Russell H. Vreeland

Isolation of a 250 million-year-old halotolerant bacterium from a primarysalt crystal

Bacteria have been found associated with a variety of ancient samples, however few studies are generally accepted due to questions about sample quality and contamination. When Cano and Borucki isolated a strain of Bacillus sphaericus from an extinct bee trapped in 25–30 million-year-old amber, careful sample selection and stringent sterilization techniques were the keys to acceptance. Here we report the isolation and growth of a previously unrecognized spore-forming bacterium (Bacillus species, designated 2-9-3) from a brine inclusion within a 250 million-year-old salt crystal from the Permian Salado Formation. Complete gene sequences of the 16S ribosomal DNA show that the organism is part of the lineage of Bacillus marismortui and Virgibacillus pantothenticus. Delicate crystal structures and sedimentary features indicate the salt has not recrystallized since formation. Samples were rejected if brine inclusions showed physical signs of possible contamination. Surfaces of salt crystal samples were sterilized with strong alkali and acid before extracting brines from inclusions. Sterilization procedures reduce the probability of contamination to less than 1 in 10 9.

Halomonas elongata, a New Genus and Species of Extremely Salt-Tolerant Bacteria

The morphological, biochemical, and physiological characteristics of nine bacterial strains isolated from a solar salt facility located on Bonaire, Netherlands Antilles are described. The bacteria were gram-negative rods which produce white, opaque colonies on solid media. During the log phase of growth, the cultures consisted of single and paired cells with polar flagella predominating. Older cultures characteristically produced highly elongated, flexible rods. All of these strains reduced NO3 to NO2, grew anaerobically in the presence of NO3, and fermented glucose but oxidized sucrose, glycerol, mannose, and cellobiose. All strains were ornithine and lysine decarboxylase positive, catalase positive, and cytochrome oxidase negative. Eight of the nine strains grew in a complex Casamino Acids liquid medium containing from 0 to 32% (wt/vol) solar salt at temperatures from 23 to 37°C; the ninth strain was restricted in its growth to 0 to 20% solar salt. The guanine plus cytosine content of the deoxyribonucleic acid was 61 ± 1 mol%. This combination of morphology, salt tolerance, and guanine plus cytosine content supports the establishment of a new genus, Halomonas, in Family II (Vibrionaceae) of part 8, Gram-Negative Facultatively Anaerobic Bacteria, of Bergeys Manual (8th edition). The type species of this genus is H. elongata, the type strain of which is isolate 1H9 (= ATCC 33173). Strain 1H15 is regarded as belonging to a biovar of H. elongata on the basis of its production of lophotrichous cells and its inability to grow at 37°c in the presence of 32% solar salt.

Halosimplex carlsbadense gen. nov., sp. nov., a unique halophilic archaeon, with three 16S rRNA genes, that grows only in defined medium with glycerol and acetate or pyruvate.

Abstract. A halophilic archaeon has been isolated from unsterilized salt crystals taken from the 250-million-year-old Salado formation in southeastern New Mexico. This microorganism grows only on defined media supplemented with either a combination of acetate and glycerol, glycerol and pyruvate, or pyruvate alone. The archaeon is unable to grow on complex media or to use carbohydrates, amino acids, fats, proteins, or nucleic acids for growth. Unlike other halophilic microbes, this organism possesses four glycolipids, two of which may be novel. The microbe is unique in that it has three dissimilar 16S rRNA genes. Two of the three genes show only 97% similarity to one another, while the third gene possesses only 92%–93% similarity to the other two. Inferred phylogenies indicate that the organism belongs to a deep branch in the line of Haloarcula and Halorhabdus. All three lines of taxonomic evidence: phenotype, lipid patterns, and phylogeny, support creation of a new genus and species within the halophilic Archaea. The name suggested for this new genus and species is Halosimplex carlsbadense. The type strain is 2–9-1T (= ATCC BAA-75 and JCM 11222) as written in the formal description.

Halogeometricum borinquense gen. nov., sp. nov., a novel halophilic archaeon from Puerto Rico

A novel extremely halophilic archaeon was isolated from the solar salterns of Cabo Rojo, Puerto Rico. The organism is very pleomorphic, motile and requires at least 8% (w/v) NaCl to grow. Polar lipid composition revealed the presence of a novel non-sulfate-containing glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate. The G + C content of the DNA is 59 mol%. On the basis of 16S rRNA sequence data, the new isolate cannot be classified in one of the recognized genera, but occupies a position that is distantly related to the genus Haloferax. All these features justify the creation of a new genus and a new species for the family Halobacteriaceae, order Halobacteriales. The name Halogeometricum borinquense gen. nov., sp. nov. is proposed. The type strain is ATCC 700274T.

Distribution and diversity of halophilic bacteria in a subsurface salt formation.

Abstract The Waste Isolation Pilot Plant (WIPP) is a salt mine constructed 650 meters below the ground surface by the United States Department of Energy. The facility will be used for permanent disposal of transuranic wastes. This underground repository has been constructed in the geologically stable Permian age Salado salt formation. Of the wastes to be placed into the facility, 85% will be biodegradable cellulose. A 3-year survey of the bacterial populations existing within the facility was conducted. Bacterial populations were found to be heterogeneously distributed throughout the mine. Populations in some mine areas reached as high as 1.0 × 104 colony-forming units per gram of NaCl. The heterogeneous distribution of bacteria within the mine did not follow any recognizable pattern related to either age of the workings or to human activity. A biochemical comparison between ten known species of halophilic bacteria, and strains isolated from both the mine and nearby surface hypersaline lakes, showed the presence of extreme halophiles with wide biochemical diversity, some of which could prove to represent previously undescribed groups. The halophilic bacteria isolated from the mine were found to degrade cellulose and a wide variety of other carbon compounds. When exposed to two types of common laboratory paper, the cellulose-degrading halophiles attached to the substrate within 30 minutes of inoculation. Cultures enriched directly from a brine seep in the mine easily destroyed both papers and produced detectable amounts of oxalacetic and pyruvic acids. The combination of heterogeneity in the distribution of organisms, the presence of a physiologically diverse community, and the relatively slow metabolism of cellulose may explain several long-standing debates about the existence of microorganisms in ancient underground salt formations.

New evidence for 250 Ma age of halotolerant bacterium from a Permian salt crystal

The purported oldest living organism, the spore-forming bacterium Virgibacillus sp. Perm- ian strain 2-9-3, was recently cultured from a brine inclusion in halite of the 250 Ma Permian Salado Formation. However, the antiquity of Virgibacillus sp. 2-9-3 has been chal- lenged; it has been argued that the halite crystal and the fluid inclusion from which the bacterial spores were extracted may be younger than the Permian Salado salts. Here we report that brine inclusions from the same layer of salt that housed Virgibacillus sp. 2-9-3 are composed of evaporated Late Permian seawater that was trapped in halite cement crys- tals precipitated syndepositionally from shallow groundwater brines at temperatures of 17- 37 8C. These results support the 250 Ma age of the fluid inclusions, and by inference, the long-term survivability of microorganisms such as Virgibacillus sp. 2-9-3.

Phylum All. Euryarchaeota phy. nov.

The phylum currently consists of seven classes: the Methanobacteria, the Methanococci, the Halobacteria, the Thermoplasmata, the Thermococci, the Archaeoglobi, and the Methanopyri. With the sole exception of the Methanococci, which is subdivided into three orders, each class contains a single order. The Euryarchaeota are morphologically diverse and occur as rods, cocci, irregular cocci, lancet-shaped, spiral-shaped, disk-shaped, triangular, or square cells. Cells stain Gram-positive or Gram-negative based on the presence or absence of pseudomurein in cell walls. In some classes, cell walls consist entirely of protein or may be completely absent (Thermoplasmata). Five major physiological groups have been described previously: the methanogenic Archaea, the extremely halophilic Archaea, Archaea lacking a cell wall, sulfate reducing Archaea, and the extremely thermophilic S0 metabolizers.

Haloterrigena thermotolerans sp. nov., a halophilic archaeon from Puerto Rico.

An extremely halophilic Archaeon belonging to the order Halobacteriales was isolated from the solar salterns of Cabo Rojo, Puerto Rico. The organism, designated strain PR5T, is rod-shaped, non-motile and requires at least 12% (w/v) NaCl to grow. The strain is highly thermotolerant: its temperature optimum is 50 degrees C and growth is possible up to 60 degrees C. Polar lipid analysis revealed the presence of the bis-sulfated glycolipid S2-DGD-1 as sole glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate. Both C20,C20 and C20,C25 core lipids are present. The G+C content of the DNA is 63.3 mol%. According to 16S rDNA sequence data, strain PR5T is closely related to the representatives of the genera Haloterrigena and Natrinema, but on the basis of its phenotypic properties, 16S rDNA sequence and DNA-DNA hybridization studies, strain PR5T cannot be assigned to any of the recognized species within these genera. On the basis of its polar lipid composition, the isolate has been assigned to the genus Haloterrigena. The creation of a new species, Haloterrigena thermotolerans, is therefore proposed to accommodate this isolate. The type strain is strain PR5T (= DSM 11552T = ATCC 700275T).

Isolation of Live Cretaceous (121–112 Million Years Old) Halophilic Archaea from Primary Salt Crystals

Recent reports have described the isolation and analysis of living microbes and/or DNA fragments from halite crystals of significant geological age. This manuscript describes the isolation of six living strains of halophilic Archaea from Cretaceous (121–112 MYA) halite crystals. These 6 live strains represent the oldest Archaea isolated to date. This manuscript also presents the first isolation of representatives from two different archaeal genera in a single event. The data presented show that the organisms that inhabited these hypersaline environments today are similar to those present during the Cretaceous age. Considering the number of ancient samples that have now yielded living microbes or DNA fragments the evidence for long-term survival of microbes (at least within halite) is becoming increasingly definitive. While there are obviously still other trapped microbes to find, it may now be time to begin investigating the implications of these ancient microbes and the mechanisms that foster long-term survival.

Phenotypic comparison of halotolerant bacteria: Halomonas halodurans sp. nov., nom. rev., comb. nov.

Several strains of halotolerant bacteria from North America were compared for 125 characteristics. Although these organisms had several characteristics in common, they are a relatively heterogeneous group. The 12 strains could be divided into at least three and perhaps as many as five taxa. Halomonas elongata ATCC 33173T (T = type strain) and “Pseudomonas halodurans” ATCC 29686T had 79% phenotypic similarity, indicating that these organisms are probably separate species of the same genus. We conclude that this relationship is sufficient to warrant assignment of “Pseudomonas halodurans” to the genus Halomonas as Halomons halodurans sp. nov., nom rev., comb nov.