Large scale identification of pellicle and cell-free liquid phase associated proteins in Bacillus amyloliquefaciens L-17

Abstract

Bacillus amyloliquefaciens is a soil-associated and plant growth-promoting bacterium. It is the focus of numerous studies due to its ability to sporulate, form biofilms, produce antimicrobial peptides and commercial enzymes. The ability of B. amyloliquefaciens l-17 to form floating biofilm at the air-liquid interface “pellicle” was previously demonstrated. This pellicle exhibits a highly structured architecture which is provided by loosely and tightly matrix bound polysaccharides and proteins. In this study, a first large scale proteomic investigation of both the pellicle and the cell-free liquid phase of l-17 strain was performed. An approach based on physical and chemical extraction of the pellicular matrix combined with protein analysis by mass spectrometry identified 87 weakly matrix-bound proteins and 62 tightly bound proteins. A total of 131 pellicle-associated proteins were identified, including (i) the conserved proteins TasA and TapA, involved in biofilm formation and cohesion (ii) BslA, important for biofilm hydrophobicity (iii) several enzymes that make nutrients available and protect the biofilm from competitors (iv) flagellin and (v) proteins involved in the sporulation process. Proteomic characterization of the cell-free liquid phase underlying the analyzed pellicle allowed the identification of 423 proteins including 118 proteins yet identified in the matrix of the pellicle. The proteins identified specifically in the liquid phase include enzymes involved in the biosynthesis process of non-ribosomal peptides and a variety of commercial enzymes such as proteases, lipases, aminotransferases, peroxidases and phytases. This provides valuable clues to promote the industrial and agricultural application of the cell-free liquid phase of B. amyloliquefaciens l-17.