Chromosome remodelling by SMC/Condensin in B. subtilis is regulated by monomeric Soj/ParA during growth and sporulation

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC.

The stable inheritance of chromosomes is fundamental to virtually all cells. In bacteria, the lack of an overt mitotic spindle raises intriguing questions about their mechanisms of chromosome segregation and offers the possibility of targeting the process with selectively toxic antibiotics.ParABSs are major systems involved in chromosome segregation in most bacteria and on many low-copy-number plasmids (1–3). They center on multiple parS sites in DNA, which are almost always located close to oriC, the single origin of bacterial chromosome replication (4, 5). ParB proteins (Spo0J in Bacillus subtilis) are DNA-binding CTP hydrolases (6–8). ParB-CTP dimers have an open configuration that, upon specific interactions with parS, clamp shut around DNA. Closed ParB dimers are released from parS sites, allowing local spreading by lateral (and possibly bridging) interactions before CTP hydrolysis and their release from DNA (6–15). The final system component is ParA (Soj in B. subtilis), a Walker adenosine triphosphatase (ATPase). Adenosine triphosphate (ATP) binding to Apo-ParA/Soj drives a structural change that allows its dimerization and non-specific DNA binding (16–19). Interaction between ParB/Spo0J and ParA/Soj then drives ATP hydrolysis in the latter and Soj/ParA release from DNA (17, 19, 20). The extent of ParA dimer binding on the genome varies between bacterial species. For example, in Caulobacter crescentus, ParA dimers bind nonspecifically over the entire nucleoid, whereas they are restricted to the oriC region in B. subtilis (21–24). One proposed model for ParAB-mediated chromosome segregation is the DNA relay system as in C. crescentus, which drives directed motion of one chromosome origin from the stalked to the flagellated pole (21, 25, 26). In this system, ParA dimers radiate along the length of the chromosome, with the ParB-parS complex being anchored to one pole. Upon DNA replication initiation, the replicated origin (ParB-parS) stimulates ATP hydrolysis of nearby DNA-bound ParA dimers. This releases the ParA molecules from the chromosome, which then interact with PopZ at the pole. Meanwhile, the progressing ParB/parS complex interacts with the next ParA dimer on the DNA, essentially allowing the parS to follow a retreating “cloud” of ParA on the DNA. A similar mechanism has been proposed for the segregation of ParABS plasmids (13, 27–30).In B. subtilis, Soj and Spo0J are also known to have key roles in DNA replication and endospore formation in addition to chromosome segregation (22, 31, 32). For example, we have previously shown that monomer and dimer forms of Soj have opposing effects on DNA replication (inhibiting or promoting it, respectively) through direct interactions with the master initiator of DNA replication, DnaA (20, 22, 33). It then emerged that Spo0J contributes to chromosome segregation by recruiting and loading the bacterial SMC/Condensin complex (15, 34). SMC complexes align and juxtapose the left and right chromosome arms as they travel from their Spo0J-parS loading sites to the terminus regions, after which they are specifically unloaded by XerD (35–40). It is now recognized that ParB/Spo0J can load SMC complexes in a wide variety of bacterial species (15, 34, 41–43). In B. subtilis, spo0J mutants are also deficient in endospore formation because in the absence of Spo0J, Soj accumulates as an ATP dimer that promotes DNA overreplication (22), leading to a block in sporulation via a checkpoint mechanism involving the sporulation inhibitor, sda (44, 45).As well as growing vegetatively, B. subtilis can form endospores, with sporulation being one of the best characterized developmental systems in biology (46). The system we have used in the current study is that of sporulating B. subtilis since it provides a well-defined platform from which to examine the pivotal roles of Soj and Spo0J. In early sporulation, the chromosomes undergo a major reorganization to form an elongated structure termed the axial filament (or stage I) (47). A complete axial filament stretches the entire length of the cell, with origins located and anchored at cell poles and termini linked at midcell (48–51). Formation of the axial filament is critical to ensure chromosome capture upon asymmetric cell division (Fig. 1A) (32), which is required to enable the subsequent SpoIIIE (FtsK)-dependent segregation of the bisected chromosome into the tiny prespore (52–57). Despite its importance, precisely how the chromosome is reorganized to form the axial filament, as well as how origins are segregated to opposite cell poles, remains unclear. It is understood, however, that two redundant systems operate to anchor the segregated origins to opposite poles (32, 58). Both are dependent on DivIVA, a landmark protein that localizes to septa and cell poles (59). One of these involves the sporulation-specific RacA protein, which interacts with DivIVA and specific binding (ram) sites on the chromosome to the left of oriC (Fig. 1B) (32, 58, 60, 61). RacA has also been proposed to bind the chromosome and aid in its elongation across the cell (62). However, sporulation can occur via an alternate RacA-independent system, in which a number of proteins, including Soj and Spo0J, interact with DivIVA to promote capture of the parS region at the pole (Fig. 1B) (32, 63, 64). The two systems show redundancy, since only when both systems are deleted is there a near-complete abolition of polar chromosome anchoring (32, 63). Given that Soj and Spo0J are implicated in axial filament formation, and Spo0J loads SMC complexes onto the DNA, with the latter having a critical role in nucleoid organization, the ability to genetically control each step of sporulation (versus vegetative growth) offers a set of powerful tools to understand the roles of Soj-Spo0J and SMC complexes in chromosome segregation in B. subtilis (46).Open in a separate windowFig. 1.Chromosome dynamics during early sporulation. (A) Schematic representation of the changes to chromosome segregation during early sporulation. Chromosome origins (blue circles) become anchored to opposite cell poles while the terminus region (red circles) remain associated at midcell. This is followed by asymmetric division that defines the small prespore and large mother cell. SpoIIIE (green circles) resides in the asymmetric septum to prevent DNA scission and pump the mother cell localized portion of the bisected chromosome into the prespore. (B) Schematic showing the major features of the origin region required for anchoring of the chromosome to the cell poles during sporulation. There are two redundant pathways involved in capturing the origin. The first is the sporulation-specific RacA pathway, which is centered around ram sites (black arrows) to the left of oriC. The second pathway is centered around parS at oriC (red arrows). Spo0J binds parS, and along with Soj and a complex of polar proteins, tethers the origin region to the pole. (C) The Soj ATPase cycle. Upon binding to ATP, empty (Apo) Soj monomers structurally rearrange to form ATP-Soj. ATP-Soj can dimerize, which facilitates nonspecific DNA binding. Interactions between DNA-bound Soj dimers and Spo0J (bound around parS sites) trigger the ATPase activity of Soj, release from DNA, and a structural reversion back to the Apo form. The mutations K16A and G12V lock Soj in the Apo or ATP forms, respectively.In this work, we report a series of surprising findings that change our understanding of axial filament formation and the roles of ParAB and SMC complexes in B. subtilis. First, we show that ParA/Soj is active in chromosome segregation as variants, probably monomeric, that cannot bind DNA. Furthermore, we show that ParA/Soj monomers can exist in two functional states, probably corresponding to the Apo- and ATP-bound forms. The major functional difference between these forms lies in controlling SMC complex release from Spo0J-parS sites after loading. Finally, we show formation of the axial filament involves a major redistribution of SMC complexes along the chromosome and that the Apo- and ATP-forms of ParA/Soj control this, thus providing mechanistic insights into chromosome dynamics in bacteria.     

Insights into ParB spreading from the complex structure of Spo0J and parS

Spo0J (stage 0 sporulation protein J, a member of the ParB superfamily) is an essential component of the ParABS (partition system of ParA, ParB, and parS)-related bacterial chromosome segregation system. ParB (partition protein B) and its regulatory protein, ParA, act cooperatively through parS (partition S) DNA to facilitate chromosome segregation. ParB binds to chromosomal DNA at specific parS sites as well as the neighboring nonspecific DNA sites. Various ParB molecules can associate together and spread along the chromosomal DNA. ParB oligomer and parS DNA interact together to form a high-order nucleoprotein that is required for the loading of the structural maintenance of chromosomes proteins onto the chromosome for chromosomal DNA condensation. In this report, we characterized the binding of parS and Spo0J from Helicobacter pylori (HpSpo0J) and solved the crystal structure of the C-terminal domain truncated protein (Ct-HpSpo0J)-parS complex. Ct-HpSpo0J folds into an elongated structure that includes a flexible N-terminal domain for protein–protein interaction and a conserved DNA-binding domain for parS binding. Two Ct-HpSpo0J molecules bind with one parS. Ct-HpSpo0J interacts vertically and horizontally with its neighbors through the N-terminal domain to form an oligomer. These adjacent and transverse interactions are accomplished via a highly conserved arginine patch: RRLR. These interactions might be needed for molecular assembly of a high-order nucleoprotein complex and for ParB spreading. A structural model for ParB spreading and chromosomal DNA condensation that lead to chromosome segregation is proposed.The integrity of chromosomes and plasmids relies on precise DNA replication and segregation (1, 2). The initiation of DNA replication has to synchronize with the cell cycle to ensure precise chromosome segregation (3). In bacteria, the chromosome-encoded plasmid-partitioning system (Par) (4) and the structural maintenance of chromosomes (SMC) condensation complex (5) are two highly conserved systems associate with chromosome segregation and organization. SMC contributes to the overall stability and organization of genome (6–8). The partition system denoted ParABS is comprised of two proteins (ParA and ParB) and a centromere-like DNA element (parS) (9). ParB binds specifically to parS to form a complex. After binding ATP, ParA can interact with the ParB–parS complex to form a nucleoid–adaptor complex. ParB promotes the ATP hydrolysis activity of the complex to separate the chromosomes (9–13).In the bacterial chromosomal ParABS system, ParB has two functions: one is to regulate chromosome replication and sporulation (8, 12, 14) and the other is to participate in chromosome segregation (5, 15–17). ParB spreads along the chromosomal DNA by binding at specific parS and nonspecific DNA sites to form a high-order partition complex (18–20). This partition complex is required for the loading of SMC onto the chromosomal DNA (5). In addition, the N-terminal domain of ParB can interact with ParA and stimulate its ATPase activity (21). This nucleoid–adaptor complex, ParA–ParB–parS is used to drive chromosome segregation (22, 23). However, the detailed mechanism for this process is still unclear.Members of the ParB superfamily share similar functional domains: an N-terminal domain for protein–protein interactions, a central DNA-binding domain for parS binding, and a C-terminal domain for ParB dimerization (24). Two conserved N-terminal domain residues, Lys3 and Lys7, in the ParB from Bacillus subtilis (BsSpo0J), have been shown to interact with its regulatory protein BsSoj, a member of the ParA superfamily (3). The loss-of-function BsSpo0J R80A mutant was originally discovered by Autret et al. (25) and reportedly has disrupted focus formation by fluorescence microscopy. More recently, Graham et al. (20) showed that BsSpo0J bridges chromosomal DNA using single-molecule experiments. However, its R79A, R80A, and R82A mutants could not spread in vivo and did not bridge DNA in vitro. These highly conserved arginine residues were defined as an arginine patch (20). Furthermore, Broedersz et al. (26) studied the condensation and localization of ParB by computational simulation.The crystal structures of ParB superfamily proteins have been reported for a DNA-free form of TtSpo0J (from Thermus thermophilus, containing the N-terminal and the DNA-binding domains) (10) and three complexes: the RP4–KorB-O_B complex (from plasmid RP4, containing the DNA-binding domain) (27), the P1 ParB–parS complex (from Enterobacteria phage P1, containing the DNA-binding and the C-terminal domains) (28), and the F-SopB–sopC complex (from plasmid F, containing the DNA-binding domain) (29).The Helicobacter pylori ParABS system consists of HpSoj (ParA), HpSpo0J (ParB), and parS DNA (30, 31). Herein, we report the crystal structure of a C-terminal domain truncated HpSpo0J (Ct-HpSpo0J)–parS complex. The N-terminal and the DNA-binding domains are present on Ct-HpSpo0J. The structural details of the complex in combination with results from EMSAs, fluorescence anisotropy assay, and small angle X-ray scattering (SAXS) allow us to propose a model for ParB spreading as it relates to chromosome segregation.     

FokI dimerization is required for DNA cleavage

FokI is a type IIs restriction endonuclease comprised of a DNA recognition domain and a catalytic domain. The structural similarity of the FokI catalytic domain to the type II restriction endonuclease BamHI monomer suggested that the FokI catalytic domains may dimerize. In addition, the FokI structure, presented in an accompanying paper in this issue of Proceedings, reveals a dimerization interface between catalytic domains. We provide evidence here that FokI catalytic domain must dimerize for DNA cleavage to occur. First, we show that the rate of DNA cleavage catalyzed by various concentrations of FokI are not directly proportional to the protein concentration, suggesting a cooperative effect for DNA cleavage. Second, we constructed a FokI variant, FokN13Y, which is unable to bind the FokI recognition sequence but when mixed with wild-type FokI increases the rate of DNA cleavage. Additionally, the FokI catalytic domain that lacks the DNA binding domain was shown to increase the rate of wild-type FokI cleavage of DNA. We also constructed an FokI variant, FokD483A, R487A, which should be defective for dimerization because the altered residues reside at the putative dimerization interface. Consistent with the FokI dimerization model, the variant FokD483A, R487A revealed greatly impaired DNA cleavage. Based on our work and previous reports, we discuss a pathway of DNA binding, dimerization, and cleavage by FokI endonuclease.     

Emergence of hormonal and redox regulation of galectin-1 in placental mammals: implication in maternal-fetal immune tolerance

Galectin-1 is an anti-inflammatory lectin with pleiotropic regulatory functions at the crossroads of innate and adaptive immunity. It is expressed in immune privileged sites and is implicated in establishing maternal–fetal immune tolerance, which is essential for successful pregnancy in eutherian mammals. Here, we show conserved placental localization of galectin-1 in primates and its predominant expression in maternal decidua. Phylogenetic footprinting and shadowing unveil conserved cis motifs, including an estrogen responsive element in the 5′ promoter of LGALS1, that were gained during the emergence of placental mammals and could account for sex steroid regulation of LGALS1 expression, thus providing additional evidence for the role of galectin-1 in immune–endocrine cross-talk. Maximum parsimony and maximum likelihood analyses of 27 publicly available vertebrate and seven newly sequenced primate LGALS1 coding sequences reveal that intense purifying selection has been acting on residues in the carbohydrate recognition domain and dimerization interface that are involved in immune functions. Parsimony- and codon model-based phylogenetic analysis of coding sequences show that amino acid replacements occurred in early mammalian evolution on key residues, including gain of cysteines, which regulate immune functions by redox status-mediated conformational changes that disable sugar binding and dimerization, and that the acquired immunoregulatory functions of galectin-1 then became highly conserved in eutherian lineages, suggesting the emergence of hormonal and redox regulation of galectin-1 in placental mammals may be implicated in maternal–fetal immune tolerance.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids

Research on tuberculosis and leprosy was revolutionized by the development of a plasmid transformation system in the fast-growing surrogate, Mycobacterium smegmatis. This transformation system was made possible by the successful isolation of a M. smegmatis mutant strain mc²155, whose efficient plasmid transformation (ept) phenotype supported the replication of Mycobacterium fortuitum pAL5000 plasmids. In this report, we identified the EptC gene, the loss of which confers the ept phenotype. EptC shares significant amino acid sequence homology and domain structure with the MukB protein of Escherichia coli, a structural maintenance of chromosomes (SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC proteins: EptC and MSMEG_0370 both share homology with Gram-negative bacterial MukB; and MSMEG_2423 shares homology with Gram-positive bacterial SMCs, including the single SMC protein predicted for Mycobacterium tuberculosis and Mycobacterium leprae. Purified EptC was shown to bind ssDNA and stabilize negative supercoils in plasmid DNA. Moreover, an EptC–mCherry fusion protein was constructed and shown to bind to DNA in live mycobacteria, and to prevent segregation of plasmid DNA to daughter cells. To our knowledge, this is the first report of impaired plasmid maintenance caused by a SMC homolog, which has been canonically known to assist the segregation of genetic materials.The genetic bases for signature phenotypes of Mycobacterium tuberculosis, including acid fast staining, virulence, and susceptibility to tuberculosis (TB)-specific drugs were unknown before the generation of a plasmid transformation system in mycobacteria (1). Whereas recombinant DNA technology in Escherichia coli laid the foundation for modern genetic research, analysis of mycobacterial genes in E. coli was insufficient to elucidate phenotypes. The profound difference in cell wall composition, lipid metabolism, promoter recognition, and posttranslational modification between mycobacteria and E. coli greatly limited the use of E. coli as a surrogate host for mycobacterial gene analysis. Even though plasmid transformation in Streptomyces, phylogenetically closer than E. coli to Mycobacterium, was established in 1978 (2), neither Streptomyces nor E. coli is sensitive to TB-specific drugs such as isoniazid, ethambutol, ethionamide, thioacetazone, or isoxyl. Snapper et al. developed the first plasmid transformation system for mycobacteria by isolating a Mycobacterium smegmatis mutant, namely mc²155, that allowed for the replication of Mycobacterium fortuitum plasmids (1, 3). This enabled the development of efficient cloning vectors, the recreation of drug-resistant phenotypes, and important discoveries of the biology of mycobacteria. Although mc²155 was first isolated in 1988 and characterized to be a plasmid-specific phenotype in 1990 (4), the genetic basis for this efficient plasmid transformation (ept) phenotype has not been known. Here we are reporting that the mutation mediating the ept phenotype maps to a gene encoding a structural maintenance of chromosomes (SMC) homolog, which plays an important role in cell division—a fundamental cellular process across all domains of life.Cell division requires DNA replication followed by faithful segregation of genetic material to daughter cells. Eukaryotic cells rely on spindle fibers to move homologous chromosomes to the opposite ends of the cell (5, 6). Segregation in prokaryotes resembles that in eukaryotes regarding the basic mechanism and the requirement for SMC proteins. Since the discovery of SMC proteins, extensive studies have led to the understanding of their roles in chromosome condensation and segregation (7). SMC proteins were first functionally characterized in Saccharomyces cerevisiae when a mutant allele smc1-1 caused a chromosome nondisjunction (2:0 segregation) defect at least 10 times more often than observed in the WT strains (8, 9). SMC proteins are highly conserved in eukaryotes and their homologs are found in almost all Archaea, Gram-positive bacteria, and in about 40% of Gram-negative bacteria. Gram-negative organisms, such as E. coli, do not encode the canonical SMC, but rely on a functional analog MukB (10).In prokaryotes, chromosome segregation has mainly been studied by genetic and biochemical analyses of the low-copy number plasmid DNA (11–13). High-copy number plasmids rely primarily on passive diffusion for plasmid maintenance (14), which is inapplicable to chromosomal DNA. However, the accurate segregation of low-copy number plasmids requires dedicated partitioning loci consisting of three components: a cis-acting centromere site and two trans-acting proteins ParA and ParB (15). The mycobacterial pAL5000 is a low-copy number plasmid (two to five copies per cell) (16) and, noticeably, does not require a dedicated plasmid partitioning system for its segregation in M. smegmatis mc²155. Thus, maintenance of pAL5000 plasmids by mc²155, but not by its parental strain, motivated us to look for an alteration in the host machinery responsible for plasmid segregation.In this study, we report that the genetic basis of M. smegmatis mc²155’s ability to segregate episomal plasmids, and thus assume transformability, is a mutation in a gene annotated here as eptC. Biochemical and genetic analyses of EptC demonstrate that the protein belongs to the family of SMC proteins. EptC interferes with faithful segregation of pAL5000 plasmids to daughter M. smegmatis cells by directly binding to plasmids and modulating plasmid supercoiling status. The appropriate supercoiling status has been shown crucial for successful plasmid segregation (17).     

ParA2, a Vibrio cholerae chromosome partitioning protein,forms left-handed helical filaments on DNA

Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 Å pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.     

Bacterial scaffold directs pole-specific centromere segregation

Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.The bacterial cytoplasm is a complex mixture of dynamic macromolecules densely packed into a tiny compartment. Recent studies have revealed unexpected levels of organization of bacterial cytoplasmic components, including hundreds of proteins, specific lipids, mRNA molecules, and even the nucleoid itself (1). One strategy used by bacteria to generate subcellular organization of specific macromolecular complexes is active segregation by ParA-mediated molecular partitioning machines. ParA-based partitioning systems are found throughout bacteria and have been shown to spatially organize diverse macromolecular complexes to facilitate their equal distribution to progeny during cell division (2). An important question is how directionality is provided to ParA partitioning machines.One family of highly conserved ParA-based partitioning systems segregates plasmid or chromosomal centromeres to daughter cells during cell division. ParA-mediated DNA partitioning systems (Par systems) are composed of three core components: a centromeric DNA sequence parS, a site-specific DNA binding protein ParB that binds to the centromere parS sequence, and the ATPase ParA. Structural studies demonstrate that the activity of ParA is regulated by a molecular switch in which ATP-bound ParA forms dimers that bind tightly to DNA, and ParB stimulates ATP hydrolysis and release of ADP-bound ParA as monomers (3). During centromere partitioning in vivo, ATP-bound ParA assembles into a multimeric nucleoid-bound structure (4). At the centromere, ParB binds to the parS locus and nearby DNA to create a compact nucleoprotein complex (5). This ParB/parS complex binds to ParA subunits within the ParA/nucleoid structure, stimulating ATP hydrolysis and release of ParA-ADP (6–8). The multivalent ParB/parS complex has thus been proposed to bind to and shorten the ParA superstructure on the nucleoid, moving along a receding track via a dynamic disassembly mechanism (6, 8–10). The result of this process is the movement of the chromosomal centromere (parS) relative to the nucleoid bulk, and therefore to the cell itself.Whereas the fundamental operating principles of ParA-mediated movement seem conserved, how these machines target transfer to specific subcellular destinations is unknown. Many chromosomal Par systems maintain a single origin-proximal ParB/parS complex at the old cell pole and, after replication, move one newly replicated parS locus to the opposite pole (9, 11, 12). Polar protein complexes that interact with chromosome segregation factors have been identified in various bacteria, but the mechanistic consequences of these interactions have not been established (13–15). In Caulobacter, two distinct polar protein factors affect ParA-mediated centromere segregation: the new pole-specific protein TipN (16, 17) and the polar organizing protein PopZ (18, 19). TipN is a large, membrane-anchored, coiled-coil rich protein that localizes to the new pole throughout the cell cycle and, in addition to roles in localization of flagellar synthesis (16, 17), affects processive parS segregation via an unknown mechanism (6, 20).In contrast, PopZ is a small, acidic protein that forms a polymeric network at the cell pole (18, 19). In the prereplicative cell, PopZ localizes exclusively at the old cell pole, where it anchors the ParB-bound parS locus via direct interactions with ParB (18, 19). During chromosome replication initiation, PopZ releases ParB from the old pole and adopts a bipolar PopZ distribution that seems to capture ParB/parS complexes during the segregation process (18, 19). Whereas cells lacking tipN are only mildly elongated, popZ deletion causes severe filamentation (16–19), suggesting that PopZ plays a more important role in the regulation of segregation. However, the molecular mechanism by which PopZ affects segregation has remained elusive.Here we demonstrate that the multifunctional PopZ complex plays a crucial role in pole-directed movement of ParA-mediated chromosome segregation by interacting directly with ParA. We show that PopZ, but not TipN, is required for robust polar recruitment of ParA and demonstrate that a polar PopZ scaffold recruits and concentrates free ParA released during segregation. Recruitment of ParA within the PopZ matrix sequesters free ParA and locally regenerates ParA DNA binding activity. Active ParA complexes are released for recycling into nucleoid-bound structures near the cell pole, which we propose drives centromere segregation toward pole-localized PopZ. Thus, PopZ orchestrates a positive feedback mechanism that forces ParA-mediated centromere transfer to the cell pole. The polar PopZ scaffold complex creates a unique 3D microenvironment at the pole that spatially separates distinct centromere tethering and ParA-modulation activities, enabling coupling between chromosome segregation with the initiation of cell division.     

The Pseudomonas aeruginosa multidrug efflux regulator MexR uses an oxidation-sensing mechanism

MexR is a MarR family protein that negatively regulates multidrug efflux systems in the human pathogen Pseudomonas aeruginosa. The mechanism of MexR-regulated antibiotic resistance has never been elucidated in the past. We present here that two Cys residues in MexR are redox-active. They form intermonomer disulfide bonds in MexR dimer with a redox potential of −155 mV. This MexR oxidation leads to its dissociation from promoter DNA, derepression of the mexAB–oprM drug efflux operon, and increased antibiotic resistance of P. aeruginosa. We show computationally that the formation of disulfide bonds is consistent with a conformation change that prevents the oxidized MexR from binding to DNA. Collectively, the results reveal that MexR is a redox regulator that senses peroxide stress to mediate antibiotic resistance in P. aeruginosa.     

DHR3, an ecdysone-inducible early-late gene encoding a Drosophila nuclear receptor, is required for embryogenesis

Response to the steroid hormone ecdysone in Drosophila is controlled by genetic regulatory hierarchies that include eight members of the nuclear receptor protein family. The DHR3 gene, located within the 46F early-late ecdysone-inducible chromosome puff, encodes an orphan nuclear receptor that recently has been shown to exert both positive and negative regulatory effects in the ecdysone-induced genetic hierarchies at metamorphosis. We used a reverse genetics approach to identify 11 DHR3 mutants from a pool of lethal mutations in the 46F region on the second chromosome. Two DHR3 mutations result in amino acid substitutions within the conserved DNA binding domain. Analysis of DHR3 mutants reveals that DHR3 function is required to complete embryogenesis. All DHR3 alleles examined result in nervous system defects in the embryo.     

The catalytic domain of λ site-specific recombinase

The Escherichia coli phage λ integrase protein (Int) belongs to the large Int family of site-specific recombinases. It is a heterobivalent DNA binding protein that makes use of a high energy covalent phosphotyrosine intermediate to catalyze integrative and excisive recombination at specific chromosomal sites (att sites). A 293-amino acid carboxy-terminal fragment of Int (C65) has been cloned, characterized, and used to further dissect the protein. From this we have cloned and characterized a 188-amino acid, protease-resistant, carboxy-terminal fragment (C170) that we believe is the minimal catalytically competent domain of Int. C170 has topoisomerase activity and converts att suicide substrates to the covalent phosphotyrosine complexes characteristic of recombination intermediates. However, it does not show efficient binding to att site DNA in a native gel shift assay. We propose that λ Int consists of three functional and structural domains: residues 1–64 specify recognition of “arm-type” DNA sequences distant from the region of strand exchange; residues 65–169 contribute to specific recognition of “core-type” sequences at the sites of strand exchange and possibly to protein–protein interactions; and residues 170–356 carry out the chemistry of DNA cleavage and ligation. The finding that the active site nucleophile Tyr-342 is in a uniquely protease-sensitive region complements and reinforces the recently solved C170 crystal structure, which places Tyr-342 at the center of a 17-amino acid flexible loop. It is proposed that C170 is likely to represent a generic Int family domain that thus affords a specific route to studying the chemistry of DNA cleavage and ligation in these recombinases.     

Bacterial tubulin TubZ-Bt transitions between a two-stranded intermediate and a four-stranded filament upon GTP hydrolysis

Cytoskeletal filaments form diverse superstructures that are highly adapted for specific functions. The recently discovered TubZ subfamily of tubulins is involved in type III plasmid partitioning systems, facilitating faithful segregation of low copy-number plasmids during bacterial cell division. One such protein, TubZ-Bt, is found on the large pBtoxis plasmid in Bacillus thuringiensis, and interacts via its extended C terminus with a DNA adaptor protein TubR. Here, we use cryo-electron microscopy to determine the structure of TubZ-Bt filaments and light scattering to explore their mechanism of polymerization. Surprisingly, we find that the helical filament architecture is remarkably sensitive to nucleotide state, changing from two-stranded to four-stranded depending on the ability of TubZ-Bt to hydrolyze GTP. We present pseudoatomic models of both the two- and four-protofilament forms based on cryo-electron microscopy reconstructions (10.8 Å and 6.9 Å, respectively) of filaments formed under different nucleotide states. These data lead to a model in which the two-stranded filament is a necessary intermediate along the pathway to formation of the four-stranded filament. Such nucleotide-directed structural polymorphism is to our knowledge an unprecedented mechanism for the formation of polar filaments.The tubulin family of cytoskeletal proteins plays important roles in both eukaryotic and prokaryotic cells. αβ-Tubulin dimers form microtubules in eukaryotic cells that are necessary for cell division and intracellular transport. The most common prokaryotic tubulin, FtsZ, plays an essential role in cytokinesis and is found ubiquitously in bacteria and also in many archaea. Additionally, a diverse set of less-conserved tubulin family members have been identified including a variety of monomeric eukaryotic tubulins (γ-, δ-, ε-, ζ-, and η-) (1), the αβ-tubulin–like heterodimer BtubA/B (2), the prokaryotic extrachromosomal TubZs involved in plasmid segregation (3–5), and the recently discovered bacteriophage encoded tubulins, PhuZ (6, 7).Among eukaryotic αβ-tubulins, sequence identity is quite high (75–85%), as it is also among FtsZs (40–50%) (8). However, sequence identity between eukaryotic and prokaryotic family members is quite low (10–20%), and new tubulins are often discovered using only the limited number of highly conserved residues involved in nucleotide binding and hydrolysis. Despite this sequence diversity, the core structures of individual tubulin subunits are extraordinarily well conserved, and this structural conservation extends to the longitudinal interactions between monomers (9). In contrast, such striking structural conservation stops at the protofilament level: αβ-tubulin forms tubes of varying protofilament number (10), FtsZ forms a variety of straight and curved protofilament structures (11), and BtubA/B forms five protofilament structures (12). The lack of conservation of residues involved in lateral interactions has allowed the evolution of diverse higher-order filament structures, the organization and dynamics of which are precisely tuned to their cellular function.The TubZ tubulin family, discovered recently on several Bacillus virulence plasmids (4, 5) and in a Clostridial bacteriophage (3), is a group of proteins involved in bacterial plasmid partitioning (par) systems. At their core, par systems contain a polymer-forming NTPase, a DNA-binding protein, and a centromeric binding site on the DNA (13, 14). Together, these components ensure that low copy-number plasmids are efficiently segregated to both daughter cells during cell division. The TubZ found on the pBtoxis plasmid from Bacillus thurigiensis (TubZ-Bt) has been shown to treadmill (growing at one end and shrinking at the other end), and these dynamics are important for proper plasmid segregation (15). Additionally TubZ-Bt assembly has been monitored in vitro by light scattering (16) and crystal structures have been solved in the presence of GDP, GTPγS, and in the apo state (17, 18).TubZ-Bt acts in concert with the TubR helix-turn-helix DNA binding protein (18, 19), and binds to a centromeric region of the plasmid DNA, tubC, which contains seven 12-bp pseudorepeats. The TubRC complex binds in an unknown manner to the C terminus of TubZ (residues ∼407–484). Additionally, a new protein located downstream of tubZRC, named TubY, was recently implicated in the function of the Clostridium botulinum bacteriophage c-st TubZ. TubY stabilizes the assembly of TubZ filaments alone, but depolymerizes TubZ assembled in the presences of the TubRC complex. Other TubZ systems, including TubZ-Bt, have been found to have a similar protein located upstream of the operon (3), but it is unknown whether this putative TubY plays a role in pBtoxis TubZRC plasmid segregation.Here we show that the filament morphology of TubZ-Bt is linked to a nucleotide state. Untagged TubZ-Bt forms almost exclusively four-stranded helical filaments in vitro in the presence of GTP, whereas the previously observed (16–18) two-stranded filaments only accumulate under conditions where GTP hydrolysis is blocked. We present ∼11 Å and ∼7 Å resolution cryo-electron microscopy (cryo-EM) reconstructions of the helical structure of TubZ-Bt grown in the presence of GTP or GTPγS, respectively. We can unambiguously fit the TubZ-Bt crystal structure into both of these filament morphologies to form detailed pseudoatomic models. We observed that removing the extreme C terminus of the protein compromises or abrogates filament assembly, although the majority of these residues are unresolved in the reconstructions. These structures, coupled with additional biochemical and mutational studies, lead to a model in which TubZ-Bt forms an unstable two-stranded intermediate on pathway to the formation of highly stable four-stranded filaments that is only reached upon GTP hydrolysis.     

The tail of the ParG DNA segregation protein remodels ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif

The ParF protein of plasmid TP228 belongs to the ubiquitous superfamily of ParA ATPases that drive DNA segregation in bacteria. ATP-bound ParF polymerizes into multistranded filaments. The partner protein ParG is dimeric, consisting of C-termini that interweave into a ribbon-helix-helix domain contacting the centromeric DNA and unstructured N-termini. ParG stimulates ATP hydrolysis by ParF approximately 30-fold. Here, we establish that the mobile tails of ParG are crucial for this enhancement and that arginine R19 within the tail is absolutely required for activation of ParF nucleotide hydrolysis. R19 is part of an arginine finger-like loop in ParG that is predicted to intercalate into the ParF nucleotide-binding pocket thereby promoting ATP hydrolysis. Significantly, mutations of R19 abrogated DNA segregation in vivo, proving that intracellular stimulation of ATP hydrolysis by ParG is a key regulatory process for partitioning. Furthermore, ParG bundles ParF-ATP filaments as well as promoting nucleotide-independent polymerization. The N-terminal flexible tail is required for both activities, because N-terminal DeltaParG polypeptides are defective in both functions. Strikingly, the critical arginine finger-like residue R19 is dispensable for ParG-mediated remodeling of ParF polymers, revealing that the ParG N-terminal tail possesses two separable activities in the interplay with ParF: a catalytic function during ATP hydrolysis and a mechanical role in modulation of polymerization. We speculate that activation of nucleotide hydrolysis via an arginine finger loop may be a conserved, regulatory mechanism of ParA family members and their partner proteins, including ParA-ParB and Soj-Spo0J that mediate DNA segregation and MinD-MinE that determine septum localization.