Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.     

Comparative electrophoretic profiles of esterases, and of glutamate, lactate and malate dehydrogenases, from Aeromonas hydrophila, A. caviae and A. sobria

Esterases, and glutamate, lactate and malate dehydrogenases of 64 Aeromonas hydrophila, A. caviae and A. sobria strains, were analysed by polyacrylamide agarose gel electrophoresis and by thin layer isoelectrofocusing. On the basis of the isoelectric points of malate dehydrogenase from the three species and the mobility of lactate dehydrogenase from A. sobria, 8 species specific zymotypes were defined: three for A. hydrophila strains, three for A. caviae strains and two for A. sobria strains. These zymotypes correlated with previously established DNA hybridization groups. The other electrophoretic data were found to be less useful for distinction between A. hydrophila and A. sobria strains, but supported differentiation into zymotypes for A. caviae strains. The two-dimensional electrophoretic profile established by plotting isoelectric point against electrophoretic mobility of the major esterase illustrated the degree of enzyme polymorphism among the strains of the three species. Variation in electrophoretic patterns within A. hydrophila and A. caviae might provide useful epidemiological markers.     

Molecular characterisation of the species of the genus Zygosaccharomyces

The restriction fragments polymorphisms of the mitochondrial DNA and the PCR fragment that comprised the internal transcribes spacers and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 40 strains from the 10 species of the genus Zygosaccharomyces, including the new species Z. lentus were examined. The RFLP's of the ITS-5.8S region showed a specific restriction pattern for each species, including the new species Z. lentus. The only exception were the species Z. cidri and Z. fermentati that produced identical restriction profiles. The electrophoretic chromosome patterns confirmed the differences between the species of this genus, including the phylogenetic closest species Z. cidri and Z. fermentati. They present few chromosomes ranging from 3 bands (4 or 5 chromosomes) for Z. florentinus to 7 bands (8 to 10 chromosomes) for Z. cidri and Z. fermentati. The strain level resolution power of RFLP's of mtDNA of this genus enabled the characterisation of strains from the same species, even where they are isolated from the same substrate. However, in the cases of Z. bailii and Z. lentus, electrophoretic karyotyping there was considerable variation.     

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.     

Genotyping of a miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii, based on sequence analysis of the partial 26S ribosomal RNA gene and two internal transcribed spacers

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I-VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type alpha and type beta) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.     

Electrophoretic identification of roach (Rutilus rutilus L.), rudd (Scardinim erythrophthalmus L.), bream (Abramis brama L.) and their natural hybrids

Roach, rudd, bream and their natural hybrids of 2 cm standard length or larger can be definitively identified by their enzyme electrophoretic patterns. Zymograms of lactate dehydrogenase and esterases as produced by vertical starch gel electrophoretic analysis of whole fry or adult eye extracts are the most useful in this respect. The lactate dehydrogenase isozymes, containing B sub-units, migrate more anodally in rudd and bream than in roach. Due to the tetrameric structure of lactate dehydrogenase, in hybrid rudd x roach and roach x bream, eleven isozymes can be observed as compared with six in the parental patterns. Esterases show unique patterns for all species and hybrids. With the exception of one fraction in rudd x bream, the esterase patterns of hybrids show summations of the parental phenotypes.     

Species-specific PCR primers for the rapid identification of yeasts of the genus Zygosaccharomyces

Species-specific primer pairs that produce a single band of known product size have been developed for members of the Zygosaccharomyces clade including Zygosaccharomyces bailii, Zygosaccharomyces bisporus, Zygosaccharomyces kombuchaensis, Zygosaccharomyces lentus, Zygosaccharomyces machadoi, Zygosaccharomyces mellis and Zygosaccharomyces rouxii. An existing primer pair for the provisional new species Zygosaccharomyces pseudorouxii has been confirmed as specific. The HIS3 gene, encoding imidazole-glycerolphosphate dehydratase, was used as the target gene. This housekeeping gene evolves slowly and is thus well conserved among different isolates, but shows a significant number of base pair changes between even closely related species, sufficient for species-specific primer design. The primers were tested on type and wild strains of the genus Zygosaccharomyces and on members of the Saccharomycetaceae. Sequencing of the D1/D2 region of rDNA was used to confirm the identification of all nonculture collection isolates. This approach used extracted genomic DNA, but in practice, it can be used efficiently with a rapid colony PCR protocol. The method also successfully detected known and new hybrid strains of Z. rouxii and Z. pseudorouxii. The method is rapid, robust and inexpensive. It requires little expertise by the user and is thus useful for preliminary, large-scale screens.     

Genotyping of a Miso and Soy Sauce Fermentation Yeast,Zygosaccharomyces rouxii,Based on Sequence Analysis of the Partial 26S Ribosomal RNA Gene and Two Internal Transcribed Spacers

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I–VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type α and type β) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.     

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.     

Biochemical genetic comparison of the Chinese and European races of the common carp

An electrophoretic survey of the common carp revealed 33 loci. Nine were found to be polymorphic including two phosphoglucose isomerase loci, a phosphoglucomutase locus, three esterase loci, a lactate dehydrogenase locus, a malate dehydrogenase locus, and transferrin. Five of these polymorphic loci are reported for the first time, the two phosphoglucose isomerase loci, a phosphoglucomutase locus and two of the three esterase loci. Differences in allele frequencies between the European and Chinese races were found at most loci.     

The Nubians of Kom Ombo: serum and red cell protein types

Phenotype and gene frequencies are presented for eight polymorphic systems among the Nubians of South Egypt, namely, acid phosphatase, glucose-6-phosphate dehydrogenase, adenylate kinase, 6-phosphogluconate dehydrogenase, esterase D, phosphoglucomutase I, peptidase A, and haptoglobin. Eleven systems, namely, albumin, ceruloplasmin, hemoglobin, lactate dehydrogenase, isocitrate dehydrogenase, phosphohexose isomerase, malate dehydrogenase, peptidase B and C, phosphoglucomutase II, and transferrin were found to be monomorphic. A single electrophoretic variant of phosphohexose isomerase were observed.     

Use of electrophoretic enzyme patterns for streptomycete systematics

Abstract The relative electrophoretic mobilities of various enzymes from 24 different streptomycetes were determined on polyacrylamide gels in order to examine the relatedness of species and strains of the genus Streptomyces . Of 11 different enzymes tested in this study, hexokinase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase showed a limited number of constant and reproducible polymorphic enzyme patterns, by comparing which the inter-specific relationships could be examined. In contrast, glucose dehydrogenase, alcohol dehydrogenase, 3-hydroxybutyrate dehydrogenase, phosphoglucose isomerase, peroxidase and esterase exhibited either weak non-reproducible or highly heterogeneous band patterns which were suitable for dissecting the strains within a species and a cluster group.     

Molecular assessment of indigenous yeast population from traditional balsamic vinegar

AIMS: To identify and describe the indigenous yeast population involved in traditional balsamic vinegar (TBV) fermentation. METHODS AND RESULTS: Using the restriction analysis of the ribosomal region 5.8S (5.8S rRNA) and the internal transcribed spacers 1 and 2 (5.8S-ITS region) we were able to group 133 strains isolated from 17 cooked grape must samples into 10 different yeast species, included into 4 genera. Moreover, we sequenced the D1/D2 domains of the 26S rRNA and confirmed the reliability of each identification at species level. Most strains belonged to the genus Zygosaccharomyces. In particular, Zygosaccharomyces bailii was found in 41% of the samples, followed by Saccharomyces cerevisiae, Zygosaccharomyces pseudorouxii and Candida stellata. Strains belonging respectively to Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces rouxii, Hanseniaspora valbyensis, Hanseniaspora osmophila and Candida lactis-condensi species were also detected. Despite the great number of species recovered, the mtDNA restriction profiles showed low variability at strain level. Saccharomyces cerevisiae isolates with an higher degree of intraspecific variance were considered an exception. CONCLUSIONS: Many different indigenous yeast species were recovered and TBV yeasts population seems to be far more complex than what was reported in previous literature. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the indigenous yeast species of TBV cooked must.     

Molecular evidence for the existence of natural hybrids in the genus Zygosaccharomyces

26S rDNA D1/D2 sequencing was used to characterise a number of food-associated Zygosaccharomyces rouxii strains held at the National Collection of Yeast Cultures. In the course of this study, four strains (NCYC 1682, NCYC 3042, NCYC 3060 and NCYC 3061) were identified which appeared, based on their D1/D2 sequences, to belong to a novel Zygosaccharomyces species. However, subsequent sequence analysis showed that NCYC 1682, NCYC 3060 and NCYC 3061 possess two highly divergent copies of the nuclear-encoded ADE2, HIS3 and SOD2 genes, indicating these three strains are in fact hybrids. NCYC 3042, however, does appear to represent a novel species which may be hypothesized to have crossed with Z. rouxii and given rise to hybrid strains. Additional approaches to define precise taxonomic status and mechanisms of hybrid genome formation amongst yeast species are discussed.     

Characterization of enterobacteria by esterase specific-activity profiles

The spectrum of specific activities and the electrophoretic mobilities of esterases produced by 550 strains of Enterobacteriaceae belonging to 36 species and subclassified into six groups (group 1, Escherichia coli, Shigella and Escherichia hermanii; group 2, genus Salmonella and genus Citrobacter; group 3, genus Klebsiella and genus Enterobacter; group 4, genus Serratia and Serratia fonticola; group 5, genus Proteus, genus Providencia and genus Morganella; and group 6, genus Yersinia) were analysed by acrylamide/agarose gel electrophoresis using standardized methods for staining and mobility comparisons. Nineteen types of esterase were defined by their respective esterase specific-activity profile (ESAP). A multiple correspondence analysis (MCA) of the ESAP data enabled 82% of the strains in the 36 species to be correctly classified. In each group, the species were clearly delineated after MCA on both ESAP and electrophoretic mobility data. In addition, the smallest number of characters providing species identification of Yersinia strains by esterase polymorphism was identified by means of a binary segmentation tree technique.     

Some observations on the biology of five strains of Eimeria necatrix

Five laboratory strains of Eimeria necatrix were characterised with regard to the size of their oocysts, pathogenicity, reproduction, cross-immunity, ability to grow in embryonated eggs, and electrophoretic variation of enzymes. Three strains were highly pathogenic whilst two caused only few deaths and milder changes to the mean body weight gains of infected chickens. Cross-immunity was incomplete judged by scores of lesions after heterologous challenge, and electrophoretic variation of the enzymes lactate dehydrogenase and isocitrate dehydrogenase from oocysts of the five strains was also found. All the strains completed their life cycle in embryonated eggs but only a few oocysts were recovered.     

Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing

Pernin P. 1984. Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing. International Journal for Parasitology14: 459–465. The isoenzymatic patterns of different strains of Naegleria were studied by isoelectric focusing (I.E.F.) on polyacrylamide gels for seven enzymatic activities (leucine amino peptidase; lactate dehydrogenase; glucose 6 phosphate dehydrogenase; propionyl esterase; glucose phosphate isomerase; malate dehydrogenase; acid phosphatase), two of which (lactate dehydrogenase and glucose 6 phosphate dehydrogenase) were being investigated for the first time. The three pathogenic N. fowleri strains share a common pattern for most of the enzymes tested except for glucose 6 phosphate dehydrogenase, and thus form a very homogeneous species, while thermophilic nonpathogenic strains show more heterogeneity particularly for leucine amino peptidase and glucose 6 phosphate dehydrogenase.I.E.F. must be considered as a supplementary and rapid method for the identification of N. fowleri and as a powerful tool to demonstrate the complexity of different genera of free-living amoebas.     

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.     

Differetiation of the spoilage yeast Zygosaccharomyces bailii from other Zygosaccharomyces species using 18S rDNA as target for a non-radioactive ligase detection reaction

A non-radioactive PCR coupled ligase detection reaction was developed to discriminate the food spoilage yeasts Zygosaccharomyces bailii and Z. bisporus from each other and from other members of the genus. A short region of the 18S rRNA gene was amplified from boiled cell lysates and polymerase chain reaction (PCR) products used as target in the template directed ligation of two adjacent oligonucleotides. Ligated products were captured using biotin-streptavidin chemistry and detected using digoxigenin
immuno-chemiluminescence. The ligase detection reaction was able to discriminate to the species level, targeting a single base deletion. The specificity of the reaction was assessed using seven species of the genus Zygosaccharomyces . Only strains of Z. bailii and Z. bisporus gave positive results with their respective primer sets. The lower detection limit of the strategy was 10pg (3 times 10⁷ targets) of amplified product.     

Genetic diversity of the Pichia membranifaciens strains revealed from rRNA gene sequencing and electrophoretic karyotyping, and the proposal of Candida californica comb. nov

The genetic diversity of the types or authentic strains of 20 facultative synonyms of Pichia membranifaciens (E.C. Hansen) E.C. Hansen was revealed on the basis of large-subunit (26S) rDNA D1/D2 domain and internal transcribed spacer region sequencing and electrophoretic karyotyping. At least five separate species were recognized among the strains studied. Fourteen strains were confirmed to belong to P. membranifaciens. Strain CBS 241, an authentic strain of Zygosaccharomyces chevalieri Guilliermond var. fermentati Saito, should be assigned to Pichia manshurica Santa María. Strain CBS 243, an authentic strain of Zygopichia chiantigiana Castelli, is conspecific with CBS 2287, the type strain of Pichia fluxuum (Phaff & Knapp) Kreger-van Rij. Strain CBS 1367, the type of Zygosaccharomyces bisporus Anderson, belongs to Pichia kluyveri Bedford var. kluyveri. Strain CBS 989, the type of Cryptococcus californicus Anderson & Skinner, represents a distinct species, for which a new combination, Candida californica comb. nov., is proposed. The taxonomic status of strains CBS 189, the type of Pichia calliphorae Kl?cker, and CBS 214, the type of Pichia derossii Castelli, remain to be studied further. Their D1/D2 sequences and chromosomal DNA banding patterns were similar to those of P. membranifaciens, but their internal transcribed spacer sequences differed significantly.