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Evolutionary processes acting on populations of organized molecules preceded the origin of living organisms. These prebiotic entities were independently and repeatedly produced [i.e. (re)-produced] by the assembly of their components, following an iterative process giving rise to nearly but not fully identical replicas, allowing for a prebiotic form of Darwinian evolution. Natural selection favored the more persistent assemblies, some possibly modifying their own internal structure, or even their environment, thereby acquiring function. We refer to these assemblies as proto-organs. In association with other assemblies (e.g. in a coacervate or encapsulated within a vesicle), such proto-organs could evolve and acquire a role within the collective when their coexistence favored the selection of the ensemble. Along millions of years, an extraordinarily small number of successful combinations of those proto-organs co-occurring in spatially individualizing compartments might have co-evolved forming a proto-metabolic and proto-genetic informative network, eventually leading to the selfreplication of a very few. Thus, interactions between encapsulated proto-organs would have had a much higher probability of evolving into proto-organisms than interactions among simpler molecules. Multimolecular forms evolve functions; thus, functional organs would have preceded organisms., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

On 9-13 July 2023, the 10th FEMS Congress took place in Hamburg, Germany. As part of this major event in European microbiology, the European Academy of Microbiology (EAM) organized two full sessions. One of these sessions aimed to highlight the research of four recently elected EAM fellows and saw presentations on bacterial group behaviours and development of resistance to antibiotics, as well as on new RNA viruses including bacteriophages and giant viruses of amoebae. The other session included five frontline environmental microbiologists who showcased real-world examples of how human activities have disrupted the balance in microbial ecosystems, not just to assess the current situation but also to explore fresh approaches for coping with external disturbances. Both sessions were very well attended, and no doubt helped to gain the EAM and its fellows more visibility., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB) and as a physical repository of plasmid vectors for genetic analysis and engineering of Gram-negative bacteria with a structure and nomenclature that follows a strict, fixed architecture of functional DNA segments. While the current update keeps the basic features of earlier versions, the platform has been upgraded not only with many more ready-to-use plasmids but also with features that expand the range of target species, harmonize DNA assembly methods and enable new applications. In particular, SEVA 4.0 includes (i) a sub-collection of plasmids for easing the composition of multiple DNA segments with MoClo/Golden Gate technology, (ii) vectors for Gram-positive bacteria and yeast and [iii] off-the-shelf constructs with built-in functionalities. A growing collection of plasmids that capture part of the standard-but not its entirety-has been compiled also into the DB and repository as a separate corpus (SEVAsib) because of its value as a resource for constructing and deploying phenotypes of interest. Maintenance and curation of the DB were accompanied by dedicated diffusion and communication channels that make the SEVA platform a popular resource for genetic analyses, genome editing and bioengineering of a large number of microorganisms., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Boolean NOR gates have been widely implemented in Escherichia coli as transcriptional regulatory devices for building complex genetic circuits. Yet, their portability to other bacterial hosts/chassis is generally hampered by frequent changes in the parameters of the INPUT/OUTPUT response functions brought about by new genetic and biochemical contexts. Here, we have used the circuit design tool CELLO for assembling a NOR gate in the soil bacterium and the metabolic engineering platform Pseudomonas putida with components tailored for E. coli. To this end, we capitalized on the functional parameters of 20 genetic inverters for each host and the resulting compatibility between NOT pairs. Moreover, we added to the gate library three inducible promoters that are specific to P. putida , thus expanding cross-platform assembly options. While the number of potential connectable inverters decreased drastically when moving the library from E. coli to P. putida , the CELLO software was still able to find an effective NOR gate in the new chassis. The automated generation of the corresponding DNA sequence and in vivo experimental verification accredited that some genetic modules initially optimized for E. coli can indeed be reused to deliver NOR logic in P. putida as well. Furthermore, the results highlight the value of creating host-specific collections of well-characterized regulatory inverters for the quick assembly of genetic circuits to meet complex specifications., (© The Author(s) 2021. Published by Oxford University Press.)

The environmental fate of many functional molecules that are produced on a large scale as precursors or as additives to specialty goods (plastics, fibers, construction materials, etc.), let alone those synthesized by the pharmaceutical industry, is generally unknown. Assessing their environmental fate is crucial when taking decisions on the manufacturing, handling, usage, and release of these substances, as is the evaluation of their toxicity in humans and other higher organisms. While this data are often hard to come by, the experimental data already available on the biodegradability and toxicity of many unusual compounds (including genuinely xenobiotic molecules) make it possible to develop machine learning systems to predict these features. As such, we have created a predictor of the "risk" associated with the use and release of any chemical. This new system merges computational methods to predict biodegradability with others that assess biological toxicity. The combined platform, named BiodegPred (https://sysbiol.cnb.csic.es/BiodegPred/), provides an informed prognosis of the chance a given molecule can eventually be catabolized in the biosphere, as well as of its eventual toxicity, all available through a simple web interface. While the platform described does not give much information about specific degradation kinetics or particular biodegradation pathways, BiodegPred has been instrumental in anticipating the probable behavior of a large number of new molecules (e.g. antiviral compounds) for which no biodegradation data previously existed., (© The Author(s) 2020. Published by Oxford University Press.)

The Standard European Vector Architecture 3.0 database (SEVA-DB 3.0, http://seva.cnb.csic.es) is the update of the platform launched in 2013 both as a web-based resource and as a material repository of formatted genetic tools (mostly plasmids) for analysis, construction and deployment of complex bacterial phenotypes. The period between the first version of SEVA-DB and the present time has witnessed several technical, computational and conceptual advances in genetic/genomic engineering of prokaryotes that have enabled upgrading of the utilities of the updated database. Novelties include not only a more user-friendly web interface and many more plasmid vectors, but also new links of the plasmids to advanced bioinformatic tools. These provide an intuitive visualization of the constructs at stake and a range of virtual manipulations of DNA segments that were not possible before. Finally, the list of canonical SEVA plasmids is available in machine-readable SBOL (Synthetic Biology Open Language) format. This ensures interoperability with other platforms and affords simulations of their behaviour under different in vivo conditions. We argue that the SEVA-DB will remain a useful resource for extending Synthetic Biology approaches towards non-standard bacterial species as well as genetically programming new prokaryotic chassis for a suite of fundamental and biotechnological endeavours., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Exploitation of biofilms for industrial processes requires them to adopt suitable physical structures for rendering them efficient and predictable. While hydrodynamics could be used to control material features of biofilms of the platform strain Pseudomonas putida KT2440 there is a dearth of experimental data on surface-associated growth behavior in such settings. Millimeter scale biofilm patterns formed by its parental strain P. putida mt-2 under different Reynolds numbers (Re) within laminar regime were analyzed using an upscale experimental continuous cultivation assembly. A tile-scan image acquisition process combined with a customized image analysis revealed patterns of dense heterogeneous structures at Re = 1000, but mostly flattened coverings sparsely patched for Re < 400. These results not only fix the somewhat narrow hydrodynamic regime under which P. putida cells form stable coatings on surfaces destined for large-scale processes, but also provide useful sets of parameters for engineering catalytic biofilms based on this important bacterium as a cell factory.

The XylR/Pu regulatory node of the m-xylene biodegradation pathway of Pseudomonas putida mt-2 is one of the most intricate cases of processing internal and external cues into a single controlling element. Despite this complexity, the performance of the regulatory system is determined in vivo only by the occupation of Pu by m-xylene-activated XylR and σ(54)-RNAP. The stoichiometry between these three elements defines natural system boundaries that outline a specific functional space. This space can be expanded artificially following different strategies that involve either the increase of XylR or σ(54) or both elements at the same time (each using a different inducer). In this work we have designed a new regulatory architecture that drives the system to reach a maximum performance in response to one single input. To this end, we first explored using a simple mathematical model whether the output of the XylR/Pu node could be amended by simultaneously increasing σ(54) and XylR in response to only natural inducers. The exacerbation of Pu activity in vivo was tested in strains bearing synthetic transposons encoding xylR and rpoN (the σ(54) coding gene) controlled also by Pu, thereby generating a P. putida strain with the XylR/Pu output controlled by two intertwined feed forward loops (FFLs). The lack of a negative feedback loop in the expression node enables Pu activity to reach its physiological maximum in response to a single input. Only competition for cell resources might ultimately check the upper activity limit of such a rewired m-xylene sensing device.

The Standard European Vector Architecture 2.0 database (SEVA-DB 2.0, http://seva.cnb.csic.es) is an improved and expanded version of the platform released in 2013 (doi: 10.1093/nar/gks1119) aimed at assisting the choice of optimal genetic tools for de-constructing and re-constructing complex prokaryotic phenotypes. By adopting simple compositional rules, the SEVA standard facilitates combinations of functional DNA segments that ease both the analysis and the engineering of diverse Gram-negative bacteria for fundamental or biotechnological purposes. The large number of users of the SEVA-DB during its first two years of existence has resulted in a valuable feedback that we have exploited for fixing DNA sequence errors, improving the nomenclature of the SEVA plasmids, expanding the vector collection, adding new features to the web interface and encouraging contributions of materials from the community of users. The SEVA platform is also adopting the Synthetic Biology Open Language (SBOL) for electronic-like description of the constructs available in the collection and their interfacing with genetic devices developed by other Synthetic Biology communities. We advocate the SEVA format as one interim asset for the ongoing transition of genetic design of microorganisms from being a trial-and-error endeavor to become an authentic engineering discipline., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In this review, we examine how bacterial metabolism is shaped by chemical constraints acting on the material and dynamic layout of enzymatic networks and beyond. These are moulded not only for optimisation of given metabolic objectives (e.g. synthesis of a particular amino acid or nucleotide) but also for curbing the detrimental reactivity of chemical intermediates. Besides substrate channelling, toxicity is avoided by barriers to free diffusion (i.e. compartments) that separate otherwise incompatible reactions, along with ways for distinguishing damaging vs. harmless molecules. On the other hand, enzymes age and their operating lifetime must be tuned to upstream and downstream reactions. This time dependence of metabolic pathways creates time-linked information, learning and memory. These features suggest that the physical structure of existing biosystems, from operon assemblies to multicellular development may ultimately stem from the need to restrain chemical damage and limit the waste inherent to basic metabolic functions. This provides a new twist of our comprehension of fundamental biological processes in live systems as well as practical take-home lessons for the forward DNA-based engineering of novel biological objects., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Environmental plasmids often expand the metabolic repertoire of bacteria that carry them, but they also interfere with the biochemical and genetic network of the host. The pWW0 plasmid born by Pseudomonas putida mt-2 encodes the TOL pathway for degradation of toluene/m-xylene through production of intermediate compounds benzoate/3-methylbenzoate. These can be also recognized as substrates by the chromosomally encoded ben and cat gene products, thereby creating a manifest regulatory and biochemical conflict. In this context, we have investigated how the introduction of the pWW0 plasmid into P. putida affects behaviour of the promoter of the ben pathway (Pb) in single cells. Using a series of standardized transcriptional fusions to green fluorescent protein, we found that acquisition of the TOL pathway switches the activation course of the Pb promoter from unimodal/graded to bimodal/stochastic when cells were exposed to benzoate. This behaviour was propagated downstream into the Pc promoter of the cat gene cluster, which responds to the benzoate-degradation intermediate cis,cis-muconate. The TOL plasmid thus imposes expression of the chromosomal Pb with a stochastic behaviour likely to result in biochemical heterogeneity of the otherwise genetically clonal population when exposed to benzoate as a growth substrate.

Although DNA is the ultimate repository of biological information, deployment of its instructions is constrained by the metabolic and physiological status of the cell. To this end, bacteria have evolved intricate devices that connect exogenous signals (e.g. nutrients, physicochemical conditions) with endogenous conditions (metabolic fluxes, biochemical networks) that coordinately influence expression or performance of a large number of cellular functions. The phosphoenolpyruvate:carbohydrate-phosphotransferase system (PTS) is a bacterial multi-protein phosphorylation chain which computes extracellular (e.g. sugars) and intracellular (e.g. phosphoenolpyruvate, nitrogen) signals and translates them into post-translational regulation of target activities through protein-protein interactions. The PTS of Pseudomonas putida KT2440 encompasses one complete sugar (fructose)-related system and the three enzymes that form the so-called nitrogen-related PTS (PTS(N) (tr) ), which lacks connection to transport of substrates. These two PTS branches cross-talk to each other, as the product of the fruB gene (a polyprotein EI-HPr-EIIA) can phosphorylate PtsN (EIIA(N) (tr) ) in vivo. This gives rise to a complex actuator device where diverse physiological inputs are ultimately translated into phosphorylation or not of PtsN (EIIA(N) (tr) ) which, in turn, checks the activity of key metabolic and regulatory proteins. Such a control of bacterial physiology highlights the prominence of biochemical homeostasis over genetic ruling -and not vice versa., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

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

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

Aspergillus sp. P37 is an arsenate-resistant fungus isolated from a river with a long history of heavy metal contamination. Its hypertolerant mechanism relies on an efficient arsenate reduction system in conjunction with increased coupling capabilities to glutathione and translocation of these complexes into the vacuolar system. Here, the comparison of arsenic resistance in two Aspergillus species and Saccharomyces cerevisiae in the presence or absence of different osmolytes revealed a link between tolerance to osmotic stress and tolerance to arsenic in Aspergillus strains. Under osmotic stress generated by NaCl or KCl, both Aspergillus strains were less resistant to arsenite and arsenate. In the presence of sorbitol, Aspergillus strains were more resistant to arsenite. Intracellular accumulation of arsenic was affected by the presence of the osmolytes (NaCl or sorbitol), suggesting a major role for vacuoles in the detoxification of arsenic in Aspergillus.

Bioremediation, the exploitation of biological catalysts (mostly microorganisms) for removing pollutants from the environment, requires the integration of huge amounts of data from different sources. We have developed MetaRouter, a system for maintaining heterogeneous information related to bioremediation in a framework that allows its query, administration and mining (application of methods for extracting new knowledge). MetaRouter is an application intended for laboratories working in biodegradation and bioremediation, which need to maintain and consult public and private data, linked internally and with external databases, and to extract new information from it. Among the data-mining features is a program included for locating biodegradative pathways for chemical compounds according to a given set of constraints and requirements. The integration of biodegradation information with the corresponding protein and genome data provides a suitable framework for studying the global properties of the bioremediation network. The system can be accessed and administrated through a web interface. The full-featured system (except administration facilities) is freely available at http://pdg.cnb.uam.es/MetaRouter. Additional material: http://www.pdg.cnb.uam.es/biodeg&#95;net/MetaRouter.

The binding of the transcriptional regulator XylR to its cognate upstream activating sequences (UAS) of the sigma54-dependent promoter Pu of Pseudomonas putida has been examined in vivo in single copy gene dose and stoichiometry. To this end, we have employed a novel in vivo genomic footprinting procedure that uses short exposures of bacterial cells to diffuse high intensity UV light that causes formation of TT or TC dimers. In contrast to simpler models for activation of sigma54-dependent promoters, our results clearly indicate that the XylR protein is not permanently bound in vivo to its target sites in Pu. On the contrary, the UAS appear to be mostly unoccupied at all growth stages. This is in contrast to the integration host factor (IHF), which binds Pu strongly in vivo at stationary phase, as also revealed by UV footprinting. Only overexpression of XylR altered the photoreactivity of the corresponding DNA region to report stable binding of the regulator to the UAS. However, the presence of aromatic XylR inducers reversed the forced occupation caused by increased levels of the activator. These results are compatible with the notion that XylR interacts very transiently with the UAS and detaches from the promoter during transcriptional activation of Pu.

The threat of heavy metal pollution to public health and wildlife has led to an increased interest in developing systems that can remove or neutralise its toxic effects in soil, sediments and wastewater. Unlike organic contaminants, which can be degraded to harmless chemical species, heavy metals cannot be destroyed. Remediating the pollution they cause can therefore only be envisioned as their immobilisation in a non-bioavailable form, or their re-speciation into less toxic forms. While these approaches do not solve the problem altogether, they do help to protect afflicted sites from noxious effects and isolate the contaminants as a contained and sometimes recyclable residue. This review outlines the most important bacterial phenotypes and properties that are (or could be) instrumental in heavy metal bioremediation, along with what is known of their genetic and biochemical background. A variety of instances are discussed in which valuable properties already present in certain strains can be combined or improved through state-of-the-art genetic engineering. In other cases, knowledge of metal-related reactions catalysed by some bacteria allows optimisation of the desired process by altering the physicochemical conditions of the contaminated area. The combination of genetic engineering of the bacterial catalysts with judicious eco-engineering of the polluted sites will be of paramount importance in future bioremediation strategies.

Based on genomic data and on the phenotypes of an FlhF mutant of Pseudomonas putida, the alternative sigma factor sigma(N) (sigma(54)) has been proposed to play a key role in survival to various nutritional and environmental stresses in this bacterium. Quite in contrast, we show that unlike sigma(S) (sigma(38)) the loss of sigma(N) does not impair to any significant extent the ability of P. putida to survive long-term starvation. rpoN mutants (lacking sigma(N)) are indistinguishable from the wild-type with respect to solvent tolerance, resistance to heat shock or sensitivity to hydrogen peroxide. These data suggest that while sigma(N) is a key component of expression of alternative biodegradative pathways for unusual carbon sources (i.e. m-xylene or dimethylphenols), its loss does not compromise bacterial endurance to gross types of environmental stress. Moreover, these results point out the limitations, if not the deception, of genomic predictions when confronted with experimental data.

Cascade regulatory circuits have been described that control numerous cell processes, and may provide models for the design of artificial circuits with novel properties. Here we describe the design of a transcriptional regulatory cascade to amplify the cell response to a given signal. We used the salicylate-responsive activators of Pseudomonas putida NahR of the naphthalene degradation plasmid NAH7 and XylS2, a mutant regulator of the TOL plasmid for catabolism of m-xylene and their respective cognate promoters Psal and PM: Control of the expression of xylS2 with the nahR/Psal system permitted either their selective activation with specific effectors for each protein or the simultaneous activation of both of them with salicylate. When cells face the common effector of the two regulators, both the increase in XylS2 concentration and the stimulation of its activity act synergistically on the PM: promoter, amplifying the gene expression capacity by at least one order of magnitude with respect to the individual systems. By changing the hierarchy of regulators, we showed that the specific features of the downstream regulator were crucial for the amplification effect. Directed changes in the effector profile of the regulators allowed the extension of the amplifying system to other molecular signals.

An artificial maturation pathway for increasing the solubility in vivo of recombinant proteins overproduced in Escherichia coli is reported, which is based on the proteolytic processing of viral polyproteins. The gene product of interest is expressed as a fusion to a heterologous moiety (i.e. the maltose binding protein, MalE) in order to increase the overall solubility of the hybrid. The hinge region between the two fusion partners contains a cleavage site for the NIa protein, a very specific protease from the plum pox potyvirus, as well as an affinity tag. After production, the soluble hybrid is cleaved in vivo by the protease, that is encoded by a plasmid harboured by a specialized E.coli host. The released protein remains soluble and can be purified from cell extracts by means of an affinity tag (a poly-His group) that becomes present after the cleavage. The solubilization and purification of XylR, a xylene-responsive transcriptional factor of Pseudomonas, with this method are reported.