Partition of the linear plasmid N15: interactions of N15 partition functions with the sop locus of the F plasmid

A locus close to one end of the linear N15 prophage closely resembles the sop operon which governs partition of the F plasmid; the promoter region contains similar operator sites, and the two putative gene products have extensive amino acid identity with the SopA and -B proteins of F. Our aim was to ascertain whether the N15 sop homologue functions in partition, to identify the centromere site, and to examine possible interchangeability of function with the F Sop system. When expressed at a moderate level, N15 SopA and -B proteins partly stabilize mini-F which lacks its own sop operon but retains the sopC centromere. The stabilization does not depend on increased copy number. Likewise, an N15 mutant with most of its sop operon deleted is partly stabilized by F Sop proteins and fully stabilized by its own. Four inverted repeat sequences similar to those of sopC were located in N15. They are distant from the sop operon and from each other. Two of these were shown to stabilize a mini-F sop deletion mutant when N15 Sop proteins were provided. Provision of the SopA homologue to plasmids with a sopA deletion resulted in further destabilization of the plasmid. The N15 Sop proteins exert effective, but incomplete, repression at the F sop promoter. We conclude that the N15 sop locus determines stable inheritance of the prophage by using dispersed centromere sites. The SopB-centromere and SopA-operator interactions show partial functional overlap between N15 and F. SopA of each plasmid appears to interact with SopB of the other, but in a way that is detrimental to plasmid maintenance.     

Mapping of functional domains in F plasmid partition proteins reveals a bipartite SopB-recognition domain in SopA

Active partition of the F plasmid to dividing daughter cells is assured by interactions between proteins SopA and SopB, and a centromere, sopC. A close homologue of the sop operon is present in the linear prophage N15 and, together with sopC-like sequences, it ensures stability of this replicon. We have exploited this sequence similarity to construct hybrid sop operons with the aim of locating specific interaction determinants within the SopA and SopB proteins that are needed for partition function and for autoregulation of sopAB expression. Centromere binding was found to be specified entirely by a central 25 residue region of SopB strongly predicted to form a helix-turn-helix structure. SopB protein also carries a species-specific SopA-interaction determinant within its N-terminal 45 amino acids, and, as shown by Escherichia coli two-hybrid analysis, a dimerization domain within its C-terminal 75 (F) or 97 (N15) residues. Promoter-operator binding specificity was located within an N-terminal 66 residue region of SopA, which is predicted to contain a helix-turn-helix motif. Two other regions of SopA protein, one next to the ATPase Walker A-box, the other C-terminal, specify interaction with SopB. Yeast two-hybrid analysis indicated that these regions contact SopB directly. Evidence for the involvement of the SopA N terminus in autoinhibition of SopA function was obtained, revealing a possible new aspect of the role of SopB in SopA activation.     

Sop proteins can cause transcriptional silencing of genes located close to the centromere sites of linear plasmid N15

Stable inheritance of bacterial chromosomes and low copy number plasmids is ensured by accurate partitioning of replicated molecules between the daughter cells at division. Partitioning of the prophage of the temperate bacteriophage N15, which exists as a linear plasmid molecule with covalently closed ends, depends on the sop locus, comprising genes sopA and sopB, as well as four centromere sites in different regions of the N15 genome essential for replication and the control of lysogeny. We found that binding of SopB to the centromere could silence centromere-proximal promoters, presumably due to subsequent polymerization of SopB along the DNA. Close to the IR4 centromere site we identified a promoter, P59, which was able to drive the expression of phage late genes encoding structural proteins of virion. We found that, following binding to IR4, the N15 Sop proteins could induce repression of this promoter. The repression depended on SopB and was enhanced in the presence of SopA. Sop-dependent silencing of centromere-proximal promoters may control gene expression in phage N15, particularly preventing undesired expression of late genes in the N15 prophage. Thus, the phage N15 sop system not only ensures plasmid partitioning but is also involved in the genetic network controlling prophage replication and the maintenance of lysogeny.     

Disruption of the F plasmid partition complex in vivo by partition protein SopA

The SopA protein plays an essential, though so far undefined, role in partition of the mini-F plasmid but, when overproduced, it causes loss of mini-F from growing cells. Our investigation of this phenomenon has revealed that excess SopA protein reduces the linking number of mini-F. It appears to do so by disturbing the partition complex, in which SopB normally introduces local positive supercoiling upon binding to the sopC centromere, as it occurs only in plasmids carrying sopC and in the presence of SopB protein. SopA-induced reduction in linking number is not associated with altered sop promoter activity or levels of SopB protein and occurs in the absence of changes in overall supercoil density. SopA protein mutated in the ATPase nucleotide-binding site (K120Q) or lacking the presumed SopB interaction domain does not induce the reduction in linking number, suggesting that excess SopA disrupts the partition complex by interacting with SopB to remove positive supercoils in an ATP-dependent manner. Destabilization of mini-F also depends on sopC and SopB, but the K120Q mutant retains some capacity for destabilizing mini-F. SopA-induced destabilization thus appears to be complex and may involve more than one SopA activity. The results are interpreted in terms of a regulatory role for SopA in the oligomerization of SopB dimers bound to the centromere.     

Subcellular positioning of F plasmid mediated by dynamic localization of SopA and SopB

SopA, SopB proteins and the cis-acting sopC DNA region of F plasmid are essential for partitioning of the plasmid, ensuring proper subcellular positioning of the plasmid DNA molecules. We have analyzed by immunofluorescence microscopy the subcellular localization of SopA and SopB. The majority of SopB molecules formed foci, which localized frequently with F plasmid DNA molecules. The foci increased in number in proportion to the cell length. Interestingly, beside the foci formation, SopB formed a spiral structure that was dependent on SopA, which also formed a spiral structure, independent of the presence of SopB, and these two structures partially overlapped. On the basis of these results and previous biochemical studies together with our simulations, we propose a theoretical model named "the reaction-diffusion partitioning model", using reaction-diffusion equations that explain the dynamic subcellular localization of SopA and SopB proteins and the subcellular positioning of F plasmid. We hypothesized that sister copies of plasmid DNA compete with each other for sites at which SopB multimer is at the optimum concentration. The plasmid incompatibility mediated by the Sop system might be explained clearly by this hypothesis.     

Identification of a partition region carried by the plasmid QpH1 of Coxiella burnetii

Coxiella burnetii is an intracellular bacterial pathogen which causes Q fever in humans and other animals. Most of the isolates found carry plasmids which share considerable homology. Unfortunately all of these plasmids remain cryptic. Initial attempts to look for secreted or membrane proteins encoded by these plasmids using TnphoA mutagenesis revealed an open reading frame on the EcoRI-fragment C of the plasmid QpH1. Upstream DNA sequencing of the TnphoA insertions revealed a deduced peptide sequence with homology to the SopA protein which is encoded by the F plasmid in Escherichia coli. Maxi-cell analysis showed that fragment C encoded two proteins: one was 43.5 kDa in size and designated QsopA, and a second was 38 kDa in size. These proteins are similar in molecular weight to the SopA and SopB proteins, which are essential components of the partition mechanism of the F plasmid. The region appears to be conserved in plasmids QpRS, QpDV, and QpDG, but is absent in a plasmidless isolate in which plasmid sequences have integrated into the chromosomal DNA. Complementation studies demonstrated that fragment C has a plasmid partitioning function and can restore maintenance stability of the partition-defective mini-F plasmid. These data suggest that fragment C carries the plasmid partition region of the plasmid QpH1.     

Polymerization of SopA partition ATPase: regulation by DNA binding and SopB

In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for most ATPases (the Walker-box type) the mechanism is unknown. Also unknown is how the host cell contributes to partition. We have examined the effects of non-sequence-specific DNA on in vitro self-assembly of the SopA partition ATPase of plasmid F. SopA underwent polymerization provided ATP was present. DNA inhibited this polymerization and caused breakdown of pre-formed polymers. Centromere-binding protein SopB counteracted DNA-mediated inhibition by itself binding to and masking the DNA, as well as by stimulating polymerization directly. The results suggest that in vivo, SopB smothers DNA by spreading from sopC, allowing SopA-ATP polymerization which initiates plasmid displacement. We propose that SopB and nucleoid DNA regulate SopA polymerization and hence partition.     

Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids

Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability.     

N15: the linear phage-plasmid

The lambdoid phage N15 of Escherichia coli is very unusual among temperate phages in that its prophage is not integrated into chromosome but is a linear plasmid molecule with covalently closed ends. Upon infection the phage DNA circularises via cohesive ends, then phage-encoded enzyme, protelomerase, cuts at an inverted repeat site and forms hairpin ends (telomeres) of the linear plasmid prophage. Replication of the N15 prophage is initiated at an internally located ori site and proceeds bidirectionally resulting in formation of duplicated telomeres. Then the N15 protelomerase cuts duplicated telomeres generating two linear plasmid molecules with hairpin telomeres. Stable inheritance of the plasmid prophage is ensured by partitioning operon similar to the F factor sop operon. Unlike F sop, the N15 centromere consists of four inverted repeats dispersed in the genome. The multiplicity and dispersion of centromeres are required for efficient partitioning of a linear plasmid. The centromeres are located in N15 genome regions involved in phage replication and control of lysogeny, and binding of partition proteins at these sites regulates these processes. Two N15-related lambdoid Siphoviridae phages, φKO2 in Klebsiella oxytoca and pY54 in Yersinia enterocolitica, also lysogenize their hosts as linear plasmids, as well as Myoviridae marine phages VP882 and VP58.5 in Vibrio parahaemolyticus and ΦHAP-1 in Halomonas aquamarina. The genomes of all these phages contain similar protelomerase genes, lysogeny modules and replication genes, as well as plasmid-partitioning genes, suggesting that these phages may belong to a group diverged from a common ancestor.     

Purification and characterization of SopA and SopB proteins essential for F plasmid partitioning

Mini-F plasmid has the trans-acting genes sopA and sopB and the cis-acting site sopC which are essential for accurate partitioning of plasmid DNA molecules into both daughter cells. In this study, we purified independently SopA and SopB proteins, analyzed the in vitro DNA-binding activity of these proteins by the gel retardation assay, and determined the precise binding sites of DNA by the footprinting method. SopA binds to four repeated sequences (CTTTGC) located in the promoter-operator region of the sopAB operon. The SopA binding activity is enhanced by the addition of SopB protein. SopB protein itself does not bind to this DNA region. These results suggest that the complex of SopA and SopB proteins autoregulate the expression of the sopA-sopB operon. On the other hand, SopB protein binds to the sopC region, in which 12 direct repeats of 43-base pairs nucleotides exist. SopB protein recognizes the inverted repeats of 7 base pairs in each direct repeats. SopA protein does not affect the SopB binding activity to the sopC DNA segment.     

Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

Accurate DNA segregation is essential for genome transmission. Segregation of the prototypical F plasmid requires the centromere-binding protein SopB, the NTPase SopA and the sopC centromere. SopB displays an intriguing range of DNA-binding properties essential for partition; it binds sopC to form a partition complex, which recruits SopA, and it also coats DNA to prevent non-specific SopA–DNA interactions, which inhibits SopA polymerization. To understand the myriad functions of SopB, we determined a series of SopB–DNA crystal structures. SopB does not distort its DNA site and our data suggest that SopB–sopC forms an extended rather than wrapped partition complex with the SopA-interacting domains aligned on one face. SopB is a multidomain protein, which like P1 ParB contains an all-helical DNA-binding domain that is flexibly attached to a compact (β₃–α)₂ dimer-domain. Unlike P1 ParB, the SopB dimer-domain does not bind DNA. Moreover, SopB contains a unique secondary dimerization motif that bridges between DNA duplexes. Both specific and non-specific SopB–DNA bridging structures were observed. This DNA-linking function suggests a novel mechanism for in trans DNA spreading by SopB, explaining how it might mask DNA to prevent DNA-mediated inhibition of SopA polymerization.     

Autoregulation of the partition genes of the mini-F plasmid and the intracellular localization of their products in Escherichia coli

The sopAB operon and the sopC sequence, which acts as a centromere, are essential for stable maintenance of the mini-F plasmid. Immunoprecipitation experiments with purified SopA and SopB proteins have demonstrated that these proteins interact in vitro. Expression studies using the lacZ gene as a reporter revealed that the sopAB operon is repressed by the cooperative action of SopA and SopB. Using immunofluorescence microscopy, we found discrete fluorescent foci of SopA and SopB in cells that produce both SopA and SopB in the presence of the sopC DNA segment, but not in the absence of sopC, suggesting the SopA-SopB complex binds to sopC segments. SopA was exclusively found to colocalize with nucleoids in cells that produced only SopA, while, in the absence of SopA, SopB was distributed in the cytosolic spaces.     

Autoregulation of the partition genes of the mini-F plasmid and the intracellular localization of their products in Escherichia coli

The sopAB operon and the sopC sequence, which acts as a centromere, are essential for stable maintenance of the mini-F plasmid. Immunoprecipitation experiments with purified SopA and SopB proteins have demonstrated that these proteins interact in vitro. Expression studies using the lacZ gene as a reporter revealed that the sopAB operon is repressed by the cooperative action of SopA and SopB. Using immunofluorescence microscopy, we found discrete fluorescent foci of SopA and SopB in cells that produce both SopA and SopB in the presence of the sopC DNA segment, but not in the absence of sopC, suggesting the SopA-SopB complex binds to sopC segments. SopA was exclusively found to colocalize with nucleoids in cells that produced only SopA, while, in the absence of SopA, SopB was distributed in the cytosolic spaces. Received: 14 July 1997 / Accepted: 3 October 1997     

Probing plasmid partition with centromere-based incompatibility

Low-copy number plasmids of bacteria rely on specific centromeres for regular partition into daughter cells. When also present on a second plasmid, the centromere can render the two plasmids incompatible, disrupting partition and causing plasmid loss. We have investigated the basis of incompatibility exerted by the F plasmid centromere, sopC, to probe the mechanism of partition. Measurements of the effects of sopC at various gene dosages on destabilization of mini-F, on repression of the sopAB operon and on occupancy of mini-F DNA by the centromere-binding protein, SopB, revealed that among mechanisms previously proposed, no single one fully explained incompatibility. sopC on multicopy plasmids depleted SopB by titration and by contributing to repression. The resulting SopB deficit is proposed to delay partition complex formation and facilitate pairing between mini-F and the centromere vector, thereby increasing randomization of segregation. Unexpectedly, sopC on mini-P1 exerted strong incompatibility if the P1 parABS locus was absent. A mutation preventing the P1 replication initiation protein from pairing (handcuffing) reduced this strong incompatibility to the level expected for random segregation. The results indicate the importance of kinetic considerations and suggest that mini-F handcuffing promotes pairing of SopB-sopC complexes that can subsequently segregate as intact aggregates.     

The secreted effector protein of Salmonella dublin, SopA, is translocated into eukaryotic cells and influences the induction of enteritis

Salmonella -induced enteritis is associated with the induction of an acute intestinal inflammatory response and net fluid secretion into the lumen of infected mucosa. Proteins secreted by the Inv/Spa type III secretion system of Salmonella play a key role in the induction of these responses. We have demonstrated recently that the Inv/Spa-secreted SopB and SopD effector proteins are translocated into eukaryotic cells via a Sip dependent pathway and act in concert to mediate inflammation and fluid secretion in infected ileal mucosa. Mutations of both sopB and sopD significantly reduced, but did not abrogate, the enteropathogenic phenotype. This indicated that other virulence factors are involved in the induction of enteritis. In this work, we characterize SopA, a secreted protein belonging to the family of Sop effectors of Salmonella dublin . We demonstrate that SopA is translocated into eukaryotic cells and provide evidence suggesting that SopA has a role in the induction of enteritis.