Differentiation of six sibling species in the Saccharomyces sensu stricto complex by multilocus enzyme electrophoresis and UP-PCR analysis

UP-PCR analysis and multilocus enzyme electrophoresis were used to characterize 37 strains of the sibling species Saccharomyces cerevisiae, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae and S. paradoxus. The results demonstrate that both molecular approaches are useful for discriminating between these phenotypically indistinguishable Saccharomyces species. The data obtained are in excellent agreement with previously reported genetic analyses, sequencing of the 18S rRNA and ITS regions, and DNA-DNA reassociation data. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic heterogeneity among Lactobacillus acidophilus strains

Physiological characteristics, DNA base composition (% GC) and DNA-DNA reassociation values were determined for 138 Lactobacillus acidophilus strains. Twenty seven strains were received from various culture collections and 111 strains were freshly isolated during a study on the composition of the intestinal lactic microflora of piglets and suckling calves.All strains had physiological characteristics which were substantially similar. The strains isolated from pigs were unable to ferment trehalose. The % GC ranged from 35.8 to 43.4.On the basis of the results of DNA-DNA hybridization the strains were divided into four genetic groups.This study was partially supported by grants from the Consiplio Nazionale Ricerche (C.N.R.).     

Evidence that Bacterial Blight of Kiwifruit, Caused by a Pseudomonas sp., was Introduced into New Zealand From China

Strains of the bacterium, Pseudomonas sp., causing blight of kiwifruit, isolated from affected vines in New Zealand and in the People's Republic of China, were compared using DNA-DNA hybridization and repetitive-PCR (rep-PCR) and biolog. DNA of each strain was hybridized to DNA of reference strains representing P. viridiflava, P. savastanoi and the kiwifruit pathogen. Strains of the pathogen from New Zealand and China shared reassociation values exceeding 76%. Strains from both countries shared reassociation values of more than 66% (average 82%) with DNA of the type strain of P. savastanoi, and less than 51% with DNA of P. viridiflava (average 33.5%). A comparison by rep-PCR using the primers REP, ERIC and BOX showed that strains from New Zealand and China were indistinguishable. Comparison of endonuclease restriction patterns of strains from New Zealand showed that those gathered over a 20-year period from different growing regions were more diverse than strains gathered in one orchard in a single season. The ecological implications of these data and the source of the pathogen in New Zealand are discussed. It is concluded that the pathogen originated in China and was introduced into New Zealand in vegetative kiwifruit material, and not as was previously thought, in two small seed samples.     

Divergence in the level of DNA hybridization and formation of sibling species in the lactic acid bacteria Streptococcus thermophilus

Previously, five distinct groups with 80-90% intragroup DNA homology values were revealed among 19 lactic acid-producing bacterial strains. The study of 39 new strains of thermophilic streptococci in the present work allowed us to reveal the sixth DNA homology group. The nine strains of this group are similar, at 55-70% DNA homology levels, to the type strain S. thermophilus ATCC 19258. Group VI showed a low level of DNA-DNA reassociation (20-30%) with the DNA homology groups I, II, III, and V. The intergroup DNA-DNA reassociation values determined from DNA renaturation rates varied from 20 to 50%. These data were interpreted as indicative of the existence of at least four sibling species among the thermophilic streptococci studied.     

Deoxyribonucleic acid reassociation in the classification of coagulase-positive staphylococci

DNA-DNA reassociation studies were performed with coagulase-positive staphylococci belonging to the biotypes A, B, C, D, E and F. These studies present genetic evidence for the existence of at least two distinct species within this group of organisms. The common Staphylococcus aureus strains were represented by organisms from biotypes A to D, and their DNA revealed over 80% nucleotide sequence homology under restrictive conditions. Less than 15% DNA homology was detected between strains from biotypes A to D (S. aureus) and those from biotypes E and F. The DNA of organisms from either the biotypes E or F displayed over 70% homology. Together, both biotypes are considered to represent the species S. intermedius. However, DNA homology values dropped to 50–65% between strains from different biotypes. This may justify the separation of S. intermedius biotypes E and F on a subspecies level.Abbreviations O.D. optical density - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) This work was supported by Deutsche Forschungsgemeinschaft     

Assignment ofKluyveromyces cellobiovorus nomen nudum toCandida intermedia (Ciferri & Ashford) Langeron et Guerra

A yeast culture isolated in Japan from soil and invalidly described asKluyveromyces cellobiovorus nom. nud. DBVPG 6286 (CBS 7153) was compared for its physiological and morphological properties and by nDNA-nDNA reassociation experiments with the type strains of several species of the genusKluyveromyces as well as of variousCandida species exhibiting similar phenotypic profiles. DBVPG 6286 was found to be conspecific with the type strain ofCandida intermedia (Ciferri & Ashford) Langeron et Guerra (1938).     

Four new species of the genusSporobolomyces isolated from leaves in Thailand

Thirteen undescribed strains of ballistoconidium-forming yeasts, isolated from leaves collected in the suburbs and along the southeast seacoast of Bangkok, Thailand, were divided into four different groups in the genusSporobolomyces on the basis of morphological, physiological, biochemical, and chemotaxonomical characteristics, and analyses of the sequences of 18S rDNA and internal transcribed spacer regions. DNA-DNA reassociation experiments with related species revealed that these four groups were four new distinct species.Sporobolomyces nylandii sp. nov.,S. poonsookiae sp. nov.,S. blumeae sp. nov. andS. vermiculatus sp. nov. are proposed for these strains.     

Killer yeasts of Kluyveromyces and Hansenula genera with potential application in fermentation and therapy

The killing/immunity interactions among killer strains of the genera Kluyveromyces, Hansenula and Saccharomyces from the Czechoslovak Collection of Yeasts were studied with the aim to find the strains with broad specificity and killer activity targeted against a range of undesirable wild yeasts causing stuck fermentations. Among 49 tested Kluyveromyces strains, five strains were found, and among 55 Hansenula strains, ten yeast strains were found with activity against a sensitive strain of Saccharomyces. Hansenula mrakii CCY 38-7-1 and Hansenula saturnus var. subsufficiens CCY 38-4-2 showed exceptional activity against the wine contaminants, Zygosaccharomyces bailii, as well as against pathogenic Candida species within a broad range of pH 2.9–5.1. Their potential biotechnological application is discussed.     

Analysis of ribosomal DNA restriction patterns in the genusKluyveromyces

Riboprinting was used to determine the relationships among strains belonging to 15 species of the genusKluyveromyces. The small subunit ribosomal RNA gene (SSU rDNA) was amplified using the Polymerase Chain Reaction (PCR) and subjected to a battery of nine restriction enzymes. Similarity coefficients between strains were calculated based on shared and unique restriction fragments. Cluster analysis revealed three major groups that generally correlated with previously reported relationships based on other molecular data. Variations in SSU rDNA restriction fragments may be used for differentiation of theKluyveromyces strains included in this study.The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Intraspecies Genomic Groups in Enterococcus faecium and Their Correlation with Origin and Pathogenicity

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.     

Evolution of muntjac DNA

The extent of nuclear single-copy DNA divergence between Muntiacus reevesi and Muntiacus muntjak vaginalis (Cervidae), a species pair showing extreme karyotype differences but striking morphological similarity, is 2%, as judged from the thermal stability of interspecific DNA-DNA hybrids. A comparison of the total nuclear DNA reassociation kinetics of the two species indicates a reduction of lowly repetitive sequences in M. m. vaginalis.     

Genetic relatedness of clinical and environmental isolates of lactose-positiveVibrio vulnificus

Environmental isolates of lactose-positiveVibrio vulnificus from different geographic areas were compared with clinical strains ofV. vulnificus on the basis of their phenotypic traits, virulence, DNA base composition, and DNA-DNA reasociation. EnvironmentalV. vulnificus strains were phenotypically indistinguishable from clinical isolates. These strains had a DNA base composition of 47–48 mol% guanine + cytosine and 85% reassociation at stringent temperature with DNA from clinicalV. vulnificus strains. These result indicateV. vulnificus strains from widely separated regions of the marine environment are indistinguishable from strains that have been agents of septicemia associated with shellfish consumption and of wound infections associated with seawater exposure.     

Molecular genetic evidence for the transfer of Oerskovia species into the genus Cellulomonas

A close genetic relationship among strains of Oerskovia turbata, O. xanthineolytica and various coryneforms is indicated by DNA-DNA reassociation studies. O. xanthineolytica shares high homology values (over 60%) with Cellulomonas cartae, Nocardia cellulans, Brevibacterium fermentans and Corynebacterium manihot. O. turbata and other cellulomonads show lower DNA homology values (20–25%) which are still high enough, however, to indicate a relationship at the genus level. Based on these data and supported by comparative analysis of the ribosomal 16 S RNA and the similarity of peptidoglycan types, the transfer of Oerskovia species into the genus Cellulomonas is justified.This paper is respectively dedicated to our teacher and mentor, Professor Dr. O. Kandler, on the occasion of his 60th birthday.     

Systematics of the species of the yeast genus Saccharomyces associated with the fermentation industry

Summary The so-called wine yeasts Saccharomyces cerevisiae, S. chevalieri, S. bayanus, S. italicus and S. uvarum are characterized by high ethanol tolerance and fermentation velocity. They are ecologically related, being predominantly associated with grape must and wine, and are taxonomically indistinguishable. The only significant physiological differences are between the ability to ferment certain sugars. A taxonomic revision of more than 1,000 strains isolated during the past 50 years and belonging to the above species showed extreme instability in the ability to ferment different sugars. The relationships between these yeasts were examined for DNA base composition and DNA-DNA reassociation. The G+C ranged from 37.6% to 39.0% while optical reassociation experiments defined a first group of species (Saccharomyces cerevisiae, S. chevalieri and S. italicus) exhibiting high base sequence complementarity (>90%). S. bayanus and S. uvarum also showed a high degree of relatedness. Low homology values (30%) indicate that the two groups of species are not closely related. While it is proposed to combine S. cerevisiae, S. chevalieri and S. italicus into one single species under the oldest epithet Saccharomyces cerevisiae, a study of a larger number of strains is recommended before considering the taxonomic position of S. bayanus and S. uvarum.     

Taxonomic status of atypicalLactobacillus saké andLactobacillus curvatus strains associated with vacuum-packaged meat spoilage

The taxonomic status of nine typical and atypicalLactobacillus saké andLactobacillus curvatus strains associated with vacuum-packaged meat spoilage was investigated by DNA-DNA homology and compared with four alternative identification methods. Phenotype-based identification methods as well as 23S rRNA targeted probes produced ambiguous results in the case of atypical strains. DNA-DNA hybridization indicated homologies of 63–78% between typical strains and the corresponding type strain, whereas values ranged from 38% to 54% for atypical strains. This apparent spectrum of relatedness observed was ascribed to the recent phylogenetic divergence of the two species. Atypical strains should, therefore, be designated as such and not assigned to particular species based on results of identification methods other than DNA-DNA hybridization.     

Phylogenetic Relationships of Species of the Genus Kluyveromyces van der Walt (Saccharomycetaceae) Deduced from the Partial Sequences of 18S and 26S Ribosomal RNAs

In order to clarify the phylogenetic relationships of the species classified in the genus Kluyveromyces (Saccharomycetaceae), three partial base sequences of 18S and 26S rRNAs of eighteen strains were determined. The regions determined of the strains corresponded to positions 1451 through 1618 (168 bases) of 18S rRNA and to positions 1611 through 1835 (225 bases) and 493 through 622 (130 bases) of a strain (IFO 2376) of Saccharomyces cerevisiae. The analyses of the partial base sequences suggested that the genus Kluyveromyces is phylogenetically heterogeneous, ranging from the strains that are quite close to the strain of S. cerevisiae to the strains that are distinct enough to be classified in genera separate from the genus Saccharomyces. From our sequence data, we concluded that the extent of the genus Kluyveromyces should be restricted to only one species, K. polysporus, the type species of the genus. Kluyveromyces phaffii was also distinct enough to deserve another genus. Kluyveromyces cellobiovorus was not close to any of the strains of Kluyveromyces species examined, and should be excluded from the genus. Most of the strains of the species examined were fairly close to the strain of S. cerevisiae.     

Ballistosporous yeasts found on the surface of plant materials collected in New Zealand

In the present study, strains from the surface of plant materials collected in New Zealand that belong to the genera Bensingtonia and Bullera are classified. One strain of Bensingtonia was assigned to Ben. ingoldii, while the remaining strain was assigned to Ben. naganoensis based on DNA-DNA reassociation experiment. Twenty-one of 28 Bullera strains were assigned to B. alba (11 strains), B. crocea (6 strains) and B. variabilis (4 strains). The remaining seven strains could not be assigned to any previously known species and were described as the new species, B. coprosmaensis (1 strain), B. hannae (1 strain), B. huiaensis (1 strain), B. mrakii (3 strains) and B. unica (1 strain).Abbreviations B Bullera - Ben Bensingtonia - Sp. Sporobolomyces - G+C guanine plus cytosine     

Interfertility as basis for the delimitation of Kluyveromyces marxianus

Hybridization studies between strains of Kluyveromyces marxianus and the remaining species of the genus involving the use of auxotrophic mutants, are reported. K. marxianus was found to be interfertile with K. bulgaricus, K. cicerisporus, K. dobzhanskii, K. drosophilarum, K. fragilis, K. lactis, K. phaseolosporus, K. vanudenii and K. wikenii. Accepting interfertility as criterion for conspecificity, these nine syngamous taxa are relegated to the status of biotypes or physiologic races of a single species K. marxianus.     

Intraspecific DNA divergence in Drosophila: a study on parthenogenetic D. mercatorum

Drosophila mercatorum is a species that can give rise to totally homozygous parthenogenetic strains. Using the technique of DNA-DNA hybridization, we have assessed the overall single-copy DNA differences among three independently derived strains that represent three independent genomes. Among strains, the average difference between homoduplex and heteroduplex median melting temperatures is 1.3 degrees C. This represents greater than or equal to 1.3% base-pair mismatch. Normalized percent of reassociation indicates further genetic differences, probably reflecting insertion/deletion differences and/or regions of the genome that are highly variable. This overall intraspecific genetic variation is higher than generally is thought to exist but is consistent with growing evidence of extensive DNA diversity within species of invertebrates. High intraspecific DNA variation may be correlated with rapid phyletic rates of evolution. Because of this high level of variation, the technique of DNA-DNA hybridization may be used to study intraspecific variation in invertebrates but is limited in its usefulness for higher systematic studies.