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Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

Briers Y, Staubli T, Schmid MC, Wagner M, Schuppler M, Loessner MJ - PLoS ONE (2012)

Bottom Line: Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release.Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition.Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species.

View Article: PubMed Central - PubMed

Affiliation: Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland.

ABSTRACT
Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

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Related in: MedlinePlus

Enterococcus L-form cells.E. faecium (A) and E. faecalis (B) L-form cells were generated and stained with Rho123 (10 µg ml−1) to indicate charged membranes. In contrast to Listeria L-forms, the intracellular vesicles of Enterococcus L-forms still enclosed in the mother cell cytoplasm frequently feature stronger phase contrast (upper panels) and more intense Rho123 accumulation and fluorescence (lower panels).
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pone-0038514-g007: Enterococcus L-form cells.E. faecium (A) and E. faecalis (B) L-form cells were generated and stained with Rho123 (10 µg ml−1) to indicate charged membranes. In contrast to Listeria L-forms, the intracellular vesicles of Enterococcus L-forms still enclosed in the mother cell cytoplasm frequently feature stronger phase contrast (upper panels) and more intense Rho123 accumulation and fluorescence (lower panels).

Mentions: An interesting aspect was to determine whether intracellular vesicle formation as a mechanism for multiplication may be more generally used, i.e., by other members of the genus Listeria and related bacteria. Towards this end, strains of all 8 recognized species (L. monocytogenes, L. innocua, L. grayi, L. seeligeri, L. welshmeri, L. ivanovii, L. marthii, and L. rocourtiae) could be successfully transformed into stable L-forms. Moreover L-form lines obtained from different members of the genes Enterococcus (E. faecalis, E. faecium, E. hirae, and E. durans) (Table S1, Figure S2) also showed a similar phenotype and multiplication properties. While the Enterococcus L-form morphology with respect to shape and arrangement is highly similar to Listeria, the number of intracellular vesicles per mother cell is significantly higher, and growth in soft agar medium occurs much faster. Colonies appear within 2 days, compared to 6 days with Listeria. Interestingly, a majority of the Enterococcus faecium (Figure 7A) and faecalis (Figure 7B) L-forms feature intracellular vesicles with high cytoplasmic density [16], which often appears darker than the mother cell cytoplasm. We also found that Enterococcus daughter vesicles frequently established a membrane potential (Rho123 accumulation) while still being encased by the mother cell. The larger number of intracellular vesicles, together with the higher maturation frequency inside the maternal L-form cells, may correlate to their faster growth compared to Listeria L-forms.


Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

Briers Y, Staubli T, Schmid MC, Wagner M, Schuppler M, Loessner MJ - PLoS ONE (2012)

Enterococcus L-form cells.E. faecium (A) and E. faecalis (B) L-form cells were generated and stained with Rho123 (10 µg ml−1) to indicate charged membranes. In contrast to Listeria L-forms, the intracellular vesicles of Enterococcus L-forms still enclosed in the mother cell cytoplasm frequently feature stronger phase contrast (upper panels) and more intense Rho123 accumulation and fluorescence (lower panels).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368840&req=5

pone-0038514-g007: Enterococcus L-form cells.E. faecium (A) and E. faecalis (B) L-form cells were generated and stained with Rho123 (10 µg ml−1) to indicate charged membranes. In contrast to Listeria L-forms, the intracellular vesicles of Enterococcus L-forms still enclosed in the mother cell cytoplasm frequently feature stronger phase contrast (upper panels) and more intense Rho123 accumulation and fluorescence (lower panels).
Mentions: An interesting aspect was to determine whether intracellular vesicle formation as a mechanism for multiplication may be more generally used, i.e., by other members of the genus Listeria and related bacteria. Towards this end, strains of all 8 recognized species (L. monocytogenes, L. innocua, L. grayi, L. seeligeri, L. welshmeri, L. ivanovii, L. marthii, and L. rocourtiae) could be successfully transformed into stable L-forms. Moreover L-form lines obtained from different members of the genes Enterococcus (E. faecalis, E. faecium, E. hirae, and E. durans) (Table S1, Figure S2) also showed a similar phenotype and multiplication properties. While the Enterococcus L-form morphology with respect to shape and arrangement is highly similar to Listeria, the number of intracellular vesicles per mother cell is significantly higher, and growth in soft agar medium occurs much faster. Colonies appear within 2 days, compared to 6 days with Listeria. Interestingly, a majority of the Enterococcus faecium (Figure 7A) and faecalis (Figure 7B) L-forms feature intracellular vesicles with high cytoplasmic density [16], which often appears darker than the mother cell cytoplasm. We also found that Enterococcus daughter vesicles frequently established a membrane potential (Rho123 accumulation) while still being encased by the mother cell. The larger number of intracellular vesicles, together with the higher maturation frequency inside the maternal L-form cells, may correlate to their faster growth compared to Listeria L-forms.

Bottom Line: Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release.Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition.Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species.

View Article: PubMed Central - PubMed

Affiliation: Institute of Food, Nutrition and Health, ETH Zurich, Zurich, Switzerland.

ABSTRACT
Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

Show MeSH
Related in: MedlinePlus