1株抗多杀性巴氏杆菌融合魏斯氏菌的全基因组测序及其产细菌素的理化性质分析

doi:10.16431/j.cnki.1671-7236.2025.07.032

摘要/Abstract

摘要： 【目的】筛选出一种抗多杀性巴氏杆菌的乳酸菌细菌素，完成乳酸菌全基因组测序，分析其细菌素理化特性和杀菌机制。【方法】将健康猫粪便接种于MRS肉汤培养基，37 ℃培养12 h分离细菌。以多杀性巴氏杆菌为指示菌，通过牛津杯琼脂扩散法测定分离的乳酸菌的抑菌活性，通过16S rRNA基因扩增鉴定分离的乳酸菌，并对其进行全基因组序列测定及KEGG功能注释。采用蛋白酶敏感、热稳定性、酸碱稳定性试验测定分离菌产抑菌物质的生物学特性，运用扫描电镜技术探究抑菌物质的抗菌特性。【结果】筛选出1株产抑菌物质的乳酸菌，其菌落表面光滑，形态较小；革兰染色后呈阳性球菌。16S rRNA测序分析显示，分离菌与融合魏斯氏菌相似性为97%，鉴定为融合魏斯氏菌，命名为JLP1。全基因组测序结果显示，融合魏斯氏菌JLP1基因组大小为2 350 892 bp，有效平均GC含量为44.94%，检测到2 020个编码基因。在KEGG数据库中共有1 051个基因得到注释，分别为环境信息处理、新陈代谢和遗传信息处理系统三大功能，共有49条代谢通路。排酸和排过氧化氢试验结果表明，融合魏斯氏菌JLP1抑菌物质并非有机酸和过氧化氢，经蛋白酶K处理后抑菌活性明显下降，推测其抑菌物质为细菌素。在40～80 ℃内融合魏斯氏菌JLP1所产细菌素抑菌活性稳定，100 ℃处理10 min不失活，具有良好的热稳定性；在pH 3.0～8.0内具有良好抑菌活性，酸碱耐受范围较大。电子显微镜扫描结果显示，融合魏斯氏菌JLP1所产细菌素攻击多杀性巴氏杆菌的细胞膜，形成孔隙，细胞表面皱缩。【结论】融合魏斯氏菌JLP1新陈代谢和氨基酸生物合成基因较多，菌株所产细菌素理化特性稳定且抑菌效果良好，为防治多杀性巴氏杆菌感染提供有利参考。

关键词: 多杀性巴氏杆菌; 全基因组测序; 细菌素

Abstract: 【Objective】 The experiment aimed to screen out a bacteriocin of lactic acid bacteria agaist Pasteurella multocida,complete the whole genome sequencing of the isolated lactic acid bacteria,and analyze the physicochemical properties and bactericidal mechanism of its bacteriocin. 【Method】 Healthy cat feces were inoculated in MRS broth medium and cultured at 37 ℃ for 12 h to isolate bacteria.With Pasteurella multocida as the indicator bacteria,the antibacterial activity of the isolated lactic acid bacteria was determined by Oxford cup agar diffusion method and the isolated lactic acid bacteria were identified by amplifying 16S rRNA gene.The whole genome sequence was determined and annotated in the KEGG database.The biological characteristics of the antibacterial substances produced by the isolated bacteria were determined using protease sensitivity,thermal stability and acid-base stability tests.The antibacterial properties of antibacterial substances were explored by using scanning electron microscopy technology. 【Result】 One strain of lactic acid bacteria producing antibacterial substances was screened out.Morphological observation showed that the surface of the colony was smooth and the morphology was small.Gram staining showed positive cocci.16S rRNA sequencing analysis showed that the similarity between the isolated strain and Weisseria confusa was 97%,and it was identified as Weisseria confusa,named JLP1.The results of whole genome sequencing showed that the genome size of Weisseria confusa JLP1 was 2 350 892 bp,with an effective average GC content of 44.94% and 2 020 coding genes.There were a total of 1 051 genes annotated in the KEGG database,which were the three major functions of environmental information processing,metabolism,and genetic information processing systems,with a total of 49 metabolic pathways.The results of acid excretion and hydrogen peroxide excretion test showed that the antibacterial substance of Weisseria confusa JLP1 was not organic acid and hydrogen peroxide,and the antibacterial activity decreased significantly after proteinase K treatment,so it was presumed that the antibacterial substance was bacteriocin.The antibacterial activity of bacteriocin produced by Weisseria confusa JLP1 was stable within 40 to 80 ℃,and did not lose activity after 10 minutes of treatment at 100 ℃,which had good thermal stability.It had good antibacterial activity within pH 3.0-8.0 and a wide range of acid-base tolerance.The scanning results of the electron microscope showed that the bacteriocin produced by Weisseria confusa JLP1 attacked the cell membrane of Pasteurella multocida,forming pores and causing the cell surface to shrink. 【Conclusion】 Weisseria confusa JLP1 had many metabolism and amino acid biosynthesis genes,and the bacteriocin produced by the strain had stable physicochemical properties and good antibacterial effect,providing a beneficial reference for preventing Pasteurella multocida infection.

Key words: Pasteurella multocida; whole genome sequencing; bacteriocin

中图分类号:

S852.61

蒋吕鹏, 潘果, 涂会鑫, 孙思佳, 苏惠, 董文龙, 沙万里, 闫满. 1株抗多杀性巴氏杆菌融合魏斯氏菌的全基因组测序及其产细菌素的理化性质分析[J]. 中国畜牧兽医, 2025, 52(7): 3321-3329.

JIANG Lyupeng, PAN Guo, TU Huixin, SUN Sijia, SU Hui, DONG Wenlong, SHA Wanli, YAN Man. Analysis of Whole Genome Sequencing and Physicochemical Properties of Bacteriocin of a Weisseria confusa Strain Anti-Pasteurella mulcida[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3321-3329.

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