A simple method to produce Synechocystis PCC6803 biofilm under laboratory conditions for electron microscopic and functional studies

PLoS One. 2020 Jul 30;15(7):e0236842. doi: 10.1371/journal.pone.0236842. eCollection 2020.

Abstract

Cyanobacteria can form biofilms in nature, which have ecological roles and high potential for practical applications. In order to study them we need biofilm models that contain healthy cells and can withstand physical manipulations needed for structural studies. At present, combined studies on the structural and physiological features of axenic cyanobacterial biofilms are limited, mostly due to the shortage of suitable model systems. Here, we present a simple method to establish biofilms using the cyanobacterium Synechocystis PCC6803 under standard laboratory conditions to be directly used for photosynthetic activity measurements and scanning electron microscopy (SEM). We found that glass microfiber filters (GMF) with somewhat coarse surface features provided a suitable skeleton to form Synechocystis PCC6803 biofilms. Being very fragile, untreated GMFs were unable to withstand the processing steps needed for SEM. Therefore, we used polyhydroxybutyrate coating to stabilize the filters. We found that up to five coats resulted in GMF stabilization and made possible to obtain high resolution SEM images of the structure of the surface-attached cells and the extensive exopolysaccharide and pili network, which are essential features of biofilm formation. By using pulse-amplitude modulated variable chlorophyll fluorescence imaging, it was also demonstrated that the biofilms contain photosynthetically active cells. Therefore, the Synechocystis PCC6803 biofilms formed on coated GMFs can be used for both structural and functional investigations. The model presented here is easy to replicate and has a potential for high-throughput studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biofilms / growth & development*
  • Cell Membrane / metabolism*
  • Cell Membrane / ultrastructure
  • Microscopy, Electron, Scanning / methods*
  • Polysaccharides, Bacterial / metabolism*
  • Polysaccharides, Bacterial / ultrastructure
  • Synechocystis / growth & development*
  • Synechocystis / metabolism
  • Synechocystis / ultrastructure*

Substances

  • Polysaccharides, Bacterial

Grants and funding

I.M., P.K., F.B., P.B.K., and I.V. were supported by the Hungarian Ministry for National Economy (https://www.kormany.hu/en) GINOP-2.3.2-15-2016-00001. M.S. was supported by the Hungarian Academy of Sciences (www.mta.hu), MTA Premium Postdoctoral Research Program (grant ID: PREMIUM-2017-38) and by the National Research, Development and Innovation Office (https://nkfih.gov.hu/, grant ID: NKFIH FK 128977). I.D. was supported by the Hungarian Ministry for National Economy (https://www.kormany.hu/en) GINOP-2.3.2-15-2016-00058. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.