Does RNA polymerase help drive chromosome segregation in bacteria?

Abstract

In contrast to eukaryotic cells, bacteria segregate their chromosomes without a conspicuous mitotic apparatus. Replication of bacterial chromosomes initiates bidirectionally from a single site (oriC), and visualization of the region of the chromosome containing oriC in living cells reveals that origins rapidly move apart toward opposite poles of the cell during the cell cycle. The motor that drives this poleward movement is unknown. An attractive candidate is RNA polymerase, which is a powerful and abundant molecular motor. If, as has been suggested for other macromolecular complexes, the movement of RNA polymerase is restricted in the cell, then transcription would translocate the DNA template, thereby providing the motive force to separate replicating chromosomes. A coordinated effect of many transcribing RNA polymerases could result from the widely conserved global bias of gene orientation away from oriC. By using fluorescence microscopy of living Bacillus subtilis cells, we demonstrate that an inhibitor of RNA polymerase acts to inhibit separation of newly duplicated DNAs near the origin of replication. We propose that the force exerted by RNA polymerase contributes to chromosome movement in bacteria, and that this force, coupled with the biased orientation of transcription units, helps to drive chromosome segregation.

Fig 1.

oriC movement in the presence of inhibitors of protein synthesis and transcription. (A) After a temperature shift from 45°C to 30°C, replication initiates at oriC (blue) and the tetO cassette at dacA (green dot) is replicated and the daughter copies undergo progressive separation. Note that the cartoon is a simplification of the in vivo situation where the replication forks colocalize in the replisome (as is illustrated in Fig. 3). (Ba) tetO foci in cells of strain JDB799 held at 45°C. (b) tetO foci in cells of JDB799 that had been downshifted to 30°C and incubated for 45 min in the absence of drug. (c) tetO foci in cells of JDB799 that had been downshifted to 30°C and incubated for 45 min in the presence of kanamycin (kan). (d) tetO foci in cells of JDB799 that had been downshifted to 30°C and incubated for 45 min in the presence of streptolydigin (str). (e) tetO foci in cells of the Str^R strain JDB800 that had been downshifted to 30° and incubated for 45 min in the presence of streptolydigin. (Bar = 1 μm.) (C) Interfocal distances between tetO foci in kanamycin-treated cells of JDB799 (green bars, n = 244), streptolydigin-treated cells of JDB799 (blue bars, n = 221), and streptolydigin-treated cells of the Str^R strain JDB800 (red bars, n = 117) as described above for B.

Fig 2.

LSTer movement in the presence of inhibitors of protein synthesis and transcription. (A) After a temperature shift from 45°C to 30°C in the presence of arginine hydroxamate (Arg-HX), replication initiates at oriC (blue) but then stalls at LSTer (orange) and RSTer (not shown). Removal of Arg-HX allows replication of the tetO cassette at hutM (green dot) and separation of the daughter copies. (Ba) tetO foci in downshifted cells of JDB792. (b) tetO foci in downshifted cells of JDB792 that been treated with Arg-HX; (c) tetO foci in downshifted- and Arg-HX-treated cells of JDB792 that were washed to remove Arg-HX and then incubated for 30 min. (d) tetO foci in downshifted- and Arg-HX-treated cells of JDB792 that were washed to remove Arg-HX and then incubated for 30 min with kanamycin (kan). (e) tetO foci in downshifted- and Arg-HX-treated cells of JDB792 that were washed to remove Arg-HX and then incubated for 30 min with streptolydigin (str). (f) tetO foci in downshifted- and Arg-HX-treated cells of the Str^R strain JDB871 that were washed to remove Arg-HX and then incubated for 30 min with streptolydigin. (Bar = 1 μm.) Listed below the images are the corresponding percentages of cells exhibiting doublets of tetO foci (defined as having an interfocal distance of <1 μm), based on the numbers of cells indicated.

Fig 3.

RNA polymerase-mediated DNA movement during chromosome segregation. The biased orientation of transcription units away from the origin (exaggerated in the cartoon) drives movement of newly synthesized daughter DNA molecules away from the replisome.