Recombinant S-layer production induces disordered cell division in E. coli filaments

The rod-shaped bacterium Escherichia coli is one of the best studied microorganism with a size of 1.1-1.5 µm x 2.0-6.0 µm. We used E. coli BL21 (DE3), one of the most widely used host in genetic engineering, for heterologous expression of surface layer (S-layer) proteins to enable fast and efficient protein production.
S-layer are proteins which cover the outermost of many prokaryotes and are probably the basic and oldest forms of bacterial envelope. These proteins are mostly composed of protein and glycoprotein monomers and have the ability to self-assemble into two-dimensional arrays on interfaces. Several characteristics like their work as molecular sieve, as virulence factor or the protection of the cell from toxic heavy metal ions make S-layer proteins interesting for their usage as ultrafiltration membranes, drug microcontainers, filter materials or patterning structures in nanotechnology.
Surprisingly, the heterologous expression of S-layer proteins of the uranium mining waste pile isolate Lysinibacillus sphaericus JG-A12 induced drastic morphological changes of E. coli BL21 (DE3) single cells to filaments and single cell enclosing tubes of >100 µm in length. The assumed secretion of tube-stabilizing S-layer proteins was investigated with SDS-PAGE and ß-galactosidase assay. These analyses result in a high S-layer appearance without significant ß-galactosidase activity in the supernatant and the periplasm. The origin and composition of filaments and tubes were analysed by membrane stain studies. We identified that filaments in the exponential growth phase form a continuous intracellular space without partitioning. To investigate the mechanism of filament and tube formation we analyzed GFP/S-layer expressing E. coli with DAPI-stain. The staining showed >50 µm long DNA-fibres that cross the filaments and “DNA-free” areas, the latter exhibiting strong GFP-expression. Our results point to a disordered cell division in E. coli filaments which is effected by recombinant S-layer expression.

[1] Lederer et al. (2010) Heterologous expression of the surface-layer-like protein SllB induces the formation of long filaments of Escherichia coli consisting of protein-stabilized outer membrane. Microbiology 156,3584-95.