Clostridium thermosulfurogenes EM1 formed
blebs, i.e., protrusions still in contact with the cytoplasmic membrane, that originated from the cytoplasmic membrane during growth in batch culture and continuous culture. They could be observed squeezed between the cell wall and cytoplasmic membrane in cells with seemingly intact wall layers (surface layer and
peptidoglycan layer) as well as in cells with wall layers in different states of degradation caused by
phosphate limitation or high dilution rates.
Blebs were found to turn into membrane vesicles by constriction in cases when the cell wall was heavily degraded.
Bleb and vesicle formation was also observed in the absence of substrates that induce
alpha-amylase and
pullulanase synthesis. No correlations existed between
bleb formation and the presence of active
enzyme. Similar
blebs could also be observed in a number of other gram-positive bacteria not producing these
enzymes, but they were not observed in gram-negative bacteria. For immunoelectron-microscopic localization of
alpha-amylase and
pullulanase in C. thermosulfurogenes EM1, two different
antisera were applied. One was raised against the
enzymes isolated from the culture fluid; the other was produced against a
peptide synthesized, as a defined
epitope, in analogy to the N-terminal amino acid sequence (21
amino acids) of the native extracellular
alpha-amylase. By using these
antisera,
alpha-amylase and
pullulanase were localized at the cell periphery in samples taken from continuous culture or batch culture. In samples prepared for electron microscopy by freeze substitution followed by ultrathin sectioning,
blebs could be seen, and the immunolabel pinpointing
alpha-amylase enzyme particles was seen not only randomly distributed in the cell periphery, but also lining the surface of the cytoplasmic membrane and the
blebs. Cells exhibiting high or virtually no
enzyme activity were labeled similarly with both
antisera. This finding strongly suggests that
alpha-amylase and
pullulanase may occur in both active and inactive forms, depending on growth conditions.