Recently, one of the Journal of Bacteriology Classic Spotlight series highlighted the numerous studies on bacterial spores that have been published in the journal throughout the years. Bacterial endospores, the resilient and relatively quiescent bacterial structures first identified in the 1800s, have had their genetic regulation, immunological properties, and biochemical makeup investigated for decades. The structures are incredibly resistant and produced by select members of the Gram-positive Firmicutes phylum. Despite many rigorous studies investigating these biological structures, new research published in Applied and Environmental Microbiology shows that there's always something new to learn in microbiology, including aspects that appear as straightforward as morphology.
One of the methods that has proved extremely useful in studying spore morphology is transmission electron microscopy (TEM). Some of the original research described in the Classic Spotlight utilized this technique to investigate the structural changes that occur during outgrowth, as a spore germinates to become a vegetative cell again. This is technology allowed scientists to define spore structures: the outermost, electron-dense layer called the exosporium; the laminated underlying spore coat; a thick cortex; and an inner spore coat with a cytoplasmic membrane and germ cell wall (see figure, right).
This same microscopy technique is still used fifty years later for structural studies. Marjorie Pizarro-Guajardo made her initial TEM observations, which revealed two different morphologies in spores from the same Clostridium difficile culture: one with a thick and one with a thin exosporium layer (see image, left). The spores from this original report were purified with different techniques and from different biofilm development timepoints, and so the research team set out to determine which of these factors was behind the different spore types with a follow-up study, now published in AppEnvMicro.
To minimize variables that might affect spore morphology, the team recovered purified C. difficile spores from a single culture and examined the morphology by TEM. The team observed a both thick and thin exosporium morphologies from biofilm and nonbiofilm cultures when using the same strain (hypervirulent strain R20291), but saw that the ratio of thick:thin exosporium changed when the strain was grown as a sporulating culture versus when the strain was grown as an in vitro biofilm (see figure below right).
The biofilms used for experimentation, though still an in vitro growth condition, more closely mimic what researchers have seen during C. difficile infection in a mouse. Finding that biofilm growth affects spore morphology brings up questions of how a mixed species biofilm might affect sporulation . The exosporium outer layer may affect attachment/detachment to the tissue surface or other bacterial cells, and in fact spore morphology could be regulated by biofilm formation itself. A previous Journal of Bacteriology report from a separate group found a transcriptional regulator that regulates sporulation, Spo0A, is also required for normal biofilm formation, suggesting these two processes may be related.
The ability C. difficile to resist antibiotic action through spore formation is one reason this pathogen causes such terrible illness. If scientists can better understand regulation or development of this outer spore layer, this knowledge may provide clues on how to manipulate the spore formation or germination processes. The exosporium layer is also the first layer to interact with the outside environment, and these insights introduce questions regarding its regulation and development. Spore biology, though a classic in many senses, still holds many scientific mysteries, with important applications if and when solved.
-- Julie Wolf