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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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Complementation of E. coli strains by expression of the BirA N-terminal deletion proteins.A. Complementation of E. coli BirA mutant strain BM4092. B. Complementation of E. coli ΔbirA strain VC618. Strains were grown on M9 minimal medium containing different biotin concentrations (1.6 nM, 4.1 nM, 41 nM and 1.6 µM) and X-gal. The blue color indicates transcription of bioF-lacZ fusion. The white colonies indicate transcriptional repression of the biotin operon by BirA binding at bioO. Note that B. subtilis wild type BirA does not complement the regulatory function of E. coli BirA and thus gives blue colonies.
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pone-0096757-g007: Complementation of E. coli strains by expression of the BirA N-terminal deletion proteins.A. Complementation of E. coli BirA mutant strain BM4092. B. Complementation of E. coli ΔbirA strain VC618. Strains were grown on M9 minimal medium containing different biotin concentrations (1.6 nM, 4.1 nM, 41 nM and 1.6 µM) and X-gal. The blue color indicates transcription of bioF-lacZ fusion. The white colonies indicate transcriptional repression of the biotin operon by BirA binding at bioO. Note that B. subtilis wild type BirA does not complement the regulatory function of E. coli BirA and thus gives blue colonies.

Mentions: To determine if another Group II BPL, that of B. subtilis, requires such communication of the catalytic and N-terminal domains for full ligase activity, we constructed genes encoding several N-terminal B. subtilis BirA deletion proteins. The deletion endpoints were based on structural modeling of B. subtilis BirA based on the crystal structure of S. aureus BirA (PDB 4DQ2) [7] (Fig. 6). BirA deletions Δ2-63 and Δ2-65 eliminated the predicted N-terminal domain whereas BirA deletions Δ2-74 and Δ1-81 also cut into the predicted central catalytic domain. Complementation assays using the E. coli birA1 mutant strain BM4092 and the E. coli ΔbirA deletion strain VC618 were used to test the ligase activities of the B. subtilis BirA N-terminally deleted proteins. These assays showed that upon expression of the B. subtilis Δ2-63 BirA, Δ2-65 BirA and wild type proteins in strain BM4092 all three BPLs supported growth equally well on medium containing 1.6 nM biotin (the minimal level allowing growth of E. coli) (Fig. 7A). These results indicated that the ligase activities of these two deletion proteins were essentially normal. In contrast, upon expression of the E. coli BirA lacking its amino terminus (Δ2-65 BirA), growth of the transformed strain required a biotin concentration that was 1000-fold greater. The complementation activities of the other two B. subtilis deletion proteins, BirA Δ2-74 and BirA Δ1-81 were either partially (BirA Δ2-74) or totally (BirA Δ1-81) compromised (Fig. 7A). To ensure that residual activity of the host BirA1 protein did not play a role in growth restoration we also performed complementation of the ΔbirA E. coli strain VC618 and obtained a similar complementation pattern (Fig. 7B).


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

Henke SK, Cronan JE - PLoS ONE (2014)

Complementation of E. coli strains by expression of the BirA N-terminal deletion proteins.A. Complementation of E. coli BirA mutant strain BM4092. B. Complementation of E. coli ΔbirA strain VC618. Strains were grown on M9 minimal medium containing different biotin concentrations (1.6 nM, 4.1 nM, 41 nM and 1.6 µM) and X-gal. The blue color indicates transcription of bioF-lacZ fusion. The white colonies indicate transcriptional repression of the biotin operon by BirA binding at bioO. Note that B. subtilis wild type BirA does not complement the regulatory function of E. coli BirA and thus gives blue colonies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4016012&req=5

pone-0096757-g007: Complementation of E. coli strains by expression of the BirA N-terminal deletion proteins.A. Complementation of E. coli BirA mutant strain BM4092. B. Complementation of E. coli ΔbirA strain VC618. Strains were grown on M9 minimal medium containing different biotin concentrations (1.6 nM, 4.1 nM, 41 nM and 1.6 µM) and X-gal. The blue color indicates transcription of bioF-lacZ fusion. The white colonies indicate transcriptional repression of the biotin operon by BirA binding at bioO. Note that B. subtilis wild type BirA does not complement the regulatory function of E. coli BirA and thus gives blue colonies.
Mentions: To determine if another Group II BPL, that of B. subtilis, requires such communication of the catalytic and N-terminal domains for full ligase activity, we constructed genes encoding several N-terminal B. subtilis BirA deletion proteins. The deletion endpoints were based on structural modeling of B. subtilis BirA based on the crystal structure of S. aureus BirA (PDB 4DQ2) [7] (Fig. 6). BirA deletions Δ2-63 and Δ2-65 eliminated the predicted N-terminal domain whereas BirA deletions Δ2-74 and Δ1-81 also cut into the predicted central catalytic domain. Complementation assays using the E. coli birA1 mutant strain BM4092 and the E. coli ΔbirA deletion strain VC618 were used to test the ligase activities of the B. subtilis BirA N-terminally deleted proteins. These assays showed that upon expression of the B. subtilis Δ2-63 BirA, Δ2-65 BirA and wild type proteins in strain BM4092 all three BPLs supported growth equally well on medium containing 1.6 nM biotin (the minimal level allowing growth of E. coli) (Fig. 7A). These results indicated that the ligase activities of these two deletion proteins were essentially normal. In contrast, upon expression of the E. coli BirA lacking its amino terminus (Δ2-65 BirA), growth of the transformed strain required a biotin concentration that was 1000-fold greater. The complementation activities of the other two B. subtilis deletion proteins, BirA Δ2-74 and BirA Δ1-81 were either partially (BirA Δ2-74) or totally (BirA Δ1-81) compromised (Fig. 7A). To ensure that residual activity of the host BirA1 protein did not play a role in growth restoration we also performed complementation of the ΔbirA E. coli strain VC618 and obtained a similar complementation pattern (Fig. 7B).

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.

Show MeSH
Related in: MedlinePlus