The Centromere Site of the Segregation Cassette of Broad-Host-Range Plasmid RA3 Is Located at the Border of the Maintenance and Conjugative Transfer Modules

Abstract

RA3 is a low-copy-number, broad-host-range (BHR) conjugative plasmid of the IncU incompatibility group isolated originally from Aeromonas spp. A 4.9-kb fragment of RA3 is sufficient to stabilize an otherwise unstable replicon in Escherichia coli. This fragment specifies the korA-incC-korB-orf11 operon coding for an active partition system related to the central control operon of IncP-1 plasmids and found also in BHR environmental plasmids recently classified as the PromA group. All four genes in the cassette are necessary for segregation. IncC and KorB of RA3 belong to the ParA and ParB families of partitioning proteins, respectively. In contrast with IncP-1 plasmids, neither KorB nor IncC are involved in transcriptional autoregulation. Instead, KorA exerts transcriptional control of the operon by binding to a palindromic sequence that overlaps the putative ؊35 promoter motif of the cassette. The Orf11 protein is not required for regulation, but its absence decreases the stabilization potential of the segregation module. A region discontiguous from the cassette harbors a set of unrelated repeat motifs distributed over ϳ300 bp. Dissection of this region identified the centromere sequence that is vital for partitioning. The ϳ300-bp fragment also encompasses the origin of conjugative transfer, oriT, and the promoter that drives transcription of the conjugative transfer operon. A similar set of cis-acting motifs are evident in the PromA group of environmental plasmids, highlighting a common evolutionary origin of segregation and conjugative transfer modules in these plasmids and members of the IncU group.Plasmids from the IncU incompatibility group have been isolated from the fish pathogens Aeromonas salmonicida and Aeromonas hydrophila in worldwide locations (4,5,6,12,19,20,44), as well as from clinical isolates of Escherichia coli and A. hydrophila (40,53). The archetypal IncU plasmid is the low-copy-number, conjugative RA3 plasmid (45,909 bp) isolated from A. hydrophila in Japan in 1979 (5), which confers resistance to sulfonamides, streptomycin, and chloramphenicol. RA3 transfers with high frequency from E. coli to Pseudomonas putida (Gammaproteobacteria) and Ralstonia eutropha (Betaproteobacteria) and with three-magnitude-lower efficiency between E. coli and Agrobacterium tumefaciens (Alphaproteobacteria). RA3 is stably maintained in all tested representatives of these three classes (31). The nucleotide sequence of RA3 (GenBank accession no. DQ401103) (31) revealed the existence of putative functional modules involved in replication, maintenance, and conjugative transfer that are almost identical to those of the tetracycline-resistant IncU plasmid pFBAOT6 (41). The sequence confirms a high degree of conservation in the backbone functions of IncU plasmids suggested previously, on the basis of restriction enzyme analysis (47). Particularly interesting features of the RA3/pFBAOT6 backbone include a replication module with similarity to several unrelated environmental plasmids from a wide diversity of beta-and gamm...

Identification of Three Noncontiguous Regions on Bacillus anthracis Plasmid pXO1 That Are Important for Its Maintenance

“…This site is imperfect in having two discrepancies from the consensus parS1 (27), specifically C instead of G in position 9 and transversion of AC in positions 13 and 14: TGTTTCACCTGAACAA (parS1*) versus TGTTTCACGTGAAACA (parS1). We also searched upstream of ORF 84 for adjacent direct repeats (DRs) that could play the role of alternative cis-acting centromere sites (28). While no adjacent DRs were found, there are noncontiguous DRs of 16 bp (AGGTG TGTCCTTTTTT) and 17 bp (ATATAATATAAAAATAA) within ORFs 69 to 84.…”

Novel broad-host-range vehicles for cloning and shuffling of gene cassettes

“…Some of the plasmids have evolved highly complicated so called "global" regulatory networks to simultaneously control the expression of several modules or even coordinate the expression of the whole genome (Delver and Belogurov, 1997;FernandezLopez et al, 2006;Macartney et al, 1997;Pansegrau et al, 1994;Zatyka et al, 1997). Others seem to rely on simple autoregulatory mechanisms (partition cassettes, TA-toxin-antidote operons) or autoregulatory mechanisms enhanced by additional regulatory circuits, but still encompassed within the boundaries of the functional module (replication cassettes) (Chattoraj et al, 1988;del Solar et al, 1998;Dunham et al, 2009;Friedman and Austin, 1988;Hirano et al, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 1998; Kulinska et al, 2011;Magnuson et al, 1996;Mukhopadhyay and Chattoraj, 2000;RuizEchevarría et al, 1991). Growing knowledge of the interplay between the modules and understanding of their gene expression control make more realistic the perspective of linking functional modules of different origin into a new entity, so called "synthetic plasmid".…”

Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family)

“…1). The korB gene is part of the korA-incC-korB-orf11 active partition operon, which is followed by a parS sequence (18). The genetic organization of the RA3 segregation cassette and the structural features of the encoded partition proteins, IncC and KorB, identify it as subtype Ia, an active partition system widely distributed among bacterial plasmids and chromosomes (21)(22)(23)(24)(25).…”

“…DQ401103) (10), and individual functional modules have been analyzed (18,19). The plasmid encodes a number of autoregulators, i.e., RepA and RepB in the replication module (10), KfrA and KorA in the maintenance module (18,20), and MobC in the conjugative transfer unit (19). KorC, encoded in the stability module, plays the role of a global transcriptional regulator controlling replication, maintenance, and conjugative transfer functions (10,20).…”

“…Its DNA sequence has been established (GenBank accession no. DQ401103) (10), and individual functional modules have been analyzed (18,19). The plasmid encodes a number of autoregulators, i.e., RepA and RepB in the replication module (10), KfrA and KorA in the maintenance module (18,20), and MobC in the conjugative transfer unit (19).…”