Candida albicans is a dimorphic fungus that is pathogenic for humans. No sexual cycle has been reported for this fungus, and earlier reports have differed on whether typical strains of C. albicans are haploid or diploid. Previous estimates of the DNA content of C. albicans varied by one order of magnitude. We used three independent methods to measure the kinetic complexity of the single-copy DNA from a typical strain of C. albicans (strain H317) to determine the DNA content per haploid genote; we obtained values of 15 and 20 fg per cell by using S1 nuclease and hydroxyapatite assays, respectively. Optical assays for DNA reassociation kinetics, although not definitive in themselves, yielded values in this range. Chemical measurements of the DNA content of several typical strains, including strain H317, yielded values clustered about a mean of 37 fg per cell. We concluded that these strains are diploid.Candida albicans is a dimorphic fungus, classified among the fungi imperfecti because no sexual cycle has been observed in strains of the species. Most members of the species exist in the yeast form, but the mycelial form may develop under certain conditions (23,24). Both forms of the species are pathogenic for human hosts, although the details of the pathogenicity of the two forms may be very different. Candida is the most common fungal pathogen of humans (1); clinical reports concerning the genus number in the hundreds each year (23).In contrast to the clinical reports, biochemical studies of Candida are relatively sparse. In particular, little has been established concerning the nature and organization of Candida genomes, including those of the most extensively studied species, C. albicans. Even the ploidy of C. albicans is in dispute (27,32,35,46). Several recent reports (27,28,45,46) conclude that at least some C. albicans strains are diploid, whereas results from other reports (32,35), are more simply interpreted in terms of haploidy.There have also been confficting reports of the DNA content of C. albicans strains; even values published during the last 2 years (35,39,46) disagree by nearly one order of magnitude. We present direct measurements of the total DNA content and sequence complexity of C. albicans.The determination reported here was designed specifically to include a large number of measurements to ensure a high degree of precision (2) or Stewart (41); the former procedure was modified by replacing the 0.2 M PCA wash with a 0.2 M PCA extraction (10 to 15 min on ice). We refer to these two extraction procedures as method A and method B, respectively. The supernatants of the hot PCA extractions from both methods were centrifuged at 20,000 x g for 30 min to remove substances that interfered with the diphenylamine assay. DNA was assayed colorimetrically with a diphenylamine reagent (5) and, for some experiments, also with diaminobenzoic acid reagent (37). Highly polymerized calf thymus DNA (type V, Sigma Chemical Co., St. Louis, Mo.) was used as a standard. The DNA was dried by heating at 70°C overnight...

Genetic similarities and differences between type I and type II Candida stellatoidea were 'txied. The electrophoretic karyotype, mitochondrial DNA (mtDNA) restriction patterns, and midrepe ence of nuclear DNA in type I C. stellatoidea were clearly distinguishable from those of a reference cul Of Candida albicans. The karyotype and the major bands of the midrepeat sequence of type II C.s

A cloned DNA fragment from Candida albicans containing the gene for the protein actin was used to probe the molecular structure of the actin gene of several medically important yeasts (C. albicans, Candida stellatoidea, Candida tropicalis, Candida pseudotropicalis, Candida krusei, Candida parapsilosis, Candida guilliermondii, and Torulopsis glabrata). Whole-cell DNA from each species was digested with restriction endonucleases, electrophoresed on agarose gels, and transferred to nitrocellulose. Radioactively labeled C. albicans actin gene was hybridized to the DNA fragments on the nitrocellulose. The C. albicans probe produced a strong signal with all of the Candida DNAs tested, indicating considerable conservation of this gene. In addition, the actin genes of all of the species tested were found to have no internal EcoRI or Sall restriction sites. With the exception of C. guilliermondii, all of the species tested had a single internal HindIII recognition site. However, the location of flanking restriction sites was found to be species specific. For all of the enzymes tested, the locations of the flanking restriction sites in C. albicans and C. stellatoidea were identical; all of the other strains yielded fragments clearly distinct from one another. These differences provide a molecular tool for the differentiation of medically important Candida species.

Deoxyribonucleic acid (DNA) homalogy was used to examine genetic relatedness among 25 yellow-pigmented strains of group D streptococci and to clarify the possible genetic relatedness of these strains to Streptococcus faecium and Streptococcus faecalis. In all cases, the DNA of the yellow-pigmented strains hybridized with the DNA of S. faecium and S. faecalis at a level of less than 25%. Based on median thermal dissociation temperatures (Tm's) and hybridization tests, the yellow-pigmented strains were divided into two groups (I and 11). DNAs of strains in group I had relatively low Tm's and did not exhibit significant homology with DNA of the herein designated type strain (ATCC 25788) of S. faecium subsp. casseliflavus. The DNAs of group I1 strains, on the other hand, exhibited high Tm'S and had a high degree of homology with the DNA of the type strain of the above-mentioned subspecies. Three physiological traits were found to be peculiar to group I1 organisms: ability to grow in the presence of 6.5% sodium chloride, inability to ferment sorbitol, and inability to decarboxylate tyrosine. It is proposed that the group I1 strains constitute a separate and distinct species. Because the strains presently placed in this species include the type strain of S. faecium subsp. casseliflavus Mundt and Graham, the name of this species, according to the rules of the Bacteriological Code, is Streptococcus casseliflavus (Mundt and Graham) comb. nov.Mundt and Graham (11) proposed the name Streptococcus faecium subsp. casseliflavus for group D yellow-pigmented streptococci which were isolated from plants (11, 12). Taken into consideration for the establishment of this subspecies were its pigmentation, physiology, and ecology, which set this subspecies apart from Streptococcus faecalis and the other subspecies of Streptococcus faecium.

A molecular approach was used to examine the genetic relatedness of 19 Frankia isolates by measuring the extent of deoxyribonucleic acid-deoxyribonucleic acid homology and the fidelity of hybrid duplex molecules. The Frankia isolates examined were divided into two groups based on the results of hybridization tests. The members of genogroup 1, consisting of isolates from Alnus, Myrica, and Comptonia host plants, exhibited high levels of homology (67.4 to 94.1%) with strain ArI4, an isolate from Alnus rubra. Isolates from Elaeagnus, Ceanothus, Purshia, and Casuarina and isolate Air12 from A l m s did not exhibit significant homology (less than 39%) with strain ArI4. None of the strains showed a high degree of homology with strain EuIla (less than 33%), an isolate from Elaeagnus umbellata. Among the isolates not belonging to genogroup 1, subgroupings seemed to exist, as evidenced by a very high level of homology (97%) between two isolates from Casuarina, strains D11 and the G2, but a low level of homology (27%) between other strains and strain G2. Thermal stability studies of the hybrid deoxyribonucleic acids which showed high levels of homology revealed an average mismatch of 3%, whereas the low-homology duplexes exhibited about 5% mismatch. The genome molecular weights of two probe Frankia strains, strains Ar14 and EuIla, were 8.3 X lo9 and 6.0 X lo9, respectively.Since the first confirmed isolation of a nitrogen-fixing Frankia endophyte from Comptonia root nodules (ll), more than SO strains from 20 different plant species have been isolated (2). The availability of increasing numbers of isolates has made it possible to examine various aspects of these endophytes grown in vitro.Morphologically, there appear to be no fixed distinctive differences among the isolates other than pigmentation and the occurrence of vesicles in culture. The size, shape, and number of the sporangia, as well as the overall density of the colonies, depend largely on the culture conditions (7, 16). Biochemical studies (18) also have demonstrated a uniformity among Frankia isolates in terms of cell wall composition (type 111) and phospholipid pattern (type I), although their cell sugar patterns vary widely. Based on serological (3), physiological (19), and cross-inoculation studies (5, 7, 16), the isolates can be divided into at least two groups. Generally, the isolates from Alnus, Myrica, and Comptonia belong to the same group (serogroup I, physiological group B), whereas the isolates from Elaeagnus, Ceanothus, Purshia, and Casuarina form a separate group (serogroup 11, physiological group A).Despite these studies, there are no consistently useful taxonomic criteria for proper classification of Frankia isolates. Accordingly, the principal purpose of this study was to use a molecular approach to examine the genetic relatedness of Frankia isolates. This approach was based on the base composition of deoxyribonucleic acid (DNA) (l), the degree of DNA-DNA hybridization, and the thermal stability of the hybrid DNA duplexes. MATERIALS AND METHODSO...