White Derrick, Singh Raghuveer, Rudrappa Deepak, Mateo Jackie, Kramer Levi, Freese Laura, Blum Paul
School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
Appl Environ Microbiol. 2017 Feb 1;83(4). doi: 10.1128/AEM.02631-16. Print 2017 Feb 15.
Thermotoga maritima ferments a broad range of sugars to form acetate, carbon dioxide, traces of lactate, and near theoretic yields of molecular hydrogen (H). In this organism, the catabolism of pentose sugars such as arabinose depends on the interaction of the pentose phosphate pathway with the Embden-Myerhoff and Entner-Doudoroff pathways. Although the values for H yield have been determined using pentose-supplemented complex medium and predicted by metabolic pathway reconstruction, the actual effect of pathway elimination on hydrogen production has not been reported due to the lack of a genetic method for the creation of targeted mutations. Here, a spontaneous and genetically stable pyrE deletion mutant was isolated and used as a recipient to refine transformation methods for its repair by homologous recombination. To verify the occurrence of recombination and to assess the frequency of crossover events flanking the deleted region, a synthetic pyrE allele, encoding synonymous nucleotide substitutions, was used. Targeted inactivation of araA (encoding arabinose isomerase) in the pyrE mutant was accomplished using a divergent, codon-optimized Thermosipho africanus pyrE allele fused to the T. maritima groES promoter as a genetic marker. Mutants lacking araA were unable to catabolize arabinose in a defined medium. The araA mutation was then repaired using targeted recombination. Levels of synthesis of H using arabinose-supplemented complex medium by wild-type and araA mutant cell lines were compared. The difference between strains provided a direct measurement of H production that was dependent on arabinose consumption. Development of a targeted recombination system for genetic manipulation of T. maritima provides a new strategy to explore H formation and life at an extremely high temperature in the bacterial domain.
We describe here the development of a genetic system for manipulation of Thermotoga maritima T. maritima is a hyperthermophilic anaerobic bacterium that is well known for its efficient synthesis of molecular hydrogen (H) from the fermentation of sugars. Despite considerable efforts to advance compatible genetic methods, chromosome manipulation has remained elusive and hindered use of T. maritima or its close relatives as model hyperthermophiles. Lack of a genetic method also prevented efforts to manipulate specific metabolic pathways to measure their contributions to H yield. To overcome this barrier, a homologous chromosomal recombination method was developed and used to characterize the contribution of arabinose catabolism to H formation. We report here a stable genetic method for a hyperthermophilic bacterium that will advance studies on the basic and synthetic biology of Thermotogales.
嗜热栖热菌能发酵多种糖类，生成乙酸盐、二氧化碳、微量乳酸，并能近乎理论产率地生成分子氢（H₂）。在这种生物中，戊糖（如阿拉伯糖）的分解代谢依赖于磷酸戊糖途径与糖酵解途径和恩特纳-杜德洛夫途径的相互作用。尽管已使用添加戊糖的复合培养基测定了H₂产率的值，并通过代谢途径重建进行了预测，但由于缺乏创建靶向突变的遗传方法，尚未报道途径消除对氢气产生的实际影响。在此，分离出一个自发且遗传稳定的pyrE缺失突变体，并将其用作受体，以优化通过同源重组进行修复的转化方法。为了验证重组的发生并评估缺失区域两侧交叉事件的频率，使用了一个编码同义核苷酸替换的合成pyrE等位基因。在pyrE突变体中，使用与嗜热栖热菌groES启动子融合的、密码子优化的非洲嗜热栖热菌pyrE等位基因作为遗传标记，实现了araA（编码阿拉伯糖异构酶）的靶向失活。缺乏araA的突变体在限定培养基中无法分解代谢阿拉伯糖。然后使用靶向重组修复araA突变。比较了野生型和araA突变体细胞系在添加阿拉伯糖的复合培养基上H₂的合成水平。菌株之间的差异提供了对依赖阿拉伯糖消耗的H₂产生的直接测量。开发用于嗜热栖热菌基因操作的靶向重组系统，为探索细菌域中极端高温下H₂形成和生命提供了一种新策略。
我们在此描述了一种用于嗜热栖热菌操作的遗传系统的开发。嗜热栖热菌是一种嗜热厌氧细菌，以其从糖类发酵中高效合成分子氢（H₂）而闻名。尽管为推进兼容的遗传方法付出了巨大努力，但染色体操作仍然难以实现，阻碍了将嗜热栖热菌或其近亲用作嗜热菌模型。缺乏遗传方法也阻碍了操纵特定代谢途径以测量它们对H₂产率贡献的努力。为克服这一障碍，开发了一种同源染色体重组方法，并用于表征阿拉伯糖分解代谢对H₂形成的贡献。我们在此报告了一种用于嗜热细菌的稳定遗传方法，这将推动对嗜热栖热菌纲基础生物学和合成生物学的研究。
