title: Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation.
abstract: 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 Tyr(109), 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.
authors.affiliation: Department of Plant Pathology, China Agricultural University, Beijing 100193, China. Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Canhua
authors.initials: C
authors.lastname: Lu
authors.affiliation: The Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Ernesto S
authors.initials: ES
authors.lastname: Nakayasu
authors.affiliation: Department of Plant Pathology, China Agricultural University, Beijing 100193, China. zhanglq@cau.edu.cn luoz@purdue.edu.
authors.forename: Li-Qun
authors.initials: LQ
authors.lastname: Zhang
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. zhanglq@cau.edu.cn luoz@purdue.edu.
authors.forename: Zhao-Qing
authors.initials: ZQ
authors.lastname: Luo
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D020816:Amino Acid Motifs; D002384:Catalysis; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D027081:DNA Gyrase / Q000378:metabolism*; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D015151:Immunoblotting; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C482598:fic protein, E coli; D000249:Adenosine Monophosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004264:DNA Topoisomerases, Type I; D027081:DNA Gyrase