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. 2012 Jul;194(13):3426-36.
doi: 10.1128/JB.00041-12. Epub 2012 Apr 20.

Centromere Binding and Evolution of Chromosomal Partition Systems in the Burkholderiales

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Free PMC article

Centromere Binding and Evolution of Chromosomal Partition Systems in the Burkholderiales

Fanny M Passot et al. J Bacteriol. .
Free PMC article

Abstract

How split genomes arise and evolve in bacteria is poorly understood. Since each replicon of such genomes encodes a specific partition (Par) system, the evolution of Par systems could shed light on their evolution. The cystic fibrosis pathogen Burkholderia cenocepacia has three chromosomes (c1, c2, and c3) and one plasmid (pBC), whose compatibility depends on strictly specific interactions of the centromere sequences (parS) with their cognate binding proteins (ParB). However, the Par systems of B. cenocepacia c2, c3, and pBC share many features, suggesting that they arose within an extended family. Database searching revealed seven subfamilies of Par systems like those of B. cenocepacia. All are from plasmids and secondary chromosomes of the Burkholderiales, which reinforces the proposal of an extended family. The subfamily of the Par system of B. cenocepacia c3 includes plasmid variants with parS sequences divergent from that of c3. Using electrophoretic mobility shift assay (EMSA), we found that ParB-c3 binds specifically to centromeres of these variants, despite high DNA sequence divergence. We suggest that the Par system of B. cenocepacia c3 has preserved the features of an ancestral system. In contrast, these features have diverged variably in the plasmid descendants. One such descendant is found both in Ralstonia pickettii 12D, on a free plasmid, and in Ralstonia pickettii 12J, on a plasmid integrated into the main chromosome. These observations suggest that we are witnessing a plasmid-chromosome interaction from which a third chromosome will emerge in a two-chromosome species.

Figures

Fig 1
Fig 1
Extra parS sites inhibit growth in B. cenocepacia J2315. (A) Growth inhibition induced by wild-type parS sequences. Each sector shows three colonies of J2315-Mex1 4 days after transformation with the pMMB206 vector or recombinant derivatives carrying parS-c1, -c2, -c3, or -pBC in single copy or the parS-c2 and -pBC clusters (denoted parS+). (B) Mutated parS-c1 and parS-c3 sequences tested for growth inhibition. Each single-base change in parS-c1 and parS-c3 (left top and middle boxes) is shown above its corresponding wild-type base (numbered), in blue when silent (i.e., still growth inhibitory) or in red when leading to loss of function (i.e., allowing normal growth). Doubly mutated parS-c1 and parS-c3 (right boxes) are shown above the wild-type sequence, with changed bases in blue or red as described above. The chimeric parS-c1/c3 sequence, a loss-of-function mutant, is shown (bottom left), with the noncomplementary parS-c1 and parS-c3 ends indicated in red. Arrows, inverted repeated sequences.
Fig 2
Fig 2
EMSA of the Par systems of B. cenocepacia. (A) Binding of each B. cenocepacia parS probe was tested with increasing amounts (0.2, 1, 5, and 25 μg) of crude extracts of E. coli cells overproducing its cognate ParB (▲, c3; ×, pBC; ■, c2; ◆, c1). (B) Radiolabeled parS probes, denoted above each group of lanes, were incubated with 10 mg of crude extract protein containing ParB-c1 (lanes c1), -c2 (lanes c2), -c3 (lanes c3), or -pBC (lanes p) or no ParB (lanes 0). The cognate ParB-parS interactions produce a single shifted complex in the case of c1, c2, and pBC and a three-band pattern in the case of c3 (arrowheads). The parS-c3 fragment is also retarded by an unknown E. coli protein (*). (C) Protein extracts containing ParB-c1 or no ParB, indicated by “c1” and “0” above the lane groups, were incubated with the radiolabeled 26-bp probes indicated below each lane as follows: negative-control sequence CTAGTCGTACGACTAG (lanes 0), wild-type parS-c1 (lanes WT), and parS-c1 mutated as indicated (other lanes). The mutated parS-c1 T1G-A16C fragment is shifted by an unknown E. coli protein (*). (D) Cell extracts containing no ParB, ParB-c3, or ParB-c1 (as indicated) were incubated with the hybrids indicated below each lane.
Fig 3
Fig 3
Bsr families and corresponding parS palindromes. Each color corresponds to one of the seven Bsr families. ParB proteins and loci are listed in Table S1 in the supplemental material. For clarity, only the bootstrap values superior to 800 concerning the deep branches are indicated (*). RepB proteins from Rhizobiales megaplasmids and chromosome 1 ParB proteins from Burkholderiales are indicated as outgroups. Protein and parS variations allow identification of 13 Bsr subtypes. Each subtype is exemplified (right) by one replicon: B. cenocepacia J2315 plasmid pBC (J2315-pBC), B. cenocepacia J2315 chromosome 2 (J2315-c2), B. cenocepacia J2315 chromosome 3 (J2315-c3), P. naphthalenivorans CJ2 plasmid 1 (pNAP1), P. naphthalenivorans CJ2 plasmid 2 (pNAP2), plasmid integrated in chromosome 1 of R. pickettii 12J (12J-c1::p), R. pickettii 12D plasmid 1 (12D-p1), B. vietnamiensis G4 plasmid 2 (G4-p2), R. solanacearum GMI 1000 megaplasmid (Rsol-Mp), Polaromonas sp. strain JS666 plasmid 1 (JS666-p1), Polaromonas sp. strain JS666 plasmid 2 (JS666-p2), R. ferrireducens T118 plasmid 1 (T118-p1), and Burkholderia glumae BGR1 plasmid 4 (BGR1-p4). The minimal 14-bp palindrome (parS) near the parAB operon is indicated. In the case of pNAP1 parS, underlined letters indicate positions of degeneracy. The structure of each parAB operon is shown (black line, minimal palindrome; first arrow, parA; second arrow, parB). ParA proteins vary from 217 residues (BGR1-p4) to 242 (JS666-p2), and ParB proteins vary from 290 (J2315-pBC) to 376 (JS666-p2).
Fig 4
Fig 4
Cross-reactions in Bsr1 and Bsr3 families. (A) EMSA of Bsr1 ParB proteins and parS sites. Samples are grouped according to parS, indicated below: 0, negative-control sequence TAGTCGTACGACTA; wt, parS Bsr1, TTGGCTCGAGCCAA, common to pBC (Bsr1-a) and pNAP1 (Bsr1-b); T6G and G10T, parS derivatives exclusive to pNAP1. ParB proteins are denoted above parS as follows: 0, no ParB; 1, ParB-pBC; 2, ParB-pNAP1. (B) Bsr1 interaction summary. Arrows indicate that ParB in the box binds the parS pointed to. Curved and straight arrows represent cognate and noncognate binding, respectively. Black, gray, and dashed arrows correspond, respectively, to strong, medium, and weak binding relative to the specific binding. pNAP1-parS sequences are shown with bases changed relative to Bsr1 underlined and numbered. (C) EMSA of Bsr3 ParBs and parS sites. Samples are grouped according to parS: 0, negative-control sequence CTAGTCGTACGACTAG; c3, parS chromosome 3; G4, parS plasmid 2 of B. vietnamiensis G4; 12D, parS plasmid 1 of R. pickettii 12D; 12J #, secondary parS of R. pickettii 12J; 12J, main parS of R. pickettii 12J. ParB proteins are denoted as follows: 0, no-ParB extract; 1, ParB-c3; 2, ParB-G4; 3, ParB-12D; 4, ParB-12J. The parS-c3 fragment is also retarded by an unknown E. coli protein (*). (D) Bsr3 interaction summary. Arrows signify interactions as described for panel B. parS sequences are shown below each box, with bases changed relative to parS-c3 underlined.
Fig 5
Fig 5
Binding of ParBc3 to parS-c3, parS-12D, parS-Bsr7, and related or mutated sequences was analyzed by EMSA. Positions 4, 5, and 6 and 11, 12, and 13 are underlined in parS-c3. Underlined bases in the other parS sequences differ from those in parS-c3. The characteristic three-band pattern of ParB-c3 is indicated by arrowheads.
Fig 6
Fig 6
Modifications in a putative HTH domain of ParB-Bsr3. (A) Comparison of the putative HTH domains of ParB-c3, -12J, -G4, and -12D. Amino acids identical to ParB-c3 are in black, others in red. Positions are numbered and presumed helices are shown. (B) EMSA of ParB proteins with the HTH domain exchanged (from −3 to 20 according to the numbering in panel A) or modified as shown above each panel, e.g., for c3A5K, ParB-c3 with Lys substituted for Ala at position 5. The parS probes are denoted below each lane as follows: 0, negative-control sequence CTAGTCGTACGACTAG; c3, parS-c3; D, parS Ralstonia pickettii 12D plasmid 1; J, main parS Ralstonia pickettii 12J. The parS-c3 fragment is also retarded by an unknown E. coli protein (*). Note that the three-band pattern typical of ParB-c3, possibly resulting from protein cleavage at sites distant from the HTH, is replaced by a single band after substitution with the 12D HTH. This exchange would not be expected to eliminate protease sensitivity of two sites, suggesting that the three-band pattern could have another explanation, such as formation by ParB-c3 of three types of complex with parS sites, each with a different gel electrophoretic mobility.
Fig 7
Fig 7
Evolution of an ancestral Bsr3-Par system. The Par system of Burkholderia cepacia complex chromosome 3 (C3) is proposed to be the most likely ancestor. Its ParB has a relatively wide binding specificity, and its parS comprises the features of the other systems: GTT ends (red circles), CACGTG core (red rectangle), and symmetry (red convergent arrows). An ancestral plasmid form would have been captured and frozen by C3 but would have spread on other plasmid replicons and diverged to generate the Par systems 12J, 12D, and G4, each with a parS having lost one feature. Where evolution has proceeded in different species, without the requirement for compatibility, ParB-parS cross-reaction can be detected (arrows). When managing two replicons in the same species (e.g., B. vietnamiensis G4 and R. pickettii 12D), Par systems must diverge for the replicons to be compatible and ParB-parS cross-reaction is detected barely or not at all (ball lines).
Fig 8
Fig 8
Ralstonia pickettii 12J chromosome 1 compared to R. pickettii 12D plasmid 2. Ralstonia pickettii 12J chromosome 1, nucleotides 1,560,000 to 1,950,000, is represented at the top, the whole Ralstonia pickettii 12D plasmid 2 at the bottom. They share extensive sequence homology and gene order. Conserved blocks with 99% identity are shown in red. Blue blocks correspond to inversions with sequence conservation above 80%. Duplications of 6 kb, presumably arising during plasmid integration, are highlighted as yellow blocks. (See also Materials and Methods.)

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