Deng Peng, Jia Jiayuan, Foxfire Adam, Baird Sonya M, Smith Leif J, Lu Shi-En
Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, 32 Creelman St., Mississippi State, MS 39762.
Department of Biology, Texas A&M University, TAMU 3258, College Station, TX 77843.
Phytopathology. 2023 Jan;113(1):11-20. doi: 10.1094/PHYTO-03-22-0106-R. Epub 2023 Jan 13.
MS14, isolated from a soil sample in Mississippi, is known for producing the novel antifungal compound occidiofungin. In addition, MS14 exhibits a broad range of antibacterial activities against common plant pathogens. Random mutagenesis and gene complementation indicate that four genes are required for antibacterial activity of strain MS14 against the fire blight pathogen . With the aim of finding the biosynthetic gene cluster for the unknown antibacterial compound, we used RNA-seq to analyze the transcriptome of MS14 wild type and mutants lacking antibacterial activity. The twofold lower expressed genes in all mutants were studied, and a polyketide synthase (PKS) gene cluster was predicted to be directly involved in MS14 antibacterial activities. The -resistance cassette and CRISPR-Cas9 systems were used to mutate the PKS gene cluster. Plate bioassays showed that either insertion or frame-shifting one of the PKS genes resulted in a loss of antibacterial activity. Considering that the antibacterial-defective mutants maintain the same antifungal activities as the wild-type strain, the results suggest that this PKS gene cluster is highly likely to be involved in or directly responsible for the production of MS14 antibacterial activity. Purification efforts revealed that the antibacterial activity of the compound synthesized by the gene cluster is sensitive to UV radiation. Nevertheless, these findings have provided more insights to understand the antibacterial activity of strain MS14.
MS14是从密西西比州的一份土壤样本中分离出来的，以产生新型抗真菌化合物杀稻瘟菌素而闻名。此外，MS14对常见的植物病原体具有广泛的抗菌活性。随机诱变和基因互补表明，菌株MS14对梨火疫病病原体的抗菌活性需要四个基因。为了找到未知抗菌化合物的生物合成基因簇，我们使用RNA测序来分析MS14野生型和缺乏抗菌活性的突变体的转录组。研究了所有突变体中表达量降低两倍的基因，并预测一个聚酮合酶（PKS）基因簇直接参与MS14的抗菌活性。使用抗性盒和CRISPR-Cas9系统对PKS基因簇进行突变。平板生物测定表明，PKS基因中的任何一个插入或移码都会导致抗菌活性丧失。鉴于抗菌缺陷型突变体与野生型菌株保持相同的抗真菌活性，结果表明该PKS基因簇极有可能参与或直接负责MS14抗菌活性的产生。纯化工作表明，由该基因簇合成的化合物的抗菌活性对紫外线辐射敏感。尽管如此，这些发现为理解菌株MS14的抗菌活性提供了更多的见解。
