A genetic system for Archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors

Abstract

New methods that allow, for the first time, genetic analysis in Archaea of the genus Methanosarcina are presented. First, several autonomously replicating plasmid shuttle vectors have been constructed based on the naturally occurring plasmid pC2A from Methanosarcina acetivorans. These vectors replicate in 9 of 11 Methanosarcina strains tested and in Escherichia coli. Second, a highly efficient transformation system based upon introduction of DNA by liposomes has been developed. This method allows transformation frequencies of as high as 2 x 10(8) transformants per microgram of DNA per 10(9) cells or approximately 20% of the recipient population. During the course of this work, the complete 5467-bp DNA sequence of pC2A was determined. The implications of these findings for the future of methanoarchaeal research are also discussed.

Figure 1

The DNA sequence of pC2A. The complete 5467-bp DNA sequence is shown (also see Fig. 2). Selected restriction sites are underlined. Four ORFs, ssrA, repA, orf1, and orf2, of greater than 120 aa were observed in the sequence. Their orientation is shown by the small arrows. The amino acid sequences of the putative proteins encoded by these ORFs are shown below the corresponding DNA sequences. The HUHUU (U = bulky hydrophobic residues) motif conserved among the Rep proteins of rolling circle plasmids (31) was identified in the RepA protein (heavy underline). Two long direct repeats of 64 bp (A) and 56 bp (B), and a 20-bp perfect inverted repeat which may play a role in plasmid replication were noted in the intergenic region between ssrA and repA.

Figure 2

Plasmids used in the study. pC2A is a naturally occurring plasmid from Methanosarcina acetivorans. The genes shown are described in the text. pJK3 is a pBluescript derivative carrying a modified pac gene cassette that confers Pur^R upon methanoarchaea. The promoter (pmcrB) and terminator (tmcr) of the Methanococcus voltae methyl reductase operon regulate expression of the puromycin acetyltransferase gene (pac) from Streptomyces alboniger in methanoarchaea. pWM307, pWM311, and pWM321 are Methanosarcina–E. coli shuttle vectors. The origin of replication from the plasmid R6K allows maintenance in E. coli, and manipulation of copy number by choice of an appropriate host strain. Each also carries the entire pC2A replicon for replication in Methanosarcina. Plasmids pWM309, pWM313, pWM315, pWM317, and pWM319 (not shown) are like pWM311 but carry different polylinkers within the lacZα gene. The lacZα gene allows blue-white screening of recombinant clones in E. coli for pWM309, pWM311, pWM313, pWM315, pWM317, and pWM319, but not in pWM321. The β-lactamase gene (bla) encodes resistance to penicillin derivatives in E. coli.

Figure 3

Transformation of Methanosarcina species by pWM307. Total DNA was isolated from each strain and from Pur^R clones obtained from each after transformation with pWM307. EcoRI-digested (A) or undigested (B) DNA was electrophoresed, blotted, and hybridized to labeled pXS2 as described. Plasmid pXS2 hybridizes to both the chromosomal serC gene and the pWM307 bla gene. A band corresponding to the chromosomal serC locus is seen in both transformed and untransformed strains, whereas a band corresponding to pWM307 is seen only in the transformed strains. The pWM307 band migrates with a mobility much higher than the chromosomal serC band in undigested DNA, indicating that pWM307 is replicating as a plasmid in these transformants. The strains examined were Methanosarcina spp. WH1 (WH1), Methanosarcina spp. WH2 (WH2), Methanosarcina barkeri MS (MS), Methanosarcina barkeri Fusaro (Fusaro), Methanosarcina barkeri W (W), Methanosarcina thermophila TM1 (TM1), Methanosarcina siciliae C2J (C2J), Methanosarcina mazei S-6 (S-6), and Methanosarcina acetivorans C2A (C2A). +, Pur^R clones; −, untransformed parental strains. The 1.6-kbp band of the molecular weight markers (M_r) hybridizes to pXS2 vector sequences.