一株海洋源皮特不动杆菌的分离鉴定及其全基因测序分析

Isolation, identification, and whole genome sequencing of a marine-derived strain of Acinetobacter pittii

摘要:

【背景】海洋是一个巨大的微生物资源库，其造就了适应高盐、高压和低温环境的微生物，从中可以分离出对动物病原菌具有抑菌活性的微生物。【目的】广东省湛江市近海底泥中分离出对动物病原菌有拮抗作用的海洋细菌，对抑菌活性强的细菌进行全基因组序列分析，以期挖掘其产细菌素等功能特性基因。【方法】采用M10培养基、P3培养基和BHI固体培养基从广东省湛江市海底泥样品中分离细菌，采用固体琼脂打孔法从分离细菌中筛选对金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、蜡样芽胞杆菌(Bacillus cereus)和乙型副伤寒沙门氏菌(Salmonella paratyphi B)等动物病原菌有拮抗作用的细菌，PCR扩增拮抗菌的16S rRNA基因并进行测序分析。选择抑菌效果最好的细菌进行全基因组测序分析，通过序列组装、基因预测、功能注释、anti-SMASH挖掘潜在的细菌素基因簇及潜在的作用机制，分析其产细菌素的潜能。【结果】从广东省湛江市近海底泥中分离得到317株有抑菌效果的菌株，包括芽孢杆菌属(Bacillus)、不动杆菌属(Acinetobacter)、肠杆菌属(Enterobacter)、克雷伯氏菌属(Klebsiella)、假单胞菌属(Pseudomonas)、弧菌属(Vibrio)等；筛选抑菌效果最好的菌株AM3-2；全基因组测序鉴定菌株AM3-2为皮特不动杆菌(Acinetobacter pittii)，基因组大小为3 426 922 bp，G+C含量为39.84%。共预测到3 631个蛋白质编码基因、6个rRNA、64个tRNA；基因组中含有6个与抗氧化活性相关基因，以及8个与调控免疫和炎症信号通路相关的基因；预测AM3-2存在2个相关细菌素合成基因簇。【结论】菌株AM3-2是一株潜在的产细菌素菌株，为进一步研究其抗菌特性提供了理论基础。

Abstract:

[Background] The ocean is a huge microbial resource bank, which creates microorganisms adapted to high salt, high pressure and low temperature environment, from which microorganisms with antibacterial activity against animal pathogens can be isolated. [Objective] To isolate marine bacteria with antagonistic effects on animal pathogens from the offshore sediment of Zhanjiang, Guangdong and analyze the whole genome sequences of the strains with strong antibacterial activity to mine the functional genes involved in bacteriocin production and other activities. [Methods] We used the M10 medium, P3 medium, and BHI solid medium to isolate bacterial strains from the sediment samples. The bacteria with antagonism to Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Salmonella paratyphi B were screened by the disk diffusion test. The 16S rRNA gene of the antagonistic strains was amplified by PCR and analyzed by sequencing. The strain with the strongest bacteriostatic effect was selected for whole genome sequencing, and its bacteriocin production potential was analyzed by sequence assembly, gene prediction, and functional annotation, as well as by mining of potential biosynthetic gene clusters (BGCs) for bacteriocin and deciphering of potential mechanism through anti-SMASH. [Results] A total of 317 strains with antibacterial effects were isolated from the offshore sediment in Zhanjiang, Guangdong, including Bacillus, Acinetobacter, Enterobacter, Klebsiella, Pseudomonas, and Vibrio. The strain AM3-2 with the strongest bacteriostatic effect was screened out. The whole genome sequencing identified strain AM3-2 as Acinetobacter pittii, which had a genome size of 3 426 922 bp and the G+C content of 39.84%. The genome of this strain was predicted to carry a total of 3 631 protein-coding genes, 6 rRNAs and 64 tRNAs. The genome contained 6 genes related to antioxidant activity and 8 genes associated with the regulation of immune and inflammatory signaling pathways. In addition, the strain was predicted to carry 2 BGCs for bacteriocin. [Conclusion] AM3-2 was a potential bacteriocin-producing strain, which provided a theoretical basis for further study of its antibacterial properties