Research ArticleMicrobiology

Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation

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Science Signaling  26 Jan 2016:
Vol. 9, Issue 412, pp. ra11
DOI: 10.1126/scisignal.aad0446

AMPing down DNA replication

Bacterial Fic proteins catalyze diverse posttranslational modifications, such as AMPylation, phosphorylation, and UMPylation, and are involved in regulating bacterial filament formation. Lu et al. found that DNA yield from bacterial cultures expressing plasmids encoding Fic-1 was reduced compared to DNA yield from bacteria encoding Fic-2 or Fic-3 plasmids. This suggested that Fic-1 is involved in inhibiting DNA replication. Fic-1 AMPylated the DNA topoisomerase, needed for bacterial DNA replication, GyrB, which inhibited GyrB activity and induced SOS response. Self-AMPylation of Fic-1 inhibited its activity. The authors also identified a Fic-1 antitoxin, which they named AntF. Fic-1 expression also resulted in filament formation in bacteria through a mechanism independent of the SOS response. Thus, this study identifies one molecular target for the inhibition of DNA replication by Fic-1 and suggests that additional targets mediate the effect of Fic-1 on bacterial filament formation.


The morphology of bacterial cells is important for virulence, evasion of the host immune system, and coping with environmental stresses. The widely distributed Fic proteins (filamentation induced by cAMP) are annotated as proteins involved in cell division because of the presence of the HPFx[D/E]GN[G/K]R motif. We showed that the presence of Fic-1 from Pseudomonas fluorescens significantly reduced the yield of plasmid DNA when expressed in Escherichia coli or P. fluorescens. Fic-1 interacted with GyrB, a subunit of DNA gyrase, which is essential for bacterial DNA replication. Fic-1 catalyzed the AMPylation of GyrB at Tyr109, a residue critical for binding ATP, and exhibited auto-AMPylation activity. Mutation of the Fic-1 auto-AMPylated site greatly reduced AMPylation activity toward itself and toward GyrB. Fic-1–dependent AMPylation of GyrB triggered the SOS response, indicative of DNA replication stress or DNA damage. Fic-1 also promoted the formation of elongated cells when the SOS response was blocked. We identified an α-inhibitor protein that we named anti–Fic-1 (AntF), encoded by a gene immediately upstream of Fic-1. AntF interacted with Fic-1, inhibited the AMPylation activity of Fic-1 for GyrB in vitro, and blocked Fic-1–mediated inhibition of DNA replication in bacteria, suggesting that Fic-1 and AntF comprise a toxin-antitoxin module. Our work establishes Fic-1 as an AMPylating enzyme that targets GyrB to inhibit DNA replication and may target other proteins to regulate bacterial morphology.

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