Activity of MukBEF for chromosome management in E. coli and its inhibition by MatP

Reviewer #1 (Public Review):

Summary:
In this manuscript, Seba et al., investigate the mechanism of chromosome organization by the MukBEF complex in E. coli. They use a combination of Hi-C and ChIP analysis to understand the steps of MukBEF regulation: its unloading from DNA (how MukBEF activity is prevented in the terminus regions of the chromosome by MatP), and its loading onto DNA (how DNA replication influences MukBEF association with the chromosome). Seba et al., induce chromosomal rearrangements to flip the sections of the ter region, thus perturbing matS site numbers and position. They find that MukBEF activity is prevented around matS sites, and that higher matS density has greater effect on MukBEF. Separately, using replication mutants and inducible MukBEF expression, they find that MukBEF can associate with the chromosome even in the absence of replication (as seen by the emergence of long-range contacts). However, ChIP data suggests that MukBEF binding to DNA is enriched on newly replicated DNA.

Strengths:
Altogether, this work provides a valuable and comprehensive view of MukBEF-mediated chromosome organization, with insights on the mechanism of the exclusion of MukBEF from the terminus region of the chromosome. The use of the programmed genetic rearrangements is powerful, and allows the authors to provide clear and convincing evidence for MukBEF exclusion from ter by matS sites. It is particularly striking to see that MukBEF can promote long-range contacts even in chromosomal regions between two matS, but the complex is excluded from the matS 'zones'. Experiments using cells blocked for replication show that MukBEF can influence chromosome organization in the absence of replication as well. While previous studies have reported some evidence in support of both of the above conclusions, the experiments described here offer clear and direct demonstration of the same.

Limitations:
A few control experiments are required to strengthen conclusions. Additionally, the discussion section is lacking many references and key papers have not been cited (paragraph 1 of discussion for example has no references). The possibility that SMC-ScpAB and MukBEF can act independent of replication has been suggested previously, but are not cited or discussed. Similarly, there is some evidence for SMC-ScpAB association with newly replicated DNA (PMID 21923769).

Reviewer #2 (Public Review):

Summary:
Chromosome organization in E. coli and related species ('transversal') deviates starkly from the pattern more commonly found in bacteria ('longitudinal'). The underlying mechanisms and the physiological roles, however, are not well understood. This manuscript by Seba et al. investigates the activity and regulation of MukBEF in chromosome folding in E. coli. Using a construct for inducible expression of MukBEF, the authors first demonstrate that the initiation of long-range chromosome contacts (likely by loop extrusion) is not restricted to few positions on the chromosome and rather widely distributed but excluding the replication terminus region. Using ChIP-Seq, the authors show that the distribution of MukBEF over the chromosome is consistent with widely distributed loading and moreover indicate a connection of chromosome folding and DNA replication with newly replicated DNA shower an increased tendency for MukBEF binding. To dissect this further, they then redistribute matS sites on the chromosome by a clever strategy based on large-scale transpositions. The results reveal that matS-free DNA segments undergo MukBEF dependent folding regardless of their position relative to the origin of replication, being consistent with a broad distributed loading of MukBEF. By fine-mapping with smaller transposition events, they show that few matS sites are sufficient to impede MukBEF activity. Surprisingly, however, E. coli and most related genomes harbor many matS sites, which are particularly highly concentrated near the chromosome dimer resolution dif site (Fig. 5).

Strengths:
This is a well-executed and well-presented study. The findings show that the MatP/matS system acts locally and independently of DNA replication to restrict MukBEF in the replication terminus region. Few of the many matS sites are sufficient for MukBEF restriction. The main conclusions of the work are clear and well supported by the data.

Weaknesses:
The biological relevance of MukBEF restriction from the replication terminus region remains unresolved. The authors could speculate on possible functions.

Reviewer #3 (Public Review):

Seba et al. investigate whether chromosomal recruitment of the E. coli SMC complex MukBEF is initiated at a single site, how MukBEF activity is excluded from the replication terminus region, and whether its recruitment and activity depend on DNA replication. Upon induction of MukBEF, the authors find that chromosomal long-range contacts increase globally rather than from a single site. Using large-scale chromosome rearrangements, they show that matS sites can insulate separate areas of high MukBEF activity from each other. This suggests that MukBEF loads at multiple sites in the genome. Finally, the authors propose that MukBEF associates preferentially with newly replicated DNA, based on ChIP-seq experiments after DNA replication arrest.

The conclusions of the paper are mostly well supported by the data. The ratiometric contact analyses and range-of-contact analyses are compelling and nicely show the interplay between MukBEF and its proposed unloader MatP/matS. I particularly enjoyed the chromosome re-arrangement experiments, which lend strong support to the idea that MukBEF activity is independent of a centralized loading site.

The enrichment of MukBEF in newly replicated regions is somewhat less convincing, as the effect sizes are rather small and the background signal is unknown. The conclusion that matS density controls MukBEF activity is appealing, but would likely need additional support from more systematic studies. It is based on a comparison of only two strains (looking at different combinations of three matS sites), and the effect size is small. As it is, differences in matS sequence composition and genomic context cannot be factored out.

Overall, the work is an important advance in our understanding of bacterial chromosome organization. It will be of broad interest to chromosome biologists and bacterial cell biologists.