细菌纤维素(BC)是一种可再生的生物材料,具有独特的特性,有望用于生物技术和生物医学。KomagataeibacterhanseniiATCC53,582是行业中使用的特征良好的BC高产生产商。它的基因组编码三种不同的纤维素合酶(CS),bcsAB1,bcsAB2和bcsAB3与辅助蛋白的基因一起组织在不同复杂性的操纵子中。通过构建CSs和两种预测的调节性二鸟苷酸环化酶(DGC)的染色体框内缺失,研究了其高纤维素生产表型的遗传基础,dgcA和dgcB。蛋白质组学表征表明,BcsAB1是决定性的CS,因为它的高表达及其对微晶纤维素形成的独家贡献。通过扫描电子显微镜判断,BcsAB2显示出较低的表达水平,但对BC的拉伸强度有显着贡献,并且显着改变了纤维直径。然而,静态培养后,未从该操纵子中鉴定出明显的细胞外聚合物(EPS)。尽管观察到bcsAB3的转录,蛋白质的表达低于蛋白质组分析的检测限。与BcsAB2类似,BcsAB3的缺失导致纤维素纤维直径的可见减小。大量的BcsD和辅助蛋白CmcAx,CcpAx,和BglxA强调它们对于纤维素网络的适当形成的重要性。缺乏DGC基因dgcA和dgcB的缺失突变体的表征表明了K.hanseniiATCC53,582中纤维素合成和细胞运动的新调节机制。我们的发现为合理定制BC特征奠定了基础。关键点:•BcsAB1诱导微晶纤维素纤维的形成。•BcsAB2和BcsAB3的改性改变纤维素纤维的直径。•DGC对纤维素薄膜形成和运动性的复杂调节网络。
Bacterial cellulose (BC) represents a renewable biomaterial with unique properties promising for biotechnology and biomedicine. Komagataeibacter hansenii ATCC 53,582 is a well-characterized high-yield producer of BC used in the industry. Its genome encodes three distinct cellulose synthases (CS), bcsAB1, bcsAB2, and bcsAB3, which together with genes for accessory proteins are organized in operons of different complexity. The genetic foundation of its high cellulose-producing phenotype was investigated by constructing chromosomal in-frame deletions of the CSs and of two predicted regulatory diguanylate cyclases (DGC), dgcA and dgcB. Proteomic characterization suggested that BcsAB1 was the decisive CS because of its high expression and its exclusive contribution to the formation of microcrystalline cellulose. BcsAB2 showed a lower expression level but contributes significantly to the tensile strength of BC and alters fiber diameter significantly as judged by scanning electron microscopy. Nevertheless, no distinct extracellular polymeric substance (EPS) from this operon was identified after static cultivation. Although transcription of bcsAB3 was observed, expression of the protein was below the detection limit of proteome analysis. Alike BcsAB2, deletion of BcsAB3 resulted in a visible reduction of the cellulose fiber diameter. The high abundance of BcsD and the accessory proteins CmcAx, CcpAx, and BglxA emphasizes their importance for the proper formation of the cellulosic network. Characterization of deletion mutants lacking the DGC genes dgcA and dgcB suggests a new regulatory mechanism of cellulose synthesis and cell motility in K. hansenii ATCC 53,582. Our findings form the basis for rational tailoring of the characteristics of BC. KEY POINTS: • BcsAB1 induces formation of microcrystalline cellulose fibers. • Modifications by BcsAB2 and BcsAB3 alter diameter of cellulose fibers. • Complex regulatory network of DGCs on cellulose pellicle formation and motility.