Anucleate cell formation in muk mutant cells. Cells were grown in LB broth at 23°C. (A) DBP924 wild-type. (B) DPB923, topA10. (C) CC4208, ΔmukB∷kan. (D) CC4207, topA10 ΔmukB∷kan. (E) CC4218, mukE∷kan. (F) CC4217, topA10 mukE∷kan mutant cells. White arrows highlight some anucleate cells. Black arrows highlight filamentous cells with “DNA-free bays.”

Temperature-resistance of topA10 ΔmukB∷kan is reversed by coumermycin. Cells were streaked out on prewarmed plates containing DMSO (controls) or coumermycin at 1 μg/ml at the indicated temperatures. 30°C plates were incubated for 24 hours, and 23°C plates were incubated for 48 hours. Strains were DPB924, wild type (a), DPB923, topA10 (b), CC4207, topA10 ΔmukB∷kan (c), and CC4208, ΔmukB∷kan (d).

Directed condensation model for E. coli chromosome segregation. The model is based on that of Lemon and Grossman (43). (A) The nucleoid is supercoiled and further condensed to some unspecified compact form (represented as stacked loops). During initiation, the origin (circle) is attached to a site (triangles) at the cell center. (B) The new origins (circles) attach to new sites at the 1/4 and 3/4 positions. The DNA replication forks remain attached to the central site. (C) Newly replicated DNA (black lines) is supercoiled by DNA gyrase and remodeled into the compact form by the action of the Muk proteins. As the origins are tethered in opposite halves of the cell, the nascent nucleoids form as separate masses. (D) On completion of replication, the daughter nucleoids are sufficiently separated to allow cell division. (E) In the absence of the Muk system, the supercoiled loops of the completed nucleoids are disorganized and the resulting masses fail to separate. (F) The additional negative supercoiling caused by unrestrained DNA gyrase activity in topA mutant cells increases superhelical density and allows for a more reliable separation.

Suppression of muk mutant temperature sensitivity. Colonies grown at 23°C were streaked onto LB-agar plates at the indicated temperatures and were grown for 24 hours. Strain numbers starting with “WT” and proceeding clockwise are DPB924, CC4211, CC4213, CC4207, DPB923, CC4212, CC4214, and CC4208. CC4211, CC4212, CC4213, and CC4214 contain the plasmid pAX804 that produces wild-type MukB protein. At 30°C, the wild-type and topA10 and topA10 ΔmukB∷kan mutant strains had generation times of 35, 34, and 43 min, respectively, whereas the ΔmukB∷kan strain failed to grow.

Supercoiling of plasmid pBR322 isolated from wild-type and topA10 mutant cells. A 1% agarose gel containing 5 μg/ml chloroquine was used to separate topoisomers, as outlined in Materials and Methods. More highly supercoiled species migrate further than less supercoiled species. The change in the topoisomer ladders seen when 15 μg/ml chloroquine was used (data not shown) indicates that all of the topoisomers produced from the topA and topA muk mutant cells are negatively supercoiled species in this gel. Likewise, most if not all of the bands produced by the top⁺ cells are negatively supercoiled. Thus, the topA suppressed cells produce plasmid DNA that contains on average nine or more additional negative supercoils relative to their top⁺ parents. DNA was isolated from pBR322 transformants of DPB924, DPB923, CC4207, CC4208, CC4215, and CC4216, from left to right, respectively.

Coumermycin reverses suppression of ΔmukB∷kan by topA10. Cells were grown with 1 μg/ml coumermycin for 6 hours and were 4′,6-diamidino-2-phenylindole-stained for microscopy. (A) DPB923, topA10. (B) CC4207, topA10 ΔmukB∷kan. White arrows highlight some examples of anucleate cells.