Max Chavarría

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.

Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

ABSTRACT Two branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacterium Pseudomonas putida KT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFru or PTSNtr genes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtr protein of the PTSNtr thus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology. IMPORTANCE This study demonstrates that EIIANtr influences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars by P. putida. These findings indicate that the EIIANtr protein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks. This study demonstrates that EIIANtr influences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars by P. putida. These findings indicate that the EIIANtr protein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks.

The metabolic cost of flagellar motion in Pseudomonas putida KT2440

Although the flagellar machinery of environmental bacteria endows cells with a phenomenal survival device, it also consumes much of the metabolic currency necessary for fuelling such a vigorous nano-motor. The physiological cost of flagella-related functions of the soil bacterium Pseudomonas putida KT2440 was examined and quantified through the deletion of a ≈ 70 kb DNA segment of the genome (≈ 1.1%), which includes relevant structural and regulatory genes in this micro-organism. The resulting strain lacked the protruding polar cords that define flagella in the wild-type P. putida strain and was unable of any swimming motility while showing a significant change in surface hydrophobicity. However, these deficiencies were otherwise concomitant with clear physiological advantages: rapid adaptation of the deleted strain to both glycolytic and gluconeogenic carbon sources, increased energy charge and, most remarkably, improved tolerance to oxidative stress, reflecting an increased NADPH/NADP(+) ratio. These qualities improve the endurance of non-flagellated cells to the metabolic fatigue associated with rapid growth in rich medium. Thus, flagellar motility represents the archetypal tradeoff involved in acquiring environmental advantages at the cost of a considerable metabolic burden.

Endogenous Stress Caused by Faulty Oxidation Reactions Fosters Evolution of 2,4-Dinitrotoluene-Degrading Bacteria

Environmental strain Burkholderia sp. DNT mineralizes the xenobiotic compound 2,4-dinitrotoluene (DNT) owing to the catabolic dnt genes borne by plasmid DNT, but the process fails to promote significant growth. To investigate this lack of physiological return of such an otherwise complete metabolic route, cells were exposed to DNT under various growth conditions and the endogenous formation of reactive oxygen species (ROS) monitored in single bacteria. These tests revealed the buildup of a strong oxidative stress in the population exposed to DNT. By either curing the DNT plasmid or by overproducing the second activity of the biodegradation route (DntB) we could trace a large share of ROS production to the first reaction of the route, which is executed by the multicomponent dioxygenase encoded by the dntA gene cluster. Naphthalene, the ancestral substrate of the dioxygenase from which DntA has evolved, also caused significant ROS formation. That both the old and the new substrate brought about a considerable cellular stress was indicative of a still-evolving DntA enzyme which is neither optimal any longer for naphthalene nor entirely advantageous yet for growth of the host strain on DNT. We could associate endogenous production of ROS with likely error-prone repair mechanisms of DNA damage, and the ensuing stress-induced mutagenesis in cells exposed to DNT. It is thus plausible that the evolutionary roadmap for biodegradation of xenobiotic compounds like DNT was largely elicited by mutagenic oxidative stress caused by faulty reactions of precursor enzymes with novel but structurally related substrates-to-be.

The interplay of the EIIANtr component of the nitrogen-related phosphotransferase system (PTSNtr) of Pseudomonas putida with pyruvate dehydrogenase ☆

BACKGROUNDnPseudomonas putida KT2440 is endowed with a variant of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS(Ntr)), which is not related to sugar transport but believed to rule the metabolic balance of carbon vs. nitrogen. The metabolic targets of such a system are largely unknown.nnnMETHODSnDielectric breakdown of P. putida cells grown in rich medium revealed the presence of forms of the EIIA(Ntr) (PtsN) component of PTS(Ntr), which were strongly associated to other cytoplasmic proteins. To investigate such intracellular partners of EIIA(Ntr), a soluble protein extract of bacteria bearing an E epitope tagged version of PtsN was immunoprecipitated with a monoclonal anti-E antibody and the pulled-down proteins identified by mass spectrometry.nnnRESULTSnThe E1 subunit of the pyruvate dehydrogenase (PDH) complex, the product of the aceE gene, was identified as a major interaction partner of EIIA(Ntr). To examine the effect of EIIA(Ntr) on PDH, the enzyme activity was measured in extracts of isogenic ptsN(+)/ptsN(-)P. putida strains and the role of phosphorylation was determined. Expression of PtsN and AceE proteins fused to different fluorescent moieties and confocal laser microscopy indicated a significant co-localization of the two proteins in the bacterial cytoplasm.nnnCONCLUSIONnEIIA(Ntr) down-regulates PDH activity. Both genetic and biochemical evidence revealed that the non-phosphorylated form of PtsN is the protein species that inhibits PDH.nnnGENERAL SIGNIFICANCEnEIIA(Ntr) takes part in the node of C metabolism that checks the flux of carbon from carbohydrates into the Krebs cycle by means of direct protein-protein interactions with AceE. This type of control might connect metabolism to many other cellular functions. This article is part of a Special Issue entitled: Systems Biology of Microorganisms.

Association of dnt genes of Burkholderia sp. DNT with the substrate-blind regulator DntR draws the evolutionary itinerary of 2,4-dinitrotoluene biodegradation

The regulation of the DNT pathway for biodegradation of 2,4‐dinitrotoluene of Burkholderia sp. DNT has been examined by exporting each of its components to Pseudomonas putida KT2440. The cognate regulator DntR does not respond to the pathway substrate, but to the non‐substrate salicylate. In order to examine whether such a response to an unrelated inducer was specific or rather a vestige of a previous evolutionary stage, the complete dnt complement or parts of it were expressed functionally for accumulation of various metabolic intermediates. Their effect on expression of dnt genes was then followed both biochemically and by means of a luminescent reporter engineered in the surrogate host. DntR was not only unresponsive to DNT biodegradation products, but it also failed to influence expression of dnt genes at all. Comparison of the dntR/dntA divergent promoter region with similar ones found in various catabolic systems indicated that the leading segment of the DNT biodegradation pathway evolved from a matching portion of naphthalene biodegradation routes existing in other bacteria. That a useless but still active transcriptional factor occurs along enzymes that have already evolved a new substrate specificity suggests that emergence of novel catalytic abilities precedes their submission to cognate regulatory devices, not vice versa.

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

Pseudomonas putidau2005mt-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.u2009putidau2005mt-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.u2009putidau2005KT2440 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.

An Electro-optical Device from a Biofilm Structure Created by Bacterial Activity

This work was supported by grants of the Ministerio de Ciencia e Innovacion (Grant No. MAT2008-00010/NAN) and by contracts of the Autonomous Community of Madrid and the European Union. EC and MC are grateful to Universidad de Costa Rica and the CSIC for doctoral scholarships.

The IHF regulon of exponentially growing Pseudomonas putida cells

Integration host factor (IHF) sites are largely absent from intergenic regions of ORFs encoding central metabolic functions in Pseudomonas putida mt-2. To gain an insight into this unequal distribution of otherwise abundant IHF-binding sequences, the transcriptome of IHF-plus and IHF-minus cells growing exponentially on glucose as sole carbon source was examined. In parallel, the cognate metabolic fluxes of the wild-type P. putida strain and its ihfA derivative were determined by culturing cells to a steady-state physiological regime with (13)C-labelled glucose. While expression of many transcripts was altered by the lack of IHF, flux balance analysis revealed that the ihfA mutation did not influence central carbon metabolism. Identification of multiple IHF sites adjacent to genes responsive to the factor allowed a refinement of the consensus and the mapping of the preferred binding positions for activation or repression of associated promoters. That few (if any) of the genes affected by IHF involved core pathways suggested that the central carbon metabolism tolerates the loss of the factor. Instead, IHF controlled various cell surface-related functions and downregulated genes encoding ribosomal proteins, the alpha subunit of RNA polymerase and components of the ATP synthase. These results were confirmed with lacZ fusions to a suite of promoters detected in the transcriptome as affected by IHF. Taken together, the data suggest that IHF plays a role in the physiological shift that sets P. putida for entering stationary phase.

Modeling and analysis of flux distributions in the two branches of the phosphotransferase system in Pseudomonas putida.

BackgroundSignal transduction plays a fundamental role in the understanding of cellular physiology. The bacterial phosphotransferase system (PTS) together with the PEP/pyruvate node in central metabolism represents a signaling unit that acts as a sensory element and measures the activity of the central metabolism. Pseudomonas putida possesses two PTS branches, the C-branch (PTSFru) and a second branch (PTSNtr), which communicate with each other by phosphate exchange. Recent experimental results showed a cross talk between the two branches. However, the functional role of the crosstalk remains open.ResultsA mathematical model was set up to describe the available data of the state of phosphorylation of PtsN, one of the PTS proteins, for different environmental conditions and different strain variants. Additionally, data from flux balance analysis was used to determine some of the kinetic parameters of the involved reactions. Based on the calculated and estimated parameters, the flux distribution during growth of the wild type strain on fructose could be determined.ConclusionOur calculations show that during growth of the wild type strain on the PTS substrate fructose, the major part of the phosphoryl groups is provided by the second branch of the PTS. This theoretical finding indicates a new role of the second branch of the PTS and will serve as a basis for further experimental studies.