Figure 1.
The biotin protein ligase (BPL) reaction.
Attachment of biotin to acceptor proteins occurs in a two-step reaction. First, BirA binds biotin and ATP to synthesize Bio-5′-AMP (biotinoyl-5'-adenylate) with release of pyrophosphate. In the second step the conserved lysine residue of the acceptor protein acts as a nucleophile and attacks the mixed anhydride bond to give the biotinylated acceptor protein plus AMP.
Figure 2.
Sequence alignments of S. aureus BPL, B. subtilis BirA and E. coli BirA.
B. subtilis BirA has 31% amino acid identity to S. aureus BPL and 27% amino acid identity to E. coli BirA. Conserved residues are in white text and highlighted in red and similar residues are in red text and boxed in blue. The S. aureus BPL secondary structure (PDB: 4DQ2) is shown above the amino acid sequence.
Table 1.
Bacterial strains.
Table 2.
Plasmids.
Table 3.
Oligonucleotides utilized.
Figure 3.
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.
Figure 4.
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/).
Figure 5.
In vitro biotinylation of the B. subtilis biotin acceptor proteins.
A. Sequence alignment of the B. subtilis biotinylated proteins. Conserved residues are in white text and highlighted in red and similar residues are in red text and boxed in blue. The black arrow indicates the conserved lysine residue that becomes biotinylated. B. Mass spectrometry values for purified acceptor proteins AccB-86, PyC-77, and BLAP. C. Thin layer chromatographic analysis of B. subtilis BirA ligase reaction: synthesis of Bio-5′-AMP and transfer of biotin to AccB-86, PyC-77, and BLAP.
Figure 6.
Model of B. subtilis BirA based on the S. aureus BirA crystal structure (PDB 4DQ2) [7].
The UCSF Chimera package [31] was used to create the image. Residues corresponding to the N-terminal deletion end points are given. Modeled domains are indicated.
Figure 7.
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.
Figure 8.
In vitro biotinylation analyses of the BirA N-terminal deletion proteins.
A. Thin layer chromatographic analysis of wild type B. subtilis BirA and B. subtilis BirA N-terminal deletions with and without the addition of acceptor protein AccB-86. B. Quantitation of Bio-5′-AMP synthesis by wild type B. subtilis BirA and the B. subtilis BirA N-terminal deletion proteins. The results show the average of three independent experiments, and the error bars denote standard error of the mean. C. Quantitation of biotin transfer to AccB-86 by wild type B. subtilis BirA and B. subtilis BirA N-terminal deletions. The results show the average of three independent experiments, and the error bars denote standard error of the mean. D. Thin layer chromatographic analysis of wild type E. coli BirA, E. coli Δ2-65 BirA, wild type B. subtilis BirA, and B. subtilis Δ2-65 BirA. E. Quantitation of Bio-5′-AMP synthesis by wild type E. coli BirA, E. coli Δ2-65 BirA, wild type B. subtilis BirA, and B. subtilis Δ2-65 BirA. The results show the average of three independent experiments, and the error bars denote standard error of the mean. F. Quantitation of biotin transfer to E. coli AccB-87 or B. subtilis AccB-86 by wild type E. coli BirA, E. coli Δ2-65 BirA, wild type B. subtilis BirA and B. subtilis Δ2-65 BirA. The results show the average of three independent experiments, and the error bars denote standard error of the mean.
Figure 9.
Chemical crosslinking of the B. subtilis wild type and Δ2-65 BirA proteins.
A. Wild type BirA. B. The Δ2-65 BirA. Note that efficient dimer formation requires the presence of both biotin and ATP. The EGS concentrations are in mM.
Figure 10.
β-Galactosidase assays of the effect of AccB-86 levels on bioO-dependent transcription.
The bioW::lacZ spac accB-86 strain SKH002 was grown in defined medium supplemented with the indicated concentrations of biotin plus or minus IPTG addition to induce synthesis of the AccB-86 acceptor protein. The results are the average of three independent experiments and the error bars denote standard error of the mean.