Figure 1.
The E. coli biotin synthetic pathway.
The biotin synthetic pathway is initiated (a) by BioC-catalyzed and S-adenosyl-L-methionine (SAM) mediated methylation of malonyl-ACP. The methyl group is red. The malonyl ACP methyl ester enters the fatty acid synthetic cycle as the primer. (b) Following for two rounds of the fatty acid chain elongation cycle the resulting pimeloyl-ACP methyl ester is then (c) hydrolyzed by BioH to form pimeloyl-ACP which is a substrate for BioF to begin assembly of the biotin rings (d). Abbreviations: SAH, S-adenosyl-homocysteine; AON, 8-amino-7-oxononanoate.
Figure 2.
The differing configurations of the biotin genes of diverse bacteria.
E. coli and P. aeruginosa both contain bioH but P. aeruginosa has bioH within its bio operon upstream of bioC whereas the E. coli bioH is located elsewhere on the chromosome. H. influenzae and C. jejuni have bioG within their bio operons upstream of bioC. N. meningitidis has both bioH and bioG upstream of separate copies of bioC. B. fragilis encodes a fusion of BioC and BioG. In place of BioG or BioH, most BioC-containing cyanobacteria carrying, such as Synechococcus spp. and P. marinus, have bioK upstream of bioC.
Table 1.
Bacterial strains, genomic DNAs and plasmids.
Figure 3.
Sequence alignments putative biotin synthetic esterases show conserved catalytic triad residues.
Homologues of BioH, BioG and another putative isozymes, BioK, were obtained from the SEED database (http://theseed.uchicago.edu/FIG/index.cgi). Shown are some of the sequences from a MUSCLE [18] alignment with a −1 extension gap penalty. The putative catalytic sites are shaded in yellow. The residue numbers (given in Italics) are those of E. coli BioH, H. influenzae BioG and P. marinus BioK.
Figure 4.
Expression of the bioG and bioK genes in E. coli replaces bioH function in vivo.
E. coli strain STL24 (ΔbioH) was transformed with derivative of pBAD322 carrying various bioG or bioK genes. The transformants were streaked on M9 agar plates in the pattern shown on the plate diagram containing the carbon source shown in either the presence or absence of biotin (bio). All plates were incubated at 37°C except those expressing P. marinus bioK which were incubated at 25°C. To prevent cross-feeding plates divided into three zones by plastic walls were used. Panel A. Arabinose as carbon source, STL24 ΔbioH transformed with pBAD322 carrying no insert (lower left third), expressing bioG or bioK (top third of each plate) and the wild type strain transformed with pBAD322 (lower right of each plate). Panel B. The inoculation pattern was the same as Panel A and glycerol was the carbon source in place of arabinose. Panel C. The streaking pattern is given by the plate diagram. Arabinose was the carbon source and the test strain was E. coli strain STL25 (ΔbioCΔbioH) transformed with pBAD322 carrying no insert (lower left third), bioGC (top third) or the wild type strain transformed with the vector pBAD322 (lower right third).
Figure 5.
Purification of His6-tagged BioG proteins.
Samples (10 µl) of each eluted fraction were analyzed by electrophoresis on 10% SDS-polyacrylamide gels. The lysate and soluble fractions are given in lanes L and S, respectively. The protein was eluted from the Ni-NTA column with a buffer containing 200 mM imidazole. The fractions shown were pooled and dialyzed as described in Experimental Procedures. Low range molecular weight markers are shown in lanes marked M.
Table 2.
Properties of the purified hexahistidine-tagged BioG and BioGC proteins.
Figure 6.
The BioG proteins cleave the ester group of pimeloyl-ACP methyl ester.
The reaction mixtures containing pimeloyl-ACP methyl ester were mixed with purified BioH as a positive control or a purified BioG from one of four different bacteria as shown. Lane 1 lacked enzyme added. Following incubation for 1 h 10 µl of the reaction mixture was analyzed by electrophoresis on a 20% polyacrylamide gel containing 2.5 M urea.
Figure 7.
Loss of BioG function upon substitution of the putative active site serine with alanine.
Panel A. E. coli strain STL24 (ΔbioH) was transformed with plasmids encoding H. influenzae BioG S65A (right), wild type H. influenzae BioG domain (top) or the empty pET28b+ vector (left). The transformants were streaked onto M9 plates containing 0.2% glucose. Panel B Purification of the S65A BioG. Eluted fractions (10 µl) were analyzed by electrophoresis on a 10% SDS-polyacrylamide gel. The lysate and soluble fractions are shown in lanes L and S, respectively. Panel C. The H. influenzae BioG S65A protein was assayed for esterase activity as in Figure 6.
Figure 8.
Assay of the purified BioG on shorter and longer analogues of pimeloyl-ACP methyl ester.
Reaction mixtures each containing an acyl-ACP methyl ester were either left untreated (−) or treated (+) with one of the BioG proteins as described in Materials and Methods.
Figure 9.
BioH, BioG and BioK are evolutionarily distinct.
The evolutionary relationship between sequences from several α,β-hydrolase families was inferred using the Mega5 [32]. Sequences from other families α,β-hydrolases were obtained from the Pfam database [22]. The bootstrap percentage values for 1000 replicates are shown next to the branches. The optimal tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances (the number of amino acid residue substitutions per site). The scale represents a 50% difference in compared residues per length. The analysis involved 23 amino acid sequences. All positions containing gaps and missing data were eliminated. The final dataset had a total of 148 positions. Bootstrap values lower than 80% are not shown.
Table 3.
Overlapping Coding Sequences.