Isolation of Halobacterium salinarum retrieved directly from halite brine inclusions

Abstract

Halite crystals were selected from a 186 m subsurface core taken from the Badwater salt pan, Death Valley, California to ascertain if halophilic Archaea and their associated 16S rDNA can survive over several tens of thousands of years. Using a combined microscope microdrill/micropipette system, fluids from brine inclusions were aseptically extracted from primary, hopper texture, halite crystals from 8 and 85 metres below the surface (mbls). U-Th disequilibrium dating indicates that these halite layers were deposited at 9,600 and 97,000 years before present (ybp) respectively. Extracted inclusions were used for polymerase chain reaction (PCR) analysis with haloarchaea-specific 16S rDNA primers or placed into haloarchaea culture medium. Enrichment cultures were obtained from 97 kyr halite crystal inclusion fluid and haloarchaea-containing prepared crystals (positive controls), whereas inclusions from crystals of 9.6 kyr halite and the haloarchaea-free halite crystals (negative controls) resulted in no growth. Phylogenetic analysis (16S rDNA) of the 97 kyr isolate, designated BBH 001, revealed a homology of 100% with Halobacterium salinarum. DNA-DNA hybridization experiments confirmed that BBH 001 was closely related to H. salinarum (81-75% hybridization) and its ascription to this haloarchaea species. The described method of retrieving particle-containing brine from fluid inclusions offers a robust approach for assessing the antiquity of microorganisms associated with evaporites.

Acidophilic Halophilic Microorganisms in Fluid Inclusions in Halite from Lake Magic, Western Australia

“…For example, Mormile et al (2003) isolated a bacterium from fluid inclusions in 97,000-year-old halite from Death Valley. Vreeland et al (2000) claimed to have identified two strains of viable bacteria from a large, isolated fluid inclusion in the Permian Salado Formation halite from New Mexico.…”

Entrapment of Bacteria in Fluid Inclusions in Laboratory-Grown Halite

“…The bacterium isolated from the Salado Formation (Vreeland et al, 2000) has been shown to be 99% genetically identical to Salibacillus marismortui, a modern strain from the Dead Sea (Maughan et al, 2002). Stringent sterilization techniques and isolation of cells directly from fluid inclusions (Vreeland et al, 2000;Mormile et al, 2003), however, should prevent laboratory contamination.…”

“…Entrapment within fluid inclusions can protect cells against desiccation, biochemical degradation (Mormile et al, 2003), and the effects of radiation (Kminek et al, 2003). Entrapment within fluid inclusions also could provide a mode of transport for microorganisms, both here on Earth and in the vacuum of space.…”

Microbial Diversity and Its Relationship to Planetary Protection

“…Ronto et al (55) pointed out that the current Martian UV environment is still quite severe from a biological viewpoint but also showed that substantial protection can be afforded to microbial spores under dust and ice. Based on these data and previously published (controversial) descriptions of ancient but dormant life forms on Earth (13,22,27,28,43,71), we concluded that if highly resistant endospores such as those studied at JPL (36,68,69) were delivered to Mars, they may remain viable for many years or even indefinitely. Considering the spores within the JPL spacecraft assembly facility that we know about, which are far more robust than B. subtilis spores, we clearly need to understand the true diversity of all microbial forms present, even the forms we cannot grow.…”