# Insights in bacterial genome folding

Fares Osam Yáñez-Cuna, Romain Koszul

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Fares Osam Yáñez-Cuna, Romain Koszul. Insights in bacterial genome folding. Current Opinion in Structural Biology, 2023, 82, pp.102679. 10.1016/j.sbi.2023.102679. hal-04951528

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# Insights in bacterial genome folding

Fares Osam Yáñez-Cuna and Romain Koszul

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## Abstract

Chromosomes in all domains of life are well-defined structural entities with complex hierarchical organization. The regulation of this hierarchical organization and its functional interplay with gene expression or other chromosome metabolic processes such as repair, replication, or segregation is actively investigated in a variety of species, including prokaryotes. Bacterial chromosomes are typically gene-dense with few non-coding sequences and are organized into the nucleoid, a membrane-less compartment composed of DNA, RNA, and proteins (nucleoid-associated proteins or NAPs). The continuous improvement of imaging and genomic methods has put the organization of these Mb-long molecules at reach, allowing to disambiguate some of their highly dynamic properties and intertwined structural features. Here we review and discuss some of the recent advances in the field of bacterial chromosome organization.

## Addresses

Institut Pasteur, CNRS UMR 3525, Université Paris Cité, Unité Régulation Spatiale des Génomes, 75015, Paris, France

Corresponding author: Koszul, Romain (romain.koszul@pasteur.fr)

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(Koszul R.)

### Current Opinion in Structural Biology 2023, 82:102679

This review comes from a themed issue on 3D Genome Chromatin Organization and Regulation (2023)

Edited by Genevieve Almouzni and Juanma Vaquerizas

For complete overview of the section, please refer the article collection - 3D Genome Chromatin Organization and Regulation (2023)

Available online 19 August 2023

https://doi.org/10.1016/j.sbi.2023.102679

0959-440X/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

Since pioneering cytological studies in *Escherichia coli* [1] and *Bacillus subtilis* [2], numerous works have explored the functional organization of the genome of replicating bacteria. As in eukaryotes, investigations are conducted through a combination of genetics, imaging [3], and genomic approaches such as capture of chromosome conformation (Hi-C) [4,5]. In most bacteria investigated so far, at least one megabase (Mb)-sized chromosomal region stands out from the rest of the genome either through genetic isolation, enrichment in specific

proteins, dynamics, 3D folding or a combination of these features, such as the Ter macrodomain of *E. coli* [6] or the Ori domain of *B. subtilis* [7] (Figure 1a, b). Another common feature, revealed by Hi-C genome-wide contact maps, is the bridging of the two replicohores (i.e., chromosome arms), as originally found in *Caulobacter crescentus* [4,5] and *B. subtilis* [7,8] (Figure 1b), and now identified in dozens of species as unveiled by metagenomics Hi-C studies [9–11] (Figure 1c). The condensin complex of the structural maintenance of chromosome (SMC) family was shown to bridge replicohores following initiation of replication in *B. subtilis*, traveling from the origin to the terminus of replication [7,12]. Enterobacteria, such as *E. coli*, are notable exceptions to this rule (Figure 1a). In addition, nucleoids labeled with fluorescent proteins fused to NAPs were visualized using conventional [13–15] or super-resolution imaging [7,16,17], revealing a loose helical shape.

Hi-C genomic contact maps have also pointed at other types of structural features in the last ten years, the most notable being chromosome (self-)interacting domains (CIDs), ranging in size from ~30 to 200 kb and delimited by long, highly-expressed genes [18]. The potential functional relevance of CIDs, first unveiled in *C. crescentus* [5] and then identified in all genomes investigated so far, including *B. subtilis* [7], *E. coli* [19], *Pseudomonas aeruginosa* [20] and other species, remains unclear. Past and recent works have revealed that CIDs are not monolithic and can change depending on the metabolism of the cell's conditions, as exemplified in contact maps of *C. crescentus* and *Streptomyces ambofaciens* comparing exponential growth with starvation [18] and during spore differentiation [21], respectively. CIDs boundaries are proposed to be caused by the unwinding and decompaction of DNA regions such that a physical separation between self-interacting domains is created [18]. In addition, CID boundaries are not affected by SMC condensin [5]. A recent work brings a new perspective on the common interpretation of CID signal as a delimitation of self-interacting domains (below; Figure 2a). Finally, some, but not all, Hi-C maps display discrete dots that can be interpreted as loops bridging distant loci. First pointed at in *B. subtilis*, where condensin-dependent DNA loops bridge ParB/parS sites [7], reminiscent patterns are observed in other species as well, such as *Muribaculum intestinale*, *Enterococcus faecalis*, etc. [11] In *B. subtilis*, the NAP Rok, which binds preferentially and non-specifically to AT-rich sequences
<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>

(a) Normalized Hi-C contact map of *Escherichia coli* (bin: 5 kb). The Ori and Ter macrodomains are represented as a pink and black lines, respectively. Black triangle: oriC. Blue and red lines: left and right replicosomes. A magnification of the region surrounding the Ter macrodomain (dotted square) is shown under the map, near an illustration of the macrodomain. ZapAB links the terminus of the chromosome to the Z ring, the cell division site organized by FtsZ.

(b) Normalized Hi-C contact map of *Bacillus subtilis* (bin: 5 kb). The Ori region, and the left and right replicosomes are represented in pink, blue and red, respectively. A magnification of the region surrounding the origin of replication and containing looping signals is shown underneath. Gray triangles: positions of parS sites. An illustration of the Ori domain is indicated on the right, with the main hairpin (light green area) bridging both arms of the chromosome together. The intra-arm harpin (light blue) brings regions on the left side of the chromosome close to the origin causing a bow-shaped signal on the contact map [7]. The NAP Rok clusters at different locations on the chromosome and around the origin where they can form a supercluster [25].

(c) Normalized Hi-C maps of bacterial species from different genus isolated from the mouse gut: *Ruminococcus*, *Blautia*, *Clostridium*, and *Prevotella* (from metagenomic Hi-C [9]). (d) Schematic representation of the ring-shaped structure of the MukBEF complex. The SMC MukB, the kleisin MukF and the dimeric KITE protein MukE associate to make the MukB<sub>2</sub>E<sub>2</sub>F complex. (e) Bacterial SMC-type homologues. (f) Simplified schematic representation of the *E. coli* genome. Blue and red: left and right replicosomes, respectively. Pink: Ori domain. Black: Ter domain. White circle: replication origin. Green circles: MukBEF. Right panel: schematic representation depicting the *E. coli* genome condensation forming a discontinuous axial core mediated by MukBEF as proposed in Ref. [39]. (g) Cartoon of *B. subtilis* chromosome. Blue and red: left and right replicosomes, respectively. Pink: Ori domain. Grey bars: ParS sites. Green circles: SMC-ScpAB. Right panel: schematic representation showing SMC-mediated bridging of replicosomes, starting with the Ori region, during replication. Light green: area involving the primary hairpin. Light blue: intra-arm hairpin mediated by distant parS sites.

(g) Cartoons of *B. subtilis* chromosome. Blue and red: left and right replicosomes, respectively. Pink: Ori domain. Grey bars: ParS sites. Green circles: SMC-ScpAB. Right panel: schematic representation showing SMC-mediated bridging of replicosomes, starting with the Ori region, during replication. Light green: area involving the primary hairpin. Light blue: intra-arm hairpin mediated by distant parS sites.

- commonly found on regions acquired by horizontal gene transfer and often targeted by NAPs (for a recent review look [22–24]), appears to be required to delineate the base of the loops in this species [7,25].

However, it remains unknown whether the loops observed in genomic contact maps of other bacteria are strictly dependent on NAPs, such as Rok, or whether they may result from other processes.
<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>

(a) Schematic representation of Hi-C contacts along a chromosome region. Transcription induced domains are represented along the map, consisting in dense regions of short range contacts. Highly transcribed genes generate the strongest TIDs and generate boundaries in the contact maps that were used to define CIDs. (b) During transcription, the unwinding of DNA by RNA polymerases generates positive and negative supercoils ahead and behind the complex, respectively ("twin domain" model) [47]. Topoisomerases alleviate torsional stress and keeps cell supercoil homeostasis. Topol, TopolII, and TopolV can relax negative supercoils. Gyrase and TopolV relaxes positive supercoils [69]. (c) During replication, the unwinding of DNA creates positive supercoil at the front of the complex [47]. Positive supercoils can then be alleviated, besides to the use of topoisomerases, by transforming them into precatenanes on the replicated region. Topo III and TopolV can remove precatenanes [69]. Additionally, Gyrase, TopolII, and TopolV can decatenate circular DNA molecules.

SMC-mediated regulation of bacteria
chromosome folding

SMCs ring complexes play significant roles in regulating
chromosome organization across domains of life [26]. In
prokaryotes, three members of the SMC family have

been characterized: the nearly ubiquitous Smc-ScpAB
complex, present in most bacteria but enterobacteria
and a few other gamma-proteobacteria like *Vibrionales*
(*Vibrio cholerae*), *Enterobacterales* (*Yersinia pestis*, *Klebsiella
pneumoniae*), and *Pasteurellales* (*Pasteurella multocida*,
*Hemophilus influenzae* ) [27]; MukBEF, found in gamma-
proteobacteria lacking Smc-ScpAB; and MksBEF,
often found alongside one of the other SMC com-
plexes [28].

It is proposed that SMC-ScpAB works with the ParABS
system to promote proper chromosome compaction and
segregation. In short, a dimer of ParB recognizes and
binds *parS* sites located in multiple copies mostly
around the replication origin [29,30]. Multiple ParB
dimers then spread along the neighboring chromosomal
region. ParB-*parS* recruits ParA promoting the loading of
the SMC complex near the origin [31]. This SMC-ParB-
*parS* organization of the Ori region fosters efficient
chromosome segregation [32]. The SMC bridges newly
replicated arms and actively translocates down to the
replication terminus through a loop extrusion process.
This activity brings both arms of the chromosome
together [7,12] (Figure 1g). In *B. subtilis*, colliding
SMCs are able to bypass each other while progressing
through the chromosome [33]. Upon reaching the ter-
minus region, XerD helps unload the SMC complex
after recognition of multiple XerD-specific binding
sites [34].

In most bacteria, chromosome segregation occurs concomitantly or soon after replication. In others, these processes can take place separately [35]. In *Streptomyces* species, the linear chromosomes change from an open conformation at the end of vegetative growth and while entering the sporulation stage of development, to one where opposite arms of the linear chromosome are bridged together by SMCs during sporogenic cell division [35] or during metabolic differentiation [21].

It is commonly estimated that ~10% of bacteria ge-
nomes have at least one additional large replicon
(>350 kb) alongside the main chromosome. These
replicons can be either in the form of secondary chro-
mosomes (circular or linear), chromids, or megaplasmids
[36]. *Agrobacterium tumefaciens* has two chromosomes, a
circular and a linear one, and two large plasmids. Both
chromosomes have their arms joined together by the
action of SMCs allowing their arms to interact with each
other. Notably, chromosome arms interact between
chromosomes along their lengths. All four replicons have
their Ori regions bundled together [29]. Whether these
interchromosomal interactions play any regulatory roles
remains to be determined.

Hi-C contact map of *V. cholerae*'s two circular chromo-
somes shows an enrichment of intrachromosomal con-
tacts between replicores of each chromosome, most
prominent around the Ori region and fainter interactions
towards the Ter, with the two Ter regions colocalizing in
space. The chromosomes complete replication syn-
chronously despite their different sizes, as the replica-
tion of chromosome 2 is triggered only once the *crtS*

locus along chromosome 1 is replicated, through an
interaction with the chromosome 2 replication initiator
RctB [37]. An enrichment in contacts between the ori2
and the crtS loci is clearly observed in high-resolution
Hi-C maps [38], though the functional links between
these contacts and crtS control remain unknown.

In *E. coli*, the SMC MukBEF complex appears involved
in promoting long-/middle range contacts over the
chromosome [19], but without bridging replichores
together as SMC-ScpAB does (Figure 1g). MukBEF has
been proposed to form a discontinuous axial core, from
which DNA emanates as loops through active ATP-
dependent extrusion [39] (Figure 1f). The role of this
folding remains to be fully characterized but could be
involved in keeping chromosome arms separated. MatP,
which binds to *matS* sites located at the terminus region
of the chromosome to insulate the Ter macrodomain
from the rest of the genome [6,19], displaces MukBEF
[40,19]. The Ter region, therefore, lacks such long-range
contacts (Figure 1a). This observation can be inter-
preted either as a lack of compaction, or as the behavior
of loci physically constrained apart, or else. The mech-
anism used by MukBEF contrasts from SMC-ScpAB in
that its loading does not appear to be site-specific nor
Ori proximal, and does not bring chromosome arms
together but keeps them separated. On the opposite,
SMC-ScpAB is loaded at site-specific positions near the
ori. How MukB-dependent loops are able to process
through potential roadblocks along the genome, espe-
cially highly expressed genes that are known to interfere
with SMC progression, remain unknown.

The MksBEF complex is often associated with either Smc-ScpAB or MukBEF. In *P. aeruginosa*, whose genome encodes both Smc-ScpAB and MksBEF, the latter complex participates in chromosome organization [20] by promoting long-range cis contacts on chromosome arms reminiscent of those promoted by MukBEF in *E. coli*. Its activity, however, can only be revealed in absence of SMC-ScpAB-directed chromosome organization or in altered chromosomes where the *parS* site has been moved away from *oriC* [20]. MksBEF can achieve chromosome segregation in the absence of a functional ParABS system. A hierarchy of condensin activities was therefore proposed in this species, where the participation of MksBEF depends on its ability to act on newly replicated regions before ScpAB recognizes *parS* and joins the two arms of the chromosome [20].

MksBEFG, also known as WadjetABCD, represents a
fourth class of bacterial condensin. It does not play a role
in chromosome organization but rather in plasmid con-
trol [28,30]. This complex was indeed originally
described as a protection against foreign plasmid trans-
formation. Unlike other SMCs, it is often located within
defense islands but is not often present within strains of
the same species [41]. It is suggested that the system
can be acquired by transposon-mediated horizontal gene transfer [42]. JetD (*mksG*), a nuclease, associates itself with a distant homologue of MksBEF (JetABC) which allows it to recognize and cleave circular double-stranded DNA molecules [43]. An intriguing possibility is that the complex may detect the topology of the target DNA through a loop extrusion process that would recognize the circularity of the molecule, causing the complex to stop and promote a DNA break [44]. JetABCD also appears to discriminate DNA molecules by size. Smaller plasmids (10–35 kb) are more efficiently targeted than bigger ones (>50 kb) [45].

## Transcription-mediated structuration of bacterial genomes

Early works demonstrated that the nucleoid was composed of supercoiled microdomains separated by stochastic borders determined by a combination of gene expression and topoisomerase activity [46]. Transcription is responsible for the formation of positive and negative supercoils, which can impact chromosome dynamics and activity [47]. The movement of a polymerase generates positive supercoils at the front of the fork and similar, albeit negative, ones behind (aka the “twin domain model”) (Figure 2b) [47,48]. Highly transcribed regions, such as ribosomal operons, affect up to 24 kb upstream and downstream of the transcription unit. The interplays between transcription and bacterial genome organization have been increasingly supported, notably through the correlation found between the high levels of gene expression recovered at the level of CIDs’ boundaries in Hi-C contact maps [5]. In addition, imaging studies have revealed that inhibition of *E. coli* transcription with the drug rifampicin results in rapid nucleoid decompaction [3]. A recent high-resolution Hi-C protocol for bacteria shows that the *E. coli* chromosome is organized into a succession of short, self-interacting domains corresponding to actively transcribed sequences, that are separated by non-transcribed regions depleted in short-range contacts [49]. A subset of these short transcription-induced domains (called TIDs), those with the highest level of transcription, correspond to CIDs boundaries, but overall, all transcribed regions appear to behave as small discrete domains [49] (Figure 2a). Therefore, CIDs may correspond to the transcription signal resulting from the most visible positions in the original Hi-C experiments [49]. TIDs from all sizes would be associated with transcription-induced supercoiling, that could directly impact chromosome dynamics, making transcription one, if not the primary, structuring factor of the DNA fiber at the local scale.

Transcription also appears to modulate chromosome folding by directly impacting condensin-dependent activity. Shifting condensin loading *parS* sites or disrupting the orientation of transcribed genes in *B. subtilis* [8]

and *C. crescentus* [50] leads to asymmetrical Hi-C maps, where the secondary diagonal is now highly distorted compared to the original perpendicular position to the main one. This shift is compatible with a decrease in the speed of loop extrusion progression as it encounters an active transcription locus, with a bigger effect when a collision occurs head-to-head compared to head-to-tail [51]. The alteration could result from steric constraints imposed by TIDs, transcription-induced supercoils, or most likely both. The orientation of essential and highly transcribed genes co-directionally with the progression of the replication fork, preventing head-on collisions between the replication and transcription machinery [52], may also directly reflect the need to avoid collisions between RNA polymerases and condensin-mediated extrusion.

In *E. coli*, replication-induced supercoiling patterns can also be seen in the chromosome, with negative supercoil enrichment in earlier replicated regions compared to later regions [47]. The Ter region is an exception as it is richer in negative supercoils [47]. The left and right sides of the chromosome have mostly symmetrical supercoil patterns relative to the replication origin [47]. Supercoil patterns vary over large regions of the chromosome (macrodomains), with highly transcribed genes likely to prevent supercoil diffusion [47,18]. The supercoiling patterns change with conditions (osmotic stress, etc.) and could themselves influence gene expression, as an estimated 7% [53] to half [54] of *E. coli* genes are sensitive to supercoiling, making supercoiling one of the most important regulators of gene expression. Each organism has a different genome supercoiling tolerance [55] and the overall supercoil homeostasis corresponds to a balance of topoisomerases’ activities [56]. Surprisingly, depleting topoisomerases have relatively minor effects on the global chromosome architecture [57]. For instance, and despite their opposite actions, the absence of gyrase (relaxes positive supercoiled DNA) and TopoI (relaxes negative supercoiled DNA) (Figure 2) have similar though modest consequences on chromosome folding as assessed through Hi-C, resulting in a genome-wide decrease in short-range contacts [57] TopoIII has an opposite, although still modest, effect of increasing short-range contacts [57].

## Resolving sister chromatids entanglements

The positive supercoil caused by the progressive unwinding of DNA through replication prevents further replication unless it is alleviated by topoisomerases or distributed to the back of the fork as sister chromatid intertwining (precatenates) [58] (Figure 2c). To date, prokaryotic SMCs, unlike their eukaryotic counterparts, have not been found to bridge sister chromatids [59]. This role was proposed to be fulfilled by precatenanes [60]. Sister chromatid contacts are not uniformly
distributed nor maintained along the genome, and im-
aging revealed dynamics movements and heterogeneity
between replication and segregation timing in E. coli
[15,61]. A technique aiming at quantifying genome-
wide sister-chromatid contacts (Hi-SC2) was applied
to V. cholerae and pointed at cohesive clusters surround-
ing the origin and terminal regions of the chromosome
[62]. In that work, Barre and colleagues further identi-
fied the H-NS NAP in promoting a more cohesive region
in the V. cholerae pathogenicity island [62]. In E. coli and
other bacteria, separation of sister loci depends on
TopoIV activity. TopoIII also participates in sister chro-
matid decatenation, albeit poorly. Mutation in TopoIII
does not affect overall genome folding other than
causing a slight increase in short-range contacts [57].
Changes in TopoIV concentration affect the cohesion
times between sister origins [60]. Its depletion results
in a genome-wide spatial reorganization, with an accu-
mulation of Hi-C contacts extending between the Ter
domain extremities up to replication origin (something
that resembles a butterfly pattern in contact maps) [57],
and an increase in short-range contacts over chromo-
some arms. One possible explanation for this behavior is
that unresolved catenates accumulate at the Ter
boundaries, resulting in increased long-distance con-
tacts along each chromosome arm. Indeed, TopoIV as-
sociates in vivo with MukBEF through an interaction
between the MukB hinge domain and TopoIV ParC
subunit [63]. This interaction stimulates its activity on
negative (right-hand) supercoiled DNA [64], which has
similar chirality as replication catananes, and places
TopoIV in the same regions where MukBEF is active.
There is competition between MatP and TopoIV for
binding to MukB. Such interaction not only keeps
MukBEF from the terminus region but also explains
TopoIV relative absence from that region [65]. The
resulting increase in cohesion at Ter region can be
relevant for chromosome segregation and coordinate
decatenation with the cell cycle [65]. TopoIV cell
localization also follows a similar pattern to SeqA and the
replication machinery throughout the cell cycle [66]. Its
relaxation and decatenation activities are stimulated by
SeqA protein [67]. In this model, TopoIV trails SeqA
and the replication machinery through binding with
SeqA's origin-proximal side, although it does it at a dis-
tance [66]. Sister chromosomes are mostly disentangled
by the time of cell division's septum constriction except
near the *dif* site, a site located at the terminus region of
the chromosome that helps solve multimeric forms of
the chromosome into monomers [62]. In E. coli, FtsK-
induced *dif*-XerCD recombination is responsible for
the resolution of the chromosomes [68]. It is unclear if
other SMCs, like SMC-ScpAB, associate themselves
with topoisomerases and how they regulate the deca-
tenation process.

Conclusion

Overall, this field of research is progressing quickly, with
an ever-growing number of clades being investigated
through powerful experimental methods and theoretical
approaches. Many of the questions hinted throughout
this review are therefore expected to find answers in the
next few years. We are confident that in the relatively
near future, the causal relationships between the
structure and function of bacterial genetic elements will
be substantially disambiguated.

Resources

Accession number of reads used to generate *B. subtilis* [7]
maps: PRJNA289589.

Declaration of competing interest

The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in
this paper.

Data availability

No data was used for the research described in
the article.

Acknowledgments

We are grateful to Olivier Espeli and Martial Marbouty for comments. This research was supported by the European Research Council under the Horizon 2020 Program (ERC grant agreement 771813) to RK.

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