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Two novel strains of halophilic archaea, DX253T and GY252, were isolated from Zodletone Spring, a low-salt, sulfide- and sulfur-rich spring in south-western Oklahoma, USA. The cells were cocci or coccobacilli and occurred singly or in pairs. The two strains grew in a wide range of salt concentrations (0.8–5.1 M) and required at least 5 mM Mg2+ for growth. The pH range for growth was 5–7.5 and the temperature range was 25–45 °C. In addition to having the capacity to grow at relatively low salt concentrations, cells remained viable in distilled water after prolonged incubation. The two diether phospholipids that are typical of members of the order Halobacteriales, phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, were present. Phosphatidylglycerol sulfate and two unidentified glycolipids were also detected. Each strain had two distinct 16S rRNA gene sequences that were only 89.5–90.8 % similar to sequences from the most closely related cultured and recognized species within the order Halobacteriales. The DNA G+C content of the type strain was found to be 60.5 mol%. The closest relatives were clones and uncharacterized isolates obtained from coastal salt-marsh sediments with salinities equivalent to that of seawater. The physiological, biochemical and phylogenetic differences between strains DX253T and GY252 and other previously described genera of extremely halophilic archaea suggest that these novel strains represent a novel species and genus within the family Halobacteriaceae, for which the name Haladaptatus paucihalophilus gen. nov., sp. nov. is proposed. The type strain is DX253T (=JCM 13897T=DSM 18195T=ATCC BAA-1313T=KCTC 4006T).

We evaluated the ability of the native microbiota in a low-temperature, sulfidic natural hydrocarbon seep (Zodletone) to metabolize short-chain hydrocarbons. n-Propane and n-pentane were metabolized under sulfate-reducing conditions in initial enrichments and in sediment-free subcultures. Carbon isotope analysis of residual propane in active enrichments showed that propane became enriched in (13)C by 6.7 (+/-2.0) per thousand, indicating a biological mechanism for propane loss. The detection of n-propylsuccinic and isopropylsuccinic acids in active propane-degrading enrichments provided evidence for anaerobic biodegradation via a fumarate addition pathway. A eubacterial 16S rRNA gene survey of sediment-free enrichments showed that the majority of the sequenced clones were phylogenetically affiliated within the Deltaproteobacteria. Such sequences were most closely affiliated with clones retrieved from hydrocarbon-impacted marine ecosystems, volatile fatty acid metabolizers, hydrogen users, and with a novel Deltaproteobacterial lineage. Other cloned sequences were affiliated with the Firmicutes and Chloroflexi phyla. The sequenced clones were only distantly (<95%) related to other reported low-molecular-weight alkane-degrading sulfate-reducing populations. This work documents the potential for anaerobic short-chain n-alkane metabolism for the first time in a terrestrial environment, provides evidence for a fumarate addition mechanism for n-propane activation under these conditions, and reveals microbial community members present in such enrichments.

Halosarcina pallida gen. nov., sp. nov., a halophilic archaeon from a low-salt, sulfide-rich spring A novel halophilic archaeon, strain BZ256 T , was isolated from Zodletone Spring, a sulfide-and sulfur-rich spring in south-western Oklahoma, USA. Cells were non-motile, non-flagellated cocci that divided along two axes, resulting in the formation of sarcina-like clusters. Strain BZ256 T grew at salt concentrations ranging from 1.3 to 4.3 M NaCl, with optimum growth at approximately 3.4 M, and required at least 1 mM Mg 2+ for growth. The pH range for growth was 5.0 to at least 8.5, and the temperature range for growth was 25-45 6C. The two diether phospholipids that are typical of members of the order Halobacteriales, namely phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, were present in strain BZ256 T , as were two glycolipids chromatographically identical to S-DGD-1 and DGD-1. The 16S rRNA gene sequence of strain BZ256 T showed 96.8 % similarity to that of the type strain of Halogeometricum borinquense, the closest recognized species within the order Halobacteriales. The DNA G+C content of strain BZ256 T was 65.4 mol%. Microscopic, physiological, biochemical and phylogenetic comparisons between strain BZ256 T and recognized genera of extremely halophilic archaea suggest that this strain represents a member of a novel genus and species within the family Halobacteriaceae, for which the name Halosarcina pallida gen. nov., sp. nov. is proposed. The type strain of Halosarcina pallida is BZ256 T (5KCTC 4017 T 5JCM 14848 T ).Members of the family Halobacteriaceae, the single family recognized within the order Halobacteriales, have long been identified as the most abundant micro-organisms in hypersaline environments (Oren, 1994). At the time of writing, the family Halobacteriaceae comprised 25 recognized genera and 101 recognized species. Recently, the extent of the family has been undergoing rapid expansion, with the description (as of June 2007) of five new genera and 20 novel species since 2006. This expansion has been due not only to the identification of novel taxa isolated from well-known hypersaline environments, such as the genera Halovivax, Halostagnicola, Haloplanus and Haloquadratum (Castillo et al., 2006a, b;Bardavid et al., 2007;Burns et al., 2007), but also to the recognition that members of the Halobacteriales can grow within saline microniches in non-saline environments at relatively low salt concentrations (Elshahed et al., 2004a;Savage et al., 2007). In addition, recent studies have reported the isolation of novel halobacterial strains (Purdy et al., 2004;Fukushima et al., 2007) or the detection of novel Halobacteriales-affiliated 16S rRNA gene sequences from moderate-to low-salinity systems (Munson et al., 1997;Takai et al., 2001;Walsh et al., 2005). These studies have shown that the order Halobacteriales is more diverse than previously believed. In the present study, we report on the isolation and characterization of strain BZ256 T from a sulfur-and sulfide-rich spring in south-...

A pleomorphic, extremely halophilic archaeon (strain M6 T ) was isolated from a sulfide-and sulfur-rich spring in south-western Oklahoma (USA). It formed small (0?8-1?0 mm), salmon pink, elevated colonies on agar medium. The strain grew in a wide range of NaCl concentrations (6 % to saturation) and required at least 1 mM Mg 2+ for growth. Strain M6 T was able to reduce sulfur to sulfide anaerobically. 16S rRNA gene sequence analysis indicated that strain M6 T belongs to the family Halobacteriaceae, genus Haloferax; it showed 96?7-98?0 % similarity to other members of the genus with validly published names and 89 % similarity to Halogeometricum borinquense, its closest relative outside the genus Haloferax. Polar lipid analysis and DNA G+C content further supported placement of strain M6 T in the genus Haloferax. DNA-DNA hybridization values, as well as biochemical and physiological characterization, allowed strain M6 T to be differentiated from other members of the genus Haloferax. A novel species, Haloferax sulfurifontis sp. nov., is therefore proposed to accommodate the strain. The type strain is M6 T