New insights into enterocin CRL35: mechanism of action and immunity revealed by heterologous expression in Escherichia coli

The role of the class IIa bacteriocin membrane receptor protein remains unclear, and the following two different mechanisms have been proposed: the bacteriocin could interact with the receptor changing it to an open conformation or the receptor might act as an anchor allowing subsequent bacteriocin insertion and membrane disruption. Bacteriocin-producing cells synthesize an immunity protein that forms an inactive bacteriocin–receptor–immunity complex. To better understand the molecular mechanism of enterocin CRL35, the peptide was expressed as the suicidal probe EtpM-enterocin CRL35 in Escherichia coli, a naturally insensitive microorganism since it does not express the receptor. When the bacteriocin is anchored to the periplasmic face of the plasma membrane through the bitopic membrane protein, EtpM, E. coli cells depolarize and die. Moreover, co-expression of the immunity protein prevents the deleterious effect of EtpM-enterocin CRL35. The binding and anchoring of the bacteriocin to the membrane has demonstrated to be a sufficient condition for its membrane insertion. The final step of membrane disruption by EtpM-enterocin CRL35 is independent from the receptor, which means that the mannose PTS might not be involved in the pore structure. In addition, the immunity protein can protect even in the absence of the receptor.Fil: Barraza, Daniela Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Ríos Colombo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Galván, Adriana Emilce. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Acuña, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Minahk, Carlos Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Bellomio, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

The case for class II bacteriocins: A biophysical approach using “suicide probes” in receptor-free hosts to study their mechanism of action

Class II bacteriocins are unmodified membrane-active peptides that act over a narrow spectrum of target bacteria. They bind a specific receptor protein on the membrane to form a pore, leading to membrane permeabilization and cell death. However, little is known about the molecular events triggering the pore formation after the bacteriocin recognizes the receptor. It is not clear yet if the pore is the same receptor forced into an open conformation or if the pore results from the bacteriocin insertion and oligomeric assembly in the lipid bilayer. In order to reveal which model is more suitable to explain the toxicity mechanism, in this work we use chimeric peptides, resulting from the fusion of the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35, pediocin PA-1 and microcin V. E. coli strains lacking the specific receptors for these bacteriocins were chosen as expression hosts. As these constructs display a lethal effect when they are heterologously expressed, they are called “suicide probes”. The results suggest that, indeed, the specific receptor would act as a docking molecule more than as a structural piece of the pore, as long as the bacteriocin is somehow anchored to the membrane. These set of chimeric peptides also represent an in vivo system that allows to study the interaction of the bacteriocins with real bacterial membranes, instead of model membranes. Hence, the effects of these suicide probes in membrane fluidity and transmembrane potential were also assessed, using fluorescence spectroscopy. The data show that the different suicide probes are able to increase phospholipid order and depolarize the membranes of receptor-free bacterial cells.Fil: Ríos Colombo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Dupuy, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Gonzalez, Claudio Fabricio. University of Florida; Estados UnidosFil: Bellomio, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentin

The killer yeast Wickerhamomyces anomalus Cf20 exerts a broad anti-Candida activity through the production of killer toxins and volatile compounds

Candidiasis is a group of opportunistic infections caused by yeast of the genus Candida. The appearance of drug resistance and the adverse effects of current antifungal therapies require the search for new, more efficient therapeutic alternatives. Killer yeasts have aroused as suitable candidates for mining new antifungal compounds. Killer strains secrete antimicrobial proteins named killer toxins, with promissory antifungal activity. Here we found that the killer yeast Wickerhamomyces anomalus Cf20 and its cell-free supernatant (CFS) inhibited six pathogenic strains and one collection strain of Candida spp. The inhibition is mainly mediated by secreted killer toxins and, to a lesser extent, by volatile compounds such as acetic acid and ethyl acetate. A new large killer toxin (>180 kDa) was purified, which exerted 70-74% of the total CFS anti-Candida activity, and the previously described glucanase KTCf20 was inhibitory in a lesser extent as well. In addition, we demonstrated that Cf20 possesses the genes encoding for the β-1,3-glucanases WaExg1 and WaExg2, proteins with extensively studied antifungal activity, particularly WaExg2. Finally, the 10-fold concentrated CFS exerted a high candidacidal effect at 37°C, completely inhibiting the fungal growth, although the nonconcentrated CFS (RCF 1) had very limited fungistatic activity at this temperature. In conclusion, W. anomalus Cf20 produces different low and high molecular weight compounds with anti-Candida activity that could be used to design new therapies for candidiasis and as a source for novel antimicrobial compounds as well.Fil: Fernandez de Ullivarri, Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Bulacios, Gabriela Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Navarro, Silvia Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Lanza, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Mendoza, Lucia Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

Co-expression and characterization of enterocin CRL35 and its mutant in Escherichia coli Rosetta

Even though many sequences and structures of bacteriocins from lactic acid bacteria have been fully characterized so far, little information is currently available about bacteriocins heterologously produced by Escherichia coli. For this purpose, the structural gene of enterocin CRL35, munA, was PCR-amplified using specific primers and cloned downstream PelB sequence in the pET22b (+) expression vector. E. coli Rosetta (DE3) pLysS was chosen as the host for production and enterocin was purified by an easy two-step protocol. The bacteriocin was correctly expressed with the expected intramolecular disulfide bond. Nevertheless, it was found that a variant of the enterocin, differing by 12 Da from the native polypeptide, was co-expressed by E. coli Rosetta in comparable amount. Indeed, the mutant bacteriocin contained two amino acid substitutions that were characterized by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) and HPLC-electrospray (ESI)-Q-TOF tandem mass spectrometry (MS/MS) sequencing. This is the first report regarding the production of mutants of pediocin-like bacteriocins in the E. coli expression system.Fil: Masias, Ruth Emilse. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Picariello, Gianluca. Consiglio Nazionale delle Ricerche (CNR). Istituto di Scienze dell’Alimentazione; ItaliaFil: Acuña, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia Para Lactobacilos (i); ArgentinaFil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Saavedra, Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia Para Lactobacilos (i); Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Minahk, Carlos Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentin

In vivo systems to study class II bacteriocins toxicity and immunity

Class II bacteriocins are membrane-active peptides that act over a narrowspectrum of bacterial targets and have a great potential application as antibioticsin medical sciences. They act on the cytoplasmic membrane dissipating thetransmembrane potential by forming pores. There is solid evidence thatmembrane receptor proteins are necessary for their function, however the preciserole of this receptor and the nature of the pore remain elusive. The most acceptedmodel suggest that bacteriocins bind the receptor to change its conformation,creating a channel that remains open. Nonetheless, several studies support asecond model in which the bacteriocin is able to disrupt the membrane itself andthe receptor might act just as an anchor allowing the subsequent bacteriocininsertion to form the pore. In order to reveal whether or not the pore structureinvolves the specific receptor, we designed chimeric peptides fusing themembrane protein EtpM with different class II bacteriocins. We chose E. coli as areceptor-free expression host. The fusion EtpM-bacteriocin anchors eachbacteriocin to the membrane and kills the expressing host cell, even in theabsence of the specific receptor. These results are in line with the second model inwhich the pore is formed through a receptor-independent interaction with the lipidbilayer. The effect of these interactions was also analyzed, through a fluorophorethat changes its fluorescence intensity according to transmembrane potential.On the other hand, an immunity protein protects the producer strain against itsown bacteriocin. For antimicrobials under investigation for clinical applications, thepotential emergence of resistant pathogens and the study of immune mechanismsare a primary concern. Though no direct in vitro interaction bacteriocin-immunityhas been reported before, by using an in vivo system, we present evidence thatthis binding might occur, not in aqueous solution but in a membrane inserted .Fil: Ríos Colombo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Galván, Adriana Emilce. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Navarro, Silvia Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Lanza, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Barraza, Daniela Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Acuña, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Fernandez de Ullivarri, Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Minahk, Carlos Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Bellomio, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaXLVII Reunión Anual de la Sociedad Argentina de BiofísicaLa PlataArgentinaSociedad Argentina de Biofísic

Features and applications of Ent35-MccV hybrid bacteriocin: current state and perspectives

Bacteriocins are peptides of ribosomal synthesis that are active against bacteria related to the producing strain. They have been widely used in the food industry as biopreservatives. The generation of hybrid peptides by combining the genes that encode two different bacteriocins has made it possible to study the mechanisms of action of the bacteriocins that compose them and also develop new peptides with improved biotechnological applications. Hybrid bacteriocins may be obtained in several ways. In our laboratory, by combining enterocin CRL35 and microcin V (Ent35-MccV), we obtained a broad-spectrum peptide that is active against both Gram-positive and Gram-negative bacteria. Ent35-MccV is sensitive to the action of intestinal proteases and is heat resistant, which makes it a good candidate for use as a biopreservative. For this reason, the peptide was tested in skim milk and beef burgers as food models. We also obtained more potent variants of the hybrid by modifying the central amino acid of the hinge region that connects the two bacteriocins. This review also discusses future applications and perspectives regarding the Ent35-MccV and other hybrid peptides. Key Points • Ent35-MccV is a new broad-spectrum bacteriocin. • The mechanism of action of bacteriocins can be studied using hybrid peptides. • Genetic engineering allows obtaining improved bacteriocin derivatives. • Hybrid peptides can be used in the food, pharmaceutical, and veterinary applications.Fil: Navarro, Silvia Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Lanza, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Acuña, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Bellomio, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

Pediocin-like bacteriocins: new perspectives on mechanism of action and immunity

This review attempts to analyze the mechanism of action and immunity of class IIa bacteriocins. These peptides are promising alternative food preservatives and they have a great potential application in medical sciences. Class IIa bacteriocins act on the cytoplasmic membrane of Gram-positive cells dissipating the transmembrane electrical potential by forming pores. However, their toxicity and immunity mechanism remains elusive. Here we discuss the role of the mannose phosphotransferase system (man-PTS) as the receptor for class IIa bacteriocins and the influence of the membrane composition on the activity of these antimicrobial peptides. A model that is consistent with experimental results obtained by different researchers involves the non-specific binding of the bacteriocin to the negatively charged membrane of target bacteria. This step would facilitate a specific binding to the receptor protein, altering its functionality and forming an independent pore in which the bacteriocin is inserted in the membrane. An immunity protein could specifically recognize and block the pore. Bacteriocins function in bacterial ecosystems and energetic costs associated with their production are also discussed. Theoretical models based on solid experimental evidence are vital to understand bacteriocins mechanism of action and to promote new technological developments.Fil: Ríos Colombo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Navarro, Silvia Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Bellomio, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

Microbiological culture broth designed from food waste

The current trend of increasing air, water, and soil pollution is, in part, due to inadequate management of municipal solid waste (MSW). The relationship between public health and the collection, storage and improper disposal of solid waste has encouraged several studies and the results were attributed to the spread of over twenty human and animal diseases due to this interrelationship. The term single cell protein (SCP) refers to microbial biomass used as a dietary additive. It has high nutritional value because of its high content of vitamins, lipids, and proteins of biological quality (the presence of all essential amino acids) (Lal, 2005). The aim of this work was to design a culture media for microbiological assays and to produce SCP for animal feeding, using nutrients contained in organic waste. In order to compare the effectiveness of food waste (FW) and LAPTg media, different strains of Lactobacillus, Enterococcus, Staphylococcus, Shigella, Salmonella, Saccharomyces and Schizosaccharomyces were studied. In all cases, the growth obtained from FW and LAPTg culture media were not significantly different (p > 0.05). In addition, the growth of Saccharomyces cerevisiae was studied in order to produce SCP for animal feeding. Comparative experiments involving molasses broth, FW broth, and basal broth were carried out. The biomass yield calculated at 24 h from FW broth was 13% lower than from molasses broth. The FW broth provided a significantly lower biomass yield; however, it can be very useful in areas where molasses are not available. FW broth can be elaborated at low cost, in any populated region of the world because its ingredients are wastes generated by humans. It has great versatility, allowing the development of a wide variety of microorganisms, both Gram negative and Gram positive bacteria as well as yeasts. The production of safe protein additives, with high biological quality and low cost, is necessary due to the increasing global demand for food for humans and animals.Fil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán; ArgentinaFil: Terán, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); ArgentinaFil: Arena, Mario Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto de Quimica del Noroeste; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: Oliszewky, Ruben. Universidad Nacional de Tucumán. Facultad de Agronomía y Zootecnia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán; ArgentinaFil: Gonzalez, Silvia Nelina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán; Argentin

Tyrosine 9 is the key amino acid in microcin J25 superoxide overproduction

Escherichia coli microcin J25 (MccJ25) is a lasso-peptide antibiotic comprising 21 l-amino acid residues (G1-G-A-G-H5-V-P-E-Y- F10-V-G-I-G-T15-P-I-S-F-Y20-G). MccJ25 has two independent substrates: RNA-polymerase (RNAP) and the membrane respiratory chain. The latter is mediated by oxygen consumption inhibition together with an increase of superoxide production. In the present paper, the antibiotic MccJ25 was engineered by substituting Tyr9 or Tyr20 with phenylalanine. Both mutants were well transported into the cells and remained active on RNAP. Only the Y9F mutant lost the ability to overproduce superoxide and inhibit oxygen consumption. The last results confirm that the Tyr 9, and not Tyr20, is involved in the MccJ25 action on the respiratory chain target.Fil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Bellomio, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Solbiati, José O.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Farias, Ricardo Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Vincent, Paula Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

Cytochromes bd-I and bo3 are essential for the bactericidal effect of microcin J25 on Escherichia coli cells

Microcin J25 has two targets in sensitive bacteria, the RNA polymerase, and the respiratory chain through inhibition of cellular respiration. In this work, the effect of microcin J25 in E. coli mutants that lack the terminal oxidases cytochrome bd-I and cytochrome bo3 was analyzed. The mutant strains lacking cytochrome bo3 or cytochrome bd-I were less sensitive to the peptide. In membranes obtained from the strain that only expresses cytochrome bd-I a great ROS overproduction was observed in the presence of microcin J25. Nevertheless, the oxygen consumption was less inhibited in this strain, probably because the oxygen is partially reduced to superoxide. There was no overproduction of ROS in membranes isolated from the mutant strain that only express cytochrome bo3 and the inhibition of the cellular respiration was similar to the wild type. It is concluded that both cytochromes bd-I and bo3 are affected by the peptide. The results establish for the first time a relationship between the terminal oxygen reductases and the mechanism of action of microcin J25.Fil: Galván, Adriana Emilce. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Schurig Briccio, Lici Ariane. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. University of Illinois at Urbana; Estados UnidosFil: Salomon, Raul Armando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Minahk, Carlos Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Gennis, R. B.. University of Illinois; Estados UnidosFil: Bellomio, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin