Article Figures & Data
Figures
- FIG. 1.
Location of parAB loci and parS sites. At left, arrows indicate the position of each parA homologue start codon relative to the GC skew of the 10th (0.95 to 0.05) of the replicon centered on the GC-skew minimum (c1 to c3) or of the whole replicon (p1). Genetic map sketches are aligned at parA homologue start codons and show only genes characteristic of chromosome origin regions or relevant to plasmid maintenance. orf5 is a gene of unknown function, named for its position in the gid-par operon. repA (c2 and c3) signifies generic resemblance to plasmid replication control genes; trfA indicates homology with the corresponding RK2 gene. S, parS sites (c1) or parS-like palindromes (c2, c3, and p1); S*, degenerate parS-like sequences; P, predicted promoter internal to parA of c3; A, predicted DnaA box motifs based on the consensus 5′-TTATCCAC.
- FIG. 2.
Parts of the aligned ParA (A) and ParB (B) amino acid sequences that distinguish subgroups. Residues that are identical (A) or nearly so (B) in all examples shown are in red. Numbers above show distances, not coordinates, and double hatch marks denote sequences not shown (18 residues just after the A box in A). The Walker A (nucleotide binding), A′ (catalytic), and B (Mg binding) motifs constitute the ATPase active site. ParBII is a motif identified previously (52). The helix-turn-helix (HTH) motifs are shaded green. The HTH of c3 is weakly predicted relative to the others. Note the short A′-B-box interval in the c2, c3, and p1 ParAs, very similar to the pTAR and TP228 homologues, and the absence of all HTH region homology in the latter ParBs. (C) Aligned parS and parS-like sequences with fully conserved bases in red. Note the dissimilarity of the pTAR and TP228 par region repeats.
- FIG. 3.
Phylogeny of partition proteins of bacteria with multipartite genomes. Predicted amino acid sequences of ParA homologues (A) and ParB homologues (B) were aligned and phylogenetically compared using ClustalX. Large blocks of heterology, such as N-terminal extensions present in some ParAs but absent from others, were discarded from the analysis. The option “tree-correct for multiple substitutions” was used. Phylogenetic trees were viewed using Treeview. Homologues of individual multipartite genomes or closely related groups are indicated in color, and others are single-chromosome species included as markers. All branches are of the correct length, but some are extended by dotted lines for clarity. Abbreviations for full species names are as follows: Atum, Agrobacterium tumefaciens; Bbur, Borrelia burgdorferi; Bcc, Burkholderia cenocepacia; Bpsm, Burkholderia pseudomallei; Bsub, Bacillus subtilis; Bsui, Brucella suis; Bxen, Burkholderia xenovorans; Ccre, Caulobacter crescentus; Drad, Deinococcus radiodurans; Eco, Escherichia coli; Mbov, Mycobacterium bovis; Paer, Pseudomonas aeruginosa; Pput, Pseudomonas putida; Rsol, Ralstonia solenacearum; Scoe, Streptomyces coelicolor; Smel, Sinorhizobium meliloti; Spne, Streptococcus pneumoniae; Vcho, Vibrio cholerae. The group comprising the largest chromosome of every species, denoted as c or c1, forms a distinct cluster, as shown by the shaded area. Secondary replicons are shown as c2 and c3 or in the case of megaplasmids by their given names or p1 and p2. Large and small replicons of B. xenovorans were named chromosomes 2 and 1, respectively (Joint Genome Institute) and are thus shown in quote marks here. The Borrelia burgdorferi secondary replicon ParBs are less well defined than the ParA group and are not shown. In B, the canonical parS sequence is shown next to the large chromosome group and the parS-like palindromes of B. cenocepacia and R. solanacearum are shown beside their replicons, with conserved bases in red.
- FIG. 4.
Partition activity of B. cenocepacia parABS elements in E. coli. Shown is the effect of pBBRmcs5 carrying parAB of (A) c1, (B) c2, (C) c3, or (D) p1 on the loss rates of mini-F vector pDAG203 carrying a single parS-like unit of c1 (○), c2 (▵), c3 (□), p1 (⋄), or none (* [shown only in A]). Slight differences in slope, e.g., parS of c2 and p1 in A, are not significant. The y axis in C is broken to accommodate the accelerated loss of the c3 parS plasmid caused by its own ParAB. (E) Effects of modifying parAB and parS elements on mini-F stabilization. Empty and shaded bars show loss rates for plac-parAB at high (pBBRmcs5) and low (pAM238) copy numbers (c.n.), respectively; dotted-line bars indicate that growth with selection for the mini-F was so perturbed and variable that loss rates could not be reproducibly measured; the horizontal dotted line shows the spontaneous loss rate of the mini-F vector; below the bar numbers, mutations in parS* (g8a), parB promoter in parA (PpA), and parS (c7t) are shown. Variability of loss rate was ≤18% (standard deviation). Cognate ParAB-parS interactions shown by thicker lines in A to D are represented by columns 3, 6, 12, and 18, respectively.
- FIG. 5.
parABS-mediated silencing. Exponential-phase cultures of strains carrying parAB on a plasmid and a parS-pldc::lacZ module in an integrated λ prophage were assayed for β-galactosidase, and specific activities were normalized to those of the same strains without parAB (∼250 Miller units). Error bars are standard deviations. ABs− and ABp− carry the mutations shown in Fig. 4.
Tables
- TABLE 1.
Plasmids
Plasmid Relevant characteristics Source or reference(s) pDAG203 Mini-F Δ(sopOPABC) cat+ (6.67 kb) 30 pDAG123 pRS551 (reppMB1bla+ KanrlacZYA) carrying pldc (12.6 kb) 31, 47 pAM238 rep pSC101 aadA; polylinker of pUC18 (4.3 kb) 8 pBBR1MCS5 rep pBBR1 mob RK2 Genr (4.77 kb) 24 pDAG551 pDAG203 with single c1 parS between AflIII-SexAI This work pDAG552 pDAG203 with single c2 parS between AflIII-Bsu36I This work pDAG553 pDAG203 with single c3 parS between AflIII-Bsu36I This work pDAG554 pDAG203 with single p1 parS between AflIII-Bsu36I This work pDAG555 pDAG203 with c2 parS cluster between PmlI-Bsu36I This work pDAG556 pDAG203 with c3 parS cluster between PmlI-Bsu36I This work pDAG557 pDAG203 with p1 parS cluster between PmlI-Bsu36I This work pDAG558 pAM238 with c1 parAB fragment (including 91 bp upstream of parA) between XbaI-SphI This work pDAG559 pAM238 with c2 parAB fragment (including 81 bp upstream of parA) between XbaI-HindIII This work pDAG560 pAM238 with c3 parAB fragment (including 141 bp upstream of parA) between XbaI-SphI This work pDAG561 pAM238 with p1 parAB fragment (including 126 bp upstream of parA) between XbaI-SphI This work pDAG562 pBBR1MCS5 with c1 parAB fragment (including 256 bp upstream of parA) between SpeI-HindIII This work pDAG563 pBBR1MCS5 with c2 parAB fragment (including 81 bp upstream of parA) between XbaI-BamHI This work pDAG564 pBBR1MCS5 with c3 parAB fragment (including 267 bp upstream of parA) between XbaI-BamHI This work pDAG565 pBBR1MCS5 with p1 parAB fragment (including 126 bp upstream of parA) between XbaI-BamHI This work pDAG566 pDAG563 with G→A in central CG of internal parS* This work pDAG567 pDAG565 with C→T in central CG of internal parS This work pDAG568 pDAG564 with T→A mutation of promoter −10 (TAAAATa) in c3 parA This work pDAG571 pDAG123 with single c1 parS at EcoRI upstream of pldc::lacZYA This work pDAG572 pDAG123 with single c2 parS at EcoRI upstream of pldc::lacZYA This work pDAG573 pDAG123 with single c3 parS at EcoRI upstream of pldc::lacZYA This work pDAG574 pDAG123 with single p1 parS at EcoRI upstream of pldc::lacZYA This work ↵ a The mutated base is underlined.
- TABLE 2.
parS oligonucleotides
Replicon Oligonucleotide name (FOP no.) Sequencea (5′→3′ top, 3′←5′ bottom) Restriction site(s) for: Insertion Screeningb c1 11 cgtgTGTTTCACGTGAAACA AflIII- PmlI 12 ACAAAGTGCACTTTGTggacc -SexAI c2 22 cgtgcccGTTTATGCGCATAAACccc AflIII- FspAI 23 gggCAAATACGCGTATTTGgggagt -Bsu36I c3 24 cgtgcccGTTGTCACGTGACAACccc AflIII- PmlI 25 gggCAACAGTGCACTGTTGgggagt -Bsu36I p1 26 cgtggggCTTGGCTCGAGCCAAGggg AflIII- XhoI 27 cccGAACCGAGCTCGGTTCcccagt -Bsu36I
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Files in this Data Supplement:
- Supplemental file 1
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Table S1, Oligonucleotides used for PCR amplification of parS clusters. Table S2, Oligonucleotides used for PCR amplification of parAB loci. Fig. S1, Vector maps.
Zipped PDF document, 106K.
- Supplemental file 1
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Table S1, Oligonucleotides used for PCR amplification of parS clusters. Table S2, Oligonucleotides used for PCR amplification of parAB loci. Fig. S1, Vector maps.
