Summary: During a normal cell cycle, chromosomes are exposed to many biochemical reactions that require specific types of DNA movement. Separation forces move replicated chromosomes into separate sister cell compartments during cell division, and the contemporaneous acts of DNA replication, RNA transcription and cotranscriptional translation of membrane proteins cause specific regions of DNA to twist, writhe and expand or contract. Recent experiments indicate that a dynamic and stochastic mechanism creates supercoil DNA domains soon after DNA replication. Domain structure is subsequently reorganized by RNA transcription. Examples of transcription‐dependent chromosome remodelling are also emerging from eukaryotic cell systems.

Gyrase catalyzes negative supercoiling of DNA in an ATP-dependent reaction that helps condense bacterial chromosomes into a compact interwound “nucleoid.” The supercoil density (σ) of prokaryotic DNA occurs in two forms. Diffusible supercoil density (σ_D) moves freely around the chromosome in 10 kb domains, and constrained supercoil density (σ_C) results from binding abundant proteins that bend, loop, or unwind DNA at many sites. Diffusible and constrained supercoils contribute roughly equally to the total in vivo negative supercoil density of WT cells, so σ = σ_C+σ_D. Unexpectedly, Escherichia coli chromosomes have a 15% higher level of σ compared to Salmonella enterica. To decipher critical mechanisms that can change diffusible supercoil density of chromosomes, we analyzed strains of Salmonella using a 9 kb “supercoil sensor” inserted at ten positions around the genome. The sensor contains a complete Lac operon flanked by directly repeated resolvase binding sites, and the sensor can monitor both supercoil density and transcription elongation rates in WT and mutant strains. RNA transcription caused (−) supercoiling to increase upstream and decrease downstream of highly expressed genes. Excess upstream supercoiling was relaxed by Topo I, and gyrase replenished downstream supercoil losses to maintain an equilibrium state. Strains with TS gyrase mutations growing at permissive temperature exhibited significant supercoil losses varying from 30% of WT levels to a total loss of σ_D at most chromosome locations. Supercoil losses were influenced by transcription because addition of rifampicin (Rif) caused supercoil density to rebound throughout the chromosome. Gyrase mutants that caused dramatic supercoil losses also reduced the transcription elongation rates throughout the genome. The observed link between RNA polymerase elongation speed and gyrase turnover suggests that bacteria with fast growth rates may generate higher supercoil densities than slow growing species. A 9-kb module called the “supercoil sensor” was used to measure supercoil density at 10 positions in the 4.8-Mb Salmonella Typhimurium chromosome. The sensor includes a Lac operon flanked by a pair of directly repeated DNA–binding sites for the γδ recombinase. Measurements of chromosomal supercoil levels and the RNA polymerase elongation rates were made at various positions within the 6 potential macrodomains of the chromosome. Transcription and gyrase catalytic rates were mechanistically linked. Gyrase mutants with impaired activity caused the loss of from 30% to >95% of the diffusible supercoiling throughout most of the chromosome, while treatment with rifampicin that temporarily blocked transcription restored most of the lost supercoils in gyrase mutants. A gyrase defect also caused transcription elongation rates to decrease across the chromosome, and a mutation that reduced RNA polymerase efficiency increased average chromosome supercoiling levels. A model in which topoisomerases act close to highly transcribed operons to equilibrate the supercoil flux generated by transcription suggests that matched rates of gyrase turnover and transcription elongation speed determine the average supercoil density in bacterial chromosomes.

The bacterial chromosome is organized into multiple independent domains, each capable of constraining the plectonemic negative supercoil energy introduced by DNA gyrase. Different experimental approaches have estimated the number of domains to be between 40 and 150. The site-specific resolution systems of closely related transposons Tn3 and γδ are valuable tools for measuring supercoil diffusion and analysing bacterial chromosome dynamics in vivo. Once made, the wild-type resolvase persists in cells for time periods greater than the cell doubling time. To examine chromosome dynamics over shorter time frames that are more closely tuned to processes like inducible transcription, we constructed a set of resolvases with cellular half-lives ranging from less than 5 min to 30 min. Analysing chromosomes on different time scales shows domain structure to be dynamic. Rather than the 150 domains detected with the Tn3 resolvase, wild-type cells measured over a 10 min time span have more than 400 domains per genome equivalent, and some gyrase mutants exceed 1000.

Escherichia coli and Salmonella enterica serovar Typhimurium share high degrees of DNA and amino acid identity for 65% of the homologous genes shared by the two genomes. Yet, there are different phenotypes for null mutants in several genes that contribute to DNA condensation and nucleoid formation. The mutant R436-S form of the GyrB protein has a temperature-sensitive phenotype in Salmonella , showing disruption of supercoiling near the terminus and replicon failure at 42°C. But this mutation in E. coli is lethal at the permissive temperature. A unifying hypothesis for why the same mutation in highly conserved homologous genes of different species leads to different physiologies focuses on homeotic supercoil control. During rapid growth in mid-log phase, E. coli generates 15% more negative supercoils in pBR322 DNA than Salmonella . Differences in compaction and torsional strain on chromosomal DNA explain a complex set of single-gene phenotypes and provide insight into how supercoiling may modulate epigenetic effects on chromosome structure and function and on prophage behavior in vivo.

Summary: Bacteria differ from eukaryotes by having the enzyme DNA gyrase, which catalyses the ATP‐dependent negative supercoiling of DNA. Negative supercoils are essential for condensing chromosomes into an interwound (plectonemic) and branched structure known as the nucleoid. Topo‐1 removes excess supercoiling in an ATP‐independent reaction and works with gyrase to establish a topological equilibrium where supercoils move within 10 kb domains bounded by stochastic barriers along the sequence. However, transcription changes the stochastic pattern by generating supercoil diffusion barriers near the sites of gene expression. Using supercoil‐dependent Tn3 and γδ resolution assays, we studied DNA topology upstream, downstream and across highly transcribed operons. Whenever two Res sites flanked efficiently transcribed genes, resolution was inhibited and the loss in recombination efficiency was proportional to transcription level. Ribosomal RNA operons have the highest transcription rates, and resolution assays at the rrnG and rrnH operons showed inhibitory levels 40–100 times those measured in low‐transcription zones. Yet, immediately upstream and downstream of RNA polymerase (RNAP) initiation and termination sites, supercoiling characteristics were similar to poorly transcribed zones. We present a model that explains why RNAP blocks plectonemic supercoil movement in the transcribed track and suggests how gyrase and TopA control upstream and downstream transcription‐driven supercoiling.

Transposable elements have influenced the genetic and physical composition of all modern organisms. Defining how different transposons select target sites is critical for understanding the biochemical mechanism of this type of recombination and the impact of mobile genes on chromosome structure and function. Phage Mu replicates in Gram-negative bacteria using an extremely efficient transposition reaction. Replicated copies are excised from the chromosome and packaged into virus particles. Each viral genome plus several hundred base pairs of host DNA covalently attached to the prophage right end is packed into a virion. To study Mu transposition preferences, we used DNA microarray technology to measure the abundance of >4,000 Escherichia coli genes in purified Mu phage DNA. Insertion hot- and cold-spot genes were found throughout the genome, reflecting >1,000-fold variation in utilization frequency. A moderate preference was observed for genes near the origin compared to terminus of replication. Large biases were found at hot and cold spots, which often include several consecutive genes. Efficient transcription of genes had a strong negative influence on transposition. Our results indicate that local chromosome structure is more important than DNA sequence in determining Mu target-site selection.