皮氏不动杆菌LSQ 3对贝莱斯芽孢杆菌LSQ 19成膜能力的影响及其基因组特性分析

Influence of Acinetobacter pittii LSQ 3 on the biofilm formation of Bacillus velezensis LSQ 19 and its genomic characteristics

摘要:

目的 探究菌株皮氏不动杆菌(Acinetobacter pittii) LSQ 3对贝莱斯芽孢杆菌(Bacillus velezensis) LSQ 19成膜能力的影响，以及菌株LSQ 3的基因组特性。方法 采用结晶紫染色法、细胞表面特性分析、苯酚硫酸法、XTT还原法和扫描电镜(scanning electron microscope, SEM)观察等方法分析LSQ 3无细胞上清液(cell-free supernatant, CFS)对LSQ 19成膜能力的影响；采用全基因组学测序明确菌株LSQ 3的分类学地位，并通过全基因组数据预测其次级代谢产物基因簇。结果 菌株LSQ 3的CFS显著抑制LSQ 19的生物膜形成，10 μL菌液/190 μL CFS混合确定为最小抑菌浓度(minimum inhibitory concentration, MIC)。与对照组相比，经菌株LSQ 3的CFS处理(MIC)后LSQ 19细胞的表面疏水性、黏附力、胞外聚合物(extracellular polymeric substances, EPS)产率和生物膜代谢活性均显著降低；自聚集能力与对照组相比显著升高。扫描电镜观察表明，在MIC条件下菌株LSQ 19在玻璃表面未形成生物膜。经全基因组学鉴定，菌株LSQ 3为Acinetobacter pittii，其基因组大小为3 939 365 bp，G+C含量为38.82%，含有3 601个DNA编码序列。其基因组中存在多个与生物膜合成相关的基因及毒力因子。通过antiSMASH分析发，菌株LSQ 3的基因组中含有7种次级代谢物的生物合成基因簇。结论 A. pittii LSQ 3的无细胞上清液可抑制B. velezensis LSQ 19生物膜的形成，本研究从生物膜的角度为合成菌群的构建提供了理论依据和参考。

Abstract:

Objective To explore the influence of Acinetobacter pittii LSQ 3 on the biofilm formation of Bacillus velezensis LSQ 19 and analyze the genomic characteristics of strain LSQ 3.Methods The effect of the cell-free supernatant (CFS) of LSQ 3 on the biofilm formation of LSQ 19 was analyzed by crystal violet staining, cell surface property analysis, the phenol-sulfuric acid method, XTT reduction assay, and scanning electron microscopy (SEM). Whole-genome sequencing was employed to determine the taxonomic status of strain LSQ 3, and the biosynthetic gene clusters for secondary metabolites were predicted based on the whole-genome data.Results The CFS of strain LSQ 3 significantly inhibited the biofilm formation of strain LSQ 19, and the volume ratio of 10 μL bacterial suspension to 190 μL CFS was determined as the minimum inhibitory concentration (MIC). Compared with the control, the CFS of strain LSQ 3 at MIC significantly reduced the surface hydrophobicity, adhesion, extracellular polymeric substances (EPS) production rate, and biofilm metabolic activity, while significantly improving the self-aggregation ability of LSQ 19 cells. In the presence of the CFS at MIC, LSQ 19 failed to form a biofilm on the glass surface. Strain LSQ 3 was identified as A. pittii based on whole-genome sequencing data. Its genome size was 3 939 365 bp, with the G+C content of 38.82% and 3 601 DNA coding sequences. The genome contained multiple genes involved in biofilm formation and virulence factors. The antiSMASH analysis showed that the genome of strain LSQ 3 contained seven biosynthetic gene clusters for secondary metabolites.Conclusion The CFS of A. pittii LSQ 3 can inhibit the biofilm formation of B. velezensis LSQ 19. This study provides a theoretical basis and reference for the construction of synthetic bacterial communities from the perspective of biofilms.