William D. Rosenzweig

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.

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.

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.

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.

Haloarchaeal diversity in 23, 121 and 419 MYA salts

DNA was extracted from surface-sterilized salt of different geological ages (23, 121, 419 million years of age, MYA) to investigate haloarchaeal diversity. Only Haloarcula and Halorubrum DNA was found in 23 MYA salt. Older crystals contained unclassified groups and Halobacterium. The older crystals yielded a unique 55-bp insert within the 16S rRNA V2 region. The secondary structure of the V2 region completely differed from that in haloarchaea of modern environments. The DNA demonstrates that unknown haloarchaea and the Halobacterium were key components in ancient hypersaline environments. Halorubrum and Haloarcula appear to be a dominant group in relatively modern hypersaline habitats.

Development of a Protocol to Retrieve Microorganisms from Ancient Salt Crystals

Previous research has indicated that halophilic microorganisms are associated with salt crystals from ancient formations. However,this research has generally failed to convincingly demonstrate that these organisms were originally trapped inside the crystals during their deposition and were not recent surface contaminants. This paper presents techniques for crystal selection; verifiable, noninvasive, surface sterilization of salt crystals; and sterile extraction of biological material from the crystals. Immersing salt crystals in 10 M NaOH for 5 min; rinsing with sterile, saturated brine before immersing for 5 min in 10 M HCl; and rinsing with saturated brine is an effective method for surface sterilization. No growth has resulted from contamination tests of 216 faces from 36 natural salt crystals exposed to these treatments. Pure culture experiments using the halotolerant eubacteria, Halomonas elongata and Bacillus sp. (2-9-3), and the archeon, Halogeometricium borinquense, showed that exposure to either 10 M HCl or NaOH reduced the population of these organisms by a factor of 107 to 108 colony-forming units/ml. The fluid is extracted from inclusions by drilling into surface-sterilized salt crystals with a variable-speed drill using sterilized 0.5-mm-diameter carbide drill bits and extracting the fluid with a sterilized microliter syringe. Drilling and extraction are performed in a Class II biosafety cabinet. All procedures are carried out in a biosafety level 3 facility. All personnel involved wear cleanroom coveralls, shoe covers, hair caps, and gloves. The above protocol has resulted in the isolation of a Bacillus sp. from a Permian-age salt crystal.Previous research has indicated that halophilic microorganisms are associated with salt crystals from ancient formations. However,this research has generally failed to convincingly demonstrate that these organisms were originally trapped inside the crystals during their deposition and were not recent surface contaminants. This paper presents techniques for crystal selection; verifiable, noninvasive, surface sterilization of salt crystals; and sterile extraction of biological material from the crystals. Immersing salt crystals in 10 M NaOH for 5 min; rinsing with sterile, saturated brine before immersing for 5 min in 10 M HCl; and rinsing with saturated brine is an effective method for surface sterilization. No growth has resulted from contamination tests of 216 faces from 36 natural salt crystals exposed to these treatments. Pure culture experiments using the halotolerant eubacteria, Halomonas elongata and Bacillus sp. (2-9-3), and the archeon, Halogeometricium borinquense, showed that exposure to either ...

Biogeology: How old are bacteria from the Permian age?

Hazen and Roedder touch on several geological issues raised by our limited description of the Permian Salado Formation. In our study, we sampled coarse halite in dissolution pipes for testing for viable Permian-age bacteria.

Fatty acid and DNA analyses of Permian bacteria isolated from ancient salt crystals reveal differences with their modern relatives

The isolation of living microorganisms from primary 250-million-year-old (MYA) salt crystals has been questioned by several researchers. The most intense discussion has arisen from questions about the texture and age of the crystals used, the ability of organisms to survive 250 million years when exposed to environmental factors such as radiation and the close similarity between 16S rRNA sequences in the Permian and modern microbes. The data in this manuscript are not meant to provide support for the antiquity of the isolated bacterial strains. Rather, the data presents several comparisons between the Permian microbes and other isolates to which they appear related. The analyses include whole cell fatty acid profiling, DNA–DNA hybridizations, ribotyping, and random amplified polymorphic DNA amplification (RAPD). These data show that the Permian strains, studied here, differ significantly from their more modern relatives. These differences are accumulating in both phenotypic and molecular areas of the cells. At the fatty acid level the differences are approaching but have not reached separate species status. At the molecular level the variation appears to be distributed across the genome and within the gene regions flanking the highly conserved 16S rRNA itself. The data show that these bacteria are not identical and help to rule out questions of contamination by putatively modern strains.

Survival of Halophilic Bacteria in Ancient Salts: Possibilities and Potentials

The Earth currently contains a large number of underground salt formations of significant size. These formations range in age from Jurassic (60,000,000 Million year of age [Ma]) to Cambrian (570,000,000 Ma). Most are Permian in age (230 — 280 Ma) (Javor 1989). Salt formations are distributed across nearly every continent, with the largest number in the northern hemisphere (Zharkov 1981)