Spo0J regulates the oligomeric state of Soj to trigger its switch from an activator to an inhibitor of DNA replication initiation

Control of DNA replication initiation is essential for bacterial cells to co-ordinate the faithful replication and segregation of their genetic material. The Bacillus subtilis ATPase Soj is a dynamic protein that regulates DNA replication initiation by either inhibiting or activating the DNA replication initiator protein DnaA. Here we report that the key event which switches Soj regulatory activity is a transition in its oligomeric state from a monomer to an ATP-dependent homodimer capable of DNA binding. We show that the DNA binding activity of the Soj dimer is required both for activation of DNA replication initiation and for interaction with Spo0J. Finally, we demonstrate that Spo0J inhibits Soj dimerization by stimulating Soj ATPase activity. The data provide a molecular explanation for the dichotomous regulatory activities of Soj, as well as assigning unique Soj conformations to distinct cellular localization patterns. We discuss how the regulation of Soj ATPase activity by Spo0J could be utilized to control the initiation of DNA replication during the cell cycle.     

Control of development by altered localization of a transcription factor in B. subtilis

In B. subtilis, the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) regulate the initiation of sporulation. Soj is a negative regulator of sporulation gene expression, and Spo0J antagonizes Soj function. Using fusions of Soj to green fluorescent protein, we found that Soj localized near the cell poles and upon entry into stationary phase oscillated from pole to pole. In the absence of Spo0J, Soj was associated predominantly with DNA. By in vivo cross-linking and immunoprecipitation, we found that Soj physically associates with developmentally regulated promoters, and this association increased in the absence of Spo0J. These results show that Soj switches localization and function depending on the chromosome partitioning protein Spo0J. We further show that mutations in the Soj ATPase domain disrupt localization and function and render Soj insensitive to regulation by Spo0J.     

Bacterial sporulation: pole-to-pole protein oscillation

Sporulating bacteria need to temporally coordinate DNA replication, chromosome partitioning and sporulation initiation. Recent work has shown that one aspect of this coordination lies with the interdependent subcellular localization of two proteins, Spo0J and Soj, and in the Spo0J-dependent spatial oscillation of Soj.     

Characterization of the Soj/Spo0J chromosome segregation proteins and identification of putative parS sequences in Helicobacter pylori

The Soj and Spo0J proteins, together with one or more parS sequences, are crucial to chromosome segregation and the progression of cell cycle in many bacteria. In Helicobacter pylori, genes coding for Soj and a plasmid replication-partition-related protein containing a Spo0J or ParB conserved domain, together with two putative parS sites identified in this study, were found to be located within the origin-proximal 20-30% of the circular chromosome. Recombinant H. pylori Spo0J bound specifically to the two putative parS sequences and that of Bacillus subtilis. In addition, hydrolysis of ATP by H. pylori Soj was accelerated in the presence of parS and/or Spo0J. Protein-protein interactions, intracellular levels, and subcellular localization of Soj and Spo0J were analyzed through polyclonal antibodies directed against recombinant Soj and Spo0J. This study was the first implication of the existence of a functional parABS system in H. pylori.     

The chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) contribute to accurate chromosome partitioning, separation of replicated sister origins, and regulation of replication initiation in Bacillus subtilis

Soj (ParA) and Spo0J (ParB) of Bacillus subtilis belong to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. Unlike most Par systems, for which intact copies of both parA and parB are required for the Par system to function, inactivating soj does not cause a detectable chromosome partitioning phenotype whereas inactivating spo0J leads to a 100-fold increase in the production of anucleate cells. This suggested either that Soj does not function like other ParA homologues, or that a cellular factor might compensate for the absence of soj. We found that inactivating smc, the gene encoding the structural maintenance of chromosomes (SMC) protein, unmasked a role for Soj in chromosome partitioning. A soj null mutation dramatically enhanced production of anucleate cells in an smc null mutant. To look for effects of a soj null on other phenotypes perturbed in a spo0J null mutant, we analysed replication initiation and origin positioning in (soj-spo0J)+, Deltasoj, Deltaspo0J and Delta(soj-spo0J) cells. All of the mutations caused increased initiation of replication and, to varying extents, affected origin positioning. Using a new assay to measure separation of the chromosomal origins, we found that inactivating soj, spo0J or both led to a significant defect in separating replicated sister origins, such that the origins remain too close to be spatially resolved. Separation of a region outside the origin was not affected. These results indicate that there are probably factors helping to pair sister origin regions for part of the replication cycle, and that Soj and Spo0J may antagonize this pairing to contribute to timely separation of replicated origins. The effects of Deltasoj, Deltaspo0J and Delta(soj-spo0J) mutations on origin positioning, chromosome partitioning and replication initiation may be a secondary consequence of a defect in separating replicated origins.     

RacA and the Soj-Spo0J system combine to effect polar chromosome segregation in sporulating Bacillus subtilis

Sporulating cells of Bacillus subtilis undergo a highly polarized cell division and possess a specialized mechanism to move the oriC region of the chromosome close to the cell pole before septation. DivIVA protein, which localizes to the cell pole, and the Soj and Spo0J proteins, which associate with the chromosome, are part of the mechanism that delivers the chromosome to the cell pole. A sporulation-specific protein, RacA, encodes a third DNA-binding protein, which acts in conjunction with Soj and Spo0J to effect efficient polar chromosome segregation. divIVA mutants and soj racA double mutants have an unexpected phenotype in which specific markers to the left and right of oriC can be captured in the prespore compartment but the central oriC region is efficiently excluded. This 'residual' trapping requires Spo0J protein. We suggest that the Soj RacA DivIVA system is required to extract the oriC region from its position determined by the vegetative chromosome segregation machinery and anchor it to the cell pole.     

A fixed distance for separation of newly replicated copies of oriC in Bacillus subtilis: implications for co-ordination of chromosome segregation and cell division

The Spo0J protein of Bacillus subtilis is required for normal chromosome segregation and forms discrete subcellular assemblies closely associated with the oriC region of the chromosome. Here we show that duplication of Spo0J foci occurs early in the DNA replication cycle and that this requires the initiation of DNA replication at oriC but not elongation beyond the nearby STer sites. Soon after duplication, sister oriC /Spo0J foci move rapidly apart to achieve a fixed separation of about 0.7 μm, reminiscent of the segregation of eukaryotic chromosomes on the mitotic spindle. The magnitude of the fixed separation distance may explain how chromosome segregation is kept in close register with cell growth and the initiation mass for DNA replication. It could also explain how segregation can proceed accurately in the absence of cell division. The kinetics of focal separation suggest that one role of Spo0J protein may be to facilitate formation of separate sister oriC complexes that can be segregated.     

Structural analysis of the chromosome segregation protein Spo0J from Thermus thermophilus

Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The plasmid partitioning loci encode two proteins, ParA and ParB, and a cis-acting centromere-like site denoted parS. The chromosomally encoded homologues of ParA and ParB, Soj and Spo0J, play an active role in chromosome segregation during bacterial cell division and sporulation. Spo0J is a DNA-binding protein that binds to parS sites in vivo. We have solved the X-ray crystal structure of a C-terminally truncated Spo0J (amino acids 1-222) from Thermus thermophilus to 2.3 A resolution by multiwavelength anomalous dispersion. It is a DNA-binding protein with structural similarity to the helix-turn-helix (HTH) motif of the lambda repressor DNA-binding domain. The crystal structure is an antiparallel dimer with the recognition alpha-helices of the HTH motifs of each monomer separated by a distance of 34 A corresponding to the length of the helical repeat of B-DNA. Sedimentation velocity and equilibrium ultracentrifugation studies show that full-length Spo0J exists in a monomer-dimer equilibrium in solution and that Spo0J1-222 is exclusively monomeric. Sedimentation of the C-terminal domain of Spo0J shows it to be exclusively dimeric, confirming that the C-terminus is the primary dimerization domain. We hypothesize that the C-terminus mediates dimerization of Spo0J, thereby effectively increasing the local concentration of the N-termini, which most probably dimerize, as shown by our structure, upon binding to a cognate parS site.     

Identification of a polar targeting determinant for Bacillus subtilis DivIVA

The Bacillus subtilis protein DivIVA controls both the positioning of the vegetative cell division site and the polar attachment of the chromosome during sporulation. In vegetative growth DivIVA attracts the bipartite cell division inhibitor MinCD away from the cell centre and towards the cell pole. This process ensures the inactivation of old polar division sites and leaves the cell centre free for the assembly of a new cell division complex. During sporulation MinCD and DivIVA levels fall, but DivIVA remains at the cell poles and becomes involved in the migration of the chromosomes to the pole. In order to investigate polar targeting of DivIVA, we undertook a mutational analysis of the 164-amino-acid protein. These studies identified one mutant (divIVA(R18C)) that could not localize to the cell pole but which retained the ability to support both vegetative growth and 50% sporulation efficiency. Further analysis revealed that, in the absence of polar targeting, DivIVA(R18C) localized to the nucleoid during vegetative growth in a Spo0J/Soj-dependent manner and required Spo0J/Soj and MinD to orientate the chromosomes correctly during sporulation. We demonstrate that polar targeting of DivIVA(R18C) is not essential during vegetative growth because the mutant can recognize the cell division site and influences the localization of MinD. Similarly we show that DivIVA(R18C) can function during sporulation because it can support the Spo0J/Soj orientation of the chromosome. In addition, we establish that both residues 18 and 19 constitute a DivIVA polar targeting determinant.     

Effects of the chromosome partitioning protein Spo0J (ParB) on oriC positioning and replication initiation in Bacillus subtilis

Spo0J (ParB) of Bacillus subtilis is a DNA-binding protein that belongs to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. We found that Spo0J contributes to the positioning of the chromosomal oriC region, but probably not by recruiting the origin regions to specific subcellular locations. In wild-type cells during exponential growth, duplicated origin regions were generally positioned around the cell quarters. In a spo0J null mutant, sister origin regions were often closer together, nearer to midcell. We found, by using a Spo0J-green fluorescent protein [GFP] fusion, that the subcellular location of Spo0J was a consequence of the chromosomal positions of the Spo0J binding sites. When an array of binding sites (parS sites) were inserted at various chromosomal locations in the absence of six of the eight known parS sites, Spo0J-GFP was no longer found predominantly at the cell quarters, indicating that Spo0J is not sufficient to recruit chromosomal parS sites to the cell quarters. spo0J also affected chromosome positioning during sporulation. A spo0J null mutant showed an increase in the number of cells with some origin-distal regions located in the forespore. In addition, a spo0J null mutation caused an increase in the number of foci per cell of LacI-GFP bound to arrays of lac operators inserted in various positions in the chromosome, including the origin region, an increase in the DNA-protein ratio, and an increase in origins per cell, as determined by flow cytometry. These results indicate that the spo0J mutant produced a significant proportion of cells with increased chromosome content, probably due to increased and asynchronous initiation of DNA replication.     

Novel Fission Yeast Cdc7-Dbf4-Like Kinase Complex Required for the Initiation and Progression of Meiotic Second Division

Cdc7, a conserved serine/threonine protein kinase, controls initiation of DNA replication. A regulatory subunit, Dbf4, stimulates the kinase activity of Cdc7 and recruits it to the replication origins. Schizosaccharomyces pombe has a homologous kinase complex, composed of Hsk1 and Dfp1/Him1. Here, we report a novel protein kinase of S. pombe, Spo4, which shares common structural features with the Cdc7 kinases. In spite of the structural similarities, Spo4 is dispensable for mitotic growth and premeiotic DNA replication. Intriguingly, spo4 null mutants are defective in initiation and progression of the second meiotic division. Spindles for meiosis II are often fragmented. Spo4 kinase activity is markedly enhanced when the enzyme is associated with its regulatory subunit, Spo6, a Dbf4-like protein. Expression of Spo4 is specifically induced during meiosis. Spo4 is preferentially present in nuclei, but this nuclear localization does not require Spo6. These results suggest that Spo4 is a Cdc7 kinase whose primary role is in meiosis, not in DNA replication. This is the first report of an organism which has two Cdc7-related kinase complexes with different biological functions.     

Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci

Regulation of DNA replication and segregation is essential for all cells. Orthologs of the plasmid partitioning genes parA, parB, and parS are present in bacterial genomes throughout the prokaryotic evolutionary tree and are required for accurate chromosome segregation. However, the mechanism(s) by which parABS genes ensure proper DNA segregation have remained unclear. Here we report that the ParA ortholog in B. subtilis (Soj) controls the activity of the DNA replication initiator protein DnaA. Subcellular localization of several Soj mutants indicates that Soj acts as a spatially regulated molecular switch, capable of either inhibiting or activating DnaA. We show that the classical effect of Soj inhibiting sporulation is an indirect consequence of its action on DnaA through activation of the Sda DNA replication checkpoint. These results suggest that the pleiotropy manifested by chromosomal parABS mutations could be the indirect effects of a primary activity regulating DNA replication initiation.