S layer regeneration in Methanococcus voltae protoplasts

Abstract

The regeneration of the paracrystalline proteinaceous cell wall (S layer) of the methanogen Methanococcus woltae was examined by electron microscopy. The S layer was removed from the cell surface by exposing the cells to a protoplasting buffer but was completely regenerated by 60-80 min resuspension in a regeneration medium by some repair mechanism (normal cell generation time is 16 h). The protoplast surface appeared in freeze-fracture to be initially smooth and featureless and remained so for the first 20 min in regeneration medium. Thereafter, nascent S layer appeared on the surface in the form of paracrystalline patches of various sizes that were distributed over the entire surface. The patches were closely associated with disturbances of the cell surface curvature which may have been caused by electrostatic interactions. The steps leading to the complete regeneration of the S layer could not be followed due t o the sampling times used, but presumably the patches grew large enough to contact neighbouring patches and somehow fused into a coherent layer. Other ultrastructural changes occurred to the cell envelope during S layer regeneration, most notably the recurrence of the intramembrane particles of the concave fracture face of the plasma membrane. Flagella remained attached to the protoplasts and were apparently still functional. The major changes to the protein profile of untreated cells, protoplasts and regenerating protoplasts included the loss and resynthesis of polypeptides at the 76 kDa (5 layer polypeptide) and 60 kDa (unknown polypeptide) positions. SDS-PAGE evidence indicated that new S layer polypeptides were synthesized 20 min prior to their appearance as patches on the cell surface. The S layer polypeptide may have been shed from the surface during the early stages of regeneration to form the extracellular sheet-like material observed in large quantity in the regeneration medium. The response of the cell to changes that occur at the cell surface indicates that some feedback mechanism gears the synthesis of S layer and associated envelope components.

CRISPR-mediated gene silencing reveals involvement of the archaeal S-layer in cell division and virus infection

“…However, as live-dead staining before thin sectioning showed that both large and regularly-sized cells were indeed viable and contained DNA ( Supplementary Fig. 10), we conclude that cell wall depletion destabilized the cells to such a degree that they became more susceptible to damages during the preparation for electron microscopy, as previously observed in other S-layer mutants 47 . When silenced and control cultures were exposed to increasing NaCl concentrations, a general decrease of the average cell size was observed, which was slightly higher in SB3×6 (23% diameter decrease) than in Ctrl cultures (19% diameter decrease) (Fig.…”

Expression, secretion and antigenic variation of bacterial S‐layer proteins

“…An additional S-layer is formed on the inner face of cell walls after disruption of Bacillus stearothermophilus cells and isolation of cellwall fragments (Breitwieser et al, 1992). S-protein overproduction also explains the fast regeneration of the S-layer after it has been extracted by non-lethal procedures, as noted for Lactobacillus helveticus (Lortal et al, 1992), and Methanococcus voltae (Firtel et al, 1995). In contrast Corynebacterium glutamicum cells have patches of uncovered cell wall when grown on liquid medium to stationary phase (Chami et al, 1995).…”

Surface proteins and a novel transcription factor regulate the expression of the S‐layer gene in Thermus thermophilus HB8

“…In addition to this function, these 5ЈUTR of the S-layer protein mRNAs could also be implicated in the stability of their transcripts (Fisher et al, 1988;Chu et al, 1993;, as has been demonstrated for the E. coli OmpA mRNA (Melefors and Gabian, 1988). These two mechanisms would help to explain the fast regeneration of the S-layer observed under some experimental conditions (Lortal et al, 1992;Firtel et al, 1995).…”