Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27

Appl Environ Microbiol. 2015 Dec 11;82(4):1249-1255. doi: 10.1128/AEM.03524-15. Print 2016 Feb 15.

Abstract

We developed a counterselectable deletion system for Thermus thermophilus HB27 based on cytosine deaminase (encoded by codA) from Thermaerobacter marianensis DSM 12885 and the sensitivity of T. thermophilus HB27 to the antimetabolite 5-fluorocytosine (5-FC). The deletion vector comprises the pUC18 origin of replication, a thermostable kanamycin resistance marker functional in T. thermophilus HB27, and codA under the control of a constitutive putative trehalose promoter from T. thermophilus HB27. The functionality of the system was demonstrated by deletion of the bglT gene, encoding a β-glycosidase, and three carotenoid biosynthesis genes, CYP175A1, crtY, and crtI, from the genome of T. thermophilus HB27.

MeSH terms

  • Clostridiales / enzymology
  • Clostridiales / genetics
  • Cytosine Deaminase / genetics
  • Cytosine Deaminase / metabolism*
  • Flucytosine / toxicity*
  • Gene Deletion
  • Gene Knockout Techniques / methods*
  • Genetics, Microbial / methods*
  • Selection, Genetic*
  • Thermus thermophilus / genetics*
  • beta-Glucosidase / genetics

Substances

  • Flucytosine
  • beta-Glucosidase
  • Cytosine Deaminase