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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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Lon degradation of HipB in vitro.0.6 µM His6-Lon and 0.48 µM His6-HipB were incubated in reaction buffer at 37°C (50 mM Tris-HCl (pH 8.0), 4 mM ATP, 7.5 mM MgCl2) for indicated times with or without the component specified and subjected to SDS-PAGE and silver staining followed by analysis (at least 3 independent experiments were used to calculate HipB turn over). (A) In vitro degradation of His6-HipB by His6-Lon. (B) ATP or MgCl2 were omitted in the assay. Closed squares, no ATP; open squares no MgCl2. (C) Addition of an oligodeoxynucleotide encompassing the 21 bp hip operator (closed squares) or control oligo (open squares) and (D) addition of His6-HipA (closed squares) or control protein (lysoszyme) (open squares) to the degradation assay.
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pone-0039185-g003: Lon degradation of HipB in vitro.0.6 µM His6-Lon and 0.48 µM His6-HipB were incubated in reaction buffer at 37°C (50 mM Tris-HCl (pH 8.0), 4 mM ATP, 7.5 mM MgCl2) for indicated times with or without the component specified and subjected to SDS-PAGE and silver staining followed by analysis (at least 3 independent experiments were used to calculate HipB turn over). (A) In vitro degradation of His6-HipB by His6-Lon. (B) ATP or MgCl2 were omitted in the assay. Closed squares, no ATP; open squares no MgCl2. (C) Addition of an oligodeoxynucleotide encompassing the 21 bp hip operator (closed squares) or control oligo (open squares) and (D) addition of His6-HipA (closed squares) or control protein (lysoszyme) (open squares) to the degradation assay.

Mentions: To determine whether HipB is directly recognized and degraded by Lon we purified His6-Lon and His6-HipB for in vitro degradation studies. Lon degraded HipB with a t1/2 of ≈74 min in vitro confirming our findings obtained in vivo (Fig. 3A). The HipB decay however was much slower in the in vitro assay than in vivo. A difference between in vivo and in vitro degradation rate was also described for the antitoxin RelB which was degraded rapidly (t½≈15 min) in a Lon dependent manner in vivo, whereas the in vitro half life time was >60 min [38], [39]. Substrate degradation by Lon requires ATP hydrolysis and Mg2+[40]. HipB degradation was indeed dependent on the presence of ATP and MgCl2 in the reaction buffer demonstrating that degradation of HipB is specific to the addition of active enzyme to the buffer (Fig. 3B). The half-life of HipB in the in vitro assay without either MgCl2 or ATP was 250 min and 168 min, respectively. There was some residual degradation in the absence of either ATP or Mg2+; however, both factors were clearly required for Lon-dependent degradation of HipB.


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)

Lon degradation of HipB in vitro.0.6 µM His6-Lon and 0.48 µM His6-HipB were incubated in reaction buffer at 37°C (50 mM Tris-HCl (pH 8.0), 4 mM ATP, 7.5 mM MgCl2) for indicated times with or without the component specified and subjected to SDS-PAGE and silver staining followed by analysis (at least 3 independent experiments were used to calculate HipB turn over). (A) In vitro degradation of His6-HipB by His6-Lon. (B) ATP or MgCl2 were omitted in the assay. Closed squares, no ATP; open squares no MgCl2. (C) Addition of an oligodeoxynucleotide encompassing the 21 bp hip operator (closed squares) or control oligo (open squares) and (D) addition of His6-HipA (closed squares) or control protein (lysoszyme) (open squares) to the degradation assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376134&req=5

pone-0039185-g003: Lon degradation of HipB in vitro.0.6 µM His6-Lon and 0.48 µM His6-HipB were incubated in reaction buffer at 37°C (50 mM Tris-HCl (pH 8.0), 4 mM ATP, 7.5 mM MgCl2) for indicated times with or without the component specified and subjected to SDS-PAGE and silver staining followed by analysis (at least 3 independent experiments were used to calculate HipB turn over). (A) In vitro degradation of His6-HipB by His6-Lon. (B) ATP or MgCl2 were omitted in the assay. Closed squares, no ATP; open squares no MgCl2. (C) Addition of an oligodeoxynucleotide encompassing the 21 bp hip operator (closed squares) or control oligo (open squares) and (D) addition of His6-HipA (closed squares) or control protein (lysoszyme) (open squares) to the degradation assay.
Mentions: To determine whether HipB is directly recognized and degraded by Lon we purified His6-Lon and His6-HipB for in vitro degradation studies. Lon degraded HipB with a t1/2 of ≈74 min in vitro confirming our findings obtained in vivo (Fig. 3A). The HipB decay however was much slower in the in vitro assay than in vivo. A difference between in vivo and in vitro degradation rate was also described for the antitoxin RelB which was degraded rapidly (t½≈15 min) in a Lon dependent manner in vivo, whereas the in vitro half life time was >60 min [38], [39]. Substrate degradation by Lon requires ATP hydrolysis and Mg2+[40]. HipB degradation was indeed dependent on the presence of ATP and MgCl2 in the reaction buffer demonstrating that degradation of HipB is specific to the addition of active enzyme to the buffer (Fig. 3B). The half-life of HipB in the in vitro assay without either MgCl2 or ATP was 250 min and 168 min, respectively. There was some residual degradation in the absence of either ATP or Mg2+; however, both factors were clearly required for Lon-dependent degradation of HipB.

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