Integration of foreign DNA in an intergenic region of the archaeon Methanosarcina mazei without effect on transcription of adjacent genes

Abstract

Transformation systems for methanogenic archaea are scarce, none has been reported for the genus Methanosarcina, and plasmids useful as vectors for cloning foreign DNA into methanogens that stably replicate as extrachromosomal elements are not available. We developed an integration vector for transformation of a member of the genus Methanosarcina, i.e. Methanosarcina mazei, using a segment (Int alpha; 1015 bp) which encompasses the intergenic region (431 bp) between the stress (heat-shock) genes grpE and dnaK. This segment also includes the 3' end (270 bp) of the grpE protein-coding region and the 5' end (314 bp) of the dnaK protein-coding region. Int alpha has an EcoRI site, useful for cloning, situated in the 3' direction beyond the grpE transcription termination region, and far upstream from the dnaK promoter. This location of the site, and the monocistronic mode of transcription of grpE and dnaK in M. mazei, suggested to us that a foreign insert in the site would not affect transcription of either flanking gene. A puromycin-resistance cassette (pac cassette) was inserted in the EcoRI site of Int alpha already inserted in pUC18, to obtain a vector which integrated the pac cassette in the chromosome between grpE and dnaK. The pac gene was transcribed and the transformants acquired puromycin resistance. Constitutive and heat-shock-induced transcription of grpE and dnaK in the transformants was the same as in wild-type cells. The two vectors found with transforming ability differed in the orientation of the pac cassette but both had M. mazei's DNA on each flank of the cassette, with the same orientation as that of the homologous segments in the chromosome.