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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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Multiple sequence alignment of selected HipB proteins from a variety of Gram-negative bacteria.CLUSTALW and CLC Main Workbench were used for the alignment and graphic representation, respectively. HipB sequences were downloaded from NCBI database.
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pone-0039185-g006: Multiple sequence alignment of selected HipB proteins from a variety of Gram-negative bacteria.CLUSTALW and CLC Main Workbench were used for the alignment and graphic representation, respectively. HipB sequences were downloaded from NCBI database.

Mentions: The possibility that the last sixteen residues of HipB had additional functions beyond their role in protein stability was considered especially in light of the finding that the C-terminal residue of HipB, W88, which is universally conserved (Fig. 6), interacted with a small surface pocket of HipA [41]. We first tested the effect of changing residue 88 to an alanine on HipB-DNA affinity. Using a fluorescence polarization-based assay and the hipBA O1O2 operator site, we determined that wild type HipB bound this sequence with a Kd=0.6±0.1 nM (Fig. 7A, 1). As anticipated from the HipA-HipB-DNA crystal structure [15], the HipB(W88A) protein bound this DNA with wild type HipB affinity (Kd=0.9±0.4 nM). Deletion of the last sixteen residues of HipB, (HipB72) also showed no change in DNA binding affinity (Kd=0.4±0.1 nM) (Fig. 7A, Table 1).


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)

Multiple sequence alignment of selected HipB proteins from a variety of Gram-negative bacteria.CLUSTALW and CLC Main Workbench were used for the alignment and graphic representation, respectively. HipB sequences were downloaded from NCBI database.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039185-g006: Multiple sequence alignment of selected HipB proteins from a variety of Gram-negative bacteria.CLUSTALW and CLC Main Workbench were used for the alignment and graphic representation, respectively. HipB sequences were downloaded from NCBI database.
Mentions: The possibility that the last sixteen residues of HipB had additional functions beyond their role in protein stability was considered especially in light of the finding that the C-terminal residue of HipB, W88, which is universally conserved (Fig. 6), interacted with a small surface pocket of HipA [41]. We first tested the effect of changing residue 88 to an alanine on HipB-DNA affinity. Using a fluorescence polarization-based assay and the hipBA O1O2 operator site, we determined that wild type HipB bound this sequence with a Kd=0.6±0.1 nM (Fig. 7A, 1). As anticipated from the HipA-HipB-DNA crystal structure [15], the HipB(W88A) protein bound this DNA with wild type HipB affinity (Kd=0.9±0.4 nM). Deletion of the last sixteen residues of HipB, (HipB72) also showed no change in DNA binding affinity (Kd=0.4±0.1 nM) (Fig. 7A, Table 1).

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