Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community

Abstract

In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3-to 7-fold in EPS. Also, EPS proteins were more basic by ϳ2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as ␤-N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.

Biofilm Matrix Proteins

“…Several studies have been carried out to identify the matrix proteome of several microorganisms, including Vibrio cholerae (19), Pseudomonas aeruginosa (20), Myxococcus xanthus (21) and natural biofilm communities of acid mine drainage (22). These studies revealed that, in addition to secreted proteins, the biofilm matrix also contains large numbers of periplasmic, cytoplasmic, inner, and outer membrane proteins.…”

Floc Formation Reduces the pH Stress Experienced by Microorganisms Living in Alkaline Environments

“…In addition to EPS, inorganic materials (minerals) may also be incorporated to provide structural support where physical stresses may impact survival (13). Biofilm formation is known to enhance survival against a range of environmental stresses, such as pH shifts (14), with some biofilm communities creating more favorable growth conditions by secreting specific components of EPS (15,16). In these cases, biofilm formation enables microbial propagation in extreme environments (16,17).…”

New Technologies for Studying Biofilms

“…Shotgun proteomics has now been applied to biofilm samples for metaproteomics studies of cellular and extracellular proteins (197, 198) and for characterizing the effects of biofilm growth, environmental conditions, and intercellular signaling of individual strains of biofilm organisms (e.g., 199–203). Shotgun proteomics uses direct digestion of a complex protein sample with trypsin and LCMS analysis of the ensuing peptide mixture (204).…”