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Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genetic information. Direct visualization of chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the shape of unconfined bacterial chromosome in real-time in live Bacillus subtilis cells that are expanded in volume. We show that the chromosomes predominantly exhibit crescent shapes with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins - the key drivers of chromosomal organization - along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure., (© 2024. The Author(s).)

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 ParAB S system is essential for prokaryotic chromosome segregation. After loading at parS on the genome, ParB (partition protein B) proteins rapidly redistribute to distances of ~15 kilobases from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA loaded with hundreds of proteins. Using in vitro single-molecule fluorescence imaging, we show that ParB from Bacillus subtilis can load onto DNA distantly of parS , as loaded ParB molecules themselves are found to be able to recruit additional ParB proteins from bulk. Notably, this recruitment can occur in cis but also in trans, where, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by molecular dynamics simulations, which show that cooperative ParB-ParB recruitment can enhance spreading. ParS -independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation, which is vital for the bacterial cell cycle.

ParABS systems facilitate chromosome segregation and plasmid partitioning in bacteria and archaea. ParB protein binds centromeric parS DNA sequences and spreads to flanking DNA. We show that ParB is an enzyme that hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP). parS DNA stimulates cooperative CTP binding by ParB and CTP hydrolysis. A nucleotide cocrystal structure elucidates the catalytic center of the dimerization-dependent ParB CTPase. Single-molecule imaging and biochemical assays recapitulate features of ParB spreading from parS in the presence but not absence of CTP. These findings suggest that centromeres assemble by self-loading of ParB DNA sliding clamps at parS ParB CTPase is not related to known nucleotide hydrolases and might be a promising target for developing new classes of antibiotics., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Site-directed mutagenesis is a key tool in the analysis of biological mechanisms. We have established an efficient and systematic gene targeting strategy for Bacillus subtilis based on the Golden Gate cloning methodology. Our approach permits the introduction of single or multiple point mutations or of heavily engineered alleles into the endogenous gene locus in a single step using a 96-well microtiter plate format. We have successfully applied this system for high-throughput functional screening of resized variants of the Structural Maintenance of Chromosome (Smc) protein and for exhaustive cysteine cross-linking mutagenesis. Here we describe, in detail, the experimental setup for high-throughput introduction of modifications into the B. subtilis chromosome. With minor modifications, the approach should be applicable to other bacteria and yeast.

mNeonGreen fluorescent protein is capable of photo-switching, hence in principle applicable for super-resolution imaging. However, difficult-to-control blinking kinetics that lead to simultaneous emission of multiple nearby mNeonGreen molecules impedes its use for PALM. Here, we determined the on- and off- switching rate and the influence of illumination power on the simultaneous emission. Increasing illumination power reduces the probability of simultaneous emission, but not enough to generate high quality PALM images. Therefore, we introduce a simple data post-processing step that uses temporal and spatial information of molecule localizations to further reduce artifacts arising from simultaneous emission of nearby emitters. We also systematically evaluated various sample preparation steps to establish an optimized protocol to preserve cellular morphology and fluorescence signal. In summary, we propose a workflow for super-resolution imaging with mNeonGreen based on optimization of sample preparation, data acquisition and simple post-acquisition data processing. Application of our protocol enabled us to resolve the expected double band of bacterial cell division protein DivIVA, and to visualize that the chromosome organization protein ParB organized into sub-clusters instead of the typically observed diffraction-limited foci. We expect that our workflow allows a broad use of mNeonGreen for super-resolution microscopy, which is so far difficult to achieve.

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., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 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.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

The article presents a study that establishes a physical assay to determine whether the binding of condensin to native chromosomes in Bacillus subtilis involves entrapment of DNA by the Smc-ScpAB ring.

Summary Segregation of replicated chromosomes is an essential process in all organisms. How bacteria, such as the oval-shaped human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes is poorly understood. Here we show that the pneumococcal homologue of the DNA-binding protein ParB recruits S. pneumoniae condensin (SMC) to centromere-like DNA sequences ( parS) that are located near the origin of replication, in a similar fashion as was shown for the rod-shaped model bacterium Bacillus subtilis. In contrast to B. subtilis, smc is not essential in S. pneumoniae, and Δ smc cells do not show an increased sensitivity to gyrase inhibitors or high temperatures. However, deletion of smc and/or parB results in a mild chromosome segregation defect. Our results show that S. pneumoniae contains a functional chromosome segregation machine that promotes efficient chromosome segregation by recruitment of SMC via ParB. Intriguingly, the data indicate that other, as of yet unknown mechanisms, are at play to ensure proper chromosome segregation in this organism. [ABSTRACT FROM AUTHOR]