Isoenzyme patterns: a valuable molecular tool for the differentiation of Zygosaccharomyces species and detection of misidentified isolates

Electrophoretic analysis of esterase, acid phosphatase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase isoenzymes was performed in 39 strains classified into six species of the yeast genus Zygosaccharomyces. The electrophoretic profiles obtained allowed the clear separation of Z. bailii, Z. bisporus, Z. florentinus, Z. lentus, Z. mellis and Z. rouxii, strains of the latter species clustering into two subgroups. Furthermore, this methodology enabled the detection of misidentified strains, as subsequently confirmed by DNA-DNA reassociation and sequencing of the D1/D2 domain of the 26S rRNA gene. Cluster analysis of the global electrophoretic data and those obtained using only two of the isoenzyme systems, esterase and lactate dehydrogenase, yielded similar grouping of the strains examined, indicating that these enzymes are good markers for the differentiation of Zygosaccharomyces species.     

Molecular characterisation of Hanseniaspora species

The sequences of the internal transcribed spacers (ITS regions) and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 27 strains representative of the six species belonging to the genus Hanseniaspora, were examined. From the analysis of the 5.8S rRNA gene and the ITS regions, the genus Hanseniaspora is monophyletic and can be divided into two subgroups. This subdivision was supported by electrophoretic chromosome patterns. Hanseniaspora guilliermondii, H. uvarum and H. valbyensis show 6–7 bands (8 to 9 chromosomes), while the second group comprises the species H. occidentalis, H. osmophila and H. vineae which have only 5 chromosomes.     

Molecular systematics of Lachancea yeasts

This work presents the results of molecular-genetic investigation of a new yeast genus, Lachancea Kurtzman (2003). Analysis of rRNA sequences and molecular karyotyping have shown genetic homogeneity of the genus Lachancea. Yeasts of this genus have an identical haploid number of chromosomes equal to eight, whereas limiting chromosome sizes significantly differ in various species. The largest range of chromosome bands was registered in L. cidri strains (400-2800 kb), while the smallest was found in L. waltii (1400-2800 kb). The intra- and interspecies polymorphism of Lachancea chromosomes is discussed.     

Intergeneric hybrids of Saccharomyces cerevisiae and Zygosaccharomyces fermentati obtained by protoplast fusion

To obtain strains that are able to efficiently produce ethanol from different carbohydrates, mainly cellulose hydrolysates, several species of the genus Candida and a Zygosaccharomyces fermentati strain were examined for their ability to utilize cellobiose and produce ethanol, as well as for their thermotolerance and the possibility of genetic manipulation. Candida obtusa and Zygosaccharomyces fermentati tolerated the maximal temperature for growth, possessed the highest cellobiase activity, and offered the possibility of genetic manipulation, although neither of them proved to be a good producer of ethanol. Intergeneric hybrids of Saccharomyces cerevisiae and Z. fermentati were obtained after protoplast fusion. They were selected as prototrophic strains, after isolation of auxotrophic mutants from Z. fermentati and fusion with an S. cerevisiae strain which was also auxotrophic. The hybrids, which appeared at a frequency of 2 X 10(-7), presented characteristics of both parents, such as resistance to certain drugs and the ability to grow with either cellobiose or lactic acid as the sole carbon source; they were very stable, even under nonselective conditions. These hybrids may have important industrial applications as good fermenting strains.     

Zygosaccharomyces kombuchaensis,a new ascosporogenous yeast from 'Kombucha tea'

A new ascosporogenous yeast, Zygosaccharomyces kombuchaensis sp. n. (type strain NRRL YB-4811, CBS 8849), is described; it was isolated from Kombucha tea, a popular fermented tea-based beverage. The four known strains of the new species have identical nucleotide sequences in domain D1/D2 of 26S rDNA. Phylogenetic analysis of D1/D2 and 18S rDNA sequences places Z. kombuchaensis near Zygosaccharomyces lentus. The two species are indistinguishable on standard physiological tests used for yeast identification, but can be recognized from differences in restriction fragment length polymorphism patterns obtained by digestion of 18S-ITS1 amplicons with the restriction enzymes DdeI and MboI.     

Intergeneric hybrids of Saccharomyces cerevisiae and Zygosaccharomyces fermentati obtained by protoplast fusion.

To obtain strains that are able to efficiently produce ethanol from different carbohydrates, mainly cellulose hydrolysates, several species of the genus Candida and a Zygosaccharomyces fermentati strain were examined for their ability to utilize cellobiose and produce ethanol, as well as for their thermotolerance and the possibility of genetic manipulation. Candida obtusa and Zygosaccharomyces fermentati tolerated the maximal temperature for growth, possessed the highest cellobiase activity, and offered the possibility of genetic manipulation, although neither of them proved to be a good producer of ethanol. Intergeneric hybrids of Saccharomyces cerevisiae and Z. fermentati were obtained after protoplast fusion. They were selected as prototrophic strains, after isolation of auxotrophic mutants from Z. fermentati and fusion with an S. cerevisiae strain which was also auxotrophic. The hybrids, which appeared at a frequency of 2 X 10(-7), presented characteristics of both parents, such as resistance to certain drugs and the ability to grow with either cellobiose or lactic acid as the sole carbon source; they were very stable, even under nonselective conditions. These hybrids may have important industrial applications as good fermenting strains.     

Identification of species in the genus Pichia by restriction of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal DNA gene

In this study, the variability within the ribosomal DNA region spanning the internal transcribed spacers ITS1 and ITS2 and the 5.8S gene (5.8S-ITS rDNA) was used to differentiate species in the genus Pichia. The 5.8S-ITS rDNA region was PCR-amplified and the PCR product digested with the enzymes CfoI, HinfI, and HaeIII. The variability in the size of the amplified product and in the restriction patterns enabled differentiation between species in the genus Pichia, and between Pichia species and yeast species from other genera in the Yeast-id database (). Moreover, the restriction fragment length polymorphism (RFLP) patterns of the 5.8S-ITS enabled misidentified strains to be detected and revealed genetic heterogeneity between strains within the Pichia membranifaciens and Pichia nakazawae species. Ultimately, the RFLP patterns of the 5.8S-ITS rDNA failed to differentiate between some Pichia and Candida species that could be distinguished on the basis of the sequence of the 5.8S-ITS rRNA region or the sequence of the D1/D2 domain of the 26S rDNA gene.     

A Novel ZZ/ZW Sex Chromosome System for the Genus Leporinus (Pisces, Anostomidae, Characiformes)

A wide range of sex chromosome mechanisms, including simple and multiple chromosome systems is characteristic of fishes. The Leporinus genus represent a good model to study sex chromosome mechanisms, because an unambiguous ZZ/ZW sex chromosome system was previously described for seven species, while the remaining studied species of the genus do not show differentiated sex chromosomes. The occurrence of sex chromosomes in Leporinus trifasciatus and Leporinus sp2 from the Araguaia river, Amazon basin, Brazil, was here investigated. ZZ/ZW sex chromosomes were detected for both species. The Z and W chromosome morphology of L. trifasciatus is the same as described for other species of the genus Leporinus. However, the Z and W chromosomes of L. sp2 were quite different in their morphology and banding pattern suggesting that the ZW system of this species have originated independently from the ZW system previously described for other Leporinus.     

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.Non-standard abbreviations bp base pairs - kbp kilobase pairs - RFLP's restriction fragment length polymorphisms     

Zygosaccharomyces kombuchaensis: the physiology of a new species related to the spoilage yeasts Zygosaccharomyces lentus and Zygosaccharomyces bailii

Zygosaccharomyces kombuchaensis was recently discovered in the 'tea fungus' used to make fermented tea. Z. kombuchaensis was shown by ribosomal DNA sequencing to be a novel species, and a close relative of Zygosaccharomyces lentus, from which it could not be distinguished by conventional physiological tests. Z. lentus was originally established as a new taxon by growth at 4 degrees C, sensitivity for heat and oxidative stress, and lack of growth in aerobic shaken culture at temperatures above 25 degrees C. Subsequent analysis of Z. kombuchaensis reveals that this species shares these unusual characteristics, confirming its close genealogical relationship to Z. lentus. Detailed physiological data from a number of Z. kombuchaensis and Z. lentus strains clearly demonstrate that these two species can in fact be distinguished from one another based on their differing resistance/sensitivity to the food preservatives benzoic acid and sorbic acid. The spoilage yeasts Zygosaccharomyces bailii and Z. lentus are resistant to both acetic acid and sorbic acid, whereas Z. kombuchaensis is resistant to acetic acid but sensitive to sorbic acid. This would indicate that Z. kombuchaensis strains lack the mechanism for resistance to sorbic acid, but possess the means of resistance to acetic acid. This observation would therefore suggest that these two resistance mechanisms are different, and that in all probability acetic and sorbic acids inhibit yeast growth by different modes of action. Z. kombuchaensis strains were also sensitive to benzoic acid, again suggesting inhibition dissimilar from that to acetic acid.     

Molecular genetic characterization of the yeast Lachancea kluyveri

A comparative study of Lachancea kluyveri strains isolated in Europe, North America, Japan, and the Russian Far East was performed using restriction analysis, sequencing of non-coding rDNA regions, molecular karyotyping, and the phylogenetic analysis of the alpha- galactosidase MEL genes. This study showed a close genetic relatedness of these L. kluyveri strains. The chromosomal DNAs of the L. kluyveri strains were found to range in size from 980 to 3100 kb. The haploid number of chromosomes is equal to eight. The IGS2 restriction patterns and single nucleotide substitutions in the ITS1/ITS2 rDNA region correlate neither with geographic origin nor with the source of the strains. The L. kluyveri strains isolated from different sources have a high degree of homology (79-100%) of their MEL genes. The phylogenetic analysis of all of the known alpha-galactosidases in the "Saccharomyces" clade showed that the MEL genes of the yeasts L. kluyveri. L. cidri, Saccharomyces cerevisiae, S. paradoxus, S. bayanus, and S. mikatae are species specific.     

Zygosaccharomyces lentus: a significant new osmophilic, preservative-resistant spoilage yeast, capable of growth at low temperature

Zygosaccharomyces lentus is a yeast species recently identified from its physiology and 18S ribosomal sequencing (Steels et al. 1999).The physiological characteristics of five strains of this new yeast so far isolated were investigated, particularly those of technical significance for a spoilage yeast, namely temperature range, pH range, osmotolerance, sugar fermentation, resistance to food preservatives such as sorbic acid, benzoic acid and dimethyldicarbonate (DMDC; Velcorin). Adaptation to benzoic acid, and growth in shaking and static culture were also investigated. Zygosaccharomyces lentus strains grew over a wide range of temperature (4-25 degrees C) and pH 2.2-7.0. Growth at 4 degrees C was significant. Zygosaccharomyces lentus strains grew at 25-26 degrees C in static culture but were unable to grow in aerobic culture close to their temperature maximum. All Z. lentus strains grew in 60% w/v sugar and consequently, are osmotolerant. Zygosaccharomyces lentus strains could utilize sucrose, glucose or fructose as a source of fermentable sugar, but not galactose. Zygosaccharomyces lentus strains were resistant to food preservatives, growing in sorbic acid up to 400 mg l-1 and benzoic acid to 900 mg l-1 at pH 4.0. Adaptation to higher preservative concentrations was demonstrated with benzoic acid. Resistance to DMDC was shown to be greater than that of Z. bailii and Saccharomyces cerevisiae. This study confirms that Z. lentus is an important food spoilage organism potentially capable of growth in a wide range of food products, particularly low pH, high sugar foods and drinks. It is likely to be more significant than Z. bailii in the spoilage of chilled products.     

RFLP analysis of the ribosomal internal transcribed spacers and the 5.8S rRNA gene region of the genus Saccharomyces: a fast method for species identification and the differentiation of flor yeasts

The PCR amplification and subsequent restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was applied to the identification of yeasts belonging to the genus Saccharomyces. This methodology has previously been used for the identification of some species of this genus, but in the present work, this application was extended to the identification of new accepted Saccharomyces species (S. kunashirensis, S. martiniae, S. rosinii, S. spencerorum, and S. transvaalensis), as well as to the differentiation of an interesting group of Saccharomyces cerevisiae strains, known as flor yeasts, which are responsible for ageing sherry wine. Among the species of the Saccharomyces sensu lato complex, the high diversity observed, either in the length of the amplified region (ranged between 700 and 875 bp) or in their restriction patterns allows the unequivocal identification of these species. With respect to the four sibling species of the Saccharomyces sensu stricto complex, only two of them, S. bayanus and S. pastorianus, cannot be differentiated according to their restriction patterns, which is in accordance with the hybrid origin (S. bayanus × S. cerevisiae) of S. pastorianus. The flor S. cerevisiae strains exhibited restriction patterns different from those typical of the species S. cerevisiae. These differences can easily be used to differentiate this interesting group of strains. We demonstrate that the specific patterns exhibited by flor yeasts are due to the presence of a 24-bp deletion located in the ITS1 region and that this could have originated as a consequence of a slipped-strand mispairing during replication or be due to an unequal crossing-over. A subsequent restriction analysis of this region from more than 150 flor strains indicated that this deletion is fixed in flor yeast populations.     

Molecular genetic characterization of the yeast <Emphasis Type="Italic">Lachancea kluyveri</Emphasis>

A comparative study of Lachancea kluyveri strains isolated in Europe, North America, Japan, and the Russian Far East was performed using restriction analysis, sequencing of non-coding rDNA regions, molecular karyotyping, and the phylogenetic analysis of the α-galactosidase MEL genes. This study showed a close genetic relatedness of these L. kluyveri strains. The chromosomal DNAs of the L. kluyveri strains were found to range in size from 980 to 3100 kb. The haploid number of chromosomes is equal to eight. The IGS2 restriction patterns and single nucleotide substitutions in the ITS1/ITS2 rDNA region correlate neither with geographic origin nor with the source of the strains. The L. kluyveri strains isolated from different sources have a high degree of homology (79–100%) of their MEL genes. The phylogenetic analysis of all of the known α-galactosidases in the “Saccharomyces” clade showed that the MEL genes of the yeasts L. kluyveri, L. cidri, Saccharomyces cerevisiae, S. paradoxus, S. bayanus, and S. mikatae are species specific.     

Higher alcohol and acetoin production by Zygosaccharomyces wine yeasts

Seventy strains of Zygosaccharomyces isolated from grape musts were investigated for their ability to produce higher alcohols and acetoin in synthetic medium and grape must. The Zygosaccharomyces strains produced generally low amounts of higher alcohols. Within this genus, Z. fermentati behaved differently from Z. bailii producing less isobutanol in synthetic medium and more amyl alcohols and isobutanol in grape must. Zygosaccharomyces fermentati did not form detectable amounts of acetoin in any conditions whereas Z. bailii produced it both in synthetic medium and in grape must. These strains were found to contribute to aroma and taste of wine.     

Electrophoretic karyotyping as a taxonomic tool in the genusSaccharomyces

Summary The electrophoretic karyotypes of strains of the ten species of the yeast genusSaccharomyces (sensu Vaughan-Martini & Martini 1992) were determined by the CHEF (contour-clamped homogeneous electric field) system of pulsed field gel electrophoresis. The number of bands was found to vary from 6 to 17 and the calculated molecular weights of haploid genomes ranged from 7.9 to 14.6 Mbp. The type strains ofS. exiguus and the four species of theSaccharomyces sensu stricto complex (S. bayanus, S. cerevisiae, S. paradoxus andS. pastorianus) have genomes comprised of chromosomes of all three size classes: light (< 500 kb), medium (500–1000 kb) and heavy (> 1,000 kb).Saccharomyces kluyveri DNA has only heavy bands, while the remaining species exhibit medium and heavy chromosomes. When more than one strain of each species was examined, it was seen that while the speciesS. bayanus, S. castellii, S. cerevisiae, S. kluyveri, S. paradoxus andS. pastorianus showed uniform karyotypes,S. dairensis, S. exiguus, S. servazzii andS. unisporus comprise heterogeneous taxa.     

Rhodococcus aetherivorans sp. nov., a new species that contains methyl t-butyl ether-degrading actinomycetes

The taxonomic positions of two actinomycetes, strains Bc663 and 10bc312T, provisionally assigned to the genus Rhodococcus were determined using a combination of genotypic and phenotypic properties. The organisms have phenotypic properties typical of members of the genus Rhodococcus and were assigned to the 16S rRNA subgroup which contains Rhodococcus rhodochrous and closely related species. The two strains, which have many phenotypic features in common, belong to the same genomic species albeit one readily separated from Rhodococcus ruber with which they form a distinct phyletic line. The organisms were also distinguished from all of the species classified in the R. rhodochrous subgroup, including R. ruber, using a combination of phenotypic properties. The genotypic and phenotypic data show that strains Bc663 and 10bc312T merit recognition as a new species of Rhodococcus. The name proposed for the new species is Rhodococcus aetherivorans (10bc312T = DSM 44752T = NCIMB 13964T).     

Identification of species of the genus Candida by analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers

The PCR amplification and subsequent restriction analysis of the ribosomal region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene is applied to the identification of yeasts belonging to the genus Candida. This methodology has previously been used for the identification of some species of this genus, but in the present work this application has been applied to the identification and characterisation of a greater number of species of the genus Candida, with a special survey of species of clinical and biotechnological interest. Among the species of the genus Candida, the high variability observed, both in the length of the amplified region (ranging between 390 and 900 bp) and in their restriction patterns, allows the unequivocal identification to the species level, with the exception of the group of species that comprises C. membranifaciens, C. conglobata, C. atlantica, C. atmosphaerica, and C. oleophila, that required the sequencing of the D1/D2 domain of the 26S rRNA gene or the 5.8S-ITS region for their proper differentiation. The 5.8S-ITS restriction analysis also failed in the differentiation of species within the pairs C.aaseri/C.butyri,C.fructus/C.musae,C.santamariae var. santamariae / C. beechii and C. zeylanoides / C. krissii. In this case, the high sequence similarities obtained for their 26S D1/D2 domain and the 5.8S-ITS region indicate that each pair of species should be considered as a single species. The main purpose of this work is to generate a database for a high number of yeast species, of both biotechnological and clinical interest, and to facilitate their easy, fast, and reliable identification. The present work improves the database available online at the IATA web page (http://motor.edinfo.es/iata/) with the patterns of 75 species belonging to the genus Candida.     

Chromosome polymorphism in the yeast Saccharomyces

The variability of chromosomal band patterns was determined by pulse electrophoresis. The natural strains differed by the quantity and electrophoretic mobility of chromosomal DNA bands. The strains of independent genetic stocks originated from the XII race of Saccharomyces cerevisiae showed less significant difference in band patterns than the strains of different species of the Saccharomyces genus. The progeny of among strains with different karyotypes hybrid showed non-regular segregation of parental bands, the occurrence of new bands and the bands with altered mobility. Reverse crosses of hybrid progeny with strains of Peterhoff genetic stocks of S. cerevisiae led to decrease in chromosomal polymorphism. Homozygotization for ski5 allele and selection for increasing the copy number of killer plasmids was accompanied with repeated splash of polymorphism in 1-2 generations of intratetrad and intrafamily crossed hybrid progeny. Subsequent stabilization of electrophoretic karyotype took place, excluding the mendelian dimorphism of chromosome III, with was a stable trait of the last 6 generations of that progeny.     

Pulsed-field gel electrophoresis of the genomic restriction fragments of coagulase-negative staphylococci

Abstract The genomes of 47 coagulase-negative staphylococcal strains assigned to different species were analysed by pulsed-field electrophoresis. The strains were clustered on the basis of their similarity in the Sma I restriction patterns into various groups, each group consisting of the type strain and the strains whose Sma I restriction patterns were similar to that of the type strain. The Sma I restriction groups seem to correspond to the following species: Staphylococcus warneri, S. hominis, S. xylosus, S. lugdunensi, S. kloosii, S. haemolyticus, S. lentus, S. cohnii, S. equorum, S. chromogenes, S. saprophyticus, S. simulans, S. carnosus, S. capitis and S. auricularis . The species S. sciuri, S. caseolyticus, S. gallinarum, S. epidermidis and S. schleiferi were represented only by their type strains and showed no similarity in their Sma I restriction patterns neither to each other nor to all the species investigated here. Thus, the classification of coagulase-negative staphylococcal strains into the above species seems to be confirmed also by genome restriction analysis carried out by pulsed-field gel electrophoresis.