Huijboom Linda, Tempelaars Marcel, Fan Mingzhen, Zhu Yourong, Boeren Sjef, van der Linden Erik, Abee Tjakko
Food Microbiology, Wageningen University & Research, Bornse Weilanden 9, 6708, WG, Wageningen, the Netherlands.
Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, Wageningen, 6708, WE, the Netherlands.
Res Microbiol. 2023 Jul-Aug;174(6):104072. doi: 10.1016/j.resmic.2023.104072. Epub 2023 Apr 18.
Bacillus cereus is a food-borne pathogen capable of producing biofilms. Following analysis of biofilm formation by B. cereus ATCC 14579 transposon mutants in defined medium (DM), a deletion mutant of bc2939 (Δbc2939) was constructed that showed decreased crystal violet biofilm staining and biofilm cell counts. In addition, Δbc2939 also produced smaller colony biofilms with lower cell counts and loss of wrinkly morphology. The bc2939 gene encodes for Prephenate dehydrogenase, which converts Prephenate to 4-Hydroxy-phenylpyruvate (4-HPPA) in the l-tyrosine branch of the Shikimate pathway. While growth of the mutant and WT in DM was similar, addition of l-tyrosine was required to restore WT-like (colony) biofilm formation. Comparative proteomics showed reduced expression of Tyrosine-protein kinase/phosphatase regulators and extracellular polysaccharide cluster 1 (EPS1) proteins, aerobic electron transfer chain cytochrome aa3/d quinol oxidases, and iso-chorismate synthase involved in menaquinone synthesis in DM grown mutant biofilm cells, while multiple oxidative stress-related catalases and superoxide dismutases were upregulated. Performance in shaking cultures showed a 100-fold lower concentration of menaquinone-7 and reduction in cell counts of DM grown Δbc2939 indicating increased oxygen sensitivity. Combining all results, points to an important role of Tyrosine-modulated EPS1 production and menaquinone-dependent aerobic respiration in B. cereus ATCC 14579 (colony) biofilm formation.
蜡样芽孢杆菌是一种能够产生生物膜的食源性病原体。在对蜡样芽孢杆菌ATCC 14579转座子突变体在限定培养基（DM）中形成生物膜进行分析后，构建了bc2939基因的缺失突变体（Δbc2939），该突变体表现出结晶紫生物膜染色减少和生物膜细胞计数降低。此外，Δbc2939还产生了较小的菌落生物膜，细胞数量减少且失去了褶皱形态。bc2939基因编码预苯酸脱氢酶，该酶在莽草酸途径的L-酪氨酸分支中将预苯酸转化为4-羟基苯丙酮酸（4-HPPA）。虽然突变体和野生型在DM中的生长相似，但需要添加L-酪氨酸来恢复野生型样（菌落）生物膜的形成。比较蛋白质组学显示，在DM中生长的突变体生物膜细胞中，酪氨酸蛋白激酶/磷酸酶调节剂和细胞外多糖簇1（EPS1）蛋白、有氧电子传递链细胞色素aa3/d泛醇氧化酶以及参与甲基萘醌合成的异分支酸合酶的表达降低，而多种与氧化应激相关的过氧化氢酶和超氧化物歧化酶上调。摇瓶培养结果显示，DM中生长的Δbc2939的甲基萘醌-7浓度降低了100倍，细胞计数减少，表明其对氧气的敏感性增加。综合所有结果，表明酪氨酸调节的EPS1产生和甲基萘醌依赖性有氧呼吸在蜡样芽孢杆菌ATCC 14579（菌落）生物膜形成中起重要作用。
