Fig. 1
Replication analysis in wild-type (WT) and pykA null cells growing exponentially in MC medium. A Schematic representation of CCM. Glyco: Glycolysis; Gluco: gluconeogenesis; PPP: pentose phosphate pathway; TCA: tricarboxylic acid cycle; O: overflow pathway; G3P: glyceraldehyde 3-phosphate; PEP: phosphoenolpyruvate; Gray arrows: carbon flux; Thick bars: CCM area genetically linked to replication genes [42, 44]. B Growth rate and pyruvate kinase activity. Cells were grown exponentially for more than 20 generations using successive dilutions. Growth was assessed by spectrophotometry (OD650nm, a typical experiment is shown) and pyruvate kinase activity was determined in crude extracts (mean from six independent experiments). C Ori/ter ratio: Ori/ter ratios were determined by qPCR using as template total DNA extracted from growing cells. Numbers in brackets stand for the number of independent measurements. The Mann-Whitney U test showed that the ratios in wild-type and ΔpykA cells are significantly different at p < .05 (Table S1). D DNA elongation: Parameters of DNA elongation were determined using a marker frequency analysis by qPCR. The nearly monotonous decrease of marker frequencies from the origin to the terminus showed that there is no pause site along the chromosome (a typical experiment is shown). Numbers in brackets refer to C period (mean and SD from at least 3 experiments) and mean fork speed. E Number of origins per cell: To determine the number of origins/cell, chloramphenicol was added to exponentially growing cells. The drug inhibits replication initiation and cell division but allows completion of ongoing rounds of replication. After 4 h of drug treatment (runout experiment), cells were analyzed by flow cytometry after DNA staining. Panels: typical runout DNA histograms with the % of cells containing 4 and 8 chromosomes. Numbers in brackets stand for the number of independent reiterations of the experiment. The number of origins/cell (mean and SD) is given below the strain name. See “Methods” for details