Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes.
Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. DeltaleuB and DeltatrpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.
Allers
Thorsten
T
Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom. thorsten.allers@nottingham.ac.uk
Ngo
Hien-Ping
HP
Mevarech
Moshe
M
Lloyd
Robert G
RG
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D017353:Gene Deletion; D005819:Genetic Markers; D019622:Haloferax volcanii / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism; D014367:Tryptophan Synthase / Q000235:genetics* / Q000378:metabolism
D019843:Archaeal Proteins; D003470:Culture Media; D005819:Genetic Markers; D012965:Sodium Chloride; D014364:Tryptophan; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase; D014367:Tryptophan Synthase; D007930:Leucine