Supporting Information

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The Principle of Marker Frequency (MF) Analysis. In an asynchronously growing population, cells in all stages of chromosome replication are present. In some, replication has not initiated, whereas in others, the replication forks have progressed to different positions around the chromosome, from the origin to the terminus (Fig. 5A). A chromosome region (marker) located close to a replication origin will be present at a higher average copy number in the population than a region near a termination site, because the origin region always is replicated first. In MF analysis, the copy number of different chromosome markers is plotted against the marker positions on the chromosome, and the resulting graph is used to identify replication origin and termination sites (Fig. 5B). Through the use of whole-genome DNA microarrays, several thousand markers may be included in the analysis. High-quality arrays with a high degree of gene coverage allow very small differences in marker ratios to be detected, which results in a high level of sensitivity and resolution.

Fig. 5. The principle of MF analysis. (A) Circular chromosome divided into 24 markers. The chromosome is assumed to replicate bidirectionally from a single origin, and the markers in the left (red) and right (blue) chromosome arms are shaded darker the closer they are to the terminus. Three pairs of replication forks (bidirectional arrows) from different cells, assumed to have reached different positions along the chromosome at the time of sampling, are indicated. (B) MF distribution in an asynchronously growing population, showing the successively lower relative copy number of the markers the further the distance from the origin.

Fig. 6. Cell size and DNA content distributions from exponentially growing (rows 1 and 3) and stationary phase (rows 2 and 4) cultures of (A) S. acidocaldarius and (B) S. solfataricus. The flow cytometry analyses were performed on separate occasions with different instrument settings, and the peak positions in the S. acidocaldarius and S. solfataricus distributions are therefore not directly comparable. The position of the two-chromosome peak is shifted toward the left in stationary-phase samples, as compared to actively growing cells. This is due to changes in chromatin organization as the cells enter the resting stage that, in turn, affect the DNA staining properties (1).

1. Bernander, R. & Poplawski, A. (1997) J. Bacteriol. 179, 4963-4969.