post-evolution growth assays of sulfolobus acidocaldarius ancestral and evolved lineages

<meta charset="utf8"></head><body>Protocole d’évolution expérimental pour l’adaptation thermophile à l’aide de thermomélangeurs à faible coût

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.
The understanding of microbial evolutionary dynamics in extreme environments remains limited despite its critical importance. In contrast, a significant body of knowledge about evolution in mesophilic environments has been acquired through the application of a technique known as experimental evolution. Experimental evolution involves observing evolutionary change under laboratory conditions2,3,4,5. Often, this involves a defined change environment (e.g., temperature, salinity, introduction of a toxin or a competitor organism)7,8,9. When combined with whole-genome sequencing, experimental evolution has enabled us to test key aspects of evolutionary processes, including parallelism, repeatability, and the genomic basis for adaptation. However, to date, the bulk of experimental evolution has been performed with mesophilic microbes (including bacteria, fungi, and viruses2,3,4,5, but largely excluding archaea). A method for experimental evolution applicable to thermophilic microbes would enable us to better understand how they evolve and contribute to a more comprehensive understanding of evolution. This has potentially wide-ranging implications, from deciphering the origins of thermophilic life on Earth to biotechnological applications involving &#39;extremozymes&#39; used in high-temperature bioprocesses10 and astrobiological research11.
The archaeon Sulfolobus acidocaldarius is an ideal candidate as a model organism for developing experimental evolution techniques for thermophiles. S. acidocaldarius reproduces aerobically, with an optimal growth temperature at 75 &#176;C (range 55 &#176;C to 85 &#176;C) and high acidity (pH 2-3)4,6,12,13,14. Remarkably, despite its extreme growth conditions, S. acidocaldarius maintains population densities and mutation rates comparable to mesophiles7,15,16,17,18. In addition, it possesses a relatively small, well-annotated genome (strain DSM639: 2.2 Mb, 36.7% GC, 2,347 genes)12; S. acidocaldarius also benefits from robust genome engineering tools, allowing for a direct assessment of the evolutionary process through targeted gene knockouts19. A notable example of this is the availability of genetically modified strains of S. acidocaldarius, such as the uracil auxotrophic strains of MW00119 and SK-120, which can serve as selectable markers.
There are significant challenges with conducting experimental evolution with thermophiles like S. acidocaldarius. Extended incubation at high temperatures required for these studies imposes considerable evaporation for both liquid and solid culturing techniques. Extended operation at high temperatures can also damage the traditional shaking incubators that are commonly used in experimental evolution in liquid media. Exploring multiple temperatures necessitates a substantial financial investment for acquiring and maintaining several incubators. Furthermore, the high energy consumption required raises significant environmental and financial concerns.
This work introduces a method to address the challenges encountered in performing experimental evolution with thermophiles like S. acidocaldarius. Building upon a technique developed by Baes et al. for investigating heat shock response14,21, the method developed here utilizes bench-top thermomixers for consistent and reliable high-temperature incubation. Its scalability allows for the simultaneous assessment of multiple temperature treatments, with reduced costs in acquiring additional incubation equipment. This enhances experimental efficiency, enabling robust statistical analysis and extensive investigation of factors influencing evolutionary dynamics in thermophiles22. Moreover, this approach significantly reduces the financial initial investment and energy consumption compared to traditional incubators, offering a more sustainable and environmentally friendly alternative.
Our method lays the groundwork for experimentally investigating evolutionary dynamics in environments characterized by extreme temperatures, which may have played a key role during the early stages of the diversification of life on Earth. Thermophilic organisms have unique properties, but their extreme growth conditions and specialized requirements have often limited their accessibility as a model system. Overcoming these barriers not only expands research opportunities for investigating evolutionary dynamics but also enhances the broader utility of thermophiles as model systems in scientific research.

<meta charset="utf8"></head><body>Pleins feux sur l’auteur : Comprendre l’adaptation des microbes à l’aide de techniques innovantes pour explorer l’évolution thermophile

Adaptation aux extrêmes de la vie : évolution expérimentale avec l’archéon extrêmophile Sulfolobus acidocaldarius

Essais de croissance post-évolution des lignées ancestrales et évoluées de Sulfolobus acidocaldarius

post-evolution-growth-assays-sulfolobus-acidocaldarius-ancestral

Research

JoVE Journal

Biology

Post-Evolution Growth Assays of Sulfolobus acidocaldarius Ancestral and Evolved Lineages

27 vues. Pour commencer, incubez les cultures ancestrales et évoluées de Sulfolobus acidocaldarius aux températures finales. Une fois l’incubation terminée, mesurez leurs densités optiques à 600 nanomètres. Ensuite, ajoutez 1,5 millilitre de BBM plus dans six séries de sept tubes pour chaque culture.Ajoutez ensuite 15 microlitres de chaque culture dans les six ensembles de tubes. Réglez trois thermomélangeurs à 75 degrés Celsius et trois thermomé...

Vidéo: Essais de croissance post-évolution des lignées ancestrales et évoluées de Sulfolobus acidocaldarius

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

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.

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 &#176;C.

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures ...