The flux of Na+, Cl- and H+ ions and the accumulation of glycerol in cells of a salt-tolerant yeast, Zygosaccharomyces rouxii, was examined during the initial stages of salt stress. Although the intracellular accumulation of glycerol that was required for osmoregulation began immediately after exposure of the cells to salt stress, rapid influx of Na+ and Cl- ions into cells also occurred. These ions were subsequently extruded from the cells and the intracellular levels of both ions were maintained much lower than those in the external medium. Furthermore, a rapid efflux of protons was induced by activation of the plasma membrane ATPase (PM- ATPase) immediately after the start of salt stress and then the influx (re-influx) of protons into cells occurred. The addition of a specific inhibitor of PM-ATPase, diethylstilbestrol (DES), and of inhibitors of the energy-generating system, namely sodium azide, antimycin A and dinitrophenol, inhibited the intracellular accumulation of glycerol and activation of PM-ATPase, and they retarded the rapid efflux of protons and the extrusion of Na+ and Cl- ions. The re-influx of protons into cells was inhibited by amphotericin B, probably because of the destruction of the structure of the plasma membrane. In mutants in which rapid activation of PM-ATPase was not observed immediately after the start of salt stress, the efflux of protons was markedly retarded and extrusion of Na+ and Cl- ions was also suppressed during salt stress. Amiloride, an inhibitor of the Na+/H+-antiporter, inhibited the re-influx of protons and the efflux of Na+ and Cl- ions, but this inhibitor did not inhibit the accumulation of glycerol, activation of PM-ATPase or the rapid efflux of protons. These results suggest that the Na+/H+-antiporter may play a significant role in extrusion of the ions that enter cells during the initial stages of salt stress.
The new strains of aerobic chemoorganotrophic rhodoquinone-containing bacteria previously isolated from activated sludge were studied from taxonomic and phylogenetic viewpoints. These strains were Gram- negative, nonmotile coccobacilli, had a strictly respiratory type of metabolism with oxygen or nitrate as the terminal acceptor, produced catalase and oxidase, and contained both ubiquinone-8 and rhodoquinone-8 as major quinones. DNA-DNA reassociation studies revealed that the new strains were highly related to each other at hybridization levels of more than 74%, suggesting the genetic coherency of the isolates as a single species. The 16S rRNA gene from one of the isolates, strain AS-P1, was amplified in vitro and sequenced directly. Sequence comparisons and a distance matrix tree analysis revealed that strain AS-P1 was most closely related to Comamonas testosteroni, a representative of the beta subclass of the Proteobacteria, but the level of sequence similarity between the two appeared to be low enough to warrant different generic allocations. The strains were differentiated from related organisms by a number of phenotypic and chemotaxonomic properties. Thus, we conclude that the isolates should be placed in a new genus and species of the beta subclass of the Proteobacteria, for which we propose the name Brachymonasdenitrificans. Evolutionary relationships of rhodoquinone producers to bacterial species with various quinone classes were discussed on the basis of 16S rRNA sequence information.
The multicellular filamentous green bacteria, Chloroflexus species, inhabit natural hot springs. For the isolation of these bacteria, a new isolation method was developed using an organic medium and culture under alternating aerobic and anaerobic conditions. The improved method reliably provided a high rate of isolation of the thermophilic photoheterotrophs. Ten strains that may belong to this genus were isolated from Japanese hot springs in various areas. All these new isolates were thermophilic photosynthetic bacteria that were morphologically similar to Chloroflexus aurantiacus. The isolates were also similar to C. aurantiacus in photosynthetic pigments and guanine plus cytosine (G+C) contents. The results of DNA-DNA hybridization and quinone composition analysis indicated that eight of the new strains belong to C. aurantiacus, while two strains (MD-66 and YI-9) which have the ability to form bacterial mat-like dense aggregates, were significantly different from C. aurantiacus.
Small subunit ribosomal RNA gene sequences were determined in nine species of the ballistoconidium-forming yeast genus Bensingtonia. The phylogenetic trees were constructed for the species of the genus Bensingtonia and related taxa containing Sporobolomyces and Bullera species by neighbor joining, maximum parsimony and maximum likelihood methods. The phylogenetic trees showed that the basidiomycetous yeasts were divided into two main clusters, which were correlated well with the presence or absence of xylose in the cells. In the xylose-lacking basidiomycetous yeasts, seven species out of nine of the genus Bensingtonia constituted a distinct cluster. Bensingtonia ciliata, the type species of the genus, was included in this cluster. The remaining two species, B. intermedia and B. yamatoana, were located in the cluster which contained Rhodosporidium toruloides, Sporidiobolus johnsonii, Sporobolomyces roseus and Leucosporidium scottii. Erythrobasidium hasegawianum was distinctly located in the xylose-lacking basidiomycetous cluster. The molecular phylogeny showed clearly that the genus Bensingtonia was not monophyletic.
A new species of ballistoconidium-forming yeast, Bensingtonia musae, was isolated from a dead leaf of Musa paradisiaca collected in the southeast seacoast of Bangkok, Thailand. B. musae showed physiological and biochemical characteristics similar to B. ingoldii and B. intermedia. DNA-DNA reassociation experiments, however, showed that it was distinct from these two species. B. musae is easily distinguished from B. ingoldii by the assimilation of sucrose, cellobiose, lactose, melezitose, soluble starch and nitrate, and from B. intermedia by the assimilation of cellobiose, L-arabinose, erythritol and salicin, and the requirement of p-aminobenzoic acid and pyridoxine. In the phylogenetic tree constructed based on small subunit rRNA gene sequences, B. musae was located at a cluster which was composed of B. ciliata, the type species of the genus, B. ingoldii, B. miscanthi, B. naganoensis, B. phylladus, B. subrosea and B. yuccicola. Among these species, B. musae was the most closely related to B. ingoldii.