Identification of a megaplasmid centromere reveals genetic structural diversity within the repABC family of basic replicons

Abstract

The basic replication unit of many plasmids and second chromosomes in the alpha-proteobacteria consists of a repABC locus that encodes the trans- and cis-acting components required for both semiautonomous replication and replicon maintenance in a cell population. In terms of physical genetic organization and at the nucleotide sequence level, repABC loci are well conserved across various genera. As with all repABC-type replicons that have been genetically characterized, the 1.4 Mb pSymA and 1.7 Mb pSymB megaplasmids from the plant endosymbiont Sinorhizobium meliloti encode strong incompatibility (inc) determinants. We have identified a novel inc sequence upstream of the repA2 gene in pSymA that is not present on pSymB and not reported in other repABC plasmids that have been characterized. This region, in concert with the repA and repB genes, stabilizes a test plasmid indicating that it constitutes a partitioning (par) system for the megaplasmid. Purified RepB binds to this sequence and binding may be enhanced by RepA. We have isolated 19 point mutations that eliminate incompatibility, reduce RepB binding or the stabilization phenotype associated with this sequence and all of these map to a 16-nucleotide palindromic sequence centred 330 bp upstream of the repA2 gene. An additional five near-perfect repeats of this palindrome are located further upstream of the repA2 gene and we show that they share some conservation with known RepB binding sites in different locations on other repABC plasmids and to two sequences found on the tumour inducing plasmid of Agrobacterium tumefaciens. These additional palindromes also bind RepB but one of them does not display obvious incompatibility effects. A heterogenic distribution of par sequences demonstrates unexpected diversity in the structural genetic organization of repABC loci, despite their obvious levels of similarity.

Neo-formation of chromosomes in bacteria

“…The SERs appear to share many characteristic features with chromosomes: enrichment in Dam methylation sites of the replication origin 9,18 , presence of initiator titration sites 9,19 , synchronization of the replication with the cell cycle 9,20-28 , KOPS-guided FtsK translocation 29 , FtsK-dependent dimer resolution system 29 , MatP/matS macrodomain organisation system 30 , and similar fine-scale segregation dynamics 22,28 . Furthermore, although initiated at different time points [22][23][24][25][26][27][28] using replicon-specific systems 7,[9][10][11]31,32 , the replications of the chromosome and the SER(s) within a multipartite genome are coordinated, hence maintaining the genome stoichiometry 21,22,25,26,28 . In the few species where this was studied, the replication of the SER is initiated after that of the chromosome [22][23][24][25][26][27][28] .…”

Rhizobial plasmids — replication, structure and biological role

“…They are incompatible with their respective parental plasmid when provided in trans, similar to centromere-like sequences from many other plasmids [33,34], and they are the binding sites for RepB proteins [31]. Single-point mutations in the parS sequence of the S. meliloti pSymA plasmid eliminated incompatibility and reduced RepB binding to this sequence [20]. The number of parS sites may vary significantly among known repABC replicons.…”

“…The differences may be related to transcriptional regulatory elements, the presence of peptide-encoding minigenes within the repABC operon [31] and, especially, the number and position of centromerelike parS sequences [20,32] (Figure 1). The latter ones usually consist of one or more copies of a 16-bp palindromic consensus sequence.…”

“…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).…”

“…So far, the repABC replicons are the only identified plasmids in which partitioning and replication genes are expressed as a single transcriptional unit ( Figure 1). In all other plasmids, partitioning and replication genes are independently expressed from different promoters [20]. The operons comprise three genes -repA, repB and repC, always in the same relative order.…”