A Francisella virulence factor catalyses an essential reaction of biotin synthesis

Abstract

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.

Expression and Activity of the BioH Esterase of Biotin Synthesis is Independent of Genome Context

“…2c). Note that despite its low sequence identity with E. coli BioH the P. aeruginosa protein is clearly a BioH and not one of the other pimeloyl-ACP methyl ester esterases because those nonorthologous proteins cannot be aligned with E. coli BioH even given very permissive alignment parameters 24, 25, 30 .

Figure 2Sequence alignments, purification and modeling of BioH proteins. (A), sequence alignment of the BioH proteins of E. coli and P. aeruginosa PAO1.

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Dissemination and Mechanism for the MCR-1 Colistin Resistance

“…Similarly, the recombinant plasmid pBAD24:: lptA was constructed through cloning of the Neisseria LptA-encoding gene into pBAD24 (S1 Table). Using the pBAD24:: mcr - 1 plasmid as the template, The experiments of site-directed PCR mutagenesis were conducted as we earlier described [47]. All the acquired plasmids were verified by PCR assays and direct DNA sequencing.…”

Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase

“…Genetic studies have revealed important roles for biotin biosynthesis in Mycobacterium tuberculosis during growth, infection and the latency phase, thus establishing this pathway as a potential drug target for new anti-tuberculosis therapies [3], [8], [9], [10], [11] and [12]. In addition, biotin biosynthesis has been intensively studied in other bacteria and the enzymes involved in the pathway have been implicated in bacterial virulence during infection and intracellular replication [13].…”