Biology subjects

Dvorak, P.

Publications and source records attributed to Dvorak, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Characterisation of PHA synthase (PhaC) from the thermophilic bacterium Caldimonas thermodepolymerans DSM15344

Polyhydroxyalkanoates (PHAs) are microbial polyesters that represent a sustainable alternative to fossil fuel-based plastics. These biodegradable polymers have properties similar to conventional plastics and can be produced from waste materials. The key enzyme in PHA synthesis is PHA synthase (PhaC) and despite its importance, only a few PhaCs from extremophilic microorganisms have been characterised in detail. Here we characterised PHA synthase enzyme from the thermophilic bacterium Caldimonas thermodepolymerans DSM15344, an attractive candidate for PHA production from lignocellulosic residues due to its unique properties such as utilisation of xylose-rich substrates and high PHA yields on this pentose sugar. PhaC was successfully expressed in the surrogate host E. coli BL21 (DE3) GOLD, purified to homogeneity and its enzymatic activity was confirmed in vitro. The optimal pH range and temperature were determined as 6.3-6.9 and 50 {degrees}C, respectively. The melting temperature of PhaC was detected at 57.83 {+/-} 0.04 {degrees}C and the enzyme storage stability was assessed. The tertiary structure, including the enzyme active site, was predicted using available bioinformatic tools and the predicted catalytic residues were supported by site-directed mutagenesis. This study represents the first biochemical characterisation of PHA synthase from C. thermodepolymerans, expanding the limited set of characterised thermophilic PhaC enzymes.

biochemistry↗

bioRxiv2026

Engineering Binding Efficiency and Interaction Stability of a Thermostable Cohesin-Dockerin Pair on the Bacterial Cell Surface

Efficient conversion of polymeric feedstocks for sustainable bioprocessing requires robust strategies for enzyme assembly and cell-surface attachment. In nature, cellulosomes achieve highly efficient lignocellulosic polysaccharide deconstruction through scaffoldin-mediated organization of carbohydrate-active enzymes via specific cohesin-dockerin interactions. These modular binding pairs are therefore attractive tools for synthetic biology and engineered whole-cell biocatalysis, yet their performance has been studied mainly in vitro or in yeast or Gram-positive bacteria. The factors governing their function on the microbial surfaces - particularly those of Gram-negative bacteria - remain incompletely understood. Here, we investigated the binding efficiency and interaction stability of two thermophilic cohesin-dockerin pairs from Acetivibrio thermocellus and Acetivibrio clariflavus displayed on the surface of the genome-streamlined strain Pseudomonas putida EM371 using an Ag43-based display system from Escherichia coli and a dockerin-tagged fluorescent reporter. We show that binding efficiency is strongly affected by the temperature at which the cohesin-dockerin complex is formed. We further demonstrate that the interaction stability of the A. clariflavus pair can be substantially improved by targeted amino acid substitutions in the dockerin domain guided by molecular dynamics simulations and free-energy calculations. These results identify key parameters controlling the performance of thermophilic cohesin-dockerin modules on living bacterial cell surfaces and establish a computation-guided strategy for engineering more stable cellulosome-derived assembly interfaces, advancing the development of modular whole-cell platforms for sustainable biotechnology applications. TOC graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/743725v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@1a0d637org.highwire.dtl.DTLVardef@4fc620org.highwire.dtl.DTLVardef@15882f0org.highwire.dtl.DTLVardef@1fc6aa3_HPS_FORMAT_FIGEXP M_FIG C_FIG Cohesin-dockerin pairs provide strong and modular non-covalent interactions for synthetic biology and biotechnology applications. We establish an experimental and computational pipeline to improve their two key properties - binding efficiency and interaction stability - on the surface of Pseudomonas putida, enabling more robust cell-surface assembly systems.

synthetic biology↗

bioRxiv2026

Stomatal movement in Arabidopsis is driven by guard cell-localized and copper-insensitive CSD1 splice variant

Copper (Cu) is an essential micronutrient whose bioavailability is strongly affected by soil physicochemical properties. During evolution, plants have developed mechanisms to flexibly adjust their metabolism to Cu status. Superoxide dismutases (SODs), including Cu/ZnSOD1 (CSD1) and FeSOD1 (FSD1), are key antioxidant enzymes regulated in Cu dependent manner in Arabidopsis thaliana. Examination of CSD1 cellular distribution and activity revealed that CSD1 is a nuclear and cytosolic SOD whose abundance and activity respond to Cu availability inversely to FSD1. Combined microscopic and biochemical analyses of Cu-dependent dynamics revealed that, unlike FSD1, CSD1 localization in guard cells (GCs) remains independent of Cu availability. CSD1 escapes miR398-mediated regulation in GCs through a cell type-specific splice variant (CSD1.2) that carries an altered miR398-binding site. In silico analyses indicate that this mechanism is also present in crop species. Functionally, the csd1 mutant showed reduced sensitivity to abscisic acid (ABA)-induced stomatal closure, a phenotype rescued by reintroducing CSD1. Biochemical and reactive oxygen species (ROS) level analyses indicate that CSD1.2 most likely acts independently of its canonical enzymatic activity in GCs and functions upstream of the ROS burst in the ABA signaling pathway. Together, we present a novel, cell-type-specific mechanism that safeguards ABA-driven stomatal closure under fluctuating Cu supply.

plant biology↗

bioRxiv2025

Protein and genetic interactions between RACK1A and FSD1 modulate plant development and stress granule-dependent response to salt in Arabidopsis.

The generation of reactive oxygen species (ROS) and their regulation by antioxidant enzymes such as IRON SUPEROXIDE DISMUTASE 1 (FSD1) are critical for managing plant responses to salt stress. However, the protein networks modulating ROS levels during salt stress remain incompletely understood. Our co-immunoprecipitation analysis identified the FSD1 as an interaction partner of the scaffolding protein RECEPTOR FOR ACTIVATED C KINASE 1A (RACK1A). Bimolecular fluorescence complementation analyses revealed that RACK1A interacts with FSD1 predominantly in the cytoplasm. Despite elevated FSD1 activity in rack1a mutants, the abundance of FSD1 protein remained unchanged. Computational predictions of interaction interfaces suggested that RACK1A may interfere with the catalytic site of FSD1. Advanced fluorescence microscopy and genetic studies further confirmed localization and relocation patterns of RACK1A and FSD1 during salt stress responses. Additionally, we found that the RACK1A-FSD1 module was involved in root hair tip growth, highlighting the developmental significance of this interaction. While rack1a mutants exhibited salt resilience, fsd1-1 rack1a-1 double mutant displayed reduced salt stress resistance compared to rack1a single mutant, which was substantiated by deregulation of ROS levels. RACK1A and FSD1 accumulated in cycloheximide-sensitive structural condensates in the cytoplasm that colocalized with the stress granule marker protein TSN in roots. However, RACK1A knock-out completely abolished salt-stress-dependent relocation of FSD1 to structural condensates, suggesting that RACK1A recruits FSD1 to these stress granules. Thus, this study uncovers an entirely novel mechanism for the regulation of RACK1/FSD1-dependent antioxidant defense in response to salt stress in Arabidopsis.

plant biology↗

bioRxiv2024

Building a syntrophic Pseudomonas putida consortium with reciprocal substrate processing of lignocellulosic disaccharides

Synthetic microbial consortia can leverage their expanded enzymatic reach to tackle biotechnological challenges too complex for single strains, such as lignocellulose valorisation. The benefit of metabolic cooperation comes with a catch - installing stable interactions between consortium members. We constructed a syntrophic consortium of Pseudomonas putida strains for lignocellulosic disaccharide processing. Two strains were engineered to hydrolyse and metabolise lignocellulosic sugars: one grows on xylose and hydrolyses cellobiose to produce glucose, while the other grows on glucose and cleaves xylobiose to produce xylose. This specialisation allows each strain to provide essential growth substrate to its partner, establishing a stable mutualistic interaction, which we term reciprocal substrate processing. Key enzymes from Escherichia coli (xylose isomerase pathway) and Thermobifida fusca (glycoside hydrolases) were introduced into P. putida to broaden its carbohydrate utilisation capabilities and arranged in a way to install the strain cross-dependency. A mathematical model of the consortium assisted in predicting the effects of substrate composition, strain ratios, and protein expression levels on population dynamics. Our results demonstrated that modulating extrinsic factors such as substrate concentration can optimise growth and balance fitness disparities between the strains, but achieving this by altering intrinsic factors such as glycoside hydrolase expression levels is much more challenging. This study underscores the potential of synthetic microbial consortia to facilitate the bioconversion of lignocellulosic sugars and offers insights into overcoming the challenges of establishing synthetic microbial cooperation.

synthetic biology↗

bioRxiv2024

An initial genome editing toolset for Caldimonas thermodepolymerans, the first model of thermophilic polyhydroxyalkanoates producer

The limited number of well-characterized model bacteria cannot address all the challenges in a circular bioeconomy. Therefore, there is a growing demand for new production strains with enhanced resistance to extreme conditions, versatile metabolic capabilities, and the ability to utilize cost-effective renewable resources while efficiently generating attractive biobased products. Particular thermophilic microorganisms fulfill these requirements. Non-virulent Gram-negative Caldimonas thermodepolymerans DSM15344 is one such attractive thermophile that efficiently converts a spectrum of plant biomass sugars into high quantities of polyhydroxyalkanoates (PHA) - a fully biodegradable substitutes for synthetic plastics. However, to enhance its biotechnological potential, the bacterium needs to be "domesticated". In this study we established effective homologous recombination and transposon-based genome editing systems for C. thermodepolymerans. By optimizing the electroporation protocol and refining counterselection methods, we achieved significant improvements in genetic manipulation and constructed the AI01 chassis strain with improved transformation efficiency and a {Delta}phaC mutant that will be used to study the importance of PHA synthesis in Caldimonas. The advances described herein highlight the need for tailored approaches when working with thermophilic bacteria and provide a springboard for further genetic and metabolic engineering of C. thermodepolymerans, which can be considered the first model of thermophilic PHA producer.

bioengineering↗

bioRxiv2024

Population genomics resolves cryptic species of the ecologically flexible genus Laspinema (cyanobacteria)

Cyanobacterial taxonomy is entering the genomic era, but only a few taxonomic studies have employed population genomics, which provides a framework and a multitude of tools to understand species boundaries. Phylogenomic and population genomic analyses previously suggested that several cryptic lineages emerged within the genus Laspinema. Here, we apply population genomics to define boundaries between these lineages and propose new cryptic species, Laspinema olomoucense and Laspinema palackyanum. Moreover, we sampled soil and puddles across Central Europe and sequenced the 16S rRNA and 16S-23S ITS of the isolated Laspinema strains. Together with database mining of 16S rRNA sequences, we found that the genus Laspinema has a cosmopolitan distribution and inhabits a wide variety of habitats, including freshwater, saline water, mangroves, soil crusts, soils, puddles, and the human body.

microbiology↗

bioRxiv2023

The longest intron rule

Despite the fact that long introns mean an energy and time burden for eukaryotic cells, they play an irreplaceable role in the diversification and regulation of protein production. A general feature is the accumulation of the longest introns closer to the start of protein-coding genes. Our work aimed to more closely characterize the genes in which the longest intron is only located in the second or third third of the gene. Data on the lengths of all introns in genes were extracted from the genomes of 4 vertebrates (human, mouse, chicken and zebrafish), nematode worm and yeast. With the genes divided according to the relative position of the longest intron, gene set enrichment analyses were performed, the results of which were then confronted with the results of randomly generated gene sets of the same size. The analyses show that among the genes associated with biological processes of ion transport across membranes, cell signaling or the development of multicellular organisms, there is a greater representation of genes with the longest intron in the first third. Conversely, among the genes associated with the processes of RNA processing and ribosome biogenesis, there are more genes with the longest intron in the second or third third. It is a newly discovered characteristic with more general validity among multicellular organisms.

genomics↗

bioRxiv2023

Genomic and Phenotypic Comparison of Polyhydroxyalkanoates Producing Strains of genus Caldimonas/Schlegelella

Polyhydroxyalkanoates (PHAs) have emerged as an ecologically friendly alternative to conventional polyesters. In this study, we present a comprehensive analysis of the genomic and phenotypic characteristics of three non-model thermophilic bacteria known for their ability to produce PHAs: Schlegelella aquatica LMG 23380T, Caldimonas thermodepolymerans DSM 15264, and C. thermodepolymerans LMG 21645 accompanied by a comparison with the type strain C. thermodepolymerans DSM 15344T. We have assembled the first complete genomes of these three bacteria and performed the structural and functional annotation. This analysis has provided valuable insights into the biosynthesis of PHAs and has allowed us to propose a comprehensive scheme for the carbohydrate metabolism in the studied bacteria. Through phylogenomic analysis, we have confirmed the synonymity between Caldimonas and Schlegelella genera, and further demonstrated that S. aquatica and S. koreensis, currently classified as orphan species, belong to the Caldimonas genus. SummaryThe genomic and phenotypic analysis of Schlegelella aquatica LMG 23380T and Caldimonas thermodepolymerans DSM 15264 and LMG 21645 sheds light on the production of sustainable polyesters known as polyhydroxyalkanoates (PHAs). The genome assembly and functional annotation highlight key genes related to PHA production and other important traits. Notably, C. thermodepolymerans stands out with its unique xyl operon, making it a highly promising candidate for biotechnological PHA production from xylose-rich lignocellulosic resources. The study emphasizes the importance of a polyphasic approach combining genotypic and phenotypic analyses in prokaryotic taxonomy, emphasizing the need for exploration in the genomic era. By uncovering the key traits of these bacteria, this research opens new horizons towards sustainable production of environmentally friendly polyesters.

microbiology↗

bioRxiv2023

Remodelling of cytoskeleton and plasma membrane proteins contributes to drought sensitivity of Arabidopsis rhd2 mutant

NADPH oxidases are enzymes localised in the plasma membrane and emitting superoxide to the extracellular space. By production of superoxide as one type of reactive oxygen species (ROS), they exert pleiotropic functions in plant development and various stress responses. Arabidopsis thaliana RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN C/ROOT HAIR DEFECTIVE 2 (AtRBOHC/RHD2) is an NADPH oxidase with preferential gene expression in roots. Polar localisation and ROS production by this enzyme are essential for root hair elongation. However, the proteome-wide and physiological consequences of RBOHC/RHD2 mutations are unknown. To find out potential new functions of AtRBOHC/RHD2, we employed a differential proteomic analysis of Arabidopsis rhd2-1 mutant, carrying a loss-of-function mutation in RBOHC/RHD2. Proteomic analyses that were validated with independent biochemical, phenotypical and advanced microscopy methods, showed quantitative deregulation of proteins involved in abiotic and biotic stress response, metabolism, vesicular transport and cell wall modification. Considerable differences in the differential proteomes between roots and above-ground parts were found in the mutant. The altered abundance of aquaporins and homeostasis of transmembrane pumps and transporters most likely determine the higher sensitivity of Arabidopsis rhd2-1 mutant to drought. HighlightProteomics and advanced microscopy reveal that the drought sensitivity of Arabidopsis mutant in ROOT HAIR DEFECTIVE 2 is linked to altered homeostasis of plasma membrane proteins and cytoskeleton remodelling.

plant biology↗

bioRxiv2023

Genomic and metabolic plasticity drive alternative scenarios for adapting Pseudomonas putida to non-native substrate D-xylose.

D-Xylose, a major constituent of plant biomass and second most abundant sugar on Earth, holds a considerable potential as a substrate for sustainable bio-production. Pseudomonas putida KT2440 is an attractive bacterial host for valorizing biogenic feedstocks but lacks a xylose utilization pathway. While several attempts to engineer P. putida for growth on xylose have been reported, a comprehensive understanding of xylose metabolism in this bacterium is lacking, hindering its further improvement and rational tailoring for specific biotechnological purposes. In this study, we elucidated the xylose metabolism in the genome-reduced P. putida strain, EM42, endowed with xylose isomerase pathway (xylAB) and transporter (xylE) from Escherichia coli and used the obtained knowledge in combination with adaptive laboratory evolution to accelerate the bacteriums growth on the pentose sugar. Carbon flux analyses, targeted gene knock-outs, and in vitro enzyme assays portrayed xylose assimilation in P. putida and confirmed a partially cyclic upper xylose metabolism. Deletion of the local transcriptional regulator gene hexR de-repressed genes of several key catabolic enzymes and reduced the lag phase on xylose. Guided by metabolic modeling, we augmented P. putida with additional heterologous pentose phosphate pathway genes and subjected rationally prepared strains to adaptive laboratory evolution (ALE) on xylose. The descendants showed accelerated growth and reduced growth lag. Genomic and proteomic analysis of engineered and evolved mutants revealed the importance of a large genomic re-arrangement, transaldolase overexpression, and balancing gene expression in the synthetic xylABE operon. Importantly, omics analyses found that similar growth characteristics of two superior mutants were achieved through distinct evolutionary paths. This work provides a unique insight into how cell metabolism adjusts to a non-native substrate; it highlights the remarkable genomic and metabolic plasticity of P. putida and demonstrates the power of combining knowledge-driven engineering with ALE in generating desirable microbial phenotypes. HighlightsO_LIElucidated xylose catabolism via exogenous isomerase pathway in P. putida EM42. C_LIO_LIDeletion of transcriptional regulator HexR improved growth on xylose. C_LIO_LIKnowledge-guided interventions and adaptive evolution accelerated growth. C_LIO_LIOmics analyses of selected mutants highlighted the genomic and metabolic plasticity of P. putida. C_LIO_LITwo mutants with superior characteristics emerged from distinct evolutionary paths. C_LI

bioengineering↗

bioRxiv2023

Spatiotemporal distribution of ROS production, delivery and utilization in Arabidopsis root hairs

Fluorescent selective probes for reactive oxygen species (ROS) detection in living cells are versatile tools for the documentation of ROS production in plant developmental or stress reactions. We employed high-resolution live-cell imaging and semi-quantitative analysis of Arabidopsis thaliana stained with CM-H2DCFDA, CellROXTM Deep Red and AmplexTM Red for functional characterization of spatiotemporal mode of ROS production, delivery and utilization during root hair formation. Cell viability marker fluorescein diacetate served as a positive control for dye-loading and undisturbed tip growth after staining. Colocalization analysis with subcellular molecular markers and utilization of two root hair mutants with similar phenotype of non-elongating root hairs, but with contrast reasons for this impairment, we found that: i) CM-H2DCFDA is a sensitive probe for ROS generation in cytoplasm, ii) CellROXTM Deep Red labels ROS in mitochondria, iii) AmplexTM Red labels apoplastic ROS and mitochondria, and shows high selectivity to root hairs, iv) rhd2-1 mutant with nonfunctional AtRBOHC/RHD2 has a low level of CM-H2DCFDA-reactive ROS in cytoplasm and lacks AmplexTM Red-reactive ROS in apoplast, v) ACTIN2-deficient der1-3 mutant is not altered in these aspects. The sensitivity of CellROXTM Deep Red was documented by discrimination between larger ROS-containing mitochondria and small, yet ROS-free pre-mature mitochondria in the growing tip of root hairs. We characterized spatial changes in ROS production and compartmentalization induced by external ROS modulators, ethylene precursor 1-aminocyclopropane-1-carboxylic acid and ionophore valinomycin. This dynamic and high-resolution study of ROS production and utilization opens new opportunities for precise speciation of particular ROS involved in the root hair formation. One sentence summary: High-resolution live-cell imaging of ROS production and subcellular localization in bulges and growing root hairs of Arabidopsis using CM-H2DCFDA, CellROXTM Deep Red and AmplexTM Red selective probes.

plant biology↗

bioRxiv2022

Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model

Pseudomonas putida KT2440 is an attractive bacterial host for biotechnological production of valuable chemicals from renewable lignocellulosic feedstocks as it can valorize lignin-derived aromatics or cellulosic glucose. P. putida EM42, a genome-reduced variant of P. putida KT2440 endowed with advantageous physiological properties, was recently engineered for growth on cellobiose, a major cellooligosaccharide product of enzymatic cellulose hydrolysis. Co-utilization of cellobiose with glucose was achieved in a mutant lacking periplasmic glucose dehydrogenase Gcd (PP_1444). However, the cause of the observed co-utilization was not understood and the {Delta}gcd strain suffered from a significant growth defect. In this study, we aimed to investigate the basis of the simultaneous uptake of the two sugars and accelerate the growth of P. putida EM42 {Delta}gcd mutant for the bioproduction of valuable compounds from glucose and cellobiose. We show that the gcd deletion abolished the inhibition of the exogenous {beta}-glucosidase BglC from Thermobifida fusca by the intermediates of the periplasmic glucose oxidation pathway. The additional deletion of the hexR gene, which encodes a repressor of the upper glycolysis genes, failed to restore the rapid growth on glucose. The reduced growth rate of the {Delta}gcd mutant was partially compensated by the implantation of heterologous glucose (Glf from Zymomonas mobilis) and cellobiose (LacY from Escherichia coli) transporters. Remarkably, this intervention resulted in the accumulation of pyruvate in aerobic P. putida cultures. We demonstrated that the excess of this key metabolic intermediate can be redirected to the enhanced biosynthesis of ethanol and lactate. The overproduction of pyruvate was then unveiled by an upgraded genome-scale metabolic model constrained with proteomic and kinetic data. The model pointed to the saturation of glucose catabolism enzymes due to unregulated substrate uptake and it predicted improved bioproduction of pyruvate-derived chemicals by the engineered strain. This work sheds light on the co-metabolism of cellulosic sugars in an attractive biotechnological host and introduces a novel strategy for pyruvate overproduction in bacterial cultures under aerobic conditions. HighlightsO_LICo-utilization of glucose and cellobiose achieved in P. putida EM42 {Delta}gcd mutant. C_LIO_LIGrowth defect of the mutant compensated by implanting exogenous sugar transporters. C_LIO_LIEnhanced influx of carbon caused aerobic overproduction of pyruvate and acetate. C_LIO_LICarbon from excess pyruvate streamed into ethanol or L-lactate. C_LIO_LIPyruvate overproduction unveiled by a mathematical model of P. putida metabolism. C_LI

bioengineering↗

bioRxiv2021

Arabidopsis iron superoxide dismutase 1 protects against methyl viologen-induced oxidative stress in a copper-dependent manner

Iron superoxide dismutase 1 (FSD1) was recently characterized as a plastidial, cytoplasmic, and nuclear superoxide dismutase with osmoprotective and antioxidative functions. However, its role in oxidative stress tolerance is not well understood. Here, we characterized the role of FSD1 in response to methyl viologen (MV)-induced oxidative stress in Arabidopsis thaliana. The findings demonstrated that the antioxidative function of FSD1 depends on the availability of Cu2+ in growth media. Prolonged MV exposure led to a decreased accumulation rate of superoxide, higher levels of hydrogen peroxide production, and higher protein carbonylation in the fsd1 mutants and transgenic plants lacking a plastidial pool of FSD1, compared to the wild type. MV led to a rapid increase in FSD1 activity, followed by a decrease. Chloroplastic localization of FSD1 is necessary for these changes. Proteomic analysis showed that the sensitivity of the fsd1 mutants coincided with decreased abundance of ferredoxin and light PSII harvesting complex proteins, with altered levels of signaling proteins. Collectively, the study provides evidence for the conditional antioxidative function of FSD1 and its possible role in signaling.

plant biology↗

bioRxiv2021

An updated structural model of the A domain of the Pseudomonas putida XylR regulator exposes a distinct interplay with aromatic effectors

A revised model of the aromatic binding A domain of the {sigma}54-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 the large 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. 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. Proteomic analyses of the thereby tagged peptides confirmed the predicted in/out distribution of residues but also suggested that the fully-folded protein is not accessible by externally added effectors. The data thus suggested that XylR inducers assist the folding and/or the structuring of the A domain in an intramolecular non-repressive form rather than interacting dynamically with the aromatic partner once a fully structured protein is shaped. Originality-Significance StatementXylR is a transcriptional regulator of Pseudomonas putida strain mt-2 which activates the upper TOL pathway promoter Pu for catabolism of toluene and m-xylene upon binding of these aromatic effectors to its N-terminal A domain. While this feature has made XylR a popular platform for the development of whole-cell biosensors for aromatic compounds, the difficulty to crystallize the A domain --let alone the whole-length protein-- has made structural comprehension of the effector-regulator binding quite problematic. To overcome this impasse, we have combined homology-based structural predictions of the A domain of XylR with biochemical probing of exposed amino acids on the surface of the protein, both in vivo and in vitro. The results generally matched the effects of mutations known from previous genetic/phenotypic analyses of the protein. However, the data also suggested an intriguing mechanism of activation of XylR by effectors in which the inducer assists the shaping of the regulator in an active conformation rather than interacting a posteriori with an already formed protein invitro. This may in fact explain the longstanding failure to purify the protein in an effector-responsive form.

microbiology↗

bioRxiv2020

Surface display of designer protein scaffolds on genome-reduced strains of Pseudomonas putida

The bacterium Pseudomonas putida KT2440 is gaining considerable interest as a microbial platform for biotechnological valorization of polymeric organic materials, such as waste lignocellulose or plastics. However, P. putida on its own cannot make much use of such complex substrates, mainly because it lacks an efficient extracellular depolymerizing apparatus. We seek to meet this challenge by adopting a recombinant cellulosome strategy for this attractive host. Here, we report an essential step in this endeavor – a display of designer enzyme-anchoring protein “scaffoldins”, encompassing cohesin binding domains from divergent cellulolytic bacterial species on the P. putida surface. Two P. putida chassis strains, EM42 and EM371, with streamlined genomes and substantial differences in the composition of the outer membrane were employed in this study. Scaffoldin variants were delivered to their surface with one of four tested autotransporter systems (Ag43 from Escherichia coli), and the efficient display was confirmed by extracellular attachment of chimeric β-glucosidase and fluorescent proteins. Our results highlight the importance of cell surface engineering for display of recombinant proteins in Gram-negative bacteria and pave the way towards designer cellulosome strategies, tailored for P. putida.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

FSD1: a plastidial, nuclear and cytoplasmic enzyme relocalizing to the plasma membrane under salinity

Here, we aimed to resolve the developmental expression and subcellular localization of Arabidopsis iron superoxide dismutase FSD1, which belongs to the family of superoxide dismutases (SODs), prominent enzymes decomposing superoxide anion and determining abiotic stress tolerance. We found that fsd1 knockout mutants exhibit reduced lateral root number and that this phenotype was complemented by proFSD1::GFP:FSD1 and proFSD1::FSD1:GFP constructs. Light sheet fluorescence microscopy revealed a temporary accumulation of FSD1-GFP at the site of endosperm rupture during seed germination. In emerged roots, FSD1-GFP showed the highest abundance in cells of the lateral root cap, columella, and endodermis/cortex initials. The largest subcellular pool of FSD1-GFP was localized in the plastid stroma, while it was also located in the nuclei and cytoplasm. FSD1 is crucial for seed germination and salt stress tolerance, which is tightly coupled with FSD1-GFP subcellular relocation to the plasma membrane. FSD1 is most likely involved in superoxide decomposition in the periplasm. This study suggests a new osmoprotective function of SODs in plants.

plant biology↗

bioRxiv2019

Growth-(un)coupled biotransformation of D-xylose to D-xylonic acid by Pseudomonas putida EM42.

Co-production of two or more desirable compounds from low-cost substrates by a single microbial catalyst could greatly improve the economic competitiveness of many biotechnological processes. However, reports demonstrating the adoption of such co-production strategy are still scarce. In this study, the ability of genome-edited strain Psudomonas putida EM42 to simultaneously valorise D-xylose and D-cellobiose -two important lignocellulosic carbohydrates -by converting them into the platform chemical D-xylonic acid and medium chain length polyhydroxyalkanoates, respectively, was investigated. Biotransformation experiments performed with P. putida resting cells showed that promiscuous periplasmic glucose oxidation route can efficiently generate extracellular xylonate with high yield reaching 0.97 g per g of supplied xylose. Xylose oxidation was subsequently coupled to the growth of P. putida with cytoplasmic {beta}-glucosidase BglC from Thermobifida fusca on D-cellobiose. This disaccharide turned out to be a better co-substrate for xylose-to-xylonate biotransformation than monomeric glucose. This was because unlike glucose, cellobiose did not block oxidation of the pentose by periplasmic glucose dehydrogenase Gcd, but, similarly to glucose, it was a suitable substrate for polyhydroxyalkanoate formation in P. putida. Co-production of extracellular xylose-born xylonate and intracellular cellobiose-born medium chain length polyhydroxyalkanoates was established in proof-of-concept experiments with P. putida grown on the disaccharide. This study highlights the potential of P. putida EM42 as a microbial platform for the production of xylonic acid, identifies cellobiose as a new substrate for mcl-PHA production, and proposes a fresh strategy for the simultaneous valorisation of xylose and cellobiose.

bioengineering↗