title: VapC-1 of nontypeable Haemophilus influenzae is a ribonuclease.
abstract: Nontypeable Haemophilus influenzae (NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the vapBC family. Here, we have characterized the function of one of the NTHi alleles, vapBC-1. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an Escherichia coli expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both E. coli and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi vapBC-1 gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage.
authors.affiliation: Department of Surgery, School of Medicine, University of California, Davis Medical Center, Sacramento, CA 95817, USA. dadaines@ucdavis.edu
authors.forename: Dayle A
authors.initials: DA
authors.lastname: Daines
authors.forename: Mack H
authors.initials: MH
authors.lastname: Wu
authors.forename: Sarah Y
authors.initials: SY
authors.lastname: Yuan
mesh_terms: D000483:Alleles; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics / Q000378:metabolism*; D009876:Operon; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012260:Ribonucleases