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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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Inhibition halos and lytic activity of CLP from Bacillus megaterium pL6 against Bacillus cereus.On panel (a), the inhibition halo B against Bacillus cereus confluent layer C is induced by Bacillus megaterium pL6 cultivated inside the hole A. On panel (b), the withdrawal of the antibiotic activity produced by B. megaterium by cells of B. cereus is a function of the interaction time (50 µL of a cyclolipopeptides solution at 10 mg/mL were incubated at 37°C with 2 mL stationary cell culture of B. cereus for 15, 30 and 120 minutes before withdrawing 70 µL samples for centrifugation and determination of supernatant residual activity against B. cereus from inhibition haloes); the control was performed in the absence of B. cereus cells. On panel (c), the withdrawal of the antibiotic activity from B. cereus is seen in the presence of two different ionic strengths (0.05 and 0.4 M NaCl). Bacillus cereus cells were grown up to stationary phase, concentrated 10 times before adding 100 µL of 10 mg/mL cyclolipopeptides solution at pH 8.0 to 2 mL of the concentrated B. cereus suspension LB for 0.5 or 45 minutes interaction; thereafter mixtures were centrifuged and resuspended in water for removal of biosurfactant excess; 70 µL of the pelleted cells were resuspended in the NaCl solutions and centrifuged again before withdrawal of 60 µL of each supernatant for determining the inhibition haloes against B. cereus after incubation for 9 h at 37°C. Controls were 60 µL of the Bsf solution (10 mg/mL) or 0.4 M NaCl solution only. On panel (d), the pH of the biosurfactant solution affects the inhibition halo of B. cereus. The pH was adjusted to 2, 4, 6 and 8 by adding 1N HCl or 1N NaOH to 0.2 mL of biosurfactant solutions before loading 0.1 mL of each solution onto the holes of a Petri dish containing B. cereus; the control was 0.1 mL milli Q water adjusted to pH 2.
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pone-0097261-g002: Inhibition halos and lytic activity of CLP from Bacillus megaterium pL6 against Bacillus cereus.On panel (a), the inhibition halo B against Bacillus cereus confluent layer C is induced by Bacillus megaterium pL6 cultivated inside the hole A. On panel (b), the withdrawal of the antibiotic activity produced by B. megaterium by cells of B. cereus is a function of the interaction time (50 µL of a cyclolipopeptides solution at 10 mg/mL were incubated at 37°C with 2 mL stationary cell culture of B. cereus for 15, 30 and 120 minutes before withdrawing 70 µL samples for centrifugation and determination of supernatant residual activity against B. cereus from inhibition haloes); the control was performed in the absence of B. cereus cells. On panel (c), the withdrawal of the antibiotic activity from B. cereus is seen in the presence of two different ionic strengths (0.05 and 0.4 M NaCl). Bacillus cereus cells were grown up to stationary phase, concentrated 10 times before adding 100 µL of 10 mg/mL cyclolipopeptides solution at pH 8.0 to 2 mL of the concentrated B. cereus suspension LB for 0.5 or 45 minutes interaction; thereafter mixtures were centrifuged and resuspended in water for removal of biosurfactant excess; 70 µL of the pelleted cells were resuspended in the NaCl solutions and centrifuged again before withdrawal of 60 µL of each supernatant for determining the inhibition haloes against B. cereus after incubation for 9 h at 37°C. Controls were 60 µL of the Bsf solution (10 mg/mL) or 0.4 M NaCl solution only. On panel (d), the pH of the biosurfactant solution affects the inhibition halo of B. cereus. The pH was adjusted to 2, 4, 6 and 8 by adding 1N HCl or 1N NaOH to 0.2 mL of biosurfactant solutions before loading 0.1 mL of each solution onto the holes of a Petri dish containing B. cereus; the control was 0.1 mL milli Q water adjusted to pH 2.

Mentions: Figure 2 (a) exhibits the inhibition halo induced by Bacillus megaterium pL 6 cells against a highly sensitive Bacillus cereus strain. The Petri dish was mounted with two layers of Luria Broth agar (LB) with 1.5% agar at the bottom and 0.8% agar on the top. The hole in the plate center (A) in the top layer was seeded with 0.1 mL of a fresh suspension of B. megaterium pL 6 cells cultured up to the stationary phase. The top agar layer surrounding the central hole was previously mixed with 0.3 mL of a fresh stationary B. cereus LB culture. After incubation at 37°C overnight, a visible inhibition halo (B) contrasted with the growth of B. cereus in the region (C) of the Petri dish. The inhibition halo indicates that B. megaterium pL6 cells release substances which diffuse through the agar preventing the growth of B. cereus. The radial diffusion occurs roughly in two dimensions, differently from the tridimensional process taking place in nature when species of microorganisms compete for the same ecological niche [24]. Figure 2 (b) shows the withdrawal of the CLP by B. cereus over a range of interaction times. Increasing the interaction time between CLP and B. cereus cells, leads to a reduction of CLP in the supernatant as visualized by the reduction of the inhibition halo diameter. From 15 minutes interaction time, the limiting withdrawal of the CLP by the cells is achieved as seen from the constant halo diameter from 15 up to 120 minutes. In Figure 2 (c), the release of CLP with time at two different ionic strengths (0.05 and 0.4 M NaCl) increases with time at 0.05 M NaCl and remains constant with time at 0.4 M NaCl. At 30 s of interaction time between B. cereus and CLP, CLP release from the cells occurs for the two different ionic strengths. However, at 45 minutes of interaction time and 0.05 M NaCl, CLP release is larger than the one induced by the 0.4 M NaCl solution. The release of antibiotic activity is less efficient at high ionic strength, suggesting the occurrence of CLP/cell interactions different from the electrostatic one which should be screened in accordance with the magnitude of the ionic strength. In agreement with observations made for certain lipoplexes, which are lipid-DNA complexes intended to transfect DNA into eukaryotic cells, a high salt concentration triggers some lipid-DNA dissociation although the vast majority of the complexes remain as such [25], possibly due to the hydrophobic interaction between lipid and DNA [26]. Probably, the electrostatic interaction is not the sole driving force of CLP into the cell envelope structures. Figure 2 (d) shows the increase in CLP activity against B. cereus with pH: CLP mixture is more active over the 7–8 pH range. The antibiotic activity detected against B. cereus cells may be related primarily to deprotonated carboxyls or negatively charged CLP. Ionizable groups in these CLP are: a) carboxyl from glutamic acid residues or aspartic acid residues; b) amino group from ornitine residues. Surprisingly, negatively charged bacterial cells interact with similarly charged CLP. However, local charges on the CPL molecules, varying from polar to fully charged groups depending on pH and on the group nature and polarizability [27], certainly play a crucial role on the interaction between the CLP and cells. These CLP molecules are doubtless, well equipped to disassemble the cell envelope of several kinds of microorganisms. Since both the cell wall and the CLP have a net negative charge, electrostatic interaction between CLP and cell wall may occur at local positively charged groups as is the case of protruding section of proteins embedded into the peptideglycan layer charged groups. The disassembly of CLPs themselves due to dissociation of negatively charged moieties at pH 6–8 and intermolecular electrostatic repulsion presented in this work later on further supports this explanation for the increase in activity of the non aggregated CLP molecules.


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)

Inhibition halos and lytic activity of CLP from Bacillus megaterium pL6 against Bacillus cereus.On panel (a), the inhibition halo B against Bacillus cereus confluent layer C is induced by Bacillus megaterium pL6 cultivated inside the hole A. On panel (b), the withdrawal of the antibiotic activity produced by B. megaterium by cells of B. cereus is a function of the interaction time (50 µL of a cyclolipopeptides solution at 10 mg/mL were incubated at 37°C with 2 mL stationary cell culture of B. cereus for 15, 30 and 120 minutes before withdrawing 70 µL samples for centrifugation and determination of supernatant residual activity against B. cereus from inhibition haloes); the control was performed in the absence of B. cereus cells. On panel (c), the withdrawal of the antibiotic activity from B. cereus is seen in the presence of two different ionic strengths (0.05 and 0.4 M NaCl). Bacillus cereus cells were grown up to stationary phase, concentrated 10 times before adding 100 µL of 10 mg/mL cyclolipopeptides solution at pH 8.0 to 2 mL of the concentrated B. cereus suspension LB for 0.5 or 45 minutes interaction; thereafter mixtures were centrifuged and resuspended in water for removal of biosurfactant excess; 70 µL of the pelleted cells were resuspended in the NaCl solutions and centrifuged again before withdrawal of 60 µL of each supernatant for determining the inhibition haloes against B. cereus after incubation for 9 h at 37°C. Controls were 60 µL of the Bsf solution (10 mg/mL) or 0.4 M NaCl solution only. On panel (d), the pH of the biosurfactant solution affects the inhibition halo of B. cereus. The pH was adjusted to 2, 4, 6 and 8 by adding 1N HCl or 1N NaOH to 0.2 mL of biosurfactant solutions before loading 0.1 mL of each solution onto the holes of a Petri dish containing B. cereus; the control was 0.1 mL milli Q water adjusted to pH 2.
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Related In: Results  -  Collection

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

pone-0097261-g002: Inhibition halos and lytic activity of CLP from Bacillus megaterium pL6 against Bacillus cereus.On panel (a), the inhibition halo B against Bacillus cereus confluent layer C is induced by Bacillus megaterium pL6 cultivated inside the hole A. On panel (b), the withdrawal of the antibiotic activity produced by B. megaterium by cells of B. cereus is a function of the interaction time (50 µL of a cyclolipopeptides solution at 10 mg/mL were incubated at 37°C with 2 mL stationary cell culture of B. cereus for 15, 30 and 120 minutes before withdrawing 70 µL samples for centrifugation and determination of supernatant residual activity against B. cereus from inhibition haloes); the control was performed in the absence of B. cereus cells. On panel (c), the withdrawal of the antibiotic activity from B. cereus is seen in the presence of two different ionic strengths (0.05 and 0.4 M NaCl). Bacillus cereus cells were grown up to stationary phase, concentrated 10 times before adding 100 µL of 10 mg/mL cyclolipopeptides solution at pH 8.0 to 2 mL of the concentrated B. cereus suspension LB for 0.5 or 45 minutes interaction; thereafter mixtures were centrifuged and resuspended in water for removal of biosurfactant excess; 70 µL of the pelleted cells were resuspended in the NaCl solutions and centrifuged again before withdrawal of 60 µL of each supernatant for determining the inhibition haloes against B. cereus after incubation for 9 h at 37°C. Controls were 60 µL of the Bsf solution (10 mg/mL) or 0.4 M NaCl solution only. On panel (d), the pH of the biosurfactant solution affects the inhibition halo of B. cereus. The pH was adjusted to 2, 4, 6 and 8 by adding 1N HCl or 1N NaOH to 0.2 mL of biosurfactant solutions before loading 0.1 mL of each solution onto the holes of a Petri dish containing B. cereus; the control was 0.1 mL milli Q water adjusted to pH 2.
Mentions: Figure 2 (a) exhibits the inhibition halo induced by Bacillus megaterium pL 6 cells against a highly sensitive Bacillus cereus strain. The Petri dish was mounted with two layers of Luria Broth agar (LB) with 1.5% agar at the bottom and 0.8% agar on the top. The hole in the plate center (A) in the top layer was seeded with 0.1 mL of a fresh suspension of B. megaterium pL 6 cells cultured up to the stationary phase. The top agar layer surrounding the central hole was previously mixed with 0.3 mL of a fresh stationary B. cereus LB culture. After incubation at 37°C overnight, a visible inhibition halo (B) contrasted with the growth of B. cereus in the region (C) of the Petri dish. The inhibition halo indicates that B. megaterium pL6 cells release substances which diffuse through the agar preventing the growth of B. cereus. The radial diffusion occurs roughly in two dimensions, differently from the tridimensional process taking place in nature when species of microorganisms compete for the same ecological niche [24]. Figure 2 (b) shows the withdrawal of the CLP by B. cereus over a range of interaction times. Increasing the interaction time between CLP and B. cereus cells, leads to a reduction of CLP in the supernatant as visualized by the reduction of the inhibition halo diameter. From 15 minutes interaction time, the limiting withdrawal of the CLP by the cells is achieved as seen from the constant halo diameter from 15 up to 120 minutes. In Figure 2 (c), the release of CLP with time at two different ionic strengths (0.05 and 0.4 M NaCl) increases with time at 0.05 M NaCl and remains constant with time at 0.4 M NaCl. At 30 s of interaction time between B. cereus and CLP, CLP release from the cells occurs for the two different ionic strengths. However, at 45 minutes of interaction time and 0.05 M NaCl, CLP release is larger than the one induced by the 0.4 M NaCl solution. The release of antibiotic activity is less efficient at high ionic strength, suggesting the occurrence of CLP/cell interactions different from the electrostatic one which should be screened in accordance with the magnitude of the ionic strength. In agreement with observations made for certain lipoplexes, which are lipid-DNA complexes intended to transfect DNA into eukaryotic cells, a high salt concentration triggers some lipid-DNA dissociation although the vast majority of the complexes remain as such [25], possibly due to the hydrophobic interaction between lipid and DNA [26]. Probably, the electrostatic interaction is not the sole driving force of CLP into the cell envelope structures. Figure 2 (d) shows the increase in CLP activity against B. cereus with pH: CLP mixture is more active over the 7–8 pH range. The antibiotic activity detected against B. cereus cells may be related primarily to deprotonated carboxyls or negatively charged CLP. Ionizable groups in these CLP are: a) carboxyl from glutamic acid residues or aspartic acid residues; b) amino group from ornitine residues. Surprisingly, negatively charged bacterial cells interact with similarly charged CLP. However, local charges on the CPL molecules, varying from polar to fully charged groups depending on pH and on the group nature and polarizability [27], certainly play a crucial role on the interaction between the CLP and cells. These CLP molecules are doubtless, well equipped to disassemble the cell envelope of several kinds of microorganisms. Since both the cell wall and the CLP have a net negative charge, electrostatic interaction between CLP and cell wall may occur at local positively charged groups as is the case of protruding section of proteins embedded into the peptideglycan layer charged groups. The disassembly of CLPs themselves due to dissociation of negatively charged moieties at pH 6–8 and intermolecular electrostatic repulsion presented in this work later on further supports this explanation for the increase in activity of the non aggregated CLP molecules.

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