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. 1. Introduction Methanogenic Archaea, a monophyletic group of strictly anaerobic archaea, are responsible for the vast majority of biologically produced methane. The process, methanogenesis, is not only highly relevant for anthropocentric concerns, such as climate change, sustainable energy strategies, waste treatment, and agriculture but also plays an essential role in the global carbon cycle because it recycles organic matter from anaerobic to aerobic environments [1] (and references therein). Methanogens convert intermediates of anaerobic biomass degradation, like H2+CO2, formate, acetate, and methylated compounds to methane via distinct yet overlapping pathways, and couple this process to energy conservation via a chemiosmotic mechanism [2, 3]. In Methanosarcina species, methanogenesis from methylated compounds, such as methanol or methylamines, proceeds by transfer of the methyl-group to coenzyme M (CoM) via substrate-specific methyltransferases [4] and subsequent reduction of
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