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Several vaccines targeting bacterial pathogens show reduced efficacy upon concurrent viral infection, indicating that a new vaccinology approach is required. To identify antigens for the human pathogen Streptococcus pneumoniae that are effective following influenza infection, we performed CRISPRi-seq in a murine model of superinfection and identified the conserved lafB gene as crucial for virulence. We show that LafB is a membrane-associated, intracellular protein that catalyzes the formation of galactosyl-glucosyl-diacylglycerol, a glycolipid important for cell wall homeostasis. Respiratory vaccination with recombinant LafB, in contrast to subcutaneous vaccination, was highly protective against S. pneumoniae serotypes 2, 15A, and 24F in a murine model. In contrast to standard capsule-based vaccines, protection did not require LafB-specific antibodies but was dependent on airway CD4+ T helper 17 cells. Healthy human individuals can elicit LafB-specific immune responses, indicating LafB antigenicity in humans. Collectively, these findings present a universal pneumococcal vaccine antigen that remains effective following influenza infection. [Display omitted] • CRISPRi-seq in pneumococcal superinfection identified lafB as crucial for virulence • LafB catalyzes the formation of a glycolipid important for cell wall homeostasis • Intranasal vaccination with LafB protects against pneumococcal non-vaccine serotypes • Nasal vaccine-induced protection depends on lung Th17 lymphocytes with TRM features Liu et al. identify lafB as crucial for Streptococcus pneumoniae replication in vivo using CRISPRi-seq. Intranasal vaccination with flagellin-adjuvanted LafB induces lung Th7 lymphocytes that protect against superinfections with various pneumococcal serotypes in mice. Healthy individuals can elicit LafB-specific immune responses, suggesting that LafB is a universal, capsule-independent pneumococcal antigen. [ABSTRACT FROM AUTHOR]

Summary: Condensin—an SMC-kleisin complex—is essential for efficient segregation of sister chromatids in eukaryotes [1–4]. In Escherichia coli and Bacillus subtilis, deletion of condensin subunits results in severe growth phenotypes and the accumulation of cells lacking nucleoids [5, 6]. In many other bacteria and under slow growth conditions, however, the reported phenotypes are much milder or virtually absent [7–10]. This raises the question of what role prokaryotic condensin might play during chromosome segregation under various growth conditions. In B. subtilis and Streptococcus pneumoniae, condensin complexes are enriched on the circular chromosome near the single origin of replication by ParB proteins bound to parS sequences [11, 12]. Using conditional alleles of condensin in B. subtilis, we demonstrate that depletion of its activity results in an immediate and severe defect in the partitioning of replication origins. Multiple copies of the chromosome remain unsegregated at or near the origin of replication. Surprisingly, the growth and chromosome segregation defects in rich medium are suppressed by a reduction of replication fork velocity but not by partial inhibition of translation or transcription. Prokaryotic condensin likely prevents the formation of sister DNA interconnections at the replication fork or promotes their resolution behind the fork. [ABSTRACT FROM AUTHOR]

Summary: Proper segregation of DNA replication products is essential in all cells. In Bacillus subtilis, two protein complexes have been implicated in this process: the ParAB homologs, Soj and Spo0J, and the bacterial Smc/ScpAB complex, also called condensin. Here we demonstrate that Smc is highly enriched in the region around the origin of replication, specifically near parS sites to which Spo0J binds and at highly transcribed genes. Furthermore, we find that efficient recruitment of Smc to a large region around the origin of replication depends on the presence of Spo0J. We show that Spo0J performs two independent functions: regulation of initiation of DNA replication via Soj and promotion of chromosome segregation by Smc recruitment. Our results demonstrate a direct functional interaction between two widely conserved systems involved in chromosome replication and segregation. [Copyright &y& Elsevier]

Summary: Cohesin is a multisubunit complex that mediates sister-chromatid cohesion. Its Smc1 and Smc3 subunits possess ABC-like ATPases at one end of 50 nm long coiled coils. At the other ends are pseudosymmetrical hinge domains that interact to create V-shaped Smc1/Smc3 heterodimers. N- and C-terminal domains within cohesin''s kleisin subunit Scc1 bind to Smc3 and Smc1 ATPase heads respectively, thereby creating a huge tripartite ring. It has been suggested that cohesin associates with chromosomes by trapping DNA within its ring. Opening of the ring due to cleavage of Scc1 by separase destroys sister-chromatid cohesion and triggers anaphase. We show that cohesin''s hinges are not merely dimerization domains. They are essential for cohesin''s association with chromosomes, which is blocked by artificially holding hinge domains together but not by preventing Scc1''s dissociation from SMC ATPase heads. Our results suggest that entry of DNA into cohesin''s ring requires transient dissociation of Smc1 and Smc3 hinge domains. [Copyright &y& Elsevier]

Background: A multi-subunit protein complex called cohesin is involved in holding sister chromatids together after DNA replication. Cohesin contains four core subunits: Smc1, Smc3, Scc1, and Scc3. Biochemical studies suggest that Smc1 and Smc3 each form 50 nm-long antiparallel coiled coils (arms) and bind to each other to form V-shaped heterodimers with globular ABC-like ATPases (created by the juxtaposition of N- and C-terminal domains) at their apices. These Smc “heads” are connected by Scc1, creating a tripartite proteinaceous ring.Results: To investigate the role of Smc1 and Smc3''s ATPase domains, we engineered smc1 and smc3 mutations predicted to abolish either ATP binding or hydrolysis. All mutations abolished Smc protein function. The binding of ATP to Smc1, but not Smc3, was essential for Scc1''s association with Smc1/3 heterodimers. In contrast, mutations predicted to prevent hydrolysis of ATP bound to either head abolished cohesin''s association with chromatin but not Scc1''s ability to connect Smc1''s head with that of Smc3. Inactivation of the Scc2/4 complex had a similar if not identical effect; namely, the production of tripartite cohesin rings that cannot associate with chromosomes.Conclusions: Cohesin complexes whose heads have been connected by Scc1 must hydrolyze ATP in order to associate stably with chromosomes. If chromosomal association is mediated by the topological entrapment of DNA inside cohesin''s ring, then ATP hydrolysis may be responsible for creating a gate through which DNA can enter. We suggest that ATP hydrolysis drives the temporary disconnection of Scc1 from Smc heads that are needed for DNA entrapment and that this process is promoted by Scc2/4. [Copyright &y& Elsevier]

The cohesin complex is essential for sister chromatid cohesion during mitosis. Its Smc1 and Smc3 subunits are rod-shaped molecules with globular ABC-like ATPases at one end and dimerization domains at the other connected by long coiled coils. Smc1 and Smc3 associate to form V-shaped heterodimers. Their ATPase heads are thought to be bridged by a third subunit, Scc1, creating a huge triangular ring that could trap sister DNA molecules. We address here whether cohesin forms such rings in vivo. Proteolytic cleavage of Scc1 by separase at the onset of anaphase triggers its dissociation from chromosomes. We show that N- and C-terminal Scc1 cleavage fragments remain connected due to their association with different heads of a single Smc1/Smc3 heterodimer. Cleavage of the Smc3 coiled coil is sufficient to trigger cohesin release from chromosomes and loss of sister cohesion, consistent with a topological association with chromatin. [Copyright &y& Elsevier]

This second of two SnapShots on SMC proteins depicts their roles at different stages of the eukaryotic cell cycle. The composition and architecture of SMC protein complexes and their regulators appear in SMC Protein Complexes Part I (available at http://www.cell.com/cell/pdf/S0092-8674%2815%2901690-6.pdf ). To view this SnapShot, open or download the PDF. [ABSTRACT FROM AUTHOR]

DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the mechanisms for plasmid recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET nuclease cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as molecular signature for plasmid recognition and non-self DNA elimination. We moreover elucidate key elements of SMC loop extrusion, including the motor direction and the structure of a DNA-holding state. [Display omitted] • SMC-based Wadjet defense systems directly survey DNA circularity by loop extrusion • Cryo-EM structures reveal a Wadjet cleavage state with distinct SMC motor geometry • Mechanical DNA bending by JetABC facilitates DNA kinking and cleavage by JetD • A single large DNA roadblock stalls SMC DNA translocation but not cleavage The JET nuclease (Wadjet) is a prokaryotic SMC-based defense system that exclusively cleaves plasmid DNA. Roisné-Hamelin et al. determine structures of plasmid-borne JET, elucidating DNA bending/kinking prior to cleavage. Together with biochemical work, this supports a model of plasmid recognition by SMC motor stalling when circular DNA is completely extruded. [ABSTRACT FROM AUTHOR]

Multi-subunit SMC ATPases control chromosome superstructure and DNA topology, presumably by DNA translocation and loop extrusion. Chromosomal DNA is entrapped within the tripartite SMCkleisin ring. Juxtaposed SMC heads ("J heads") or engaged SMC heads ("E heads") split the SMCkleisin ring into "S" and "K" sub-compartments. Here, we map a DNA-binding interface to the S compartment of E heads SmcScpAB and show that head-DNA association is essential for efficient DNA translocation and for traversing highly transcribed genes in Bacillus subtilis. We demonstrate that in J heads, SmcScpAB chromosomal DNA resides in the K compartment but is absent from the S compartment. Our results imply that the DNA occupancy of the S compartment changes during the ATP hydrolysis cycle. We propose that DNA translocation involves DNA entry into and exit out of the S compartment, possibly by DNA transfer between compartments and DNA segment capture. • Hinge-DNA association is dispensable for Smc function and DNA translocation • DNA association by engaged Smc heads is essential and supports translocation • DNA is excluded from the interarm space in J heads SmcScpAB • DNA transfer into and out of the interarm space may promote DNA translocation By mapping the location of chromosomal DNA in the bacterial SMC complex, Vazquez Nunez et al. reveal that juxtaposed heads exclude DNA from the interarm space. Transient DNA occupancy in the interarm space occurs through essential head-DNA association. DNA transfer into and out of the interarm space is proposed to promote DNA translocation. [ABSTRACT FROM AUTHOR]

Structural maintenance of chromosome (SMC) complexes fold DNA by loop extrusion to support chromosome segregation and genome maintenance. Wadjet systems (JetABCD/MksBEFG/EptABCD) are derivative SMC complexes with roles in bacterial immunity against selfish DNA. Here, we show that JetABCD restricts circular plasmids with an upper size limit of about 100 kb, whereas a linear plasmid evades restriction. Purified JetABCD complexes cleave circular DNA molecules, regardless of the DNA helical topology; cleavage is DNA sequence nonspecific and depends on the SMC ATPase. A cryo-EM structure reveals a distinct JetABC dimer-of-dimers geometry, with the two SMC dimers facing in opposite direction—rather than the same as observed with MukBEF. We hypothesize that JetABCD is a DNA-shape-specific endonuclease and propose the "total extrusion model" for DNA cleavage exclusively when extrusion of an entire plasmid has been completed by a JetABCD complex. Total extrusion cannot be achieved on the larger chromosome, explaining how self-DNA may evade processing. [Display omitted] • SMC JetABCD complexes restrict plasmids independent of DNA uptake route and sequence • Larger and linear plasmids evade restriction by JetABCD • Purified JetABCD cleaves circular DNA molecules, regardless of DNA helical topology • A cryo-EM structure reveals an SMC-like architecture with a unique dimer geometry DNA-targeting immunity systems must discriminate self-DNA from nonself-DNA. Liu et al. show that bacterial Wadjet systems (JetABCD) combine nuclease with SMC DNA motor functions to specifically restrict smaller circular plasmids by DNA cleavage. Complete DNA extrusion prior to any cleavage may exclude larger chromosomal and linear DNA molecules from restriction. [ABSTRACT FROM AUTHOR]

Summary SMC condensin complexes are central modulators of chromosome superstructure in all branches of life. Their SMC subunits form a long intramolecular coiled coil, which connects a constitutive “hinge” dimerization domain with an ATP-regulated “head” dimerization module. Here, we address the structural arrangement of the long coiled coils in SMC complexes. We unequivocally show that prokaryotic Smc-ScpAB, eukaryotic condensin, and possibly also cohesin form rod-like structures, with their coiled coils being closely juxtaposed and accurately anchored to the hinge. Upon ATP-induced binding of DNA to the hinge, however, Smc switches to a more open configuration. Our data suggest that a long-distance structural transition is transmitted from the Smc head domains to regulate Smc-ScpAB’s association with DNA. These findings uncover a conserved architectural theme in SMC complexes, provide a mechanistic basis for Smc’s dynamic engagement with chromosomes, and offer a molecular explanation for defects in Cornelia de Lange syndrome. [ABSTRACT FROM AUTHOR]

Summary: Background: Cohesin, a multisubunit protein complex conserved from yeast to humans, holds sister chromatids together from the onset of replication to their separation during anaphase. Cohesin consists of four core subunits, namely Smc1, Smc3, Scc1, and Scc3. Smc1 and Smc3 proteins are characterized by 50-nm-long anti-parallel coiled coils flanked by a globular hinge domain and an ABC-like ATPase head domain. Whereas Smc1 and Smc3 heterodimerize via their hinge domains, the kleisin subunit Scc1 connects their ATPase heads, and this results in the formation of a large ring. Biochemical studies suggest that cohesin might trap sister chromatids within its ring, and genetic evidence suggests that ATP hydrolysis is required for the stable association of cohesin with chromosomes. However, the precise role of the ATPase domains remains enigmatic. Results: Characterization of cohesin''s ATPase activity suggests that hydrolysis depends on the binding of ATP to both Smc1 and Smc3 heads. However, ATP hydrolysis at the two active sites is not per se cooperative. We show that the C-terminal winged-helix domain of Scc1 stimulates the ATPase activity of the Smc1/Smc3 heterodimer by promoting ATP binding to Smc1''s head. In contrast, we do not detect any effect of Scc1''s N-terminal domain on Smc1/Smc3 ATPase activity. Conclusions: Our studies reveal that Scc1 not only connects the Smc1 and Smc3 ATPase heads but also regulates their ATPase activity. [Copyright &y& Elsevier]

Summary Multi-subunit SMC complexes control chromosome superstructure and promote chromosome disjunction, conceivably by actively translocating along DNA double helices. SMC subunits comprise an ABC ATPase “head” and a “hinge” dimerization domain connected by a 49 nm coiled-coil “arm.” The heads undergo ATP-dependent engagement and disengagement to drive SMC action on the chromosome. Here, we elucidate the architecture of prokaryotic Smc dimers by high-throughput cysteine cross-linking and crystallography. Co-alignment of the Smc arms tightly closes the interarm space and misaligns the Smc head domains at the end of the rod by close apposition of their ABC signature motifs. Sandwiching of ATP molecules between Smc heads requires them to substantially tilt and translate relative to each other, thereby opening up the Smc arms. We show that this mechanochemical gating reaction regulates chromosome targeting and propose a mechanism for DNA translocation based on the merging of DNA loops upon closure of Smc arms. [ABSTRACT FROM AUTHOR]

Summary SMC proteins support vital cellular processes in all domains of life by organizing chromosomal DNA. They are composed of ATPase “head” and “hinge“ dimerization domains and a connecting coiled-coil “arm.” Binding to a kleisin subunit creates a closed tripartite ring, whose ∼47-nm-long SMC arms act as barrier for DNA entrapment. Here, we uncover another, more active function of the bacterial Smc arm. Using high-throughput genetic engineering, we resized the arm in the range of 6–60 nm and found that it was functional only in specific length regimes following a periodic pattern. Natural SMC sequences reflect these length constraints. Mutants with improper arm length or peptide insertions in the arm efficiently target chromosomal loading sites and hydrolyze ATP but fail to use ATP hydrolysis for relocation onto flanking DNA. We propose that SMC arms implement force transmission upon nucleotide hydrolysis to mediate DNA capture or loop extrusion. [ABSTRACT FROM AUTHOR]

Chromosomes readily unlink and segregate to daughter cells during cell division, highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes promote DNA organization by loop extrusion. In most bacteria, chromosome folding initiates at dedicated start sites marked by the ParB/parS partition complexes. Whether SMC complexes recognize a specific DNA structure in the partition complex or a protein component is unclear. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate foreign chromosomes. Using chimeric proteins and chemical cross-linking, we find that ParB directly binds the Smc subunit. We map an interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex, presumably to initiate DNA loop extrusion., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Multi-subunit SMC ATPases control chromosome superstructure apparently by catalyzing a DNA-loop-extrusion reaction. SMC proteins harbor an ABC-type ATPase "head" and a "hinge" dimerization domain connected by a coiled coil "arm." Two arms in a SMC dimer can co-align, thereby forming a rod-shaped particle. Upon ATP binding, SMC heads engage, and arms are thought to separate. Here, we study the shape of Bacillus subtilis Smc-ScpAB by electron-spin resonance spectroscopy. Arm separation is readily detected proximal to the heads in the absence of ligands, and separation near the hinge largely depends on ATP and DNA. Artificial blockage of arm opening eliminates DNA stimulation of ATP hydrolysis but does not prevent basal ATPase activity. We report an arm contact as being important for controlling the transformations. Point mutations at this arm interface eliminated Smc function. We propose that partially open, intermediary conformations provide directionality to SMC DNA translocation by (un)binding suitable DNA substrates., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosome-presumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

SMC/kleisin complexes form elongated annular structures, which are critical for chromosome segregation, genome maintenance, and the regulation of gene expression. We describe marked structural similarities between bacterial and eukaryotic SMC/kleisin partner proteins (designated here as "kite" proteins for kleisin interacting tandem winged-helix (WH) elements of SMC complexes). Kite proteins are integral parts of all prokaryotic SMC complexes and Smc5/6 but not cohesin and condensin. They are made up of tandem WH domains, form homo- or heterodimers via their amino-terminal WH domain, and they associate with the central part of a kleisin subunit. In placental mammals, the kite subunit NSE3 gave rise to several (>60) kite-related proteins, named MAGE, many of which encode tumor- and testis-specific antigens. Based on architectural rather than sequence similarity, we propose an adapted model for the evolution of the SMC protein complexes and discuss potential functional similarities between bacterial Smc/ScpAB and eukaryotic Smc5/6., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Disjunction of maternal and paternal centromeres during meiosis I requires crossing over between homologous chromatids, which creates chiasmata that hold homologs together. It also depends on a mechanism ensuring that maternal and paternal sister kinetochore pairs attach to oppositely oriented microtubules. Proteolytic cleavage of cohesin's Rec8 subunit by separase destroys cohesion between sister chromatid arms at anaphase I and thereby resolves chiasmata. The Spo12 and Slk19 proteins have been implicated in regulating meiosis I kinetochore orientation and/or in preventing cleavage of Rec8 at centromeres. We show here that the role of these proteins is instead to promote nucleolar segregation, including release of the Cdc14 phosphatase required for Cdk1 inactivation and disassembly of the anaphase I spindle. Separase is also required but surprisingly not its protease activity. It has two mechanistically different roles during meiosis I. Loss of the protease-independent function alone results in a second meiotic division occurring on anaphase I spindles in spo12delta and slk19delta mutants.