Altering under-represented DNA sequences elevates bacterial transformation efficiency.
Altering under-represented DNA sequences elevates bacterial transformation efficiency.
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细菌分子生物学的基石是对所研究的微生物进行基因操纵的能力。许多细菌难以进行遗传操作，其表型部分归因于新获得的 DNA 的强力去除，例如通过限制性修饰 (R-M) 系统。在此，我们报告了可显着提高细菌转化效率的方法，这些方法使用因表达许多 R-M 系统而难以转化的微生物（幽门螺杆菌）进行试验。最初，我们确定了抑制几个 R-M 系统表达并随之提高转化效率的条件。然后我们确定了一种可以广泛保护新获得的 DNA 的方法。我们通过计算预测了幽门螺杆菌基因组中代表性不足的短 DNA 序列，认为这些序列反映了基于序列的监视（例如 R-M 系统）的目标。然后，我们利用这些信息来修改和消除抗生素耐药性盒中的此类位点，创建“隐形”版本。与未修饰的盒相比，以这种方式修饰抗生素抗性盒会产生显着更高的转化效率，这是一种与基因组位点无关的反应。我们的结果表明，通过修改代表性不足的 DNA 序列或转化条件来避免 R-M 系统，是增强 DNA 转化的有效方法。我们识别代表性不足的序列的方法适用于任何具有已测序基因组的微生物。操纵细菌基因组对于许多领域都至关重要。这种操作是通过基因工程进行的，通常需要将新的 DNA 片段添加到基因组中。细菌拥有强大的系统来识别和降解新 DNA，其中一些系统依赖于限制性酶。这些酶以特定序列切割 DNA。我们发现了细菌基因组中通常缺失的一组 DNA 序列，其数量超出了我们偶然的预期。从新的 DNA 片段中消除这些序列使其能够以比包含这些序列的新 DNA 更高的频率整合到细菌基因组中。删除这些序列似乎可以让新的DNA以“隐形”模式在细菌雷达下飞行。这种转型改进方法应用起来很简单，并且可能广泛适用。
A cornerstone of bacterial molecular biology is the ability to genetically manipulate the microbe under study. Many bacteria are difficult to manipulate genetically, a phenotype due in part to robust removal of newly acquired DNA, for example, by restriction-modification (R-M) systems. Here, we report approaches that dramatically improve bacterial transformation efficiency, piloted using a microbe that is challenging to transform due to expression of many R-M systems, Helicobacter pylori. Initially, we identified conditions that dampened expression of several R-M systems and concomitantly enhanced transformation efficiency. We then identified an approach that would broadly protect newly acquired DNA. We computationally predicted under-represented short DNA sequences in the H. pylori genome, with the idea that these sequences reflect targets of sequence-based surveillance such as R-M systems. We then used this information to modify and eliminate such sites in antibiotic resistance cassettes, creating a “stealth” version. Modifying antibiotic resistance cassettes in this way resulted in significantly higher transformation efficiency compared to non-modified cassettes, a response that was genomic loci independent. Our results suggest that avoiding R-M systems, via modification of under-represented DNA sequences or transformation conditions, is a powerful method to enhance DNA transformation. Our approach to identify under-represented sequences is applicable to any microbe with a sequenced genome. Manipulating the genomes of bacteria is critical to many fields. Such manipulations are made by genetic engineering, which often requires new pieces of DNA to be added to the genome. Bacteria have robust systems for identifying and degrading new DNA, some of which rely on restriction enzymes. These enzymes cut DNA at specific sequences. We identified a set of DNA sequences that are missing normally from a bacterium’s genome, more than would be expected by chance. Eliminating these sequences from a new piece of DNA allowed it to be incorporated into the bacterial genome at a higher frequency than new DNA containing the sequences. Removing such sequences appears to allow the new DNA to fly under the bacterial radar in “stealth” mode. This transformation improvement approach is straightforward to apply and likely broadly applicable.
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