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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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L-form cells feature intracellular viable progeny vesicles.The viability of progeny vesicles was assessed by GFP synthesis and fluorescence (A–C), spatial distribution of Rho123 (D–F), and reductive metabolism of a tetrazolium dye (G–I). Intracellular vesicles either showed no signals, faint signals, or strong signals, in some cases stronger than the mother cell. Panel J shows baseline corrected and normalized Raman spectra of the average background control (lower curve, n = 10), mother cell cytoplasm with a strong GFP signal (middle curve, n = 13) and non-fluorescent large intracellular vesicles (upper curve, n = 18). The different spectral curves have been offset but the scale was retained. Relevant peaks are indicated by red boxes, and correspond to vibration signals from RNA/DNA (785 cm−1, 1583 cm−1), and proteins (1003 cm−1, 1583 cm−1 and 1666 cm−1). These molecules are present both in the mother cell cytoplasm and, albeit less pronounced, inside the intracellular progeny vesicles. Panel K shows a box plot of Raman intensities measured for the background (buffer), mother cell cytoplasm, and intracellular vesicles at 1666 cm−1, which reflects protein content. The median intensity is lower in intracellular vesicles compared to the mother cell cytoplasm, indicating a slightly lower protein content in the progeny vesicles.
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pone-0038514-g003: L-form cells feature intracellular viable progeny vesicles.The viability of progeny vesicles was assessed by GFP synthesis and fluorescence (A–C), spatial distribution of Rho123 (D–F), and reductive metabolism of a tetrazolium dye (G–I). Intracellular vesicles either showed no signals, faint signals, or strong signals, in some cases stronger than the mother cell. Panel J shows baseline corrected and normalized Raman spectra of the average background control (lower curve, n = 10), mother cell cytoplasm with a strong GFP signal (middle curve, n = 13) and non-fluorescent large intracellular vesicles (upper curve, n = 18). The different spectral curves have been offset but the scale was retained. Relevant peaks are indicated by red boxes, and correspond to vibration signals from RNA/DNA (785 cm−1, 1583 cm−1), and proteins (1003 cm−1, 1583 cm−1 and 1666 cm−1). These molecules are present both in the mother cell cytoplasm and, albeit less pronounced, inside the intracellular progeny vesicles. Panel K shows a box plot of Raman intensities measured for the background (buffer), mother cell cytoplasm, and intracellular vesicles at 1666 cm−1, which reflects protein content. The median intensity is lower in intracellular vesicles compared to the mother cell cytoplasm, indicating a slightly lower protein content in the progeny vesicles.

Mentions: We monitored viability and metabolic processes of the intracellular vesicles using several reporters. Synthesis and maturation/oxidation of GFP was used as an indicator for active transcription and translation processes (Figure 3A-C). Spatial distribution of the cationic membrane-permeable dye Rhodamine123 (Rho123) served as indicator for an existing membrane potential, as well as charge and polarity of intracellular vesicle membranes [12]. Rho123 is only retained in compartments with a “negative inside” membrane potential, and distributes across lipid compartments according to the highest polarity (Figure 3D-F). In addition, metabolic reduction of a colorless tetrazolium salt into a red formazan dye was used as indicator for respiratory activity and oxidative metabolism (Figure 3G-I) [12]. While a majority of the intracellular vesicles did not appear to be positive for these indicators, others showed clear signs of respiratory and metabolic activities, and presence of a membrane potential. This suggests that only a fraction of the intracellular vesicles inside the vesiculated L-forms may be viable and active, while most of them seem to be inactive and show no attributes of life.


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)

L-form cells feature intracellular viable progeny vesicles.The viability of progeny vesicles was assessed by GFP synthesis and fluorescence (A–C), spatial distribution of Rho123 (D–F), and reductive metabolism of a tetrazolium dye (G–I). Intracellular vesicles either showed no signals, faint signals, or strong signals, in some cases stronger than the mother cell. Panel J shows baseline corrected and normalized Raman spectra of the average background control (lower curve, n = 10), mother cell cytoplasm with a strong GFP signal (middle curve, n = 13) and non-fluorescent large intracellular vesicles (upper curve, n = 18). The different spectral curves have been offset but the scale was retained. Relevant peaks are indicated by red boxes, and correspond to vibration signals from RNA/DNA (785 cm−1, 1583 cm−1), and proteins (1003 cm−1, 1583 cm−1 and 1666 cm−1). These molecules are present both in the mother cell cytoplasm and, albeit less pronounced, inside the intracellular progeny vesicles. Panel K shows a box plot of Raman intensities measured for the background (buffer), mother cell cytoplasm, and intracellular vesicles at 1666 cm−1, which reflects protein content. The median intensity is lower in intracellular vesicles compared to the mother cell cytoplasm, indicating a slightly lower protein content in the progeny vesicles.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368840&req=5

pone-0038514-g003: L-form cells feature intracellular viable progeny vesicles.The viability of progeny vesicles was assessed by GFP synthesis and fluorescence (A–C), spatial distribution of Rho123 (D–F), and reductive metabolism of a tetrazolium dye (G–I). Intracellular vesicles either showed no signals, faint signals, or strong signals, in some cases stronger than the mother cell. Panel J shows baseline corrected and normalized Raman spectra of the average background control (lower curve, n = 10), mother cell cytoplasm with a strong GFP signal (middle curve, n = 13) and non-fluorescent large intracellular vesicles (upper curve, n = 18). The different spectral curves have been offset but the scale was retained. Relevant peaks are indicated by red boxes, and correspond to vibration signals from RNA/DNA (785 cm−1, 1583 cm−1), and proteins (1003 cm−1, 1583 cm−1 and 1666 cm−1). These molecules are present both in the mother cell cytoplasm and, albeit less pronounced, inside the intracellular progeny vesicles. Panel K shows a box plot of Raman intensities measured for the background (buffer), mother cell cytoplasm, and intracellular vesicles at 1666 cm−1, which reflects protein content. The median intensity is lower in intracellular vesicles compared to the mother cell cytoplasm, indicating a slightly lower protein content in the progeny vesicles.
Mentions: We monitored viability and metabolic processes of the intracellular vesicles using several reporters. Synthesis and maturation/oxidation of GFP was used as an indicator for active transcription and translation processes (Figure 3A-C). Spatial distribution of the cationic membrane-permeable dye Rhodamine123 (Rho123) served as indicator for an existing membrane potential, as well as charge and polarity of intracellular vesicle membranes [12]. Rho123 is only retained in compartments with a “negative inside” membrane potential, and distributes across lipid compartments according to the highest polarity (Figure 3D-F). In addition, metabolic reduction of a colorless tetrazolium salt into a red formazan dye was used as indicator for respiratory activity and oxidative metabolism (Figure 3G-I) [12]. While a majority of the intracellular vesicles did not appear to be positive for these indicators, others showed clear signs of respiratory and metabolic activities, and presence of a membrane potential. This suggests that only a fraction of the intracellular vesicles inside the vesiculated L-forms may be viable and active, while most of them seem to be inactive and show no attributes of life.

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