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High local substrate availability stabilizes a cooperative trait.

Bachmann H, Molenaar D, Kleerebezem M, van Hylckama Vlieg JE - ISME J (2010)

Bottom Line: Cooperative behavior is widely spread in microbial populations.By using multiple experimental approaches, as well as modeling population dynamics, we demonstrate that the persistence of the proteolytic trait is determined by the fraction of the generated peptides that can be captured by the cell before diffusing away from it.The mechanism described is likely to be relevant for the evolutionary stability of many extracellular substrate-degrading enzymes.

View Article: PubMed Central - PubMed

Affiliation: NIZO Food Research, Health Department, Ede, The Netherlands.

ABSTRACT
Cooperative behavior is widely spread in microbial populations. An example is the expression of an extracellular protease by the lactic acid bacterium Lactococcus lactis, which degrades milk proteins into free utilizable peptides that are essential to allow growth to high cell densities in milk. Cheating, protease-negative strains can invade the population and drive the protease-positive strain to extinction. By using multiple experimental approaches, as well as modeling population dynamics, we demonstrate that the persistence of the proteolytic trait is determined by the fraction of the generated peptides that can be captured by the cell before diffusing away from it. The mechanism described is likely to be relevant for the evolutionary stability of many extracellular substrate-degrading enzymes.

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Related in: MedlinePlus

Localized peptide concentrations measured by intracellular luciferase-based peptide sensing. Mixed batch cultures of prt+ and prt− strains were grown in reconstituted skimmed milk. The fraction of the prt+ strains in the co-culture were 90% (a, b), 50% (c, d) and 10% (e, f). Intracellular peptide concentrations were measured via the dppA-controlled luminescence signal and are given in arbitrary units (y axis, top panel). The dppA reporter construct resides either in the prt+ (dashed line) or the prt− (solid line) host strain. The presented data is corrected for the relative abundance of the strain carrying the luciferase reporter. The slopes of the luminescence traces are given on the y axis of the lower panels. Each curve represents the average of four biological replicates. The results show that at a high relative abundance of the prt+ strain, the dppA expression levels are similar, indicating little or no difference in peptide availability for the two strains (a–d). At low frequencies of the prt+ strain, the intracellular amino-acid levels are higher in the prt+ strains (e, f), which is detected by the downregulation of dppA expression in that strain. For the lower panels, the standard error is shaded in gray. The dip in luminescence activity between 400 and 500 min on the x axis is an intrinsic property of the luminescence reporter, which is linked to changes in metabolic activity when cells go into stationary phase (Bachmann et al., 2007).
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fig1: Localized peptide concentrations measured by intracellular luciferase-based peptide sensing. Mixed batch cultures of prt+ and prt− strains were grown in reconstituted skimmed milk. The fraction of the prt+ strains in the co-culture were 90% (a, b), 50% (c, d) and 10% (e, f). Intracellular peptide concentrations were measured via the dppA-controlled luminescence signal and are given in arbitrary units (y axis, top panel). The dppA reporter construct resides either in the prt+ (dashed line) or the prt− (solid line) host strain. The presented data is corrected for the relative abundance of the strain carrying the luciferase reporter. The slopes of the luminescence traces are given on the y axis of the lower panels. Each curve represents the average of four biological replicates. The results show that at a high relative abundance of the prt+ strain, the dppA expression levels are similar, indicating little or no difference in peptide availability for the two strains (a–d). At low frequencies of the prt+ strain, the intracellular amino-acid levels are higher in the prt+ strains (e, f), which is detected by the downregulation of dppA expression in that strain. For the lower panels, the standard error is shaded in gray. The dip in luminescence activity between 400 and 500 min on the x axis is an intrinsic property of the luminescence reporter, which is linked to changes in metabolic activity when cells go into stationary phase (Bachmann et al., 2007).

Mentions: To directly measure the proposed localized substrate availability around prt+ cells, we transcriptionally fused the bacterial luciferase reporter genes (luxAB) to the lactococcal promoters of the metE and dppA genes (Bachmann et al., 2010) that respond to intracellular peptide and amino-acid levels (den Hengst et al., 2005), whereby luciferase activity acts as an intracellular amino-acid and peptide sensor (Supplementary Table 1). The luxAB reporter constructs were transformed into L. lactis MG1363, a prt− strain, and MG610, a derivative of MG1363, which contains two copies of the protease gene prtMP stably integrated into the genome (Leenhouts et al., 1991). Subsequently, it was established in pure cultures that expression of both reporter constructs is downregulated when residing in the prt+ strain as compared with the prt− strain (Supplementary Figure S5). Mixed cultures with different prt+/prt− ratios and containing the reporters for intracellular amino-acid availability in either of the two host strains were studied (Supplementary Figure S1 and Supplementary Table 2). If the proposed model of a host-dependent localized peptide availability is correct, a cell density-dependent and host-specific response of the peptide and amino-acid sensor system is expected throughout the growth of a batch culture. This host-specific response can be measured as the slope of a sliding window for each consecutive measurement of the luminescence time series. In cultures with a relatively high proportion of prt+ cells (90% and 50% prt+), the luminescence signals differed very little, irrespective of whether the reporter was in the prt+ or the prt− strain (Figures 1a–d). This indicated that in these mixed cultures, the level of extracellular protease activity is apparently sufficient to generate peptide levels that allow repression of the dppA and metE promoters, also in the prt− strain of a co-culture. In contrast, when the culture contained only 10% prt+ cells, a distinct difference was measured between the dppA- (Figures 1e and f) or metE- (Supplementary Figure S6) sensor activity in the prt+ and prt− strains. Initially, a rapid increase of the luciferase signal, coinciding with the depletion of the free amino acids and peptides in milk, is observed in both strains. However, the subsequent phase is characterized by a steady increase of the luminescence signal when the reporter resides in the prt− strain, whereas a decreasing relative expression per cell is observed for the reporter residing in the prt+ strain. The high dppA- and metE-sensor activity in the prt− relative to the prt+ host indicates that the level of intracellular peptides is lower in the prt− host. Because these measurements were made in mixed cultures, they support the proposed high local peptide availability for the prt+ host. DppA expression is controlled by the pleiotropic transcriptional repressor CodY, which is activated at low concentrations of branched chain amino acids and described to have over 100 potential targets (Guedon et al., 2005). The presented data therefore suggests that the complete CodY regulon might be differentially regulated in the prt+ cells as compared with the prt− cells. It should be noted that L. lactis is a non-motile bacterium and that all measurements were made in static cultures, leaving diffusion as the determining factor of peptide movement (Supplementary Figure S2).


High local substrate availability stabilizes a cooperative trait.

Bachmann H, Molenaar D, Kleerebezem M, van Hylckama Vlieg JE - ISME J (2010)

Localized peptide concentrations measured by intracellular luciferase-based peptide sensing. Mixed batch cultures of prt+ and prt− strains were grown in reconstituted skimmed milk. The fraction of the prt+ strains in the co-culture were 90% (a, b), 50% (c, d) and 10% (e, f). Intracellular peptide concentrations were measured via the dppA-controlled luminescence signal and are given in arbitrary units (y axis, top panel). The dppA reporter construct resides either in the prt+ (dashed line) or the prt− (solid line) host strain. The presented data is corrected for the relative abundance of the strain carrying the luciferase reporter. The slopes of the luminescence traces are given on the y axis of the lower panels. Each curve represents the average of four biological replicates. The results show that at a high relative abundance of the prt+ strain, the dppA expression levels are similar, indicating little or no difference in peptide availability for the two strains (a–d). At low frequencies of the prt+ strain, the intracellular amino-acid levels are higher in the prt+ strains (e, f), which is detected by the downregulation of dppA expression in that strain. For the lower panels, the standard error is shaded in gray. The dip in luminescence activity between 400 and 500 min on the x axis is an intrinsic property of the luminescence reporter, which is linked to changes in metabolic activity when cells go into stationary phase (Bachmann et al., 2007).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig1: Localized peptide concentrations measured by intracellular luciferase-based peptide sensing. Mixed batch cultures of prt+ and prt− strains were grown in reconstituted skimmed milk. The fraction of the prt+ strains in the co-culture were 90% (a, b), 50% (c, d) and 10% (e, f). Intracellular peptide concentrations were measured via the dppA-controlled luminescence signal and are given in arbitrary units (y axis, top panel). The dppA reporter construct resides either in the prt+ (dashed line) or the prt− (solid line) host strain. The presented data is corrected for the relative abundance of the strain carrying the luciferase reporter. The slopes of the luminescence traces are given on the y axis of the lower panels. Each curve represents the average of four biological replicates. The results show that at a high relative abundance of the prt+ strain, the dppA expression levels are similar, indicating little or no difference in peptide availability for the two strains (a–d). At low frequencies of the prt+ strain, the intracellular amino-acid levels are higher in the prt+ strains (e, f), which is detected by the downregulation of dppA expression in that strain. For the lower panels, the standard error is shaded in gray. The dip in luminescence activity between 400 and 500 min on the x axis is an intrinsic property of the luminescence reporter, which is linked to changes in metabolic activity when cells go into stationary phase (Bachmann et al., 2007).
Mentions: To directly measure the proposed localized substrate availability around prt+ cells, we transcriptionally fused the bacterial luciferase reporter genes (luxAB) to the lactococcal promoters of the metE and dppA genes (Bachmann et al., 2010) that respond to intracellular peptide and amino-acid levels (den Hengst et al., 2005), whereby luciferase activity acts as an intracellular amino-acid and peptide sensor (Supplementary Table 1). The luxAB reporter constructs were transformed into L. lactis MG1363, a prt− strain, and MG610, a derivative of MG1363, which contains two copies of the protease gene prtMP stably integrated into the genome (Leenhouts et al., 1991). Subsequently, it was established in pure cultures that expression of both reporter constructs is downregulated when residing in the prt+ strain as compared with the prt− strain (Supplementary Figure S5). Mixed cultures with different prt+/prt− ratios and containing the reporters for intracellular amino-acid availability in either of the two host strains were studied (Supplementary Figure S1 and Supplementary Table 2). If the proposed model of a host-dependent localized peptide availability is correct, a cell density-dependent and host-specific response of the peptide and amino-acid sensor system is expected throughout the growth of a batch culture. This host-specific response can be measured as the slope of a sliding window for each consecutive measurement of the luminescence time series. In cultures with a relatively high proportion of prt+ cells (90% and 50% prt+), the luminescence signals differed very little, irrespective of whether the reporter was in the prt+ or the prt− strain (Figures 1a–d). This indicated that in these mixed cultures, the level of extracellular protease activity is apparently sufficient to generate peptide levels that allow repression of the dppA and metE promoters, also in the prt− strain of a co-culture. In contrast, when the culture contained only 10% prt+ cells, a distinct difference was measured between the dppA- (Figures 1e and f) or metE- (Supplementary Figure S6) sensor activity in the prt+ and prt− strains. Initially, a rapid increase of the luciferase signal, coinciding with the depletion of the free amino acids and peptides in milk, is observed in both strains. However, the subsequent phase is characterized by a steady increase of the luminescence signal when the reporter resides in the prt− strain, whereas a decreasing relative expression per cell is observed for the reporter residing in the prt+ strain. The high dppA- and metE-sensor activity in the prt− relative to the prt+ host indicates that the level of intracellular peptides is lower in the prt− host. Because these measurements were made in mixed cultures, they support the proposed high local peptide availability for the prt+ host. DppA expression is controlled by the pleiotropic transcriptional repressor CodY, which is activated at low concentrations of branched chain amino acids and described to have over 100 potential targets (Guedon et al., 2005). The presented data therefore suggests that the complete CodY regulon might be differentially regulated in the prt+ cells as compared with the prt− cells. It should be noted that L. lactis is a non-motile bacterium and that all measurements were made in static cultures, leaving diffusion as the determining factor of peptide movement (Supplementary Figure S2).

Bottom Line: Cooperative behavior is widely spread in microbial populations.By using multiple experimental approaches, as well as modeling population dynamics, we demonstrate that the persistence of the proteolytic trait is determined by the fraction of the generated peptides that can be captured by the cell before diffusing away from it.The mechanism described is likely to be relevant for the evolutionary stability of many extracellular substrate-degrading enzymes.

View Article: PubMed Central - PubMed

Affiliation: NIZO Food Research, Health Department, Ede, The Netherlands.

ABSTRACT
Cooperative behavior is widely spread in microbial populations. An example is the expression of an extracellular protease by the lactic acid bacterium Lactococcus lactis, which degrades milk proteins into free utilizable peptides that are essential to allow growth to high cell densities in milk. Cheating, protease-negative strains can invade the population and drive the protease-positive strain to extinction. By using multiple experimental approaches, as well as modeling population dynamics, we demonstrate that the persistence of the proteolytic trait is determined by the fraction of the generated peptides that can be captured by the cell before diffusing away from it. The mechanism described is likely to be relevant for the evolutionary stability of many extracellular substrate-degrading enzymes.

Show MeSH
Related in: MedlinePlus