Epifluorescence, SEM, TEM and nanoSIMS image analysis of the cold phenotype of Clostridium psychrophilum at subzero temperatures.

We have applied an image-based approach combining epifluorescence microscopy, electron microscopy and nanoscale secondary ion mass spectrometry (nanoSIMS) with stable isotope probing to examine directly the characteristic cellular features involved in the expression of the cold phenotype in the Antarctic bacterium Clostridium psychrophilum exposed to a temperature range from +5 to -15 °C under anoxic conditions. We observed dramatic morphological changes depending on temperature. At temperatures below -10 °C, cell division was inhibited and consequently filamentous growth predominated. Bacterial cells appeared surrounded by a remarkably thick cell wall and a capsule formed of long exopolysaccharide fibres. Moreover, bacteria were entirely embedded within a dense extracellular matrix, suggesting a role both in cryo-protection and in the cycling of nutrients and genetic material. Strings of extracellular DNA, transient cell membrane permeability and release of membrane vesicles were observed that suggest that evolution via transfer of genetic material may be especially active under frozen conditions. While at -5 °C, the bacterial population was metabolically healthy, at temperatures below -10 °C, most cells showed no sign of active metabolism or the metabolic flux was extremely slowed down; instead of being consumed, carbon was accumulated and stored in intracellular granules as in preparation for a long-term survival.