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A general model for toxin-antitoxin module dynamics can explain persister cell formation in E. coli.

Gelens L, Hill L, Vandervelde A, Danckaert J, Loris R - PLoS Comput. Biol. (2013)

Bottom Line: Conditional cooperativity and increasing the number of binding sites on the operator serves to reduce the metabolic burden of the cell by reducing the total amounts of proteins produced.The amplitude of these spikes determines the duration of the persister state.Finally, increases in the antitoxin degradation rate and decreases in the bacterial growth rate cause a rise in the amount of persisters during nutritional stress.

View Article: PubMed Central - PubMed

Affiliation: Applied Physics Research Group APHY, Vrije Universiteit Brussel, Brussels, Belgium. lendert.gelens@vub.ac.be

ABSTRACT
Toxin-Antitoxin modules are small operons involved in stress response and persister cell formation that encode a "toxin" and its corresponding neutralizing "antitoxin". Regulation of these modules involves a complex mechanism known as conditional cooperativity, which is supposed to prevent unwanted toxin activation. Here we develop mathematical models for their regulation, based on published molecular and structural data, and parameterized using experimental data for F-plasmid ccdAB, bacteriophage P1 phd/doc and E. coli relBE. We show that the level of free toxin in the cell is mainly controlled through toxin sequestration in toxin-antitoxin complexes of various stoichiometry rather than by gene regulation. If the toxin translation rate exceeds twice the antitoxin translation rate, toxins accumulate in all cells. Conditional cooperativity and increasing the number of binding sites on the operator serves to reduce the metabolic burden of the cell by reducing the total amounts of proteins produced. Combining conditional cooperativity and bridging of antitoxins by toxins when bound to their operator sites allows creation of persister cells through rare, extreme stochastic spikes in the free toxin level. The amplitude of these spikes determines the duration of the persister state. Finally, increases in the antitoxin degradation rate and decreases in the bacterial growth rate cause a rise in the amount of persisters during nutritional stress.

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Toxin-antitoxin models for one or more binding sites on the operator based on the repression.(A) A toxin-antitoxin module typically consists of a promoter/operator region, followed by the genes for the antitoxin and the toxin. After transcription of the polycistronic mRNA, the toxin and antitoxin are translated. These proteins can form two non-toxic complexes, AT and TAT. The degradation (represented by ) of antitoxin and mRNA is more intense (indicated in black) than degradation of toxin and both complexes AT and TAT, in which case the degradation rate corresponds to dilution by cell division. (B) Molecular mechanism of conditional cooperativity in the “independent binding sites” model as experimentally observed for relBE. Two AT complexes complexes can bind independently next to each other. Addition of a third toxin to form a TAT complex leads to release of this entity from the operator. (C) Molecular mechanism in the “interacting binding sites” model. In this case, as observed for ccdAB and phd/doc, the toxin has two binding sites for an antitoxin and is thus able to bridge two antitoxin dimers on the operator via a low and a high affinity interaction. Addition of an additional toxin leads to a switch from a low to a high affinity interaction, and the resulting TAT complex again is released from the operator.
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pcbi-1003190-g001: Toxin-antitoxin models for one or more binding sites on the operator based on the repression.(A) A toxin-antitoxin module typically consists of a promoter/operator region, followed by the genes for the antitoxin and the toxin. After transcription of the polycistronic mRNA, the toxin and antitoxin are translated. These proteins can form two non-toxic complexes, AT and TAT. The degradation (represented by ) of antitoxin and mRNA is more intense (indicated in black) than degradation of toxin and both complexes AT and TAT, in which case the degradation rate corresponds to dilution by cell division. (B) Molecular mechanism of conditional cooperativity in the “independent binding sites” model as experimentally observed for relBE. Two AT complexes complexes can bind independently next to each other. Addition of a third toxin to form a TAT complex leads to release of this entity from the operator. (C) Molecular mechanism in the “interacting binding sites” model. In this case, as observed for ccdAB and phd/doc, the toxin has two binding sites for an antitoxin and is thus able to bridge two antitoxin dimers on the operator via a low and a high affinity interaction. Addition of an additional toxin leads to a switch from a low to a high affinity interaction, and the resulting TAT complex again is released from the operator.

Mentions: We model TA modules based on all essential interactions in three well-studied systems: the F-plasmid ccdAB, bacteriophage P1 phd/doc and E. coli relBE operon (Figure 1). Common to these systems is that the toxin and antitoxin can form complexes with distinct stoichiometries and DNA binding properties. In the figure the free antitoxin (A) and the free toxin (T) correspond to the biologically relevant species and are typically dimers for the antitoxin, but can be monomers (, ) or dimers () for the toxin, depending on the TA module considered. The AT complex (corresponding to the molecular species -, - and -) has a higher affinity for the operator sites than the isolated antitoxin. The TAT species consists of two toxins flanking a single antitoxin dimer - corresponding to --, -- and -- species. As the DNA binding properties of this species are dependent on the TA module considered, they will be discussed below. The TA operator has one or more binding sites (denoted with in Figure 1A) for A, AT and/or TAT. It is assumed that transcription is halted when at least one molecule (A) or complex (AT or TAT) is bound on the operator. When no proteins are bound on the operator, the genes coding for the toxin and antitoxin are transcribed. Translation of the mRNA leads to the creation of toxin and antitoxin. In TA modules the translation rate for the antitoxin has been found to be larger than the one for the toxin [17]. Therefore, when the toxin-antitoxin operator is being freely expressed, initially more antitoxin than toxin will be created. However, the antitoxin is degraded faster than the much more stable toxin, influencing the steady state toxin∶antitoxin ratio. The degradation of the antitoxins also extends to those within the complexes AT and TAT. Although the bound toxins protect the antitoxin from proteolytic degradation, this protection is not complete and the decay of antitoxin within complexes allows for the release of the attached toxins.


A general model for toxin-antitoxin module dynamics can explain persister cell formation in E. coli.

Gelens L, Hill L, Vandervelde A, Danckaert J, Loris R - PLoS Comput. Biol. (2013)

Toxin-antitoxin models for one or more binding sites on the operator based on the repression.(A) A toxin-antitoxin module typically consists of a promoter/operator region, followed by the genes for the antitoxin and the toxin. After transcription of the polycistronic mRNA, the toxin and antitoxin are translated. These proteins can form two non-toxic complexes, AT and TAT. The degradation (represented by ) of antitoxin and mRNA is more intense (indicated in black) than degradation of toxin and both complexes AT and TAT, in which case the degradation rate corresponds to dilution by cell division. (B) Molecular mechanism of conditional cooperativity in the “independent binding sites” model as experimentally observed for relBE. Two AT complexes complexes can bind independently next to each other. Addition of a third toxin to form a TAT complex leads to release of this entity from the operator. (C) Molecular mechanism in the “interacting binding sites” model. In this case, as observed for ccdAB and phd/doc, the toxin has two binding sites for an antitoxin and is thus able to bridge two antitoxin dimers on the operator via a low and a high affinity interaction. Addition of an additional toxin leads to a switch from a low to a high affinity interaction, and the resulting TAT complex again is released from the operator.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757116&req=5

pcbi-1003190-g001: Toxin-antitoxin models for one or more binding sites on the operator based on the repression.(A) A toxin-antitoxin module typically consists of a promoter/operator region, followed by the genes for the antitoxin and the toxin. After transcription of the polycistronic mRNA, the toxin and antitoxin are translated. These proteins can form two non-toxic complexes, AT and TAT. The degradation (represented by ) of antitoxin and mRNA is more intense (indicated in black) than degradation of toxin and both complexes AT and TAT, in which case the degradation rate corresponds to dilution by cell division. (B) Molecular mechanism of conditional cooperativity in the “independent binding sites” model as experimentally observed for relBE. Two AT complexes complexes can bind independently next to each other. Addition of a third toxin to form a TAT complex leads to release of this entity from the operator. (C) Molecular mechanism in the “interacting binding sites” model. In this case, as observed for ccdAB and phd/doc, the toxin has two binding sites for an antitoxin and is thus able to bridge two antitoxin dimers on the operator via a low and a high affinity interaction. Addition of an additional toxin leads to a switch from a low to a high affinity interaction, and the resulting TAT complex again is released from the operator.
Mentions: We model TA modules based on all essential interactions in three well-studied systems: the F-plasmid ccdAB, bacteriophage P1 phd/doc and E. coli relBE operon (Figure 1). Common to these systems is that the toxin and antitoxin can form complexes with distinct stoichiometries and DNA binding properties. In the figure the free antitoxin (A) and the free toxin (T) correspond to the biologically relevant species and are typically dimers for the antitoxin, but can be monomers (, ) or dimers () for the toxin, depending on the TA module considered. The AT complex (corresponding to the molecular species -, - and -) has a higher affinity for the operator sites than the isolated antitoxin. The TAT species consists of two toxins flanking a single antitoxin dimer - corresponding to --, -- and -- species. As the DNA binding properties of this species are dependent on the TA module considered, they will be discussed below. The TA operator has one or more binding sites (denoted with in Figure 1A) for A, AT and/or TAT. It is assumed that transcription is halted when at least one molecule (A) or complex (AT or TAT) is bound on the operator. When no proteins are bound on the operator, the genes coding for the toxin and antitoxin are transcribed. Translation of the mRNA leads to the creation of toxin and antitoxin. In TA modules the translation rate for the antitoxin has been found to be larger than the one for the toxin [17]. Therefore, when the toxin-antitoxin operator is being freely expressed, initially more antitoxin than toxin will be created. However, the antitoxin is degraded faster than the much more stable toxin, influencing the steady state toxin∶antitoxin ratio. The degradation of the antitoxins also extends to those within the complexes AT and TAT. Although the bound toxins protect the antitoxin from proteolytic degradation, this protection is not complete and the decay of antitoxin within complexes allows for the release of the attached toxins.

Bottom Line: Conditional cooperativity and increasing the number of binding sites on the operator serves to reduce the metabolic burden of the cell by reducing the total amounts of proteins produced.The amplitude of these spikes determines the duration of the persister state.Finally, increases in the antitoxin degradation rate and decreases in the bacterial growth rate cause a rise in the amount of persisters during nutritional stress.

View Article: PubMed Central - PubMed

Affiliation: Applied Physics Research Group APHY, Vrije Universiteit Brussel, Brussels, Belgium. lendert.gelens@vub.ac.be

ABSTRACT
Toxin-Antitoxin modules are small operons involved in stress response and persister cell formation that encode a "toxin" and its corresponding neutralizing "antitoxin". Regulation of these modules involves a complex mechanism known as conditional cooperativity, which is supposed to prevent unwanted toxin activation. Here we develop mathematical models for their regulation, based on published molecular and structural data, and parameterized using experimental data for F-plasmid ccdAB, bacteriophage P1 phd/doc and E. coli relBE. We show that the level of free toxin in the cell is mainly controlled through toxin sequestration in toxin-antitoxin complexes of various stoichiometry rather than by gene regulation. If the toxin translation rate exceeds twice the antitoxin translation rate, toxins accumulate in all cells. Conditional cooperativity and increasing the number of binding sites on the operator serves to reduce the metabolic burden of the cell by reducing the total amounts of proteins produced. Combining conditional cooperativity and bridging of antitoxins by toxins when bound to their operator sites allows creation of persister cells through rare, extreme stochastic spikes in the free toxin level. The amplitude of these spikes determines the duration of the persister state. Finally, increases in the antitoxin degradation rate and decreases in the bacterial growth rate cause a rise in the amount of persisters during nutritional stress.

Show MeSH