The physiological function of
biotin requires
biotin protein ligase activity in order to attach the
coenzyme to its cognate
proteins, which are
enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative
biotin protein ligases rather than the usual single
enzyme. F. novicida BirA has a
ligase domain as well as an N-terminal
DNA-binding regulatory domain, similar to the prototypical BirA
protein in E. coli. However, the second
ligase, which we name BplA, lacks the N-terminal
DNA binding motif. It has been unclear why a bacterium would encode these two disparate
biotin protein ligases, since F. novicida contains only a single biotinylated
protein. In vivo complementation and
enzyme assays demonstrated that BirA and BplA are both functional
biotin protein ligases, but BplA is a much more efficient
enzyme. BirA, but not BplA, regulated transcription of the
biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida
infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct
enzymes with specific roles; BplA possesses the major
ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of
biotin. During
infection BplA seems primarily employed to maximize the efficiency of
biotin utilization without limiting the expression of
biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens.
IMPORTANCE: Our findings show that Francisella novicida has evolved two functional
biotin protein ligases, BplA and BirA. BplA is a much more efficient
enzyme than BirA, and its expression is significantly induced upon
infection of macrophages. Only BplA is required for F. novicida pathogenicity, whereas BirA prevents wasteful
biotin synthesis. These data demonstrate that the atypical occurrence of two
biotin protein ligases in F. novicida is linked to distinct roles in virulence and
biotin metabolism.