Rapid identification of Candida albicans and other human pathogenic yeasts by using short oligonucleotides in a PCR.

A PCR system that can quickly and accurately identify 14 species of human pathogenic yeasts was developed. The procedure distinguished between nine species of a closely related clade, Lodderomyces elongisporus, Candida parapsilosis, a new Candida sp., C. sojae, C. tropicalis, C. maltosa, C. viswanathii, C. albicans, and C. dubliniensis and between another five more divergent species, Pichia guilliermondii, C. glabrata, C. zeylanoides, C. haemulonii, and C. haemulonii type II. A rapid DNA extraction procedure that yields purified DNA in about 1 h is also described. The system uses uniform conditions with four primers for each reaction, two 40- to 50-mer universal primers that serve as a positive control and two 23- to 30-mer species-specific primers. Species-specific primers were derived from a 600-nucleotide variable region (D1/D2) at the 5' end of the large-subunit (26S) ribosomal DNA gene and were generally designed to use mismatches at the 3' end. Universal primers were developed from conserved nucleotide sequences in the small-subunit (18S) rRNA gene. In this system, a control 1,200- to 1,300-base DNA fragment was produced in all reactions and a smaller 114- to 336-base DNA fragment was produced if the chromosomal DNA from the target species was present. The PCR procedure is rapid and easy to interpret and may be used with mixed cultures.

Construction and characterization of a DNA probe for distinguishing strains of Aspergillus flavus.

Repetitive DNA sequences have proven useful and reliable characters in evaluating genetic relatedness of strains at different levels of taxonomic classification. A DNA probe was constructed to distinguish among strains of Aspergillus flavus by DNA fingerprinting techniques. Chromosomal DNA of A. flavus var. flavus NRRL 6541 was partially digested with EcoRI and ligated to a Lambda Dash bacteriophage vector. Four lambda clones were identified which displayed multiple and distinct bands when hybridized with chromosomal DNA from seven strains of A. flavus var. flavus digested with either EcoRI or PstI. One of these clones was chosen for further analysis and was subcloned into pUC19. The subclone, pAF28, contained a 6.2-kb chromosomal DNA insert and was able to distinguish among strains characterized by K. E. Papa (Mycologia 78:98-101, 1986) as belonging to 22 different vegetative compatibility groups. The subclone identified unique banding patterns when hybridized to genomic DNA digested with PstI. The cloned probe may be species specific as it hybridized with the DNA of all isolates of A. flavus tested in addition to strains recognized as varieties of A. flavus (e.g., A. flavus var. oryzae, A. flavus var. parasiticus, and A. flavus var. sojae). pAF28 hybridized to a single band on a Southern blot with Aspergillus nomius DNA but did not hybridize with the DNA of other fungal species tested including Aspergillus ochraceus, Aspergillus auricomus, Aspergillus alliaceus, Fusarium moniliforme, and Penicillium thomii.

Physical and genetic characterization of linear DNA plasmids from the heterothallic yeast Saccharomycopsis crataegensis.

Five strains of the heterothallic yeast Saccharomycopsis crataegensis have been previously shown to contain DNA and/or RNA plasmidlike molecules (Shepherd et al. 1987). Three DNA plasmids, designated pScrl-1, -2 and -3, were found in strain NRRL Y-5902, while two were identified in each of NRRL strains Y-5903 and Y-5904. DNA plasmids were not identified in S. crataegensis strains Y-5910 or YB-192. Four S. crataegensis strains (Y-5903, Y-5904, Y-5910 and YB-192) were also shown to possess double-stranded RNA (dsRNA) molecules not found in strain Y-5902 (Shepherd et al. 1987). Hybridization studies now demonstrate the DNA plasmids in Y-5903 and Y-5904 to be highly homologous to their respective size counterparts (pScrl-1 and pScrl-2) in Y-5902 and to show some homology to pScrl-3. Restriction endonuclease mapping studies confirm the linear nature of each plasmid and establish identical restriction maps for a 1.4 kilobase (kb) region in pScrl-2 and -3. This 1.4 kb region accounts for the hybridization homology of pScrl-2 and pScrl-3 noted by Shepherd et al. (1987) and for homology of the plasmids of Y-5903 and Y-5904 to pScrl-3 of Y-5902. The pScrl plasmids show no homology to the dsRNA molecules of S. crataegensis, the 2 microM circular DNA of Staccharomyces cerevisiae, the 'killer' plasmids of Kluyveromyces lactis, or the linear DNA plasmids of Pichia inositovora. In crosses between linear DNA plasmid-containing and dsRNA-containing strains, only progeny containing the pScrl plasmids were recovered. Poor spore viability and a lack of complete tetrad recovery limited the extent of the analysis, but the findings suggest a cytoplasmic mode of inheritance for these linear DNAs.

Taxonomic relationships among strains of the anaerobic bacterium Bacteroides ruminicola determined by DNA and extracellular polysaccharide analysis.

DNA and extracellular polysaccharide (EPS) analyses were performed on 14 strains of Bacteroides ruminicola. The guanine-plus-cytosine (G+C) base contents, determined from the buoyant densities of chromosomal DNAs, showed a broad range of values, from 37.6 to 50.9 mol%. DNA hybridization showed generally low DNA relatedness among the strains. Seven strains formed two groups of closely related bacteria consisting of five (group 1) and two (group 2) strains, and another strain, E42g, showed moderate relatedness to group 1 strains. However, the remaining six strains were not related to any of the other strains. DNA reassociation indicates that the strains constitute a genetically diverse group representing as many as nine separate species. EPS analysis showed that the strains produced EPS with rather uniform sugar compositions, which did not correlate with strain relationships determined by DNA analysis. Four strains had EPS with acidic sugars or unknown compounds. The EPS of strain 20-63 contained the unusual acidic sugar 4-O-(1-carboxyethyl)-rhamnose. This monosaccharide has been shown to occur in nature in only one other bacterial species.

Cloning, sequencing, and expression of a xylanase gene from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens.

A gene coding for xylanase activity, xynA, from the anaerobic ruminal bacterium Butyrivibrio fibrisolvens 49 was cloned into Escherichia coli JM83 by using plasmid pUC19. The gene was located on a 2.3-kilobase (kb) DNA insert composed of two adjacent EcoRI fragments of 1.65 and 0.65 kb. Expression of xylanase activity required parts of both EcoRI segments. In E. coli, the cloned xylanase enzyme was not secreted and remained cell associated. The enzyme exhibited no arabinosidase, cellulase, alpha-glucosidase, or xylosidase activity. The isoelectric point of the cloned protein was approximately 9.8, and optimal xylanase activity was obtained at pH 5.4. The nucleotide sequence of the 1,535-base-pair EcoRV-EcoRI segment from the B. fibrisolvens chromosome that included the xynA gene was determined. An open reading frame was found that encoded a 411-amino-acid-residue polypeptide of 46,664 daltons. A putative ribosome-binding site, promoter, and leader sequence were identified. Comparison of the XynA protein sequence with that of the XynA protein from alkalophilic Bacillus sp. strain C-125 revealed considerable homology, with 37% identical residues or conservative changes. The presence of the cloned xylanase gene in other strains of Butyrivibrio was examined by Southern hybridization. The cloned xylanase gene hybridized strongly to chromosomal sequences in only two of five closely related strains.

Isolation and Characterization of Xylan-Degrading Strains of Butyrivibrio fibrisolvens from a Napier Grass-Fed Anaerobic Digester.

Six new xylanolytic bacterial strains have been isolated from a Napier grass-fed anaerobic digester. These strains were identified as Butyrivibrio fibrisolvens and were similar in many respects to ruminal isolates described previously. The new isolates exhibited a high degree of DNA homology with several ruminal strains of B. fibrisolvens. Xylan or xylose was required to induce the production of enzymes for xylan degradation, xylanase and xylosidase. Production of these enzymes was repressed in the presence of glucose. Xylanase activity was predominantly extracellular, while that of xylosidases was cell associated. The new isolates of B. fibrisolvens grew well in defined medium containing xylan as the sole carbon source and did not produce obvious slime or capsular layers. These strains may be useful for future genetic investigations.

Genetic basis of the complementary DpnI and DpnII restriction systems of S. pneumoniae: an intercellular cassette mechanism.

Cells of S. pneumoniae contain either DpnI, a restriction endonuclease that cleaves only the methylated DNA sequence 5'-GmeATC-3', or DpnII, which cleaves the same sequence when not methylated. A chromosomal DNA segment containing DpnII genes was cloned in S. pneumoniae. Nucleotide sequencing of this segment revealed genes encoding the methylase and endonuclease and a third protein of unknown function. When the plasmid was introduced into DpnI cells, recombination during chromosomal facilitation of its establishment substituted genes encoding the DpnI endonuclease and another protein in place of the DpnII genes. DNA hybridization and sequencing showed that the DpnI and DpnII segments share homology on either side but not between themselves or with other regions of the chromosome. Thus, the complementary restriction systems are found on nonhomologous and mutually exclusive cassettes that can be inserted into a particular point in the chromosome of S. pneumoniae on the basis of neighboring homology.

Nucleotide sequence of the Dpn II DNA methylase gene of Streptococcus pneumoniae and its relationship to the dam gene of Escherichia coli.

The structural gene (dpnM) for the Dpn II DNA methylase of Streptococcus pneumoniae, which is part of the Dpn II restriction system and methylates adenine in the sequence 5'-G-A-T-C-3', was identified by subcloning fragments of a chromosomal segment from a Dpn II-producing strain in an S. pneumoniae host/vector cloning system and demonstrating function of the gene also in Bacillus subtilis. Determination of the nucleotide sequence of the gene and adjacent DNA indicates that it encodes a polypeptide of 32,903 daltons. A putative promoter for transcription of the gene lies within a hundred nucleotides of the polypeptide start codon. Comparison of the coding sequence to that of the dam gene of Escherichia coli, which encodes a similar methylase, revealed 30% of the amino acid residues in the two enzymes to be identical. This homology presumably reflects a common origin of the two genes prior to the divergence of Gram-positive and Gram-negative bacteria. It is suggested that the restriction function of the gene is primitive, and that the homologous restriction system in E. coli has evolved to play an accessory role in heteroduplex DNA base mismatch repair.

Ectopic integration of chromosomal genes in Streptococcus pneumoniae.

When a DNA fragment containing a marker gene was ligated to random chromosomal fragments of Streptococcus pneumoniae and used to transform a recipient strain lacking that gene, the gene was integrated at various locations in the chromosome. Such ectopic integration was demonstrated for the malM gene, and its molecular basis was analyzed with defined donor molecules consisting of ligated fragments containing the malM and sul genes of S. pneumoniae. In a recipient strain deleted in the mal region of its chromosome, these constructs gave Mal+ transformants in which the malM and sul genes were now linked, with malM located between duplicate sul segments. Ectopic integration was unstable under nonselective conditions; mal(sul) ectopic insertions were lost at a rate of 0.05% per generation. Several possible mechanisms of ectopic integration were examined. The donor molecule is most likely to be a circular form of ligated homologous and nonhomologous fragments that, after entry into the cell, undergoes circular synapsis with the recipient chromosome at the site of homology, followed by repair and additive integration.

Transformation in pneumococcus: nuclease resistance of deoxyribonucleic acid in the eclipse complex.

Donor deoxyribonucleic acid strands in the eclipse phase of genetic transformation of pnuemococcus (Streptococcus pneumoniae) are purified as a complex with a cf the deoxyribonucleic acid strand in this complex to digestion by nucleases was shown to be 50- to 1,000-fold less than that of uncomplexed single strands of deoxyribonucleic acid. Deoxyribonuclease I, micrococcal nuclease, Neurospora endonuclease, nuclease P1, and the major endogenous nuclease of cell-free extracts were studied. Sensitivity to nuclease attack was not uniform along the deoxyribonucleic acid strand; sequences of strongly protected bases were separated by more sensitive regions. The minimum size of protected fragments was about 70 bases. A complex of protein with the protected deoxyribonucleic acid segments was obtained after partial digestion. The sizes of these complexes, of the protected deoxyribonucleic acid segments, and of the protein subunit released by complete nuclease digestion, are all approximately identical, as determined by gel exclusion chromatography. Deoxyribonucleic acid strands of eclipse complex were also shown to be particularly well protected from attack by the major pneumococcal endonuclease in cell extracts.