Improved vectors for transcriptional/translational signal screening in corynebacteria using the melC operon from Streptomyces glaucescens as reporter

Sirin A I Adham, Sonia Rodríguez, Angelina Ramos, Ramón I. Santamaría, José A. Gil

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

The tyrosinase operon (melC) from Streptomyces glaucescens was cloned and functionally expressed in Brevibacterium lactofermentum and Corynebacterium glutamicum under the control of the promoter of the kan gene from Tn5. Recombinant corynebacterial cells containing the tyrosinase operon produced melanin on agar plates and in liquid culture when supplemented with copper and tyrosine. A conjugative bifunctional replacement vector for transcriptional/translational signal screening (pEMel-1) was constructed using expression of the melC operon from S. glaucescens, which can be used for cloning promoter sequences as EcoRI-NdeI fragments. When the DNA fragments with promoter activity such as cspBp or trpp were inserted into pEMel-1, B. lactofermentum harboring the chimeric plasmids produced melanin at different stages of growth, allowing temporal detection of promoter activity. The vector was also used to detect the activity of a Streptomyces promoter (xysAp), which was inactive in B. lactofermentum, after PCR mutagenesis. The melC operon can be used for the visual, inexpensive (compared to the high price of starch azure for amylase detection), and non-selective (in contrast to the kan or cat genes) screening of several thousand clones at high colony density without killing of the transformants due to the presence of iodine (as in the case of amylase assay).

Original languageEnglish
Pages (from-to)53-59
Number of pages7
JournalArchives of Microbiology
Volume180
Issue number1
DOIs
Publication statusPublished - Jul 1 2003

Keywords

  • Brevibacterium lactofermentum
  • Corynebacterium glutamicum
  • cspB
  • melC
  • Mutagenic PCR
  • Promoters
  • Streptomyces
  • trp
  • xysA

ASJC Scopus subject areas

  • Microbiology

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