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. 2019 Jul 17;116(31):15661–15670. doi: 10.1073/pnas.1906119116

Fig. 6.

Fig. 6.

Polar sequestration enhances cell cycle robustness by maintaining the hemimethylated (HM) state of newly synthesized chromosome. (A) Cells expressing single copy or multicopy of eyfp-ccrM under the control of Pxyl were grown in M2G + 0.3% xylose to exponential phase and imaged by phase contrast and epifluorescence microscopy. Expression of a higher amount of eYFP-CcrM impairs its polar localization. (B) Spot dilutions of strains in A. Wild-type C. crescentus NA1000 and strains harboring a single copy of Pxyl-eyfp-ccrM (S), multicopies of Pxyl-eyfp-ccrM (M), single copy of empty vector (pXYFPN2), and multicopies of empty vector (pBMCS2) were diluted to an OD600 of 0.03, serially diluted, and spotted onto the PYE agar plate supplied with either 0.2% glucose or 0.3% xylose and incubated at 30 °C for 2 d before photography. Induced expression of multicopy eyfp-ccrM resulted in impaired growth. (C) Schematic of the Caulobacter chromosome showing position 1 and position 2 where the CYFP reporter cassettes are inserted. Chromosome methylation states are shown as a function of DNA replication and cell cycle progression. Fully methylated (FM, solid line) state position 1 is progressively converted to a HM (dotted line) state following the SW-to-ST cell transition and the initiation of DNA replication. Fully methylated position 2 is converted to a hemimethylated state at the end of DNA replication in the predivisional cell (PD). (D) Relative CYFP expressions in synchronized stalked cells treated with 0.2% glucose (eYFP-CcrM repression) or 0.3% xylose (eYFP-CcrM induction). The CYFP reporter cassettes driven by either PdnaA or PctrA1 were inserted at 2 different positions on the chromosome of strains harboring single copy of Pxyl-eyfp-ccrM or multicopies of Pxyl-eyfp-ccrM. The qPCR assays were performed using primers targeting the transcribed promotor region of PdnaA or PctrA1 and CYFP linker, so that the assayed transcriptional levels represent the expression of CYFP gene, not eYFP-CcrM. Results suggest that eYFP-CcrM polar sequestration prevents remethylation of daughter chromosome in the strain constitutively expressing eYFP-CcrM. ****P < 0.0001, by unpaired Student’s t test.