生命极端的适应：极端微生物 Sulfolobus acidocaldarius 的实验进化

摘要

在这里，我们提出了一种实验进化方案，用于利用低成本、节能的台式热混合器作为培养箱在嗜热菌中适应。该技术通过对 Sulfolobus acidocaldarius 的温度适应表征来证明，Sulfolobus acidocaldarius 是一种最佳生长温度为 75 °C 的古细菌。

摘要

古细菌 Sulfolobus acidocaldarius 已成为一种很有前途的嗜热模式系统。研究嗜热菌如何适应温度变化是一项关键要求，不仅对于理解基本的进化过程，而且对于开发 S. acidocaldarius 作为生物工程的底盘。使用嗜热菌进行实验进化的一个主要障碍是传统培养箱用于高温生长的设备维护和能源使用费用。为了应对这一挑战，提出了一种利用低成本和节能的台式热混合器在 S. acidocaldarius 中进行实验进化的综合实验方案。该方案涉及体积相对较小的分批培养技术 （1.5 mL），能够追踪多个独立谱系的适应。通过使用额外的热混合器，这种方法很容易扩展。这种方法通过减少与实验研究相关的初始投资和持续成本，增加了 S. acidocaldarius 作为模型系统的可及性。此外，该技术可转移到其他微生物系统，以探索对不同环境条件的适应。

引言

地球上的早期生命可能起源于极端环境，例如热液喷口，其特点是极高的温度和酸度¹。微生物继续栖息在极端环境中，包括温泉和火山 solfatara。描述在这些极端条件下发生的进化动力学可能会阐明在这些条件下生存的特殊生理过程。这可能具有广泛的影响，从我们对生物多样性起源的理解到开发具有生物技术应用的新型高温酶。

尽管极端环境中的微生物进化动力学至关重要，但对它的理解仍然有限。相比之下，通过应用一种称为实验进化的技术，获得了关于嗜温环境中进化的重要知识体系。实验进化包括在实验室条件下观察进化变化 2,3,4,5。通常，这涉及确定的变化环境（例如，温度、盐度、引入毒素或竞争对手的生物体）^7,8,9。当与全基因组测序相结合时，实验进化使我们能够测试进化过程的关键方面，包括平行性、可重复性和适应的基因组基础。然而，迄今为止，大部分实验进化....

研究方案

1. S. acidocaldarius 生长培养基 （BBM⁺） 的制备

注：为了培养 S. acidocaldarius，该方案使用 Basal Brock 培养基 （BBM⁺）²³。这是通过首先混合下面概述的无机储备液来制备 BBM− 来制备的，BBM⁻ 可以提前制备。然后根据需要通过将有机储备溶液添加到 BBM ^- 中来制备 BBM⁺。储备溶液配方也见表 1。所有培养基和储备液均应在双蒸 H₂O （ddH₂O） 中制备。

1. 生长培养基无机储备液的制备
   1. 制备痕量元素储备液。
      1. 向 ddH₂O 中加入 9 g/L 十水合四硼酸钠 （Na₂B₄O₇·10H₂O），然后滴加 1：1 H₂SO₄ 直至溶解，以制备微量元素储备液。
      2. 依次加入 0.44 g/L 七水硫酸锌 （ZnSO_4....

讨论

这项工作开发了一种嗜热菌的实验进化方案，这里为古细菌 S. acidocaldarius 量身定制，但适用于其他具有高温生长要求的微生物。该协议建立在最初为嗜温细菌设计的技术之上，但经过专门修改以克服与高温需氧生长相关的技术挑战 2,4,5,24。这种方法的创建将能够探索?.......

材料

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.