Reduced Binding of the Endolysin LysTP712 to Lactococcus lactis ΔftsH Contributes to Phage Resistance

Absence of the membrane protease FtsH in Lactococcus lactis hinders release of the bacteriophage TP712. In this work we have analyzed the mechanism responsible for the non-lytic phenotype of L. lactis ΔftsH after phage infection. The lytic cassette of TP712 contains a putative antiholin–pinholin system and a modular endolysin (LysTP712). Inducible expression of the holin gene demonstrated the presence of a dual start motif which is functional in both wildtype and L. lactis ΔftsH cells. Moreover, simulating holin activity with ionophores accelerated lysis of wildtype cells but not L. lactis ΔftsH cells, suggesting inhibition of the endolysin rather than a role of FtsH in holin activation. However, zymograms revealed the synthesis of an active endolysin in both wildtype and L. lactis ΔftsH TP712 lysogens. A reporter protein was generated by fusing the cell wall binding domain of LysTP712 to the fluorescent mCherry protein. Binding of this reporter protein took place at the septa of both wildtype and L. lactis ΔftsH cells as shown by fluorescence microscopy. Nonetheless, fluorescence spectroscopy demonstrated that mutant cells bound 40% less protein. In conclusion, the non-lytic phenotype of L. lactis ΔftsH is not due to direct action of the FtsH protease on the phage lytic proteins but rather to a putative function of FtsH in modulating the architecture of the L. lactis cell envelope that results in a lower affinity of the phage endolysin to its substrate.This work has been supported by grant BIO2013-46266R (MINECO, Spain). BM, PG, and AR also acknowledge funding by GRUPIN14-139 Plan de Ciencia, Tecnología e Innovación 2013-2017 (Principado de Asturias, Spain) and FEDER EU funds.Peer reviewe

Evaluation of the Potential of Lactobacillus paracasei Adjuncts for Flavor Compounds Development and Diversification in Short-Aged Cheddar Cheese

peer-reviewedThe non-starter microbiota of Cheddar cheese mostly comprises mesophilic lactobacilli, such as Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and Lactobacillus plantarum. These bacteria are recognized for their potential to improve Cheddar cheese flavor when used as adjunct cultures. In this study, three strains of L. paracasei (DPC2071, DPC4206, and DPC4536) were evaluated for their contribution to the enhancement and diversification of flavor in short-aged Cheddar cheese. The strains were selected based on their previously determined genomic diversity, variability in proteolytic enzyme activities and metabolic capability in cheese model systems. The addition of adjunct cultures did not affect the gross composition or levels of lipolysis of the cheeses. The levels of free amino acids (FAA) in cheeses showed a significant increase after 28 days of ripening. However, the concentrations of individual amino acids in the cheeses did not significantly differ except for some amino acids (aspartic acid, threonine, serine, and tryptophan) at Day 14. Volatile profile analysis revealed that the main compounds that differentiated the cheeses were of lipid origin, such as long chain aldehydes, acids, ketones, and lactones. This study demonstrated that the adjunct L. paracasei strains contributed to the development and diversification of compounds related to flavor in short-aged Cheddar cheeses

Implementación de pruebas ECOE para la evaluación de competencias en el Grado de Farmacia

Memoria ID2022-214 Ayudas de la Universidad de Salamanca para la innovación docente, curso 2022-2023

Mecanismos moleculares de respuesta al estrés sobre la pared celular en Lactococcus lactis

El éxito de las fermentaciones en la industria láctea depende de la aptitud tecnológica de las cepas iniciadoras basada, entre otras características, en la resistencia a condiciones de estrés. El objetivo de esta Tesis Doctoral ha sido identificar mecanismos moleculares desarrollados por Lactococcus lactis como respuesta y resistencia al estrés ocasionado sobre la pared celular que, en último término, sirvan como base para optimizar su papel industrial como cultivo iniciador en quesería. La lactococina 972 (Lcn972), una bacteriocina que no forma poros en la membrana sino que bloquea la síntesis de peptidoglicano en el septo, y exclusivamente en Lactococcus, ha sido utilizada como herramienta para provocar estrés sobre la pared celular. Las líneas de actuación para cumplir el objetivo planteado han sido: a) determinar la contribución a la supervivencia de L. lactis de genes que responden al estrés sobre su pared celular; b) identificar mecanismos de resistencia a Lcn972, y c) estudiar el papel de la proteasa FtsH en el ciclo infectivo del bacteriofago TP712. En primer lugar se generaron mutantes multicopia y no funcionales del gen llmg0169 y del operón llmg2164-2163, regulados por el sistema de dos componentes CesSR, y que presentan el mayor grado de inducción como respuesta a estrés sobre la pared celular. Los mutantes se sometieron a condiciones de estrés tecnológico (acidez, temperatura, sal, liofilización, compuestos antimicrobianos y bacteriófagos), lo que permitió demostrar que estos genes eran necesarios, pero no suficientes, para garantizar la viabilidad de L. lactis en esas condiciones. En el caso del operón llmg2164-2163, se demostró que era, además, un determinante de resistencia a Lcn972. Por otro lado, la caracterización genética y fenotípica de mutantes de L. lactis resistentes a Lcn972 permitió identificar mecanismos implicados en dicha resistencia. Entre ellos destacó la modificación de la composición del peptidoglicano en las cepas resistentes, con un mayor contenido de muropéptidos tripéptido, en detrimento de los que contienen pentapéptido.Estos mutantes fueron, a su vez, resistentes a otros compuestos antimicrobianos y a la infección por los bacteriófagos c2 y sk1, características deseables en cepas de uso industrial. El análisis genético de los mutantes resistentes a Lcn972 puso de manifiesto una deleción de 22,6 kpb incluyendo, entre otros, genes relacionados con el metabolismo de la maltosa, con la infección del bacteriófago c2 y el sistema de dos componentes F, aunque no se pudo demostrar la implicación directa de ninguno de ellos en la resistencia a Lcn972. También se detectó la activación del gen llmg2447, mediada por la inserción en su promotor de la secuencia de inserción IS981. La sobreexpresión de llmg2447 en L. lactis confirió específicamente resistencia a Lcn972. Por su entorno genético, llmg2447 podría haber formado parte de un sistema ancestral anti-sigma/sigma ECF y haber evolucionado hacia un elemento con función protectora. Por último, se estudió el papel de la proteasa de membrana FtsH, cuyo gen también pertenece al regulón de CesR, en la inducción de profagos como respuesta al daño de la pared celular en L. lactis. Los resultados demostraron que, en ausencia de FtsH, el bacteriófago TP712 no es capaz de propagarse en L. lactis debido a su incapacidad de degradar la pared celular de su hospedador. A esta conclusión se llegó tras descartar experimentalmente la implicación de FtsH en otras fases del ciclo biológico del fago como adsorción, integración como profago, escisión y replicación del ADN fágico, o ensamblaje de las partículas virales. Se demostró que estos viriones eran infectivos, pero permanecían retenidos en el citoplasma tras la inducción del profago en L. lactis ¿ftsH. No se ha podido demostrar su papel en la activación de la holina y se postula su implicación en la regulación de la actividad de la endolisina.Cell Wall-active Bacteriocins and Their Applications Beyond Antibiotic Activity. http://dx.doi.org/10.1007/s12602-012-9116-9 . http://hdl.handle.net/10261/80726Contribution of the CesR-regulated genes llmg0169 and llmg2164-2163 to Lactococcus lactis fitness. http://dx.doi.org/10.1016/j.ijfoodmicro.2009.06.002 . http://hdl.handle.net/10261/51415Isolation of Lactococcus lactis mutants simultaneously resistant to the cell wall-active bacteriocin Lcn972, lysozyme, nisin, and bacteriophage c2. http://dx.doi.org/10.1128/AEM.00795-12 . http://hdl.handle.net/10261/81013The putative lactococcal extracytoplasmic function anti-sigma factor Llmg2447 determines resistance to the cell wall-active bacteriocin Lcn972. http://dx.doi.org/10.1128/AAC.01206-12 . http://hdl.handle.net/10261/81156Lack of the host membrane protease FtsH hinders release of the Lactococcus lactis bacteriophage TP712. http://dx.doi.org/10.1099/vir.0.057182-0 . http://hdl.handle.net/10261/93382Peer Reviewe

Cell Wall-active Bacteriocins and Their Applications Beyond Antibiotic Activity

Este artículo forma parte de la tesis de clara Roces. Mecanismos moleculares de respuesta al estrés sobre la pared celular en Lactococcus lactis. http://hdl.handle.net/10261/109851Microorganisms synthesize several compounds with antimicrobial activity in order to compete or defend themselves against others and ensure their survival. In this line, the cell wall is a major protective barrier whose integrity is essential for many vital bacterial processes. Probably for this reason, it represents a 'hot spot' as a target for many antibiotics and antimicrobial peptides such as bacteriocins. Bacteriocins have largely been recognized by their pore-forming ability that collapses the selective permeability of the cytoplasmic membrane. However, in the last few years, many bacteriocins have been shown to inhibit cell wall biosyntheis alone, or in a concerted action with pore formation like nisin. Examples of cell wall-active bacteriocins are found in both Gram-negative and Gram-positive bacteria and include a wide diversity of structures such as nisin-like and mersacidin-like lipid II-binding bacteriocins, two-peptide lantibiotics, and non-modified bacteriocins. In this review, we summarize the current knowledge on these antimicrobial peptides as well as the role, composition, and biosynthesis of the bacterial cell wall as their target. Moreover, even though bacteriocins have been a matter of interest as natural food antimicrobials, we propose them as suitable tools to provide new means to improve biotechnologically relevant microorganisms. © 2012 Springer Science+Business Media New York.Peer Reviewe

Bacteriocins produced by wild Lactococcus lactis strains isolated from traditional, starter-free cheeses made of raw milk

Sixty bacterial strains were encountered by random amplification of polymorphic DNA (RAPD) and repetitive extragenic palindromic (REP) typing in a series of 306 Lactococcus lactis isolates collected during the manufacturing and ripening stages of five traditional, starter-free cheeses made from raw milk. Among the 60 strains, 17 were shown to produce bacteriocin-like compounds in both solid and liquid media. At a genotypic level, 16 of the strains were identified by molecular methods as belonging to L. lactis subsp. lactis and one to L. lactis subsp. cremoris. Among the L. lactis subsp. lactis strains, phenotypic and genetic data determined that eleven produced either nisin A (nine strains) or nisin Z (two strains), and that five produced lactococcin 972. Variable levels of the two bacteriocins were produced by different strains. In addition, nisin was shown to be produced in inexpensive, dairy- and meat-based media, which will allow the practical application of its producing strains in industrial processes. Specific PCR and nucleotide and deduced amino acid sequence analysis identified the inhibitor produced by the single L. lactis subsp. cremoris isolate as a lactococcin G-like bacteriocin. Beyond the use of bacteriocins as functional ingredients for the biopreservation of foods, the newly identified bacteriocin-producing L. lactis strains from traditional cheeses may also be useful for designing starter cultures with protective properties and/or adjunct cultures for accelerating cheese ripening. © 2010 Elsevier B.V.Research was supported by a project from the Spanish Ministry of Science and Innovation (MICINN) (Ref. AGL2007-61869-ALI). A. Alegría was awarded a scholarship from FICYT (Severo Ochoa program, Ref. BP08-053). S. Delgado was supported by a research contract from MICINN (Juan de la Cierva program, Ref. JCI-2008-02391).Peer Reviewe

Cultivos Iniciadores en Quesería: Tradición y Modernidad

El uso consciente de los cultivos iniciadores comenzó cuando el hombre adicionaba una pequeña porción de leche coagulada a un lote de leche fresca para obtener un producto fermentado con propiedades organolépticas mejoradas. Posteriormente, los avances en biología y tecnología han permitido le diseño de iniciadores para su uso industrial de una forma estandarizada.Peer Reviewe

Mecanismos implicados en la resistencia de Lactococcus lactis a la bacteriocina Lcn972

Comunicación presentada en la 6ª reunión de la Red temática BAL (Participación de las Bacterias Lácticas en la Salud Humana y en la Calidad Alimentaria), celebrada el 28 y 29 de junio de 2012 en Tarragona.Peer Reviewe

Bacteriocinas del BAL contra la pared... celular

Trabajo presentado en la 4º Reunión de la Red Temática (Participación de las bacterias lácticas en la salud humana y en la calidad alimentaria), celebrada en Granada del 12 al 14 de noviembre de 2009.Financiada por la Acción Complementaria Modalidad B del Ministerio de Ciencia e Innovación AGL2009-06415-EALI.Peer reviewe