Genomic comparison of archaeal conjugative plasmids from Sulfolobus

Abstract

All of the known self-transmissable plasmids of the Archaea have been found in the genus Sulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self-transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence analysis with two earlier sequenced plasmids, pNOB8 and pING1. The analyses revealed three conserved and functionally distinct sections in the genomes. Section A is considered to encode the main components of the conjugative apparatus, where two genes show low but significant sequence similarity to sections of genes encoding bacterial conjugative proteins. A putative origin of replication is located in section B, which is highly conserved in sequence and contains several perfect and imperfect direct and inverted repeats. Further downstream, in section C, an operon encoding six to nine smaller proteins is implicated in the initiation and regulation of replication. Each plasmid carries an integrase gene of the type that does not partition on integration, and there is strong evidence for their integration into host chromosomes, where they may facilitate intercellular exchange of chromosomal genes. Two plasmids contain hexameric short regularly spaced repeats (SRSR), which have been implicated in plasmid maintenance, and each plasmid carries multiple recombination motifs, concentrated in the variable regions, which likely provide sites for genomic rearrangements.

Figure 1.

Comparative gene map of the Sulfolobus self-transmissable plasmids. Thick, black horizontal lines delineate the genomic sections A, B and C. The main conserved open reading frames (ORFs) in section A, the highly conserved ORF153 and ORF196, the integrase and PlrA are labeled for pKEF9. Homologs in the different plasmids can be identified by color and pattern. Open reading frames shown as an empty box have no homologs in the other plasmids. The short regularly spaced repeat (SRSR) clusters are indicated by green rectangular boxes on the linear genomes of pKEF9 and pNOB8-33. Recombination motifs are represented by yellow boxes on the linear genomes. The location of the deletion in pNOB8-33 is downstream from ORF617. The deletion variant plasmid pING2 extends from downstream of ORF1044 to the center of the operon in section C.

Figure 2.

Comparison of motif patterns in ORF1044 and ORF620 of Sulfolobus and the bacterial TraG and TrbE homologs, respectively. The conserved domains 1 and 2, and the motifs III, IV and V are labeled. ATPI indicates a Walker A motif and NTPII represents a Walker B motif. Black boxes denote putative transmembrane spanning helices. (A) ORF1044. Conserved domains and motifs were identified by alignment of the Sulfolobus ORFs with 19 different bacterial TraG homologs (Schröder et al. 2002). (B) ORF620. Conserved motifs were identified by alignment with 19 different TrbE homologs of the VirB4 family (Rabel et al. 2003).

Figure 3.

(A) Putative replication origin. Red boxes = 10–11 bp perfect direct repeats; blue boxes = 16 bp imperfect direct repeats; dark grey arrows = inverted repeats conserved in all plasmids; green arrows = inverted repeats conserved in pKEF9, pING1, pARN3 and pARN4; and light grey arrows = inverted repeats conserved in pHVE14 and pNOB8. Black arrows = plasmid-specific inverted repeats. (B) The 170 bp conserved region. The 10–11 bp repeats are shown in red and 16 bp imperfect repeats are shown in blue. The total number of these imperfect repeats is: pKEF9 = 13, pHVE14 = 10; pNOB8 = 6; pING1 = 12; pARN3 = 9; and pARN4 = 8. Inverted repeats are shown in green.

Figure 4.

(A) Putative replication operon from pKEF9, pING1 and pARN4. Homologous open reading frames (ORFs) are shown in identical colors and the ORF sizes are given. Positions of the recombination motifs are indicated by yellow boxes on the linear genomes. The region of the operon present in the deletion derivative pING2 is mapped below pING1. (B) Promoter regions for the regulatory site of the putative CopG homolog on pKEF9 and pARN4 are aligned. The start codon of the first gene in the operon is underlined in red. Inverted repeats are marked by arrows. Direct repeats are underlined.

Figure 5.

Alignment of short regularly spaced repeat (SRSR) sequences of pKEF9 (red). Conserved sequence positions are indicated. The genome nucleotide positions of the SRSR clusters are: pKEF9 = 19,067–18,720; and pNOB8-33 = 32,855–33,199.