Modeling the Initiation Phase of the Catalytic Cycle in the Glycyl-Radical Enzyme Benzylsuccinate Synthase.

The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site.

Piceatannol: a renaissance in antibacterial innovation unveiling synergistic potency and virulence disruption against serious pathogens.

In the relentless battle against multi-drug resistant Gram-negative bacteria, piceatannol emerges as a beacon of hope, showcasing unparalleled antibacterial efficacy and a unique ability to disrupt virulence factors. Our study illuminates the multifaceted prowess of piceatannol against prominent pathogens-Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. Notably, piceatannol demonstrated a remarkable ability to inhibit biofilm formation, reduce bacterial mobility, and diminish extracellular enzyme synthesis.

Lipid A in outer membrane vesicles shields bacteria from polymyxins.

The continuous emergence of multidrug-resistant bacterial pathogens poses a major global healthcare challenge, with Klebsiella pneumoniae being a prominent threat. We conducted a comprehensive study on K. pneumoniae's antibiotic resistance mechanisms, focusing on outer membrane vesicles (OMVs) and polymyxin, a last-resort antibiotic.

Framework for exploring the sensory repertoire of the human gut microbiota.

Bacteria sense changes in their environment and transduce signals to adjust their cellular functions accordingly. For this purpose, bacteria employ various sensors feeding into multiple signal transduction pathways. Signal recognition by bacterial sensors is studied mainly in a few model organisms, but advances in genome sequencing and analysis offer new ways of exploring the sensory repertoire of many understudied organisms.

Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans.

Plasmid-encoded type IV-A CRISPR-Cas systems lack an acquisition module, feature a DinG helicase instead of a nuclease, and form ribonucleoprotein complexes of unknown biological functions. Type IV-A3 systems are carried by conjugative plasmids that often harbor antibiotic-resistance genes and their CRISPR array contents suggest a role in mediating inter-plasmid conflicts, but this function remains unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 system co-opts the type I-E adaptation machinery from its host, Klebsiella pneumoniae (K.

Modular Low-Copy-Number Plasmid Vectors for Rhodobacterales with Extended Host Range in Alphaproteobacteria.

Members of the alphaproteobacterial order Rhodobacterales are metabolically diverse and highly abundant in the ocean. They are becoming increasingly interesting for marine biotechnology, due to their ecological adaptability, wealth of versatile low-copy-number plasmids, and their ability to produce secondary metabolites. However, molecular tools for engineering strains of this bacterial lineage are limited.

Rationally designed chromosome fusion does not prevent rapid growth of Vibrio natriegens.

DNA replication is essential for the proliferation of all cells. Bacterial chromosomes are replicated bidirectionally from a single origin of replication, with replication proceeding at about 1000 bp per second. For the model organism, Escherichia coli, this translates into a replication time of about 40 min for its 4.

Methodological advances enabled by the construction of a synthetic yeast genome.

The international Synthetic Yeast Project (Sc2.0) aims to construct the first synthetic designer eukaryote genome. Over the past few years, the Sc2.

Plasmid partitioning driven by collective migration of ParA between nucleoid lobes.

The ParABS system is crucial for the faithful segregation and inheritance of many bacterial chromosomes and low-copy-number plasmids. However, despite extensive research, the spatiotemporal dynamics of the ATPase ParA and its connection to the dynamics and positioning of the ParB-coated cargo have remained unclear. In this study, we utilize high-throughput imaging, quantitative data analysis, and computational modeling to explore the in vivo dynamics of ParA and its interaction with ParB-coated plasmids and the nucleoid.

Emergence of fractal geometries in the evolution of a metabolic enzyme.

Fractals are patterns that are self-similar across multiple length-scales. Macroscopic fractals are common in nature; however, so far, molecular assembly into fractals is restricted to synthetic systems. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpiński triangles.

A mRNA panel for differentiation between acute exacerbation or pneumonia in COPD patients.

Introduction: Patients suffering from chronic obstructive pulmonary disease (COPD) are prone to acute exacerbations (AECOPD) or community acquired pneumonia (CAP), both posing severe risk of morbidity and mortality. There is no available biomarker that correctly separates AECOPD from COPD. However, because CAP and AECOPD differ in aetiology, treatment and prognosis, their discrimination would be important.

Getting to the point: unipolar growth of Hyphomicrobiales.

The governing principles and suites of genes for lateral elongation or incorporation of new cell wall material along the length of a rod-shaped cell are well described. In contrast, relatively little is known about unipolar elongation or incorporation of peptidoglycan at one end of the rod. Recent work in three related model systems of unipolar growth (Agrobacterium tumefaciens, Brucella abortus, and Sinorhizobium meliloti) has clearly established that unipolar growth in the Hyphomicrobiales order relies on a set of genes distinct from the canonical elongasome.

Infrared spectroscopy reveals metal-independent carbonic anhydrase activity in crotonyl-CoA carboxylase/reductase.

The conversion of CO by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many biological processes. However, methods following the microscopic details of CO conversion at the active site are limited. Here, we used infrared spectroscopy to study the interaction of CO, water, bicarbonate, and other reactants with β-carbonic anhydrase from (CA) and crotonyl-CoA carboxylase/reductase from (Ccr), two of the fastest CO-converting enzymes in nature.

Ferredoxin reduction by hydrogen with iron functions as an evolutionary precursor of flavin-based electron bifurcation.

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO and were chemolithoautotrophs that obtained their energy and electrons from H. The acetyl-CoA pathway of CO fixation is central to that view because of its antiquity: Among known CO fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin-the physiological donor of electrons in the acetyl-CoA pathway-with electrons from H.

Polyketide Trimming Shapes Dihydroxynaphthalene-Melanin and Anthraquinone Pigments.

Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii are produced by a modular type II polyketide synthase system. A key enzyme involved in AQ biosynthesis is PlAntI, which catalyzes the hydrolysis of the bicyclic-intermediate-loaded acyl carrier protein, polyketide trimming, and assembly of the aromatic AQ scaffold.

Enhanced assembly of bacteriophage T7 produced in cell-free reactions under simulated microgravity.

On-demand biomanufacturing has the potential to improve healthcare and self-sufficiency during space missions. Cell-free transcription and translation reactions combined with DNA blueprints can produce promising therapeutics like bacteriophages and virus-like particles. However, how space conditions affect the synthesis and self-assembly of such complex multi-protein structures is unknown.

In Silico Design, In Vitro Construction, and In Vivo Application of Synthetic Small Regulatory RNAs in Bacteria.

Small regulatory RNAs (sRNAs) are short non-coding RNAs in bacteria capable of post-transcriptional regulation. sRNAs have recently gained attention as tools in basic and applied sciences, for example, to fine-tune genetic circuits or biotechnological processes. Even though sRNAs often have a rather simple and modular structure, the design of functional synthetic sRNAs is not necessarily trivial.

Alterations in the molecular control of mitochondrial turnover in COPD lung and airway epithelial cells.

Abnormal mitochondria have been observed in bronchial- and alveolar epithelial cells of patients with chronic obstructive pulmonary disease (COPD). However, it is unknown if alterations in the molecular pathways regulating mitochondrial turnover (mitochondrial biogenesis vs mitophagy) are involved. Therefore, in this study, the abundance of key molecules controlling mitochondrial turnover were assessed in peripheral lung tissue from non-COPD patients (n = 6) and COPD patients (n = 11; GOLDII n = 4/11; GOLDIV n = 7/11) and in both undifferentiated and differentiated human primary bronchial epithelial cells (PBEC) from non-COPD patients and COPD patients (n = 4-7 patients/group).

A MoClo-Compatible Toolbox of ECF Sigma Factor-Based Regulatory Switches for Proteobacterial Chassis.

The construction of complex synthetic gene circuits with predetermined and reliable output depends on orthogonal regulatory parts that do not inadvertently interfere with the host machinery or with other circuit components. Previously, extracytoplasmic function sigma factors (ECFs), a diverse group of alternative sigma factors with distinct promoter specificities, were shown to have great potential as context-independent regulators, but so far, they have only been used in a few model species. Here, we show that the alphaproteobacterium , which has been proposed as a plant-associated bacterial chassis for synthetic biology, has a similar phylogenetic ECF acceptance range as the gammaproteobacterium .

Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in .

Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and hence are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated.

Structure of the GDP-bound state of the SRP GTPase FlhF.

The GTPase FlhF, a signal recognition particle (SRP)-type enzyme, is pivotal for spatial-numerical control and bacterial flagella assembly across diverse species, including pathogens. This study presents the X-ray structure of FlhF in its GDP-bound state at a resolution of 2.28 Å.