Mn2+促进Bacillus altitudinis LZP02生物膜形成

doi:10.13560/j.cnki.biotech.bull.1985.2023-0888

摘要/Abstract

摘要：

【目的】 Bacillus altitudinis LZP02是一株水稻根际促生菌（PGPR），能定殖于水稻根部，形成生物膜，且Mn²⁺对LZP02菌株生物膜形成具有促进作用，但调控机制尚不明确，旨在探究Mn²⁺对LZP02菌株生物膜形成的促进机制。【方法】 采用结晶紫染色法进行生物膜定量分析、蒽酮-硫酸法测定胞外多糖产量、扫描电镜（SEM）观察LZP02菌株在水稻根际定殖情况、转录组学测序技术分析差异表达基因（DEGs）。【结果】 添加4 mmol/L Mn²⁺和8 mmol/L Mn²⁺显著提高了LZP02菌株的成膜能力和胞外多糖产量，扫描电镜发现4 mmol/L Mn²⁺和8 mmol/L Mn²⁺提高了LZP02菌株在水稻根际的定殖能力，随着Mn²⁺浓度的增加，DEGs数量显著增多，其主要富集在芽孢形成、毒素代谢途径和双组分系统中。1 mmol/L Mn²⁺和4 mmol/L Mn²⁺处理组发现skf操纵子中基因的表达均上调。扫描电镜观察发现在1 mmol/L Mn²⁺和4 mmol/L Mn²⁺处理组中LZP02菌体存在损伤。4 mmol/L Mn²⁺处理组双组分系统中KinE和Spo0A基因均显著上调。【结论】 Mn²⁺通过“嗜食同类”和双组分系统中KinE基因激活Spo0A~P提高了菌株LZP02的成膜能力。

关键词: Bacillus altitudinis LZP02, 生物膜, Mn²⁺

Abstract:

【Objective】 Bacillus altitudinis LZP02 is a plant growth-promoting rhizobacteria（PGPR）in the rice rhizosphere, which can colonize rice rhizosphere to form biofilm, and Mn²⁺ can promote the biofilm formation of LZP02 strain, but the regulation mechanism is not clear. The purpose of this study is to explore the mechanism of Mn²⁺ promoting the biofilm formation of LZP02 strain. 【Method】 Crystal violet staining and anthrone-sulfuric acid method were used to perform quantitative analysis of biofilm and exopolysaccharide yield, respectively. Scanning electron microscope（SEM）was used to observe LZP02 colonization in rice rhizosphere. And differently expression genes（DEGs）were analyzed by transcriptome sequencing technology.【Result】 The addition of 4 mmol/L Mn²⁺and 8 mmol/L Mn²⁺ significantly enhanced the film-forming ability of LZP02 strain and the production of exopolysaccharide, scanning electron microscopy showed that 4 mmol/L Mn²⁺ and 8 mmol/L Mn²⁺ improved the colonization ability of LZP02 strain in rice rhizosphere. The number of DEGs increased significantly with the increase of Mn²⁺ concentration, which was mainly enriched in spore formation, toxin metabolism pathway and two-component system. The expression of skf operon gene was up-regulated in 1 mmol/L Mn²⁺and 4 mmol/L Mn²⁺ treatments. Scanning electron microscope observation showed that there was damage in LZP02 cells in 1 mmol/L Mn²⁺ and 4 mmol/L Mn²⁺ treatments. The genes of KinE and Spo0A in the two-component system of 4 mmol/L Mn²⁺treatment group were significantly up-regulated. 【Conclusion】 Mn²⁺ can improve the biofilm-forming ability of strain LZP02 by “cannibalism” and KinE gene activates Spo0A~P in two-component system.

Key words: Bacillus altitudinis LZP02, biological film, Mn²⁺

单馨, 黄东慧, 徐伟慧, 王志刚, 陈文晶, 胡云龙. Mn²⁺促进Bacillus altitudinis LZP02生物膜形成[J]. 生物技术通报, 2024, 40(3): 251-260.

SHAN Xin, HUANG Dong-hui, XU Wei-hui, WANG Zhi-gang, CHEN Wen-jing, HU Yun-long. Mn²⁺ Promotes the Biofilm Formation of Bacillus altitudinis LZP02[J]. Biotechnology Bulletin, 2024, 40(3): 251-260.

图/表 9

表1 引物序列

Table 1 Primer sequences

Gene	Primer sequence（5'-3'）	Length/bp
Spo0A	F-GCTCGGCAGCATTACAAA	215
Spo0A	R-TCAGCTTATCCGCAACCA	215
Spo0F	F-CCCGACCTTGTGCTGTTG	172
Spo0F	R-GCTTGGCAAAGTGCGTCA	172
KinB	F-TTCCGCTCCAGTGTTCTT	272
KinB	R-TTTGTGATAATGCCTTGC	272
KinE	F-TTAAAGCGACTTGGTGAA	159
KinE	R-TCTGACAGGAAGCGTAAT	159
KapB	F-CTTATGACGGTGAAGTGC	117
KapB	R-CCAGATTATGAAGGGAGC	117
16S rRNA	F-TGTGTAGCGGTGAAATGCG	140
16S rRNA	R-CATCGTTTACGGCGTGGAC	140

图1 不同浓度的Mn2+对LZP02生物膜形成的影响 A: 固-气表面的菌落形态；B: 液-气表面菌落形态；C: 生物膜在OD540处的吸收值；D: 胞外多糖含量。不同小写字母表示处理间差异显著（P < 0.05）

Fig. 1 Effect of different Mn2+ concentration on the biofilm formation of LZP02 A: The colony architectures on solid-gas surface. B: The colony architectures on liquid-gas surface. C: Absorbance of biofilm at OD540. D: Extracellular polysaccharide content. The different lowercase letters indicate significant differences among treatments（P < 0.05）

图2 不同浓度Mn2+对LZP02菌株在水稻根际上定殖及成膜的影响

Fig. 2 Effect of different Mn2+ concentration on the colonization and film formation of LZP02 strain in rice rhizosphere

图3 差异基因表达量火山图

Fig. 3 Volcano plot of differential gene expression

图4 差异基因富集分析 A、B、C: GO富集分析；D: KEGG富集分析

Fig. 4 Enrichment analysis of differential genes A, B, C: GO enrichment analysis. D: KEGG enrichment analysis

图5 LZP02菌株双组分系统分析 A: LZP02菌株双组分系统流程图；B: 转录组中双组分系统相关基因的Log2FC

Fig. 5 Two-component system analysis in LZP02 strain A: Two-component system flow chart in LZP02 strain; B: Log2FC of genes related to two-component system in transcriptome

图6 LZP02菌株芽孢形成途径分析 A: 不同处理组显著下调基因数量；B: 在M-1处理组中显著下调基因；C: M-4处理组和M-8处理组下调基因Venn图；D: M-4处理组和M-8处理组共同下调基因表达量热图；E: 不同浓度的Mn2+对LZP02 芽孢形成的影响. ***P<0.001

Fig. 6 Pathway analysis of spore formation in strain LZP02 A: The number of significantly down-regulated genes in different treatment groups. B: Significantly down-regulated genes in M-1 treatment group. C: The Venn map of down-regulated genes in M-4 and M-8 treatment groups. D: The heatmap of co-downregulating genes in M-4 and M-8 treatment groups. E: Effect of different Mn2+ concentrations on the formation of LZP02 spore. ***P<0.001

图7 LZP02菌株毒素代谢途径分析 A: 毒素途径差异基因热图；B: skf基因簇；C: 转录组中相关基因的Log2FC；D: LZP02中破损细胞的扫描电镜观察

Fig. 7 Analysis of toxin metabolic pathways in strain LZP02 A: The heatmap of DEGs in toxin biosynthesis pathway. B: The skf gene cluster. C: Log2FC of related genes in transcriptome. D: Electron microscopic observation of damaged cells in LZP02

图8 差异表达基因RT-qPCR验证 A: 转录组中5个差异基因的Log2FC值；B: RT-qPCR中5个差异基因的表达

Fig. 8 RT-qPCR verification of differential expressed genes A: Log2FC values of five DGEs in the transcriptome. B: Gene expression of five DGEs in RT-qPCR

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Mn²⁺促进Bacillus altitudinis LZP02生物膜形成

Mn²⁺ Promotes the Biofilm Formation of Bacillus altitudinis LZP02

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