Reclassification of Pichia membranifaciens sensu Kurtzman

Forty strains which were stocked as Pichia membranifaciens sensu Kurtzman and 9 strains stocked as Candida valida, anamorphs of P. membranifaciens, in the Institute for Fermentation, Osaka (IFO) were reclassified based on the data of base composition of nuclear DNA, DNA/DNA hybridization, coenzyme Q system, electrophoretic karyotype, and base sequence of 18S rDNA. P. membranifaciens complex was assigned into 3 groups: (I) P. membranifaciens group, including 25 strains with high DNA homologies to the type strain of P. membranifaciens (72–98%); (II) P. manshurica group, including 18 strains with high DNA homology of 79–95% to the type strain of P. manshurica; and a group including the remaining 6 strains, which had low DNA homology to the above two species. GC content was 42.9–45.3 mol% for the P. membranifaciens group, 40.0–42.0 mol% for the P. manshurica group, and 27.2–44.7 mol% for the remaining group. All three groups had ubiquinone Co Q-7. Of the 6 anomalous strains, IFO 0162 was identified as Pichia deserticola, and IFO 0839 and IFO 0840 were identified as Issatchenkia occidentalis; but IFO 0842, IFO 0843, and IFO 1788 were thought to be unknown strains.     

Identity of the xerophilic species Aspergillus penicillioides: Integrated analysis of the genotypic and phenotypic characters

We examined the identity of Aspergillus penicillioides, the typical xerophilic and strictly anamorphic species, using an integrated analysis of the genotypic and phenotypic characters. Our experimental methods on two genotypic characters, i.e., DNA base composition using the HPLC method and DNA relatedness using the nitrocellulose filter hybridization technique between A. flavus, A. oryzae, and their close relations revealed a good agreement with the values by buoyant density (for DNA base composition) and spectrophotometric determination (for DNA relatedness) reported by Kurtzman et al. in 1986. On the basis of these comparisons, we examined DNA base composition and DNA relatedness of six selected strains of A. penicillioides, including IFO 8155 (originally described as A. vitricola), one strain of A. restrictus, and the respective strains from Eurotium amstelodami, E. repens, and E. rubrum. As a result, five strains within A. penicillioides, including the neotype strain NRRL 4548, had G+C contents of 46 to 49 mol%, whereas IFO 8155 had 50 mol%. A. restrictus had 52 mol%, and three Eurotium species ranged from 46 to 49 mol%. The DNA relatedness between A. penicillioides (five strains), except for IFO 8155, exhibited values greater than 70%, but the DNA complementarity between four strains and IFO 8155 in A. penicillioides revealed values of less than 40%. DNA relatedness values between three species of Eurotium were 65 to 72%. We determined 18S, 5.8S, and ITS rDNA sequences as other genotypic characters from A. penicillioides (six strains), A. restrictus, and related teleomorphic species of Eurotium. In three phylogenetic trees inferred from these sequences, five strains of A. penicillioides, including the neotype strain, were closely related to each other, whereas IFO 8155 was distantly related and grouped with other xerophilic species. Our results have suggested that A. penicillioides typified by NRRL 4548 and A. penicillioides IFO 8155 (ex holotype of A. vitricola) are not conspecific. The enzyme patterns as a genotypic character and general morphology and conidial ornamentation types as phenotypic characters supported this conclusion. Therefore the name A. vitricola Ohtsuki, typified by the holotype strain IFO 8155, should be revived. Evolutionary affinities among Aspergillus species and related teleomorphs, including the xerophilic taxa, are discussed.     

Changes in the intracellular concentrations of the adenosine phosphates and nicotinamide adenine dinucleotides ofSaccharomyces cerevisiae during batch fermentation

Significant changes in the intracellular concentrations of adenosine phosphates and nicotinamide adenine dinucleotides were observed during fermentation of grape must by three different strains ofSaccharomyces cerevisiae: S. cerevisiae var.cerevisiae, a typical fermentative yeast strain and two flor-veil-forming strains,S. cerevisiae var.bayanus andS. cerevisiae var.capensis. The intracellular concentration of ATP was always higher inS. cerevisiae var.cerevisiae than in the flor-veil-forming strains. NAD⁺ and NADP⁺ concentrations decreased at faster rates in the flor-veil-forming yeasts than in the other yeast but NADH concentration was the same in all yeasts for the first 10 days of fermentation. NADPH concentration was always lower inS. cerevisiae var.cerevisiae than in the other yeasts and this yeast also showed higher rates of growth and fermentation during the early stages of the fermentation and the presence of non-viable cells at the end of fermentation. In contrast, the flor-veil-forming strains maintained growth and fermentation capabilities for a relatively long time and viable cells were present throughout the entire fermentation process (31 days).The authors are with the Department of Microbiology, Faculty of Sciences, University of Cordoba, Avda. San Alberto Magno s/n, 14004-Córdoba, Spain     

Reidentification of facultatively alkaliphilic Bacillus firmus OF4 as Bacillus pseudofirmus OF4

With a view toward verifying the original classification of alkaliphilic Bacillus firmus OF4, physiological and biochemical characteristics were more extensively catalogued than in original studies, and this catalog was supplemented with 16S rDNA sequence homology and more extensive DNA–DNA hybridization analyses. Phylogenetic analysis of this alkaliphile based on the comparison of multiple 16S rDNA sequences from Bacillus species indicated that this strain is most closely related to Bacillus pseudofirmus. Consistently, in the DNA–DNA hybridization analysis of the alkaliphile and Bacillus reference strains, the highest level of DNA–DNA relatedness (96%) was found between the alkaliphile and the B. pseudofirmus type strain (DSM 8715^T). The findings support the conclusion that this alkaliphile strain is more closely related to B. pseudofirmus than to B. firmus, and we propose the future use of the designation B. pseudofirmus OF4. Received: April 20, 1999 / Accepted: August 31, 1999     

Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans

Alkaliphilic Bacillus sp. AH-101 was characterized in terms of physiological and biochemical characteristics, and 16S rDNA sequence homology and DNA–DNA hybridization analyses were performed. Phylogenetic analysis of strain AH-101 based on comparison of 16S rDNA sequences revealed that this strain is closely related to Bacillus halodurans. DNA–DNA hybridization of AH-101 and related Bacillus reference strains showed that the highest level of DNA–DNA relatedness (88%) was found between strain AH-101 and the B. halodurans type strain (DSM497). Our findings demonstrate that strain AH-101 is a member of the species B. halodurans. Received: June 10, 1999 / Accepted: August 6, 1999     

Chromosomal location of Lg-FLO1 in bottom-fermenting yeast and the FLO5 locus of industrial yeast

Aims: To determine the chromosomal location and entire sequence of Lg-FLO1, the expression of which causes the flocculation of bottom-fermenting yeast. Methods and Results: Two cosmid clones carrying DNA from a bottom-fermenting yeast chromosome VIII right-arm end were selected by colony hybridization. Sequencing revealed that the clones contained DNA derived from a Saccharomyces cerevisiae type chromosome VIII and a Saccharomyces bayanus type chromosome VIII, both from bottom-fermenting yeast. Conclusions: Lg-FLO1 is located on the S. cerevisiae type chromosome VIII at the same position as the FLO5 gene of the laboratory yeast S. cerevisiae S288c. The unique chromosome VIII structure of bottom-fermenting yeast is conserved among other related strains. FLO5 and Lg-FLO1 promoter sequences are identical except for the presence of three 42 bp repeats in the latter, which are associated with gene activity. Flocculin genes might have been generated by chromosomal recombination at these repeats. Significance and Impact of the Study: This is the first report of the exact chromosomal location and entire sequence of Lg-FLO1. This information will be useful in the brewing industry for the identification of normal bottom-fermenting yeast. Moreover, variations in the FLO5 locus among strains are thought to reflect yeast evolution.     

New Hybrids between Saccharomyces Sensu Stricto Yeast Species Found among Wine and Cider Production Strains

Two yeast isolates, a wine-making yeast first identified as a Mel⁺ strain (ex. S. uvarum) and a cider-making yeast, were characterized for their nuclear and mitochondrial genomes. Electrophoretic karyotyping analyses, restriction fragment length polymorphism maps of PCR-amplified MET2 gene fragments, and the sequence analysis of a part of the two MET2 gene alleles found support the notion that these two strains constitute hybrids between Saccharomyces cerevisiae and Saccharomyces bayanus. The two hybrid strains had completely different restriction patterns of mitochondrial DNA as well as different sequences of the OLI1 gene. The sequence of the OLI1 gene from the wine hybrid strain appeared to be the same as that of the S. cerevisiae gene, whereas the OLI1 gene of the cider hybrid strain is equally divergent from both putative parents, S. bayanus and S. cerevisiae. Some fermentative properties were also examined, and one phenotype was found to reflect the hybrid nature of these two strains. The origin and nature of such hybridization events are discussed.     

DNA relatedness among strains of the sweet potato pathogen Streptomyces ipomoea (Person and Martin 1940) Waksman and Henrici 1948.

DNA relatedness among 28 putative strains of Streptomyces ipomoea from geographically diverse locations and the type strain, NRRL B-12321, was determined spectrophotometrically. The data confirm that these 28 strains are not closely related genetically to the plant-pathogenic species Streptomyces scabies (39% DNA relatedness) or Streptomyces acidiscabies (17% DNA relatedness) or any other major blue-spored Streptomyces species (less than 30% DNA relatedness). Of the 29 strains examined, 4 could be clearly distinguished from S. ipomoea on the basis of morphological criteria, i.e., they had gray rather than blue spores and produced melanin pigment, and their low DNA relatedness to authentic S. ipomoea strains confirmed their original misidentification. The remaining 25 S. ipomoea strains exhibited high DNA relatedness among themselves (76 to 100% homology), even though they had been isolated in different locations throughout the United States and Japan. The avirulent type strain, NRRL B-12321, exhibited slightly lower DNA relatedness with the virulent strains of S. ipomoea (85% average DNA relatedness) than was observed among the virulent strains (average of 96% DNA relatedness).     

DNA-DNA homology studies among strains ofHaloferax and other halobacteria

Deoxyribonucleic acid (DNA)-DNA hybridization was used to determine the relationships of the type strains of the three species of the genusHaloferax to 21 halobacterial strains representing isolates from hypersaline habitats and collection strains. Two genotypic groups with high DNA homology were obtained, belonging to the speciesHaloferax gibbonsii andHaloferax mediterranei. The highest DNA relatedness (74%) was obtained in theHalobacterium salinarium group.Halobacterium denitrificans showed very low DNA homology with the other halobacteria investigated. The G+C content of the eight strains ofHaloferax tested ranged between 59.1 and 65.5 mol%.     

DNA relatedness among strains of the sweet potato pathogen Streptomyces ipomoea (Person and Martin 1940) Waksman and Henrici 1948.

DNA relatedness among 28 putative strains of Streptomyces ipomoea from geographically diverse locations and the type strain, NRRL B-12321, was determined spectrophotometrically. The data confirm that these 28 strains are not closely related genetically to the plant-pathogenic species Streptomyces scabies (39% DNA relatedness) or Streptomyces acidiscabies (17% DNA relatedness) or any other major blue-spored Streptomyces species (less than 30% DNA relatedness). Of the 29 strains examined, 4 could be clearly distinguished from S. ipomoea on the basis of morphological criteria, i.e., they had gray rather than blue spores and produced melanin pigment, and their low DNA relatedness to authentic S. ipomoea strains confirmed their original misidentification. The remaining 25 S. ipomoea strains exhibited high DNA relatedness among themselves (76 to 100% homology), even though they had been isolated in different locations throughout the United States and Japan. The avirulent type strain, NRRL B-12321, exhibited slightly lower DNA relatedness with the virulent strains of S. ipomoea (85% average DNA relatedness) than was observed among the virulent strains (average of 96% DNA relatedness).     

Differentiation of ruminal and human Streptococcus bovis strains by DNA homology and 16s rRNA probes

Streptococcus bovis is commonly present in the rumen, but strains of S. bovis have also occasionally been isolated from human blood or fecal samples. Studies were undertaken with 16s rRNA gene sequences and DNA hybridizations to define the genetic relationships between these two groups of strains. Ruminal strains were found to yield genomic DNA restriction endonuclease digest patterns different from human strains when either the 16s rRNA gene amplified from ruminal S. bovis strain JB1 or a conserved universal 23s rRNA fragment was used as probes. A DNA probe based on the V1 region of the 16s rRNA of S. bovis JB1 was found to hybridize to DNAs of other ruminal S. bovis strains K27FF4, 21-09-6C, five new ruminal isolates, and weak hybridization was found with DNAs from S. bovis 33317 (type strain), S. equinus 9812, and six other ruminal isolates. No hybridization occurred with strains representing different major human biotypes/homology groups (43143, 43144, 27960, V1387). All ruminal S. bovis strains had a guanosine plus cytosine DNA content of 37.4–38.8 mol% and, based on DNA-DNA genomic hybridizations, could be separated into two homology groups, one of which included S. equinus 9812 and S. bovis 33317. Both ruminal groups had less than 38% DNA homology to the human strains, indicating ruminal strains are clearly two separate species distinct from the human strains.     

Centromeric DNA from Saccharomyces uvarum is functional in Saccharomyces cerevisiae

Previous comparisons of centromeric DNA sequences in laboratory strains of Saccharomyces cerevisiae have revealed conserved sequences within 120 base pairs (bp) which appear to be essential for centromere function. We wanted to find out whether centromeric DNA in Saccharomyces strains with different degrees of DNA sequence divergence carry the same conserved sequences or not. Bam HI DNA fragments from two S. cerevisiae strains and one Saccharomyces uvarum strain were cloned into a centromere selection vector and tested for centromere function in a S. cerevisiae laboratory strain. Fragments having centromere function were obtained at approximately equal frequencies from all three strains. Two of the S. uvarum centromeric DNAs and two of the S. cerevisiae centromeric DNAs were sequenced and shown to carry in a 120 bp region sequences essentially like those of centromeric DNA in S. cerevisiae laboratory strains. DNA hybridization to separated chromosomal DNAs revealed that the two newly determined S. cerevisiae centromeric DNA sequences belong to chromosomes V and XIII, respectively. On leave from: Department of Cell and Tumor Biology, Roswell Park Memorial Institute, Buffalo, NY 14263, USA; On leave from: The Biological Laboratories, University of Leiden, The Netherlands     

Production of Hormoconis resinae glucoamylase P by a stable industrial strain of Saccharomyces cerevisiae

A stable strain of Saccharomyces cerevisiae secreting glucoamylase (EC 3.2.1.3) with high debranching activity was constructed using recombinant DNA technology. An expression cassette without bacterial sequences, containing Hormoconis resinae glucoamylase P cDNA and the dominant selection marker MEL1 was integrated into the yeast chromosome using ARS1 homology. The glucoamylase expression level of the integrant yeast strain was increased by chemical mutagenesis. The yeast strains secreting glucoamylase were able to grow on soluble starch (5%, w/v) and ferment it to ethanol.Correspondence to: A. Vainio     

Whole Genome Phage Display Selects for Proline-rich Boi Polypeptides against Bem1p

Interaction selection by biopanning from a fragmented yeast proteome displayed on filamentous phage particles was successful in identifying proline-rich fragments of Boi1p and Boi2p. These proteins bind to the second ``src homology region 3' (SH3) domain of Bem1p, a protein of Saccharomyces cerevisiae involved in bud formation. Target Bem1p was a doubly-tagged recombinant, Bem1_{[Asn142-Ile551]}, which strongly interacts in ELISA with a C-terminal 75 amino acids polypeptide from Cdc24p exposed on phage. The whole yeast genomic display library contained ~7.7 × 10⁷ independent clones of sheared S. cerevisiae genomic DNA fused to a truncated M13 gene III. This study corroborates the value of fragmented-proteome display to identify strong and direct interacting protein modules.     

MARINICHLORELLA KAISTIAE GEN. ET SP. NOV. (TREBOUXIOPHYCEAE,CHLOROPHYTA) BASED ON POLYPHASIC TAXONOMY1

We describe three coccoid green algal strains belonging to a new genus and species, Marinichlorella kaistiae Z. Aslam, W. Shin, M. K. Kim, W.‐T. Im et S.‐T. Lee, in seawater samples from the South Sea of Korea. These strains were maintained at 25°C–30°C under a 12:12 light:dark (L:D) photoregime in an ASN‐III medium at a pH of 7.5. These strains were tolerant of high salinity (7.5% NaCl) (w/v) and temperature (40°C). Molecular phylogenetic analyses using 18S rRNA gene sequence data resolved these organisms to a clade separate from green coccoid algae with similar morphology. The DNA–DNA hybridization results demonstrated very low relatedness of these organisms to phylogenetically related species of the genera Chlorella and Parachlorella. The molar guanine + cytosine content (G + C mol%) of the genomic DNA of these organisms ranged from 64.7 to 69.1 mol%. Based on molecular phylogeny, DNA–DNA hybridization, and other morphological studies, we propose a new taxon, Marinichlorella kaistiae, to describe these strains and classify them in the family Chlorellaceae. The type strain is KAS007^T (= KCTC AG10303^T = IAM C‐620^T).     

<Emphasis Type="Italic">Bacillus rhizosphaerae</Emphasis> sp. nov., an novel diazotrophic bacterium isolated from sugarcane rhizosphere soil

A Gram-positive, non-pigmented, rod-shaped, diazotrophic bacterial strain, designated SC-N012^T, was isolated from rhizosphere soil of sugarcane and was subjected to a polyphasic taxonomic study. The strain exhibited phenotypic properties that included chemotaxonomic characteristics consistent with its classification in the genus Bacillus. Sequence analysis of the 16S rRNA gene of SC-N012^T revealed the closest match (98.9% pair wise similarity) with Bacillus clausii DSM 8716^T. However, DNA–DNA hybridization experiments indicated low levels of genomic relatedness (32%) with this strain. The major components of the fatty acid profile are iso-C_15:0, anteiso-C_15:0, iso-C_17:0 and anteiso-C_17:0. The diagnostic cell-wall diamino acid was meso-diaminopimelic acid. The G+C content of the genomic DNA is 43.0 mol%. The lipids present in strain SC-N012^T are diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol and two unknown phospholipids. Their predominant respiratory quinone was MK-7. Studies of DNA-DNA relatedness, morphological, physiological and chemotaxonomic analyses and phylogenetic data based on 16S rRNA gene sequencing allowed strain SC-N012^T to be described as members of novel species of the genus Bacillus, for which the name Bacillus rhizosphaerae sp. nov. is proposed. The type strain is SC-N012^T (=DSM 21911^T = NCCB 100267^T).     

Paenibacillus sinensis sp. nov., a nitrogen-fixing species isolated from plant rhizospheres

Two strains HN-1^T and 39 were isolated from rhizospheres of different plants grown in different regions of PR China. The two strains exhibited high nitrogenase activities and possessed almost identical 16S rRNA gene sequences. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between the two strains were 99.9 and 99.8%, respectively, suggesting that they belong to one species. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strains HN-1^T and 39 are the members of the genus Paenibacillus and both strains exhibited 99.5% similarity to Paenibacillus stellifer DSM 14472^T and the both strains represented a separate lineage from all other Paenibacillus species. However, the ANI of type strain HN-1^T with P. stellifer DSM 14472^T was 90.69, which was below the recommended threshold value (<?95–96% ANI). The dDDH showed 42.1% relatedness between strain HN-1^T and P. stellifer DSM 14472^T, which was lower than the recommended threshold value (dDDH?<?70%). The strain HN-1^T contain anteiso-C_15:0 as major fatty acids and MK-7 as predominant isoprenoid quinone. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four aminophospholipids and an unidentified glycolipid. Unlike the most closely related P. stellifer DSM 14472^T, strain HN-1^T or 39 was positive for catalase reaction. Distinct phenotypic and genomic characterisations from previously described taxa support the classification of strains HN-1^T or 39 as representatives of a novel species of the genus Paenibacillus, for which the name Paenibacillus sinensis is proposed, with type strains HN-1^T (=CGMCC 1.18902, JCM 34,620), and reference strain 39 (=CGMCC 1.18879, JCM 34,616), respectively.

     

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.     

Nonsense suppression in Schizosaccharomyces pombe: the S. pombe Sup3-e tRNASerUGA gene is active in S. cerevisiae

Summary The gene encoding the efficient UGA suppressor sup3-e of Schizosaccharomyces pombe was isolated by in vivo transformation of Saccharomyces cerevisiae UGA mutants with S. pombe sup3-e DNA. DNA from a clone bank of EcoRI fragments from a S. pombe sup3-e strain in the hybrid yeast vector YRp17 was used to transform the S. cerevisiae multiple auxotroph his4-260 leu2-2 trp1-1 to prototrophy. Transformants were isolated at a low frequency; they lost the ability to grow in minimal medium after passaging in non-selective media. This suggested the presence of the suppressor gene on the non-integrative plasmid. Plasmid DNA, isolated from the transformed S. cerevisiae cells and subsequently amplified in E. coli, transformed S. cerevisiae his4-260 leu2-2 trp1-1 to prototrophy. In this way a 2.4 kb S. pombe DNA fragment carrying the sup3-e gene was isolated. Sequence analysis revealed the presence of two tRNA coding regions separated by a spacer of only seven nucleotides. The sup3-e tRNA _Ser ^UGA tRNA gene is followed by a sequence coding for the initiator tRNA^Met. The transformation results demonstrate that the cloned S. pombe UGA suppressor is active in S. cerevisiae UGA mutant strains.     

Deoxyribonucleic acid relatedness among strains of the moderately halophilic speciesDeleya halophila

The genomic relatedness among 16 strains assigned to the moderately halophilic speciesDeleya halophila and other 20 representative strains of halophilic and nonhalophilic species was estimated by determination of deoxyribonucleic acid (DNA) base composition and by DNA-DNA hybridization studies. The guanine-plus-cytosine (G + C) base contents, determined from the melting temperature of DNAs ofD. halophila strains, were 66.0–68.8 mol %. DNA-DNA homology studies, determined by membrane filter technique, indicate that the 16 strains ofD. halophila comprise a genetically homogeneous group. High homology (70–100%) was obtained between the type strainD. halophila CCM 3662 and the otherD. halophila strains studied; however, very low DNA relatedness was found between the representative strains ofD. halophila and otherDeleya species (13-0%), as well as other moderately halophilic, marine, or nonhalophilic bacteria investigated.