Improved vectors for transcriptional signal screening in corynebacteria.
New promoter-probe and terminator-probe shuttle vectors for Escherichia coli and corynebacteria were constructed. These vectors, working with the uidA gene of E. coli as reporter gene, are very useful for the cloning and subsequent analysis of transcriptional regulatory signals. The beta-glucuronidase encoding activity of uidA can be easily detected on agar plates containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-gluc). In the terminator-probe vector pUT2, uidA is expressed from a promoter of the Brevibacterium lactofermentum cryptic plasmid pBL1. Multiple cloning sites (MCS) located immediately upstream of uidA allow introduction and selection of terminators or regulatory signals. In the promoter-probe vector pUT3, transcription readthrough from vector promoters is prevented by a terminator of B. lactofermentum isolated using pUT2. We have successfully used pUT2 and pUT3 to isolate several terminators and promoter regions active in B. lactofermentum and shown that the E. coli strong terminator cartridge omega appears less efficient in corynebacteria.
Bardonnet
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Laboratorie de Microbiologie, INSA, Villeurbanne, France.
Blanco
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C
D001483:Base Sequence; D001951:Brevibacterium / Q000235:genetics; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000235:genetics*; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012321:DNA-Directed RNA Polymerases