Development of β-lactamase as a tool for monitoring conditional gene expression by a tetracycline-riboswitch in Methanosarcina acetivorans

  • The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β -galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β -lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β -lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.

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Metadaten
Author:Shemsi Demolli, Miriam M. Geist, Julia E. WeigandORCiDGND, Nicole Matschiavelli, Beatrix SüßGND, Michael RotherORCiDGND
URN:urn:nbn:de:hebis:30:3-333501
DOI:https://doi.org/10.1155/2014/725610
ISSN:1472-3646
ISSN:1472-3654
Pubmed Id:https://pubmed.ncbi.nlm.nih.gov/24678266
Parent Title (English):Archaea
Publisher:Hindawi
Place of publication:New York, NY
Document Type:Article
Language:English
Date of Publication (online):2014/01/20
Date of first Publication:2014/01/20
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2014/04/02
Volume:2014
Issue:Article ID 725610
Page Number:11
First Page:1
Last Page:10
Note:
Copyright © 2014 Shemsi Demolli et al. This is an open access article distributed under the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/ , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
HeBIS-PPN:364409096
Institutes:Biowissenschaften / Biowissenschaften
Medizin / Medizin
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Sammlungen:Sammlung Biologie / Sondersammelgebiets-Volltexte
Licence (German):License LogoCreative Commons - Namensnennung 3.0