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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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Purification of the wild type and N-terminal deletion BirA proteins and the biotin acceptor proteins.The proteins were purified as described in Materials and Methods and subjected to SDS-electrophoresis on a 4–20% polyacrylamide gel. M: molecular weight standards (Precision Plus Protein Standard Kaleidoscope from BioRad). Lane 1: B. subtilis N-terminally hexahistidine-tagged BirA (38.9 kDa). Lanes 2-5. B. subtilis N-terminally hexahistidine-tagged Δ2-63 BirA (31.8 kDa), Δ2-65 BirA (31.6 kDa), Δ2-74 BirA (30.4 kDa) and Δ1-81 BirA (29.7 kDa), respectively. Lanes 6-11 are the B. subtilis acceptor proteins AccB-86 (9.4 kDa), PyC-77 (8.3 kDa) and biotin lipoyl attachment protein (BLAP) (8.73 kDa). Lane 10 is E. coli C-terminal hexahistidine-tagged BirA. Lane 11 is E. coli C-terminal hexahistidine tagged Δ2-65 BirA (29.18 kDa) and lane 11 is E. coli AccB-87.
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pone-0096757-g003: Purification of the wild type and N-terminal deletion BirA proteins and the biotin acceptor proteins.The proteins were purified as described in Materials and Methods and subjected to SDS-electrophoresis on a 4–20% polyacrylamide gel. M: molecular weight standards (Precision Plus Protein Standard Kaleidoscope from BioRad). Lane 1: B. subtilis N-terminally hexahistidine-tagged BirA (38.9 kDa). Lanes 2-5. B. subtilis N-terminally hexahistidine-tagged Δ2-63 BirA (31.8 kDa), Δ2-65 BirA (31.6 kDa), Δ2-74 BirA (30.4 kDa) and Δ1-81 BirA (29.7 kDa), respectively. Lanes 6-11 are the B. subtilis acceptor proteins AccB-86 (9.4 kDa), PyC-77 (8.3 kDa) and biotin lipoyl attachment protein (BLAP) (8.73 kDa). Lane 10 is E. coli C-terminal hexahistidine-tagged BirA. Lane 11 is E. coli C-terminal hexahistidine tagged Δ2-65 BirA (29.18 kDa) and lane 11 is E. coli AccB-87.

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)

Purification of the wild type and N-terminal deletion BirA proteins and the biotin acceptor proteins.The proteins were purified as described in Materials and Methods and subjected to SDS-electrophoresis on a 4–20% polyacrylamide gel. M: molecular weight standards (Precision Plus Protein Standard Kaleidoscope from BioRad). Lane 1: B. subtilis N-terminally hexahistidine-tagged BirA (38.9 kDa). Lanes 2-5. B. subtilis N-terminally hexahistidine-tagged Δ2-63 BirA (31.8 kDa), Δ2-65 BirA (31.6 kDa), Δ2-74 BirA (30.4 kDa) and Δ1-81 BirA (29.7 kDa), respectively. Lanes 6-11 are the B. subtilis acceptor proteins AccB-86 (9.4 kDa), PyC-77 (8.3 kDa) and biotin lipoyl attachment protein (BLAP) (8.73 kDa). Lane 10 is E. coli C-terminal hexahistidine-tagged BirA. Lane 11 is E. coli C-terminal hexahistidine tagged Δ2-65 BirA (29.18 kDa) and lane 11 is E. coli AccB-87.
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pone-0096757-g003: Purification of the wild type and N-terminal deletion BirA proteins and the biotin acceptor proteins.The proteins were purified as described in Materials and Methods and subjected to SDS-electrophoresis on a 4–20% polyacrylamide gel. M: molecular weight standards (Precision Plus Protein Standard Kaleidoscope from BioRad). Lane 1: B. subtilis N-terminally hexahistidine-tagged BirA (38.9 kDa). Lanes 2-5. B. subtilis N-terminally hexahistidine-tagged Δ2-63 BirA (31.8 kDa), Δ2-65 BirA (31.6 kDa), Δ2-74 BirA (30.4 kDa) and Δ1-81 BirA (29.7 kDa), respectively. Lanes 6-11 are the B. subtilis acceptor proteins AccB-86 (9.4 kDa), PyC-77 (8.3 kDa) and biotin lipoyl attachment protein (BLAP) (8.73 kDa). Lane 10 is E. coli C-terminal hexahistidine-tagged BirA. Lane 11 is E. coli C-terminal hexahistidine tagged Δ2-65 BirA (29.18 kDa) and lane 11 is E. coli AccB-87.
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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