Adaptation at the Extremes of Life: Experimental Evolution with the Extremophile Archaeon Sulfolobus acidocaldarius

Summary

Here, we present an experimental evolution protocol for adaptation in thermophiles utilizing low-cost, energy-efficient bench-top thermomixers as incubators. The technique is demonstrated through the characterization of temperature adaptation in Sulfolobus acidocaldarius, an archaeon with an optimal growth temperature of 75 °C.

Abstract

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures is a key requirement, not only for understanding fundamental evolutionary processes but also for developing S. acidocaldarius as a chassis for bioengineering. One major obstacle to conducting experimental evolution with thermophiles is the expense of equipment maintenance and energy usage of traditional incubators for high-temperature growth. To address this challenge, a comprehensive experimental protocol for conducting experimental evolution in S. acidocaldarius is presented, utilizing low-cost and energy-efficient bench-top thermomixers. The protocol involves a batch culture technique with relatively small volumes (1.5 mL), enabling tracking of adaptation in multiple independent lineages. This method is easily scalable through the use of additional thermomixers. Such an approach increases the accessibility of S. acidocaldarius as a model system by reducing both initial investment and ongoing costs associated with experimental investigations. Moreover, the technique is transferable to other microbial systems for exploring adaptation to diverse environmental conditions.

Introduction

Early life on Earth may have originated in extreme environments, such as hydrothermal vents, which are characterized by extremely high temperatures and acidity¹. Microbes continue to inhabit extreme environments, including hot springs and volcanic solfatara. Characterizing the evolutionary dynamics that occur under these extreme conditions may shed light on the specialized physiological processes that enable survival under these conditions. This may have wide-ranging implications, from our understanding of the origins of biological diversity to the development of novel high-temperature enzymes with biotechnological applications.

Protocol

1. Preparation of S. acidocaldarius growth medium (BBM⁺)

NOTE: To cultivate S. acidocaldarius, this protocol uses Basal Brock Medium (BBM⁺)²³. This is prepared by first combining the inorganic stock solutions outlined below to create BBM⁻, which may be prepared in advance. BBM⁺ is then prepared as needed by adding the organic stock solutions to BBM⁻. Stock solution recipes are also presented in Table 1. All media and stock solutions should be prepared in double-distilled H₂O (ddH₂

Results

Growth curve measurements
Growth curves for S. acidocaldarius DSM639 are shown in Figure 3A. Growth was found to be similar when comparing incubation using thermomixers with that in conventional incubators. Average growth rate parameters were estimated by fitting a logistic curve to each replicated growth curve and calculating the mean and standard error. Times to mid-exponential phase on the thermomixer and incubator were 27.2 h ± 1.1 h and 31.1 h ± 1.9 h, resp.......

Discussion

This work has developed an experimental evolution protocol for thermophiles, here tailored for the archaeon S. acidocaldarius, but adaptable to other microbes with high-temperature growth requirements. This protocol builds on techniques initially designed for mesophilic bacteria but is specifically modified to overcome the technical challenges associated with high-temperature aerobic growth^2,4,5,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm syringe-driven membrane filters	StarLab	E4780-1226	For filter sterilising media components that cannot be autoclaved.
1 μL inoculation loops	Greiner	731161, 731165, or 731101	For inoculating cultures. Other loops can be used.
1000 μL pipette tips	StarLab	S1111-6811	Other pipette tips can be used.
2 mL microcentrifuge tubes	StarLab	S1620-2700	For culturing S. acidocaldarius in thermomixers.
200 μL pipette tips	StarLab	S1111-0816	Other pipette tips can be used.
50 mL polystyrene tubes with conical bottom	Corning	430828 or 430829	Other tubes may be used. Check performance at 75 °C. Tubes with plug seal caps may not allow sufficient aeration; check before using.
50 mL syringe	BD plastipak	300865	For use with syringe-driven filters.
96 well microtitre plates (non-treated, flat bottom)	Nunc	260860	For measuring OD at 600 nm in spectrophotometer.
Adjustable width multichannel pipette	Pipet-Lite	LA8-300XLS	Optional, but saves time when transferring between microcentrifuge and 96 well plates.
Ammonium sulfate ((NH4)2SO4)	Millipore	168355	For Brock stock solution I.
Autoclave	Priorclave	B60-SMART or SV100-BASE	Other autoclaves can also be used.
Breathe-EASY gas permeable sealing membrane	Sigma-Aldrich	Z763624-100EA	Cut to size to use on pierced microcentrifuge tubes. If substituting other gas permeable memrbanes, ensure performance is adequate at 75 °C
Calcium chloride dihydrate (CaCl2·2H2O)	Sigma-Aldrich	C3306	For Brock stock solution I.
CELLSTAR Six well plates (suspension/non-treated)	Greiner	M9062	Other manufacturers' six well plates can likely be substituted. Check performance at high temperatures.
Cobalt(II) sulfate heptahydrate (CoSO4·7H2O)	Supelco	1025560100	For Trace element stock solution.
Copper(II) chloride dihydrate (CuCl2·2H2O)	Sigma-Aldrich	307483	For Trace element stock solution.
D-(+)-glucose anhydrous (C6H12O6)	Thermo Scientific Chemicals	11462858	Other pentose and hexose sugars may also be used (e.g. D-xylose, D-arabinose). Glucose is not a preferred carbon source for S. acidocaldarius (SV Albers, personal communication)
Double-distilled water (ddH2O)
Gelrite	Duchefa Biochemie	G1101.1000	Gelrite (gellan gum) is used in place of agar to make solid media due to its higher melting point.
Glass 100 mm Petri dishes	Brand	BR455742	Glass Petri dishes are used because most standard polystyrene 90 mm Petri dishes deform at 75 °C (brand-dependent). Alternatively, six well plates can be used as these do not deform at high temperatures.
Incubator	New Brunswick	Innnova 42R	Other incubators can also be used. Check the operating temperature for equipment prior to purchase/use, as many incubators are not capable of temperatures higher than 65°C.
Iron(III) chloride hexahydrate (FeCl3·6H2O)	Supelco	103943	For Fe Stock Solution
Magnesium sulfate heptahydrate (Epsom salt) (MgSO4·7H2O)	Sigma-Aldrich	230391	For Brock stock solution I.
Manganese(II) chloride tetrahydrate (MnCl2·4H2O)	Sigma-Aldrich	SIALM5005-100G	For Trace element stock solution.
Mini Smart Wi-Fi Socket, Energy Monitoring	Tapo	Tapo P110	To monitor energy consumtion
N-Z-Amine A - Casein enzymatic hydrolysate	Sigma-Aldrich	C0626-500G	N-Z-Amine-A is used as a source of amino acids.
Paper clip (or other sturdy wire)	none	none	For piercing 2 mL microcentrifuge tubes.
Potassium dihydrogen phosphate (Monopotassium phosphate) (KH2PO4)	Sigma-Aldrich	P0662	For Brock stock solution I.
Promega Wizard Genomic DNA Purification Kit	Promega	A1120	Optional, to extract genomic DNA in the lab
Sodium molybdate dihydrate (Na2MoO4·2H2O)	Sigma-Aldrich	M1651-100G	For Trace element stock solution.
Sodium tetraborate decahydrate (Borax) (Na2B4O7·10H2O)	Sigma-Aldrich	S9640	For Trace element stock solution.
Spectrophotometer	BMG	SPECTROstar OMEGA	For measuring OD at 600 nm. Other spectrophotometers that can read OD at 600 nm can be used.
Sulfuric acid (Diluted in a 1:1 ratio with water) (H2SO4)	Thermo Scientific Chemicals	11337588	Used to adjust pH of Brock stock solution II/III to a final pH of 2–3.
Thermomixer	DLab	HM100-Pro	Other thermomixers can also be used; key consideration is the ability to maintain 65–75 °C temperatures and 400 RPM
Uracil (C4H4N2O2)	Sigma-Aldrich	U0750	Deletion of pyrE is a common genetic marker used in S. acidocaldarius. Deletion strains must be supplemented with uracil for growth. Supplementation is not strictly required for the DSM639 wild-type strain, but is included here as future experiments may involve deletion strains.
Vanadyl sulfate dihydrate (VOSO4·2H2O)	Sigma-Aldrich	204862	For Trace element stock solution.
Zinc sulfate heptahydrate (ZnSO4·7H2O)	Sigma-Aldrich	221376	For Trace element stock solution.