Genome-wide coorientation of replication and transcription reduces adverse effects on replication in Bacillus subtilis

Abstract

In many bacteria, there is a strong bias for genes to be encoded on the leading strand of DNA, resulting in coorientation of replication and transcription. In Bacillus subtilis, transcription of the majority of genes (75%) is cooriented with replication. By using genome-wide profiling of replication with DNA microarrays, we found that this coorientation bias reduces adverse effects of transcription on replication. We found that in wild-type cells, transcription did not appear to affect the rate of replication elongation. However, in mutants with reversed transcription bias for an extended region of the chromosome, replication elongation was slower. This reduced replication rate depended on transcription and was limited to the region in which the directions of replication and transcription are opposed. These results support the hypothesis that the strong bias to coorient transcription and replication is due to selective pressure for processive, efficient, and accurate replication.

Fig. 1.

Organization of replication and transcription in wild-type (A, oriC at 0°) and mutants (B, oriC at 257° and C, oriC at 94°). oriC, origin of replication; ter, terminus of replication; big gray arrows, replication in regions where the bulk of transcription is codirectional; big black arrows, replication in regions where the bulk of transcription is head-on; small arrows, symbolic representation of orientation of bulk transcription units; arrowheads, ribosomal RNA operons with the indicated directionality (only rrnB is labeled). There are 10 rrn operons: rrnO at 1°; rrnA at 3°; rrnJ and rrnW at 7–8°; rrnH, rrnI, and rrnG at 13–15°; rrnE at 54°; rrnD at 80°; and rrnB at 271°. For clarity, only some are indicated.

Fig. 2.

Replication elongation was not affected by transcription in cells with the wild-type chromosomal organization. KPL151 (oriC at 0°; dnaB134ts) was grown in minimal glucose medium at 30°C, shifted to 45°C for 30 min, then back to 30°C to allow initiation of replication. Relative gene dosage was determined by cohybridization of replicating and preinitiation reference DNA to the microarrays, and plotting the ratio on the y axis (log₂) against the corresponding gene positions on the x axis. 0°/360° is located in the middle; 172° (the terminus) is to both the left and the right. Each data point represents dosage of a single ORF. Lines are drawn for the rolling averages. (A) DNA profile 20 min after initiation of replication. (B and C) DNA profiles 40 min after initiation of replication without (B) or with (C) rifampicin (0.25 mg/ml; to inhibit transcription) added 20 min after initiation of replication. The slight increase in gene dosage near the terminus is probably due to incomplete replication in the preinitiation reference sample. (D) Overlay of the averaged genomic profiles from A to C. A, gray; B, blue; C, orange.

Fig. 3.

Replication elongation is slowed by head-on transcription. Replication was monitored by DNA microarrays and the rolling average of data points was plotted as in Fig. 2. dnaBts strains KPL151 (oriC at 0°) (A–C) and JDW207 (oriC at 257°) (D–F) were grown in minimal fumarate medium at 30°C, shifted to 45°C for 60 min, then back to 30°C to allow initiation of replication. The efficiency of replication initiation was typically lower than that of cells grown in glucose (Fig. 2), perhaps due to the decreased growth rate in fumarate. We have not explored this difference. (A and D) Microarray profiles 20 min after initiation of replication. Vertical bars indicate the position of oriC. (B and E) Microarray profiles 20 min after initiation of replication, with rifampicin (0.25 mg/ml) added 4 min after temperature shift-down. (C and F) Overlay of microarray profiles without (black; −rif) and with (gray; +rif) rifampicin.

Fig. 4.

DNA content in asynchronously growing cells. Cells were grown in minimal fumarate medium at 37°C (A and B) or 30°C (C). co, regions where replication and the bulk of transcription are cooriented; h, regions where replication and the bulk of transcription are head-on. The slopes of each segment of the plots are indicated (±2 × standard error). 0°, oriC, ter, and rrnB are indicated. The strain with oriC at 94° grows much more poorly than the others and there is much more scatter in the data. (A) Wild-type cells with oriC at 0° (JH642). (B) 257°::oriC ΔoriC-L (MMB703). (C) 94°::oriC ΔoriC-L dnaB134ts (JDW258).

Fig. 5.

DNA content in asynchronously growing cells whose replication initiates from oriN. Cells were grown in minimal fumarate medium at 37°C. Notations are as in Fig. 4. These strains have a deviation in sequence from approximately ypjG (201°) to approximately hepT (204°), causing a drop in the signals for most genes in this region relative to the reference strain JH642 (27). (A) 359°::oriN ΔoriC-S (MMB208). (B) 257°::oriN ΔoriC-S (MMB700).