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

Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we find that C. glutamicum cells are at least diploid under all conditions tested and that these organisms have overlapping C-periods during replication with both origins initiating replication simultaneously. Based on experimentally obtained data we propose growth rate dependent cell cycle models for C. glutamicum.


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Bacterial chromosome organization is highly regulated, where replication coincides 35 with segregation of sister nucleoids and is tightly coordinated with cell division (1). 36 Cell cycle control mechanisms exist, which ensure constant DNA content throughout 37 cell generations. In particular, the action of the key replication initiator protein DnaA is 38 timed by various regulatory systems, for instance via the CtrA protein cascade in 39 Caulobacter crescentus or SeqA in Escherichia coli (2-6). Upon replication initiation 40 DnaA binds to the origin of replication (oriC) and mediates duplex unwinding prior to 41 loading of the replication machinery (7,8). The two evolving replication forks migrate 42 along the left and right arm of the circular chromosome towards the terminus of 43 replication (terC), where FtsK-dependent XerCD recombinases resolve decatenated 44 chromosomes as a final step (9,10). Replication usually takes place within defined 45 cellular regions via stably assembled protein complexes, namely replisomes, of 46 rather static or dynamic nature (11,12). 47 The bacterial cell cycle can be divided in different stages illustrated in Figure 1. The 48 time of DNA-replication is termed C-period, which is followed by a time interval 49 necessary for cell division executed by the divisiome (D-period). Several bacteria like 50 Mycobacterium smegmatis and C. crescentus replicate their genome once within a 51 generation, where C-periods are temporally separated from each other (13,14). At 52 slow growing conditions a non-replicative state termed B-period precedes the C-53 period (not shown), thus the bacterial cell cycle resembles in some aspects the 54 eukaryotic cell cycle (G1, S, G2 phases). Contrary to this, fast growing organisms 55 such as Bacillus subtilis, E. coli and Vibrio cholerae can overlap C-periods during fast 56 growth, a phenomenon termed multifork replication (15)(16)(17). Under these conditions a 57 new round of replication is reinitiated before termination of the previous one. 58 Therefore, generation times are considerably shorter than the duration of the C-59 period. However, only one round of replication is initiated per cell cycle and usually 60 one C-period is completed at the time point of cell division (18). Many bacteria 61 contain only one copy of the chromosome. However, several bacteria and archaea 62 can have increased DNA contents due to oligo-or polyploidy (19). Polyploid cells 63 harbor multiple, fully replicated chromosome copies throughout their life cycle, which 64 has been frequently found in prokaryotes including certain gram-positive bacteria, oriC towards the opposite cell half (33,45,46). The precise mechanism of the 94 ParABS-mediated DNA segregation has been under debate, however, to date 95 dynamic diffusion-ratchet and DNA-relay models are favored, where nucleoid and 96 plasmid movement is mediated along a ParA gradient caused by local ParB-97 stimulated depletion of . Deletion of this partitioning system 98 has mild effects in B. subtilis and V. cholerae cells, but causes severe chromosome 99 segregation defects in other organisms and is essential for viability in C. crescentus 100 and Myxococcus xanthus (46,(50)(51)(52)(53)(54)(55)(56). 101 Here we present the cell cycle and spatiotemporal organization of oriCs and 102 replisomes in C. glutamicum, a rod-shaped polar growing actinobacterium. It is 103 closely related to pathogens like C. diphteriae and M. tuberculosis, the latter being 104 amongst the top ten causes of fatal infections worldwide (57). Besides this, C. 105 glutamicum is of great economic importance as an amino acid and vitamin producer 106 and extensive efforts in metabolic engineering are being carried out concerning 107 metabolite production and yield increase (58). Although, its metabolism is one of the 108 best studied amongst model organisms the underlying cell cycle parameters and 109 chromosome organization patterns had so far not been analyzed in detail. C. 110 glutamicum relies on a ParABS system to segregate their nucleoids prior to cell 111 division (51,59,60). Chromosome segregation influences division site selection and, 112 hence, growth and chromosome organization are tightly coupled in C. glutamicum 113 (59). This may in part explain why protein machineries that have been described in 114 various bacterial species like the Min system or a nucleoid occlusion system, both 115 being involved in division septum placement are absent in C. glutamicum (61). 116 In this study, we tracked in vivo fluorescently labeled centromer-binding protein ParB  Oligonucleotides, plasmids and bacterial strains 132 All primers, plasmids and bacterial strains used in this study are listed in Table S1 133 and S2, respectively.

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Integration plasmids were constructed with 500 bp homologous regions upstream 136 and downstream of the 3' end of the gene to be labeled with a fluorophore sequence 137 in between. For pK19mobsacB-parB-eYFP and pK19mobsacB-parB-mCherry2 138 plasmids the upstream and downstream fragments were PCR amplified from the C. ParB-XbaI-D-F/ ParB-Bam-D-R. eYFP or mCherry2 sequences were amplified using 141 primer pairs eYFP-SalI-F/ eYFP-XbaI-R or mCherry2-SalI-F/ mCherry2-XbaI-R. In 142 order to construct pK19mobsacB-DnaN-mCherry upstream and downstream 143 homologous regions were amplified via primer pairs  R and DnaN-XbaI-D-F/ DnaN-BamHI-D-R. The mCherry sequence was amplified 145 using mCherry-SalI-F/ mCherry-XbaI-R primer pairs. The resulting PCR fragments 146 were digested with the respective restriction enzymes and consecutively ligated into 147 pK19mobsacB vectors. Plasmid cloning was performed using DH5α. C. glutamicum 148 were transformed via electroporation and selected for integration of the fluorophore 149 as described before (62) triplicates. Oligonucleotides used are listed in Table S1 and results were analyzed 189 using the 2 -∆CT method (65).

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Culture samples were fixed 1:9 (v/v) in 70% ethanol and stored at 4°C until use. Cells 192 were pelleted at 5000 rpm for 5 min and washed once in phosphate-buffered saline 193 (PBS). The DNA staining procedure was adopted from a protocol described before

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Origin numbers correlate with cell length in a ParA-independent way. 239 We have shown before that the C. glutamicum partitioning protein ParB localizes at  like growth and cell lengths for this strain were confirmed (Fig. S1). The dependence 352 of ParB-eYFP and DnaN-mCherry foci numbers on cell length is shown in Figure 4D. replication were initiated at polar as well as at midcell positioned origins (Fig. S3).

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Overlapping replication periods allow for fast growth.

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The observation of multiple DnaN and ParB foci suggest the possibility that C. conditions a ParB focus is stably attached to each pole, suggesting an ori-ter-ter-ori 470 orientation of the chromosomes (Fig. 2 & 3). Interestingly, newly replicated origins 471 segregate to midcell were they remain until cytokinesis is completed and they stay 472 tethered to the newly forming cell poles (Fig. 3). Up to now it is unclear by which 473 molecular mechanism the placement of ParB and the chromosomal origin is directed.

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Earlier work has shown that ParB and the cell division protein FtsZ interact (51).