Comparative sequence analysis of plasmids pME2001 and pME2200 of Methanothermobacter marburgensis strains Marburg and ZH3

Comparison of the updated complete nucleotide sequences of the two related plasmids pME2001 and pME2200 from the thermophilic archaeon Methanothermoba cter marburgensis (formerly, Methanobacterium thermoautotrophicum) strains Marbug and ZH3, respectively, revealed an almost identical common backbone structure and five plasmid-specific inserted fragments (IFs), four of which are flanked by perfect or nearly perfect direct repeats 25-52 bp in length . A 4354-bp minimal replicon was derived from the alignment of the two plas mids, which encodes one putative antisense RNA related to replication contr ol and five open reading frames (ORFs) organized in two operons. The first operon consists of four ORFs, the third of which, i.e. ORF3, contains a hel ix-turn-helix motif and a purine NTP-binding motif often found in proteins involved in DNA metabolic processes. The database search results suggest th at ORF3 might function as a replication initiator protein. The large putati ve Rep protein encoded by pME2001 was overexpressed in Escherichia coli as an N-terminal His-tagged version using pET28a and a compatible helper plasm id that coexpresses minor tRNAs, argU and ileX to compensate for codon usag e difference, ORFs 1, 2, and 3 are organized in a sequence reminiscent of t hat described in E. coli plasmids of die R1 family, cop-tap-rep. ORF6 encod ed by IF1, one of the pME2200-specific elements, showed significant similar ity to ORF6 encoded by archaeal phage Psi M2 of marburgensis strain Marburg and may confer the apparent immunity of its host strain ZH3 to infection b y phage Psi M2. Our data indicate that M. marburgensis plasmids may evolve by a series of gene duplication and excision events. (C) 2001 Academic Pres s.