全转录组分析和基因缺失以了解氯霉素抗性机制，并开发一种用于黄色粘球菌同源重组的筛选方法。

Whole transcriptome analysis and gene deletion to understand the chloramphenicol resistance mechanism and develop a screening method for homologous recombination in Myxococcus xanthus.

机构信息

Marine Agriculture Research Center, Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao, 266101, Shandong, China.

State Key Laboratory of Microbial Technology, Shandong University, Qingdao, 266237, Shandong, China.

出版信息

Microb Cell Fact. 2019 Jul 10;18(1):123. doi: 10.1186/s12934-019-1172-3.

DOI:10.1186/s12934-019-1172-3

PMID:31291955

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6617876/

Abstract

BACKGROUND

Myxococcus xanthus DK1622 is a model system for studying multicellular development, predation, cellular differentiation, and evolution. Furthermore, it is a rich source of novel secondary metabolites and is widely used as heterologous expression host of exogenous biosynthetic gene clusters. For decades, genetic modification of M. xanthus DK1622 has mainly relied on kanamycin and tetracycline selection systems.

RESULTS

Here, we introduce an alternative selection system based on chloramphenicol (Cm) to broaden the spectrum of available molecular tools. A chloramphenicol-resistant growth phase and a chloramphenicol-susceptible growth phase before and after chloramphenicol-induction were prepared, and later sequenced to identify specific genes related to chloramphenicol-repercussion and drug-resistance. A total of 481 differentially expressed genes were revealed in chloramphenicol-resistant Cm5_36h and 1920 differentially expressed genes in chloramphenicol-dormant Cm_8h. Moreover, the gene expression profile in the chloramphenicol-dormant strain Cm_8h was quite different from that of Cm5_36 which had completely adapted to Cm, and 1513 differentially expression genes were identified between these two phenotypes. Besides upregulated acetyltransferases, several transporter encoding genes, including ABC transporters, major facilitator superfamily transporters (MFS), resistance-nodulation-cell division (RND) super family transporters and multidrug and toxic compound extrusion family transporters (MATE) were found to be involved in Cm resistance. After the knockout of the most highly upregulated MXAN_2566 MFS family gene, mutant strain DK-2566 was proved to be sensitive to Cm by measuring the growth curve in the Cm-added condition. A plasmid with a Cm resistance marker was constructed and integrated into chromosomes via homologous recombination and Cm screening. The integration efficiency was about 20% at different concentrations of Cm.

CONCLUSIONS

This study provides a new antibiotic-based selection system, and will help to understand antibiotic resistance mechanisms in M. xanthus DK1622.

摘要

背景

粘球菌 DK1622 是研究细胞多态性分化、细胞分化、细胞吞噬和进化的模式生物。此外，它还是新型次生代谢物的丰富来源，被广泛用作外源生物合成基因簇的异源表达宿主。几十年来，粘球菌 DK1622 的遗传修饰主要依赖于卡那霉素和四环素选择系统。

结果

在这里，我们引入了一种基于氯霉素（Cm）的替代选择系统，以拓宽可用分子工具的范围。制备了氯霉素抗性生长阶段和氯霉素诱导前后氯霉素敏感生长阶段，然后进行测序以鉴定与氯霉素反应和耐药性相关的特定基因。在氯霉素抗性 Cm5_36h 中发现了 481 个差异表达基因，在氯霉素休眠 Cm_8h 中发现了 1920 个差异表达基因。此外，氯霉素休眠菌株 Cm_8h 的基因表达谱与完全适应 Cm 的 Cm5_36h 非常不同，这两种表型之间鉴定出 1513 个差异表达基因。除了上调的乙酰转移酶外，还发现了几种转运蛋白编码基因，包括 ABC 转运蛋白、主要易化因子超家族转运蛋白（MFS）、耐药结节细胞分裂（RND）超家族转运蛋白和多药和毒性化合物外排家族转运蛋白（MATE），这些基因都与 Cm 抗性有关。敲除表达量最高的 MXAN_2566 MFS 家族基因后，通过在添加 Cm 的条件下测量生长曲线，证明突变株 DK-2566 对 Cm 敏感。构建了一个带有 Cm 抗性标记的质粒，并通过同源重组和 Cm 筛选将其整合到染色体上。在不同浓度的 Cm 下，整合效率约为 20%。