Analysis of cellulose synthesis in a high-producing acetic acid bacterium Komagataeibacter hansenii

Abstract

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.

Implementation of a Novel Method for Processing Proteins from Acetic Acid Bacteria via Liquid Chromatography Coupled with Tandem Mass Spectrometry

“…Other authors have used proteomic procedures [ 16 , 19 , 25 , 44 , 54 ], as well as those based on other omics technologies [ 20 , 27 , 55 , 56 , 57 ], to determine key metabolic processes of AAB, such as incomplete oxidation of ethanol to acetic acid, the metabolism of biomolecules (amino acids, nucleic acids, and fatty acids), energy generation related-pathways (TCA cycle and pentose phosphate pathway), and membrane-dependent high acidity stress response mechanisms, among others. LC–MS/MS allowed the determination of key metabolic processes and strategies for vinegar production and other biotechnological applications of interest to AAB [ 39 ].…”