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The self-assembly of a cyclic lipopeptides mixture secreted by a B. megaterium strain and its implications on activity against a sensitive Bacillus species.

Pueyo MT, Mutafci BA, Soto-Arriaza MA, Di Mascio P, Carmona-Ribeiro AM - PLoS ONE (2014)

Bottom Line: Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH.The aggregates are inactive per se at the pH of the culture medium which is around 6 or below.The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo SP, Brazil.

ABSTRACT
Cyclic lipopeptides are produced by a soil Bacillus megaterium strain and several other Bacillus species. In this work, they are detected both in the Bacillus intact cells and the cells culture medium by MALDI-TOF mass spectrometry. The cyclic lipopeptides self-assemble in water media producing negatively charged and large aggregates (300-800 nm of mean hydrodynamic radius) as evaluated by dynamic light scattering and zeta-potential analysis. The aggregate size depends on pH and ionic strength. However, it is not affected by changes in the osmolarity of the outer medium suggesting the absence of an internal aqueous compartment despite the occurrence of low molecular weight phospholipids in their composition as determined from inorganic phosphorus analysis. The activity against a sensitive Bacillus cereus strain was evaluated from inhibition halos and B. cereus lysis. Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH. The aggregates are inactive per se at the pH of the culture medium which is around 6 or below. The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.

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MALDI-TOF mass spectra of cyclic lipopeptides from Bacillus megaterium pL6 and other Bacillus species.The spectra for CLP secreted in the culture medium by Bacillus megaterium pL6 as a mixture of surfactins, iturins, lichenysins and fengycins and other low molecular weight substances (600–900 m/z) (a) are also found in intact cells (b). In (c), more than 30 spectra from culture supernatant (labeled A) and whole cells (labeled B) from several Bacillus (B. megaterium, B. cereus, B. lentus, B. laterosporus, B. polymyxa, B. circulans, B. pumilus) were analyzed to obtain the frequency of MALDI-TOF MS peaks versus average m/z of low molecular weight substances (600–900 m/z). The ion 714 m/z was detected practically in all the spectra from whole cells but not found in the supernatant as expected for a cell wall component; compounds found both in the cell culture supernatant and detected in whole cells are assumed to be exported substances (c).
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pone-0097261-g001: MALDI-TOF mass spectra of cyclic lipopeptides from Bacillus megaterium pL6 and other Bacillus species.The spectra for CLP secreted in the culture medium by Bacillus megaterium pL6 as a mixture of surfactins, iturins, lichenysins and fengycins and other low molecular weight substances (600–900 m/z) (a) are also found in intact cells (b). In (c), more than 30 spectra from culture supernatant (labeled A) and whole cells (labeled B) from several Bacillus (B. megaterium, B. cereus, B. lentus, B. laterosporus, B. polymyxa, B. circulans, B. pumilus) were analyzed to obtain the frequency of MALDI-TOF MS peaks versus average m/z of low molecular weight substances (600–900 m/z). The ion 714 m/z was detected practically in all the spectra from whole cells but not found in the supernatant as expected for a cell wall component; compounds found both in the cell culture supernatant and detected in whole cells are assumed to be exported substances (c).

Mentions: The MALDI-TOF mass spectrum of a CLP mixture isolated from the supernatant of a late stationary phase culture of B. megaterium (Figure 1a) is similar to the one obtained for intact cells (Figure 1b). These two spectra however, are different in the sense that the exposition of substances in solution to the laser beam allows their proper ionization but, in the cell, they may not become ionized. The peaks in Figure 1b probably are due to exposed substances at the cell wall surface which are readily ionized by the laser beam. MALDI-TOF MS spectra from intact cells are possibly composed of ions derived from some cell envelope components [23]. Probably, the efficiently ionized substances having high signal intensity are the most exposed to the laser beam. Those released to the culture medium by the bacillus are likely quite exposed whereas those in the cells are the ones caught by the laser beam on the verge of being secreted to the medium. The substances having molecular weights in the 900–1600 m/z range were identified as exported CLP that are always detected both in intact cells and the culture supernatant. Figure 1c shows the gathering of 30 mass spectra from Bacillus megaterium pL6 and other Bacillus species over the 500–850 m/z range with peaks systematically detected in the culture supernatant (labeled as A) and in the washed cells (labeled as B). One should notice that some peaks were detected both for the supernatant samples and the washed cells (A/B). The 655, 656, 664, 671, 688 m/z peaks were obtained both for the supernatant and the cells whereas the 714 and 843 m/z peaks were detected for intact cells only. All these compounds were thought to be lipids due to their behavior in the extraction procedure. For tentative identification, the Lipid Molecular Structure Database (http://www.lmsd.tcd.ie/) was queried suggesting their glycerophospholipidic nature with different head groups. The CLP mixture quoted throughout this work possibly contains a set of these low molecular weight substances. Furthermore, the quantitative determination of phosphorus content for CLP lyophilized samples yielded 176 nanomols of Pi per mg indicating that this may be a correct assumption.


The self-assembly of a cyclic lipopeptides mixture secreted by a B. megaterium strain and its implications on activity against a sensitive Bacillus species.

Pueyo MT, Mutafci BA, Soto-Arriaza MA, Di Mascio P, Carmona-Ribeiro AM - PLoS ONE (2014)

MALDI-TOF mass spectra of cyclic lipopeptides from Bacillus megaterium pL6 and other Bacillus species.The spectra for CLP secreted in the culture medium by Bacillus megaterium pL6 as a mixture of surfactins, iturins, lichenysins and fengycins and other low molecular weight substances (600–900 m/z) (a) are also found in intact cells (b). In (c), more than 30 spectra from culture supernatant (labeled A) and whole cells (labeled B) from several Bacillus (B. megaterium, B. cereus, B. lentus, B. laterosporus, B. polymyxa, B. circulans, B. pumilus) were analyzed to obtain the frequency of MALDI-TOF MS peaks versus average m/z of low molecular weight substances (600–900 m/z). The ion 714 m/z was detected practically in all the spectra from whole cells but not found in the supernatant as expected for a cell wall component; compounds found both in the cell culture supernatant and detected in whole cells are assumed to be exported substances (c).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016289&req=5

pone-0097261-g001: MALDI-TOF mass spectra of cyclic lipopeptides from Bacillus megaterium pL6 and other Bacillus species.The spectra for CLP secreted in the culture medium by Bacillus megaterium pL6 as a mixture of surfactins, iturins, lichenysins and fengycins and other low molecular weight substances (600–900 m/z) (a) are also found in intact cells (b). In (c), more than 30 spectra from culture supernatant (labeled A) and whole cells (labeled B) from several Bacillus (B. megaterium, B. cereus, B. lentus, B. laterosporus, B. polymyxa, B. circulans, B. pumilus) were analyzed to obtain the frequency of MALDI-TOF MS peaks versus average m/z of low molecular weight substances (600–900 m/z). The ion 714 m/z was detected practically in all the spectra from whole cells but not found in the supernatant as expected for a cell wall component; compounds found both in the cell culture supernatant and detected in whole cells are assumed to be exported substances (c).
Mentions: The MALDI-TOF mass spectrum of a CLP mixture isolated from the supernatant of a late stationary phase culture of B. megaterium (Figure 1a) is similar to the one obtained for intact cells (Figure 1b). These two spectra however, are different in the sense that the exposition of substances in solution to the laser beam allows their proper ionization but, in the cell, they may not become ionized. The peaks in Figure 1b probably are due to exposed substances at the cell wall surface which are readily ionized by the laser beam. MALDI-TOF MS spectra from intact cells are possibly composed of ions derived from some cell envelope components [23]. Probably, the efficiently ionized substances having high signal intensity are the most exposed to the laser beam. Those released to the culture medium by the bacillus are likely quite exposed whereas those in the cells are the ones caught by the laser beam on the verge of being secreted to the medium. The substances having molecular weights in the 900–1600 m/z range were identified as exported CLP that are always detected both in intact cells and the culture supernatant. Figure 1c shows the gathering of 30 mass spectra from Bacillus megaterium pL6 and other Bacillus species over the 500–850 m/z range with peaks systematically detected in the culture supernatant (labeled as A) and in the washed cells (labeled as B). One should notice that some peaks were detected both for the supernatant samples and the washed cells (A/B). The 655, 656, 664, 671, 688 m/z peaks were obtained both for the supernatant and the cells whereas the 714 and 843 m/z peaks were detected for intact cells only. All these compounds were thought to be lipids due to their behavior in the extraction procedure. For tentative identification, the Lipid Molecular Structure Database (http://www.lmsd.tcd.ie/) was queried suggesting their glycerophospholipidic nature with different head groups. The CLP mixture quoted throughout this work possibly contains a set of these low molecular weight substances. Furthermore, the quantitative determination of phosphorus content for CLP lyophilized samples yielded 176 nanomols of Pi per mg indicating that this may be a correct assumption.

Bottom Line: Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH.The aggregates are inactive per se at the pH of the culture medium which is around 6 or below.The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo SP, Brazil.

ABSTRACT
Cyclic lipopeptides are produced by a soil Bacillus megaterium strain and several other Bacillus species. In this work, they are detected both in the Bacillus intact cells and the cells culture medium by MALDI-TOF mass spectrometry. The cyclic lipopeptides self-assemble in water media producing negatively charged and large aggregates (300-800 nm of mean hydrodynamic radius) as evaluated by dynamic light scattering and zeta-potential analysis. The aggregate size depends on pH and ionic strength. However, it is not affected by changes in the osmolarity of the outer medium suggesting the absence of an internal aqueous compartment despite the occurrence of low molecular weight phospholipids in their composition as determined from inorganic phosphorus analysis. The activity against a sensitive Bacillus cereus strain was evaluated from inhibition halos and B. cereus lysis. Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH. The aggregates are inactive per se at the pH of the culture medium which is around 6 or below. The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.

Show MeSH
Related in: MedlinePlus