In this study, we characterized the effect of several stressors on C. thermocellum.
Our results show that C. thermocellum
is generally tolerant of many of the stressors that it was exposed to, such as low phosphorous, low nitrogen, and added inhibitory substances such as acetate and ethanol. C. thermocellum
was less tolerant of vitamin deficiency, exposure to oxygen and changes in the types of available carbon source, each of which triggered spore formation. The sporulation response observed as a result of alternating carbon source between cellobiose and Avicel was surprising, as C. thermocellum
can grow equally well on each. One possible explanation for this effect may be that C. thermocellum
produces a large protein complex, known as the cellulosome, which acts to break down insoluble substrates [17
]. The cellulosome is important for growth on cellulose, and its constituent parts are expressed at lower levels when C. thermocellum
is grown on soluble substrates such as cellobiose [17
]. The change in enzyme requirements and production after a change in substrate may induce enough stress to cause a sporulation response, as was observed in this study.
The response leading to the L-form resting state was more dramatic than the response that lead to spores, with approximately 98% of the cells in the culture transitioning to the L-form state when starved of cellobiose. It is interesting to note that L-forms were not observed at the end of a continuous cellulose fermentation (data not shown), indicating the dramatic exhaustion of available substrate may be an important trigger for L-forms. Once in the L-form state, no growth was detected by base addition, optical density, or viable counts, and end-product analysis via HPLC indicated no further production of ethanol, acetic acid, or lactic acid, the normal endproducts of C. thermocellum
metabolism. However, L-forms did remain viable at 108
CFU/ml at the time of formation. Additionally, once L-forms were inoculated into new media with adequate carbon source, they resumed growth as normal rod-shaped cells. The most cited definition of L-forms defines them as an alternative growth state [26
]. This is because in some cases L-forms are able to divide by a process similar to budding [25
], or via reproduction within the L-form and subsequent release after the lysis of the mother cell [36
]. Reproduction of L-forms was not observed in C. thermocellum
cultures, though many of the L-forms did have small dark protrusions, previously observed and hypothesized to be budding cells in Haemophilus influenzae
], but never conclusively determined to be such [21
]. Quantification of C. thermocellum
L-forms over time to determine how many persisted in culture indicated only decreases in cell population over time (data not shown), indicating cell death, not proliferation. However, because C. thermocellum
L-forms are induced by severe nutrient limitation, it is difficult to assess their ability to grow and divide as the necessary nutrients needed to promote normal growth are absent during C. thermocellum
L-form formation and cultivation.
Traditionally, most lab-cultured L-forms are induced by treatment with antibiotics that target the cell wall. In this case, cells may escape the deleterious effects of this treatment by transitioning to the L-form state. This has been proposed as a method for pathogenic organisms to survive in a host in spite of antibiotic treatment [38
]. However,the development of L-forms is not limited to antibiotic treatments. Other authors have postulated that the L-form state constitutes a means for cells to escape an unfavorable growth environment [30
] or as a biologically relevant state in non-lab environments [33
]. In Markova et al., E. coli
L-forms were seen to form after exposure to extreme heat stress, and after prolonged cultivation in minimal media. Several accounts of Borrelia burgdorferi
L-forms (also referred to as cysts or round-bodies) were observed after starvation conditions [31
], in which serum-minus media and water were each used for induction. In one such study, Alban et al. determined that once sufficient nutrients were supplied, up to 52% of the L-forms were able to revert back to normal cells, though this number decreased with increasing age of the culture. These instances of L-form induction and recovery closely mirror what we observe in Clostridium thermocellum
. The destruction of the cell wall, or the failure to maintain it, may be representative of a cell struggling to keep or obtain the energy needed for survival.
Once we determined that C. thermocellum L-forms were viable, we questioned why the cells would form an L-form rather than remain rod-shaped or form a spore. It seemed unlikely that L-forms were deformed or unformed spores, as defects in spore formation manifest in identifiable stages, none of which resemble the L-form. We therefore hypothesized that L-form formation provided some advantage for C. thermocellum. One potential explanation is that transitioning to an L-form requires less energy than sporulation or conserves energy overall for the cell. It is also possible that L-forms provide some advantage over spores or rod-shaped cells in terms of survival or recovery. Testing the first scenario effectively would have been technically difficult, so we went about testing the second hypothesis.
To compare spores, rod-shaped cells, and L-forms in terms of survivability and recovery, we tested how well each cell type tolerated heat and how quickly each could resume growth. C. thermocellum spores proved to be much better at tolerating heat stress than L-forms or rod-shaped cells suggesting advantages for C. thermocellum spores in prolonged survival under other stressful conditions. L-forms did not survive heat stress as well as spores, but did exhibit a shorter lag-phase upon recovery when compared with both spores and stationary phase cells, each of which took over 9 hours longer to begin exponential growth. While L-forms demonstrated faster recovery, L-form viability over time was consistent with that of stationary phase cells when subjected to prolonged starvation. This suggests that the primary advantage for C. thermocellum in forming an L-form does not lie in enhanced viability over time, but rather in the ability to recover rapidly when conditions become favorable for growth. This feature may allow for L-from cells to out-compete other non-growing cells in natural environments.What molecular or physiological triggers come into play to determine whether a cell becomes spore, an L-form or remain rod shaped remain to be explored.