The principle of uncertainty in biology: Will machine learning/artificial intelligence lead to the end of mechanistic studies?

Molecular Biology has long tried to discover mechanisms, considering that unless we understand the principles, we cannot develop applications. Now machine learning and artificial intelligence enable direct leaps to application without understanding the principles. Will this herald a decline in mechanistic studies?

Molecular biology for green recovery-A call for action.

Molecular biology holds a vast potential for tackling climate change and biodiversity loss. Yet, it is largely absent from the current strategies. We call for a community-wide action to bring molecular biology to the forefront of climate change solutions.

SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes.

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.

Water potential governs the effector specificity of the transcriptional regulator XylR of Pseudomonas putida.

The biodegradative capacity of bacteria in their natural habitats is affected by water availability. In this work, we have examined the activity and effector specificity of the transcriptional regulator XylR of the TOL plasmid pWW0 of Pseudomonas putida mt-2 for biodegradation of m-xylene when external water potential was manipulated with polyethylene glycol PEG8000. By using non-disruptive luxCDEAB reporter technology, we noticed that the promoter activated by XylR (Pu) restricted its activity and the regulator became more effector-specific towards head TOL substrates when cells were grown under water subsaturation. Such a tight specificity brought about by water limitation was relaxed when intracellular osmotic stress was counteracted by the external addition of the compatible solute glycine betaine. With these facts in hand, XylR variants isolated earlier as effector-specificity responders to the non-substrate 1,2,4-trichlorobenzene under high matric stress were re-examined and found to be unaffected by water potential in vivo. All these phenomena could be ultimately explained as the result of water potential-dependent conformational changes in the A domain of XylR and its effector-binding pocket, as suggested by AlphaFold prediction of protein structures. The consequences of this scenario for the evolution of specificities in regulators and the emergence of catabolic pathways are discussed.

The long journey towards standards for engineering biosystems: Are the Molecular Biology and the Biotech communities ready to standardise?

Synthetic biology needs to adopt sound scientific and industry-like standards in order to achieve its ambitious goals of efficient and accurate engineering of biological systems.

SEVA 3.0: an update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts.

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 faulty SOS response of Pseudomonas putida KT2440 stems from an inefficient RecA-LexA interplay.

Despite its environmental robustness Pseudomonas putida strain KT2440 is very sensitive to DNA damage and displays poor homologous recombination efficiencies. To gain an insight into this deficiency isogenic ∆recA and ∆lexA1 derivatives of prophage-free strain P. putida EM173 were generated and responses of the recA and lexA1 promoters to DNA damage tested with GFP reporter technology. Basal expression of recA and lexA1 of P. putida were high in the absence of DNA damage and only moderately induced by norfloxacin. A similar behaviour was observed when equivalent GFP fusions to the recA and lexA promoters of E. coli were placed in P. putida EM173. In contrast, all SOS promoters were subject to strong repression in E. coli, which was released only when cells were treated with the antibiotic. Replacement of P. putida's native LexA1 and RecA by E. coli homologues did not improve the responsiveness of the indigenous functions to DNA damage. Taken together, it seems that P. putida fails to mount a strong SOS response due to the inefficacy of the crucial RecA-LexA interplay largely tractable to the weakness of the corresponding promoters and the inability of the repressor to shut them down entirely in the absence of DNA damage.

An updated structural model of the A domain of the Pseudomonas putida XylR regulator poses an atypical interplay with aromatic effectors.

A revised model of the aromatic binding A domain of the σ54 -dependent regulator XylR of Pseudomonas putida mt-2 was produced based on the known 3D structures of homologous regulators PoxR, MopR and DmpR. The resulting frame was instrumental for mapping a number of mutations known to alter effector specificity, which were then reinterpreted under a dependable spatial reference. Some of these changes involved the predicted aromatic binding pocket but others occurred in distant locations, including dimerization interfaces and putative zinc binding site. The effector pocket was buried within the protein structure and accessible from the outside only through a narrow tunnel. Yet, several loop regions of the A domain could provide the flexibility required for widening such a tunnel for passage of aromatic ligands. The model was experimentally validated by treating the cells in vivo and the purified protein in vitro with benzyl bromide, which reacts with accessible nucleophilic residues on the protein surface. Structural and proteomic analyses confirmed the predicted in/out distribution of residues but also supported two additional possible scenarios of interaction of the A domain with aromatic effectors: a dynamic interaction of the fully structured yet flexible protein with the aromatic partner and/or inducer-assisted folding of the A domain.

Transcriptional control of 2,4-dinitrotoluene degradation in Burkholderia sp. R34 bears a regulatory patch that eases pathway evolution.

The dnt pathway of Burkholderia sp. R34 is in the midst of an evolutionary journey from its ancestral, natural substrate (naphthalene) towards a new xenobiotic one [2,4-dinitrotoluene (DNT)]. The gene cluster encoding the leading multicomponent ring dioxygenase (DntA) has activity on the old and the new substrate, but it is induced by neither. Instead, the transcriptional factor encoded by the adjacent gene (dntR) activates expression of the dnt cluster upon addition of salicylate, one degradation intermediate of the ancestral naphthalene route but not any longer a substrate/product of the evolved DntA enzyme. Fluorescence of cells bearing dntA-gfp fusions revealed that induction of the dnt genes by salicylate was enhanced upon exposure to bona fide DntA substrates, i.e., naphthalene or DNT. Such amplification was dependent on effective dioxygenation of these pathway-specific head compounds, which thereby fostered expression of the cognate catabolic operon. The phenomenon seems to happen not through direct binding to a cognate transcriptional factor but through the interplay of a non-specific regulator with a substrate-specific enzyme. This regulatory scenario may ease transition of complete catabolic operons (i.e. enzymes plus regulatory devices) from one substrate to another without loss of fitness during the evolutionary roadmap between two optimal specificities.

Ribonucleases control distinct traits of Pseudomonas putida lifestyle.

The role of archetypal ribonucleases (RNases) in the physiology and stress endurance of the soil bacterium and metabolic engineering platform Pseudomonas putida KT2440 has been inspected. To this end, variants of this strain lacking each of the most important RNases were constructed. Each mutant lacked either one exoribonuclease (PNPase, RNase R) or one endoribonuclease (RNase E, RNase III, RNase G). The global physiological and metabolic costs of the absence of each of these enzymes were then analysed in terms of growth, motility and morphology. The effects of different oxidative chemicals that mimic the stresses endured by this microorganism in its natural habitats were studied as well. The results highlighted that each ribonuclease is specifically related with different traits of the environmental lifestyle that distinctively characterizes this microorganism. Interestingly, the physiological responses of P. putida to the absence of each enzyme diverged significantly from those known previously in Escherichia coli. This exposed not only species-specific regulatory functions for otherwise known RNase activities but also expanded the panoply of post-transcriptional adaptation devices that P. putida can make use of for facing hostile environments.

Low CyaA expression and anti-cooperative binding of cAMP to CRP frames the scope of the cognate regulon of Pseudomonas putida.

Although the soil bacterium Pseudomonas putida KT2440 bears a bona fide adenylate cyclase gene (cyaA), intracellular concentrations of 3',5'-cyclic adenosine monophosphate (cAMP) are barely detectable. By using reporter technology and direct quantification of cAMP under various conditions, we show that such low levels of the molecule stem from the stringent regulation of its synthesis, efflux and degradation. Poor production of cAMP was the result of inefficient translation of cyaA mRNA. Moreover, deletion of the cAMP-phosphodiesterase pde gene led to intracellular accumulation of the cyclic nucleotide, exposing an additional cause of cAMP drain in vivo. But even such low levels of the signal sustained activation of promoters dependent on the cAMP-receptor protein (CRP). Genetic and biochemical evidence indicated that the phenomenon ultimately rose from the unusual binding parameters of cAMP to CRP. This included an ultratight cAMP-CrpP. putida affinity (KD of 45.0 ± 3.4 nM) and an atypical 1:1 effector/dimer stoichiometry that obeyed an infrequent anti-cooperative binding mechanism. It thus seems that keeping the same regulatory parts and their relational logic but changing the interaction parameters enables genetic devices to take over entirely different domains of the functional landscape.

Picking the right metaphors for addressing microbial systems: economic theory helps understanding biological complexity.

Any descriptive language is necessarily metaphoric and interpretative. Two somewhat overlapping-but not identical-languages have been thoroughly employed in the last decade to address the issue of regulatory complexity in biological systems: the terminology of network theory and the jargon of electric circuitry. These approaches have found many formal equivalences between the layout of extant genetic circuits and the architecture of man-made counterparts. However, these languages still fail to describe accurately key features of biological objects, in particular the diversity of signal-transfer molecules and the diffusion that is inherent to any biochemical system. Furthermore, current formalisms associated with networks and circuits can hardly face the problem of multi-scale regulatory complexity-from single molecules to entire ecosystems. We argue that the language of economic theory might be instrumental not only to portray accurately many features of regulatory networks, but also to unveil aspects of the biological complexity problem that remain opaque to other types of analyses. The main perspective opened by the economic metaphor when applied to control of microbiological activities is a focus on metabolism, not gene selfishness, as the necessary background to make sense of regulatory phenomena. As an example, we analyse and reinterpret the widespread phenomenon of catabolite repression with the formal frame of the consumer's choice theory.

Exploiting geometric similarity for statistical quantification of fluorescence spatial patterns in bacterial colonies.

BACKGROUND: Currently the combination of molecular tools, imaging techniques and analysis software offer the possibility of studying gene activity through the use of fluorescent reporters and infer its distribution within complex biological three-dimensional structures. For example, the use of Confocal Scanning Laser Microscopy (CSLM) is a regularly-used approach to visually inspect the spatial distribution of a fluorescent signal. Although a plethora of generalist imaging software is available to analyze experimental pictures, the development of tailor-made software for every specific problem is still the most straightforward approach to perform the best possible image analysis. In this manuscript, we focused on developing a simple methodology to satisfy one particular need: automated processing and analysis of CSLM image stacks to generate 3D fluorescence profiles showing the average distribution detected in bacterial colonies grown in different experimental conditions for comparison purposes. RESULTS: The presented method processes batches of CSLM stacks containing three-dimensional images of an arbitrary number of colonies. Quasi-circular colonies are identified, filtered and projected onto a normalized orthogonal coordinate system, where a numerical interpolation is performed to obtain fluorescence values within a spatially fixed grid. A statistically representative three-dimensional fluorescent pattern is then generated from this data, allowing for standardized fluorescence analysis regardless of variability in colony size. The proposed methodology was evaluated by analyzing fluorescence from GFP expression subject to regulation by a stress-inducible promoter. CONCLUSIONS: This method provides a statistically reliable spatial distribution profile of fluorescence detected in analyzed samples, helping the researcher to establish general correlations between gene expression and spatial allocation under differential experimental regimes. The described methodology was coded into a MATLAB script and shared under an open source license to make it accessible to the whole community.

The Wsp intermembrane complex mediates metabolic control of the swim-attach decision of Pseudomonas putida.

Pseudomonas putida is a microorganism of biotechnological interest that-similar to many other environmental bacteria-adheres to surfaces and forms biofilms. Although various mechanisms contributing to the swim-attach decision have been studied in this species, the role of a 7-gene operon homologous to the wsp cluster of Pseudomonas aeruginosa-which regulates cyclic di-GMP (cdGMP) levels upon surface contact-remained to be investigated. In this work, the function of the wsp operon of P. putida KT2440 has been characterized through inspection of single and multiple wsp deletion variants, complementation with Pseudomonas aeruginosa's homologues, combined with mutations of regulatory genes fleQ and fleN and removal of the flagellar regulator fglZ. The ability of the resulting strains to form biofilms at 6 and 24 h under three different carbon regimes (citrate, glucose and fructose) revealed that the Wsp complex delivers a similar function to both Pseudomonas species. In P. putida, the key components include WspR, a protein that harbours the domain for producing cdGMP, and WspF, which controls its activity. These results not only contribute to a deeper understanding of the network that regulates the sessile-planktonic decision of P. putida but also suggest strategies to exogenously control such a lifestyle switch.

ArsH protects Pseudomonas putida from oxidative damage caused by exposure to arsenic.

The two As resistance arsRBC operons of Pseudomonas putida KT2440 are followed by a downstream gene called arsH that encodes an NADPH-dependent flavin mononucleotide reductase. In this work, we show that the arsH1 and (to a lesser extent) arsH2 genes of P. putida KT2440 strengthened its tolerance to both inorganic As(V) and As(III) and relieved the oxidative stress undergone by cells exposed to either oxyanion. Furthermore, overexpression of arsH1 and arsH2 endowed P. putida with a high tolerance to the oxidative stress caused by diamide (a drainer of metabolic NADPH) in the absence of any arsenic. To examine whether the activity of ArsH was linked to a direct action on the arsenic compounds tested, arsH1 and arsH2 genes were expressed in Escherichia coli, which has an endogenous arsRBC operon but lacks an arsH ortholog. The resulting clones both deployed a lower production of reactive oxygen species (ROS) when exposed to As salts and had a superior endurance to physiological redox insults. These results suggest that besides the claimed direct action on organoarsenicals, ArsH contributes to relieve toxicity of As species by mediating reduction of ROS produced in vivo upon exposure to the oxyanion, e.g. by generating FMNH2 to fuel ROS-quenching activities.

Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene.

The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. In this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast's URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutLE36K ). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G < C:C < G:A < C:A, A:A, G:G, T:T, T:G, A:C, C:T < G:T, T:C as the one prevalent in Pseudomonas putida. This information is vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.

Environmental Performance of Pseudomonas putida with a Uracylated Genome.

A variant of the soil bacterium Pseudomonas putida with a genome containing a ∼20 % replacement of the whole of thymine (T) by uracil (U) was made by deleting genes ung (uracil DNA glycosylase) and dut (deoxyuridine 5'-triphosphate nucleotide hydrolase). Proteomic comparisons revealed that, of 281 up-regulated and 96 down-regulated proteins in the Δung Δdut cells, as compared to the wild-type, many were involved in nucleotide metabolism. Unexpectedly, genome uracylation did not greatly change the gross environmental endurance profile of P. putida, increased spontaneous mutagenesis by only twofold and supported expression of heterologous proteins well. As U-enriched DNA is potentially degraded by the base excision repair of recipients encoding a uracil DNA glycosylase, we then tested the spread potential of genetic material originating in the Δung Δdut cells either within the same species or in a commonly used Escherichia coli strain. Transformation and conjugation experiments revealed that horizontal gene transfer of U-containing plasmids fared worse than those made of standard DNA by two orders of magnitude. Although this figure does not guarantee the certainty of containment, it suggests a general strategy for curbing the dispersal of recombinant genetic constructs.

Digitalizing heterologous gene expression in Gram-negative bacteria with a portable ON/OFF module.

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.

The imbroglio of the physiological Cra effector clarified at last.

Owing to its role in controlling carbon and energy metabolism, the catabolite repressor/activator protein Cra has been one of the most studied prokaryotic regulators of the last 30 years. Yet, a key mechanistic detail of its biological function - i.e. the nature of the metabolic effector that rules its DNA-binding ability - has remained controversial. Despite the high affinity of Cra for fructose-1-phosphate (F1P), the prevailing view claimed that fructose-1,6-biphosphate (FBP) was the key physiological effector. Building on such responsiveness to FBP, Cra was proposed to act as a glycolytic flux sensor and central regulator of critical metabolic transactions. At the same time, data raised on the Cra protein of Pseudomonas putida ruled out that FBP could be an effector - but instead suggested that it was the unintentional carrier of a small contamination by F1P, the actual signal molecule. While these data on the P. putida Cra were received with skepticism - if not dismissal - by the community of the time, the paper by (Bley-Folly et al, 2018) now demonstrates beyond any reasonable doubt that the one and only effector of E. coli Cra is F1P and that every action of FBP on this regulator can be traced to its systematic mix with the authentic binder.

Evolving metabolism of 2,4-dinitrotoluene triggers SOS-independent diversification of host cells.

The molecular mechanisms behind the mutagenic effect of reactive oxygen species (ROS) released by defective metabolization of xenobiotic 2,4-dinitrotoluene (DNT) by a still-evolving degradation pathway were studied. To this end, the genes required for biodegradation of DNT from Burkholderia cepacia R34 were implanted in Escherichia coli and the effect of catabolizing the nitroaromatic compound monitored with stress-related markers and reporters. Such a proxy of the naturally-occurring scenario faithfully recreated the known accumulation of ROS caused by faulty metabolism of DNT and the ensuing onset of an intense mutagenesis regime. While ROS triggered an oxidative stress response, neither homologous recombination was stimulated nor the recA promoter activity increased during DNT catabolism. Analysis of single-nucleotide changes occurring in rpoB during DNT degradation suggested a relaxation of DNA replication fidelity rather than direct damage to DNA. Mutants frequencies were determined in strains defective in either converting DNA damage into mutagenesis or mediating inhibition of mismatch repair through a general stress response. The results revealed that the mutagenic effect of ROS was largely SOS-independent and stemmed instead from stress-induced changes of rpoS functionality. Evolution of novel metabolic properties thus resembles the way sublethal antibiotic concentrations stimulate the appearance of novel resistance genes.