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

Abstract

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.

Ribosomal RNA gene fragments from fossilized cyanobacteria identified in primary gypsum from the late Miocene, Italy

“…Although numerous chemical analyses have demonstrated that fluid inclusions retain the chemistry of their original source waters (Lowenstein & Hardie, 1985; Satterfield et al. , 2005) and preserve living micro‐organisms (Vreeland et al. , 2000, 2006, 2007; Powers et al.…”

“…Particularly suspect are fluid inclusions and their associated pore spaces, which may host post-depositional bacterial spores, so that the actual age of the microbes remains doubtful. Although numerous chemical analyses have demonstrated that fluid inclusions retain the chemistry of their original source waters (Lowenstein & Hardie, 1985;Satterfield et al, 2005) and preserve living micro-organisms (Vreeland et al, 2000(Vreeland et al, , 2006(Vreeland et al, , 2007Powers et al, 2001;Lowenstein et al, 2005;Satterfield et al, 2005), we conducted a meticulous sampling procedure to exclude samples that contained fluid inclusions. We preferred to focus on gypsum microsamples extracted from physically isolated, primarily deposited, single crystals containing abundant bacterial filaments to minimize uncertainties due to the fact that inclusions and the viable bacterial spores contained in them may in some cases represent post-depositional features (Hazen & Roedder, 2001).…”

Isolation of Live Cretaceous (121–112 Million Years Old) HalophilicArchaeafrom Primary Salt Crystals

“…That same study has still been controversial due to the similarity in the sequences of the 16S rRNA genes of the ancient isolate and those of a putatively modern microbe (Graur and Pupko 2001;Nickle et al 2002). While the molecular questions have been discussed at length, this debate is somewhat tangential since the conclusion that the bacterium was of Permian age was actually based upon the stability and age of the geological formation, the primary nature of the crystal sampled and the low level of contamination probability (1 in 10 −9 ) Vreeland et al 2000Vreeland et al , 2006Satterfield et al 2005). In a separate study, Fish et al (2002) isolated DNA from halite crystals of varying ages (11 to 425 MYA) that were first sectioned using intense lasers, analyzed for fluid inclusion chemistry, then polished and cleaned with an ultra-sonic cleaner.…”

Continental evaporites and the search for evidence of life on Mars

“…From the salt mines of the Permian and Triassic age, archaeal halophiles were isolated and characterized on the basis of lipid patterns (Norton et al, 1993) and new species were also described (Denner et al, 1994). The spore-forming bacterium Virgibacillus species 2-9-3, that is among the oldest living organisms, was detected and isolated from 250-million-year-old salt crystals from the Permian Salado Formation, New Mexico (Vreeland et al, 2000;Satterfield et al, 2005;Vreeland et al, 2006). In spite of the quick DNA decay, the discovery of the segments of haloarchaeal DNA sequences, extracted from 419-million-year-old salts (Park et al, 2009), suggests that the salt potential in terms of microbial preservation is far from exhausted.…”