Six states of Enterococcus hirae V-type ATPase reveals non-uniform rotor rotation during turnover.

The vacuolar-type ATPase from Enterococcus hirae (EhV-ATPase) is a thus-far unique adaptation of V-ATPases, as it performs Na+ transport and demonstrates an off-axis rotor assembly. Recent single molecule studies of the isolated V1 domain have indicated that there are subpauses within the three major states of the pseudo three-fold symmetric rotary enzyme. However, there was no structural evidence for these. Herein we activate the EhV-ATPase complex with ATP and identified multiple structures consisting of a total of six states of this complex by using cryo-electron microscopy. The orientations of the rotor complex during turnover, especially in the intermediates, are not as perfectly uniform as expected. The densities in the nucleotide binding pockets in the V1 domain indicate the different catalytic conditions for the six conformations. The off-axis rotor and its' interactions with the stator a-subunit during rotation suggests that this non-uniform rotor rotation is performed through the entire complex.

Degradation of indole via a two-component indole oxygenase system from Enterococcus hirae GDIAS-5.

Animal farming copiously generates indoles, which contribute to odor and pose a challenge for deodorization. While biodegradation is widely accepted, there is a lack of suitable indole-degrading bacteria for animal husbandry. In this study, we aimed to construct genetically engineered strains with indole-degrading abilities. Enterococcus hirae GDIAS-5 is a highly efficient indole-degrading bacterium, which functions via a monooxygenase YcnE presumably contributes to indole oxidation. However, the efficiency of engineered Escherichia coli expressing YcnE for indole degradation is lower than that of GDIAS-5. To improve its efficacy, the underlying indole-degradation mechanisms in GDIAS-5 were analyzed. An ido operon that responds to a two-component indole oxygenase system was identified. In vitro experiments showed that the reductase component of YcnE, YdgI, can improve the catalytic efficiency. The reconstruction of the two-component system in E. coli exhibited higher indole removal efficiency than GDIAS-5. Furthermore, isatin, the key intermediate metabolite in indole degradation, might be degraded via a novel isatin-acetaminophen-aminophenol pathway involving an amidase whose coding gene is located near the ido operon. The two-component anaerobic oxidation system, upstream degradation pathway, and engineering strains investigated in this study provide important insights into indole degradation metabolism and offer efficient resources for achieving bacterial odor elimination.

Structure of lactate oxidase from Enterococcus hirae revealed new aspects of active site loop function: Product-inhibition mechanism and oxygen gatekeeper.

l-Lactate oxidase (LOx) is a flavin mononucleotide (FMN)-dependent triose phosphate isomerase (TIM) barrel fold enzyme that catalyzes the oxidation of l-lactate using oxygen as a primary electron acceptor. Although reductive half-reaction mechanism of LOx has been studied by structure-based kinetic studies, oxidative half-reaction and substrate/product-inhibition mechanisms were yet to be elucidated. In this study, the structure and enzymatic properties of wild-type and mutant LOxs from Enterococcus hirae (EhLOx) were investigated. EhLOx structure showed the common TIM-barrel fold with flexible loop region. Noteworthy observations were that the EhLOx crystal structures prepared by co-crystallization with product, pyruvate, revealed the complex structures with "d-lactate form ligand," which was covalently bonded with a Tyr211 side chain. This observation provided direct evidence to suggest the product-inhibition mode of EhLOx. Moreover, this structure also revealed a flip motion of Met207 side chain, which is located on the flexible loop region as well as Tyr211. Through a saturation mutagenesis study of Met207, one of the mutants Met207Leu showed the drastically decreased oxidase activity but maintained dye-mediated dehydrogenase activity. The structure analysis of EhLOx Met207Leu revealed the absence of flipping in the vicinity of FMN, unlike the wild-type Met207 side chain. Together with the simulation of the oxygen-accessible channel prediction, Met207 may play as an oxygen gatekeeper residue, which contributes oxygen uptake from external enzyme to FMN. Three clades of LOxs are proposed based on the difference of the Met207 position and they have different oxygen migration pathway from external enzyme to active center FMN.

Indole metabolism mechanisms in a new, efficient indole-degrading facultative anaerobe isolate Enterococcus hirae GDIAS-5.

Indole is an inter-species and inter-kingdom signaling molecule widespread in the natural world. A large amount of indole in livestock wastes makes it difficult to be degraded, which causes serious malodor. Identifying efficient and eco-friendly ways to eliminate it is an urgent task for the sustainable development of husbandry. While bioconversion is a widely accepted means, the mechanism of indole microbial degradation is little understood, especially under anaerobic conditions. Herein, a new Enterococcus hirae isolate GDIAS-5, effectively degraded 100 mg/L indole within 28 h aerobically or 5 days anaerobically. Three intermediates (oxindole, isatin, and catechol) were identified in indole degradation, and catechol was further degraded by a meta-cleavage catabolic pathway. Two important processes for GDIAS-5 indole utilization were discovered. One is Fe(III) uptake and reduction, which may be a critical process that is coupled with indole oxidation, and the other is the entire pathway directly involved in indole oxidation and metabolism. Furthermore, monooxygenase ycnE responsible for indole oxidation via the indole-oxindole-isatin pathway was identified for the first time. Bioinformatic analyses showed that ycnE from E. hirae formed a phylogenetically separate branch from monooxygenases of other species. These findings provide new targets and strategies for synthetic biological reconstruction of indole-degrading bacteria.

Application of Homology Modeling by Enhanced Profile-Profile Alignment and Flexible-Fitting Simulation to Cryo-EM Based Structure Determination.

Application of cryo-electron microscopy (cryo-EM) is crucially important for ascertaining the atomic structure of large biomolecules such as ribosomes and protein complexes in membranes. Advances in cryo-EM technology and software have made it possible to obtain data with near-atomic resolution, but the method is still often capable of producing only a density map with up to medium resolution, either partially or entirely. Therefore, bridging the gap separating the density map and the atomic model is necessary. Herein, we propose a methodology for constructing atomic structure models based on cryo-EM maps with low-to-medium resolution. The method is a combination of sensitive and accurate homology modeling using our profile-profile alignment method with a flexible-fitting method using molecular dynamics simulation. As described herein, this study used benchmark applications to evaluate the model constructions of human two-pore channel 2 (one target protein in CASP13 with its structure determined using cryo-EM data) and the overall structure of Enterococcus hirae V-ATPase complex.

Aislamiento de cepas de Enterococcus hirae productoras de enterocinas a partir del contenido intestinal de mejillón patagónico (Mytilus edulis platensis) Aislamiento de Enterococcus hirae de mejillón patagónico / Isolation of enterocin-producing Enterococcus hirae strains from the intestinal content of the Patagonian mussel (Mytilus edulis platensis)

Resumen Se aislaron del contenido intestinal del mejillón patagónico dos cepas de bacterias ácido lácticas y se caracterizaron por pruebas fenotípicas y moleculares. Los aislamientos se identificaron como Enterococcus hirae y fueron denominados E. hirae 463Me y 471Me. Por técnicas de PCR se identificó el gen de la enterocina P en ambas cepas, mientras que solamente en la cepa 471Me se detectó la enterocina hiracin JM79. Ambas cepas resultaron sensibles a los antibióticos clínicamente importantes y entre los rasgos de virulencia investigados mediante amplificación por PCR solo se pudieron detectar los genes cylL l y cylL s , sin embargo, no se observó actividad hemolítica en la prueba de agar sangre. Los sobrenadantes libres de células resultaron activos contra todas las cepas de Listeria y Enterococcus ensayadas, contra Lactobacillus plantarum TwLb 5 y contra Vibrio anguilarum V10. En óptimas condiciones de crecimiento, ambas cepas mostraron actividad inhibitoria contra Listeria innocua ATCC 33090 después de 2h de incubación. E. hirae 471Me alcanzó una actividad inhibitoria máxima de 163.840UA/ml después de 6h de incubación, mientras que el mismo valor se registró para E. hirae 463Me después de 8h. En ambos casos, la actividad antagonista alcanzó su máximo antes de lograr la fase estacionaria y permaneció estable hasta las 24h de incubación. En nuestro conocimiento, este es el primer informe de aislamiento de cepas bacteriocinogénicas de E. hirae de mejillón patagónico. La alta actividad inhibitoria y la ausencia de rasgos de virulencia indican que estos microorganismos podrían aplicarse en áreas biotecnológicas como la biopreservación de alimentos o las formulaciones probióticas.

Abstract Two bacteriocin-producing lactic acid bacterial strains were isolated from the intestinal content of the Patagonian mussel and characterized by phenotypic and molecular tests. The isolates were identified as Enterococcus hirae and named E. hirae 463Me and 471Me. The presence of the enterocin P gene was identified in both strains by PCR techniques, while enterocin hiracin JM79 was detected only in the 471Me strain. Both strains were sensitive to clinically important antibiotics and among the virulence traits investigated by PCR amplification, only cylL l and cylL s could be detected; however, no hemolytic activity was observed in the blood agar test. Cell free supernatants were active against all Listeria and Enterococcus strains tested, Lactobacillus plantarum TwLb 5 and Vibrio anguilarum V10. Under optimal growth conditions, both strains displayed inhibitory activity against Listeria innocua ATCC 33090 after 2h of incubation. E. hirae 471Me achieved a maximum activity of 163840AU/ml after 6h of incubation, while the same value was recorded for E. hirae 463Me after 8h. In both cases, the antagonist activity reached its maximum before the growth achieved the stationary phase and remained stable up to 24h of incubation. To our knowledge, this is first report of the isolation of bacteriocinogenic E. hirae strains from the Patagonian mussel. The high inhibitory activity and the absence of virulence traits indicate that they could be applied in different biotechnological areas such as food biopreservation or probiotic formulations.

Novel enterocin E20c purified from Enterococcus hirae 20c synergised with ß-lactams and ciprofloxacin against Salmonella enterica.

BACKGROUND: An increasing rate of antibiotic resistance among Gram-negative bacterial pathogens has created an urgent need to discover novel therapeutic agents to combat infectious diseases. Use of bacteriocins as therapeutic agents has immense potential due to their high potency and mode of action different from that of conventional antibiotics. RESULTS: In this study, a novel bacteriocin E20c of molecular weight 6.5 kDa was purified and characterized from the probiotic strain of Enterococcus hirae. E20c had bactericidal activities against several multidrug resistant (MDR) Gram-negative bacterial pathogens. Flow cytometry and scanning electron microscopy studies showed that it killed the Salmonella enterica cells by forming ion-permeable channels in the cell membrane leading to enhanced cell membrane permeability. Further, checkerboard titrations showed that E20c had synergistic interaction with antibiotics such as ampicillin, penicillin, ceftriaxone, and ciprofloxacin against a ciprofloxacin- and penicillin-resistant strain of S. enterica. CONCLUSION: Thus, this study shows the broad spectrum antimicrobial activity of novel enterocin E20c against various MDR pathogens. Further, it highlights the importance of bacteriocins in lowering the minimum inhibitory concentrations of conventional antibiotics when used in combination.

Antimicrobial activity of bacteriocins produced by Enterococcus isolates recovered from Egyptian homemade dairy products against some foodborne pathogens.

The increasing mandate for fresh-like food products and the possible hazards of chemically preserved foods necessitate the search for alternatives. Bacteriocins represent a promising food biopreservative. In the present study, one hundred enterococci isolates recovered from Egyptian raw cow milk and homemade dairy products were screened for bacteriocin production. The overall detection rate was 10%. Three isolates, namely, Enterococcus faecalis (OE-7 and OE-12) and Enterococcus hirae (OE-9), showed the highest antibacterial activity with narrow spectrum against multidrug-resistant (MDR) Gram-positive foodborne bacteria: Enterococcus faecalis and Staphylococcus aureus. The antimicrobial activity was completely abolished by trypsin and proteinase K but not affected by lipase and/or amylase indicating the protein nature of the antimicrobial activity. Optimum conditions for bacteriocin production were cultivation in MRS broth at 37 °C, pH 6-6.5 for 16-24 h. The tested bacteriocins exhibited bactericidal activity on S. aureus subsp. aureus ATCC 25923; such activity was further investigated by transmission electron microscopy that revealed leakage and lysis of treated cells. Characterization of tested bacteriocins revealed high activity in a wide range of pH and temperature, storage stability, and heat resistance. PCR analysis revealed that the tested isolates produced multiple enterocins showing homology with the enterocins L50A, AS-48, and 31. Finally, this study reported potent antibacterial activity of bacteriocins derived from dairy products Enterococci against MDR foodborne and spoilage pathogens. The potency, specificity, and stability of these bacteriocins presented promising perspectives for application as biopreservatives in the food industry. The biopreservation of foods by bacteriocins produced by lactic acid bacteria recovered directly from foods remains an innovative approach.

[Isolation of enterocin-producing Enterococcus hirae strains from the intestinal content of the Patagonian mussel (Mytilus edulis platensis)]. / Aislamiento de cepas de Enterococcus hirae productoras de enterocinas a partir del contenido intestinal de mejillón patagónico (Mytilus edulis platensis).

Two bacteriocin-producing lactic acid bacterial strains were isolated from the intestinal content of the Patagonian mussel and characterized by phenotypic and molecular tests. The isolates were identified as Enterococcus hirae and named E. hirae 463Me and 471Me. The presence of the enterocin P gene was identified in both strains by PCR techniques, while enterocin hiracin JM79 was detected only in the 471Me strain. Both strains were sensitive to clinically important antibiotics and among the virulence traits investigated by PCR amplification, only cylLl and cylLs could be detected; however, no hemolytic activity was observed in the blood agar test. Cell free supernatants were active against all Listeria and Enterococcus strains tested, Lactobacillus plantarum TwLb 5 and Vibrio anguilarum V10. Under optimal growth conditions, both strains displayed inhibitory activity against Listeria innocua ATCC 33090 after 2h of incubation. E. hirae 471Me achieved a maximum activity of 163840AU/ml after 6h of incubation, while the same value was recorded for E. hirae 463Me after 8h. In both cases, the antagonist activity reached its maximum before the growth achieved the stationary phase and remained stable up to 24h of incubation. To our knowledge, this is first report of the isolation of bacteriocinogenic E. hirae strains from the Patagonian mussel. The high inhibitory activity and the absence of virulence traits indicate that they could be applied in different biotechnological areas such as food biopreservation or probiotic formulations.

Nisin Production by Enterococcus hirae DF105Mi Isolated from Brazilian Goat Milk.

The purpose of this study was to select the promising biopreservation bacteriocin producer strain from goat milk and characterize the expressed bacteriocin, related to its physiological and biochemical properties and specificity of operon encoding production and expression of antimicrobial peptide. Brazilian goat milk was used as the source for the selection of bacteriocin-producing lactic acid bacteria. One strain (DF105Mi) stood out for its strong activity against several Listeria monocytogenes strains. Selected strain was identified based on the biochemical and physiological characteristics and 16s rRNA analysis. The bacteriocin production and inhibitory spectrum of strain DF105Mi were studied, together with the evaluation of the effect of temperature, pH, and chemicals on bacteriocin stability and production, activity, and adsorption to target cells as well as to the cell surface of bacteriocin producers. Physiological and bio-molecular analyses based on targeting of different genes, parts of nisin operon were performed in order to investigate the hypothesis that the studied strain can produce and express nisin. Based on biochemical, physiological, and 16s rRNA analysis, the strain DF105Mi was classified as Enterococcus hirae. The selected strain produces a bacteriocin which is stable in a wide range of pH (2.0-12.0), temperature (up to 120 °C), presence of selected chemicals and presents adsorption affinity to different test organisms, process influenced by environmental conditions. Higher bacteriocin production by Ent. hirae DF105Mi was recorded during stationary growth phase, but only when the strain was cultured at 37 °C. The strain's genetic analysis indicated presence of the genes coding for the production of the bacteriocin nisin. This result was confirmed by cross-checking the sensitivity of the produced strain to commercial nisin A. The strong anti-Listeria activity, bacteriocin adsorption, and stability of produced bacteriocin indicate that Ent. hirae DF105Mi presents a differentiated potential application for biopreservation of fermented dairy products.

Essay on Biomembrane Structure.

Of all the macromolecular assemblies of life, the least understood is the biomembrane. This is especially true in regard to its atomic structure. Ideas on biomembranes, developed in the last 200 years, culminated in the fluid mosaic model of the membrane. In this essay, I provide a historical outline of how we arrived at our current understanding of biomembranes and the models we use to describe them. A selection of direct experimental findings on the nano-scale structure of biomembranes is taken up to discuss their physical nature, and special emphasis is put on the surprising insights that arise from atomic scale descriptions.

Physiological and molecular insights of bacteriocin production by Enterococcus hirae ST57ACC from Brazilian artisanal cheese.

The bacteriocinogenic Enterococcus hirae ST57ACC recently isolated from a Brazilian artisanal cheese was subjected here to additional analyses in order to evaluate its bacteriocin production and the potential influence of ABC transporter system in its expression. Besides these physiological and molecular aspects, the bacteriocin was evaluated for its cytotoxicity against HT-29. Differences in the inoculum size had no impact on the growth of E. hirae ST57ACC; however, the bacteriocin was only produced after 9 h of growth when the strain was inoculated at 5% or 10% (v/v), with similar levels of bacteriocin production obtained by both conventional growth and batch fermentation. Furthermore, potential expression of ABC transporters corresponding to the bacteriocin transport and sugar metabolism was identified. In terms of adverse effects, when a semi-purified fraction of the bacteriocin and the cell-free supernatant were tested against HT-29, total cell viability was similar to observed on untreated cells, indicating the absence of cytotoxic effect. Based on the obtained results, E. hirae ST57ACC can produce its bacteriocin at industrial level by using bioreactors, its bacteriocin expression is potentially influenced by the ABC transporter system, and no cytotoxic effects were observed on HT-29 cells, indicating its potential use as a bio-preservative.

Off-axis rotor in Enterococcus hirae V-ATPase visualized by Zernike phase plate single-particle cryo-electron microscopy.

EhV-ATPase is an ATP-driven Na+ pump in the eubacteria Enterococcus hirae (Eh). Here, we present the first entire structure of detergent-solubilized EhV-ATPase by single-particle cryo-electron microscopy (cryo-EM) using Zernike phase plate. The cryo-EM map dominantly showed one of three catalytic conformations in this rotary enzyme. To further stabilize the originally heterogeneous structure caused by the ATP hydrolysis states of the V1-ATPases, a peptide epitope tag system was adopted, in which the inserted peptide epitope sequence interfered with rotation of the central rotor by binding the Fab. As a result, the map unexpectedly showed another catalytic conformation of EhV-ATPase. Interestingly, these two conformations identified with and without Fab conversely coincided with those of the minor state 2 and the major state 1 of Thermus thermophilus V/A-ATPase, respectively. The most prominent feature in EhV-ATPase was the off-axis rotor, where the cytoplasmic V1 domain was connected to the transmembrane Vo domain through the off-axis central rotor. Furthermore, compared to the structure of ATP synthases, the larger size of the interface between the transmembrane a-subunit and c-ring of EhV-ATPase would be more advantageous for active ion pumping.

Low-density lipoprotein (LDL)-dependent uptake of Gram-positive lipoteichoic acid and Gram-negative lipopolysaccharide occurs through LDL receptor.

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are bacterial lipids that stimulate pro-inflammatory cytokine production, thereby exacerbating sepsis pathophysiology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates uptake of cholesterol by downregulating hepatic lipoprotein receptors, including low-density lipoprotein (LDL) receptor (LDLR) and possibly LDLR-related protein-1 (LRP1). PCSK9 also negatively regulates Gram-negative LPS uptake by hepatocytes, however this mechanism is not completely characterized and mechanisms of Gram-positive LTA uptake are unknown. Therefore, our objective was to elucidate the mechanisms through which PCSK9 regulates uptake of LTA and LPS by investigating the roles of lipoproteins and lipoprotein receptors. Here we show that plasma PCSK9 concentrations increase transiently over time in septic and non-septic critically ill patients, with highly similar profiles over 14 days. Using flow cytometry, we demonstrate that PCSK9 negatively regulates LDLR-mediated uptake of LTA and LPS by HepG2 hepatocytes through an LDL-dependent mechanism, whereas LRP1 and high-density lipoprotein do not contribute to this uptake pathway. Bacterial lipid uptake by hepatocytes was not associated with cytokine production or hepatocellular injury. In conclusion, our study characterizes an LDL-dependent and LDLR-mediated bacterial lipid uptake pathway regulated by PCSK9, and provides evidence in support of PCSK9 inhibition as a potential therapeutic strategy for sepsis.

Characterization of α-D-glucan produced by a probiont Enterococcus hirae KX577639 from feces of south Indian Irula tribals.

Exopolysaccharide (EPS) producing probiotic strain Enterococcus hirae KX577639 were isolated from the feces of South Indian Irula tribes. EPS yield was 18.57 g/L (dry weight) at 48 h in 2% sucrose supplemented MRS medium. TLC and GC-MS analysis confirmed the presence of predominant glucose monomer indicating the homopolysaccharide nature of EPS. FTIR and NMR studies revealed that the EPS were branched α-D-glucan polymer with α-(1 → 6) and α-(1 → 3) linkages. SEM analysis of glucan-EPS revealed porous and starch like cracked granules of aggregation. AFM studies proved spherical lumps and dense, grainy like network. The thermal behavior of glucan-EPS showed degradation temperature of 315.98 °C and melting point of 296.67 °C. The XRD analysis confirmed the amorphous nature of EPS with a crystalline index of 0.48. The water solubility index and water holding capacity of glucan-EPS showed 46.5% and 202.04%. These distinctive features of the glucan EPS could find its potential application in functional food products as the α-(1 → 3) linkage are resistant to human digestive enzymes and can serve as a nutrient to gut bacteria. This the first study reporting the EPS production by Enterococcus hirae.

Enterococcus hirae LcpA (Psr), a new peptidoglycan-binding protein localized at the division site.

BACKGROUND: Proteins from the LytR-CpsA-Psr family are found in almost all Gram-positive bacteria. Although LCP proteins have been studied in other pathogens, their functions in enterococci remain uncharacterized. The Psr protein from Enterococcus hirae, here renamed LcpA, previously associated with the regulation of the expression of the low-affinity PBP5 and ß-lactam resistance, has been characterized. RESULTS: LcpA protein of E. hirae ATCC 9790 has been produced and purified with and without its transmembrane helix. LcpA appears, through different methods, to be localized in the membrane, in agreement with in silico predictions. The interaction of LcpA with E. hirae cell wall indicates that LcpA binds enterococcal peptidoglycan, regardless of the presence of secondary cell wall polymers. Immunolocalization experiments showed that LcpA and PBP5 are localized at the division site of E. hirae. CONCLUSIONS: LcpA belongs to the LytR-CpsA-Psr family. Its topology, localization and binding to peptidoglycan support, together with previous observations on defective mutants, that LcpA plays a role related to the cell wall metabolism, probably acting as a phosphotransferase catalyzing the attachment of cell wall polymers to the peptidoglycan.

Biochemical Properties and Mechanism of Action of Enterocin LD3 Purified from Enterococcus hirae LD3.

Enterocin LD3 was purified using activity-guided multistep chromatography techniques such as cation-exchange and gel-filtration chromatography. The preparation's purity was tested using reverse-phase ultra-performance liquid chromatography. The specific activity was tested to be 187.5 AU µg(-1) with 13-fold purification. Purified enterocin LD3 was heat stable up to 121 °C (at 15 psi pressure) and pH 2-6. The activity was lost in the presence of papain, reduced by proteinase K, pepsin and trypsin, but was unaffected by amylase and lipase, suggesting proteinaceous nature of the compound and no role of carbohydrate and lipid moieties in the activity. MALDI-TOF/MS analysis of purified enterocin LD3 resolved m/z 4114.6, and N-terminal amino acid sequence was found to be H2NQGGQANQ-COOH suggesting a new bacteriocin. Dissipation of membrane potential, loss of internal ATP and bactericidal effect were recorded when indicator strain Micrococcus luteus was treated with enterocin LD3. It inhibited Gram-positive and Gram-negative bacteria including human pathogens such as Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Listeria monocytogenes, Escherichia coli O157:H7, E. coli (urogenic, a clinical isolate) and Vibrio sp. These properties of purified enterocin LD3 suggest its applications as a food biopreservative and as an alternative to clinical antibiotics.