No abstract

Twenty-one strains of Saccharomonospora azurea, Saccharomonospora cyanea, Saccharomonospora glauca, Saccharomonospora viridis, and "Saccharomonospora caesia" were examined to evaluate the discriminatory value of 16s ribosomal DNA (rDNA) fingerprints. The 16s rDNAs were amplified by PCR by using oligonucleotide primers complementary to 16s rRNA genes. A restriction fragment length polymorphism (RFLP) analysis of the 16s rDNAs was performed with SmaI and MZuI. The four validly described Saccharomonospora species could be differentiated on the basis of their characteristic 16s rDNA restriction patterns. The strains of "S. caesia" gave a restriction pattern identical to that of S. azurea K161T (T = type strain). This result was anticipated from the previous report that S. azurea K161T and the strains of "S. caesia" have identical 16s rRNA sequences. We found that purification of amplified 16s rDNA products following PCR was necessary for our RFLP analysis.

In order to clarify interspecific relationships and to investigate the intraspecific phylogenetic structure of the genus Saccharomonospora, 16s to 23s ribosomal DNA (16s-23s) and 23s to 5s ribosomal DNA (23s-5s) internally transcribed spacers (ITSs) were used for sequence analyses. The 16s-23s and 23s-5s ITSs from 22 Saccharomonospora strains were amplified by PCR and directly sequenced. The average levels of nucleotide similarity of the 16s-23s and 23s-5s ITSs for the four valid species were 87.6% f 3.9% and 83% +-2.2%, respectively. For the most part, intraspecific sequence differences were not found in the two ITSs; the only exception was Saccharomonospora ghuca K194, which differed from other S. ghuca strains by 1 bp in the 23s-5s ITS. The Sacchuromonospora viridis strains had a smaller 16s-23s ITS region than the other strains, which may be useful for differentiating these organisms from other Saccharomonospora species. The characteristics of the two ITS regions make them more useful than 16s rRNA sequences as a tool for defining and identifying Saccharomonospora strains. However, Sacchuromonospora azurea K161T had two types of 23s-5s ITSs; rrnB, separated by XhoI digestion, had two additional nucleotides inserted between positions 52 and 55. Most of the 16s-23s and 23s-5s ITS sequences of S. azurea K161T and strains of "Saccharomonospora caesia" were identical; the only exception was rrnB in S. azurea K161T. The lengths and levels of sequence divergence of the two ITSs of Saccharomonospora sp. strain KlSO were different from the lengths and levels of sequence divergence of the ITSs of other species. These findings suggest that a taxonomic revision of the genus Saccharomonospora is necessary. Two trees based on 16s-23s and 23s-5s ITS sequences revealed distinct interspecific relationships in the genus Saccharomonospora.The genus Saccharomonospora was proposed by Nonomura and Ohara (20) for monosporic actinomycetes containing meso-diaminopimelic acid, arabinose, and galactose in the cell wall peptidoglycan (wall chemotype IV sensu Lechevalier and Lechevalier [ 181). Representatives of this genus form nonfragmenting, branched mycelia and develop aerial hyphae that bear single spores. The additional physical and chemical markers that characterize the genus have been described elsewhere The original type species of the genus is Saccharomonospora viridis (20). In addition to S. viridis, three Saccharomonospora species, Saccharomonospora azurea (24), Saccharomonospora cyanea (25), and Saccharomonospora glauca (7), have been validly described. Although "Saccharomonospora caesia" was proposed by Greiner-Mai et al. (8) as a fifth taxon for strains previously classified as Micropolyspora caesia (9, 13), this organism was not included on the Approved Lists of Bacterial Names (28) and has not been validly published on subsequent Approved Lists or Validation Lists. The results of recent numerical phenetic studies (11) and results obtained by nucleic acid techniques (12,(39)(40)(41) suggest that the strains of "S. caesi...

Nearly complete sequences of 16s rRNA genes of representative strains of the genus Saccharomonospora were determined following the isolation and cloning of the amplified genes. The sequences were aligned with those of representatives of the family Pseudonocardiaceae, and a phylogenetic tree was inferred by the neighborjoining method. The genus Saccharomonospora formed a distinct clade within the evolutionary radiation encompassed by the family Pseudonocardiaceae. The average nucleotide similarity value found between the type strains of the four validly described Saccharomonospora species was 97.5% k 1.0%. The most distant relationship was found between Saccharomonospora azurea and Saccharomonospora viridis K73 (96.3% similarity). In contrast, Sacchuromonospora azurea K161 and "Saccharomonospora caesia" K163 had identical 16s rRNA gene sequences. The nucleotide sequence data suggest that the genus Saccharomonospora contains several new centers of variation.The genus Saccharomonospora was proposed by Nonomura and Ohara (30) for monosporic actinomycetes that contained meso-diaminopimelic acid, arabinose, and galactose in the peptidoglycan (wall chemotype IV sensu Lechevalier Lechevalier [25]). Subsequently, additional chemical markers were found to characterize the taxon, notably the presence of major amounts of iso-and anteiso-fatty acids, 2-hydroxy fatty acids, phosphatidylethanolamine, and menaquinones that were tetrahydrogenated with nine isoprene units (21,25).There are currently four validly described Saccharomonospora species, namely, Saccharomonospora azurea (32), Saccharomonospora cyanea (33), Saccharomonospora glauca (17), and Saccharomonospora viridis (30), the type species of the genus. A fifth species, "Saccharomonospora caesia," was proposed by for organisms previously classified as Micropolyspora caesia (20,22); this organism did not appear in the Approved Lists of Bacterial Names (35) and has not been added subsequently, and hence, it has not been validly published. It is important to distinguish between members of validly described and putatively new Saccharomonospora species, as some kinds of saccharomonosporae can cause hypersensitivity pneumonitis. S. viridis is strongly implicated as one the causal agents of farmer's lung disease (3,16).It is also important to determine the taxonomic structure of the genus Saccharomonospora in order to discover the relationships between the existing taxa and between them and prospective new species. It appears from preliminary 16s rRNA sequence studies that the genus Saccharomonospora forms a distinct phyletic line within the evolutionary radiation encompassed by the family Pseudonocardiaceae (5, 9, 10, 28, 36). To date, three Saccharomonospora strains have been the subject of 16s rRNA sequencing analyses, the type strain of S. viridis, the putative type strain of "S. caesia," and an isolate from stored grain, Saccharomonospora sp. strain A1206, that was tentatively considered, subject to additional sequencing studies, to be the nucleus of a new species (36). ...