Summary We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side chain. Expression of bioJ allows growth of an E. coli bioH strain on biotin-free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl-ACP methyl ester carboxyl-esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl-ACP to pimeloyl-ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel sub-clade of the α/β-hydrolase family. Structure-guided mapping combined with site-directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence.

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.

SUMMARY Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins. However, the S. aureus BirA was reported to dimerize and bind DNA in the absence of biotin or biotinoyl-AMP (Soares da Costa et al. (2014) Mol Microbiol 91: 110-120). These in vitro results argued that the protein would be unable to respond to the levels of biotin or acceptor proteins and thus would lack the regulatory properties of the other characterized BirA proteins. We tested the regulatory function of the protein using an in vivo model system and examined its DNA binding properties in vitro using electrophoretic mobility shift and fluorescence anisotropy analyses. We report that the S. aureus BirA is an effective regulator of biotin operon transcription and that the prior data can be attributed to artifacts of mobility shift analyses. We also report that deletion of the DNA binding domain of the S. aureus BirA results in loss of virtually all of its ligation activity.

Biotin (vitamin B7) is essential for all three domains of life where it acts as a cofactor for biotin-dependent enzymes, including key carboxylases, decarboxylases, and transcarboxylases of central metabolism (Satiaputra et al., 2016). Biotinylation refers to the extremely specific process whereby biotin becomes covalently attached to a single lysine residue of its cognate biotin receptor protein(s) (Chapman-Smith & Cronan, 1999). Protein biotinylation requires tight regulation since the biotinylation process is involved in protein signaling, localization, activation, and degradation (Sternicki et al., 2017). De novo biotin synthesis is a metabolically expensive process, requiring as many as 20 equivalents of ATP and at least 5 enzymes to assemble a molecule of biotin (Feng et al., 2013).Biotin protein ligase (BPL) is the enzyme responsible for covalent attachment of biotin to its cognate enzymes and is essential for growth (Chapman-Smith & Cronan, 1999;Sirithanakorn & Cronan, 2021). Biotinylation proceeds in two discrete steps (Figure 1): biotin and ATP react to form biotinyl-5′AMP (Bio-5′-AMP).The protein-bound Bio-5′-AMP intermediate is then attacked by the ε-amino group of a unique lysine residue of the acceptor protein (Chapman-Smith & Cronan, 1999). This results in the amide-linked biotin-modified protein with release of AMP. To date, three classes of BPL enzymes have been reported (Satiaputra et al., 2016). Class I BPLs are monofunctional enzymes composed only of the conserved catalytic modules required for biotinylation. In contrast, Class II BPLs, also known as BirAs, are bifunctional enzymes since they contain an additional N-terminal DNA-binding domain. The N-terminal DNA-binding domain of BirA proteins allows them to function not only in biotinylation but also in transcriptional regulation of biotin synthesis and/or transport (Cronan, 1988(Cronan, , 1989. The most extensively studied BirA is that of Escherichia coli. When biotin is limiting or unbiotinylated biotin-accepting proteins accumulate, BirA is a monomeric biotinylation enzyme. When the cell is replete with biotin and all cognate proteins have been biotinylated, the BirA-Bio-5′-AMP complex dimerizes (Beckett, 2007). The BirA-Bio-5′-AMP dimers