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Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli.

Turlin E, Heuck G, Simões Brandão MI, Szili N, Mellin JR, Lange N, Wandersman C - Microbiologyopen (2014)

Bottom Line: We also show that it is disrupted by iron chelation, which reduces the intracellular iron concentration necessary for loading iron into protoporphyrin IX (PPIX, the immediate heme precursor).The MacAB-TolC pump is required for Salmonella typhimurium survival in macrophages.We propose that PPIX is an endogenous substrate of the MacAB-TolC pump in E. coli and S. typhimurium and that this compound is produced inside bacteria when natural heme homeostasis is disrupted by iron shortage, as happens when bacteria invade the mammalian host.

View Article: PubMed Central - PubMed

Affiliation: Unité des Membranes Bactériennes, Département de Microbiologie, Institut Pasteur, 75724, Paris Cedex 15, France.

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PPIX accumulation in various strains grown with or without an iron chelator (Dip). Cells were grown overnight in LB medium and 100- 11-fold-diluted in the morning. When they reached an OD600 of 0.5, Dip was added to half of the cultures at the final concentration of 100 μmol/L. Cells were harvested, when growth stopped for the cultures grown in the presence of Dip and bacterial cultures were diluted to the same OD600 in PBS. (A) Visualization of fluorescence of the soluble fraction under near-UV light. Name of the strains and addition of iron chelator (Dip) are indicated on (A). All experiments were done five times. (B) HPLC analysis of the porphyrin content in the three iron-chelated cultures prepared as described above. The main peak corresponds to PPIX. (C) PPIX concentration in cells grown with or without iron chelator. The same cultures grown with and without Dip were harvested, diluted to the same OD600, and total intracellular porphyrin concentrations were determined by fluorescence spectrometry with a near-UV an excitation. Porphyrin concentrations were normalized so as to represent the porphyrin amount in cell lysates corresponding to a bacterial suspension with an optical density of 100 at 600 nm (OD600 100). The error bars represent the standard deviation for three experiments.
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fig02: PPIX accumulation in various strains grown with or without an iron chelator (Dip). Cells were grown overnight in LB medium and 100- 11-fold-diluted in the morning. When they reached an OD600 of 0.5, Dip was added to half of the cultures at the final concentration of 100 μmol/L. Cells were harvested, when growth stopped for the cultures grown in the presence of Dip and bacterial cultures were diluted to the same OD600 in PBS. (A) Visualization of fluorescence of the soluble fraction under near-UV light. Name of the strains and addition of iron chelator (Dip) are indicated on (A). All experiments were done five times. (B) HPLC analysis of the porphyrin content in the three iron-chelated cultures prepared as described above. The main peak corresponds to PPIX. (C) PPIX concentration in cells grown with or without iron chelator. The same cultures grown with and without Dip were harvested, diluted to the same OD600, and total intracellular porphyrin concentrations were determined by fluorescence spectrometry with a near-UV an excitation. Porphyrin concentrations were normalized so as to represent the porphyrin amount in cell lysates corresponding to a bacterial suspension with an optical density of 100 at 600 nm (OD600 100). The error bars represent the standard deviation for three experiments.

Mentions: In the absence of Dip, cultures were non-fluorescent under near-UV light. In the presence of the iron chelator, the entF strain was slightly fluorescent whereas, entF tolC and entF macAB showed strong fluorescence (Fig. 2A). The total porphyrin intracellular contents were determined by fluorescence spectrometry with a near-UV excitation (Fig. 2C). The total intracellular porphyrins in the entF increased 10 times in the presence of iron chelators as compared to the Dip free medium. This tendency was even more pronounced for the entF macAB and entF tolC strains where a boost of 20 and 25, respectively, was achieved in the absence of iron. HPLC analysis of the porphyrin content in the three iron-chelated cultures confirmed these results and showed that the main compound in the cell extract corresponds to PPIX (Fig. 2B). Therefore, in the absence of intracellular iron to ligate PPIX, there was an accumulation of porphyrins including PPIX which is expelled by the MacAB-TolC pump.


Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli.

Turlin E, Heuck G, Simões Brandão MI, Szili N, Mellin JR, Lange N, Wandersman C - Microbiologyopen (2014)

PPIX accumulation in various strains grown with or without an iron chelator (Dip). Cells were grown overnight in LB medium and 100- 11-fold-diluted in the morning. When they reached an OD600 of 0.5, Dip was added to half of the cultures at the final concentration of 100 μmol/L. Cells were harvested, when growth stopped for the cultures grown in the presence of Dip and bacterial cultures were diluted to the same OD600 in PBS. (A) Visualization of fluorescence of the soluble fraction under near-UV light. Name of the strains and addition of iron chelator (Dip) are indicated on (A). All experiments were done five times. (B) HPLC analysis of the porphyrin content in the three iron-chelated cultures prepared as described above. The main peak corresponds to PPIX. (C) PPIX concentration in cells grown with or without iron chelator. The same cultures grown with and without Dip were harvested, diluted to the same OD600, and total intracellular porphyrin concentrations were determined by fluorescence spectrometry with a near-UV an excitation. Porphyrin concentrations were normalized so as to represent the porphyrin amount in cell lysates corresponding to a bacterial suspension with an optical density of 100 at 600 nm (OD600 100). The error bars represent the standard deviation for three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig02: PPIX accumulation in various strains grown with or without an iron chelator (Dip). Cells were grown overnight in LB medium and 100- 11-fold-diluted in the morning. When they reached an OD600 of 0.5, Dip was added to half of the cultures at the final concentration of 100 μmol/L. Cells were harvested, when growth stopped for the cultures grown in the presence of Dip and bacterial cultures were diluted to the same OD600 in PBS. (A) Visualization of fluorescence of the soluble fraction under near-UV light. Name of the strains and addition of iron chelator (Dip) are indicated on (A). All experiments were done five times. (B) HPLC analysis of the porphyrin content in the three iron-chelated cultures prepared as described above. The main peak corresponds to PPIX. (C) PPIX concentration in cells grown with or without iron chelator. The same cultures grown with and without Dip were harvested, diluted to the same OD600, and total intracellular porphyrin concentrations were determined by fluorescence spectrometry with a near-UV an excitation. Porphyrin concentrations were normalized so as to represent the porphyrin amount in cell lysates corresponding to a bacterial suspension with an optical density of 100 at 600 nm (OD600 100). The error bars represent the standard deviation for three experiments.
Mentions: In the absence of Dip, cultures were non-fluorescent under near-UV light. In the presence of the iron chelator, the entF strain was slightly fluorescent whereas, entF tolC and entF macAB showed strong fluorescence (Fig. 2A). The total porphyrin intracellular contents were determined by fluorescence spectrometry with a near-UV excitation (Fig. 2C). The total intracellular porphyrins in the entF increased 10 times in the presence of iron chelators as compared to the Dip free medium. This tendency was even more pronounced for the entF macAB and entF tolC strains where a boost of 20 and 25, respectively, was achieved in the absence of iron. HPLC analysis of the porphyrin content in the three iron-chelated cultures confirmed these results and showed that the main compound in the cell extract corresponds to PPIX (Fig. 2B). Therefore, in the absence of intracellular iron to ligate PPIX, there was an accumulation of porphyrins including PPIX which is expelled by the MacAB-TolC pump.

Bottom Line: We also show that it is disrupted by iron chelation, which reduces the intracellular iron concentration necessary for loading iron into protoporphyrin IX (PPIX, the immediate heme precursor).The MacAB-TolC pump is required for Salmonella typhimurium survival in macrophages.We propose that PPIX is an endogenous substrate of the MacAB-TolC pump in E. coli and S. typhimurium and that this compound is produced inside bacteria when natural heme homeostasis is disrupted by iron shortage, as happens when bacteria invade the mammalian host.

View Article: PubMed Central - PubMed

Affiliation: Unité des Membranes Bactériennes, Département de Microbiologie, Institut Pasteur, 75724, Paris Cedex 15, France.

Show MeSH
Related in: MedlinePlus