While prokaryotic promoters controlled by signal-responding regulators typically display a range ... more While prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an OFF state of zero transcription-as ideally needed for synthetic circuits. To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation-inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropyl ketone) and shown to perform effectively in both Escherichia coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of, e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.
SummaryThe genome of the soil bacterium Pseudomonas putida KT2440 encodes singular orthologues of... more SummaryThe genome of the soil bacterium Pseudomonas putida KT2440 encodes singular orthologues of genes crp (encoding the catabolite repression protein, Crp) and cyaA (adenylate cyclase) of Escherichia coli. The levels of cAMP formed by P. putida cells were below detection with a Dictyostelium biosensor in vivo. The cyaAP.putida gene was transcribed in vivo but failed to complement the lack of maltose consumption of a cyaA mutant of E. coli, thereby indicating that cyaAP.putida was poorly translated or rendered non‐functional in the heterologous host. Yet, generation of cAMP by CyaAP.putida could be verified by expressing the cyaAP.putida gene in a hypersensitive E. coli strain. On the other hand, the crpP.putida gene restored the metabolic capacities of an equivalent crp mutant of E. coli, but not in a double crp/cyaA strain, suggesting that the ability to regulate such functions required cAMP. In order to clarify the breadth of the Crp/cAMP system in P. putida, crp and cyaA mutants were generated and passed through a battery of phenotypic tests for recognition of gross metabolic properties and stress‐endurance abilities. These assays revealed that the loss of each gene led in most (but not all) cases to the same phenotypic behaviour, indicating a concerted functionality. Unexpectedly, none of the mutations affected the panel of carbon compounds that can be used by P. putida as growth substrates, the mutants being impaired only in the use of various dipeptides as N sources. Furthermore, the lack of crp or cyaA had little influence on the gross growth fingerprinting of the cells. The poor physiological profile of the Crp–cAMP system of P. putida when compared with E. coli exposes a case of regulatory exaptation, i.e. the process through which a property evolved for a particular function is co‐opted for a new use.
bioRxiv (Cold Spring Harbor Laboratory), May 27, 2021
By building on the SEVA (Standard European Vector Architecture) format we have refactored a numbe... more By building on the SEVA (Standard European Vector Architecture) format we have refactored a number of regulatory nodes recruited from both Gram-negative and Gram-positive bacteria for rigorously comparing and parameterizing five expression devices that respond to diverse and unrelated chemical inducers, i.e. LacI q-Ptrc, XylS-Pm, AlkS-PalkB, CprK-PDB3 and ChnR-PchnB. These were assembled as cargoes following the SEVA standard within exactly the same vector backbone and bearing the different functional segments arrayed in an invariable DNA scaffold. Their performance in an Escherichia coli strain of reference was then analyzed through the readout a fluorescence reporter gene that contained strictly identical translation signal elements in all cases and in the same DNA context. This study allowed us to describe and compare the cognate expression systems with unprecedented quantitative detail. The systems under scrutiny diverged considerably in their capacity, expression noise, inducibility and OFF/ON ratios. These features, along with the absence of physiological effects caused by the inducers and the lack of crossregulation offer a panoply of choices to potential users and help interoperability of the specific constructs.
The essential molecular chaperonin GroEL is an example of a functionally highly versatile cellula... more The essential molecular chaperonin GroEL is an example of a functionally highly versatile cellular machine with a wide variety of in vitro applications ranging from protein folding to drug release. Directed evolution of new functions for GroEL is considered difficult, due to its structure as a complex homomultimeric double ring and the absence of obvious molecular engineering strategies. In order to investigate the potential to establish an orthogonal GroEL system in Escherichia coli, which might serve as a basis for GroEL evolution, we first successfully individualised groEL genes by inserting different functional peptide tags into a robustly permissive site identified by transposon mutagenesis. These peptides allowed fundamental aspects of the intracellular GroEL complex stoichiometry to be studied and revealed that GroEL single‐ring complexes, which assembled in the presence of several functionally equivalent but biochemically distinct monomers, each consist almost exclusively of only one type of monomer. At least in the case of GroEL, individualisation of monomers thus leads to individualisation of homomultimeric protein complexes, effectively providing the prerequisites for evolving an orthogonal intracellular GroEL folding machine.
The σ 54-dependent prokaryotic regulator XylR implements a one-input / one-output actuator that t... more The σ 54-dependent prokaryotic regulator XylR implements a one-input / one-output actuator that transduces the presence of the aromatic effector m-xylene into transcriptional activation of the cognate promoter Pu. Such a signal conversion involves the effector-mediated release of the intramolecular repression of the N-terminal A domain on the central C module of XylR. On this background, we set out to endow this regulator with additional signal-sensing capabilities by inserting a target site of the viral protease NIa in permissive protein locations that once cleaved in vivo could either terminate XylR activity or generate an effector-independent, constitutive transcription factor. To find optimal protein positions to this end we saturated the xylR gene DNA with a synthetic transposable element designed for randomly delivering in-frame polypeptides throughout the sequence of any given protein. This Tn5based system supplies the target gene with insertions of a selectable marker that can later be excised leaving behind the desired (poly) peptides grafted into the protein structure. Implementation of such knock-in / leave behind (KILB) method to XylR was instrumental to produce a number of variants of this TF that could compute in vivo two inputs (m-xylene and protease) into a single output following a logic that was dependent on the site of the insertion of the NIa target sequence in the TF. Such NIa-sensitive XylR specimens afforded the design of novel regulatory nodes that entered protease expression as one of signals recognized in vivo for controlling Pu. This approach is bound to facilitate the functionalization of TFs and other proteins with new traits, especially when their forward engineering is made difficult by, for example, the absence of structural data.
bioRxiv (Cold Spring Harbor Laboratory), Feb 5, 2020
Pseudomonas putida KT2440, a microorganism of interest for biotechnological purposes, is one amon... more Pseudomonas putida KT2440, a microorganism of interest for biotechnological purposes, is one amongst the many bacteria that attach to surfaces and produce biofilm. Although other mechanisms that contribute to this decision have been studied until now, a 7-genes-operon with a disposition and homology shared with the wsp operon in Pseudomonas aeruginosa remained to be investigated. In this work, we characterized the function of P. putida wsp operon by the combination of deletion mutants with complementations with P. aeruginosa's genes and with deletions of 3 other genes: the genes that code for the transcription factors fleQ and fleN and the flagellar movement regulator, fglZ. Examining mutant behaviour at 6 and 24 hours under three different carbon regimes (citrate, glucose and fructose) we saw that this complex carries out a similar function in both Pseudomonas. In P. putida, the key components are WspR, a protein that harbours the domain for producing c-di-GMP, and WspF, which controls its activity. Transformation with the equivalent proteins of P. aeruginosa had a significant impact on of P. putida mutant phenotypes and could complement their functions under some conditions. These results contribute to the deeper understanding of the complex element network that regulate lifestyle decision in P. putida
The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB... more 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-touse 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.
The features of the light-responsive cyanobacterial CcaSR regulatory node that determine interope... more The features of the light-responsive cyanobacterial CcaSR regulatory node that determine interoperability of this optogenetic device between Escherichia coli and Pseudomonas putida have been examined. For this, all structural parts (i.e. ho1 and pcyA genes for synthesis of phycobilin, the ccaS/ccaR system from Synechocystis and its cognate downstream promoter) were maintained but their expression levels and stoichiometry diversified by [i] reassembling them together in a single broad host range, standardized vector and [ii] subjecting the noncoding regulatory sequences to multiple cycles of directed evolution with random genomic mutations (DIvERGE), a recombineering method that intensifies mutation rates within discrete DNA segments. Once passed to P. putida, various clones displayed a wide dynamic range, insignificant leakiness and excellent capacity in response to green light. Inspection of the evolutionary intermediates pinpointed translational control as the main bottleneck for interoperability and suggested a general approach for easing the exchange of genetic cargoes between different species i.e. optimization of relative expression levels and upturning of subcomplex stoichiometry.
Users may download and print one copy of any publication from the public portal for the purpose... more Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
The nucleoid protein p6 of Bacillus subtilis phage f29 binds to DNA, recognizing a structural fea... more The nucleoid protein p6 of Bacillus subtilis phage f29 binds to DNA, recognizing a structural feature rather than a speci®c sequence. Upon binding to the viral DNA ends, p6 generates an extended nucleoprotein complex that activates the initiation of f29 DNA replication. Protein p6 also participates in transcription regulation, repressing the early C2 promoter and assisting the viral regulatory protein p4 in controlling the switch from early to late transcription. Proteins p6 and p4 bind cooperatively to an~200 bp DNA region located between the late A3 and the early A2c promoters, generating an extended nucleoprotein complex that helps to repress the early A2c promoter and to activate the late A3 promoter. We show that stable assembly of this complex requires interaction between protein p6 and the C-terminus of protein p4. Therefore, at this DNA region, stable polymerization of protein p6 relies on p4-speci®ed signals in addition to the structural features of the DNA. Protein p4 would de®ne the phase and boundaries of the p6±DNA complex.
The development of genetic competence in Bacillus subtilis is regulated by a complex signal trans... more The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComKbinding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type ␣-subunits and with C-terminal deletion mutants of the ␣-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.
Strategies to control the levels of key enzymes of bacterial metabolism are commonly based on the... more Strategies to control the levels of key enzymes of bacterial metabolism are commonly based on the manipulation of gene of interest within the target pathway. The development of new protocols towards the manipulation of biochemical processes is still a major challenge in the field of metabolic engineering. On this background, the FENIX (functional engineering of SsrA/NIa-based flux control) system allows for the post-translational regulation of protein levels, providing both independent control of the steady-state protein amounts and inducible accumulation of target proteins. This strategy enables an extra layer of control over metabolic fluxes in bacterial cell factories (see Graphical abstract below). The protocol detailed here describes the steps needed to design FENIX-tagged proteins and to adapt the system to virtually any pathway for fine-tuning of metabolic fluxes.
Background: Since publication in 1977 of plasmid pBR322, many breakthroughs in Biology have depen... more Background: Since publication in 1977 of plasmid pBR322, many breakthroughs in Biology have depended on increasingly sophisticated vector platforms for analysis and engineering of given bacterial strains. Although restriction sites impose a certain format in the procedures for assembling cloned genes, every attempt thus far to standardize vector architecture and nomenclature has ended up in failure. While this state of affairs may still be tolerable for traditional one-at-a-time studies of single genes, the onset of systems and synthetic biology calls for a simplification-along with an optimization-of the currently unwieldy pool of genetic tools. Results: The functional DNA sequences present in the natural bacterial transposon Tn5 have been methodically edited and refactored for the production of a multipurpose genetic tool named pBAM1, which allows a range of manipulations in the genome of Gram-negative bacteria. This all-synthetic construct enhances the power of minitransposon vectors for either de-construction or reconstruction of phenotypes á la carte by incorporating features inspired in systems engineering: modularity, re-usability, minimization, and compatibility with other genetic tools. pBAM1 bears an streamlined, restriction site-freed and narrow-host range replication frame bearing the sequences of R6K oriV, oriT and an ampicillin resistance marker. These go along with a business module that contains a hostindependent and hyperactive transposition platform for in vivo or in vitro insertion of desired DNA into the genome of the target bacterium. All functional sequences were standardized for a straightforward replacement by equivalent counterparts, if required. pBAM1 can be delivered into recipient cells by either mating or electroporation, producing transposon insertion frequencies of 1.8 × 10-3 and 1.02 × 10-7 , respectively in the soil bacterium Pseudomonas putida. Analyses of the resulting clones revealed a 100% of unique transposition events and virtually no-cointegration of the donor plasmid within the target genome. Conclusions: This work reports the design and performance of an all-synthetic mini-transposon vector. The power of the new system for both identification of new functions or for the construction of desired phenotypes is shown in a genetic survey of hyper-expressed proteins and regulatory elements that influence the expression of the σ 54dependent Pu promoter of P. putida.
bioRxiv (Cold Spring Harbor Laboratory), Sep 26, 2019
While prokaryotic promoters controlled by signal-responding regulators typically display a range ... more While prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an off state of zero transcription-as ideally needed for synthetic circuits. To overcome this problem we have modelled and implemented simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation-inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropylketone) and shown to perform effectively both in E. coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.
The biological regime of Pseudomonas putida (and any other bacterium) under given environmental c... more The biological regime of Pseudomonas putida (and any other bacterium) under given environmental conditions results from the hierarchical expression of sets of genes that become turned on and off in response to one or more physicochemical signals. In some cases, such signals are clearly defined, but in many others, cells are exposed to a whole variety of ill-defined inputs that occur simultaneously. Transcriptomic analyses of bacteria passed from a reference condition to a complex niche can thus expose both the type of signals that they experience during the transition and the functions involved in adaptation to the new scenario. In this article, we describe a complete protocol for generation of transcriptomes aimed at monitoring the physiological shift of P. putida between two divergent settings using as a simple case study the change between homogeneous, planktonic lifestyle in a liquid medium and growth on the surface of an agar plate. To this end, RNA was collected from P. putida KT2440 cells at various times after growth in either condition, and the genome-wide transcriptional outputs were analysed. While the role of individual genes needs to be verified on a case-by-case basis, a gross inspection of the resulting profiles suggested cells that are cultured on solid media consistently had a higher translational and metabolic activity, stopped production of flagella and were conspicuously exposed to a strong oxidative stress. The herein described methodology is generally applicable to other circumstances for diagnosing lifestyle determinants of interest.
Pseudomonas putida KT2440, a microorganism of interest for biotechnological purposes, is one amon... more Pseudomonas putida KT2440, a microorganism of interest for biotechnological purposes, is one amongst the many bacteria that attach to surfaces and produce biofilm. Although other mechanisms that contribute to this decision have been studied until now, a 7-genes-operon with a disposition and homology shared with the wsp operon in Pseudomonas aeruginosa remained to be investigated. In this work, we characterized the function of P. putida wsp operon by the combination of deletion mutants with complementations with P. aeruginosa's genes and with deletions of 3 other genes: the genes that code for the transcription factors fleQ and fleN and the flagellar movement regulator, fglZ. Examining mutant behaviour at 6 and 24 hours under three different carbon regimes (citrate, glucose and fructose) we saw that this complex carries out a similar function in both Pseudomonas. In P. putida, the key components are WspR, a protein that harbours the domain for producing c-di-GMP, and WspF, which controls its activity. Transformation with the equivalent proteins of P. aeruginosa had a significant impact on of P. putida mutant phenotypes and could complement their functions under some conditions. These results contribute to the deeper understanding of the complex element network that regulate lifestyle decision in P. putida
While prokaryotic promoters controlled by signal-responding regulators typically display a range ... more While prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an OFF state of zero transcription-as ideally needed for synthetic circuits. To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation-inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropyl ketone) and shown to perform effectively in both Escherichia coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of, e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.
SummaryThe genome of the soil bacterium Pseudomonas putida KT2440 encodes singular orthologues of... more SummaryThe genome of the soil bacterium Pseudomonas putida KT2440 encodes singular orthologues of genes crp (encoding the catabolite repression protein, Crp) and cyaA (adenylate cyclase) of Escherichia coli. The levels of cAMP formed by P. putida cells were below detection with a Dictyostelium biosensor in vivo. The cyaAP.putida gene was transcribed in vivo but failed to complement the lack of maltose consumption of a cyaA mutant of E. coli, thereby indicating that cyaAP.putida was poorly translated or rendered non‐functional in the heterologous host. Yet, generation of cAMP by CyaAP.putida could be verified by expressing the cyaAP.putida gene in a hypersensitive E. coli strain. On the other hand, the crpP.putida gene restored the metabolic capacities of an equivalent crp mutant of E. coli, but not in a double crp/cyaA strain, suggesting that the ability to regulate such functions required cAMP. In order to clarify the breadth of the Crp/cAMP system in P. putida, crp and cyaA mutants were generated and passed through a battery of phenotypic tests for recognition of gross metabolic properties and stress‐endurance abilities. These assays revealed that the loss of each gene led in most (but not all) cases to the same phenotypic behaviour, indicating a concerted functionality. Unexpectedly, none of the mutations affected the panel of carbon compounds that can be used by P. putida as growth substrates, the mutants being impaired only in the use of various dipeptides as N sources. Furthermore, the lack of crp or cyaA had little influence on the gross growth fingerprinting of the cells. The poor physiological profile of the Crp–cAMP system of P. putida when compared with E. coli exposes a case of regulatory exaptation, i.e. the process through which a property evolved for a particular function is co‐opted for a new use.
bioRxiv (Cold Spring Harbor Laboratory), May 27, 2021
By building on the SEVA (Standard European Vector Architecture) format we have refactored a numbe... more By building on the SEVA (Standard European Vector Architecture) format we have refactored a number of regulatory nodes recruited from both Gram-negative and Gram-positive bacteria for rigorously comparing and parameterizing five expression devices that respond to diverse and unrelated chemical inducers, i.e. LacI q-Ptrc, XylS-Pm, AlkS-PalkB, CprK-PDB3 and ChnR-PchnB. These were assembled as cargoes following the SEVA standard within exactly the same vector backbone and bearing the different functional segments arrayed in an invariable DNA scaffold. Their performance in an Escherichia coli strain of reference was then analyzed through the readout a fluorescence reporter gene that contained strictly identical translation signal elements in all cases and in the same DNA context. This study allowed us to describe and compare the cognate expression systems with unprecedented quantitative detail. The systems under scrutiny diverged considerably in their capacity, expression noise, inducibility and OFF/ON ratios. These features, along with the absence of physiological effects caused by the inducers and the lack of crossregulation offer a panoply of choices to potential users and help interoperability of the specific constructs.
The essential molecular chaperonin GroEL is an example of a functionally highly versatile cellula... more The essential molecular chaperonin GroEL is an example of a functionally highly versatile cellular machine with a wide variety of in vitro applications ranging from protein folding to drug release. Directed evolution of new functions for GroEL is considered difficult, due to its structure as a complex homomultimeric double ring and the absence of obvious molecular engineering strategies. In order to investigate the potential to establish an orthogonal GroEL system in Escherichia coli, which might serve as a basis for GroEL evolution, we first successfully individualised groEL genes by inserting different functional peptide tags into a robustly permissive site identified by transposon mutagenesis. These peptides allowed fundamental aspects of the intracellular GroEL complex stoichiometry to be studied and revealed that GroEL single‐ring complexes, which assembled in the presence of several functionally equivalent but biochemically distinct monomers, each consist almost exclusively of only one type of monomer. At least in the case of GroEL, individualisation of monomers thus leads to individualisation of homomultimeric protein complexes, effectively providing the prerequisites for evolving an orthogonal intracellular GroEL folding machine.
The σ 54-dependent prokaryotic regulator XylR implements a one-input / one-output actuator that t... more The σ 54-dependent prokaryotic regulator XylR implements a one-input / one-output actuator that transduces the presence of the aromatic effector m-xylene into transcriptional activation of the cognate promoter Pu. Such a signal conversion involves the effector-mediated release of the intramolecular repression of the N-terminal A domain on the central C module of XylR. On this background, we set out to endow this regulator with additional signal-sensing capabilities by inserting a target site of the viral protease NIa in permissive protein locations that once cleaved in vivo could either terminate XylR activity or generate an effector-independent, constitutive transcription factor. To find optimal protein positions to this end we saturated the xylR gene DNA with a synthetic transposable element designed for randomly delivering in-frame polypeptides throughout the sequence of any given protein. This Tn5based system supplies the target gene with insertions of a selectable marker that can later be excised leaving behind the desired (poly) peptides grafted into the protein structure. Implementation of such knock-in / leave behind (KILB) method to XylR was instrumental to produce a number of variants of this TF that could compute in vivo two inputs (m-xylene and protease) into a single output following a logic that was dependent on the site of the insertion of the NIa target sequence in the TF. Such NIa-sensitive XylR specimens afforded the design of novel regulatory nodes that entered protease expression as one of signals recognized in vivo for controlling Pu. This approach is bound to facilitate the functionalization of TFs and other proteins with new traits, especially when their forward engineering is made difficult by, for example, the absence of structural data.
bioRxiv (Cold Spring Harbor Laboratory), Feb 5, 2020
Pseudomonas putida KT2440, a microorganism of interest for biotechnological purposes, is one amon... more Pseudomonas putida KT2440, a microorganism of interest for biotechnological purposes, is one amongst the many bacteria that attach to surfaces and produce biofilm. Although other mechanisms that contribute to this decision have been studied until now, a 7-genes-operon with a disposition and homology shared with the wsp operon in Pseudomonas aeruginosa remained to be investigated. In this work, we characterized the function of P. putida wsp operon by the combination of deletion mutants with complementations with P. aeruginosa's genes and with deletions of 3 other genes: the genes that code for the transcription factors fleQ and fleN and the flagellar movement regulator, fglZ. Examining mutant behaviour at 6 and 24 hours under three different carbon regimes (citrate, glucose and fructose) we saw that this complex carries out a similar function in both Pseudomonas. In P. putida, the key components are WspR, a protein that harbours the domain for producing c-di-GMP, and WspF, which controls its activity. Transformation with the equivalent proteins of P. aeruginosa had a significant impact on of P. putida mutant phenotypes and could complement their functions under some conditions. These results contribute to the deeper understanding of the complex element network that regulate lifestyle decision in P. putida
The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB... more 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-touse 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.
The features of the light-responsive cyanobacterial CcaSR regulatory node that determine interope... more The features of the light-responsive cyanobacterial CcaSR regulatory node that determine interoperability of this optogenetic device between Escherichia coli and Pseudomonas putida have been examined. For this, all structural parts (i.e. ho1 and pcyA genes for synthesis of phycobilin, the ccaS/ccaR system from Synechocystis and its cognate downstream promoter) were maintained but their expression levels and stoichiometry diversified by [i] reassembling them together in a single broad host range, standardized vector and [ii] subjecting the noncoding regulatory sequences to multiple cycles of directed evolution with random genomic mutations (DIvERGE), a recombineering method that intensifies mutation rates within discrete DNA segments. Once passed to P. putida, various clones displayed a wide dynamic range, insignificant leakiness and excellent capacity in response to green light. Inspection of the evolutionary intermediates pinpointed translational control as the main bottleneck for interoperability and suggested a general approach for easing the exchange of genetic cargoes between different species i.e. optimization of relative expression levels and upturning of subcomplex stoichiometry.
Users may download and print one copy of any publication from the public portal for the purpose... more Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
The nucleoid protein p6 of Bacillus subtilis phage f29 binds to DNA, recognizing a structural fea... more The nucleoid protein p6 of Bacillus subtilis phage f29 binds to DNA, recognizing a structural feature rather than a speci®c sequence. Upon binding to the viral DNA ends, p6 generates an extended nucleoprotein complex that activates the initiation of f29 DNA replication. Protein p6 also participates in transcription regulation, repressing the early C2 promoter and assisting the viral regulatory protein p4 in controlling the switch from early to late transcription. Proteins p6 and p4 bind cooperatively to an~200 bp DNA region located between the late A3 and the early A2c promoters, generating an extended nucleoprotein complex that helps to repress the early A2c promoter and to activate the late A3 promoter. We show that stable assembly of this complex requires interaction between protein p6 and the C-terminus of protein p4. Therefore, at this DNA region, stable polymerization of protein p6 relies on p4-speci®ed signals in addition to the structural features of the DNA. Protein p4 would de®ne the phase and boundaries of the p6±DNA complex.
The development of genetic competence in Bacillus subtilis is regulated by a complex signal trans... more The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComKbinding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type ␣-subunits and with C-terminal deletion mutants of the ␣-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.
Strategies to control the levels of key enzymes of bacterial metabolism are commonly based on the... more Strategies to control the levels of key enzymes of bacterial metabolism are commonly based on the manipulation of gene of interest within the target pathway. The development of new protocols towards the manipulation of biochemical processes is still a major challenge in the field of metabolic engineering. On this background, the FENIX (functional engineering of SsrA/NIa-based flux control) system allows for the post-translational regulation of protein levels, providing both independent control of the steady-state protein amounts and inducible accumulation of target proteins. This strategy enables an extra layer of control over metabolic fluxes in bacterial cell factories (see Graphical abstract below). The protocol detailed here describes the steps needed to design FENIX-tagged proteins and to adapt the system to virtually any pathway for fine-tuning of metabolic fluxes.
Background: Since publication in 1977 of plasmid pBR322, many breakthroughs in Biology have depen... more Background: Since publication in 1977 of plasmid pBR322, many breakthroughs in Biology have depended on increasingly sophisticated vector platforms for analysis and engineering of given bacterial strains. Although restriction sites impose a certain format in the procedures for assembling cloned genes, every attempt thus far to standardize vector architecture and nomenclature has ended up in failure. While this state of affairs may still be tolerable for traditional one-at-a-time studies of single genes, the onset of systems and synthetic biology calls for a simplification-along with an optimization-of the currently unwieldy pool of genetic tools. Results: The functional DNA sequences present in the natural bacterial transposon Tn5 have been methodically edited and refactored for the production of a multipurpose genetic tool named pBAM1, which allows a range of manipulations in the genome of Gram-negative bacteria. This all-synthetic construct enhances the power of minitransposon vectors for either de-construction or reconstruction of phenotypes á la carte by incorporating features inspired in systems engineering: modularity, re-usability, minimization, and compatibility with other genetic tools. pBAM1 bears an streamlined, restriction site-freed and narrow-host range replication frame bearing the sequences of R6K oriV, oriT and an ampicillin resistance marker. These go along with a business module that contains a hostindependent and hyperactive transposition platform for in vivo or in vitro insertion of desired DNA into the genome of the target bacterium. All functional sequences were standardized for a straightforward replacement by equivalent counterparts, if required. pBAM1 can be delivered into recipient cells by either mating or electroporation, producing transposon insertion frequencies of 1.8 × 10-3 and 1.02 × 10-7 , respectively in the soil bacterium Pseudomonas putida. Analyses of the resulting clones revealed a 100% of unique transposition events and virtually no-cointegration of the donor plasmid within the target genome. Conclusions: This work reports the design and performance of an all-synthetic mini-transposon vector. The power of the new system for both identification of new functions or for the construction of desired phenotypes is shown in a genetic survey of hyper-expressed proteins and regulatory elements that influence the expression of the σ 54dependent Pu promoter of P. putida.
bioRxiv (Cold Spring Harbor Laboratory), Sep 26, 2019
While prokaryotic promoters controlled by signal-responding regulators typically display a range ... more While prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an off state of zero transcription-as ideally needed for synthetic circuits. To overcome this problem we have modelled and implemented simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation-inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropylketone) and shown to perform effectively both in E. coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.
The biological regime of Pseudomonas putida (and any other bacterium) under given environmental c... more The biological regime of Pseudomonas putida (and any other bacterium) under given environmental conditions results from the hierarchical expression of sets of genes that become turned on and off in response to one or more physicochemical signals. In some cases, such signals are clearly defined, but in many others, cells are exposed to a whole variety of ill-defined inputs that occur simultaneously. Transcriptomic analyses of bacteria passed from a reference condition to a complex niche can thus expose both the type of signals that they experience during the transition and the functions involved in adaptation to the new scenario. In this article, we describe a complete protocol for generation of transcriptomes aimed at monitoring the physiological shift of P. putida between two divergent settings using as a simple case study the change between homogeneous, planktonic lifestyle in a liquid medium and growth on the surface of an agar plate. To this end, RNA was collected from P. putida KT2440 cells at various times after growth in either condition, and the genome-wide transcriptional outputs were analysed. While the role of individual genes needs to be verified on a case-by-case basis, a gross inspection of the resulting profiles suggested cells that are cultured on solid media consistently had a higher translational and metabolic activity, stopped production of flagella and were conspicuously exposed to a strong oxidative stress. The herein described methodology is generally applicable to other circumstances for diagnosing lifestyle determinants of interest.
Pseudomonas putida KT2440, a microorganism of interest for biotechnological purposes, is one amon... more Pseudomonas putida KT2440, a microorganism of interest for biotechnological purposes, is one amongst the many bacteria that attach to surfaces and produce biofilm. Although other mechanisms that contribute to this decision have been studied until now, a 7-genes-operon with a disposition and homology shared with the wsp operon in Pseudomonas aeruginosa remained to be investigated. In this work, we characterized the function of P. putida wsp operon by the combination of deletion mutants with complementations with P. aeruginosa's genes and with deletions of 3 other genes: the genes that code for the transcription factors fleQ and fleN and the flagellar movement regulator, fglZ. Examining mutant behaviour at 6 and 24 hours under three different carbon regimes (citrate, glucose and fructose) we saw that this complex carries out a similar function in both Pseudomonas. In P. putida, the key components are WspR, a protein that harbours the domain for producing c-di-GMP, and WspF, which controls its activity. Transformation with the equivalent proteins of P. aeruginosa had a significant impact on of P. putida mutant phenotypes and could complement their functions under some conditions. These results contribute to the deeper understanding of the complex element network that regulate lifestyle decision in P. putida
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