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 [22]. The inferred diffusible supercoil density for recombination of a 9 kb interval in the Salmonella chromosome in vivo is shown on the right [13].
Table 1.
Resolution measurements and apparent σD values of WT, and TS mutants of gyrase (gyrA, gyrB) and Top IV (parC, parE).
Table 2.
TS alleles of gyrase and Topo IV decrease diffusible chromosome supercoiling at Cs 85.
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 [41]. 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 gyrB1820TS 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.
Table 3.
Rifampicin induced recovery of chromosomal σD in gyrB1820 mutants.
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).
Table 4.
Impact of a 6 amino acid rpoC deletion on Salmonella resolution efficiency.
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.