Novel chromosome organization pattern in actinomycetales–overlapping replication cycles combined with diploidy

Abstract

20Bacteria regulate chromosome replication and segregation tightly with cell division to 21 ensure faithful segregation of DNA to daughter generations. The underlying 22 mechanisms have been addressed in several model species. It became apparent 23 that bacteria have evolved quite different strategies to regulate DNA segregation and 24 chromosomal organization. We have investigated here how the actinobacterium 25 Corynebacterium glutamicum organizes chromosome segregation and DNA 26 replication. Unexpectedly, we find that C. glutamicum cells are at least diploid under 27 all conditions tested and that these organisms have overlapping C-periods during 28 replication with both origins initiating replication simultaneously. Based on 29 experimentally obtained data we propose growth rate dependent cell cycle models for 30 C. glutamicum. 31 32 65 proteobacteria, Deinococcales, cyanobacteria and also archaea (20-27).66 Besides the distinct cell cycle modes chromosome localization patterns differ 67 between model organisms. In a non-replicating, slow-growing E. coli cell the single 68 4chromosome is placed symmetrically with oriC and terC regions located at midcell 69 and the replichores spatially separated to the two cell halves (28). Upon replication 70 initiation the two sister chromosomes segregate bidirectionally to opposite cell halves 71 with replisomes positioned at midcell (29,30). Finally, oriC and terC are confined to 72 cell quarter regions. Contrary to this, the model organisms C. crescentus, Vibrio 73 cholerae and Pseudomonas aeruginosa localize their nucleoids about the 74 longitudinal axis with chromosome arms adjacent to each other (31-34). Sister 75 replichores move to the opposite cell half with the segregated oriC facing towards the 76 pole, mirroring the second chromosome at the transverse axis. The oriC region of C. 77 crescentus and V. cholera is positioned by polar landmark proteins (35,36), where 78 replisomes assemble and simultaneously move towards midcell in the course of 79 replication (12,17). For the most part, P. aeruginosa places its replication machinery 80 centrally (34). Finally, B. subtilis switches from the longitudinal chromosome 81 organization to the E. coli "left-oriC-right" configuration during replication initiation 82 (37). 83The mitotic-like ParABS system has been identified as a driving force behind 84 coordinated nucleoid partitioning for more than two third of bacterial species 85 analyzed, with exceptions specifically within the phylum of γ-proteobacteria such as 86 E. coli (38). This segregation mechanism involves components similar to the plasmid 87 encoded par genes responsible for active segregation of low-copy-number plasmids 88 (39). Thereby, the ParB protein binds a variable number of centromere-like DNA 89 sequences called parS sites in oriC-proximity (40) and spreads along the DNA 90 forming large protein-DNA complexes (41-43). Interaction of ParB with the Walker-91 type ATPase ParA mediates ATP-hydrolysis and thereby ParA detachment from DNA 92 (44), driving...

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

“…Chromosome segregation is governed by ten oriC-proximal parS sites. Previous studies on C. glutamicum chromosome partitioning have revealed two stable ParB-oriC clusters at each cell pole, whereas newly replicated origins are segregated towards a division septum formed at midcell 41 . In B. subtilis, C. crescentus and Pseudomonas aeruginosa ParAB-mediated chromosome segregation and folding depends on parS sites 18,19,21 .…”

“…For more accurate cluster estimations, PALM analysis was performed using slow-growing cells resulting in significantly fewer ParB macroclusters per cell ( Supplementary Fig. 7C) 41 . As segregation of oriC complexes might alter their DNA compaction, we focused on the two largest macroclusters per cell, stably tethered at cell poles.…”

“…Flow cytometry. DNA contents per cell were verified in C. glutamicum strains analyzed by 3C using flow cytometry as described before 41 Flow cytometry analysis was performed as described before 41 . In short, exponentially growing cultures were treated with 25 µg/ml chloramphenicol for more than 4 h, to induce replication runouts.…”

“…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, whereas newly replicated sister oriCs segregate towards division septa via the ParABS system [41][42][43] . 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 anucleate cells [44][45][46] .…”

Competition between DivIVA and the nucleoid for ParA binding promotes segrosome separation and modulates mycobacterial cell elongation

“…Markedly, for most of the cell cycle, ParB complexes are precisely localised in bacterial cells in genus-specific positions, e.g. at the pole in C. crescentus, V. cholerae and C. glutamicum; subpolarly in M. xanthus; or midcell in vegetatively growing B. subtilis (Mohl and Gober, 1997;Harms et al, 2013;Wang et al, 2013;Kleckner et al, 2014;Wang et al, 2014;Badrinarayanan et al, 2015;Bohm et al, 2017). Interestingly, in Mycobacterium cells, the ParB complex is somewhat off-centred, and the mycobacterial chromosome segregation is partially asymmetric (Trojanowski et al, 2015;Ginda et al, 2017;Hołówka et al, 2018).…”