title: Promoting resuscitation of viable but nonculturable cells of Vibrio harveyi by a resuscitation-promoting factor-like protein YeaZ.
abstract: OBJECTIVE
  To demonstrate the resuscitation-promoting activities of recombinant YeaZ from Vibrio harveyi SF-1.
  
  
  RESULTS
  The gene of resuscitation-promoting factor YeaZ was cloned from genomic DNA of V. harveyi SF-1. The gene was expressed in Escherichia coli, and the expressed protein was purified by Ni2+-affinity chromatography. A yeaZ mutant was constructed by using the suicide plasmid pNQ705 with homologous recombination. Disruption of yeaZ did not affect cell growth significantly in 2216 E broth at 28°C. The wild-type and mutant viable but nonculturable (VBNC) cells could be resuscitated by temperature upshift method. In addition, the recombinant YeaZ increased the culturable counts from 1·27 × 104CFU per ml and 1·99 × 104CFU per ml to 2·88 × 105CFU per ml and 4·59 × 105CFU per ml, respectively. After the VBNC cells of wild-type and mutant cells were maintained at 4°C for 120 days, no resuscitation was obtained by temperature upshift method, but addition of the recombinant YeaZ promoted the resuscitation of the wild-type and mutant cells, with the culturable cell counts of 1·13 × 103and 1·44 × 103CFU per ml, respectively. Disruption of yeaZ decreased the virulence of V. harveyi in zebrafish. The lethal dose 50% of the yeaZ null mutant was more than 10-fold higher than that of the wild-type cells.
  
  
  CONCLUSIONS
  The recombinant YeaZ could efficiently promote resuscitation of the wild-type and mutant cells of V. harveyi from VBNC to culturable state. The protein also promoted resuscitation of the VBNC wild-type and mutant cells, which were maintained at 4°C for 120 days and not recovered by temperature upshift method. Disruption of yeaZ decreased the virulence of V. harveyi in zebrafish.
  
  
  CONCLUSIONS
  Here, we show clear evidence of a resuscitation-promoting factor YeaZ of V. harveyi and the roles in resuscitation of the VBNC cells and its pathogenicity.
authors.affiliation: College of Energy and Power Engineering, Lanzhou University of Technology, Lanzhou, China.|School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Li
authors.affiliation: School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou, China.
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Marine Biology, College of marine Life Science, Ocean University of China, Qingdao, China.
authors.forename: M
authors.initials: M
authors.lastname: Zhao
authors.affiliation: College of Energy and Power Engineering, Lanzhou University of Technology, Lanzhou, China.|School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Yang
authors.affiliation: School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou, China.
authors.forename: L
authors.initials: L
authors.lastname: Yue
authors.affiliation: Department of Marine Biology, College of marine Life Science, Ocean University of China, Qingdao, China.
authors.forename: X
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D016207:Cytokines / Q000378:metabolism; D050296:Microbial Viability; D010802:Phylogeny; D013696:Temperature; D014733:Vibrio / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity / Q000502:physiology; D014774:Virulence; D015027:Zebrafish
chemical_list: D001426:Bacterial Proteins; D016207:Cytokines; C113704:resuscitation-promoting factor, bacteria
keywords: 
  VBNC
  ; 
  Vibrio harveyi
  ; YeaZ; resuscitation; virulence