Abstract
Higher-order chromosome folding and segregation is tightly regulated in all domains of life. In bacteria, details on nucleoid organization regulatory mechanisms and function remains poorly characterized, especially in non-model species. Here, we investigate the role of DNA partitioning protein ParB and condensin complexes, two key players in bacterial chromosome structuring, 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 a reliable chromosome segregation. Using a combination of chromatin immunoprecipitation and photoactivated single molecule localization microscopy evidences the formation of distinct ParB-nucleoprotein subclusters in dependence of parS numbers. We further identified and functionally characterized two condensin paralogs. Whereas SMC/ScpAB complexes are loaded via ParB at parS sites mediating chromosomal inter-arm contacts like in Bacillus subtilis, the MukBEF-like SMC complex MksBEFG does not contribute to chromosomal DNA-folding. Rather, the MksBEFG complex is involved in plasmid maintenance and interacts with the polar oriC-tethering factor DivIVA. These data complement current models of ParB-SMC/ScpAB crosstalk, while showing that some condensin complexes evolved functions uncoupled from chromosome folding.
Introduction
Each organism must complete genome replication and separation in the course of one cell cycle prior to cell division in concert with transcriptional processes. To this end, chromosomes are highly organized structures in terms of segregation and overall folding patterns 1. The functional organization of bacterial genomes, structured into the nucleoid, has been predominantly investigated in a limited number of model species, e.g. E. coli, V. cholerae, B. subtilis or C. crescentus, revealing diverse levels of compaction and segregation strategies2–4.
ParABS systems and condensins are two (nearly) ubiquitous bacterial enzyme machineries that contribute to chromosome homeostasis. With a few exceptions amongst γ-proteobacteria, all branches of bacteria and several Archaea harbor parS sites that recruit partitioning protein ParB 5. The ParABS system contains one or several parS sites usually in the vicinity to the chromosomal origin of replication (oriC). ParB proteins bind to these sequence-specific motives and form large nucleocomplexes by spreading and 3D-bridging between ParB dimers 6–9, resulting in large topological domains encompassing the oriC, that have been revealed by Hi-C for B. subtilis 10. In an alternative model termed nucleation and caging, ParB-nucleation at parS is stabilized by dynamic ParB dimer-dimer interactions and weak interactions with non-specific DNA generating a scaffold for locally high ParB concentrations confined around parS 11. The ParB segregation is driven by a ParA ATPase, which binds nonspecifically to the nucleoid and is released from DNA upon ATP hydrolysis triggered by transient ParB-interactions 12,13. In the course of chromosome replication ParB-oriC complexes act in combination with ParA as Brownian ratchets along dynamic DNA loci: slow ParA-DNA rebinding rates generate ParA-gradients, which serve as tracks for directed movement of partition complexes away from their sisters 14–17. Perturbation of the system by placing parS sites at ectopic, oriC-distal regions can cause severe DNA segregation phenotypes 18,19. To date, only few studies investigated the impact of chromosomal parS localization on DNA-segregation and folding 18–22.
In addition to ParABS systems, most bacteria harbor condensin complexes, members of the structural maintenance of chromosomes (SMC) family of proteins found in all kingdoms of life 23. In standard model organisms, condensins are equally essential for faithful chromosome segregation by compacting DNA into separate nucleoids 24–26. The SMC/ScpAB (structural maintenance of chromosomes) complex is well-studied in B. subtilis, where it consists of two large SMC subunits and the kleisin ScpA associated with dimeric accessory protein ScpB that assemble into a ring-like structure 27. A recent study suggests progressive extrusion of condensin-encircled DNA loops upon conformational changes in the SMC subunit, which leads to a gradual size increase of trapped DNA molecules 28. The active process(es) driving DNA extrusion 29,30 allow(s) for translocation along the chromosome with velocities of around 50 Kb/ min 31, and depend(s) on the ATPase activity of SMC 32. To be loaded on parS sites, SMC/ScpAB complexes necessitate ParB 20,22,33,34. They redistribute to distant chromosomal regions, promoting topological changes, and notably the co-alignment of right and left replichores 10,21,22,31,35. In sharp contrast with SMC/ScpAB, the E. coli condensin MukBEF does not promote the co-alignment of chromosomal arms 36,37, but facilitate cis structuration by establishing long-range contacts between loci belonging to the same replichores from stochastic chromosomal loci (except for the ter region containing the replication terminus) 37,38. Despite the importance of condensins in chromosome organization, the role of SMC homologs besides the model species B. subtilis, C. crescentus and E. coli remain largely unexplored. These species all contain a single condensin complex, yet a broad range of bacteria possesses combinations of SMC/ScpAB and MksBEFG (MukB-like SMC), for which functional characterizations are non-existent to date 39. Current work in bacteria as well as knowledge from eukaryotic studies convey the general assumption that all SMCs are likely to play role(s) in chromosome organization. In bacteria it is unknown, why some species harbor more than one type of condensin, and whether and how they would work in concert with each other and coordinate with systems such as ParABS.
In this work, we used a combination of high-resolution microscopy and genomic chromosome conformation capture (3C/Hi-C) 35 to unveil the global organization of the diploid C. glutamicum genome. C. glutamicum is a polar growing actinobacterium, whose genome encodes both SMC/ScpAB and MksBEFG. In this species, the two oriCs are continuously associated with the polar scaffold protein DivIVA, while newly replicated sister oriCs segregate towards division septa via the ParABS system 40–42. In contrast to B. subtilis, C. glutamicum ParAB are by themselves crucially important drivers of reliable nucleoid separation prior to cell division, where ParAB deletions yield in 20 % of anucleoid cells 43–45. Here, analyses of chromosomal ParB-binding patterns evince ten redundant parS sites, which mediate ParB subcluster formation at oriC. A single parS site maintains ParB propagation over 32 Kb neighboring regions, and is sufficient to promote the SMC-dependent alignment of the two chromosomal arms. Hi-C also reveal SMC-dependent long-range contacts surrounding oriC. On the contrary, we showed that the polarly positioned MksBEFG condensin acts exclusively on plasmid-transmission to daughter cells, without influencing nucleoid architecture.
Results
Chromosome segregation is governed by a cluster of ten oriC-proximal parS sites
Previous studies on C. glutamicum chromosome partitioning have revealed two stable ParB-oriC clusters at each cell pole, while newly replicated origins are segregated towards a division septa formed at midcell 40. In B. subtilis, C. crescentus and P. aeruginosa ParAB-mediated chromosome segregation and folding depends on parS sites 18,19,21. In C. glutamicum, parS positions have not been characterized yet. Initially, four to eight parS sites were predicted in Corynebacterineae 5. However, we identified ten B. subtilis-like 16 bp consensus sequences in C. glutamicum localized in one cluster within a 35 Kb region distant 73 Kb from oriC using blast (1% of the 3.21 Mb chromosome; Fig. 1A). Out of the ten parS sites, only the one located furthermost from oriC (parS1) lies within a coding sequence (trpCF). All other parS sequences (labelled parS 2-10) are located in intergenic regions. Degenerated parS sequences exhibiting at least three base-pair mismatches were also identified further away from oriC, e.g. 5’ of cg0146 or within the fusA and cg1994 coding region. To test whether these putative parS were responsible for the recruitment of ParB, α-mCherry-ChIP analyses were performed with a strain harboring a mCherry-tagged version of the native ParB (note that all mutant strains used in this study derive from clean allelic replacements and have, unless otherwise noted, a wild type-like phenotype). Distinct and very reproducible enrichment signals were obtained at ten parS sites close to oriC (parS1-10 at 3.16 MB) (Fig. 1A), whereas the imperfect parS sequences identified with blast clearly failed to recruit ParB. Additional smaller peaks were identified at highly transcribed DNA regions, in particular at ribosomal genes, tRNA gene clusters and at all of the rRNA operons (Fig. 1A). Magnification of the oriC region reveals three distinct ParB propagation zones overlapping with parS1-4, parS5-8 and parS9-10, respectively (Fig. 1B). Remarkably, those three regions seem to recruit decreasing amounts of ParB, from parS1-4 (most enriched) to parS9-10 (less enriched). Since all parS are identical in sequence, differences in ParB-recruitment might result from the number and distance of parS sequences in the context of the overall nucleoid folding patterns at the oriC-region.
Higher-order organization of the C. glutamicum chromosome
In B. subtilis, SMC-mediated chromosome folding initiates at ParB-parS clusters surrounding the origin of replication, bridging the two replichores with each other 10,21. To characterize whether C. glutamicum parS sites play a similar role in the overall organization of the chromosome, we applied a Hi-C like approach 10,46 to exponentially growing wild type cells (Material and Methods). The genome-wide contact map, displaying the average contact frequencies between all 5 Kb segments of wild type chromosomes (Fig. 1C) displayed the following 3D features. First, a strong and broad diagonal reflecting frequent local contacts between adjacent loci and observed in all Hi-C experiments. Second, chromatin interaction domains (CIDs), i.e. regions making increased contact frequencies within themselves and previously described in C. crescentus and other species 10,21,35,37,47, (Fig. 1C, S1) (11 domains detected at a 200 Kb resolution). Third, a secondary diagonal perpendicular to the main one and extending from the 35 Kb parS cluster (Fig. 1D – white dashed line) from the ori region to the terminus. This structure shows that the two replichores are bridged over their entire length, similarly to B. subtilis and, to some extent, C. crescentus 10,21,35. Interestingly, this secondary diagonal also displays discrete long-range contact enrichments (Fig 1C), which may reflect bridging of the two chromosomal arms at specific locations. Finally, the contact map also displays a faint, cross-shaped signal corresponding to contacts between the ori region and the rest of the chromosome (Fig. 1C – dark triangle on the sides of the contact matrix), a feature never described before. These contacts might represent a replication signal reflecting the translocation of the ParB-oriC complex along the nucleoid during segregation when oriCs reposition at midcell. This signal is also maximal at the parS cluster and not at oriC locus. An observation that reinforce the fact that the parS cluster is at the tip of Corynebacterium chromosome fold and is one of the main actor of chromosome segregation.
A single parS site is sufficient to maintain a wild type ParB binding region and chromosome architecture
Since all parS sites are in close proximity on the C. glutamicum chromosome, we tested the importance of ParB-parS complex titration for the overall chromosome organization. Cells with chromosomes carrying a single parS site grow and divide like wild type cells (Fig. 2A, S2). However, the removal of all 10 parS sites resulted in a cell length phenotype (Fig. S2) and 29% DNA-free mini-cells hinting to a nucleoid segregation defect similar to the ΔparB phenotype (Fig. 2A, Tab. S1). We further analyzed ParB localization in mutant strains carrying either one or none parS sites. Firstly, cellular localization of fluorescent ParB-eYFP foci is similar to wild type, positioning at cell poles and migrating to the newly formed septa (Fig. 2B, S3) 40. Interestingly, the combination of a single parS site with ParB-eYFP resulted in 7% anucleoid mini-cells (Fig. S2, Tab. S1), reflecting functional constraints of the ParB-eYFP fusion in presence of only one parS site. Therefore, the high number of chromosomal parS sites likely evolved to improve the robustness of the segregation machinery. ParB ChIP-qPCR signals of locus parS1 were similar in both wild type and mutant strains (Fig. 2C). ParB spreading around the single parS site was characterized through ChIP-seq analysis (Fig. 2D, S4), where ParB binding was maximum within 2 Kb windows on both sides of parS, while extending up to 16 Kb on either side. If each parS site promotes ParB deposition in comparable ranges to parS1, abrupt enrichment drops downstream of parS4 and parS8 would have been absent in wild type, suggesting that DNA properties accountable for the regulation of ParB deposition are independent of parS distributions along the nucleation zone. We next investigated the role of parS sites and ParB in the overall chromosome folding by performing Hi-C in mutants (Fig. 2E, 2F). The absence of ParB or all parS sites led to the disappearance of the secondary diagonal. In addition, the cross-shaped pattern resulting from contacts between the ori and the whole chromosome disappears in those mutants, also illustrated by the ratio between wild type and mutant contact maps (Fig. 2F). This result shows that parS sites and ParB are two major structural components of chromosome organization and act to recruit downstream factors that fold the chromosome emanating from the parS cluster, and bridge the two chromosomal arms together down to the replication terminus region. The contact map of the strain deleted for parS2-10 but carrying parS1 maintains a secondary diagonal, showing that a single parS site is sufficient to ensure the loading of ParB and the overall folding of the chromosome (Fig. 2E, 2F). However, some differences appeared between wild type contact matrix and the one resulting from the strain harboring only one parS site. In this mutant, the large domain surrounding oriC appears more defined than in wild type suggesting that one parS site is not sufficient to fully restore Corynebacterium chromosome folding possibly due to a slower replication and consequently a less dynamic folding (Fig. 2E, 2F). The single parS site was repositioned at different genomic regions. Cells harboring an ectopic parS site at 9.5°, 90°, 180° or 270° positions were viable (Fig. S2, S3A, B). Unlike in cells harboring parS1 at its original position, ParB-parS complexes distribute randomly along the longitudinal cell axis in all of these mutants (Fig. S3B), leading to around 25% anucleate cells (Tab. S1). Therefore, none of these parS-shifts restores controlled nucleoid segregation. The number of ParB foci nevertheless correlates well with cell length (Fig. S3C), excluding replication initiation deficiencies. ParB-binding to a parS sequence positioned at the 90° chromosomal position (locus cg0904, strain CBK037) was identified in a 9 Kb range on either side of parS (Fig. S3D, S4), approximately half the ParB-propagation distance determined for cells harboring one parS at its native locus. We also analyzed mutant CBK037 (parS at 90° chromosomal position) using Hi-C (Fig. S5). The resulting contact map presents a “bow shape” motif at the position of the aberrant parS sequence demonstrating a recruitment of the ParB protein at this location as well as a local folding of the chromosome. However, this loading appears insufficient to fold the whole chromosome and to anchor the two chromosome arms over their entire length. DNA topology, origin replication and overall chromosomal localization might hereby determine parS-distant ParB-DNA interaction. Collectively, these results show a redundancy of parS sites, yet their function is restricted to a confined oriC-proximal region.
ParB subclusters identified by PALM reflect protein enrichment at parS sites
To directly characterize oriC domain compaction via ParB, we applied photoactivated localization microscopy (PALM) to visualize individual ParB-PAmCherry molecules with nanometer resolution. PALM revealed distinct ParB-dense regions at cell poles and quarter positions regions, similar to foci observed via diffraction limited epifluorescence microscopy (Fig. 2G). These ParB-enriched regions (macro-clusters) display heterogeneous densities, with a variable number of higher density zones within sub-clusters. Macro- and sub-clusters have been identified via the Optics algorithm48,49 (see Material and Methods) and analyzed in strains harboring a single, two or all the parS sites (Fig. 2G, S6). We define a macrocluster as 32 events being localized within a maximum distance of 50 nm for macroclusters and 35 nm for subclusters. Note that high chromosome numbers promote inter-molecular oriC-colocalization in fast-growing cells. For more accurate cluster estimations, PALM analysis was performed using slow-growing cells resulting in significantly fewer ParB macro-clusters per cell (Fig. S6B) 40. Since segregation of oriC complexes might alter their DNA compaction, we focused on the two largest macroclusters per cell, stably tethered at cell poles. The amount of ParB contained within each macro-cluster in wild type is significantly higher than in cells containing a single parS site (Fig. 2H), in agreement with the ParB deposition observed via Chip-seq. A parallel between PALM and Chip-seq can also be drawn with respect to the number of sub-clusters per macro-cluster, with a higher number of sub-clusters in the wild type compared to the single parS site (Fig. 2H). These differences were not observed when comparing cells harboring all, or two parS sites (parS1,10) (Fig. S3, S4, S6). These observations could explain the differences observed between contact matrices of wild type and ∆parS 2-10 strains and the higher structuring of the oriC domain when only one parS site is present. We therefore conclude that the architecture of C. glutamicum partition complex is dependent on parS and ParB-parS nucleoprotein-complexes are visible as individual subclusters.
C. glutamicum harbors two paralogs of condensin complexes
In bacteria, the condensin paralog complexes SMC/ScpAB and, in E. coli and other enterobacteria, MukBEF, are key players of chromosome folding 10,21,35,37. MksBEF (for MukBEF-like SMC) is another condensin occasionally found in bacteria genomes 39, whose role(s) remain(s) obscure. A sequence homology search of the C. glutamicum genome pointed at the presence of both SMC/ScpAB and MksBEF. The SMC/kleisin is encoded by genes cg2265 (smc), cg1611 (scpA) and cg1614 (scpB) (Fig. 3A), while the Mks complex is encoded on a widely conserved operon39 and comprises genes cg3103-cg3106 (mksGBEF) (Fig. 3A), including MksG which has being suggested to act in complex with MksBEF 39.
To characterize condensin complex formation in vivo, mass spectrometry of pulldown experiments using SMC and MksB as baits of whole cell lysates were performed. Stability of SMC and MksB fluorescent fusions were confirmed by western blotting (Fig. S7). Kleisin subunit ScpA and ScpB co-precipitated significantly with SMC compared to the negative control, while subunits MksF and MksE, but not MksG, were substantially enriched in the MksB pulldown experiments (Fig. S7). ParB, which mediates SMC-loading onto DNA in B. subtilis and S. pneumonia 20,33,34, was not immuno-precipitated with SMC in any of the experiments. Bacterial two-hybrid analyses confirmed mass spectrometry results, pointing at the formation of SMC/ScpAB and MksBEF complex (Fig. 3B, C). No significant interactions between SMC/ScpAB and ParB were detected, and we observed ScpA-ScpA self-interaction signals well above background. Moreover, MksG connects to the MksBEF complex via interaction with MksF, while MksF and MksG subunits further interact with the C. glutamicum polar scaffold protein DivIVA.
SMC-mediated cohesion of chromosomal arms
We aimed to characterize C. glutamicum condensin SMC/ScpAB. Mutation of the SMC/ScpAB complex causes a conditionally lethal phenotype due to chromosome mis-segregation in B. subtilis 25. In contrast, a smc deletion in C. glutamicum did not result in growth defects, DNA-segregation defects or aberrant cell length distributions and morphologies compared to the wild type in minimal or complex media (Fig. S8, Tab. S1). Nonetheless, the combination of genetic backgrounds parB∷parB-eYFP and Δsmc yield a minor fraction of anucleate cells (4-5 %) (Tab. S1), indicating that SMC and ParB function in the same pathway and have a synthetic phenotype. Hence, a functional interaction of SMC and ParB proteins regulating chromosome organization is likely. In order to further determine cellular localization of SMC/ScpAB complexes, a strain harboring a fluorescently tagged version of core subunit SMC was imaged, revealing the formation of SMC clusters along the entire longitudinal axis of the cell (Fig. 3D). Clusters of SMC and ParB investigated in a strain carrying both labelled complexes (parB∷parB-mNeonGreen smc∷smc-mCherry) are often proximal but do not necessarily co-localize, while the foci numbers correlate with cell length (Fig. 3E). Up to eight SMC-mCherry foci were counted per cell. On average, cells contained fewer SMC-foci than ParB nucleoprotein complexes (Fig. S7). To further characterize the role of SMC, we generated Hi-C contact maps of the mutant (Fig. 3F). Deletion of smc abolishes the secondary diagonal in the maps (Fig. 3F). The combination of smc and parB mutations mimics a parB phenotype, again resulting in the loss of contacts between chromosomal arms and further in the loss of the segregation signal described before (Fig. 3F). Therefore, an interplay of SMC/ScpAB with ParB is responsible for replichore cohesion in C. glutamicum, similar to B. subtilis and C. crescentus each harboring only one condensin complex 10,21,22,35. Thus, ParB acts epistatic to SMC.
ParB-dependent SMC-recruitment to chromosomal loading sites
Since cellular SMC-mCherry signal hinted to distinct agglomeration clusters along the C. glutamicum chromosome, we investigated its putative binding sites via ChIP-seq. A small enrichment in SMC deposition was detected at and around the parS1-10 cluster (Fig. 4A) that disappears upon parB or parS deletion (Fig. 4A, S4, S9). In addition, comparably minor enrichment signals are present throughout the chromosome, which partially coincide with genomic loci of high transcriptional activity. Distinct SMC-mCherry foci are less frequent in the absence of ParB or parS. (Fig. S9). These findings suggest that ParB promote condensin loading onto DNA at oriC-proximal parS sites. In addition, ChIP-seq revealed that SMC concentrates at a 13 Kb region upstream parS1 (Fig. 4A). SMC enrichment in this region was lost following a partial deletion of this locus and its -reinsertion at another genomic position or following its substitution by a random DNA sequence (Fig. S9). Therefore, the accumulation of SMC at the 13 Kb region in the vicinity of parS sites points at roadblocks that trap SMC, rather than specific SMC-binding. This hypothesis is further supported by the study of the contact map of wild type cells (Fig. 1C, 1D, S1, S10). Indeed, the SMC enrichment region is clearly delimited by a strong border on its left (Fig. S1 – Directional Index at 100 Kb resolution and Fig. S10 – red dashed line). In the absence of ParB or SMC (Fig. S10), the strong border observed in HiC maps is shifted towards parS sites. Therefore, this border originates from a combination of multiple processes.
SMC is also recruited to parS inserted in ectopic positions, e.g. the 90° parS-insertion (Fig S4). Indeed, in the absence of SMC (Fig. S5), the bow shape motif is no longer present at the ectopic parS site demonstrating that chromosomal arm cohesion is SMC-dependent and that artificial loading of SMC at non-native positions is not sufficient to fold the entire chromosome. We further assayed chromosomal SMC-loading sites by making use of a well-characterized SMC ATP-hydrolysis mutant E1084Q 32,50–52. SMCE1084Q mutant strongly accumulates at parS sites in C. glutamicum, mimicking a ParB-enrichment pattern (Fig. 4A). Decreased ChIP-enrichment signals throughout the rest of the chromosome hint to an impaired SMC-migration along DNA (Fig. 4B). Conclusively, we confirm specific SMC-loading by ParB to an oriC-proximal region on the C. glutamicum chromosome.
Interestingly, ChIP-analysis of a C. glutamicum ParBR175A mutation, which leads to a loss of dimer-dimer interactions in the corresponding B. subtilis ParBR79A mutation 8, results in increased SMC-binding at ParBR175A propagation zones (Fig. 4A). Changes in in vitro dsDNA-binding affinities compared to wild type ParB could not be verified (Fig. S3), neither enhanced binding affinity for SMC/ScpAB by bacterial two-hybrid analyses (Fig. 3B). The mutation results in large fractions of DNA-free cells and growth rates and ParBR175A cluster formation are particularly affected in cells harboring a single parS site (Fig. S2, Fig. S3). ChIP-data indicate broadened and less distinct enrichment signals compared to wild type ParB in presence of all or one parS sites (Fig. 4A, S3, S4). Therefore, ParBR175A is still capable of building up weak nucleoprotein complexes around parS sites. Hi-C data of the corresponding mutant show the same tendency with a conservation of the overall chromosome architecture with the presence of a secondary diagonal and the conservation of the origin domain folding (Fig. 4C, S10). However, the signal ranging from the secondary diagonal is weak compared to the wild type one as shown by the ratio matrix (Fig. 4C). Consequently, SMC-translocation along DNA appears only partially impaired in this mutant (Fig. 4A, S4). The ParBR175A mutation either locks the translocation ability of SMC/ScpAB by a direct interaction or alterations of ParBR175A nucleoprotein complex properties lead to SMC-trapping along DNA-loops at parS. Altogether, these analyses confirm that the C. glutamicum SMC/ScpAB complex is a Bacillus-like condensin that loads and redistributes to distant chromosomal regions via an explicit ParB-crosstalk at parS.
MksB impacts on plasmid maintenance in C. glutamicum
To test whether both C. glutamicum condensins SMC and MksB are redundant in function, we generated mutants lacking the condensin core subunit ΔmksB or both Δsmc ΔmksB. Similar to Δsmc, no growth and morphology phenotypes could be detected for both mutants (Fig. S8, Tab. S1). A triple mutation ΔparB Δsmc ΔmksB did not attenuate the ΔparB phenotype, excluding redundancy of condensin functions in chromosome segregation (Fig. S8). Further, oriC-ParB foci numbers (Fig. 3D) as well as their spatiotemporal localization (Fig. S8, time-lapse microscopy not shown) remain largely unaffected upon deletion of smc and mksB. MksB fluorescence was mainly detected at the cell poles (Fig. 3D), further supporting an interaction with the polar protein DivIVA. Moreover, we applied Hi-C to characterize the role of MksB in genome folding in the different mutants (Fig. 3F). In contrast to smc, deletion of mksB had no effect on chromosome organization, as shown by the nearly white ratio map between the wild type and the mutant (Fig. 3F). Moreover, ∆smc and ∆smc∆mksB contact maps were nearly identical (Fig. 3F), showing that MksB and SMC are most likely not involved in the same process(es). ChIP-seq of MksB failed to detect specific loading sites along the C. glutamicum chromosome (Fig. S11), supporting the hypothesis that MksB, unlike other bacterial condensins studied so far, plays no direct or indirect role in C. glutamicum chromosomes organization. Therefore, we analyzed its impact on the maintenance of extrachromosomal DNA. The MksBEFG complex appears involved in plasmid maintenance, as shown by the qPCR copy number analysis of two low-copy number (pBHK18 and pWK0) and two high-copy number (pJC1 and pEK0) plasmids. In ΔmksB mutants both low-copy number plasmids were enriched up to ten-fold compared to wild type, when grown in the absence of selection marker (Fig. S11). On the contrary, the amount of high copy number vectors per cell was hardly affected. We confirmed these findings by plasmid extractions from C. glutamicum cells lacking MksB that yielded exceptionally large quantities of pBHK18 and pWK0, turning them into high copy number plasmids under these conditions (Fig. S11). By contrast, amounts of pJC1 and pEK0 did not differ notably compared to control strains. These analyses show a MksB-dependent decrease in plasmid level, specifically of low copy number plasmids.
Together, our data show that the two condensins in C. glutamicum evolved very different functions: whereas SMC/ScpAB act with ParB to promote replichore pairing and origin domain organization, MksBEFG does not organize chromosome architecture and seems involved in plasmid maintenance through a mechanism that remains to be characterized.
Discussion
Condensins are widely conserved enzyme machineries, which have been implicated in chromosome organization of pro- and eukaryotes 53. For long it was considered that bacterial genomes encode one condensin complex that would either be of the Smc/ScpAB type as found in B. subtilis and C. crescentus or the MukBEF complex encoded in E. coli and related proteobacteria 23. However, recent reports suggested the existence of two or even multiple condensin systems in a single species 39. A prominent example is P. aeruginosa, where the MksBEF complex was suggested to act in chromosome organization due to a synthetic DNA segregation phenotype in combination with SMC/ScpAB 39. Yet, the underlying mechanisms and the precise function of these two condesnin systems remained largely untested. We report here that the Gram positive actinobacterium C. glutamicum also contains SMC/ScpAB and the Muk-like MksBEFG complexes. We set out to address the individual functions of the two condensin systems. Surprisingly our data provide clear evidence that the class of MksBEFG proteins do not work as chromosomal interactors, thus the function of bacterial condensins in promoting DNA segregation to daughter cells is not generally conserved. A recent bioinformatics study predicted a role for MksBEFG complexes (termed Wadjet system) in plasmid-related defense, where heterologous complex expression conveyed protection against the uptake of a high copy number plasmid 54. However, function of MksBEFG in its native host had not been addressed before. We could show that the Mks system is indeed involved in the control of plasmid copy numbers and that there is no involvement of this system in chromosome organization. This is a fascinating similarity to specific eukaryotic condensins such as Rad50, being the closest eukaryotic relative to MukB/MksB 23,39. It was recently shown that Rad50-CARD9 complexes sense foreign cytoplasmic DNA in mammalian cells. This includes antiviral effects of Rad50 that are important for innate immune responses. Thus, it takes no wonder that several DNA-viruses have evolved strategies to inhibit Rad50 signaling 55. Also the more distantly related eukaryotic SMC5/6 complex had been shown to act in a defense mechanism against circular hepatitis B virus DNA, resembling the specific effect of prokaryotic MksBEFG on plasmids 56. Together, our data lend support to the notion that condensins function in innate immunity is an ancient mechanism and condensins complexes that diverged early in evolution have specialized functions beyond chromosome organization. However notably, we provide evidence that the MksBEFG complex is the only known condensin amongst pro- and eukaryotes that exclusively targets non-chromosomal DNA. For MksBEF systems it has been proposed that a fourth subunit, MksG is important for function in plasmid maintenance 54. We could verify that MksG is part of the MksBEF complex of C. glutamicum. The direct interaction of a Mks complex with a polar scaffold protein like the C. glutamicum DivIVA has not been described before, but may be very well in accord of its function in plasmid control. Polar plasmid localization has been described in other systems before 57. A challenging question for the future will be to determine how the MksBEFG system can identify foreign DNA and how it is loaded onto the plasmids. In vitro analysis of the related MukB has revealed that the protein preferentially binds to single stranded DNA, although also autonomous loading on double stranded DNA was observed 58. Thus, one might speculate that MksB is loaded onto plasmid DNA once replication is initiated.
We further describe here that the second condensin SMC/ScpAB is indeed the major player of replichore cohesion and chromosome organization in C. glutamicum. Like in B. subtilis, SMC is loaded onto the chromosome by a ParB/parS loading complex before spreading to the entire chromosome. The mild DNA-partitioning defects of a smc deletion in combination with a ParB-eYFP modification (Tab. S1) strongly suggest a supportive role of SMC/ScpAB in the process of nucleoid separation, yet the smc phenotype appears to be entirely compensated by ParB. Therefore, our data demonstrate that the conserved role for SMC in chromosome organization 10,20–22,34,35 is also maintained in presence of a second condensin complex. Moreover, bacterial two-hybrid analyses of SMC/ScpAB subunits evidence a self-interaction of C. glutamicum kleisin ScpA (Fig. 3), that has not been described in other organisms before. Based on this result, we speculate that C. glutamicum SMC/ScpAB might form dimers via kleisin subunits similar to E. coli MukBEF complex 59,60. These data point to a handcuffing model, where two SMC/ScpAB complexes are physically coupled together and translocate in pairs along the chromosome, similar as suggested for B. subtilis 31. We further describe a new phenotype for a ParBR175A point mutation in C. glutamicum, that blocks SMC-release from its loading site. Building on this, we observe a weak interaction signal of ParBR175A with ScpB in bacterial two-hybrid analyses. Alternatively, SMC/ScpAB remains indirectly entrapped in higher-order ParBR175A-nucleocomplexes, which possess altered DNA-folding properties. In either case, this mutant underlines the crosstalk between SMC/ScpAB and ParB nucleoprotein complexes in bacterial nucleoid organization.
Analysis of ParB complexes using 2D PALM reveals ParB-dense regions within clusters that correlate to the number of ParB enrichment zones along adjacent parS sites. In line with a current study on ParB cluster-assembly in V. cholerae 61, we suggest that these subclusters derive from independent nucleation and caging events, which merge into one ParB-macrocomplex per oriC in C. glutamicum. Presence of a single parS site leads to formation of almost globular ParB densities. Using Hi-C approaches, we further show that parS sites as well as ParB are major players of chromosome folding in C. glutamicum as previously shown in other models 10,21,22,35. C. glutamicum chromosome adopts a global folding with a strong cohesion between the two chromosomal arms as expected from a bacterium harboring a longitudinal chromosomal organization similar to B. subtilis and to a lesser extent C. crescentus (Fig. 1). Our analysis also suggests the existence of a chromosomal domain at parS sites in C. glutamicum as previously observed in B. subtilis, but with important differences: parS sites in C. glutamicum are only found on one side of the oriC locus and appeared to be at the edge of the nucleoid structure as observed in C. crescentus. A hairpin structure as it was observed in B. subtilis is absent in C. glutamicum10. Contact maps of a strain with an ectopic parS site feature a bow-shaped structure reflecting an asymmetry in arm interaction, which has been shown before in B. subtilis and C. crescentus 21,22. Zipping of the chromosome is not complete and the ectopic parS site does not reorient the entire chromosome. Therefore, additional factors are involved in chromosome localization that supplement polar ParB-parS binding to DivIVA.
Importantly, we describe ParB/parS-dependent DNA segregation signals showing that the chromosomal ori-ter configuration allows for ParAB-driven oriC-segregation trailing along the nucleoid. Based on our data we propose the following model shown in Figure 5: Organisms with polarly localized oriCs and a longitudinal chromosome organization rely on ParAB for oriC segregation, since they can use the DNA-scaffold as a track. By contrast, species with a central replication factory cannot efficiently use ParAB. B. subtilis is an exception, since here a longitudinal chromosome orientation is present during sporulation and hence, parAB (spo0J/soj) phenotypes are only obvious during spore formation. Consequently, SMC/ScpAB-mediated replichore cohesion is generally dispensable for oriC segregation in bacteria with a strict longitudinal chromosome arrangement that allows for efficient ParABS-driven chromosome partitioning.
Authors contribution
Conceptualization: Kati Böhm, Martial Marbouty, Romain Koszul, Marc Bramkamp Methodology:
- Strain constructions and analyses, epifluorescence microscopy, ChIP, bacterial two-hybrid screening, in vitro protein assays: Kati Böhm
- Chromosome Conformation Capture: Martial Marbouty
- PALM: Giacomo Giacomelli
- Mass spectrometry: Andreas Schmidt, Axel Imhof
Writing manuscript: Kati Böhm, Martial Marbouty, Andreas Schmidt (Material and Methods), Giacomo Giacomelli (Material and Methods), Romain Koszul, Marc Bramkamp
Materials and Methods
Bacterial strains, plasmids and oligonucleotides
Primers, plasmids and strains used in this study are listed in Tables S2 and S3. Detailed information on strain construction and growth conditions are provided in the Supplementary Information.
Plasmid extraction from C. glutamicum cells
C. glutamicum cells were grown in 10 ml BHI medium to exponential growth phases in presence of selection antibiotic, following incubation with 20 mg/ml lysozyme in P1 buffer (NucleoSpin® Plasmid Kit, Macherey-Nagel) overnight at 30 °C. Subsequently, plasmids were extracted by using the plasmid kit according to manufacturer’s instruction.
Protein identification via immunoprecipitation and mass spectrometry
Immunoprecipitation of SMC and MksB interaction partners was performed with strains CBK012 and CBK015, further including strain CBK052 as negative control. Lysate of exponentially grown cells was used for immunoprecipitation via magnetic RFP-Trap® agarose beads. For proteomic analysis samples were further processed and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify and quantify proteins in all samples. A detailed description of immunoprecipitation and proteomic analysis is provided in the Supplementary Information.
Bacterial two-hybrid screening
Protein interactions obtained by mass spectrometry were confirmed via bacterial two-hybrid assays 62, using compatible vectors expressing adenylate cyclase subunits T25 and T18 (pKT25/ pKNT25 and pUT18/ pUT18C). E. coli BTH101 co-transformed with respective vectors were plated on indicator medium LB/ X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 40 μg/ml) supplemented with IPTG (0.5 mM) and antibiotics kanamycin (50 μg/ml), carbenicillin (100 μg/ml) and streptomycin (100 μg/ml) and incubated at 30°C for 24 h. Interacting hybrid proteins were identified by blue-white screening (not shown) and β-galactosidase assays in a 96 well plate format as previously described 63. Cotransformants harboring empty plasmids or pUT18C-zip/ pKT25-zip plasmids served as positive and negative controls. Miller units of negative controls served as reference and were set to zero; Miller units of any other sample were normalized accordingly. All C- and N-terminal combinations of hybrid proteins were assayed and positive signals were confirmed through at least three replicates.
Fluorescence microscopy
Microscopy images were acquired on an Axio-Imager M1 fluorescence microscope (Carl Zeiss) and a Delta Vision Elite microscope (GE Healthcare, Applied Precision). Experimental procedures and technical configurations are specified in the Supplementary Information. Data analysis was performed via FIJI and MicrobeJ software 64,65.
Chromatin Immunoprecipitation (ChIP) combined with sequencing
A detailed description of this method is available in the Supplementary Information. Briefly, cells were crosslinked (1 % formaldehyde) for 30 min at room temperature and lysed. DNA was sheared by sonication, incubated with α-mCherry antibody for 2 h at 4 °C, washed subsequently and crosslinks were reverted at 65 °C o. N. DNA purification was followed by library preparation and sequencing using an Illumina MiSeq system. Reads were aligned to the C. glutamicum ATCC 13032 genome sequence (GeneBankID: BX927147.1). Further data analysis was performed using online tools 66.
Real-time PCR
DNA amplification was performed using a 2x qPCR Mastermix (KAPA SYBR®FAST, Peqlab) according to manufacturer’s instruction, where reaction volumes of 10 µl contained 200 nM oligonucleotides and 4 μl of diluted DNA, respectively. Samples were measured in technical duplicates via an iQ5 multicolor real-time PCR detection system (Bio-Rad) and CT-values were determined via the Bio Rad-IQ™5 software version 2.1. Primer efficiencies were estimated by calibration dilution curves and slope calculation 67; data were analyzed via the 2−∆CT method 68 accounting for dilution factors and sample volumes used for DNA purification. qPCR data of ChIP samples were normalized according to the ParB-mCherry2 signal obtained at locus parS1 in the wild type background, serving as reference in each experiment.
Electrophoretic mobility shift assay
DNA-ParB binding was assayed using purified protein (for protein purification, see Supplementary Information) and double-stranded DNA fragments of approximately 1100 bp length with or without two parS sites. Fragments were generated by PCRs of a C. glutamicum genomic locus surrounding parS9 and parS10 using primer pairs parS9mut-HindIII-up-F/ parS10mut-EcoRI-D-R. ParB concentrations of 0.05 to 25 µM were incubated with 100 ng DNA for 30 min at 30°C, following sample separation in native gels (3-12 % polyacrylamide, ServaGel™). DNA was stained using SYBR® Green I (Invitrogen).
Photoactivated localization microscopy (PALM)
c. glutamicum cells were fixed with 3 % of formaldehyde prior to super resolution imaging using a Zeiss ELYRA P.1 microscope with laser lines HR diode 50 mW 405 nm and HR DPSS 200 mW 561 nm and an Andor EM-CCD iXon DU 897 camera. Cellular ParB-PAmCherry signals were further analyzed using Fiji software64 and identification of distinct protein clusters was carried out by applying the OPTICS algorithm in R 48,49. A detailed description of sample preparation, parameters for detection of ParB-PAmCherry signals, PALM image calculation and analysis is available in the Supplementary Information.
Chromosome Conformation Capture Libraries
Chromosome Conformation Capture (3C/Hi-C) libraries were generated as previously described by Val et al. 47 with minor changes, as specified in the Supplementary Information.
Contact map generation
Contact maps were generated as previously described 37. Reads were aligned independently (forward and reverse) using Bowtie 2 in local and very sensitive mode and were assigned to a restriction fragment. Non-informative events (self-circularized DNA fragments, or uncut fragments) were discarded by taking into account the pair-reads relative directions and the distribution of the different configurations as described in Cournac et al 69. We then bin the genomes into regular units of 5 Kb to generate contact maps and normalized them using the sequential component normalization procedure (SCN) 69.
Contact map comparison
Ratio between contact maps was computed for each point of the map by dividing the amount of normalized contacts in one condition by the amount of normalized contacts in the other condition and by plotting the Log2 of the ratio. The color code reflects a decrease or increase of contacts in one condition compared to the other (blue or red signal, respectively). No change is represented by a white signal. Ratio maps of replicates are illustrated in Figure S12.
Identification of domains frontiers using directional index
To quantify the degree of directional preference, we applied on correlation matrices the same procedure as in Marbouty, et al. 10. For each 5 Kb bin, we extracted the vector of interactions from the correlation matrix between the studied bin and bins at regular 5 Kb intervals, up to 250 Kb in left and right directions. The two vectors were then compared with a paired t•test to assess their statistical significant difference (p=0.05). The directional preferences for the bin along the chromosome are represented as a bar plot with positive and negative t•values shown as red and green bars.
Statistical analysis
Correlation coefficients, linear regressions and analyses of nearest neighbor distance distributions were calculated using Excel, Graph Pad Prism (GraphPad Software) and R49.
Data availability
Proteomic data are available via ProteomeXchange with the project identifier PXD008916 70. Reviewer account details: username: reviewer52143{at}ebi.ac.uk, password: zyJpq85V
Genome-wide sequencing reads of ChIP-seq and chromosome conformation capture assays generated in this study are available in the Sequence Read Archive (SRA) under accession numbers PRJNA529385 and PRJNA525583.
Acknowledgements and Funding
This work was further funded by grants to M.B. from the Deutsche Forschungsgemeinschaft (BR2815/6-1 & 2815/6-2) and the Ministry of Science and Education (BMBF: 031A302 e:Bio-Modul II: 0.6 plus).This research was supported by funding to R.K. from the European Research Council under the Horizon 2020 Program (ERC grant agreement 260822).
We thank our colleagues from R.K. and M.B. labs for discussions, feedback and comments on the manuscripts and the interpretation of the data. We are thankful to Prof. Dr. Stephan Gruber (Department of Fundamental Microbiology, University of Lausanne) for inspiring discussions and for his help with ChIP-seq. The authors are grateful to Dr. Andreas Brachmann (LMU) for his help with the genome sequencing.