bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.403915; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Spatial rearrangement of the Streptomyces venezuelae linear chromosome 2 during sporogenic development 3 4 5 Szafran MJ. 1,*, Małecki T.1, Strzałka A.1, Pawlikiewicz K.1, Duława J.1, Zarek A.1, Kois- 6 Ostrowska A.1., Findlay K.2, Le TBK.2, Jakimowicz D. 1,* 7 8 1 Faculty of Biotechnology, University of Wrocław, 50-383 Wrocław, Poland 9 2 The John Innes Centre, Norwich Research Park, NR4 7UH Norwich, The United Kingdom 10 *Corresponding authors: [email protected]; [email protected] 11 12 ABSTRACT 13 Depending on the species, bacteria organize their chromosomes with either spatially 14 separated or closely juxtaposed replichores. However, in contrast to eukaryotes, 15 significant changes in bacterial chromosome conformation during the cell cycle have not 16 been demonstrated to date. Streptomyces are unique among bacteria due to their linear 17 chromosomes and complex life cycle. These bacteria develop multigenomic hyphae that 18 differentiate into chains of unigenomic exospores. Only during sporulation- associated cell 19 division, chromosomes are segregated and compacted. In this study, we show that at entry 20 to sporulation, arms of S. venezuelae chromosomes are spatially separated, but they are 21 closely aligned within the core region during sporogenic cell division. Arm juxtaposition is 22 imposed by the segregation protein ParB and condensin SMC. Moreover, we disclose that 23 the chromosomal terminal regions are organized into domains by the Streptomyces- 24 specific protein - HupS. Thus, we demonstrate chromosomal rearrangement from open to 25 close conformation during Streptomyces life cycle. 26 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.403915; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 27 INTRODUCTION 28 Chromosomal organization differs notably among the domains of life, and even 29 among bacteria, chromosomal arrangement is not uniform. While linear eukaryotic 30 chromosomes undergo dramatic condensation before their segregation, the compaction of 31 usually circular bacterial chromosomes remains relatively constant throughout the cell 32 cycle1–3. To date, chromosome conformation capture methods, applied in only a few model 33 species, have revealed that bacterial chromosomes adopt one of the two distinct 34 conformation patterns. Either both replichores of circular chromosomes align closely (as in 35 Bacillus subtilis4,5, Caulobacter crescentus6, Corynebacterium glutamicum7, Mycoplasma 36 pneumoniae8, and chromosome II of Vibrio cholerae9) or tend to adopt an “open” 37 conformation, which lacks close contacts between both chromosomal arms (e.g., in 38 Escherichia coli10 and V. cholerae chromosome I9). The cytological studies showed that 39 chromosome organisation may alter depending on the growth phase or developmental 40 stage1,11,12,14. While the most prominent change in bacterial chromosome organization was 41 observed during extensive DNA compaction that occurs during sporulation of Bacillus spp13 42 and Streptomyces spps, the details of chromosome arrangement during this process have 43 not been elucidated. 44 The folding of bacterial genomes requires a strictly controlled interplay between 45 several cellular factors that encompass: macromolecular crowding, transcription and the 46 activities of dedicated DNA-organizing proteins - topoisomerases, condensins and nucleoid- 47 associated proteins (NAPs)10,15,16. By nonspecific DNA binding, NAPs induce its bending, 48 bridging or wrapping to govern the local organization and global compaction of bacterial 49 chromosomes1,10,16. Since the subcellular repertoire of NAPs is dependent on growth 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.403915; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 50 conditions, changes in their supply account for the fluctuation of chromosome compaction 51 in response to environmental clues. While, the repertoire of NAPs also varies between 52 bacterial species, some of them, such as HU homologues, are widespread among 53 bacteria14,17,18. 54 In contrast to NAPs, condensins are the most conserved DNA-organizing proteins 55 identified in all kingdoms of life. In bacteria, the homologues of condensin are complexes of 56 SMCs or its E. coli functional analogue MukB, associated with their accessory proteins (ScpAB 57 or MukEF, respectively) 19,20,21. According to a recently proposed model, the primary activity 58 of SMC is the extrusion of large DNA loops22,23. SMC complexes were shown to move along 59 bacterial chromosomes from the centrally positioned origin of replication (oriC), leading to 60 gradual chromosome compaction and parallel alignment of both replichores5,6,24,25. 61 Importantly, although the alignment of two chromosomal arms was not detected in E. coli, 62 the MukBEF complex is still responsible for the long-range DNA interactions within 63 chromosomal domains10,26. 64 In B. subtilis, C. glutamicum, and C. crescentus, the loading of SMC in the vicinity of 65 the oriC was shown to be dependent on the segregation protein ParB4,27,28. Binding to a 66 number of parS sequences, ParB forms a large nucleoprotein complex (segrosome), that 67 interact with the partner protein ParA, ATPase non-specifically bound to nucleoid 29,30. ParB 68 interaction with ParA triggers its ATP hydrolysis and nucleoid release, generating a 69 concentration gradient that drives segrosome separation31–33. Segrosome formation was 70 shown to be a prerequisite for chromosomal arm alignment in the model Gram-positive 71 bacteria with circular chromosomes5,7,28. Importantly, there have been no reports on the 3D 72 organization of bacterial linear chromosomes to date. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.403915; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 73 The example bacteria with linear chromosomes are Streptomyces - Gram-positive 74 mycelial soil actinobacteria, which are highly valued producers of numerous antibiotics and 75 other clinically important metabolites. Notably, the production of these secondary 76 metabolites is closely correlated with Streptomyces unique and complex life cycle, which 77 encompasses vegetative growth and sporulation, as well as exploratory growth in some 78 species14,34,35. During the vegetative stage, Streptomyces grow as branched hyphae 79 composed of elongated cell compartments, each containing multiple copies of nonseparated 80 and largely uncondensed chromosomes14,36. Under stress conditions sporogenic hyphae 81 develop, and their rapid elongation is accompanied by extensive replication of 82 chromosomes37. When hyphae stop extending, the multiple chromosomes undergo massive 83 compaction and segregation to unigenomic spores14. These processes are accompanied by 84 multiple synchronized cell divisions initiated by the FtsZ protein, which assembles into a 85 ladder of regularly spaced Z-rings38,39. Chromosome segregation is driven by ParA and ParB 86 proteins, whereas nucleoid compaction is induced by concerted action of SMC and 87 sporulation-specific NAPs that include HupS - one of the two Streptomyces HU homologues, 88 structurally unique to actinobacteria due to the presence of histone-like domain. The 89 elimination of any of these proteins results in DNA decompaction in spores and affects 90 resistance to environmental factors40–44. Thus, while in unicellular bacteria chromosome 91 segregation occurs during ongoing replication, during Streptomyces sporulation, intensive 92 chromosome replication is temporarily separated from their compaction and segregation, 93 with the two latter processes occurring only during sporogenic cell division. 94 While the Streptomyces chromosomes are among the largest in bacterial kingdom, 95 the knowledge concerning their organization is scarce. Limited cytological evidence suggests 96 that during vegetative growth, both ends of the linear chromosome colocalize, and the oriC 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.09.403915; this version posted December 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 97 region of the apical chromosome is positioned at the tip-proximal edge of the nucleoid45,46. 98 The visualization of nucleoid and ParB-marked oriC regions in sporogenic hyphae showed 99 that oriCs are positioned centrally within pre-spore compartments, but did