Myxococcus xanthus DK1622 is known as a proficient producer of different kinds of secondary metabolites (SM) with various biological activities, including myxovirescin A, myxalamide A, myxochromide A and DKxanthene. Low production of SM in the wild type bacteria makes searching for production optimization methods highly desirable. Identification and induction of endogenous key molecular feature(s) regulating the production level of the metabolites remain promising, while heterologous expression of the biosynthetic genes is not always efficient because of various complicating factors including codon usage bias. This study established proteomic and molecular approaches to elucidate the regulatory roles of the ROK regulatory protein in the modification of secondary metabolite biosynthesis. Interestingly, the results revealed that rok inactivation significantly reduced the production of the SM and also changed the motility in the bacteria. Electrophoretic mobility shift assay using purified ROK protein indicated a direct enhancement of the promoters encoding transcription of the DKxanthene, myxochelin A, and myxalamide A biosynthesis machinery. Comparative proteomic analysis by two-dimensional fluorescence difference in-gel electrophoresis (2D-DIGE) was employed to identify the protein profiles of the wild type and rok mutant strains during early and late logarithmic growth phases of the bacterial culture. Resulting data demonstrated overall 130 differently altered proteins by the effect of the rok gene mutation, including putative proteins suspected to be involved in transcriptional regulation, carbohydrate metabolism, development, spore formation, and motility. Except for a slight induction seen in the production of myxovirescin A in a rok over-expression background, no changes were found in the formation of the other SM. From the outcome of our investigation, it is possible to conclude that ROK acts as a pleiotropic regulator of secondary metabolite formation and development in M. xanthus, while its direct effects still remain speculative. More experiments are required to elucidate in detail the variable regulation effects of the protein and to explore applicable approaches for generating valuable SM in this bacterium.

中文翻译：

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ROK样蛋白（Myxococcus xanthus DK1622）充当二次代谢的多效转​​录调节因子。

xanthus xanthus DK1622已知是具有各种生物活性的各种次级代谢产物（SM）的精通生产者，包括myxovirescin A，myxalamide A，myxochromide A和DKxanthene。在野生型细菌中SM的低产量使得寻找生产最优化方法非常必要。鉴定和诱导调节代谢产物产生水平的内源性关键分子特征仍然很有希望，而生物合成基因的异源表达由于包括密码子使用偏倚在内的各种复杂因素而并不总是有效的。这项研究建立了蛋白质组学和分子方法，以阐明韩国调节蛋白在次级代谢产物生物合成修饰中的调节作用。有趣的是 结果表明，rok灭活显着降低了SM的产生，还改变了细菌的运动能力。使用纯化的ROK蛋白进行的电泳迁移率变动分析表明，编码DKxanthene，粘黏螯合素A和Myxalamide A生物合成机制转录的启动子直接增强。通过二维荧光差异凝胶电泳（2D-DIGE）进行比较蛋白质组学分析，以鉴定细菌培养早期和后期对数生长期的野生型和rok突变菌株的蛋白质谱。结果数据显示，通过rok基因突变的影响，共有130种不同的蛋白质发生了变化，其中包括可能与转录调控，碳水化合物代谢，发育，孢子形成和运动。在rok过表达的背景下，除了在粘液霉素A的生产中看到轻微的诱导作用外，其他SM的形成均未发现变化。根据我们的调查结果，可以得出结论，韩国可以作为多发性调节剂来控制黄花棘枝secondary次级代谢产物的形成和发育，而其直接影响仍然是推测性的。需要更多的实验来详细阐明蛋白质的可变调节作用，并探索在这种细菌中产生有价值的SM的适用方法。可以得出结论，韩国可以作为多发性调节剂来控制黄原花次生代谢产物的形成和发展，而其直接影响仍然是推测性的。需要更多的实验来详细阐明蛋白质的可变调节作用，并探索在这种细菌中产生有价值的SM的适用方法。可以得出结论，韩国可以作为多菌种调控黄原花次生代谢产物的形成和发展，而其直接影响仍然是推测性的。需要更多的实验来详细阐明蛋白质的可变调节作用，并探索在这种细菌中产生有价值的SM的适用方法。