Chapter 17 : Archaeal Plasmids

Linear genome maps of some pRN plasmids. Blackened arrows: ORFs present in all free pRN plasmids, PlrA, CopG, and RepA. ORFA is conserved and often located adjacent to or in a similar position to CopG ORFs. White arrows: ORFs ( > 50 aa) showing no significant sequence similarity to the other plasmid ORFs. Patterned arrows indicate ORFs that are conserved in one or more pRN plasmids. (A) Free pRN plasmids. Diagonal-lined arrows indicate ORFs in pSSVx homologous to SSV2 viral ORFs. Dotted arrows indicate a conserved region with homologous ORFs shared by pDI.10 and pHF.N7. Rm denotes the putative recombination motifs. Shaded bars indicate sequence regions that show similarity to single-stranded and doublestranded origins of bacterial rolling-circle plasmids (see text). (B) Integrated pRN-type plasmids. Integrase genes responsible for the chromosome insertion of the plasmids are partitioned during the integration of pSTl and pXQI. The repA gene of pXQl contains an IS element, ISC1439. tRNA genes that function as target sites for the integrases are indicated.

Putative recombination mechanism shared by Sulfolobus plasmids. The diagram shows how the transition may occur between (A) pHEN7-type plasmids and (B) the smaller pRN-l and pRN-2-type plasmids. A deletion mechanism is depicrcd for the former where the putative cutting sites, within two loop regions, are indicated by arrows. Five conserved nucleotides bordering the recombination Rm motifs are bold-faced.

Sequence alignment of Sulfolobus PlrA proteins. The PlrA proteins are encoded in the pRN-type and conjugative plasmids listed in Tables 1 and 2 and in the S. tokodaii chromosome (S. toko.). The protein is highly conserved in both sequence and length. X's indicate conserved amino acids in a possible leucine zipper, which, in contrast to other known leucine zipper motifs, is located at the N terminus of the protein ( 27 ). The alignment was drawn using t_coffee and BOX shade software (http://www.ch.embnet.org/software/BOX_form.html) where black and gray boxes, respectively, indicate identical and a similar amino acids.

Gene maps of conjugative plasmids free and integrated in Sulfolobus genomes. ORFs are shaded and labeled where homologues are found in other bacterial or archaeal plasmids. The putative recombination sites (Rm) are shown as multiple vertical lines crossing the gene map. (A) Alignment of pNOB8 and the related integrated plasmid in the S. tokodaii genome. The location of the plasmid in the genome is indicated. The SRSR cluster in pNOB8 is shown, and arrows indicate the position of the duplicated R m sites where recombination occurs in plNG4 to yield pINGl ( 66 ). (B) Alignment of pARN4 and the plasmid in the S. acidocaldaritts genome. (C) A summary of ORFs that are present in all sequenced free conjugative plasmids superimposed on the pNOB8 gene map.

TraG sequence alignment. Partial alignment of the putative archaeal TraG proteins with selected members of the TraG superfamily. Arch, archaeal sequences; bact, bacterial sequences. Proteins of the TraG superfamily contain two functional domains that probably form a nucleotide-binding domain. Conserved motifs are denoted above the alignment. Black background, amino acids identical in at least 50% of the sequences; shaded background, amino acids similar in at least 50% of the sequences. The alignment was prepared using the BOX shade program (http://www.ch.embnet.org/software/ BOX_form.html). (A) Domain 1 contains the conserved ATP-binding motif I. (B) N-terminal part of domain 2 exhibits the conserved motifs II (NTP-binding) and III (unknown function). GenBank sequence accession numbers are: pINGl, Q9C4Y4; pNOBS, 093672 ; pARN4/2, pARN3/2, pKEF9/l, and pHVE14 (B. Greve and R. A. Garrett, unpublished results); ST1326 (Sulfolobus tokodaii), Q971N4; TraG (Thermoanaerobactertengcongensis), Q8R8F9; TrsK (pMRCOl, Lactococcus lactis), 087219; TrwB (pR388, E. coli), Q04230; VirD4 (pTiC58, Agrobacterium tumefaciens), P18594; TraG (Ralstonia solanacearum), Q8XW89; A1I8037 (Anabaena sp.), Q8YK80; MobC (Bacteroides fragilis), Q9ZF54.

SRSR alignment. Alignment of SRSR direct repeat sequences in the conjugative plasmids pNOB8 and pKEF9/1. Arrows indicate imperfect inverted repeats. Conserved sequences are shown.

Model for insertion of pXQl into the S. solfataricus P2 chromosome. The integrase target site in the plasmid and chromosome is the 45-bp att site indicated by a blackened bar ( 38 ). In the integrated form the integrase gene is partitioned into intN and intC. pXQl carries four ORFs that are homologous to those of other pRN plasmids ( Fig. 1 ). IS element ISC1439 interrupts repA ( 49 ).