Biology subjects

Calles, B.

Publications and source records attributed to Calles, B..

6 recordsLinked to original sources

bioRxiv2024

The structural permissiveness of triosephosphate isomerase (TpiA) of Escherichia coli

Triosephosphate isomerase (TpiA) is widely regarded as an example of an optimally evolved enzyme due to its essential role in biological systems, its structural conservation, and its near-perfect kinetic parameters. In this study, we investigated the structural robustness of the archetypal TpiA variant from Escherichia coli using an in vitro 5- amino acid linker scanning method. The resulting library was introduced into a tpiA mutant strain for functional complementation. From this library, 15 TpiA variants that were phenotypically indistinguishable from the wild-type enzyme were selected for further analysis. Although all variants retained enzymatic activities within the wild-type range, several insertions were found in highly structured protein domains where the linker was expected to cause significant structural perturbations. Despite these potentially disruptive additions, the enzymes maintained their activity even when expressed in a dnaK mutant, suggesting that chaperones did not compensate for structural abnormalities in vivo. Additionally, when these mutant TpiA variants were produced using the PURE in vitro transcription/translation system, they exhibited enzymatic activity comparable to, and in some cases exceeding, that of the non-mutated enzyme. AlphaFold2 revealed that insertions reconstructed the local architecture of the nearby amino acid sequences. The evolutionary implications of this remarkable structural resilience are discussed.

microbiology↗

bioRxiv2021

Parameterization of regulatory nodes for engineering broad host range heterologous gene expression

By building on the SEVA (Standard European Vector Architecture) format we have refactored a number of regulatory nodes recruited from both Gram-negative and Gram-positive bacteria for rigorously comparing and parameterizing five expression devices that respond to diverse and unrelated chemical inducers, i.e. LacIq-Ptrc, XylS-Pm, AlkS-PalkB, CprK-PDB3 and ChnR-PchnB. These were assembled as cargoes following the SEVA standard within exactly the same vector backbone and bearing the different functional segments arrayed in an invariable DNA scaffold. Their performance in an Escherichia coli strain of reference was then analyzed through the readout a fluorescence reporter gene that contained strictly identical translation signal elements in all cases and in the same DNA context. This study allowed us to describe and compare the cognate expression systems with unprecedented quantitative detail. The systems under scrutiny diverged considerably in their capacity, expression noise, inducibility and OFF/ON ratios. These features, along with the absence of physiological effects caused by the inducers and the lack of cross-regulation offer a panoply of choices to potential users and help interoperability of the specific constructs.

synthetic biology↗

bioRxiv2020

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

Pseudomonas putida KT2440, a microorganism of interest for biotechnological purposes, is one amongst the many bacteria that attach to surfaces and produce biofilm. Although other mechanisms that contribute to this decision have been studied until now, a 7-genes-operon with a disposition and homology shared with the wsp operon in Pseudomonas aeruginosa remained to be investigated. In this work, we characterized the function of P. putida wsp operon by the combination of deletion mutants with complementations with P. aeruginosas genes and with deletions of 3 other genes: the genes that code for the transcription factors fleQ and fleN and the flagellar movement regulator, fglZ. Examining mutant behaviour at 6 and 24 hours under three different carbon regimes (citrate, glucose and fructose) we saw that this complex carries out a similar function in both Pseudomonas. In P. putida, the key components are WspR, a protein that harbours the domain for producing c-di-GMP, and WspF, which controls its activity. Transformation with the equivalent proteins of P. aeruginosa had a significant impact on of P. putida mutant phenotypes and could complement their functions under some conditions. These results contribute to the deeper understanding of the complex element network that regulate lifestyle decision in P. putida

microbiology↗

bioRxiv2019

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 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/dicyclopropylketone) and shown to perform effectively both in E. 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.

synthetic biology↗

bioRxiv2019

Multiple-site diversification of regulatory sequences enables inter-species operability of genetic devices

The features of the light-responsive cyanobacterial CcaSR regulatory node that determine interoperability of this optogenetic device between Escherichia coli and Pseudomonas putida have been examined. For this, all structural parts (i.e. ho1 and pcyA genes for synthesis of phycobilin, the ccaS/ccaR system from Synechocystis and its cognate downstream promoter) were maintained but their expression levels and stoichiometry diversified by [i] reassembling them together in a single broad host range, standardized vector and [ii] subjecting the non-coding regulatory sequences to multiple cycles of directed evolution with random genomic mutations (DIvERGE), a recombineering method that intensifies mutation rates within discrete DNA segments. Once passed to P. putida, various clones displayed a wide dynamic range, insignificant leakiness and excellent capacity in response to green light. Inspection of the evolutionary intermediates pinpointed translational control as the main bottleneck for interoperability and suggested a general approach for easing the exchange of genetic cargoes between different species i.e. optimization of relative expression levels and upturning of subcomplex stoichiometry.

synthetic biology↗

bioRxiv2018

The metabolic redox regime of Pseudomonas putida tunes its evolvability towards novel xenobiotic substrates

During evolution of biodegradation pathways for xenobiotic compounds, the transition towards novel substrates of Rieske non-heme iron oxygenases borne by environmental bacteria is frequently associated with faulty reactions. Such reactions release reactive oxygen species (ROS), endowed with high mutagenic potential. The present work studies how the operation of a given metabolic network by a bacterial host may either foster or curtail the still-evolving biochemical pathway for catabolism of 2,4-dinitrotoluene (2,4-DNT). To this end, the genetically tractable strain Pseudomonas putida EM173 was chromosomally implanted with a Tn7 construct carrying the whole genetic complement (recruited from the environmental isolate Burkholderia sp. R34) necessary for complete biodegradation of 2,4-DNT. By using reporter technology and direct measurements of ROS formation, we observed that the engineered P. putida strain experienced oxidative stress when catabolizing the nitroaromatic substrate. However, ROS was neither translated into significant activation of the SOS response to DNA damage nor resulted in a mutagenic regime (unlike Burkholderia sp. R34, the original host of the pathway). To inspect whether the tolerance of P. putida to oxidative insults could be traced to its characteristic reductive redox regime, we artificially lowered the pool of NAD(P)H by conditional expression of a water forming, NADH-specific oxidase. Under the resulting low-NAD(P)H status, 2,4-DNT triggered a conspicuous mutagenic and genomic diversification scenario. These results indicate that the background biochemical network of environmental bacteria ultimately determines the evolvability of metabolic pathways. Moreover, the data explains the efficacy of some bacteria such as Pseudomonads to host and evolve new catabolic routes.\n\nIMPORTANCESome environmental bacteria evolve new capacities for aerobic biodegradation of chemical pollutants by adapting pre-existing redox reactions to recently faced compounds. The process typically starts by co-option of enzymes of an available route to act on the chemical structure of the substrates-to-be. The critical bottleneck is generally the first biochemical step and most of the selective pressure operates on reshaping the initial reaction. In Rieske non-heme iron oxygenases, the interim uncoupling of the novel substrate to the old enzymes results in production of highly mutagenic ROS. In this work, we demonstrate that the background metabolic regime of the bacterium that hosts an evolving catabolic pathway (e.g. biodegradation of the xenobiotic 2,4-DNT) determines whether the cells would either adopt a genetic diversification regime or a robust ROS-tolerant state. These results expose new perspectives to contemporary attempts for rational assembly of whole-cell biocatalysts, as pursued by present-day metabolic engineering.

microbiology↗