Biomedical subjects

J M Guillamón

Publications and source records attributed to J M Guillamón.

15 recordsLinked to original sources

PubMed2004

Application of molecular methods for the differentiation of acetic acid bacteria in a red wine fermentation.

AIMS: To apply rapid and reliable molecular techniques for typing acetic acid bacteria and studying their population dynamics during wine-making processes. METHODS AND RESULTS: We tested the usefulness of the Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and Repetitive Extragenic Palindromic-PCR (REP-PCR) techniques with reference strains of most of the species of acetic acid bacteria. We obtained exclusive patterns for each strain with the ERIC-PCR technique, proving the utility for characterizing below species level. REP-PCR technique was not as adequate for this purpose because some strains yielded identical fingerprint. One hundred twenty isolates from a commercial red wine fermentation were fingerprinted using both techniques. We detected a high degree of strain diversity in the first stage of fermentation that decreased throughout the process. However, several strains and species were dominant in the alcoholic fermentation phases. The identification of different strains or genotypes at the species level was carried out by restriction analysis of the 16S ribosomal DNA gene. Gluconobacter oxydans dominated the fresh must, while Acetobacter aceti was the only isolated species at the end of the process. Gluconacetobacter hansenii and G. liquefaciens were also isolated in significant numbers at the beginning of fermentation. CONCLUSIONS: ERIC-PCR and REP-PCR techniques proved useful for characterizing strains of acetic acid bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of molecular techniques for a fast and reliable genotypic characterization should increase our knowledge of the ecology of acetic acid bacteria and determine more accurately their growth behaviour during various stages of vinification.

Acetic Acid↗

PubMed2004

New PCR-based methods for yeast identification.

AIMS: To characterize reference yeast strains and identify indigenous strains isolated from wine fermentations by PCR methods. METHODS AND RESULTS: We compared several PCR techniques for yeast identification. We used oligonucleotide primers that are complementary to (i) intron splice sites, (ii) REP and (iii) ERIC elements to produce PCR fingerprints that display specific patterns between the different yeast species. These three techniques were used to characterize 41 reference yeast strains belonging to 15 different species and to identify 40 indigenous strains isolated from grape must and wine fermentations. Species-specific banding patterns were obtained with the three PCR-techniques with different degrees of intraspecific differentiation depending on the method. By comparing the PCR fingerprints of unknown isolates with those produced by reference strains, we identified yeast strains isolated from an industrial wine fermentation. CONCLUSIONS: All three PCR techniques are rapid, reliable and simple methods of yeast identification. As far as we know, this is the first time that the primers designed for amplifying repetitive elements in bacteria have been successfully used in yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Industry needs rapid, reliable and simple methods of yeast identification. The proposed PCR techniques will allow to achieve this objective.

DNA Fingerprinting↗

PubMed2001

Yeast population dynamics in spontaneous fermentations: comparison between two different wine-producing areas over a period of three years.

Yeast ecology, biogeography and biodiversity are important and interesting topics of research. The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years (1996 to 1998). No yeast starter cultures had been used in these wineries which therefore provided an ideal winemaking environment to investigate the dynamics of grape-related indigenous yeast populations. Non-Saccharomyces yeast species were identified by RFLPs of their rDNA, while Saccharomyces species and strains were identified by RFLPs of their mtDNA. This study confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomyces dominated towards the end of the alcoholic fermentation. However, significant differences were found with previous studies, such as the survival of non-Saccharomyces species in stages with high alcohol content and a large variability of Saccharomyces strains (a total of 112, all of them identified as Saccharomyces cerevisiae) with no clear predominance of any strain throughout all the fermentation, probably related to the absence of killer phenotype and lack of previous inoculation with commercial strains.

DNA, Mitochondrial↗

PubMed2001

The glutamate synthase (GOGAT) of Saccharomyces cerevisiae plays an important role in central nitrogen metabolism.

Central nitrogen metabolism contains two pathways for glutamate biosynthesis, glutaminases and glutamate synthase (GOGAT), using glutamine as the sole nitrogen source. GOGAT's importance for cellular metabolism is still unclear. For a further physiological characterisation of the GOGAT function in central nitrogen metabolism, a GOGAT-negative (Deltaglt1) mutant strain (VWk274 LEU(+)) was studied in glutamine-limited continuous cultures. As reference, we did the same experiments with a wild-type strain (VWk43). Intracellular and extracellular metabolites were analysed during different steady states in both strains. The redox state of the cell was taken into account and the NAD(H) and NADP(H) concentrations were determined as well as the reduced and oxidised forms of glutathione (GSH and GSSG, respectively). The results of this study confirm an earlier suggestion, based on a metabolic network model, that GOGAT may be a link between the carbon catabolic reactions (energy production) and nitrogen anabolic reactions (biomass production) by working as a shuttle between cytosol and mitochondria.

Culture Media↗

PubMed2000

Identification of acetic acid bacteria by restriction fragment length polymorphism analysis of a PCR-amplified fragment of the gene coding for 16S rRNA.

Acetic acid bacteria (AAB) irreversibly spoil wines and represent a serious problem. Limited studies on the ecology of AAB during winemaking have been done due to the lack of rapid and precise techniques for their identification. RFLP analysis of PCR-amplified fragment of 16S rDNA was performed on AAB reference strains. The amplified rDNAs were approximately 870-bp long for all AAB species while no amplicons were detected for lactic acid bacteria and yeasts. Out of the four restriction enzymes tested, TaqI was the most efficient one and divided the studied AAB into six groups. However, complete differentiation among collection strains of Acetobacter pasteurianus and Gluconoacetobacter hansenii was not possible.

Acetic Acid↗

PubMed2000

Identification of acetic acid bacteria by RFLP of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer.

DNA corresponding to 16S rDNA and the 165-23S rDNA intergenic spacer (ITS) from 22 reference strains of acetic acid bacteria, representing the diversity of the family Acetobacteraceae, and 24 indigenous acetic acid bacteria isolated from wine fermentations were analysed by PCR-RFLP. Frateuria aurantia LMG 1558T and Escherichia coli ATCC 11775T were included as outgroups. PCR-amplified products of about 1450 bp were obtained from the 16S rDNA of all the strains and products of between 675 and 800 bp were obtained from the 16S-23S rDNA ITS. PCR products were digested with 4-base-cutting restriction enzymes in order to evaluate the degree of polymorphism existing among these strains. Of the enzymes tested, Taql and Rsal were the most discriminatory and showed no intraspecific variations in the restriction patterns. Restriction analysis of the 16S rDNA with these enzymes is proposed as a rapid and reliable method to identify acetic acid bacteria at the level of genus and species (or related species group) and its applicability to identification of indigenous acetic acid bacteria was demonstrated. The same degree of distinction as that for the 16S rDNA analysis was obtained within reference strains of acetic acid bacteria by PCR-RFLP of the 16S-23S rDNA ITS. However, 16S-23S rDNA ITS restriction patterns of strains isolated from wine did not match those of any of the reference strains. Thus, PCR-RFLP of the 16S-23S rDNA ITS is not a useful method to identify isolates of acetic acid bacteria at the species level, although it may be an adequate method to detect intraspecific differentiation.

Acetobacteraceae↗

PubMed1998

Molecular analysis of yeast population dynamics: effect of sulphur dioxide and inoculum on must fermentation.

The effects of sulphur dioxide and a commercial starter inoculum on yeast population dynamics have been analysed by a molecular approach. Yeast identification from fermenting Carinyena grape musts was performed by RFLP's of mtDNA and rRNA-coding DNA. As expected, the use of a commercial inoculum speeded up the start of fermentation, while SO2 addition limited the development of non-Saccharomyces species. However, this effect was also observed with yeast inoculation. Further analysis of population dynamics could lead to a recommendation for the reduction of the dosage of SO2 by the addition of appropriate inoculum of yeasts in the must. Furthermore, the timing of inoculum addition could be modified to allow a proper contribution of non-Saccharomyces species. Molecular biology analysis of population dynamics could provide a tool to efficiently reduce the dosage of SO2 and adjust the timing of inoculum addition.

DNA, Mitochondrial↗

PubMed1998

Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribed spacer (ITS) region.

In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation.

DNA, Fungal↗

PubMed1998

Diversity of Saccharomyces strains in wine fermentations: analysis for two consecutive years.

An ecological study of Saccharomyces cerevisiae strains in spontaneous alcoholic fermentation has been conducted in the same winery for two consecutive years (1994 and 1995). Yeast cells were identified and characterized using mitochondrial DNA restriction analysis. Although a great diversity of wild strains was observed, a sequential substitution of S. cerevisiae strains during the different phases of fermentation was detected. Furthermore, the most frequent strains were encountered in both years, and the dynamic populations were not influenced by climatic conditions. Finally, the RsaI restriction enzyme produced a species-specific pattern which allowed the identification of all the isolates as S. cerevisiae.

DNA, Fungal↗

PubMed1997

Rapid characterization of wild and collection strains of the genus Zygosaccharomyces according to mitochondrial DNA patterns.

Several wild and collection strains of the genus Zygosaccharomyces were characterized using a rapid and simple method of restriction analysis of mitochondrial DNA. Patterns obtained with three endonucleases (HaeIII, HinfI and RsaI) made it possible to differentiate each species and to identify the wild strains, isolated from the same spoiled concentrated must, as belonging to the species Z. rouxii. The HinfI restriction enzyme produced a strain-specific pattern which allowed us to recognize that the seven wild isolates belonged to only three strains.

DNA, Mitochondrial↗

PubMed1996

Molecular characterization, relatedness and antifungal susceptibility of the basidiomycetous Hormographiella species and Coprinus cinereus from clinical and environmental sources.

Hormographiella-like strains, isolated from different natural substrates and producing sclerotia and occasionally basidiomata of Coprinus cinereus, were compared morphologically and using molecular techniques with clinical strains of Hormographiella aspergillata and H. verticillata. Analysis of restriction fragment length polymorphisms of ribosomal and mitochondrial-like DNA confirmed interspecific differences between H. aspergillata and H. verticillata, supporting the morphological data, and helped demonstrate that H. aspergillata is the anamorph of C. cinereus. The latter was confirmed also by crossing tests. The analysis of the mtDNA restriction profiles revealed intraspecific variability in C. cinereus, which allowed differentiation of clinical and environmental strains. Due to the implication of C. cinereus and Hormographiella in human opportunistic infections, the antifungal susceptibility test is included. Results show that all strains were susceptible to miconazole, itraconazole and ketoconazole but not to flucytosine and fluconazol. Susceptibility against amphotericin B was variable; while H. verticillata was susceptible, four out of seven C. cinereus strains tested were resistant.

Antifungal Agents↗

PubMed1996

Molecular differentiation of Keratinomyces (Trichophyton) species.

The taxonomy of the form-genus Keratinomyces (Trichophyton) within the group of the dermatophytes is based on morphological features which remain insufficient for the distinction of these anamorphic species. The three species included in the genus Keratinomyces, namely K. ajelloi, K. ceretanicus and K. longifusus were examined by means of their mitochondrial-like DNA diversity and compared to few other dermatophytes. The analysis of the mtDNA restriction fragments confirmed that the three species are different and well separate from the other dermatophytes.

Classification↗

PubMed1996

The utility of mitochondrial DNA restriction analysis in the classification of strains of Chrysosporium (hyphomycetes).

The taxonomy of the fungal genus Chrysosporium is mainly based on morphological features. In our current studies we have found several Chrysosporium species which showed intermediate morphological characteristics between several species. For this reason, we have carried out an analysis of the mitochondrial DNA restriction fragments of these strains that have permit us to classify each isolate strain in a species.

Chrysosporium↗

PubMed1994

Rapid characterization of four species of the Saccharomyces sensu stricto complex according to mitochondrial DNA patterns.

Several strains of the four sibling species of the genus Saccharomyces (S. bayanus, S. cerevisiae, S. paradoxus, and S. pastorianus) were characterized by using a rapid and simple method of restriction analysis of mitochondrial DNA. Patterns obtained with four-cutter endonucleases (such as AluI, DdeI, HinfI, and RsaI) made it possible to differentiate each species. S. cerevisiae and S. paradoxus presented a greater number of large fragments than S. pastorianus and S. bayanus with all the assay enzymes. With AluI and DdeI, species-specific bands clearly permitted differentiation between S. pastorianus and S. bayanus. To test the resolution of this method, wild Saccharomyces strains were analyzed. The correct assignment of these strains to a known taxon by this rapid method was confirmed by means of electrophoretic karyotyping.

DNA, Fungal↗

PubMed1993

Phylogenetic relationships among wine yeast strains based on electrophoretic whole-cell protein patterns.

In the present work, a phylogenetic study based on protein electrophoretic profiles of Saccharomyces strains isolated from different Spanish wine regions has been carried out. Qualitative differences between the protein electrophoregrams were found at inter- and intraspecific level, but not between electrophoregrams of strains isolated at the same ecosystem. The numerical analysis of these results allowed us to conclude that intraspecific relationships are determined by ecological factors, as well as human influences (dispersion and artificial selection). A correlation between ecological and/or geographical origin and the relationships among strains was observed.

Cluster Analysis↗