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An isolation strategy, exploring novel microorganisms in frozen enrichment cultures (ENFE), which uses a combination of enrichment culture and 16S rRNA gene clone analysis, was evaluated for isolating uncultured thermophiles from a terrestrial acidic hot spring. The procedure comprised (a) multiple enrichment cultures under various conditions, (b) cryostorage of all enrichments, (c) microbial community analyses of the enrichments using 16S rRNA gene sequences, and (d) purification of microorganisms from enrichments containing previously uncultured microorganisms. The enrichments were performed under a total of 36 conditions, and 16 of these enrichments yielded positive microbial growth with the detection of three previously uncultured archaea. Two of the three previously uncultured archaea, strains HS-1 and HS-3, were successfully isolated. Strain HS-1 and HS-3 represented a novel lineage of the order Sulfolobales and novel species of the genus Sulfolobus, respectively. Although innovative isolation methods play strategic roles in isolating previously uncultured microorganisms, the ENFE strategy showed potential for characterizing and isolating such microorganisms using conventional media and techniques.

A spherical thermoacidophilic archaeon, strain TA-2, was obtained from acidic hot springs located in Ohwaku Valley, Hakone, Japan. This isolate is an obligate aerobic chemoorganoheterotroph that grows optimally at about 75 degrees C, pH 2.8. The G + C content of DNA from TA-2 is 47 mol%. The 16S rRNA gene from TA-2 showed more than 99% similarity with those of Metallosphaera sedula and Metallosphaera prunae and less than 92% similarity with other members of the order Sulfolobales. DNA-DNA hybridization experiments showed more than 93% genomic DNA homology among TA-2, M. sedula DSM5348T, and M. prunae DSM10039T. However, TA-2 lacks calditoglycerocaldarchaeol derivatives, which are usually found in the membrane lipids of members of the order Sulfolobales. Therefore, calditoglycerocaldarchaeol may not be essential for survival in thermophilic and acidophilic environments. The isolate was deposited as Metallosphaera sedula TA-2 (JCM 9064, IFO 15160).

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27 kDa) and 248 aa (27 kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages.