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. Our research demonstrates that OMVs protect bacteria from polymyxins. OMVs derived from Polymyxin B (PB)-stressed K. pneumoniae exhibited heightened protective efficacy due to increased vesiculation, compared to OMVs from unstressed Klebsiella. OMVs also shield bacteria from different bacterial families. This was validated ex vivo and in vivo using precision cut lung slices (PCLS) and Galleria mellonella. In all models, OMVs protected K. pneumoniae from PB and reduced the associated stress response on protein level. We observed significant changes in the lipid composition of OMVs upon PB treatment, affecting their binding capacity to PB. The altered binding capacity of single OMVs from PB stressed K. pneumoniae could be linked to a reduction in the lipid A amount of their released vesicles. Although the amount of lipid A per vesicle is reduced, the overall increase in the number of vesicles results in an increased protection because the sum of lipid A and therefore PB binding sites have increased. This unravels the mechanism of the altered PB protective efficacy of OMVs from PB stressed K. pneumoniae compared to control OMVs. The lipid A-dependent protective effect against PB was confirmed in vitro using artificial vesicles. Moreover, artificial vesicles successfully protected Klebsiella from PB ex vivo and in vivo. The findings indicate that OMVs act as protective shields for bacteria by binding to polymyxins, effectively serving as decoys and preventing antibiotic interaction with the cell surface. Our findings provide valuable insights into the mechanisms underlying antibiotic cross-protection and offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug-resistant bacterial infections.

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

Fractals are patterns that are self-similar across multiple length-scales1. Macroscopic fractals are common in nature2-4; however, so far, molecular assembly into fractals is restricted to synthetic systems5-12. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpinski triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution.

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. The structure exhibits the classical N- and G-domain fold, consistent with related SRP GTPases such as Ffh and FtsY. Comparative analysis with GTP-loaded FlhF elucidates the conformational changes associated with GTP hydrolysis. These topological reconfigurations are similarly evident in Ffh and FtsY, and play a pivotal role in regulating the functions of these hydrolases.

The mysterious diadenosine tetraphosphate (AP4A).

Dinucleoside polyphosphates, a class of nucleotides found amongst all the Trees of Life, have been gathering a lot of attention in the past decades due to their putative role as cellular alarmones. In particular, diadenosine tetraphosphate (AP4A) has been widely studied in bacteria facing various environmental challenges and has been proposed to be important for ensuring cellular survivability through harsh conditions. Here, we discuss the current understanding of AP4A synthesis and degradation, protein targets, their molecular structure where possible, and insights into the molecular mechanisms of AP4A action and its physiological consequences. Lastly, we will briefly touch on what is known with regards to AP4A beyond the bacterial kingdom, given its increasing appearance in the eukaryotic world. Altogether, the notion that AP4A is a conserved second messenger in organisms ranging from bacteria to humans and is able to signal and modulate cellular stress regulation seems promising.

Diadenosine tetraphosphate regulates biosynthesis of GTP in Bacillus subtilis.

Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to humans. Nevertheless, its physiological role and the underlying molecular mechanisms are poorly characterized. We investigated the molecular mechanism by which Ap4A regulates inosine-5'-monophosphate dehydrogenase (IMPDH, a key branching point enzyme for the biosynthesis of adenosine or guanosine nucleotides) in Bacillus subtilis. We solved the crystal structure of BsIMPDH bound to Ap4A at a resolution of 2.45 Å to show that Ap4A binds to the interface between two IMPDH subunits, acting as the glue that switches active IMPDH tetramers into less active octamers. Guided by these insights, we engineered mutant strains of B. subtilis that bypass Ap4A-dependent IMPDH regulation without perturbing intracellular Ap4A pools themselves. We used metabolomics, which suggests that these mutants have a dysregulated purine, and in particular GTP, metabolome and phenotypic analysis, which shows increased sensitivity of B. subtilis IMPDH mutant strains to heat compared with wild-type strains. Our study identifies a central role for IMPDH in remodelling metabolism and heat resistance, and provides evidence that Ap4A can function as an alarmone.

Inhibition of SRP-dependent protein secretion by the bacterial alarmone (p)ppGpp.

The stringent response enables bacteria to respond to nutrient limitation and other stress conditions through production of the nucleotide-based second messengers ppGpp and pppGpp, collectively known as (p)ppGpp. Here, we report that (p)ppGpp inhibits the signal recognition particle (SRP)-dependent protein targeting pathway, which is essential for membrane protein biogenesis and protein secretion. More specifically, (p)ppGpp binds to the SRP GTPases Ffh and FtsY, and inhibits the formation of the SRP receptor-targeting complex, which is central for the coordinated binding of the translating ribosome to the SecYEG translocon. Cryo-EM analysis of SRP bound to translating ribosomes suggests that (p)ppGpp may induce a distinct conformational stabilization of the NG domain of Ffh and FtsY in Bacillus subtilis but not in E. coli.

Structural and functional characterization of the bacterial biofilm activator RemA.

Bacillus subtilis can form structurally complex biofilms on solid or liquid surfaces, which requires expression of genes for matrix production. The transcription of these genes is activated by regulatory protein RemA, which binds to poorly conserved, repetitive DNA regions but lacks obvious DNA-binding motifs or domains. Here, we present the structure of the RemA homologue from Geobacillus thermodenitrificans, showing a unique octameric ring with the potential to form a 16-meric superstructure. These results, together with further biochemical and in vivo characterization of B. subtilis RemA, suggests that the protein can wrap DNA around its ring-like structure through a LytTR-related domain.

The ups and downs of ectoine: structural enzymology of a major microbial stress protectant and versatile nutrient.

Ectoine and its derivative 5-hydroxyectoine are compatible solutes and chemical chaperones widely synthesized by Bacteria and some Archaea as cytoprotectants during osmotic stress and high- or low-growth temperature extremes. The function-preserving attributes of ectoines led to numerous biotechnological and biomedical applications and fostered the development of an industrial scale production process. Synthesis of ectoines requires the expenditure of considerable energetic and biosynthetic resources. Hence, microorganisms have developed ways to exploit ectoines as nutrients when they are no longer needed as stress protectants. Here, we summarize our current knowledge on the phylogenomic distribution of ectoine producing and consuming microorganisms. We emphasize the structural enzymology of the pathways underlying ectoine biosynthesis and consumption, an understanding that has been achieved only recently. The synthesis and degradation pathways critically differ in the isomeric form of the key metabolite N-acetyldiaminobutyric acid (ADABA). γ-ADABA serves as preferred substrate for the ectoine synthase, while the α-ADABA isomer is produced by the ectoine hydrolase as an intermediate in catabolism. It can serve as internal inducer for the genetic control of ectoine catabolic genes via the GabR/MocR-type regulator EnuR. Our review highlights the importance of structural enzymology to inspire the mechanistic understanding of metabolic networks at the biological scale.

Degradation of the microbial stress protectants and chemical chaperones ectoine and hydroxyectoine by a bacterial hydrolase-deacetylase complex.

When faced with increased osmolarity in the environment, many bacterial cells accumulate the compatible solute ectoine and its derivative 5-hydroxyectoine. Both compounds are not only potent osmostress protectants, but also serve as effective chemical chaperones stabilizing protein functionality. Ectoines are energy-rich nitrogen and carbon sources that have an ecological impact that shapes microbial communities. Although the biochemistry of ectoine and 5-hydroxyectoine biosynthesis is well understood, our understanding of their catabolism is only rudimentary. Here, we combined biochemical and structural approaches to unravel the core of ectoine and 5-hydroxy-ectoine catabolisms. We show that a conserved enzyme bimodule consisting of the EutD ectoine/5-hydroxyectoine hydrolase and the EutE deacetylase degrades both ectoines. We determined the high-resolution crystal structures of both enzymes, derived from the salt-tolerant bacteria Ruegeria pomeroyi and Halomonas elongata These structures, either in their apo-forms or in forms capturing substrates or intermediates, provided detailed insights into the catalytic cores of the EutD and EutE enzymes. The combined biochemical and structural results indicate that the EutD homodimer opens the pyrimidine ring of ectoine through an unusual covalent intermediate, N-α-2 acetyl-l-2,4-diaminobutyrate (α-ADABA). We found that α-ADABA is then deacetylated by the zinc-dependent EutE monomer into diaminobutyric acid (DABA), which is further catabolized to l-aspartate. We observed that the EutD-EutE bimodule synthesizes exclusively the α-, but not the γ-isomers of ADABA or hydroxy-ADABA. Of note, α-ADABA is known to induce the MocR/GabR-type repressor EnuR, which controls the expression of many ectoine catabolic genes clusters. We conclude that hydroxy-α-ADABA might serve a similar function.

Biosynthesis of the Stress-Protectant and Chemical Chaperon Ectoine: Biochemistry of the Transaminase EctB.

Bacteria frequently adapt to high osmolarity surroundings through the accumulation of compatible solutes. Ectoine is a prominent member of these types of stress protectants and is produced via an evolutionarily conserved biosynthetic pathway beginning with the L-2,4-diaminobutyrate (DAB) transaminase (TA) EctB. Here, we studied EctB from the thermo-tolerant Gram-positive bacterium Paenibacillus lautus (Pl) and show that this tetrameric enzyme is highly tolerant to salt, pH, and temperature. During ectoine biosynthesis, EctB converts L-glutamate and L-aspartate-beta-semialdehyde into 2-oxoglutarate and DAB, but it also catalyzes the reverse reaction. Our analysis unravels that EctB enzymes are mechanistically identical to the PLP-dependent gamma-aminobutyrate TAs (GABA-TAs) and only differ with respect to substrate binding. Inspection of the genomic context of the ectB gene in P. lautus identifies an unusual arrangement of juxtapositioned genes for ectoine biosynthesis and import via an Ehu-type binding-protein-dependent ABC transporter. This operon-like structure suggests the operation of a highly coordinated system for ectoine synthesis and import to maintain physiologically adequate cellular ectoine pools under osmotic stress conditions in a resource-efficient manner. Taken together, our study provides an in-depth mechanistic and physiological description of EctB, the first enzyme of the ectoine biosynthetic pathway.

Alkali and Alkaline Earth Metal Derivatives of Disila-Bridged Podands: Coordination Chemistry and Structural Diversity.

Within this study, the synthesis and coordination chemistry of open-chain ligands bearing disila-units is presented. Instead of basic 1:1 complexes, structural diversity was discovered in the variety of ligand and salt. Stable complexes of alkali and alkaline earth metal complexes were obtained by equimolar reactions of different salts with the disila-bridged podands 8,9-disila-EO5 (1) and 11,12-disila-EO7 (2) (EO5 = pentaethylene glycol; EO7 = heptaethylene glycol). The respective alkaline earth metal complexes of the type [Ca(8,9-disila-EO5)(OTf)2] (3), [Sr(8,9-disila-EO5)I2] (5), [Sr(11,12-disila-EO7)I]I (6), and [Ba(11,12-disila-EO7)OTf2] (7) (OTf = CF3SO3-) were characterized via single-crystal X-ray diffraction analyses. Within the reaction of the alkali metal salt NaPF6 with 1, the sodium ion acts as a template during the complexation process. Under elimination of one molecule of diethylene glycol, the dinuclear species [Na2(8,9,17,18-tetrasila-EO8)(PF6)2]·EO2 (4) (EO8 = octaethylen glycol, EO2 = diethylene glycol) is obtained, in which the sodium cations are 7-fold coordinated within a disilane-bearing framework. The reaction of 2 with CsOTf failed, leading to recrystallization of anhydrous CsOTf. By means of DFT calculations it was shown that the disila-bearing ligands are burdened with negative hyperconjugation interactions between the silicon and the oxygen atoms, but the coordination by sufficiently hard cations can easily overcompensate the competing polarization. In contrast, soft Lewis acids barely share interactions with silicon-bonded oxygen atoms. All findings are consistent with observations made in solution according to 29Si NMR spectroscopical studies.