José I. Jiménez

Aerobic degradation of aromatic compounds

Our view on the bacterial responses to the aerobic degradation of aromatic compounds has been enriched considerably by the current omic methodologies and systems biology approaches, revealing the participation of intricate metabolic and regulatory networks. New enzymes, transporters, and specific/global regulatory systems have been recently characterized, and reveal that the widespread biodegradation capabilities extend to unexpected substrates such as lignin. A completely different biochemical strategy based on the formation of aryl-CoA epoxide intermediates has been unraveled for aerobic hybrid pathways, such as those involved in benzoate and phenylacetate degradation. Aromatic degradation pathways are also an important source of metabolic exchange factors and, therefore, they play a previously unrecognized biological role in cell-to-cell communication. Beyond the native bacterial biodegradation capabilities, pathway evolution as well as computational and synthetic biology approaches are emerging as powerful tools to design novel strain-specific pathways for degradation of xenobiotic compounds.

Deciphering the genetic determinants for aerobic nicotinic acid degradation: The nic cluster from Pseudomonas putida KT2440

The aerobic catabolism of nicotinic acid (NA) is considered a model system for degradation of N-heterocyclic aromatic compounds, some of which are major environmental pollutants; however, the complete set of genes as well as the structural–functional relationships of most of the enzymes involved in this process are still unknown. We have characterized a gene cluster (nic genes) from Pseudomonas putida KT2440 responsible for the aerobic NA degradation in this bacterium and when expressed in heterologous hosts. The biochemistry of the NA degradation through the formation of 2,5-dihydroxypyridine and maleamic acid has been revisited, and some gene products become the prototype of new types of enzymes with unprecedented molecular architectures. Thus, the initial hydroxylation of NA is catalyzed by a two-component hydroxylase (NicAB) that constitutes the first member of the xanthine dehydrogenase family whose electron transport chain to molecular oxygen includes a cytochrome c domain. The Fe2+-dependent dioxygenase (NicX) converts 2,5-dihydroxypyridine into N-formylmaleamic acid, and it becomes the founding member of a new family of extradiol ring-cleavage dioxygenases. Further conversion of N-formylmaleamic acid to formic and maleamic acid is catalyzed by the NicD protein, the only deformylase described so far whose catalytic triad is similar to that of some members of the α/β-hydrolase fold superfamily. This work allows exploration of the existence of orthologous gene clusters in saprophytic bacteria and some pathogens, where they might stimulate studies on their role in virulence, and it provides a framework to develop new biotechnological processes for detoxification/biotransformation of N-heterocyclic aromatic compounds.

Comprehensive experimental fitness landscape and evolutionary network for small RNA

Significance Evolution by natural selection is driven by fitness differences, which define a “fitness landscape” in the space of all possible genetic sequences. Understanding the landscape is critical for understanding and predicting natural selection, yet very little is known about the structure of a real fitness landscape. Here we experimentally determine the complete fitness landscape of small RNA selected to interact with GTP, a building block for early life. We find that the landscape is composed of largely disconnected islands of active sequences. This scenario suggests that natural selection under these conditions would be constrained to local exploration of sequence space but that replaying the initial emergence of a functional RNA could lead to a different outcome. The origin of life is believed to have progressed through an RNA world, in which RNA acted as both genetic material and functional molecules. The structure of the evolutionary fitness landscape of RNA would determine natural selection for the first functional sequences. Fitness landscapes are the subject of much speculation, but their structure is essentially unknown. Here we describe a comprehensive map of a fitness landscape, exploring nearly all of sequence space, for short RNAs surviving selection in vitro. With the exception of a small evolutionary network, we find that fitness peaks are largely isolated from one another, highlighting the importance of historical contingency and indicating that natural selection would be constrained to local exploration in the RNA world.

Inhibition of Bacterial Conjugation by Phage M13 and Its Protein g3p: Quantitative Analysis and Model

Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes.

Isocost Lines Describe the Cellular Economy of Genetic Circuits

Genetic circuits in living cells share transcriptional and translational resources that are available in limited amounts. This leads to unexpected couplings among seemingly unconnected modules, which result in poorly predictable circuit behavior. In this study, we determine these interdependencies between products of different genes by characterizing the economy of how transcriptional and translational resources are allocated to the production of proteins in genetic circuits. We discover that, when expressed from the same plasmid, the combinations of attainable protein concentrations are constrained by a linear relationship, which can be interpreted as an isocost line, a concept used in microeconomics. We created a library of circuits with two reporter genes, one constitutive and the other inducible in the same plasmid, without a regulatory path between them. In agreement with the model predictions, experiments reveal that the isocost line rotates when changing the ribosome binding site strength of the inducible gene and shifts when modifying the plasmid copy number. These results demonstrate that isocost lines can be employed to predict how genetic circuits become coupled when sharing resources and provide design guidelines for minimizing the effects of such couplings.

Genomic Insights in the Metabolism of Aromatic Compounds in Pseudomonas

Pseudomonads are ubiquitous γ-proteobacteria with a remarkable degree of physiological and genetic adaptability. Members of the genus Pseudomonas are found in large numbers in different natural environments (soil, freshwater, marine) as well as in association with plants and animals. These bacteria are involved in important metabolic activities in the environment, being element cycling and degradation of biogenic and xenobiotic pollutants some of their major tasks56, 64, 90, 96. The metabolic versatility of Pseudomonas strains has been used for biotechnological applications, mainly to degrade waste (bioremediation) and to synthesize specialty chemicals (biocatalysis)69, 99.

Synthetic tunable amplifying buffer circuit in E. coli.

While predictable design of a genetic circuits output is a major goal of synthetic biology, it remains a significant challenge because DNA binding sites in the cell affect the concentration of available transcription factors (TF). To mitigate this problem, we propose to use a TF that results from the (reversible) phosphorylation of protein substrate as a circuits output. We demonstrate that by comparatively increasing the amounts of substrate and phosphatase, the TF concentration becomes robust to the presence of DNA binding sites and can be kept at a desired value. The circuits input/output gain can, in turn, be tuned by changing the relative amounts of the substrate and phosphatase, realizing an amplifying buffer circuit with tunable gain. In our experiments in E. coli, we employ phospho-NRI as the output TF, phosphorylated by the NRII kinase, and dephosphorylated by the NRII phosphatase. Amplifying buffer circuits such as ours could be used to insulate a circuits output from the context, bringing synthetic biology one step closer to modular design.

A second chromosomal copy of the catA gene endows Pseudomonas putida mt-2 with an enzymatic safety valve for excess of catechol

Pseudomonas putida mt-2 harbours two different routes for catabolism of catechol, namely one meta pathway encoded by the xyl genes of the TOL plasmid pWW0 and one ortho pathway determined by the chromosomal ben and cat genes. P. putida mt-2 has a second chromosomal copy of the catA gene (named catA2) located downstream of the ben operon that encodes an additional catechol-1,2-dioxygenase. The metabolic and regulatory phenotypes of strains lacking one enzyme, the other and both of them in cells with and without the TOL plasmid were evaluated. The data consistently indicated that induction of the ortho pathway by benzoate plasmid-less strain P. putida KT2440 led to catechol surplus, the toxicity of which at high concentrations being counteracted by CatA2. Cells carrying pWW0 but lacking catA2 experienced both a rapid loss of the plasmid when grown on benzoate (a substrate of the lower pathway) and a slowdown of their growth rate when cultured with benzylalcohol (a substrate converted to benzoate by the upper pathway). These data reveal the role of CatA2 as a type of metabolic safety valve for excess catechol that alleviates the metabolic conflict generated by simultaneous expression of the meta and ortho pathways, thereby facilitating their co-existence.

Resource Competition Shapes the Response of Genetic Circuits

A common approach to design genetic circuits is to compose gene expression cassettes together. While appealing, this modular approach is challenged by the fact that expression of each gene depends on the availability of transcriptional/translational resources, which is in turn determined by the presence of other genes in the circuit. This raises the question of how competition for resources by different genes affects a circuits behavior. Here, we create a library of genetic activation cascades in E. coli bacteria, where we explicitly tune the resource demand by each gene. We develop a general Hill-function-based model that incorporates resource competition effects through resource demand coefficients. These coefficients lead to nonregulatory interactions among genes that reshape the circuits behavior. For the activation cascade, such interactions result in surprising biphasic or monotonically decreasing responses. Finally, we use resource demand coefficients to guide the choice of ribosome binding site and DNA copy number to restore the cascades intended monotonically increasing response. Our results demonstrate how unintended circuits behavior arises from resource competition and provide a model-guided methodology to minimize the resulting effects.

Production of selenium nanoparticles in Pseudomonas putida KT2440

Selenium (Se) is an essential element for the cell that has multiple applications in medicine and technology; microorganisms play an important role in Se transformations in the environment. Here we report the previously unidentified ability of the soil bacterium Pseudomonas putida KT2440 to synthesize nanoparticles of elemental selenium (nano-Se) from selenite. Our results show that P. putida is able to reduce selenite aerobically, but not selenate, to nano-Se. Kinetic analysis indicates that, in LB medium supplemented with selenite (1 mM), reduction to nano-Se occurs at a rate of 0.444 mmol L−1 h−1 beginning in the middle-exponential phase and with a final conversion yield of 89%. Measurements with a transmission electron microscope (TEM) show that nano-Se particles synthesized by P. putida have a size range of 100 to 500 nm and that they are located in the surrounding medium or bound to the cell membrane. Experiments involving dynamic light scattering (DLS) show that, in aqueous solution, recovered nano-Se particles have a size range of 70 to 360 nm. The rapid kinetics of conversion, easy retrieval of nano-Se and the metabolic versatility of P. putida offer the opportunity to use this model organism as a microbial factory for production of selenium nanoparticles.