Ancient Microbes from Halite Fluid Inclusions: Optimized Surface Sterilization and DNA Extraction

Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system

Starvation-Survival in Haloarchaea

Recent studies claiming to revive ancient microorganisms trapped in fluid inclusions in halite have warranted an investigation of long-term microbial persistence. While starvation-survival is widely reported for bacteria, it is less well known for halophilic archaea—microorganisms likely to be trapped in ancient salt crystals. To better understand microbial survival in fluid inclusions in ancient evaporites, laboratory experiments were designed to simulate growth of halophilic archaea under media-rich conditions, complete nutrient deprivation, and a controlled substrate condition (glycerol-rich) and record their responses. Haloarchaea used for this work included Hbt. salinarum and isolate DV582A-1 (genus Haloterrigena) sub-cultured from 34 kyear Death Valley salt. Hbt. salinarum and DV582A-1 reacted to nutrient limitation with morphological and population changes. Starved populations increased and most cells converted from rods to small cocci within 56 days of nutrient deprivation. The exact timing of starvation adaptations and the physical transformations differed between species, populations of the same species, and cells of the same population. This is the first study to report the timing of starvation strategies for Hbt. salinarum and DV582A-1. The morphological states in these experiments may allow differentiation between cells trapped with adequate nutrients (represented here by early stages in nutrient-rich media) from cells trapped without nutrients (represented here by experimental starvation) in ancient salt. The hypothesis that glycerol, leaked from Dunaliella, provides nutrients for the survival of haloarchaea trapped in fluid inclusions in ancient halite, is also tested. Hbt. salinarum and DV582A-1 were exposed to a mixture of lysed and intact Dunaliella for 56 days. The ability of these organisms to utilize glycerol from Dunaliella cells was assessed by documenting population growth, cell length, and cell morphology. Hbt. salinarum and DV582A-1 experienced size reductions and shape transitions from rods to cocci. In the short-term, these trends more closely resembled the response of these organisms to starvation conditions than to nutrient-rich media. Results from this experiment reproduced the physical state of cells (small cocci) in ancient halite where prokaryotes co-exist with single-celled algae. We conclude that glycerol is not the limiting factor in the survival of haloarchaea for thousands of years in fluid inclusions in halite

Unrooted Maximum likelihood tree, Saline Valley ITS1 algal sequences and closest matches from extant sequences deposited in the NCBI database.

<p>Distinct clusters are observed for the 4 different time periods. Bottom right shows average evolutionary distance between sequences and their closest NCBI match.</p

Agarose gel electrophoresis, Human HV1 PCR, Product size ∼440 bp.

<p>a) Evaluation of different surface sterilization protocols. Lane 1: DNA ladder, 2: Ac, 3:Al, 4:Bl, 5:AlBl, 6:AlAc, 7:AcBl, 8:AlAcBl, 9,12:Extract control, 10:Alcohol, 11:Spike, 13: PCR -ve. b) Effectiveness of surface sterilization. Halite crystals (modern Saline Valley) spiked with different amounts of human DNA. Lane 1: DNA ladder, Lanes 2,3,10,11: 1.5 ng spike, Lanes 4,5,12,13: 3.0 ng spike, Lanes 6,7,14,15: 4.5 ng spike, Lanes 8,9,16,17: no spike. Lanes 2–9: no surface sterilization. Lanes 10–17 surface sterilized.</p

DNA quantification using PicoGreen®.

<p>a) Effectiveness of surface sterilization: average DNA yields, in ng, before and after surface sterilization for different spike amounts. b) Plot of average DNA yields in ng per gm of crystal for different extraction protocols.</p

Map showing location of Saline Valley, California and stratigraphic column of core SV-4A, showing uranium series ages (black arrows), sediment types, paleoenvironments, and sample depths (red arrows).

<p>Modified from Howe (<i>1998</i>) and Lowenstein et al. (<i>2011</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020683#pone.0020683-Lowenstein4" target="_blank">[34]</a>. Upper right shows fluid filled inclusions in halite crystals from Saline Valley with algal forms trapped inside (arrows). a) Two algal cells (probably <i>Dunaliella</i>) in fluid inclusion in modern halite collected in April 2004. The red color likely comes from <i>carotenoid</i> pigments found in the cells. b) Fluid inclusion in halite crystal from core SV-4A (21.3 m depth, ∼36 ka), with algal cells and clumps of smaller biomaterials, probably prokaryotes. c) Fluid inclusion in halite from core SV-4A (43.9 m depth, ∼64 ka). Arrow shows whole cell; above arrow, cells are in various stages of degradation including the glycocalyx (cell coat) of several ruptured algal cells. d) Fluid inclusion in halite from core SV-4A (91.4 m depth, ∼150 ka), showing many well-preserved algal cells. Scale bars are 10 microns in all images.</p

Agarose gel electrophoresis images from a) Algal ITS1 (∼100–300 bp), b) Algal ITS2 (∼300 bp) and c) <i>Archaeal</i> 16s (∼200 bp).

<p>Sample order: Lane 1: DNA ladder, 2: Modern Saline Valley, 3: Saline Valley ∼36 ka, 4: Saline Valley ∼64 ka, 5: Saline Valley ∼150 ka, and 6: PCR –ve.</p