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Regulation of the Escherichia coli HipBA toxin-antitoxin system by proteolysis.

Hansen S, Vulić M, Min J, Yen TJ, Schumacher MA, Brennan RG, Lewis K - PLoS ONE (2012)

Bottom Line: Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases.We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background.These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis.

View Article: PubMed Central - PubMed

Affiliation: Antimicrobial Discovery Center, Department of Biology, Northeastern University, Boston, Massachusetts, United States of America.

ABSTRACT
Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.

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Overview of the hipBA locus of E. coli based on Schumacher et al.(A) Model of the hipBA operon. One of four operator sites is shown. (B) View of the crystal structure of the HipB dimer bound to a 21 base pair hipBA operator site (from reference [15]. One HipB subunit is colored green and the other red. The α helices are shown as coils and the α strands as arrows. The amino termini of each subunit are labelled N and the carboxy termini, C. The 16 C-terminal residues (73–88) are unstructured and residues 75–88, which are disordered in the structure of the HipA-HipB-DNA complex, are depicted as dashes and could easily extend from the body of HipB by more than 50 Å.
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pone-0039185-g001: Overview of the hipBA locus of E. coli based on Schumacher et al.(A) Model of the hipBA operon. One of four operator sites is shown. (B) View of the crystal structure of the HipB dimer bound to a 21 base pair hipBA operator site (from reference [15]. One HipB subunit is colored green and the other red. The α helices are shown as coils and the α strands as arrows. The amino termini of each subunit are labelled N and the carboxy termini, C. The 16 C-terminal residues (73–88) are unstructured and residues 75–88, which are disordered in the structure of the HipA-HipB-DNA complex, are depicted as dashes and could easily extend from the body of HipB by more than 50 Å.

Mentions: HipB does not share homology with any of the known antitoxins. Neutralization of its cognate toxin also differs mechanistically from other TA modules. Typically, the antitoxin contains an extended C-terminal stretch, which is structured only when in complex with its toxin [31]–[36]. Contacts usually involve residues near the active site of the toxin, which can simply be blocked by the antitoxin [33], [35]–[37] or make it sterically impossible for the toxin to reach its target [31], [32], [34]. HipB, however, does not make any contacts with HipA near the active site. One HipB dimer binds two HipA molecules involving interactions with both the N and the C domain of HipA [15] (Fig. 1A). The C terminus of HipB is disordered (Fig. 1B) and remains unstructured in the presence of HipA [15]. To test whether proteolytic regulation is a shared characteristic of HipB with the typical antitoxins of the mRNA interferase and gyrase inhibitor TA modules, despite functional and structural differences, we measured the rate of in vivo degradation of HipB in wild type E. coli. Since endogenous HipB could not be detected by Western Blotting using a polyclonal antibody to HipB (data not shown), N-terminally six-his tagged HipB (His6-HipB) was expressed from a plasmid containing an IPTG inducible promoter (pBRhipB). After 60 min of induction, protein synthesis was stopped by the addition of chloramphenicol and the rate of HipB proteolysis was determined by Western blotting (Fig. 2A). His6-HipB was degraded with a t1/2 of ≈17 min in wild type cells confirming a rate of degradation characteristic for antitoxins [38], [39]. Next, we transformed pBRhipB into protease deficient strains lacking lon (KLE902); clpP (KLE903); or hslVU (KLE904) to identify a protease responsible for HipB degradation. We compared the rate of in vivo degradation of HipB in wild type to the rate of degradation in the protease deficient strains. Deletion of clpP or hslVU had a slight effect on HipB. The half life time of HipB was approximately 24 min in ΔclpP and 28 min in ΔhslVU. Deletion of lon stabilized HipB (Fig. 2) (t1/2>200 min), indicating that Lon is likely the main protease involved in HipB degradation in vivo. Since deletion of Lon protease had the strongest effect on the HipB turnover, we focused our studies on Lon dependent HipB degradation.


Regulation of the Escherichia coli HipBA toxin-antitoxin system by proteolysis.

Hansen S, Vulić M, Min J, Yen TJ, Schumacher MA, Brennan RG, Lewis K - PLoS ONE (2012)

Overview of the hipBA locus of E. coli based on Schumacher et al.(A) Model of the hipBA operon. One of four operator sites is shown. (B) View of the crystal structure of the HipB dimer bound to a 21 base pair hipBA operator site (from reference [15]. One HipB subunit is colored green and the other red. The α helices are shown as coils and the α strands as arrows. The amino termini of each subunit are labelled N and the carboxy termini, C. The 16 C-terminal residues (73–88) are unstructured and residues 75–88, which are disordered in the structure of the HipA-HipB-DNA complex, are depicted as dashes and could easily extend from the body of HipB by more than 50 Å.
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Related In: Results  -  Collection

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pone-0039185-g001: Overview of the hipBA locus of E. coli based on Schumacher et al.(A) Model of the hipBA operon. One of four operator sites is shown. (B) View of the crystal structure of the HipB dimer bound to a 21 base pair hipBA operator site (from reference [15]. One HipB subunit is colored green and the other red. The α helices are shown as coils and the α strands as arrows. The amino termini of each subunit are labelled N and the carboxy termini, C. The 16 C-terminal residues (73–88) are unstructured and residues 75–88, which are disordered in the structure of the HipA-HipB-DNA complex, are depicted as dashes and could easily extend from the body of HipB by more than 50 Å.
Mentions: HipB does not share homology with any of the known antitoxins. Neutralization of its cognate toxin also differs mechanistically from other TA modules. Typically, the antitoxin contains an extended C-terminal stretch, which is structured only when in complex with its toxin [31]–[36]. Contacts usually involve residues near the active site of the toxin, which can simply be blocked by the antitoxin [33], [35]–[37] or make it sterically impossible for the toxin to reach its target [31], [32], [34]. HipB, however, does not make any contacts with HipA near the active site. One HipB dimer binds two HipA molecules involving interactions with both the N and the C domain of HipA [15] (Fig. 1A). The C terminus of HipB is disordered (Fig. 1B) and remains unstructured in the presence of HipA [15]. To test whether proteolytic regulation is a shared characteristic of HipB with the typical antitoxins of the mRNA interferase and gyrase inhibitor TA modules, despite functional and structural differences, we measured the rate of in vivo degradation of HipB in wild type E. coli. Since endogenous HipB could not be detected by Western Blotting using a polyclonal antibody to HipB (data not shown), N-terminally six-his tagged HipB (His6-HipB) was expressed from a plasmid containing an IPTG inducible promoter (pBRhipB). After 60 min of induction, protein synthesis was stopped by the addition of chloramphenicol and the rate of HipB proteolysis was determined by Western blotting (Fig. 2A). His6-HipB was degraded with a t1/2 of ≈17 min in wild type cells confirming a rate of degradation characteristic for antitoxins [38], [39]. Next, we transformed pBRhipB into protease deficient strains lacking lon (KLE902); clpP (KLE903); or hslVU (KLE904) to identify a protease responsible for HipB degradation. We compared the rate of in vivo degradation of HipB in wild type to the rate of degradation in the protease deficient strains. Deletion of clpP or hslVU had a slight effect on HipB. The half life time of HipB was approximately 24 min in ΔclpP and 28 min in ΔhslVU. Deletion of lon stabilized HipB (Fig. 2) (t1/2>200 min), indicating that Lon is likely the main protease involved in HipB degradation in vivo. Since deletion of Lon protease had the strongest effect on the HipB turnover, we focused our studies on Lon dependent HipB degradation.

Bottom Line: Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases.We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background.These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis.

View Article: PubMed Central - PubMed

Affiliation: Antimicrobial Discovery Center, Department of Biology, Northeastern University, Boston, Massachusetts, United States of America.

ABSTRACT
Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.

Show MeSH
Related in: MedlinePlus