Molecular and functional analysis of pTAV320, a repABC-type replicon of the Paracoccus versutus composite plasmid pTAV1

Abstract

The second replicator region of the native plasmid pTAVl of Paracoccus versutus has been identified thus proving the composite nature of this replicon. The minimal replicon designated pTAV320 (43 kb) was cloned and sequenced. pTAV320 encodes three putative proteins -RepA, RepB and RepC.This replicator region shows strong structural and functional similarity t o mpABC-type rep1 icons found in several Agrobacterium and Rhizobium plasmids. The origin of replication appears to be localized within the coding sequence of the repC gene. RepC was shown t o be essential for replication. RepA and RepB were necessary for stable maintenance of the plasmid, which implies a role in active partitioning. The presence of the complete sequence of pTAV320 (in its non-replicative form) could stabilize in cis pTAV202, a rninireplicon derived from the other pTAVl replicator region. Deletions introduced into the mpC gene abolished the 'stabilizing' activity of pTAV320, suggesting that the centromere-like sequence, necessary for partitioning, might be localized within this gene. The two replicator regions of pTAV1 (pTAV320 and pTAV202) expressed incompatibility towards the parental plasmid but were compatible in trans in P. wersutus cells. The pTAV320 replicon can be maintained in several Paracoccus, Agrobacterium, Rhizobium and Rhodohcter strains in addition t o P. versutus.

Identification of a transposable genomic island of Paracoccus pantotrophus DSM 11072 by its transposition to a novel entrapment vector pMMB2

“…The E. coli-specific mobilizable vector pABW1 (Bartosik et al, 1997) was digested with PstI and ligated with the 2?9 kb entrapment cartridge from pGBG1 (Schneider et al, 2000). The resulting plasmid (pMMB1) was digested with XbaI and KpnI and ligated with a 5?6 kb linear form of the repABC-containing mini-replicon pTAV320 (Bartosik et al, 1998), recovered from shuttle plasmid pABW22A (Table 1). The resulting positive selection vector was designated pMMB2 (Fig.…”

“…1; see Methods for details). This plasmid was a fusion of: (i) an E. coli-specific vector, pABW1 (Bartosik et al, 1997); (ii) mini-replicon pTAV320, carrying a repABC-type replicon originating from low-copy-number plasmid pTAV1 (107 kb) of Paracoccus versutus UW1 (Bartosik et al, 1998); and (iii) a cI-tetA selective cartridge (Schneider et al, 2000).…”

Structural and functional genomics of plasmid pSinA of Sinorhizobium sp. M14 encoding genes for the arsenite oxidation and arsenic resistance

“…Two different-sized replicons were obtained: (i) pMSA5 -containing a 3.5-kb EcoRI restriction fragment of pSinA, and (ii) pMSA10 -containing a 4.5-kb BamHI restriction fragment of pSinA. Sequencing showed that pMSA10 carries: (i) a single replication initiation protein gene (repC) (ORF3) without other components of the typical repABC operon (Bartosik et al, 1998), (ii) a gene potentially involved in recombinational resolution of plasmid multimers (mrs; ORF4) and (iii) two additional genes: phd (ORF5) and pemK (ORF6), which probably create a putative hybrid phd-pemK addiction system. Despite the absence of the repAB genes, pMSA10 was still able to replicate in various representatives of Alphaproteobacteria, including: A. tumefaciens LBA288, Brevundimonas sp.…”

Rhizobial plasmids — replication, structure and biological role

“…The size of particular RepA proteins and their N-terminal DNA binding domain suggest that the segregation systems of most rhizobial repABC replicons are Type Ia and thus are members of the MinD/ParA superfamily [14,23]. RepA and RepB proteins in conjunction with parS, a centromere-like sequence, provide the plasmid segregation machinery, which is required for efficient maintenance of the lowcopy DNA replicons in a dividing population of rhizobial cells [18,20,24]. The third protein-encoding gene of the operon, repC, is essential for plasmid replication ( Figure 1).…”

“…The third protein-encoding gene of the operon, repC, is essential for plasmid replication ( Figure 1). RepC, an initiator protein, functions by binding to the origin of replication oriV located within its own coding sequence, close to or inside of a large A+T region [18,24,25]. The repC gene is the minimal region sufficient for replication when inserted into a non-replicating (e.g.…”