PMC

Phylogenetic tree and structural modeling of the bacterial biotin synthesis gatekeeper enzymes
The modeled structure of each member except BioH which has a known structure is shown on the right. All BioJ homologues are found in Francisella species are given in red.

Genetic organization of biotin synthesis-related genes and working model for the Francisella biotin biosynthesis pathway
A. Comparison of the Francisella biotin operon genome neighborhood to that of E. coli. Note that the two Francisella genomes are inverted due to opposite ORF numbering systems
B. Scheme of the proposed Francisella biotin synthesis pathway

Expression of Francisella philomiragia bioJ (Fphi_1796) complements growth of an E. coli ΔbioH strain
A. Expression of Francisella philomiragia bioJ (Fphi_1796) allows growth of the E. coli ΔbioH strain on biotin-free medium
B. Growth curves of the ΔbioH strain with various expression levels of plasmid-borne bioJ (Fphi_1796). The growth curves were automatically plotted using a Bioscreen C instrument. The E. coli ΔbioH strain was STL24 (). Designations: Vec, empty vector pBAD322; Fphi, F. philomiragia bioJ ; Ara, arabinose. Glycerol was the carbon source in the absence of arabinose.

The BioJ Ser-Asp-His catalytic triad is essential for growth of F. novicida in minimal media and for efficient replication in macrophages (and systemic survival in mice
The BioJ catalytic triad is required for growth of F. novicida in minimal medium, and can be alleviated by the addition of biotin (A) or pimelate (B). Wild-type strain U122 of F. novicida and the three catalytic point mutants of BioJ (S151A, D248A and H278A) are grown in Chamberlain’s medium (CHB) with/without biotin or pimelate. The OD₆₀₀ was measured every hour.
C. The catalytic triad of BioJ is required for replication in macrophages lacking exogenous biotin.
D-G. The BioJ catalytic triad residues are required for systemic survival in mice. Mice were infected sub-cutaneously with a 1:1 mixture of wild-type strain 122 of Francisella with ΔbioJ (D), S151A (E), D248A (F) or H278A (G). At 48 h post infection the, spleens and livers were harvested to quantify bacterial levels, and the competitive index (CI) was calculated. Statistical analysis was described previously (). *P<0.05, **P<0.001, ***P<0.0001.

Characterization and verification of Francisella BioJ
A. Size exclusion chromatography profile of purified BioJ
The separation was conducted using a Superdex 75 column eluted at 0.46 ml/min. The insert gel is a gradient SDS-PAGE (4-20%) analysis of BioJ collected from the peak fractions. The molecular weights of the calibration proteins kit (Biorad) are shown by the ruler at the top. The standard proteins were bovine serum albumin (67 kDa), ovalbumin (43 kDa), ribonuclease A (13.7 kDa), aprotinin (6.5 kDa) and vitamin B12 (1.35 kDa), respectively. Chemical cross-linking analyses of fatty acid metabolism FadR, a protein of known dimeric structure (B) and BioJ (C). FadR provides a control for the efficacy of cross-linking. Ethylene glycol bis-succinimidylsuccinate was the cross-linker. M: Molecular weight.
D. MS identification of the purified BioJ protein
The tryptic peptides that match the BioJ sequence are given in bold and underlined type.

A. Comparison of the putative active site of BioJ with the known catalytic residues of BioH. The key residues are in purple. S. marcescens: Serratia marcescens
Enzymatic assays of BioJ (Panel B) and the single mutant S151A (Panel C), D248A (Panel D) and H278A proteins (Panel E) by the conformationally-sensitive electrophoretic mobility shift assay.
Minus denotes no addition of BioJ (or a mutant protein) whereas the triangle on the right hand represents the protein levels in a inverse dilution series (0.034, 0.068, 0.17, 0.34, 0.7, 1.7, 3.4 pmol). The enzymatic reaction (10 μl total volume) contained 150 μM methyl pimeloyl-ACP. The reaction mixture was separated using 20% PAGE containing 2.5 M urea.
F. In vivo functional analyses of the triad residue BioJ mutant proteins (S151A, D248A & H278A)
The plates of were incubated at 37 °C for ~20 h after streaking. The host strain was the E. coli ΔbioH strain STL24. Designations: Ara, arabinose; Fphi, F. philomiragia bioJ ; Vec, medium copy arabinose-inducible plasmid pBAD322.

Bioassay of BioJ function in the overall biotin synthesis pathway
A. Both biotin and dethiobiotin (DTB) support growth of the biotin auxotrophic strain ER90 on biotin-free minimal medium
B. Evidence for the absence of biotin in cell extracts prepared from the E. coli ΔbioH strain
Strain STL24 (ΔbioH) () was used to prepare cell extracts and the biotin indicator strain was ER90 (ΔbioF bioC bioD). Either biotin or DTB as positive control or the cell extract (10 mg/ml) was spotted on the paper disk as described (). The bioassay was performed using strain ER90. The plates were kept at 30°C for ~20 h.
The red formazan deposit formed by reduction of the tetrazolium indicator denotes growth of the biotin auxotrophic strain ER90 supported by either biotin or DTB.
C. Scheme of the in vitro DTB synthesis system
D. Restoration of DTB synthesis to the ΔbioH extract by addition of pimeloyl-ACP methyl ester and BioJ
Either the purified wild type BioJ protein or the mutant BioJ versions (S151A, D248A and H278A) was added to 20 μg/ml (0.5 μM). The reaction on the upper left quadrant contained nearly all the components required for DTB synthesis with the exception of BioJ (or its mutant) enzyme whereas samples spotted on the upper right quadrant lacked cell extract, only contained both the wild type and mutant BioJ proteins. The reactions of the lower left quadrant contained all the components requied for DTB synthesis. Designations: plus, addition of BioJ protein (or its mutants); minus: no addition of BioJ protein (or its mutants) or cell extracts.

Francisella BioJ is a functional carboxylesterase
A. Schematic diagram of the enzymatic reaction catalyzed by Francisella BioJ
B. Enzymatic assays for Francisella BioJ hydrolysis of pimeloyl-ACP methyl ester to pimeloyl-ACP
The enzymatic reactions (10 μl total volume) were performed at 37°C and contained 150 μM pimeloyl-ACP methyl ester as substrate. The reaction mixtures were analyzed by 20% PAGE containing 2.5 M urea. The BioJ-reaction times (right-hand six lanes of Panel B) were 2, 5, 10, 15, 20 and 30 minutes, respectively. CK denotes a reaction containing all components except BioJ. . The triangle over the right-hand seven lanes of the bottom panel represents BioJ levels in a inverse dilution series (0.034, 0.068, 0.17, 0.34, 0.7, 1.7 and 3.4 pmol).
C. Matrix-assisted laser desorption/ionization mass spectroscopic analysis of the BioJ reaction
Upon overexpression of ACP in E. coli three forms of the protein are found due to titration of the deformylase and methionine amino peptidase that process the amino termini of nascent proteins. Form 1 is the form isolated when expression is from the chromosomal acpP gene. Abbreviations: Met, methionine; Me, methyl ester.
D. Substrate specificity of BioJ
The minus signs denote no addition of BioJ enzyme whereas the plus signs denote addition of the protein.