title: A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum.
abstract: BACKGROUND
  Targetrons are gene targeting vectors derived from mobile group II introns. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which use their combined activities to achieve highly efficient site-specific DNA integration with readily programmable DNA target specificity.
  
  
  RESULTS
  Here, we used a mobile group II intron from the thermophilic cyanobacterium Thermosynechococcus elongatus to construct a thermotargetron for gene targeting in thermophiles. After determining its DNA targeting rules by intron mobility assays in Escherichia coli at elevated temperatures, we used this thermotargetron in Clostridium thermocellum, a thermophile employed in biofuels production, to disrupt six different chromosomal genes (cipA, hfat, hyd, ldh, pta, and pyrF). High integration efficiencies (67-100% without selection) were achieved, enabling detection of disruptants by colony PCR screening of a small number of transformants. Because the thermotargetron functions at high temperatures that promote DNA melting, it can recognize DNA target sequences almost entirely by base pairing of the intron RNA with less contribution from the intron-encoded protein than for mesophilic targetrons. This feature increases the number of potential targetron-insertion sites, while only moderately decreasing DNA target specificity. Phenotypic analysis showed that thermotargetron disruption of the genes encoding lactate dehydrogenase (ldh; Clo1313_1160) and phosphotransacetylase (pta; Clo1313_1185) increased ethanol production in C. thermocellum by decreasing carbon flux toward lactate and acetate.
  
  
  CONCLUSIONS
  Thermotargetron provides a new, rapid method for gene targeting and genetic engineering of C. thermocellum, an industrially important microbe, and should be readily adaptable for gene targeting in other thermophiles.
authors.affiliation: Section of Molecular Genetics and Microbiology, Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, School of Biological Sciences, University of Texas at Austin, Austin, Texas, United States of America.
authors.forename: Georg
authors.initials: G
authors.lastname: Mohr
authors.forename: Wei
authors.initials: W
authors.lastname: Hong
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Gu-zhen
authors.initials: GZ
authors.lastname: Cui
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Yang
authors.forename: Qiu
authors.initials: Q
authors.lastname: Cui
authors.forename: Ya-jun
authors.initials: YJ
authors.lastname: Liu
authors.forename: Alan M
authors.initials: AM
authors.lastname: Lambowitz
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002876:Chromosomes, Bacterial; D048013:Acetivibrio thermocellus / Q000235:genetics* / Q000378:metabolism; D000458:Cyanobacteria / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D023061:Gene Order; D018390:Gene Targeting; D005822:Genetic Vectors; D007438:Introns; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D007763:Lac Operon; D055442:Metabolome; D055432:Metabolomics / Q000379:methods; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D010709:Phosphate Acetyltransferase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016337:RNA, Catalytic; D013696:Temperature
chemical_list: D012329:RNA, Bacterial; D016337:RNA, Catalytic; D007770:L-Lactate Dehydrogenase; D010709:Phosphate Acetyltransferase