A comparison of the 16S rRNA gene (rDNA) sequences of seven type strains belonging to different Alicyclobacillus species (i.e., five validated species, one proposed species and one genomic species) suggested that the 5′ end hypervariable region (259–273 bases in length) of 16S rDNA was specific for the respective type strains. Further phylogenetic analysis based on DNA sequences of the hypervariable region using 24 Alicyclobacillus strains revealed that the strains could be categorized into five species and the A. acidocaldarius-Alicyclobacillus genomic species 1 group. The hypervariable region was highly conserved among the five species: A. acidiphilus, A. acidoterrestris, A. cycloheptanicus, A. herbarius, and A. hesperidum. The strains in the A. acidocaldarius-Alicyclobacillus genomic species 1 group were subdivided into two clusters (Clusters I and II) based on DNA sequences in the hypervariable region. On the basis of phenotypic characteristics, chemotaxonomic and phylogenetic analyses, and DNA-DNA hybridization data, strains in Cluster I were grouped as Alicyclobacillus genomic species 1 and strains in Cluster II were re-identified as A. acidocaldarius, thereby demonstrating that the hypervariable regions were also highly conserved within these two species. These results suggest that as is the case with Bacillus, the hypervariable region is significantly species-specific in the genus Alicyclobacillus to distinguish Alicyclobacillus species by DNA sequence comparison of the hypervariable region.
Bacterial diversity among marine creatures, especially molluscs, as a source for searching out novel lineages of bacteria, was studied. Marine creatures were collected at the coasts of the Kanto area in Japan. A total of 116 strains of bacteria were isolated from the intestines of 19 species of marine creatures includings molluscs, pisces and protochordata. Partial sequencing of 16S rDNA revealed that most of the isolates belonged to the gamma subclass of the Proteobacteria and Cytophaga-Flavobacterium-Bacteroides group. The BLAST searches revealed that the complete 16S rDNA sequence of 17 strains out of 116 isolates showed less than 94% similarity with 16S rDNA sequences deposited in the database. Four strains out of the 17 isolates belonged to the Rhodobacter group, 8 strains to the Alteromonas group, and the remaining 5 strains to the Cytophaga-Flavobacterium-Bacteroides group. Phylogenetic positions of 6 strains belonging to the Alteromonas group, which were isolated from different marine creatures, were close to each other, and represented a novel 16S rDNA lineage within the gamma subclass of Proteobacteria. Therefore, it may be inferred that these 6 strains belong to a new genus of Proteobacteria. Phylogenetic positions of the other strains are also independent from neighboring taxa, and they were suggested to respectively form a novel lineage. From these results, it is clear that the biodiversity of bacteria in marine creatures is much wider than was previously thought, and unknown microbiological resources are buried in these organisms.
The aim of the present study was to perform a fast pre-selection from a great number of wine yeasts using a simple phenotypic-based methodology that allows many different strains to be simultaneously tested. A total of 150 elliptic yeasts, isolated from must and wine from black grapes of a distinctive Italian variety, were studied. Yeasts were identified to genus level by assessing their ability to ferment glucose and their production of spores on acetate agar. The Saccharomyces strains were seeded on BiGGY agar to determine their H2S production, on calcium carbonate agar to test their acetic acid production, and on grape-skin agar and on grape-seed agar to assess their interaction with phenolic compounds. The Saccharomyces strains were also examined for fermentative vigor after 2 d or 7 d both with and without the addition of 100 mg L−1 of SO2 in must at 20° brix and pH 3.20. At the end of fermentation, the wines produced by the 18 best yeasts were analyzed and the strains were studied for additional biochemical and technological characteristics. The resistance of the strains to simultaneous acid-stress and osmotic-stress was studied carrying out in duplicate winemaking tests in must at 30° brix and pH 2.60. A remarkable heterogeneity among the 150 autochthonous yeasts studied was demonstrated. The phenotypical biodiversity is particularly interesting for several technological characteristics useful in winemaking, such as fermentation vigor, acetic acid production and malic acid content of the wines. The vast majority of the elliptic wine yeasts isolated did not show suitable characteristics, so only 18 strains, 12% of the total, remained for the final tests. Many of the strains that had passed the preliminary screenings revealed some defects when they were studied for fermentation performance, both in standard winemaking and under stressors. Two strains exhibited particularly interesting performances: one strain for winemaking of normal musts and the other for winemaking of musts from dried grapes or under stressful conditions.
A slightly halophilic, extremely halotolerant, alkaliphilic, and facultatively anaerobic rod bacterium was isolated from a decomposing marine alga collected in Okinawa, Japan. The isolate, designated O15-7T, was Gram-positive, endospore-forming, catalase-positive, menaquinone-7-possessing bacterium that is motile by peritrichous flagella. The isolate was an inhabitant of marine environments; the optimum NaCl concentration for growth was 0.75–3.0% (w/v) with a range of 0–22.0%, and the optimum pH was 7.0–8.5 with a range of 5.5–9.5. Catalase was produced in aerobic cultivation but not in anaerobic cultivation. Carbohydrate, sugar alcohol or a related carbon compound was required for growth. In aerobic cultivation, the isolate produced pyruvate, acetate and CO2 from glucose, and in anaerobic cultivation, it produced lactate, formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1 for the last three products. No gas was produced anaerobically. Lactate yield per consumed glucose was markedly affected by the pH of the fermentation medium: 51% at pH 6.5 and 8% at pH 9.0. The cell-wall peptidoglycan contained meso-diaminopimelic acid. Phylogenetically, the isolate occupied an independent lineage within the group composed of the halophilic/halotolerant/alkaliphilic and/or alkalitolerant species in Bacillus rRNA group 1 with the highest 16S rRNA gene sequence similarity of 95.2% to the genus Gracilibacillus. For this isolate, Paraliobacillus ryukyuensis gen. nov., sp. nov. was proposed. The type strain, O15-7T (G+C=35.6 mol%), has been deposited in the DSMZ, IAM, NBRC, and NRIC (DSM 15140T=IAM 15001T=NBRC 10001T=NRIC 0520T).