Limits...
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.

Show MeSH

Related in: MedlinePlus

Morphological features of L. monocytogenes L-forms.(A) The size distribution of L-forms (n = 2801) (squares) and its subpopulation containing intracellular vesicles (n = 211) (circles) is represented as the relative frequency per 1 µm size classes. Cells from the whole population have an average diameter of 6.1±3.1 µm. L-forms containing intracellular vesicles have an average diameter of 9.4±4.4 μm. (B) The subpopulation with intracellular vesicles was divided in a group with 1, 2 or 3 intracellular vesicles (squares) and a group with 4 or more intracellular vesicles (circles). Cumulative frequency curves are shown for both groups in incremental steps of 1 µm. Four or more intracellular vesicles have been mainly accumulated by the larger L-form cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368840&req=5

pone-0038514-g001: Morphological features of L. monocytogenes L-forms.(A) The size distribution of L-forms (n = 2801) (squares) and its subpopulation containing intracellular vesicles (n = 211) (circles) is represented as the relative frequency per 1 µm size classes. Cells from the whole population have an average diameter of 6.1±3.1 µm. L-forms containing intracellular vesicles have an average diameter of 9.4±4.4 μm. (B) The subpopulation with intracellular vesicles was divided in a group with 1, 2 or 3 intracellular vesicles (squares) and a group with 4 or more intracellular vesicles (circles). Cumulative frequency curves are shown for both groups in incremental steps of 1 µm. Four or more intracellular vesicles have been mainly accumulated by the larger L-form cells.

Mentions: In soft agar media, L-form cells grow to distinct colonies, which consist of a core of mostly cell debris, surrounded by multiplying L-form cells in the peripheral zones (Figure S1). In contrast to the uniformly rod-shaped parental cells, L-forms are spherical and feature a highly variable size. Cells diameter shows an asymmetrical right-tailed distribution, ranging from 0.5 to 30 µm, with an average of 6.1±3.1 μm (n = 2801) (Figure 1A). To compare the volume of L-form cells with parental bacteria, dimensions of L. monocytogenes Scott A grown for 18 h (BHI, 30°C) were measured (n = 20). The rods of average length 2.3±0.4 µm and width 0.7±0.1 µm represent cylinders, with an average volume of 0.89 µm3. L-forms are largely spherical bodies, and the volume of an average vs. the largest L-form cells observed is 119 vs. 14,137 µm3, which corresponds to a volume increase of approximately 134 and 16,000 compared to the parental cells. The L-form subpopulation with intracellular daughter vesicles (n = 211, corresponding to 7.5% of the total) feature larger cells, with an average diameter of 9.4±4.4 μm. A distribution plot shows that intracellular vesicles accumulate mostly, but not exclusively, in the larger L-forms (Figure 1A). The subpopulation of L-forms containing intracellular vesicles was further subdivided in groups with up to 3, or 4 and more intracellular vesicles. Comparison of the cumulative frequency curves of both groups indicate that the number of intracellular vesicles further increases when mother cells grow larger (Figure 1B). Altogether, growing L-form cells continue to increase in size, and the larger cells may accumulate up to several tens of intracellular vesicles, which then assume most of the cytosolic volume of the mother cell. These vesicle-rich cells are most frequently observed among the distal, outermost regions of a soft-agar grown colony (Figure S1), which is consistent with the idea that L-form multiplication and colony expansion takes place at the periphery.


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)

Morphological features of L. monocytogenes L-forms.(A) The size distribution of L-forms (n = 2801) (squares) and its subpopulation containing intracellular vesicles (n = 211) (circles) is represented as the relative frequency per 1 µm size classes. Cells from the whole population have an average diameter of 6.1±3.1 µm. L-forms containing intracellular vesicles have an average diameter of 9.4±4.4 μm. (B) The subpopulation with intracellular vesicles was divided in a group with 1, 2 or 3 intracellular vesicles (squares) and a group with 4 or more intracellular vesicles (circles). Cumulative frequency curves are shown for both groups in incremental steps of 1 µm. Four or more intracellular vesicles have been mainly accumulated by the larger L-form cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038514-g001: Morphological features of L. monocytogenes L-forms.(A) The size distribution of L-forms (n = 2801) (squares) and its subpopulation containing intracellular vesicles (n = 211) (circles) is represented as the relative frequency per 1 µm size classes. Cells from the whole population have an average diameter of 6.1±3.1 µm. L-forms containing intracellular vesicles have an average diameter of 9.4±4.4 μm. (B) The subpopulation with intracellular vesicles was divided in a group with 1, 2 or 3 intracellular vesicles (squares) and a group with 4 or more intracellular vesicles (circles). Cumulative frequency curves are shown for both groups in incremental steps of 1 µm. Four or more intracellular vesicles have been mainly accumulated by the larger L-form cells.
Mentions: In soft agar media, L-form cells grow to distinct colonies, which consist of a core of mostly cell debris, surrounded by multiplying L-form cells in the peripheral zones (Figure S1). In contrast to the uniformly rod-shaped parental cells, L-forms are spherical and feature a highly variable size. Cells diameter shows an asymmetrical right-tailed distribution, ranging from 0.5 to 30 µm, with an average of 6.1±3.1 μm (n = 2801) (Figure 1A). To compare the volume of L-form cells with parental bacteria, dimensions of L. monocytogenes Scott A grown for 18 h (BHI, 30°C) were measured (n = 20). The rods of average length 2.3±0.4 µm and width 0.7±0.1 µm represent cylinders, with an average volume of 0.89 µm3. L-forms are largely spherical bodies, and the volume of an average vs. the largest L-form cells observed is 119 vs. 14,137 µm3, which corresponds to a volume increase of approximately 134 and 16,000 compared to the parental cells. The L-form subpopulation with intracellular daughter vesicles (n = 211, corresponding to 7.5% of the total) feature larger cells, with an average diameter of 9.4±4.4 μm. A distribution plot shows that intracellular vesicles accumulate mostly, but not exclusively, in the larger L-forms (Figure 1A). The subpopulation of L-forms containing intracellular vesicles was further subdivided in groups with up to 3, or 4 and more intracellular vesicles. Comparison of the cumulative frequency curves of both groups indicate that the number of intracellular vesicles further increases when mother cells grow larger (Figure 1B). Altogether, growing L-form cells continue to increase in size, and the larger cells may accumulate up to several tens of intracellular vesicles, which then assume most of the cytosolic volume of the mother cell. These vesicle-rich cells are most frequently observed among the distal, outermost regions of a soft-agar grown colony (Figure S1), which is consistent with the idea that L-form multiplication and colony expansion takes place at the periphery.

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