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Chromosomes must be highly compacted and organized within cells, but how this is achieved in vivo remains poorly understood. We report the use of chromosome conformation capture coupled with deep sequencing (Hi-C) to map the structure of bacterial chromosomes. Analysis of Hi-C data and polymer modeling indicates that the Caulobacter crescentus chromosome consists of multiple, largely independent spatial domains that are probably composed of supercoiled plectonemes arrayed into a bottle brush-like fiber. These domains are stable throughout the cell cycle and are reestablished concomitantly with DNA replication. We provide evidence that domain boundaries are established by highly expressed genes and the formation of plectoneme-free regions, whereas the histone-like protein HU and SMC (structural maintenance of chromosomes) promote short-range compaction and the colinearity of chromosomal arms, respectively. Collectively, our results reveal general principles for the organization and structure of chromosomes in vivo.

Higher-order chromosome folding and segregation are tightly regulated in all domains of life. In bacteria, details on nucleoid organization regulatory mechanisms and function remain poorly characterized, especially in non-model species. Here, we investigate the role of DNA-partitioning protein ParB and SMC condensin complexes in the actinobacterium Corynebacterium glutamicum . Chromosome conformation capture reveals SMC-mediated long-range interactions around ten centromere-like parS sites clustered at the replication origin ( oriC ). At least one oriC -proximal parS site is necessary for reliable chromosome segregation. We use chromatin immunoprecipitation and photoactivated single-molecule localization microscopy to show the formation of distinct, parS -dependent ParB-nucleoprotein subclusters. We further show that SMC/ScpAB complexes, loaded via ParB at parS sites, mediate chromosomal inter-arm contacts (as previously shown in Bacillus subtilis ). However, the MukBEF-like SMC complex MksBEFG does not contribute to chromosomal DNA-folding; instead, this complex is involved in plasmid maintenance and interacts with the polar oriC -tethering factor DivIVA. Our results complement current models of ParB-SMC/ScpAB crosstalk and show that some condensin complexes evolved functions that are apparently uncoupled from chromosome folding. The regulation of higher-order chromosome folding and segregation in bacteria is poorly understood. Here, Böhm et al. provide insights into the roles of DNA partitioning protein ParB and SMC condensin complexes in Corynebacterium glutamicum .

Structural maintenance of chromosomes (SMC) complexes play critical roles in chromosome dynamics in virtually all organisms, but how they function remains poorly understood. In the bacterium Bacillus subtilis, SMC-condensin complexes are topologically loaded at centromeric sites adjacent to the replication origin. Here we provide evidence that these ring-shaped assemblies tether the left and right chromosome arms together while traveling from the origin to the terminus (>2 megabases) at rates >50 kilobases per minute. Condensin movement scales linearly with time, providing evidence for an active transport mechanism. These data support a model in which SMC complexes function by processively enlarging DNA loops. Loop formation followed by processive enlargement provides a mechanism by which condensin complexes compact and resolve sister chromatids in mitosis and by which cohesin generates topologically associating domains during interphase.

Chromosomal loci jiggle in place between segregation events in prokaryotic cells and during interphase in eukaryotic nuclei. This motion seems random and is often attributed to Brownian motion. However, we show here that locus dynamics in live bacteria and yeast are sensitive to metabolic activity. When ATP synthesis is inhibited, the apparent diffusion coefficient decreases, whereas the subdiffusive scaling exponent remains constant. Furthermore, the magnitude of locus motion increases more steeply with temperature in untreated cells than in ATP-depleted cells. This \"superthermal\" response suggests that untreated cells have an additional source of molecular agitation, beyond thermal motion, that increases sharply with temperature. Such ATP-dependent fluctuations are likely mechanical, because the heat dissipated from metabolic processes is insufficient to account for the difference in locus motion between untreated and ATP-depleted cells. Our data indicate that ATP-dependent enzymatic activity, in addition to thermal fluctuations, contributes to the molecular agitation driving random (sub)diffusive motion in the living cell.

Bacterial chromosomes are organized by condensins, such as Structural Maintenance of Chromosomes (SMC) proteins. In Streptomyces , a genus of sporulating bacteria, SMC proteins align chromosomal arms and promote efficient compaction of chromosomal DNA during spore formation. We hypothesized that disrupting nucleoid architecture by deleting the smc gene would affect the positioning of the origin of replication ( oriC ) or the process of chromosome replication during spore germination. To test this hypothesis, we conducted marker frequency analyses and microscopy studies to observe the positioning of labelled oriC and replisomes in both wild type and Δ smc backgrounds. Additionally, we investigated the positioning of three chromosomal loci in early vegetative cells. Our results indicate that the deletion of smc impairs chromosome replication and hinders germ tube development. Furthermore, detailed analysis of chromosome organization revealed that, in the absence of SMC, the oriC region becomes mispositioned within the nucleoid. These findings underscore the important role of SMC in maintaining nucleoid architecture during the early growth stages of Streptomyces .

It has long been a question whether Staphylococcus aureus, a major human pathogen, is able to develop natural competence for transformation by DNA. We previously showed that a novel staphylococcal secondary sigma factor, SigH, was a likely key component for competence development, but the corresponding gene appeared to be cryptic as its expression could not be detected during growth under standard laboratory conditions. Here, we have uncovered two distinct mechanisms allowing activation of SigH production in a minor fraction of the bacterial cell population. The first is a chromosomal gene duplication rearrangement occurring spontaneously at a low frequency [≤10(-5)], generating expression of a new chimeric sigH gene. The second involves post-transcriptional regulation through an upstream inverted repeat sequence, effectively suppressing expression of the sigH gene. Importantly, we have demonstrated for the first time that S. aureus cells producing active SigH become competent for transformation by plasmid or chromosomal DNA, which requires the expression of SigH-controlled competence genes. Additionally, using DNA from the N315 MRSA strain, we successfully transferred the full length SCCmecII element through natural transformation to a methicillin-sensitive strain, conferring methicillin resistance to the resulting S. aureus transformants. Taken together, we propose a unique model for staphylococcal competence regulation by SigH that could help explain the acquisition of antibiotic resistance genes through horizontal gene transfer in this important pathogen.

Bacteriophages are the main vehicle for gene swapping in bacteria, notoriously of pathogenicity islands and antibiotic resistance genes. Chen et al. noticed that the Staphylococcus aureus prophages do not excise from their host's genome until very late in their life cycles (see the Perspective by Davidson). Thus, the phage DNA is amplified while embedded in the bacterial chromosome. The resulting concatemers are processively packed into virus capsules while still integrated in the host chromosome. Each virion is only set loose when the capsule has reached physical capacity—a process called “headful” packaging. In situ amplification maximizes viral replication, and the headful mechanism means adjacent bacterial-host DNA also gets grabbed to fill the capsule. This process ensures that host genes are transmitted along with the phage. Science , this issue p. 207 ; see also p. 152 Staphylococcus aureus phages amplify and package while chromosomally integrated such that host DNA becomes incorporated in the virus particle. Genetic transduction is a major evolutionary force that underlies bacterial adaptation. Here we report that the temperate bacteriophages of Staphylococcus aureus engage in a distinct form of transduction we term lateral transduction. Staphylococcal prophages do not follow the previously described excision-replication-packaging pathway but instead excise late in their lytic program. Here, DNA packaging initiates in situ from integrated prophages, and large metameric spans including several hundred kilobases of the S. aureus genome are packaged in phage heads at very high frequency. In situ replication before DNA packaging creates multiple prophage genomes so that lateral-transducing particles form during normal phage maturation, transforming parts of the S. aureus chromosome into hypermobile regions of gene transfer.

Despite recent progress in visualization experiments, the mechanism underlying chromosome segregation in bacteria still remains elusive. Here we address a basic physical issue associated with bacterial chromosome segregation, namely the spatial organization of highly confined, self-avoiding polymers (of nontrivial topology) in a rod-shaped cell-like geometry. Through computer simulations, we present evidence that, under strong confinement conditions, topologically distinct domains of a polymer complex effectively repel each other to maximize their conformational entropy, suggesting that duplicated circular chromosomes could partition spontaneously. This mechanism not only is able to account for the spatial separation per se but also captures the major features of the spatiotemporal organization of the duplicating chromosomes observed in Escherichia coli and Caulobacter crescentus.

It is commonly assumed that the horizontal transfer of most bacterial chromosomal genes is limited, in contrast to the frequent transfer observed for typical mobile genetic elements. However, this view has been recently challenged by the discovery of lateral transduction in Staphylococcus aureus , where temperate phages can drive the transfer of large chromosomal regions at extremely high frequencies. Here, we analyse previously published as well as new datasets to compare horizontal gene transfer rates mediated by different mechanisms in S. aureus and Salmonella enterica . We find that the horizontal transfer of core chromosomal genes via lateral transduction can be more efficient than the transfer of classical mobile genetic elements via conjugation or generalized transduction. These results raise questions about our definition of mobile genetic elements, and the potential roles played by lateral transduction in bacterial evolution. It is commonly thought that horizontal transfer of most bacterial chromosomal genes is limited, in comparison with the frequent transfer of mobile genetic elements. Humphrey et al. show that, actually, phage-mediated lateral transduction of core chromosomal genes can be more efficient than the transfer of mobile genetic elements via conjugation or generalized transduction.

Key Points The bacterial chromosome must be linearly compacted more than 1,000-fold to fit within the bacterial cell. The chromosome is compacted in an orderly and hierarchical fashion in lockstep with DNA replication. This condensation has a central role in organizing replicated sister chromosomes and driving their segregation. The organization of the chromosome within the bacterial cell recapitulates the genetic map. Segregation of bacterial chromosomes can be broken down into three discrete steps: separation of the newly replicated origins; bulk chromosome segregation; and resolution and transport of the replication termini at the division septum. In many bacteria, origin segregation is facilitated by a highly conserved partitioning system. Bulk chromosome segregation is principally driven by the orderly compaction of the replicated sisters along adjacent DNA segments. This lengthwise condensation is mediated by the concerted action of supercoiling, small nucleoid-associated proteins and structural maintenance of chromosome (SMC) condensin complexes. Segregation of replicated termini requires topoisomerase IV to remove catenanes and XerCD recombinase to convert chromosome dimers into monomers. Decatenation and dimer resolution are coordinated and facilitated by a septum-associated DNA translocase. Bacterial chromosomes were originally thought to be unstructured and largely unconstrained, but recent advances have supplemented historical research to reveal a highly structured and dynamic chromosome organization. This Review discusses our latest understanding of bacterial chromosome organization, including how the simultaneous nature of DNA replication and chromosome segregation in bacteria necessitates intricate interplay between these processes. The bacterial chromosome must be compacted more than 1,000-fold to fit into the compartment in which it resides. How it is condensed, organized and ultimately segregated has been a puzzle for over half a century. Recent advances in live-cell imaging and genome-scale analyses have led to new insights into these problems. We argue that the key feature of compaction is the orderly folding of DNA along adjacent segments and that this organization provides easy and efficient access for protein–DNA transactions and has a central role in driving segregation. Similar principles and common proteins are used in eukaryotes to condense and to resolve sister chromatids at metaphase.