FIG 6 .
Timing of DNA replication initiation and determination of oriC numbers per cell. (A) Marker frequency analysis of oriC and terminus regions. (Left) Schematic representation of the chromosomal positions of the oriC- and terC-proximal marker genes used. (Right) oriC-to-terminus ratios of the WT strain grown under different conditions were determined by frequency analysis of markers cg0002 and cg1702 (see Fig. S6B for cg0018/cg2361 ratios). Cells were grown in BHI medium, BHI+Gluc, or MMI supplemented with glucose. Samples were taken in the exponential (exp, white boxes) and stationary (stat, gray boxes) growth phases. Results are shown as box plots with medians indicated as solid lines and whiskers of 1.5 times the interquartile range (n = 6); outliers are depicted as open circles. ANOVA yielded significant variation among growth phase (F1,30 = 28.00, P < 0.0001) and medium (F2,30 = 3.43, P < 0.05) conditions. Letters indicate significant differences between data sets determined by post-hoc Bonferroni analysis at P < 0.05. (B) Whole-genome sequencing. Genomic DNA of WT C. glutamicum grown in BHI or MMI medium supplemented with glucose was isolated in the exponential and stationary growth phases. Data were analyzed by Illumina MiSeq shotgun sequencing and mapped to the C. glutamicum ATCC 13032 genome sequence (GenBank accession no. BX927147.1). Data are displayed as the mean gene coverage of each 50-kb sliding window as a percentage of the total mean coverage per sample. Note that the RES167 strain used in this study lacks the phage island (cg1981-cg2034) and harbors an ISCg14-mediated 5-fold tandem amplification of the tus locus (peaks at approximately 750- and 2,000-kb positions); both loci were excluded from data analysis. Stable replication progression is evidenced by the frequency of genes between the oriC (located at 0 kb) and terminus regions (at approximately 1.6 Mb). (C) Numbers of chromosomes per cell determined by flow cytometry after replication runout in BHI medium, BHI+Gluc, or MMI medium supplemented with glucose. Depending on the growth conditions, between 2 and 12 chromosomes were detected.