一株群体感应抑制活性链霉菌的筛选鉴定及其毒力因子代谢能力分析

doi:10.6048/j.issn.1001-4330.2024.04.026

新疆农业科学 ›› 2024, Vol. 61 ›› Issue (4): 1011-1020.DOI: 10.6048/j.issn.1001-4330.2024.04.026

• 植物保护·微生物·农业装备工程与机械化 • 上一篇下一篇

一株群体感应抑制活性链霉菌的筛选鉴定及其毒力因子代谢能力分析

柏晓玉¹^,²(), 朱丽英³, 包慧芳², 江凌⁴, 朱静², 顾美英², 朱艳蕾¹, 张志东¹^,²()

1.新疆师范大学生命科学学院,乌鲁木齐 830054
2.新疆农业科学院微生物应用研究所/新疆特殊环境微生物实验室,乌鲁木齐 830091
3.南京工业大学化学与分子工程学院,南京 211816
4.南京工业大学食品与轻工学院,南京 211816

收稿日期:2023-08-22 出版日期:2024-04-20 发布日期:2024-05-31
通信作者: 张志东(1977-),男,新疆乌鲁木齐人,研究员,博士,硕士生/博士生导师,研究方向为特殊环境微生物及益生菌资源挖掘与应用,(E-mail)zhangzheedong@qq.com
作者简介:柏晓玉(1997-),女,山东益都人,硕士研究生,研究方向为微生物生态(E-mail)1306439822@qq.com
基金资助:
新疆维吾尔自治区青年科技拔尖人才项目-青年科技创新人才培养(2022TSYCCX0067);新疆农业科学院科技创新重点培育专项(xjkcpy-2021002);国家自然科学基金项目(32060004);新疆维吾尔自治区自然科学基金项目“杰出青年基金”(2022D01E19)

Screening and identification of a Streptomyces strain with quorum sensing inhibitory activity and effect of the crude extracts on virulence factors of Erwinia amylovora

BAI Xiaoyu¹^,²(), ZHU Liying³, BAO Huifang², JIANG Ling⁴, ZHU Jing², GU Meiying², Zhu Yanlei¹, ZHANG Zhidong¹^,²()

1. College of Life Sciences, Xinjiang Normal University, Urumqi 830054, China
2. Institute of Microbiology, Xinjiang Academy of Agricultural Sciences/Xinjiang Key Laboratory of Special Environmental Microbiology, Urumqi 830091, China
3. School of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing 211816, China
4. College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, China

Received:2023-08-22 Published:2024-04-20 Online:2024-05-31
Correspondence author: ZHANG Zhidong(1977-),male, from Urumqi,Xinjiang,master, researcher, research direction: Special environmental microorganisms and food Microbiology,(E-mail)zhangzheedong@qq.com
Supported by:
Xinjiang Uygur Autonomous Region Young Science and Technology Top Talent project-young Science and technology innovation personnel training(2022TSYCCX0067);Key Cultivation Project of Science and Technology Innovation by Xinjiang Academy of Agricultural Sciences(xjkcpy-2021002);National Natural Science Foundation of China(32060004);Xinjiang Natural Science Foundation for Distinguished Young Scholars(2022D01E19)

摘要/Abstract

摘要：

【目的】挖掘干旱区放线菌天然产物代谢潜力,筛选群体感应抑制活性的产物,研究其对梨火疫病原菌毒力因子代谢能力的影响。【方法】利用紫色杆菌(Chromobacterium violaceum 026, CV026)群体感应抑制活性筛选模型,对新疆库木塔格沙漠周边干旱地区土壤中分离的放线菌进行发酵,采用牛津杯法对其发酵液进行活性筛选;鉴定群体感应抑制活性菌株及验证其发酵液提取和活性,分析粗提液对欧文氏菌毒力因子代谢影响,并采用液质联用(liquid chromatography and mass spectrometry, LC-MS)方法检测菌株代谢产物活性物质,挑选相似结构化合物验证群体感应抑制活性。【结果】获得一株具有明显群体感应抑制活性菌株K-9,其与Streptomyces rubradiris strain NBRC 14000^T相似性达到99.72%。该菌发酵粗提液具有明显的群体感应抑制活性,可有效降低紫色杆菌CV026产紫色素和梨火疫病原菌的游动能力,并对梨火疫病原菌生物膜、胞外多糖和胞外酶等毒力因子产生具有显著抑制活性,呈明显的浓度依赖性;从链霉菌K-9粗提液中得到635种化合物,苯基丙烯酸、4-羟基-3甲氧基苯甲酸等5种明显群体感应抑制化合物。【结论】干旱区放线菌K-9为Streptomyces rubradiris,其发酵代谢产物具有明显群体感应抑制活性,能显著抑制欧文氏菌毒力因子的产生,并发现了多种新型的群体感应抑制活性化合物。

关键词: 群体感应抑制剂; 干旱地区放线菌; 欧文氏菌; 毒力因子

Abstract:

【Objective】 To explore the potential metabolisms for natural product compounds from actinomycetes in the arid area and investigate effect of the quorum sensing inhibition on virulence factors in Erwinia amylovora. 【Methods】 Chromobacterium violaceum 026 (CV026) was used for the screening model of the quorum sensing inhibitions, the fermentation of actinomycetes isolated from soils in arid areas around the Kumutag Desert was carried out, and the quorum sensing inhibitory activity of the fermentation broth was tested using the Oxford Cup method. The strain was identified by the 16S rRNA gene sequence analysis, and effect of crude extracts from the broth on virulence factors of Erwinia amylovora was measured. In addition to that, liquid chromatography and mass spectrometry (LC-MS) was used to measure metabolites, and compounds with similar structure to reported quorum sensing inhibitors were selected to validate. 【Results】 Isolate K-9 was screened and identified to belong to the genus Streptomyces, which showed strong quorum sensing inhibitory activity and shared the highest similarities with Streptomyces rubradiris NBRC 14000^T(99.72%). The crude extracts of the broth effectively reduced the violacein production of CV026 and the swimming ability of Erwinia amylovora. Virulence factors of Erwinia amylovora such as biofilm, pyocyanin and rhamnolipid were inhabited significantly with the concentration-dependent interactions. A total of 635 compounds were identified from the crude extract by LC-MS, and 5 compounds with significant quorum sensing inhibitory activity were observed. 【Conclusion】 Streptomyces sp. K-9 is one potential new species isolated from arid areas, whose fermentation metabolites shows obvious quorum sensing inhibitory activity, and can inhibit virulence factors produced by Pseudomonas aeruginosa. Additionally, many kinds of novel quorum sensing inhibitors were obtained.

Key words: quorum sensing inhibition; actinomycete from arid area; Erwinia amylovora; virulence factors

中图分类号:

S188

柏晓玉, 朱丽英, 包慧芳, 江凌, 朱静, 顾美英, 朱艳蕾, 张志东. 一株群体感应抑制活性链霉菌的筛选鉴定及其毒力因子代谢能力分析[J]. 新疆农业科学, 2024, 61(4): 1011-1020.

BAI Xiaoyu, ZHU Liying, BAO Huifang, JIANG Ling, ZHU Jing, GU Meiying, Zhu Yanlei, ZHANG Zhidong. Screening and identification of a Streptomyces strain with quorum sensing inhibitory activity and effect of the crude extracts on virulence factors of Erwinia amylovora[J]. Xinjiang Agricultural Sciences, 2024, 61(4): 1011-1020.

图/表 11

图1 菌株K-9的筛选及菌落形态注:A: 菌株K-9不同稀释梯度的发酵液对紫色杆菌CV026紫色色素的抑制作用; B: 菌株K-9菌落形态;C: 菌株K-9菌落显微形态

Fig.1 Screening and morphological characteristics of strain K-9 Note:A: Inhibition of violacein of Chromobacterium violaceum CV026 by fermentation broth of strain K-9 with different dilution gradients; B: Colony morphology of strain K-9; C: Morphological characteristics of the colony under stereomicroscope

图2 基于菌株 K-9及相关近源菌株16S rRNA 基因序列的NJ系统进化树注:Bootstrap检验次数为1 000次;分支结点上的数字为自举值(显示≥50%的数值);标尺为0.01

Fig.2 Neighbor-Joining tree based on 16S rRNA gene sequences showing the phylogenetic relationships among K-9 strain and other related strains Note:Bootstrap values (≥50%) based on 1,000 replications are shown at branch nodes. Bar 0.01 sequence variation

图3 粗提液TLC图及活性成分生物显影注:A:粗提物层析在254 nm波长下显影结果;B:粗提物中活性成分的生物自显影

Fig.3 Analysis of crude extracts of strain K-9 by bioautography of active ingredients and TLC Note:A: TLC of crude extract on 254 nm; B: Bioautography of active components in crude extract

表1 不同浓度粗提液下菌株生长变化

Tab.1 Effect of crude extracts with different concentrations on the growth of strains

粗提液浓度 Concentration of crude extracts (mg/mL)	紫色杆菌026 Chromobacterium violaceum 026	梨火疫病原菌 Erwinia amylovora
10	-	-
5	-	W
2.5	W	+
1.25	+	+
0.6	+	+
0.3	+	+
0.15	+	+

图4 不同浓度粗提液下紫色杆菌CV026紫色素产量变化注:***:对照组和处理组差异极其显著(P<0.001);**对照组和处理组差异极显著(P<0.01),下同

Fig.4 Changes of crude extracts with different concentrations on violacein production of CV026 Note:***: means extremely significant difference between control and treatment (P<0.001); **:means highly significant difference between control and treatment (P<0. 01),the same as below

图5 不同浓度粗提液下梨火疫病原菌生物膜变化

Fig.5 Changes of crude extracts with different concentrations on biofilm production by Erwinia amylovora

图6 不同浓度粗提液下胞外多糖产量的变化注:*:对照组和处理组显著差异(P<0. 05);无*指两组无显著差异,下同

Fig.6 Changes of crude extracts with different concentrations on rhamnolipid production by Erwinia amylovora Note:* :means significant difference between control and treatment(P<0. 05); No * :means significant difference between two groups,the same as below

图7 不同浓度粗提液下梨火疫病原菌产蛋白酶变化

Fig.7 Changes of crude extracts with different concentrations on protease production by Erwinia amylovora

图8 不同浓度粗提液下梨火疫病原菌群集和泳动性变化注:A: 梨火疫病原菌群集;B:梨火疫病原菌泳动。下标1~5分别为阳性对照,0.6、1.2、2.5、5 mg/mL 粗提液

Fig.8 Changes of different crude extracts on swarming and swimming of Erwinia amylovora Note:A: Erwinia amylovora swarming; B: Erwinia amylovora swimming. Subscripts 1-5 were positive controls, 0.6, 1.2, 2.5, and 5.0 mg/mL crude extract

图9 菌株K-9粗提液总离子流注:A:粗提液正离子流;B:粗提液负离子流

Fig.9 Total ionic chromatographic fingerprints of crude extract of strain K-9 Note:A: positive ionic chromatographic fingerprints of crude extract; B: negative ionic chromatographic fingerprints of crude extract

表2 8种化合物的抑菌和群体感应抑制活性检测

Tab.2 Detection of Inhibition and Quorum sensing inhibitory activity of 8 compounds

化合物 Compounds	抑菌面积 Inhibition area (cm²)	紫色素透明圈 Violacein inhibition area (cm²)
苯基丙烯酸 Trans-Cinnamic acid	-	4.15
4-羟基-3甲氧基苯甲酸 Vanillic acid	-	3.14
吡咯-2-羧酸 Pyrrole-2-carboxylic acid	0.2	1.57
3,4-二羟苯基丙酸 3,4-Dihydroxyhydro cinnamic acid	0.2	1.54
DL-4-羟基苯乳酸 DL-4-Hydroxyphenyllactic acid	-	0.79
哌啶酸 Pipecolic acid	-	-
甘氨酸三甲胺内盐 Betaine	-	-
4-羟基-3,5-二甲氧基苯甲酸 Syringic acid	-	-

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一株群体感应抑制活性链霉菌的筛选鉴定及其毒力因子代谢能力分析

Screening and identification of a Streptomyces strain with quorum sensing inhibitory activity and effect of the crude extracts on virulence factors of Erwinia amylovora

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