Rates of gyrase supercoiling and transcription elongation control supercoil density in a bacterial chromosome

Abstract

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.

Figure 1. Mechanism of the γδ resolution reaction in vitro and in vivo showing how reaction efficiency correlates with (−) superhelix density.

A. Recombination in the Tn3/γδ resolvase system requires a pair of 114 bp sites (Res) that include three binding sites for a dimer of the resolvase. The sites are I (blue), II (red), and III (yellow.) Supercoiling is required for the formation of a synapse in which two directly repeated Res sites entrap 3 negative crossing DNA nodes. Only resolvase dimers bound to Res site I, shown as blue boxes or blue ovals for different Res sites, can catalyze strand exchange. B. Movement of the interwound DNA strands promotes formation of the three-node tangle in A that occurs by reversible branching and slithering. Recombination results in an irreversible strand exchange that leaves two molecules linked as single supercoiled catenanes. C. The dependence of (−) supercoiling for plasmid recombination in vitro is shown by the scale on the left . The inferred diffusible supercoil density for recombination of a 9 kb interval in the Salmonella chromosome in vivo is shown on the right .

Figure 2. Resolution efficiencies in the Salmonella chromosome decline in strains carrying TS mutations in gyrase and Topo IV, even when cells are grown at permissive temperature (30°).

Recombination reactions at 8 locations around the Salmonella chromosome was studied in 32 strains described in Table 1. The experiment covers the 6 macrodomains of E. coli, shown as color coded arcs superimposed on the Salmonella map: green, Ori domain; black, Right Unstructured domain; red, Right domain; purple, Ter domain with black hatches showing matS sites; blue, Left domain; and black, Left Unstructured domain . The direction of replication fork movement in replichore 1 (brown) or 2 (pink) is shown by arrows outside the circle. Each strain had a 9 kb Lac-Gn supercoil sensor inserted between consecutive genes, plus a plasmid that contains a thermo-inducible γδ resolvase with a 30 min half life (Materials and Methods). Recombination data and estimated values of apparent diffusible supercoiling for each experiment are reported in Table 1.

Figure 3. Interrupting transcription causes a dramatic rebound in resolution for strains carrying the GyrB1820 gyrase.

Recombination efficiencies of supercoil sensors at 8 positions are shown for WT (red) and gyrB1820 ^TS mutants tested without Rif (black). The purple numbers show recombination rates after rifampicin was added to cultures immediately following the 10 min induction of resolvase and rifampicin was subsequently washed out of cells 30 min later.

Figure 4. An RpoC mutant that slows transcription and mimics the stringent response in the absence of ppGpp causes global increases in resolution efficiency in the Salmonella chromosome.

Resolution assays for Lac-Gn modules around the Salmonella chromosome are shown for WT (red) and the rpoC mutant (black).

Figure 5. RNAP elongation rates at 8 chromosomal loci in WT (red) and gyrB1820 mutant strains (black) are significantly reduced by the GyrB1820 mutation.

A. 20 ml cultures growing in minimal (AB) medium with glucose were grown at 37° to an OD A₆₀₀ = 0.20. Three 0.5 ml aliquots were taken, added to ice-cold ZS buffer and saved for a base line reading. IPTG was added to a concentration 1.5 mM at the time point indicated by the arrow, and samples were removed at 10 sec intervals. The chromogenic substrate ONPG was added to each culture in timed assays that extended for 1.5–3 h, depending on the activity level. B. The mRNA elongation rate was calculated by dividing the 3072 nt lacZ mRNA by the lag time to linear increase in β-Gal, giving the rate in units of mRNA nt/sec.