Search

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates., QC 20240418

Lactic acid bacteria (LAB) are Gram positive bacteria frequently used in the food industry for fermentation, mainly transformation of carbohydrates into lactic acid. In addition, these bacteria also have the capacity to metabolize citrate, an organic acid commonly found in food products. Its fermentation leads to the production of 4-carbon compounds such as diacetyl, resulting in a buttery flavor desired in dairy products. Citrate metabolism is known to have several beneficial effects on LAB physiology. Nevertheless, a controversial effect of citrate has been described on the acid tolerance of the wine bacterium Oenococcus oeni. This observation raises questions about the effect of citrate on the capacity of O. oeni to conduct malolactic fermentation in highly acidic wines. This review aims to summarize the current understanding of citrate metabolism in LAB, with a focus on the wine bacterium O. oeni. Metabolism with the related enzymes is detailed, as are the involved genes organized in cit loci. The known systems of cit locus expression regulation are also described. Finally, the beneficial effects of citrate catabolism on LAB physiology are reported and the negative impact observed in O. oeni is discussed.

Pseudomonas aeruginosa is a ubiquitous nosocomial opportunistic pathogen that harbors many virulence determinants. Part of P. aeruginosa success colonizing a variety of habitats resides in its metabolic robustness and plasticity, which are the basis of its capability of adaptation to different nutrient sources and ecological conditions, including the infected host. Given this situation, it is conceivable that P. aeruginosa virulence might be, at least in part, under metabolic control, in such a way that virulence determinants are produced just when needed. Indeed, it has been shown that the catabolite repression control protein Crc, which together with the RNA chaperon Hfq regulates the P. aeruginosa utilization of carbon sources at the post-transcriptional level, also regulates, directly or indirectly, virulence-related processes in P. aeruginosa. Among them, Crc regulates P. aeruginosa cytotoxicity, likely by modulating the activity of the Type III Secretion System (T3SS), which directly injects toxins into eukaryotic host cells. The present work shows that the lack of Crc produces a Type III Secretion-defective phenotype in P. aeruginosa. The observed impairment is a consequence of a reduced expression of the genes encoding the T3SS, together with an impaired secretion of the proteins involved. Our results support that the impaired T3SS activity of the crc defective mutant is, at least partly, a consequence of a defective protein export, probably due to a reduced proton motive force. This work provides new information about the complex regulation of the expression and the activity of the T3SS in P. aeruginosa. Our results highlight the need of a robust bacterial metabolism, which is defective in the ∆crc mutant, to elicit complex and energetically costly virulence strategies, as that provided by the T3SS., Ministerio de Ciencia e Innovación, Depto. de Microbiología y Parasitología, Fac. de Farmacia, TRUE, pub, Descuento UCM

The solute carrier 17 (SLC17) family transports diverse organic anions using two distinct modes of coupling to a source of energy. Transporters that package glutamate and nucleotide into secretory vesicles for regulated release by exocytosis are driven by membrane potential but subject to allosteric regulation by H+ and Cl-. Other SLC17 members including the lysosomal sialic acid exporter couple the flux of organic anion to cotransport of H+. To begin to understand how similar proteins can perform such different functions, we have studied E. coli DgoT, a H+/galactonate cotransporter. A recent structure of DgoT showed many residues contacting D-galactonate, and we now find that they do not tolerate even conservative substitutions. In contrast, the closely related lysosomal H+/sialic acid cotransporter Sialin tolerates similar mutations, consistent with its recognition of diverse substrates with relatively low affinity. We also find that despite coupling to H+, DgoT transports more rapidly but with lower apparent affinity at high pH. Indeed, membrane potential can drive uptake, indicating electrogenic transport and suggesting a H+ : galactonate stoichiometry >1. Located in a polar pocket of the N-terminal helical bundle, Asp46 and Glu133 are each required for net flux by DgoT, but the E133Q mutant exhibits robust exchange activity and rescues exchange by D46N, suggesting that these two residues operate in series to translocate protons. E133Q also shifts the pH sensitivity of exchange by DgoT, supporting a central role for the highly conserved TM4 glutamate in H+ coupling by DgoT.

Pseudomonas aeruginosa is a ubiquitous nosocomial opportunistic pathogen that harbors many virulence determinants. Part of P. aeruginosa success colonizing a variety of habitats resides in its metabolic robustness and plasticity, which are the basis of its capability of adaptation to different nutrient sources and ecological conditions, including the infected host. Given this situation, it is conceivable that P. aeruginosa virulence might be, at least in part, under metabolic control, in such a way that virulence determinants are produced just when needed. Indeed, it has been shown that the catabolite repression control protein Crc, which together with the RNA chaperon Hfq regulates the P. aeruginosa utilization of carbon sources at the post-transcriptional level, also regulates, directly or indirectly, virulence-related processes in P. aeruginosa. Among them, Crc regulates P. aeruginosa cytotoxicity, likely by modulating the activity of the Type III Secretion System (T3SS), which directly injects toxins into eukaryotic host cells. The present work shows that the lack of Crc produces a Type III Secretion-defective phenotype in P. aeruginosa. The observed impairment is a consequence of a reduced expression of the genes encoding the T3SS, together with an impaired secretion of the proteins involved. Our results support that the impaired T3SS activity of the crc defective mutant is, at least partly, a consequence of a defective protein export, probably due to a reduced proton motive force. This work provides new information about the complex regulation of the expression and the activity of the T3SS in P. aeruginosa. Our results highlight the need of a robust bacterial metabolism, which is defective in the ∆crc mutant, to elicit complex and energetically costly virulence strategies, as that provided by the T3SS.

Oenococcus oeni is the main lactic acid bacterium associated with malolactic fermentation (MLF) of wines. MLF plays an important role in determining the final quality of wines. Nevertheless, due to the stressful conditions inherent to wine and especially acidity, MLF may be delayed. This study aimed to explore by adaptive evolution improvements in the acid tolerance of starters but also to gain a better understanding of the mechanisms involved in adaptation toward acidity. Four independent populations of the O. oeni ATCC BAA-1163 strain were propagated (approximately 560 generations) in a temporally varying environment, consisting in a gradual pH decrease from pH 5.3 to pH 2.9. Whole genome sequence comparison of these populations revealed that more than 45% of the substituted mutations occurred in only five loci for the evolved populations. One of these five fixed mutations affects mae, the first gene of the citrate operon. When grown in an acidic medium supplemented with citrate, a significantly higher bacterial biomass was produced with the evolved populations compared to the parental strain. Furthermore, the evolved populations slowed down their citrate consumption at low pH without impacting malolactic performance.

Tuberculosis (TB) continues to be a serious threat worldwide, especially in developing countries. Current first-line treatment for TB infections is a multidrug regimen for 4-9 months; if not taken as prescribed, drug-resistant TB can emerge. Novel drugs with unconventional targets are warranted to lessen the lengthy and four-drug treatment. Antimicrobial polymers mimicking naturally occurring antimicrobial peptides (AMPs) have gained much attention due to their enzymatic stability, tunability, cost-effectiveness, and unique mode of action - directly or indirectly - on bacterial membrane. However, selectivity and toxicity have limited their biological applications. Previously, our group synthesized a novel class of antimicrobial polymers, poly(guanylurea)s (PGUs). Unlike conventional AMP-mimics, poly (guanylurea piperazine)s have a linear architecture (i.e., no pendants) with all key functional groups along the backbone, resulting in low cytotoxicity and selectivity against mycobacteria. Here, this work explores the inherent antimycobacterial selectivity and bactericidal activity of PGU-P-8K on M. smegmatis, as a model organism for mycobacteria. PGU-P-8K showed fast-acting bactericidal activity (i.e., less than a day) at its minimum bactericidal concentration (MBC). This effect was further explored and attributed it to PGU-P-8K interfering with the bioenergetics in mycobacteria. PGU-P-8K displayed targeting the mycobacterial cell envelope by disrupting multiple intracellular processes while retaining the cell membrane integrity, confirmed by imagining of post-treatment cells and overexpression of genes related to cell envelope stress. Additionally, this project investigates how the key functionalities (e.g., aromatic substituents, amphiphilicity, rigidity) along the backbone of PGU-P-8K play a role in its antimycobacterial activity. The in-depth studies of how the physicochemical properties of PGU-P-8K affect selectivity and mode of action on mycobacteria contributes

The bacterial flagellum and the related virulence-associated injectisome system of pathogenic bacteria utilize a type III secretion system (T3SS) to export substrate proteins across the inner membrane in a proton motive force-dependent manner. The T3SS is composed of an export gate (FliPQR/FlhA/FlhB) located in the flagellar basal body and an associated soluble ATPase complex in the cytoplasm (FliHIJ). Here, we summarise recent insights into the structure, assembly and protein secretion mechanisms of the T3SS with a focus on energy transduction and protein transport across the cytoplasmic membrane., Peer Reviewed

The bacterial flagellum and the related virulence-associated injectisome system of pathogenic bacteria utilize a type III secretion system (T3SS) to export substrate proteins across the inner membrane in a proton motive force-dependent manner. The T3SS is composed of an export gate (FliPQR/FlhA/FlhB) located in the flagellar basal body and an associated soluble ATPase complex in the cytoplasm (FliHIJ). Here, we summarise recent insights into the structure, assembly and protein secretion mechanisms of the T3SS with a focus on energy transduction and protein transport across the cytoplasmic membrane., Peer Reviewed

Oxygen tension in the kidney is mostly determined by O-2 consumption (Qo(2)), which is, in turn, closely linked to tubular Na+ reabsorption. The objective of the present study was to develop a model of mitochondrial function in the proximal tubule (PT) cells of the rat renal cortex to gain more insight into the coupling between Qo(2), ATP formation (G(ATP)), ATP hydrolysis (QATP), and Na+ transport in the PH. The present model correctly predicts in vitro and in vivo measurements of Qo(2), Owns, and ATP and P-i concentrations in PT cells. Our simulations suggest that O-2 levels are not rate limiting in the proximal convoluted tubule, absent large metabolic perturbations. The model predicts that the rate of ATP hydrolysis and cytoplasmic pH each substantially regulate the G AT p-to-Qo(2) ratio, a key determinant of the number of Na+ moles actively reabsorbed per mole of O-2 consumed. An isolated increase in QATP or in cytoplasmic pH raises the GAS-to-Qo(2) ratio. Thus. variations in Na+ reabsorption and pH along the PT may, per se, generate axial heterogeneities in the efficiency of mitochondria' metabolism and Na+ transport. Our results also indicate that the G(AT)(P)-to-Qo(2) ratio is strongly impacted not only by H+ leak permeability. which reflects mitochondrial uncoupling, but also by K+ leak pathways. Simulations suggest that the negative impact of increased uncoupling in the diabetic kidney on mitochondrial metabolic efficiency is partly counterbalanced by increased rates of Na+ transport and ATP consumption. This model provides a framework to investigate the role of mitochondrial dysfunction in acute and chronic renal diseases.

Anaerobic industrial fermentation processes do not require aeration and intensive mixing and the accompanying cost savings are beneficial for production of chemicals and fuels. However, the free-energy conservation of fermentative pathways is often insufficient for the production and export of the desired compounds and/or for cellular growth and maintenance. To increase free-energy conservation during fermentation of the industrially relevant disaccharide sucrose by Saccharomyces cerevisiae, we first replaced the native yeast α-glucosidases by an intracellular sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from Phaseolus vulgaris (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 ± 0.02 h−1 (LmSPase) and 0.06 ± 0.01 h−1 (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01 h−1 and 0.08 ± 0.00 h−1, respectively. Determination of the biomass yield in anaerobic sucrose-limited chemostat cultures was used to assess the free-energy conservation of the engineered strains. Replacement of intracellular hydrolase with a phosphorylase increased the biomass yield on sucrose by 31%. Additional replacement of the native proton-coupled sucrose uptake system by PvSUF1 increased the anaerobic biomass yield by a further 8%, resulting in an overall increase of 41%. By experimentally demonstrating an energetic benefit of the combined engineering of disaccharide uptake and cleavage, this study represents a first step towards anaerobic production of compounds whose metabolic pathways currently do not conserve sufficient free-energy., QC 20180515

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Antimicrobial peptides (AMPs) are viable alternatives to the currently available antimicrobials, and numerous studies have investigated their possible use as therapeutic agents for specific clinical applications. AMPs are a diverse class of antimicrobials that often act upon the bacterial cell membrane but may exhibit additional modes of action. Identification of the multiple modes of action requires a comprehensive study at subinhibitory concentrations and careful data analysis since additional modes of action can be eclipsed by AMP action on the cell membrane. Techniques that measure the biosynthesis rate of macromolecules (e.g., DNA, RNA, protein, and cell wall) and the cytoplasmic membrane proton motive force (PMF) energy can help to unravel the diverse modes of action of AMPs. Here, we present an overview of macromolecule biosynthesis rate measurement and fluorescence spectroscopy methods to identify AMP mode(s) of action. Detailed protocols designed to measure inhibition of DNA, RNA, protein, and cell wall synthesis or membrane de-energization are presented and discussed for optimal application of these two techniques as well as to enable accurate interpretation of the experimental findings.

Ions play fundamental roles in all living cells and their gradients are often essential to fuel transports, to regulate enzyme activities and to transduce energy within and between cells. Their homeostasis is therefore an essential component of the cell metabolism. Ions must be imported from the extracellular matrix to their final subcellular compartments. Among them, the chloroplast is a particularly interesting example because there, ions not only modulate enzyme activities, but also mediate ATP synthesis and actively participate in the building of the photosynthetic structures by promoting membrane-membrane interaction. In this review, we first provide a comprehensive view of the different machineries involved in ion trafficking and homeostasis in the chloroplast, and then discuss peculiar functions exerted by ions in the frame of photochemical conversion of absorbed light energy.

Molecular motors play a central role in all known living systems. Cells use them to fight the never ending battle against entropy. Motors allow cells to organize their components in space and time to perform complex functions. In this thesis I focus on two molecular motors: the packaging motor of phi29, and the flagellar motor of Escherichia coli. These are both well-studied motors with decades of research poured into them. By leveraging past knowledge, I was able to undertake subtle studies of their function. I tortured these motors by putting them in sub-optimal operating conditions to test their limits.For the phage motor we were primarily interested in what sort of contacts it uses to engage and exert force on its DNA substrate. We queried the nature of these contacts by making a series of modified DNA substrates, with a focus on methylphosphonate substitutions that maintain the structure of DNA while removing the negative charge. Interestingly, we found that the negative charge of the phosphates was not required for the motor to translocate DNA, but that the charge stabilizes the interaction and makes the motor less likely to slip. We also discovered that the motor interacts more strongly with one of the DNA strands, and we used these modifications to identify a set of periodic contacts.For the flagellar motor I was interested in the role of proton motive force (PMF) in changing the rotational direction of the motor, which is controlled by a complex on its cytoplasmic side called the flagellar switch. The motor is powered by PMF, but does the PMF also affect the switch? We found that in fact the switch is affected by changes in PMF. This has implications for the switching mechanism. The switch is not only affected by the chemical binding signal, but is also affected by its associations with the motor and potentially its proximity to charges on the cell membrane.

Molecular motors play a central role in all known living systems. Cells use them to fight the never ending battle against entropy. Motors allow cells to organize their components in space and time to perform complex functions. In this thesis I focus on two molecular motors: the packaging motor of phi29, and the flagellar motor of Escherichia coli. These are both well-studied motors with decades of research poured into them. By leveraging past knowledge, I was able to undertake subtle studies of their function. I tortured these motors by putting them in sub-optimal operating conditions to test their limits.For the phage motor we were primarily interested in what sort of contacts it uses to engage and exert force on its DNA substrate. We queried the nature of these contacts by making a series of modified DNA substrates, with a focus on methylphosphonate substitutions that maintain the structure of DNA while removing the negative charge. Interestingly, we found that the negative charge of the phosphates was not required for the motor to translocate DNA, but that the charge stabilizes the interaction and makes the motor less likely to slip. We also discovered that the motor interacts more strongly with one of the DNA strands, and we used these modifications to identify a set of periodic contacts.For the flagellar motor I was interested in the role of proton motive force (PMF) in changing the rotational direction of the motor, which is controlled by a complex on its cytoplasmic side called the flagellar switch. The motor is powered by PMF, but does the PMF also affect the switch? We found that in fact the switch is affected by changes in PMF. This has implications for the switching mechanism. The switch is not only affected by the chemical binding signal, but is also affected by its associations with the motor and potentially its proximity to charges on the cell membrane.

17 p., Since the pioneering work by Julius Adler in the 1960's, bacterial chemotaxis has been predominantly studied as metabolism-independent. All available simulation models of bacterial chemotaxis endorse this assumption. Recent studies have shown, however, that many metabolism-dependent chemotactic patterns occur in bacteria. We hereby present the simplest artificial protocell model capable of performing metabolism-based chemotaxis. The model serves as a proof of concept to show how even the simplest metabolism can sustain chemotactic patterns of varying sophistication. It also reproduces a set of phenomena that have recently attracted attention on bacterial chemotaxis and provides insights about alternative mechanisms that could instantiate them. We conclude that relaxing the metabolism-independent assumption provides important theoretical advances, forces us to rethink some established pre-conceptions and may help us better understand unexplored and poorly understood aspects of bacterial chemotaxis.

17 p., Since the pioneering work by Julius Adler in the 1960's, bacterial chemotaxis has been predominantly studied as metabolism-independent. All available simulation models of bacterial chemotaxis endorse this assumption. Recent studies have shown, however, that many metabolism-dependent chemotactic patterns occur in bacteria. We hereby present the simplest artificial protocell model capable of performing metabolism-based chemotaxis. The model serves as a proof of concept to show how even the simplest metabolism can sustain chemotactic patterns of varying sophistication. It also reproduces a set of phenomena that have recently attracted attention on bacterial chemotaxis and provides insights about alternative mechanisms that could instantiate them. We conclude that relaxing the metabolism-independent assumption provides important theoretical advances, forces us to rethink some established pre-conceptions and may help us better understand unexplored and poorly understood aspects of bacterial chemotaxis.

17 p., Since the pioneering work by Julius Adler in the 1960's, bacterial chemotaxis has been predominantly studied as metabolism-independent. All available simulation models of bacterial chemotaxis endorse this assumption. Recent studies have shown, however, that many metabolism-dependent chemotactic patterns occur in bacteria. We hereby present the simplest artificial protocell model capable of performing metabolism-based chemotaxis. The model serves as a proof of concept to show how even the simplest metabolism can sustain chemotactic patterns of varying sophistication. It also reproduces a set of phenomena that have recently attracted attention on bacterial chemotaxis and provides insights about alternative mechanisms that could instantiate them. We conclude that relaxing the metabolism-independent assumption provides important theoretical advances, forces us to rethink some established pre-conceptions and may help us better understand unexplored and poorly understood aspects of bacterial chemotaxis.

In Escherichia coli, secretory proteins (preproteins) are translocated across the cytoplasmic membrane by the Sec system composed of a protein-conducting channel, SecYEG, and an ATP-dependent motor protein, SecA. After binding of the preprotein to SecYEG-bound SecA, cycles of ATP binding and hydrolysis by SecA are thought to drive the stepwise translocation of the preprotein across the membrane. To address how the length of a preprotein substrate affects the SecA-driven translocation process, we constructed derivatives of the precursor of the outer membrane protein A ( proOmpA) with 2, 4, 6, and 8 in-tandem repeats of the periplasmic domain. With increasing polypeptide length, an increasing delay in the time before full-length translocation was observed, but the translocation rate expressed as amino acid translocation per minute remained constant. These data indicate that in the ATP-dependent reaction, SecA drives a constant rate of preprotein translocation consistent with a stepping mechanism of translocation.

The xylP gene of Lactobacillus pentosus, the first gene of the xylPQR operon, was recently found to be involved in isoprimeverose metabolism. By expression of xylP on a multicopy plasmid in Lactobacillus plantarum 80, a strain which lacks active isoprimeverose and D-xylose transport activities, it was shown that xylP encodes a transporter. Functional expression of the XylP transporter was shown by uptake of isoprimeverose in L. plantarum 80 cells, and this transport was driven by the proton motive force generated by malolactic fermentation. XylP was unable to catalyze transport of D-xylose.

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.