJ, Yordanov B, Patange O, Phillips A, Haseloff J.
Abstract
Bidirectional intercellular signaling is an essential feature of multicellular organisms, and the engineering of complex biological systems will require multiple pathways for intercellular signaling with minimal crosstalk. Natural quorum-sensing systems provide components for cell communication, but their use is often constrained by signal crosstalk. We have established new orthogonal systems for cell-cell communication using acyl homoserine lactone signaling systems. Quantitative measurements in contexts of differing receiver protein expression allowed us to separate different types of crosstalk between 3-oxo-C6- and 3-oxo-C12-homoserine lactones, cognate receiver proteins, and DNA promoters. Mutating promoter sequences minimized interactions with heterologous receiver proteins. We used experimental data to parameterize a computational model for signal crosstalk and to estimate the effect of receiver protein levels on signal crosstalk. We used this model to predict optimal expression levels for receiver proteins, to create an effective two-channel cell communication device. Establishment of a novel spatial assay allowed measurement of interactions between geometrically constrained cell populations via these diffusible signals. We built relay devices capable of long-range signal propagation mediated by cycles of signal induction, communication and response by discrete cell populations. This work demonstrates the ability to systematically reduce crosstalk within intercellular signaling systems and to use these systems to engineer complex spatiotemporal patterning in cell populations.
Molecular Systems Biology. 2016 Jan 25;12(1):849. DOI: 10.15252/msb.20156590
Grant PK, Dalchau N, Brown JR, Federici F, Rudge TJ, Yordanov B, Patange O, Phillips A, Haseloff J.
Abstract
Bidirectional intercellular signaling is an essential feature of multicellular organisms, and the engineering of complex biological systems will require multiple pathways for intercellular signaling with minimal crosstalk. Natural quorum-sensing systems provide components for cell communication, but their use is often constrained by signal crosstalk. We have established new orthogonal systems for cell-cell communication using acyl homoserine lactone signaling systems. Quantitative measurements in contexts of differing receiver protein expression allowed us to separate different types of crosstalk between 3-oxo-C6- and 3-oxo-C12-homoserine lactones, cognate receiver proteins, and DNA promoters. Mutating promoter sequences minimized interactions with heterologous receiver proteins. We used experimental data to parameterize a computational model for signal crosstalk and to estimate the effect of receiver protein levels on signal crosstalk. We used this model to predict optimal expression levels for receiver proteins, to create an effective two-channel cell communication device. Establishment of a novel spatial assay allowed measurement of interactions between geometrically constrained cell populations via these diffusible signals. We built relay devices capable of long-range signal propagation mediated by cycles of signal induction, communication and response by discrete cell populations. This work demonstrates the ability to systematically reduce crosstalk within intercellular signaling systems and to use these systems to engineer complex spatiotemporal patterning in cell populations.
Towards an integrated model of bacterial conjugation.
FEMS Microbiology Rev. 2015 Jan;39(1):81-95. DOI: 10.1111/1574-6976.12085
Cabezón E, Ripoll-Rozada J, Peña A, de la Cruz F, Arechaga I.
Abstract
Bacterial conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in bacterial conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating expansion of antibiotic resistance.
FEMS Microbiology Rev. 2015 Jan;39(1):81-95. DOI: 10.1111/1574-6976.12085
Cabezón E, Ripoll-Rozada J, Peña A, de la Cruz F, Arechaga I.
Abstract
Bacterial conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in bacterial conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating expansion of antibiotic resistance.
Rebooting the genome: The role of negative feedback in horizontal gene transfer.
Mobile genetic elements, 2015 Feb 23, vol. 4, no 6, p. 1. DOI: 10.4161/2159256X.2014.988069
Fernandez-Lopez R., de la Cruz F.
Abstract
Horizontal Gene Transfer (HGT) is one of the key mechanisms driving bacterial evolution. Conjugative plasmids are fundamental vehicles for HGT in bacteria, playing an essential role in the spread of antibiotic resistances. Although the classical view has stressed the instrumental role of these mobile genetic elements in the dissemination of antibiotic resistance genes, plasmids contain a rich physiology devoted to horizontal and vertical reproduction. This particular lifestyle imposes specific constrains and trade-offs on plasmid physiology, and plasmids have evolved dedicated circuits to balance the opposing demands of vertical and horizontal reproduction. Recent studies on the transcriptional networks of IncW plasmids and other incompatibility groups have unveiled common architectures in the regulatory networks of different plasmid groups. Comparative studies show that negative feedback loops (NFLs) with strong gains are preferred, opening the question of a possible convergent evolution dictated by certain adaptive properties of this particular network motif. System analysis of NFLs with strong feedback gains indicate that this architecture exhibits transient overshooting after horizontal gene transfer. Since plasmid burden is dependent on the expression of plasmid functions, transcriptional overshooting results in a transient increase of the burden immediately after conjugation. We discuss the possible implications of this phenomenon on plasmid propagation, and the regulatory networks that plasmids have evolved to counteract the detrimental side effects of transient overshooting.
Mobile genetic elements, 2015 Feb 23, vol. 4, no 6, p. 1. DOI: 10.4161/2159256X.2014.988069
Fernandez-Lopez R., de la Cruz F.
Abstract
Horizontal Gene Transfer (HGT) is one of the key mechanisms driving bacterial evolution. Conjugative plasmids are fundamental vehicles for HGT in bacteria, playing an essential role in the spread of antibiotic resistances. Although the classical view has stressed the instrumental role of these mobile genetic elements in the dissemination of antibiotic resistance genes, plasmids contain a rich physiology devoted to horizontal and vertical reproduction. This particular lifestyle imposes specific constrains and trade-offs on plasmid physiology, and plasmids have evolved dedicated circuits to balance the opposing demands of vertical and horizontal reproduction. Recent studies on the transcriptional networks of IncW plasmids and other incompatibility groups have unveiled common architectures in the regulatory networks of different plasmid groups. Comparative studies show that negative feedback loops (NFLs) with strong gains are preferred, opening the question of a possible convergent evolution dictated by certain adaptive properties of this particular network motif. System analysis of NFLs with strong feedback gains indicate that this architecture exhibits transient overshooting after horizontal gene transfer. Since plasmid burden is dependent on the expression of plasmid functions, transcriptional overshooting results in a transient increase of the burden immediately after conjugation. We discuss the possible implications of this phenomenon on plasmid propagation, and the regulatory networks that plasmids have evolved to counteract the detrimental side effects of transient overshooting.
Cell shape-driven instability generates self-organised, fractal patterning of cell layers
ACS Synthetic Biology, 2:705-714, (2013) DOI: 10.1021/sb400030p
Rudge TJ, Steiner PJ, Kan A and Haseloff J.
Abstract
As a model system to study physical interactions in multicellular systems, we used layers of Escherichia coli cells, which exhibit little or no intrinsic coordination of growth. This system effectively isolates the effects of cell shape, growth, and division on spatial self-organization. Tracking the development of fluorescence-labeled cellular domains, we observed the emergence of striking fractal patterns with jagged, self-similar shapes. We then used a large-scale, cellular biophysical model to show that local instabilities due to polar cell-shape, repeatedly propagated by uniaxial growth and division, are responsible for generating this fractal geometry. Confirming this result, a mutant of E. coli with spherical shape forms smooth, nonfractal cellular domains. These results demonstrate that even populations of relatively simple bacterial cells can possess emergent properties due to purely physical interactions. Therefore, accurate physico-genetic models of cell growth will be essential for the design and understanding of genetically programmed multicellular systems.
ACS Synthetic Biology, 2:705-714, (2013) DOI: 10.1021/sb400030p
Rudge TJ, Steiner PJ, Kan A and Haseloff J.
Abstract
As a model system to study physical interactions in multicellular systems, we used layers of Escherichia coli cells, which exhibit little or no intrinsic coordination of growth. This system effectively isolates the effects of cell shape, growth, and division on spatial self-organization. Tracking the development of fluorescence-labeled cellular domains, we observed the emergence of striking fractal patterns with jagged, self-similar shapes. We then used a large-scale, cellular biophysical model to show that local instabilities due to polar cell-shape, repeatedly propagated by uniaxial growth and division, are responsible for generating this fractal geometry. Confirming this result, a mutant of E. coli with spherical shape forms smooth, nonfractal cellular domains. These results demonstrate that even populations of relatively simple bacterial cells can possess emergent properties due to purely physical interactions. Therefore, accurate physico-genetic models of cell growth will be essential for the design and understanding of genetically programmed multicellular systems.
