Chromosome organization by a conserved condensin-ParB system in the actinobacterium Corynebacterium glutamicum

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.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The custom computer algorithm used in the analysis of PALM data has been deposited in Github (https://github.com/ GiacomoGiacomelli/ParB-clustering-protein-profiling) Hi-C, flow cytometry and ChIP-Seq data have been deposited in public databases. Accession numbers are given in the main text. Whole genome data of C.

nature research | reporting summary
October 2018 Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links
May remain private before publication. glutamicum were accessed from GeneBank (GeneBankID: BX927147.1). RNA-seq data for C. glutamicum were recovered from ENA (Project PRJEB4788). The authors declare that the data supporting the findings of this study are available within the paper and raw data file of all plots and gel images have been submitted with this paper. Data can also be obtained from the corresponding authors upon request.
Experiments were at least done in biological triplicates. Sample sizes for single cell experiments image analyses were always above n=200. For flow cytometry more than 10.000 single cells were counted. Sample sizes were chosen based on standards in the field.
We did not exclude datapoints. A Source Data file with all raw data is submitted along with this paper.
Number of experimental replications are indicated in the text for every experiment. We did not observe severe differences between experimental replicates. Covariates were controlled by using identical experimental conditions, buffers and solutions as well as identically treated samples. Identical phenotypes of strains were oberserved in independent experiments.
Samples were not randomized. Single cell data (images and flow cytometry) rely on large numbers.
We did not use blinding. This was unecessary since we rely on automated image analyses and statistical data evaluation.
Specificity of antibodies was tested in western blots against cell lysate of strains expressing mCherry fusion proteins.
https://www.ncbi.nlm.nih.gov/sra/?term=prjna529385 Accession codes prjna529385 Final graphs are given in the manuscript and can be requested from the corresponding authors upon request. All raw data are given in the supplemental material file.