Juan M. Mesas

Characterization of lactic acid bacteria from musts and wines of three consecutive vintages of Ribeira Sacra

Aims:  This study was designed to isolate and characterize the lactic acid microbiota of the musts and wines of a young denomination of origin area, Ribeira Sacra in north‐west Spain.

PLASMID CURING OF OENOCOCCUS OENI

Two strains of Oenococcus oeni, RS1 (which carries the plasmid pRS1) and RS2 (which carries the plasmids pRS2 and pRS3), were grown in the presence of different curing agents and at different temperatures. Sublethal temperature together with acriflavine generated all possible types of cured strains, i.e., lacking pRS1 (from strain RS1), and lacking pRS2, pRS3, or both (from strain RS2). Sublethal temperature together with acridine orange only generated cured strains lacking pRS3. These results suggest that acriflavine is a better curing agent than acridine orange for O. oeni, and that pRS3 is the most sensitive to these curing agents. We also observed spontaneous loss of pRS2 or both pRS2 and pRS3 by electroporation. The ability to cure O. oeni strains of plasmids provides a critical new tool for the genetic analysis and engineering of this commercially important bacterium.

Synthesis, biological evaluation and SAR studies of novel bicyclic antitumor platinum(IV) complexes

The present study describes the synthesis, anticancer activity and SAR studies of novel platinum(IV) complexes having 1,2-bis(aminomethyl)carbobicyclic or oxabicyclic carrier ligands, bearing chlorido and/or hydroxido ligands in axial position and chlorido or malonato ligands in equatorial position (labile ligands). These complexes were synthetized with the aim of obtaining new anticancer principles more soluble in water and therefore more bioavailable. Several substitution patterns on the platinum atom have been designed in order to evaluate their antiproliferative activity and to establish structure-activity relationship rules. The synthesis of platinum(IV) complexes with axial hydroxyl ligands on the platinum(IV) were carried out by reaction of K2Pt(OH)2Cl4 with the corresponding diamines. The complexes with axial chlorido ligands on the platinum(IV) atom were synthesized by direct reaction of diamines with K2PtCl6. Carboxylated complexes were synthesized by the substitution reaction of equatorial chlorido ligands by silver dicarboxylates. The most actives complexes were those having malonate as a labile ligand, no matter of the structure of the carrier ligand. Regarding the influence of the structure of the non-labile 1,4-diamine carrier ligand on the cytotoxicity, it was found that the complexes having the more lipophilic and symmetrical bicyclo[2.2.2]octane framework were much more active than those having an oxygen or methylene bridge.

Low-salt restructured fish products from Atlantic mackerel (Scomber scombrus) with texture resembling turkey breast.

Atlantic mackerel (Scomber scombrus) is a pelagic and migratory species that is usually caught with other fish as bycatch. The aim of this work was to obtain low-salt restructured fish products from Atlantic mackerel resembling turkey breast using transglutaminase (0.2 U/g) as binder. NaCl concentration (0–20 g/kg), temperature (25–40 °C) and time of incubation (30–90 min) were assayed. The texture parameters (Warner–Bratzler force and Warner–Bratzler work) and expressible water were compared to those of turkey breast. Mathematical models were obtained to determine the effect of these variables on the texture of Atlantic mackerel restructured products. Optimal conditions to obtain a similar texture than turkey breast were found. The overall optimization point out that the treatment at 31.8 °C for 63.35 min using a NaCl concentration of 8.45 g/kg allowed to obtain restructured products from Atlantic mackerel with texture and expressible water similar to those of turkey breast. Color parameters (L*, a* and b*) of the product were also similar to those of turkey breast. The results showed the feasibility of producing low-salt restructured products from Atlantic mackerel resembling turkey breast using transglutaminase.

Design, synthesis and SAR studies of novel 1,2-bis(aminomethyl)cyclohexane platinum(II) complexes with cytotoxic activity. Studies of interaction with DNA of iodinated seven-membered 1,4-diaminoplatinocycles.

A selected library of nine novel platinum(II) complexes having differently functionalized 1,2-bis(aminomethyl)cyclohexane carrier ligands with a 1,4-diamino framework and iodides as labile ligands have been synthesized and evaluated in vitro for their tumor cell growth inhibitory activity, in front of one pair of human carcinoma cell lines A2780 and A2780cisR. These cell lines were chosen based on studying all the known main mechanisms of resistance of cisplatin. A2780cisR cells are resistant through a combination of reduced drug transport enhanced DNA repair/tolerance and elevated glutathione (GSH) levels with respect to the parental A2780 cells. Most platinum complexes evaluated showed a very low resistant factor, up to 16 times lower than that of cisplatin, which indicates their ability to overcome the cisplatin resistance in ovarian cancer A2780cisR cells. Structure-activity studies have been performed in order to know the influence of the several organic functionalities (CC double bond, free OH group, MeO group, etc.) and the stereochemistry on the cytotoxic activity. Moreover, studies of interaction with DNA of these complexes were performed via three techniques: circular dichroism (CD), electrophoresis on agarose gel (EF) and atomic force microscopy (AFM) in order to evaluate the modifications of secondary and tertiary structure of DNA, induced by platinum complexes. These studies allowed us to correlate the IC50 values of complexes and the intensity of interaction to DNA, the main target for these compounds.

Basic characterization and partial purification of β‐glucosidase from cell‐free extracts of Oenococcus oeni ST81

Aims:  This study was designed to characterize a β‐glucosidase of Oenococcus oeni ST81, a strain isolated from a Spanish wine of the origin appellation Ribeira Sacra.

Characterization of pRS5: a theta-type plasmid found in a strain of Pediococcus pentosaceus isolated from wine that can be used to generate cloning vectors for lactic acid bacteria.

The nucleotide sequence of pRS5 (10153bp) is reported. Through sequence analysis, 9 open reading frames (ORFs) were identified and the following features observed: a region likely involved in replication whose structural features indicate that pRS5 belongs to the pUCL287 group of theta-type replicons, and hypothetical proteins putatively involved in plasmid copy number control, restriction-modification system, toxin-antitoxin system and a putative integrase. Shuttle vectors for Escherichia coli and lactic acid bacteria (LAB) as well as a small cloning vector for direct use in LAB were constructed using the replication region of pRS5. The ability of such vectors to accept and express other genes was assessed. All pRS5-derivatives were maintained at a high rate over 200 generations without selective pressure.

pRS4: UN VECTOR DE CLONACIÓN IDÓNEO PARA BACTERIAS ÁCIDO-LÁCTICAS DE USO ALIMENTARIO pRS4: AN APPROPRIATE CLONING VECTOR FOR LACTIC-ACID BACTERIA OF FOOD USE

Abstract The ability of pRS4, a cryptic plasmid from Pediococcus pentosaceus, as a selective cloning vector for lactic acid bacteria has been analysed. The results indicate that pRS4C1, a cloning vector from pRS4 can stably transform Lactobacillus plantarum, Pediococcus acidilactici and P. pentosaceus (three lactic acid bacteria used as starters in the food industry) but it does not transform Enterococcus faecalis (a lactic acid bacteria which is undesirable due to its potential origin, mainly of faecal source). Further modifications of the pRS4C1, consisting in positional changes of the chloramphenicol resistance marker and/or single mutation into the initiation codon of the gene of the replication protein, does not generate expression of pRS4 into E. faecalis, however the ability of these derivates to transform Lactobacillus and Pediococcus remains unaltered. These results indicate that pRS4 can be used as a selective cloning vector for genetic manipulation of useful lactic acid bacteria, without risk of horizontal transfer of genetic material to undesirable lactic acid bacteria that could coexist with the formers. Resumen Se ha analizado la posibilidad de utilizar pRS4, un plásmido críptico de Pediococcus pentosaceus, como vector selectivo de clonación en bacterias ácido-lácticas. Los resultados obtenidos indican que pRS4C1, vector de clonación derivado de pRS4, transforma en forma estable Lactobacillus plantarum, Pediococcus acidilactici y P. pentosaceus, bacterias ácido-lácticas utilizadas como cultivos iniciadores en la industria alimentaria, pero no transforma Enterococcus faecalis, una bacteria ácido-láctica cuya presencia en alimentos es considerada indeseable por su habitual procedencia de origen fecal. Posteriores modificaciones de pRS4C1, consistentes en cambios de posición del marcador de resistencia a cloranfenicol y/o mutación puntual del codon de iniciación del gen que codifica para la proteína de replicación, no subsanan la carencia de expresión de pRS4 en E. faecalis, pero mantienen inalterable su capacidad de transformar y expresarse establemente en las especies de Lactobacillus y Pediococcus arriba citadas. Estos resultados indican que pRS4 puede ser utilizado como vehículo de clonación selectivo para la manipulación genética de bacterias ácido-lácticas útiles, sin riesgo de transferencia horizontal de material genético a determinadas bacterias ácido-lácticas indeseables, que puedan coexistir con las primeras en un mismo alimento. Palabras clave: Bacterias ácido-lácticas, plásmidos, vectores de clonación

The use of the replication region of plasmid pRS7 from Oenococcus oeni as a putative tool to generate cloning vectors for lactic acid bacteria.

A chimeric plasmid, pRS7Rep (6.1 kb), was constructed using the replication region of pRS7, a large plasmid from Oenococcus oeni, and pEM64, a plasmid derived from pIJ2925 and containing a gene for resistance to chloramphenicol. pRS7Rep is a shuttle vector that replicates in Escherichia coli using its pIJ2925 component and in lactic acid bacteria (LAB) using the replication region of pRS7. High levels of transformants per µg of DNA were obtained by electroporation of pRS7Rep into Pediococcus acidilactici (1.5 × 10(7)), Lactobacillus plantarum (5.7 × 10(5)), Lactobacillus casei (2.3 × 10(5)), Leuconostoc citreum (2.7 × 10(5)), and Enterococcus faecalis (2.4 × 10(5)). A preliminary optimisation of the technical conditions of electrotransformation showed that P. acidilactici and L. plantarum are better transformed at a later exponential phase of growth, whereas L. casei requires the early exponential phase for better electrotransformation efficiency. pRS7Rep contains single restriction sites useful for cloning purposes, BamHI, XbaI, SalI, HincII, SphI and PstI, and was maintained at an acceptable rate (>50%) over 100 generations without selective pressure in L. plantarum, but was less stable in L. casei and P. acidilactici. The ability of pRS7Rep to accept and express other genes was assessed. To the best of our knowledge, this is the first time that the replication region of a plasmid from O. oeni has been used to generate a cloning vector.

Plasmids from Wine-Related Lactic Acid Bacteria

This chapter presents a review of the most important plasmids isolated to date from wine-related lactic acid bacteria (LAB). The chapter is organised in four main parts dealing, respectively, with plasmids from four genera of LAB found on grapes and in must and wine (Lactobacillus, Leuconostoc, Oenococcus and Pediococcus). Some of these genera, notably Lactobacillus, include a large number of plasmid-carrying strains that have been isolated from non-wine-related sources, but this chapter focuses on plasmids of strains isolated from wine-related sources. When information on the genetics (nucleotide sequence, replication mechanism, use as cloning vectors and/or transformation procedures) of these plasmids, or their effects on phenotype, has been reported in the literature, this information is summarised here. Finally, in this chapter it is concluded that future work on wine-related-LAB plasmids will require new vectors and transformation systems, notably for Oenococcus oeni.