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

Bacterial strains, plasmids, and oligonucleotides Primers, plasmids, and strains used in this study are listed in Supplementary Data 1 and 2 . For protein–protein interaction screens, genes of interest were amplified via PCR, digested with respective enzymes, and ligated into bacterial two-hybrid vectors 71 . E. coli DH5α were utilized for plasmid cloning. Genes divIVA and parB / parB R175A were amplified using primer pairs DivIVA-XbaI-F/DivIVA-BamHI-R and ParB-XbaI-F/ParB-BamHI-R from genomic DNA or pK19mobsacB-ParBR175A, and resulting fragments were digested with XbaI/BamHI. For amplification of scpA , scpB , mksE , mksF , and mksG , primer pairs ScpA-XbaI-F/ScpA-XmaI-R, ScpB-XbaI-F/ScpB-XmaI-R, MksE-XbaI-F/MksE-XmaI-R, MksF-XbaI-F/MksF-XmaI-R, and MksG-XbaI-F/MksG-XmaI-R were utilized, followed by restriction digests with XmaI/XbaI. Primer pairs SMC-XbaI-F/SMC-KpnI-R and MksB-XmaI-F/MksB-KpnI-R were used for PCR amplification of genes smc and mksB , which were subsequently digested with XbaI/KpnI or XmaI/KpnI. To increase the distance of XmaI and KpnI restriction sites, a short sequence was inserted in between these sites by overhang PCRs using pUT18C-mcs-HindIII-F, pUT18-mcs-PvuII-F, pKNT25-mcs-NheI-F, or pKT25-mcs-HindIII-F in combination with pUT18(C)/pK(N)T25-mcs-KpnI-R for plasmids pUT18C, pUT18, pKT25, and pKNT25, respectively. Resulting fragments and corresponding vectors were digested with HindIII/KpnI, PvuII/KpnI, or NheI/KpnI and subsequently ligated, resulting in plasmids pUT18_mcs, pUT18C_mcs, pKNT25_mcs, and pKT25_mcs. All digested gene fragments mentioned above were ligated into pUT18, pUT18C, pKNT25, and pKT25 or pUT18_mcs, pUT18C_mcs, pKNT25_mcs, and pKT25_mcs, respectively. Derivatives of the suicide integration vector pK19mobsacB were used for clean allelic replacements in C. glutamicum , containing the modified genomic region of interest including its 500 bp up- and downstream homologous flanking sequences. Plasmid cloning was performed using E. coli DH5α. To construct pK19mobsacB-Δsmc 500 bp upstream and downstream of smc were PCR amplified using primer pairs Δsmc-BamHI-up-F/Δsmc-up-R and Δsmc-D-F/ Δsmc-EcoRI-D-R, respectively. Both fragments served as templates in an overhang PCR, yielding a 1000 bp fragment, which was digested with BamHI and EcoRI and subsequently ligated into pK19mobsacB. pK19mobsacB-ΔSMCload was constructed accordingly, using primer pairs ΔSMCload-HindIII-up-F/ΔSMCload-up-R and ΔSMCload-D-F/ΔSMCload-SalI-D-R, and HindIII in combination with SalI for restriction digest. For construction of pK19mobsacB-ΔmksB up-/and downstream regions of mksB were PCR amplified using primers ΔmksB-HindIII-up-F/ΔmksB-PstI-up-R and ΔmksB-PstI-D-F/ΔmksB-XbaI-D-R. Resulting 500 bp fragments were digested with HindII/ PstI and PstI/ XbaI and consecutively ligated into pK19mobsacB. Fluorescent C-terminal fusions of ParB protein with PAmCherry or mNeongreen were obtained by utilizing plasmids pK19mobsacB-parB-mNeonGreen and pK19mobsacB-parB-PAmCherry. To this end, the eYFP sequence of plasmid pK19mobsacB-parB-eYFP 41 was replaced by respective fluorophore sequences, which were amplified via PCR using PAmCherry-SalI-F/PAmCherry-XbaI-R primers and digested with SalI and XbaI. For fluorescent versions of SMC and MksB proteins plasmids pK19mobsacB-smc-mCherry, pK19mobsacB-mksB-mCherry and pK19mobsacB-mksB-PAmCherry were constructed. At first, 500 bp regions up- and downstream of the 3′-end of smc or mksB were amplified using primer pairs SMC-HindIII-up-F/SMC-SphI-up-R and SMC-BamHI-D-F/SMC-EcoRI-D-R or MksB-HindIII-up-F/MksB-Sph-up-R and MksB-BamHI-D-F/MksB-EcoRI-D-R. Fluorophore sequences were amplified with primers PAmCherry-SalI-F/mCherry-BamHI-R for SMC-mCherry and MksB-PAmCherry fusion or with primers PAmCherry-SalI-F/mCherry-XbaI-R for the MksB-mCherry fusion construct. Up- and downstream fragments were digested via HindIII/SphI and BamHI/EcoRI, whereas enzymes SalI/BamHI or SalI/XbaI were utilized for restriction digest of fluorophore sequences fused to smc or mksB , respectively. Fragments were subsequently ligated into the pK19mobsacB plasmid, starting with the corresponding downstream region, followed by the fluorophore sequence and finally the upstream region. To place part of a putative SMC binding site upstream of the parS cluster into an intergenic region 3′ of cg0177 (Supplementary Fig. 10 ), genomic sequences 500 bp up- and downstream of the insertion site were amplified using primer pairs cg0177-HindIII-up-F/cg0177-SalI-up-R and cg0177-XmaI-D-F/cg0177-EcoRI-D-R; part of the genomic SMC binding site (1.1 Kb) was amplified using primers SMCload-SalI-F and SMCload-XmaI-R. Resulting fragments were digested with HindII/SalI, SalI/XmaI, and XmaI/EcoRI, and consecutively ligated into pK19mobsacB, obtaining the plasmid pK19mobsacB-SMCload-cg0177. Plasmid pK19mobsacB-SMCload-r was constructed for the partial replacement of the SMC binding site (1.1 Kb) with a B. subtilis genomic region of identical size. For amplification of up- and downstream 500 bp regions, primer pairs ΔSMCload-HindIII-up-F/SMCload-SphI-up-R and SMCload-PstI-D-F/ΔSMCload-SalI-D-R were utilized, whereas the replacement sequence was amplified from B. subtilis genomic DNA via SMCloadr-SphI-F/SMCloadr-PstI-R. After digestion with enzymes HindIII/SphI, PstI/SalI, or SphI/PstI, fragments were successively ligated into pK19mobsacB. Further, all parS sites were mutated comprising new XmaI or SalI restriction sites (see Supplementary Fig. 2 ). For mutation of parS1 primer pairs, parS1mut-HindIII-up-F/parS1mut-XmaI-up-R and parS1mut-XmaI-D-F/parS1mut-EcoRI-D-R were utilized to mutate parS1 and to amplify sequences 500 bp up- and downstream of parS1 . Restriction digest was performed with both fragments using HindIII/XmaI or XmaI/EcoRI, respectively. Subsequent ligation into pK19mobsacB yielded plasmid pK19mobsacB-parS1mut. To mutate parS2 , parS3 , parS4 , parS7 , and parS8 , plasmid construction was performed in the same way using primers parS2mut-HindIII-up-F/parS2mut-XmaI-up-R and parS2mut-XmaI-D-F/parS2mut-EcoRI-D-R, parS3mut-HindIII-up-F/parS3mut-XmaI-up-R and parS3mut-XmaI-D-F/parS3mut-EcoRI-D-R, parS4mut-HindIII-up-F/parS4mut-XmaI-up-R and parS4mut-XmaI-D-F/parS4mut-EcoRI-D-R, parS7mut-HindIII-up-F/parS7mut-XmaI-up-R and parS7mut-XmaI-D-F/parS7mut-EcoRI-D-R, or parS8mut-HindIII-up-F/parS8mut-XmaI-up-R and parS8mut-XmaI-D-F/parS8mut-EcoRI-D-R for amplification of fragments up- and downstream of the respective parS site. Matching fragments were each digested and ligated into pK19mobsacB, as exemplified for pK19mobsacB-parS1mut construction, resulting in plasmids pK19mobsacB-parS2mut, pK19mobsacB-parS3mut, pK19mobsacB-parS4mut, pK19mobsacB-parS7mut, and pK19mobsacB-parS8mut. As parS5 and parS6 , as well as parS9 and parS10 , are localized in close proximity on the genome (