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Successful conversion of the Bacillus subtilis BirA Group II biotin protein ligase into a Group I ligase.

Henke SK, Cronan JE - PLoS ONE (2014)

Bottom Line: The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties.Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function.This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Illinois, Urbana, Illinois, United States of America.

ABSTRACT
Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that allows transcriptional regulation of biotin biosynthetic and transport genes whereas Group I BPLs lack this N-terminal domain. The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties. We report evidence that B. subtilis BirA is a Group II BPL that regulates transcription at three genomic sites: bioWAFDBI, yuiG and yhfUTS. Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function. This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity.

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Sequence alignments of B. subtilis BirA DNA binding sites and electrophoretic mobility shift assay of DNA binding by BirA.A. B. subtilis has three predicted BirA DNA binding sites: 5′ UTR of the bioWAFDBI operon, 5′ UTR of yuiG, and 5′ UTR of the yhfUTS operon. Conserved residues are highlighted in red and similar residues are highlighted in yellow. B C and D. B. subtilis BirA binding to bioO, the yuiG operator and the yhfU operator, respectively. Note that only in the presence of biotin and ATP is binding observed. E. Quantitation of DNA binding by BirA (Quantity One software). The results show the average of three independent experiments, and the error bars denote standard error of the mean. F. BirA binding to non-operator DNA (a 125 bp internal fragment of the yngHB gene that encodes BLAP). G. BirA binding to bioO without hydroxylamine treatment. Bio-5′-AMP accumulates in the active site during expression in E. coli and survives purification of BirA. H. B. subtilis BirA binding to a half site of the inverted repeat of B. subtilis bioO. Note lane 2 is positive control full-length bioO. I. B. subtilis BirA binding to E. coli bioO. A collection of all putative BirA binding sites in diverse bacteria can be found in the RegPrecise database (http://regprecise.lbl.gov/RegPrecise/).
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pone-0096757-g004: Sequence alignments of B. subtilis BirA DNA binding sites and electrophoretic mobility shift assay of DNA binding by BirA.A. B. subtilis has three predicted BirA DNA binding sites: 5′ UTR of the bioWAFDBI operon, 5′ UTR of yuiG, and 5′ UTR of the yhfUTS operon. Conserved residues are highlighted in red and similar residues are highlighted in yellow. B C and D. B. subtilis BirA binding to bioO, the yuiG operator and the yhfU operator, respectively. Note that only in the presence of biotin and ATP is binding observed. E. Quantitation of DNA binding by BirA (Quantity One software). The results show the average of three independent experiments, and the error bars denote standard error of the mean. F. BirA binding to non-operator DNA (a 125 bp internal fragment of the yngHB gene that encodes BLAP). G. BirA binding to bioO without hydroxylamine treatment. Bio-5′-AMP accumulates in the active site during expression in E. coli and survives purification of BirA. H. B. subtilis BirA binding to a half site of the inverted repeat of B. subtilis bioO. Note lane 2 is positive control full-length bioO. I. B. subtilis BirA binding to E. coli bioO. A collection of all putative BirA binding sites in diverse bacteria can be found in the RegPrecise database (http://regprecise.lbl.gov/RegPrecise/).

Mentions: To test the relative affinities of the predicted B. subtilis BirA binding sites we purified the protein (Fig. 3) and performed electrophoretic mobility shift assays (EMSAs) on DNA fragments containing the three sites (Fig. 4A). Full dependence of binding on ATP and biotin required treatment of the protein with neutral hydroxylamine to remove Bio-5′-AMP accumulated in the active site during expression in E. coli. With the treated BirA binding of the biotin biosynthetic operator (bioO) was observed only in the presence of both biotin and ATP (Fig. 4B). BirA also bound the yhfU and yuiG operators only in the presence of biotin and ATP (Fig. 4C, D). Although the three binding sites have slightly different DNA sequences (Fig. 4A), analysis of binding over a range of BirA concentrations showed that the three sites had very similar binding affinities (Fig. 4E). B. subtilis BirA failed to show non-specific DNA binding (Fig. 4F) as assayed by use of a fragment from the coding sequence of the yngHB gene. BirA preparations that had not undergone hydroxylamine treatment showed some interaction with bioO in the absence of biotin and ATP (Fig. 4G). BirA did not interact with a site that was composed of only one of the B. subtilis bioO inverted repeats suggesting that the form of BirA active in DNA binding is a dimer (Fig. 4H). B. subtilis BirA interacted only very weakly with the E. coli bioO DNA site (Fig 4I).


Successful conversion of the Bacillus subtilis BirA Group II biotin protein ligase into a Group I ligase.

Henke SK, Cronan JE - PLoS ONE (2014)

Sequence alignments of B. subtilis BirA DNA binding sites and electrophoretic mobility shift assay of DNA binding by BirA.A. B. subtilis has three predicted BirA DNA binding sites: 5′ UTR of the bioWAFDBI operon, 5′ UTR of yuiG, and 5′ UTR of the yhfUTS operon. Conserved residues are highlighted in red and similar residues are highlighted in yellow. B C and D. B. subtilis BirA binding to bioO, the yuiG operator and the yhfU operator, respectively. Note that only in the presence of biotin and ATP is binding observed. E. Quantitation of DNA binding by BirA (Quantity One software). The results show the average of three independent experiments, and the error bars denote standard error of the mean. F. BirA binding to non-operator DNA (a 125 bp internal fragment of the yngHB gene that encodes BLAP). G. BirA binding to bioO without hydroxylamine treatment. Bio-5′-AMP accumulates in the active site during expression in E. coli and survives purification of BirA. H. B. subtilis BirA binding to a half site of the inverted repeat of B. subtilis bioO. Note lane 2 is positive control full-length bioO. I. B. subtilis BirA binding to E. coli bioO. A collection of all putative BirA binding sites in diverse bacteria can be found in the RegPrecise database (http://regprecise.lbl.gov/RegPrecise/).
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pone-0096757-g004: Sequence alignments of B. subtilis BirA DNA binding sites and electrophoretic mobility shift assay of DNA binding by BirA.A. B. subtilis has three predicted BirA DNA binding sites: 5′ UTR of the bioWAFDBI operon, 5′ UTR of yuiG, and 5′ UTR of the yhfUTS operon. Conserved residues are highlighted in red and similar residues are highlighted in yellow. B C and D. B. subtilis BirA binding to bioO, the yuiG operator and the yhfU operator, respectively. Note that only in the presence of biotin and ATP is binding observed. E. Quantitation of DNA binding by BirA (Quantity One software). The results show the average of three independent experiments, and the error bars denote standard error of the mean. F. BirA binding to non-operator DNA (a 125 bp internal fragment of the yngHB gene that encodes BLAP). G. BirA binding to bioO without hydroxylamine treatment. Bio-5′-AMP accumulates in the active site during expression in E. coli and survives purification of BirA. H. B. subtilis BirA binding to a half site of the inverted repeat of B. subtilis bioO. Note lane 2 is positive control full-length bioO. I. B. subtilis BirA binding to E. coli bioO. A collection of all putative BirA binding sites in diverse bacteria can be found in the RegPrecise database (http://regprecise.lbl.gov/RegPrecise/).
Mentions: To test the relative affinities of the predicted B. subtilis BirA binding sites we purified the protein (Fig. 3) and performed electrophoretic mobility shift assays (EMSAs) on DNA fragments containing the three sites (Fig. 4A). Full dependence of binding on ATP and biotin required treatment of the protein with neutral hydroxylamine to remove Bio-5′-AMP accumulated in the active site during expression in E. coli. With the treated BirA binding of the biotin biosynthetic operator (bioO) was observed only in the presence of both biotin and ATP (Fig. 4B). BirA also bound the yhfU and yuiG operators only in the presence of biotin and ATP (Fig. 4C, D). Although the three binding sites have slightly different DNA sequences (Fig. 4A), analysis of binding over a range of BirA concentrations showed that the three sites had very similar binding affinities (Fig. 4E). B. subtilis BirA failed to show non-specific DNA binding (Fig. 4F) as assayed by use of a fragment from the coding sequence of the yngHB gene. BirA preparations that had not undergone hydroxylamine treatment showed some interaction with bioO in the absence of biotin and ATP (Fig. 4G). BirA did not interact with a site that was composed of only one of the B. subtilis bioO inverted repeats suggesting that the form of BirA active in DNA binding is a dimer (Fig. 4H). B. subtilis BirA interacted only very weakly with the E. coli bioO DNA site (Fig 4I).

Bottom Line: The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties.Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function.This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Illinois, Urbana, Illinois, United States of America.

ABSTRACT
Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that allows transcriptional regulation of biotin biosynthetic and transport genes whereas Group I BPLs lack this N-terminal domain. The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties. We report evidence that B. subtilis BirA is a Group II BPL that regulates transcription at three genomic sites: bioWAFDBI, yuiG and yhfUTS. Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function. This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity.

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