Chapter 6 : Domain Behavior and Supercoil Dynamics in Bacterial Chromosomes

Image of an E. coli chromosome. Exponential cells treated with lysozyme and Brij 58 in the presence of 1MNaCl were layered onto a 10 to 30% sucrose gradient and subjected to centrifugation. An aliquot of the fraction with highest DNA content was applied to a glow-discharged carbon-coated grid, stained with uranyl acetate, rotary shadowed, and photographed using a JEOL 1200EXII electron microscope. Micrograph by Christine Hardy, University of California at Berkeley.

Supercoil behavior in chromosomes. (A) DNA supercoils in eukaryotes are created by wrapping DNA on the surface of the highly conserved histone octamer. Eubacteria introduce supercoils enzymatically with gyrase, which causes DNA to adopt the interwound or plectonemic supercoiled conformation. Proteins like HU and H-NS restrain half of bacterial supercoiling. (B) A 114-bp res site includes three subsites, I, II, and III, that each bind a resolvase dimer. Recombination occurs within the res I site. A three-node synapse precisely juxtaposes two res I sites for catalytic exchange within a protein-DNA recombination complex. Slithering (C) and branching (D) are movements that allow synapse of two res sites.

Resolution analysis in a 100-kb interval using two resolvase enzymes, the WT Res (top curve) with a half-life of more than 1 h or the modified Res-SsrA protein (bottom curve), which is a substrate for the ClpXP protease and has a 5-min half-life in exponential cultures of E. coli or Salmonella.

Topological problems at the terminus. (A) As replication forks converge, positive supercoiling builds up to slow fork progression. (B) In WT strains, gyrase can remove the topological barrier, but gyrase mutants may fail to complete DNA synthesis due to fork regression (C) or replication-mediated linearization of one chromosome (D and E). An R loop impedes further transcription (F), exposes the displaced strand of DNA to chemical and enzymatic attack (G), and creates a barrier to replication fork progression (H).

Resolution efficiency in a 14-kb segment of the S. enterica serovar Typhimurium chromosome changes dramatically depending on the state of lacZ expression. (A) Genetic map of a 14-kb deletion interval, which leads to a deleted circle containing a Tet-regulated copy of lacZ. (B) Effect of transcription on site-specific resolution. Cultures of bacteria harboring the genetic interval shown in panel A were grown in log phase with continuous presence (open square) or absence (open circle) of 5 µg/ml CLT. At time 0, CLT was added (solid squares) or washed out (solid circles) of the medium, and cells were exposed to a 10-min expression period of a resolvase with a 5-min half-life. Recombination efficiency is plotted against the time of transcription induction or repression (see reference 32 ). (C) Model for transcription-induced domains in bacterial chromosomes. Addition of CLT causes RNA polymerase binding at promoters pR and pA and transcription of the tetR and lacZ genes. Persistent transcription induces formation of a domain in which an unknown protein(s) stabilizes a loop that isolates DNA associated with the transcribing RNA polymerases (ovals) from the rest of the genome. Inclusion of the res site adjacent to the transcription terminator (rrnB) inhibits recombination with the res site near the kan gene to the left.

Alternative explanations for RNA and membrane involvement in domain behavior. (A) RNA transcription produces novel RNA species that organize specific domains within the bacterial chromosome. Lysis releases a structure, and digestion with RNase unfolds the nucleoid by digesting "organizational" RNA. (B) Cotranscriptional translation of integral membrane proteins transiently handcuffs DNA to the membrane through a link involving RNA polymerase, mRNA, ribosomes, and nascent membrane protein. Lysis causes the membrane-attached complexes to collapse to the center, forming an inverted structure. Digestion by RNase releases DNA from the membrane, allowing nucleoid expansion.

Hypothetical use of transcription and membrane attachment to reform nucleoids after DNA replication. (A) Chromosomal replication of the parental nucleoid (large gray oval) is initiated in a DNA factory (open square) positioned at mid cell. A replication fork initiated at OriC loops out one continuously synthesized strand leftward (black) and a complementary strand rightward that is discontinuously synthesized as Okazaki fragments (gray). After DNA passes through a SeqA zone (aggregate), expression of genes encoding integral membrane proteins (black bars) or proteins exported to the periplasm and outer membrane (gray triangles) handcuff DNA to the membrane-forming domains. (B) A second replisome extrudes the second replichore rightward and leftward. The template chromosome diminishes as the replisomes approach the terminus. (C) Two new nucleoids remain intermittently and dynamically linked to the membrane.

Examples of supercoil-assisted protein assembly and disassembly. (A) Six dimers of the Res protein (triangles) form a synapse that contains three negative supercoils. The time required to form this alignment on a supercoiled 4-kb plasmid is about 1 s. (B) Phage Mu transposition requires interactions of three sites in phage DNA. These sites include the left end of the virus (attL), the right end of the virus (attR), and the internal activation sequence (IAS). The plectosome or transposition intermediate contains a tetramer of MuA protein and five plectonemic crossings of DNA. (C) Mu transposition immunity involves interactions between a Mu transpososome and a DNA complex of the target selector, MuB protein. MuB binding to DNA is highly cooperative and requires an ATP. Interactions between MuA and MuB stimulate hydrolysis and a conformation change that displaces MuB from DNA.