Novel approaches to energize microbial biocatalysts.

An efficient and cheap energization of microbial biocatalysts is essential in current biotechnological processes. A promising alternative to the use of common organic or inorganic electron donors is the semiconductor nanoparticles (SNs) that absorb light and transfer electrons (photoelectrons) behaving as artificial photosynthetic systems (biohybrid systems). Excited photoelectrons generated by illuminated SNs are highly reductive and readily accepted by membrane-bound proteins and electron shuttles to drive specific cell reduction processes and energy generation in microbes. However, the operational mechanisms of these hybrid systems are still poorly understood, especially at the material-microbe interface, and therefore the design and production of efficient biohybrids are challenging. Some major limitations/challenges and future prospects of SNs as microbial energization systems are discussed.

A Bifan Motif Shaped by ArsR1, ArsR2, and Their Cognate Promoters Frames Arsenic Tolerance of Pseudomonas putida.

Prokaryotic tolerance to inorganic arsenic is a widespread trait habitually determined by operons encoding an As (III)-responsive repressor (ArsR), an As (V)-reductase (ArsC), and an As (III)-export pump (ArsB), often accompanied by other complementary genes. Enigmatically, the genomes of many environmental bacteria typically contain two or more copies of this basic genetic device arsRBC. To shed some light on the logic of such apparently unnecessary duplication(s) we have inspected the regulation-together and by separate-of the two ars clusters borne by the soil bacterium Pseudomonas putida strain KT2440, in particular the cross talk between the two repressors ArsR1/ArsR2 and the respective promoters. DNase I footprinting and gel retardation analyses of Pars1 and Pars2 with their matching regulators revealed non-identical binding sequences and interaction patterns for each of the systems. However, in vitro transcription experiments exposed that the repressors could downregulate each other's promoters, albeit within a different set of parameters. The regulatory frame that emerges from these data corresponds to a particular type of bifan motif where all key interactions have a negative sign. The distinct regulatory architecture that stems from coexistence of various ArsR variants in the same cells could enter an adaptive advantage that favors the maintenance of the two proteins as separate repressors.

A SsrA/NIa-based Strategy for Post-Translational Regulation of Protein Levels in Gram-negative Bacteria.

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. Graphical abstract.

ArxA From Azoarcus sp. CIB, an Anaerobic Arsenite Oxidase From an Obligate Heterotrophic and Mesophilic Bacterium.

Arsenic is a toxic element widely distributed in nature, but numerous bacteria are able to resist its toxicity mainly through the ars genes encoding an arsenate reductase and an arsenite efflux pump. Some "arsenotrophic" bacteria are also able to use arsenite as energy supplier during autotrophic growth by coupling anaerobic arsenite oxidation via the arx gene products to nitrate respiration or photosynthesis. Here, we have demonstrated that Azoarcus sp. CIB, a facultative anaerobic ß-proteobacterium, is able to resist arsenic oxyanions both under aerobic and anaerobic conditions. Genome mining, gene expression, and mutagenesis studies revealed the presence of a genomic island that harbors the ars and arx clusters involved in arsenic resistance in strain CIB. Orthologous ars clusters are widely distributed in the genomes of sequenced Azoarcus strains. Interestingly, genetic and metabolic approaches showed that the arx cluster of the CIB strain encodes an anaerobic arsenite oxidase also involved in the use of arsenite as energy source. Hence, Azoarcus sp. CIB represents the prototype of an obligate heterotrophic bacterium able to use arsenite as an extra-energy source for anaerobic cell growth. The arsenic island of strain CIB supports the notion that metabolic and energetic skills can be gained by genetic mobile elements.

Further Insights into the Architecture of the PN Promoter That Controls the Expression of the bzd Genes in Azoarcus.

The anaerobic degradation of benzoate in bacteria involves the benzoyl-CoA central pathway. Azoarcus/Aromatoleum strains are a major group of anaerobic benzoate degraders, and the transcriptional regulation of the bzd genes was extensively studied in Azoarcus sp. CIB. In this work, we show that the bzdR regulatory gene and the PN promoter can also be identified upstream of the catabolic bzd operon in all benzoate-degrader Azoarcus/Aromatoleum strains whose genome sequences are currently available. All the PN promoters from Azoarcus/Aromatoleum strains described here show a conserved architecture including three operator regions (ORs), i.e., OR1 to OR3, for binding to the BzdR transcriptional repressor. Here, we demonstrate that, whereas OR1 is sufficient for the BzdR-mediated repression of the PN promoter, the presence of OR2 and OR3 is required for de-repression promoted by the benzoyl-CoA inducer molecule. Our results reveal that BzdR binds to the PN promoter in the form of four dimers, two of them binding to OR1. The BzdR/PN complex formed induces a DNA loop that wraps around the BzdR dimers and generates a superstructure that was observed by atomic force microscopy. This work provides further insights into the existence of a conserved BzdR-dependent mechanism to control the expression of the bzd genes in Azoarcus strains.

A Novel Redox-Sensing Histidine Kinase That Controls Carbon Catabolite Repression in Azoarcus sp. CIB.

We have identified and characterized the AccS multidomain sensor kinase that mediates the activation of the AccR master regulator involved in carbon catabolite repression (CCR) of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB. A truncated AccS protein that contains only the soluble C-terminal autokinase module (AccS') accounts for the succinate-dependent CCR control. In vitro assays with purified AccS' revealed its autophosphorylation, phosphotransfer from AccS'∼P to the Asp60 residue of AccR, and the phosphatase activity toward its phosphorylated response regulator, indicating that the equilibrium between the kinase and phosphatase activities of AccS' may control the phosphorylation state of the AccR transcriptional regulator. Oxidized quinones, e.g., ubiquinone 0 and menadione, switched the AccS' autokinase activity off, and three conserved Cys residues, which are not essential for catalysis, are involved in such inhibition. Thiol oxidation by quinones caused a change in the oligomeric state of the AccS' dimer resulting in the formation of an inactive monomer. This thiol-based redox switch is tuned by the cellular energy state, which can change depending on the carbon source that the cells are using. This work expands the functional diversity of redox-sensitive sensor kinases, showing that they can control new bacterial processes such as CCR of the anaerobic catabolism of aromatic compounds. The AccSR two-component system is conserved in the genomes of some betaproteobacteria, where it might play a more general role in controlling the global metabolic state according to carbon availability.IMPORTANCE Two-component signal transduction systems comprise a sensor histidine kinase and its cognate response regulator, and some have evolved to sense and convert redox signals into regulatory outputs that allow bacteria to adapt to the altered redox environment. The work presented here expands knowledge of the functional diversity of redox-sensing kinases to control carbon catabolite repression (CCR), a phenomenon that allows the selective assimilation of a preferred compound among a mixture of several carbon sources. The newly characterized AccS sensor kinase is responsible for the phosphorylation and activation of the AccR master regulator involved in CCR of the anaerobic degradation of aromatic compounds in the betaproteobacterium Azoarcus sp. CIB. AccS seems to have a thiol-based redox switch that is modulated by the redox state of the quinone pool. The AccSR system is conserved in several betaproteobacteria, where it might play a more general role controlling their global metabolic state.

A Post-translational Metabolic Switch Enables Complete Decoupling of Bacterial Growth from Biopolymer Production in Engineered Escherichia coli.

Most of the current methods for controlling the formation rate of a key protein or enzyme in cell factories rely on the manipulation of target genes within the pathway. In this article, we present a novel synthetic system for post-translational regulation of protein levels, FENIX, which provides both independent control of the steady-state protein level and inducible accumulation of target proteins. The FENIX device is based on the constitutive, proteasome-dependent degradation of the target polypeptide by tagging with a short synthetic, hybrid NIa/SsrA amino acid sequence in the C-terminal domain. Protein production is triggered via addition of an orthogonal inducer ( i.e., 3-methylbenzoate) to the culture medium. The system was benchmarked in Escherichia coli by tagging two fluorescent proteins (GFP and mCherry), and further exploited to completely uncouple poly(3-hydroxybutyrate) (PHB) accumulation from bacterial growth. By tagging PhaA (3-ketoacyl-CoA thiolase, first step of the route), a dynamic metabolic switch at the acetyl-coenzyme A node was established in such a way that this metabolic precursor could be effectively redirected into PHB formation upon activation of the system. The engineered E. coli strain reached a very high specific rate of PHB accumulation (0.4 h-1) with a polymer content of ca. 72% (w/w) in glucose cultures in a growth-independent mode. Thus, FENIX enables dynamic control of metabolic fluxes in bacterial cell factories by establishing post-translational synthetic switches in the pathway of interest.

Refactoring the λ phage lytic/lysogenic decision with a synthetic regulator.

In this work, we explore the refactoring of the circuitry of λ phage by engineering a new-to-nature regulator that responds to an ad hoc input signal that behaves orthogonal with respect to the host cell. We tailored a chimeric regulator, termed Qλ, between the CI protein of the λ phage and the BzdR repressor from Azoarcus sp. strain CIB that responds to benzoyl-CoA. When the Qλ was expressed in the appropriate Escherichia coli cells, it was able to reprogram the lytic/lysogenic λ phage decision according to the intracellular production of benzoyl-CoA. Our results are also an example of how generating new artificial regulators that respond to effectors of choice may be useful to control different cellular processes.

Functional coexistence of twin arsenic resistance systems in Pseudomonas putida†KT2440.

The genome of the soil bacterium Pseudomonas putida KT2440 bears two virtually identical arsRBCH operons putatively encoding resistance to inorganic arsenic species. Single and double chromosomal deletions in each of these ars clusters of this bacterium were tested for arsenic sensitivity and found that the contribution of each operon to the resistance to the metalloid was not additive, as either cluster sufficed to endow cells with high-level resistance. However, otherwise identical traits linked to each of the ars sites diverged when temperature was decreased. Growth of the various mutants at 15°C (instead of the standard 30°C for P. putida) uncovered that ars2 affords a much higher resistance to As (III) than the ars1 counterpart. Reverse transcription polymerase chain reaction of arsB1 and arsB2 genes as well as lacZ fusions to the Pars1 and Pars2 promoters traced the difference to variations in transcription of the corresponding gene sets at each temperature. Functional redundancy may thus be selected as a stable condition - rather than just as transient state - if it affords one key activity to be expressed under a wider range of physicochemical settings. This seems to provide a straightforward solution to regulatory problems in environmental bacteria that thrive under changing scenarios.

Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida.

Fructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (Cra(PP)) was submitted to mobility shift assays with target DNA sequences (the PfruB promoter) and candidate effectors fructose-1,6-bisphosphate (FBP), glucose 6-phosphate (G6P), and fructose-6-phosphate (F6P). 1 mM F1P was sufficient to release most of the Cra protein from its operators but more than 10 mM of FBP or G6P was required to free the same complex. However, isothermal titration microcalorimetry failed to expose any specific interaction between Cra(PP) and FBP or G6P. To solve this paradox, transcriptional activity of a PfruB-lacZ fusion was measured in wild-type and ΔfruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that PfruB activity was stimulated by fructose but not by glucose or succinate. This suggested that Cra(PP) represses expression in vivo of the cognate fruBKA operon in a fashion dependent just on F1P, ruling out any other physiological effector. Molecular docking and dynamic simulations of the Cra-agonist interaction indicated that both metabolites can bind the repressor, but the breach in the relative affinity of Cra(PP) for F1P vs FBP is three orders of magnitude larger than the equivalent distance in the Escherichia coli protein. This assigns the Cra protein of P. putida the sole role of transducing the presence of fructose in the medium into a variety of direct and indirect physiological responses.

The Standard European Vector Architecture (SEVA) plasmid toolkit.

The Standard European Vector Architecture (SEVA) toolkit is a simple and powerful resource for constructing optimal plasmid vectors based on a backbone and three interchangeable modules flanked by uncommon restriction sites. Functional modules encode several origins of replication, diverse antibiotic selection markers, and a variety of cargoes with different applications. The backbone and DNA modules have been minimized and edited for flaws in their sequence and/or functionality. A protocol for the utilization of the SEVA platform to construct transcriptional and translational fusions between a promoter under study (the arsenic-responsive Pars of Pseudomonas putida KT2440) and the reporter lacZ gene is described. The resulting plasmid collection was instrumental to measure and compare the ß-galactosidase activity that report gene expression (i.e., transcription and translation) in different genetic backgrounds.

Identification of a missing link in the evolution of an enzyme into a transcriptional regulator.

The evolution of transcriptional regulators through the recruitment of DNA-binding domains by enzymes is a widely held notion. However, few experimental approaches have directly addressed this hypothesis. Here we report the reconstruction of a plausible pathway for the evolution of an enzyme into a transcriptional regulator. The BzdR protein is the prototype of a subfamily of prokaryotic transcriptional regulators that controls the expression of genes involved in the anaerobic degradation of benzoate. We have shown that BzdR consists of an N-terminal DNA-binding domain connected through a linker to a C-terminal effector-binding domain that shows significant identity to the shikimate kinase (SK). The construction of active synthetic BzdR-like regulators by fusing the DNA-binding domain of BzdR to the Escherichia coli SKI protein strongly supports the notion that an ancestral SK domain could have been involved in the evolutionary origin of BzdR. The loss of the enzymatic activity of the ancestral SK domain was essential for it to evolve as a regulatory domain in the current BzdR protein. This work also supports the view that enzymes precede the emergence of the regulatory systems that may control their expression.

Taxonomy becoming a driving force in genome sequencing projects.

We studied the possible impact of genomic projects by comparing the number of published articles before and after the completion of the project. We found that for most species, there is no significant change in the number of citations. Also our study remarks the growing importance of taxonomy as main motivation for the sequencing of genomes.

The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes.

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

Bacterial degradation of benzoate: cross-regulation between aerobic and anaerobic pathways.

We have studied for the first time the transcriptional regulatory circuit that controls the expression of the box genes encoding the aerobic hybrid pathway used to assimilate benzoate via coenzyme A (CoA) derivatives in bacteria. The promoters responsible for the expression of the box cluster in the ß-proteobacterium Azoarcus sp., their cognate transcriptional repressor, the BoxR protein, and the inducer molecule (benzoyl-CoA) have been characterized. The BoxR protein shows a significant sequence identity to the BzdR transcriptional repressor that controls the bzd genes involved in the anaerobic degradation of benzoate. Because the boxR gene is present in all box clusters so far identified in bacteria, the BoxR/benzoyl-CoA regulatory system appears to be a widespread strategy to control this aerobic hybrid pathway. Interestingly, the paralogous BoxR and BzdR regulators act synergistically to control the expression of the box and bzd genes. This cross-regulation between anaerobic and aerobic pathways for the catabolism of aromatic compounds has never been shown before, and it may reflect a biological strategy to increase the cell fitness in organisms that survive in environments subject to changing oxygen concentrations.

The Crp regulator of Pseudomonas putida: evidence of an unusually high affinity for its physiological effector, cAMP.

Although the genome of Pseudomonas putida KT2440 encodes an orthologue of the crp gene of Escherichia coli (encoding the cAMP receptor protein), the regulatory scope of this factor seems to be predominantly co-opted in this bacterium for controlling non-metabolic functions. In order to investigate the reasons for such a functional divergence in otherwise nearly identical proteins, the Crp regulator of P. putida (Crp(P. putida)) was purified to apparent homogeneity and subject to a battery of in vitro assays aimed at determining its principal physicochemical properties. Analytical ultracentrifugation indicated effector-free Crp(P. putida) to be a dimer in solution that undergoes a significant change in its hydrodynamic shape in the presence of cAMP. Such a conformational transition was confirmed by limited proteolysis of the protein in the absence or presence of the inducer. Thermodynamic parameters calculated by isothermal titration calorimetry revealed a tight cAMP-Crp(P. putida) association with an apparent K(D) of 22.5 ± 2.8 nM, i.e. much greater affinity than that reported for the E. coli's counterpart. The regulator also bound cGMP, but with a K(D) = 2.6 ± 0.3 µM. An in vitro transcription system was then set up with purified P. putida's RNA polymerase for examining the preservation of the correct protein-protein architecture that makes Crp to activate target promoters. These results, along with cognate gel retardation assays indicated that all basic features of the reference Crp(E. coli) protein are kept in the P. putida's counterpart, albeit operating under a different set of parameters, the extraordinarily high affinity for cAMP being the most noticeable.

Biochemical characterization of the transcriptional regulator BzdR from Azoarcus sp. CIB.

The BzdR transcriptional regulator that controls the P(N) promoter responsible for the anaerobic catabolism of benzoate in Azoarcus sp. CIB constitutes the prototype of a new subfamily of transcriptional regulators. Here, we provide some insights about the functional-structural relationships of the BzdR protein. Analytical ultracentrifugation studies revealed that BzdR is homodimeric in solution. An electron microscopy three-dimensional reconstruction of the BzdR dimer has been obtained, and the predicted structures of the respective N- and C-terminal domains of each BzdR monomer could be fitted into such a reconstruction. Gel retardation and ultracentrifugation experiments have shown that the binding of BzdR to its cognate promoter is cooperative. Different biochemical approaches revealed that the effector molecule benzoyl-CoA induces conformational changes in BzdR without affecting its oligomeric state. The BzdR-dependent inhibition of the P(N) promoter and its activation in the presence of benzoyl-CoA have been established by in vitro transcription assays. The monomeric BzdR4 and BzdR5 mutant regulators revealed that dimerization of BzdR is essential for DNA binding. Remarkably, a BzdRΔL protein lacking the linker region connecting the N- and C-terminal domains of BzdR is also dimeric and behaves as a super-repressor of the P(N) promoter. These data suggest that the linker region of BzdR is not essential for protein dimerization, but rather it is required to transfer the conformational changes induced by the benzoyl-CoA to the DNA binding domain leading to the release of the repressor. A model of action of the BzdR regulator has been proposed.

Anaerobic catabolism of aromatic compounds: a genetic and genomic view.

Aromatic compounds belong to one of the most widely distributed classes of organic compounds in nature, and a significant number of xenobiotics belong to this family of compounds. Since many habitats containing large amounts of aromatic compounds are often anoxic, the anaerobic catabolism of aromatic compounds by microorganisms becomes crucial in biogeochemical cycles and in the sustainable development of the biosphere. The mineralization of aromatic compounds by facultative or obligate anaerobic bacteria can be coupled to anaerobic respiration with a variety of electron acceptors as well as to fermentation and anoxygenic photosynthesis. Since the redox potential of the electron-accepting system dictates the degradative strategy, there is wide biochemical diversity among anaerobic aromatic degraders. However, the genetic determinants of all these processes and the mechanisms involved in their regulation are much less studied. This review focuses on the recent findings that standard molecular biology approaches together with new high-throughput technologies (e.g., genome sequencing, transcriptomics, proteomics, and metagenomics) have provided regarding the genetics, regulation, ecophysiology, and evolution of anaerobic aromatic degradation pathways. These studies revealed that the anaerobic catabolism of aromatic compounds is more diverse and widespread than previously thought, and the complex metabolic and stress programs associated with the use of aromatic compounds under anaerobic conditions are starting to be unraveled. Anaerobic biotransformation processes based on unprecedented enzymes and pathways with novel metabolic capabilities, as well as the design of novel regulatory circuits and catabolic networks of great biotechnological potential in synthetic biology, are now feasible to approach.

New insights into the BzdR-mediated transcriptional regulation of the anaerobic catabolism of benzoate in Azoarcus sp. CIB.

The expression of the bzd genes involved in the anaerobic degradation of benzoate in Azoarcus sp. CIB is controlled by the specific BzdR transcriptional repressor at the P(N) promoter. This catabolic promoter is also subject to catabolite repression by some organic acids. In vivo and in vitro experiments have shown that BzdR behaves as a repressor of the P(R) promoter by overlapping the transcription initiation site as well as the -35 and -10 boxes, benzoyl-CoA being the inducer molecule. In addition, by using a P(N) : : lacZ fusion both in Azoarcus sp. CIB and in an isogenic strain lacking the bzdR gene, we have shown that the succinate-dependent catabolite repression requires participation of the BzdR repressor.