Wang Feifei, Chen Shenjia, Zhou Jinjing, Zhu Ruiyu, Chen Jian, Wang Yanbo
School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, Zhejiang, China.
Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang, China.
Appl Environ Microbiol. 2025 Jun 18;91(6):e0021825. doi: 10.1128/aem.00218-25. Epub 2025 May 7.
is a vital food spoilage bacterium and commonly spoils foods in the form of biofilms. Yet its biofilm regulation strategies have not been fully revealed. Here, we conducted a genome-wide screen of genes important for biofilm regulation using atmospheric and room temperature plasma mutagenesis together with the whole-genome resequencing technology. Three genes (, and ) encoding GGDEF-EAL domain-containing proteins were found to have different mutation manifestations between biofilm cells and free cells. On direct testing, null mutants of and especially exhibited significantly elevated cyclic di-GMP (c-di-GMP) levels. Further studies indicated that a higher level of c-di-GMP caused by the null mutant of triggered cell growth, the production of siderophore and exopolysaccharide as well as autoaggregation, and hindered cell motility, all of which together promote biofilm formation. RNA-sequencing analysis revealed the transcription profile regulated by , mostly including flagellar assembly and peptidoglycan biosynthesis pathways. Therein, the downregulated genes enriched in flagellar assembly were verified by qRT-PCR; the result of which was in agreement with the decreased cell motility.IMPORTANCEBiofilms formed by spoilage bacterium will bring about food quality and safety issues. In this study, we present the establishment of a genetic method and verified its reliability and efficiency for identifying genes associated with biofilm regulation. The genes we discovered offer new perspectives on the mechanisms of biofilm regulation in spoilage bacterium . Moreover, the gene screen method based on atmospheric and room temperature plasma mutagenesis and whole-genome resequencing-coupled technology overcomes the labor-intensive issues caused by traditional methods and should generally be suitable for identifying genes associated with biofilm formation or dispersion in other bacteria.
是一种重要的食品腐败细菌，通常以生物膜的形式使食物变质。然而，其生物膜调控策略尚未完全揭示。在此，我们利用常压室温等离子体诱变技术和全基因组重测序技术，对生物膜调控重要基因进行了全基因组筛选。发现三个编码含GGDEF-EAL结构域蛋白的基因（、和）在生物膜细胞和游离细胞之间具有不同的突变表现。直接测试表明，和尤其是的缺失突变体表现出显著升高的环二鸟苷酸（c-di-GMP）水平。进一步研究表明，缺失突变体导致的较高水平的c-di-GMP触发细胞生长、铁载体和胞外多糖的产生以及自聚集，并阻碍细胞运动，所有这些共同促进生物膜形成。RNA测序分析揭示了由调控的转录谱，主要包括鞭毛组装和肽聚糖生物合成途径。其中，通过qRT-PCR验证了鞭毛组装中下调的基因；其结果与细胞运动性降低一致。重要性腐败细菌形成的生物膜会带来食品质量和安全问题。在本研究中，我们建立了一种遗传方法，并验证了其在鉴定生物膜调控相关基因方面的可靠性和效率。我们发现的基因提供了关于腐败细菌生物膜调控机制的新视角。此外，基于常压室温等离子体诱变和全基因组重测序耦合技术的基因筛选方法克服了传统方法带来的劳动强度大的问题，通常应适用于鉴定其他细菌中与生物膜形成或分散相关的基因。
