The gene for a halophilic beta-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum.
Investigations of transcriptional regulation and the characterization of promoters in homologous expression systems are most easily performed using suitable reporter genes. Presumably because of the high internal salt concentration in halophilic Archaea, the successful application of the commonly used reporter genes has not been reported so far. Recently, the gene for an extremely halophilic beta-galactosidase (bgaH) from Haloferax alicantei has become available. After transformation of Halobacterium salinarum with a vector-carrying bgaH, the enzyme activity in cell lysates could be readily determined by a simple colorimetric assay and colonies could be screened for activity on plates containing Xgal substrate. Expression of bgaH under the control of various halobacterial promoters of known strength led to different specific beta-galactosidase activities in the lysates. Using Northern blot hybridization and semiquantitative RT-PCR, it was shown that the bgaH transcript level corresponded to the specific enzyme activity. Therefore, the bgaH gene of Haloferax alicantei appears to be a useful tool for in vivo studies of gene expression in Halobacterium salinarum and possibly other halophilic Archaea.
Patenge
N
N
Max-Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18A, 82152 Martinsried, Germany.
Haase
A
A
Bolhuis
H
H
Oesterhelt
D
D
D019848:Gene Expression Regulation, Archaeal; D019766:Genes, Archaeal; D017930:Genes, Reporter; D005822:Genetic Vectors; D006217:Halobacterium / Q000235:genetics*; D019621:Haloferax / Q000235:genetics*; D011401:Promoter Regions, Genetic; D019642:RNA, Archaeal; D012333:RNA, Messenger; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000235:genetics*
D019642:RNA, Archaeal; D012333:RNA, Messenger; D001616:beta-Galactosidase