New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes forMethanosarcinaspecies

Adam M Guss,Jun Kai Zhang,Michael Rother,William W Metcalf,Gargi Kulkkarni

doi:10.1155/2008/534081

Adam M Guss, Jun Kai Zhang + Show 3 more

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https://doi.org/10.1155/2008/534081

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Abstract

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage phiC31. Host strains expressing the phiC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.

Highlights

Methanoarchaea are a unique group of organisms that are responsible for the vast majority of biologically mediated methane production
Cloned DNA can be introduced into Methanosarcina spp. with autonomously replicating plasmid vectors (Metcalf et al 1997); this approach often introduces experimental artifacts owing to the higher plasmid copy number
Escherichia coli strains were transformed by electroporation using an E. coli Gene Pulser (Bio-Rad, Hercules, CA) as recommended

Summary

Introduction

Methanoarchaea are a unique group of organisms that are responsible for the vast majority of biologically mediated methane production. Methanogenesis plays a critical role in the carbon cycle, global warming, alternative energy strategies, waste treatment and agriculture, but the experimental study of methanoarchaea is laborious. They are oxygen-sensitive anaerobes and, until recently, methods for their genetic manipulation were scarce. This has begun to change, in particular for members of the genus Methanosarcina (reviewed in Sowers and Schreier (1999) and Rother and Metcalf (2005)). One useful site-specific recombinase system utilizes the Streptomyces bacteriophage φC31 integrase (Thorpe and Smith 1998)

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