Figure 1.
Cre recombination assay and loxP recombination as a function of distance.
(A) Schematic shows directly oriented loxP sites flanking prophage Mu attL and attR (L/R in text), and the deletion products of Cre recombination. The excised Mu prophage will be lost during cell growth. Amounts of chromosomal starting substrate and product were assessed by qPCR across the loxP sites in the substrate (primer pairs 1 or 2) and product (primer pair 3). (B) RE of loxP sites as a function of distance on the E. coli chromosome (see Figure S2A). RE (recombination efficiency) calculation is described under Materials and Methods. RE of the 190 bp loxP pair was arbitrarily set at 1 (this pair is designated malF in Figure 1C). Inset: log (base e) RE values were plotted against loxP distance, and fitted to a straight line equation , where
; the slope (m) is ∼7 kbp. The plot is not quite linear because it includes two data points that fall within the plateau region of the graph (25 kbp and 37 kbp). A plot excluding the 37 kbp value is shown in Figure S2D. (C) RE of loxP sites placed ∼70 bp outside each end of the wild-type malF::Mu prophage in ZL524 (MU) is set at 1, and compared to the smallest (190 bp) (malF, ZL582) and largest (37 kbp) (malF/yjcF, ZL592 and purH/malF, ZL594) loxP pairs within the malF locus of the parent non-Mu strain (see Figure S2A). Bottom panel, precise fold differences in RE. Error bars are standard deviation from the mean.
Figure 2.
Importance of SGS and DNA supercoiling to Mu end synapsis.
(A) RE of loxP sites flanking Mu in SGS-deleted, SGS-displaced to left (L) or right (R) of center, and reduced-Mu genome strains. MU (ZL524), ΔSGS (ZL562), SGS(L) (ZL573), SGS(R) (ZL578), 17 kb Mu (RS020), 17 kb ΔSGS (RS025). (B) RE of loxP sites flanking a Muc+ lysogen (ZL911) and its isogenic gyrBts strain (ZL941) measured at 26°C and 37°C, using the rhamnose-inducible Cre plasmid as described under Materials and Methods. Other descriptions as in Figure 1.
Figure 3.
Propagation of the Mu domain outside Mu ends.
(A) Position of loxP-site pairs inside (In) or outside (Out) Mu. The exact location of the sites is given in Table S1. (B) RE of loxP-site pairs diagrammed in A. In-5 kb, Out-5 kb etc. refers to RE of pairs of symmetrically placed sites within and outside the L and R ends of Mu in different strains at the indicated distances. MU (ZL524), In-5 kb (ZL732), Out-5 kb (ZL720), Out-10 kb (ZL724), Out-25 kb (ZL728).
Figure 4.
Interaction of prophage Mu ends probed by 3C methodology.
(A) Experimental design (see text and Materials and Methods). Blue line, Mu DNA; black line, E. coli DNA; purple and green dots, PstI and EcoRI sites, respectively; small arrows, primers used to amplify the DNA ligation product; red circle, paired L and R ends. (B) PCR products of ligation. Left: Primers were designed to produce a 155 bp fragment after PstI digestion-ligation (lane 2, arrowhead), and a 195 bp fragment after EcoRI digestion-ligation (lane 6, arrowhead); the fainter bands above the specific products in lanes 2 and 6 could not be re-amplified, hence are non-specific. The specific products were not observed in uncrosslinked (lanes 1, 5) and unligated (lanes 4, 8) samples. The band migrating at ∼100 bp in these lanes is non-specific. Lane 3, 7 DNA size marker ladder. (C) Quantitation of the ligation products. The qPCR signal obtained from wild-type MU ligation was set at 1. Crosslinking efficiency is defined as the ratio of qPCR signal from the ligation product in the ΔSGS strain compared to that in its wild-type parent. NL is the signal obtained from the non-ligated, crosslinked product in the wild-type reactions shown in lanes 4 and 8, and ΔMu is a similar control in a strain where Mu has been excised from ZL524 via recombination of the flanking loxP sites. The same set of primer pairs were used for all strains in either the PstI or the EcoRI panels. MU (ZL524), ΔSGS (ZL562), ΔMu (ZL580). (D) In vitro Cre-loxP recombination of the cross-linked DNA from the indicated strains before digestion with restriction enzymes.
Figure 5.
Cis- and trans-acting Mu transposition factors required for Mu domain formation, and discovery of early rightward transcripts in the prophage.
(A) RE of loxPs in ΔattL (ZL552) and ΔattR (ZL556) strains compared to their wild-type parent MU. (B) RE of loxPs in strains deleted for MuA (ZL536) and MuB (ZL530) genes, and MuΔB strain complemented with MuB from plasmid pJG8 (pMuB). (C) Expression of EGFP-MuB in a wild-type (WT) Mu lysogen and isogenic strains carrying SGS and early promoter deletions. Excitation and emission wavelengths were 488 nm and 507 nm, respectively. Fluorescence values were subtracted from the background fluorescence of the parental Mu strain without the EGFP-MuB (MP1999), and expressed as arbitrary units (AU). Error bars are standard deviation from the mean. Strains: Wild-type Mucts prophage expressing EGFP-MuB (RS033) and its deletion derivatives ΔPe (RS048), ΔPe* (RS102), ΔPe ΔPe* (RS103), ΔSGS (RS088), ΔSGS ΔPe (RS106), ΔSGS ΔPe* (RS107) and EGFP-MuE (RS101). (D) RE of loxPs in Mu prophage strains carrying early promoter deletions. ΔPe (RS053), ΔPe* (RS092), ΔPe ΔPe* (RS093), ΔRep ΔPe ΔPe* (ZL951). In panels A, B and D, MU is ZL524.
Figure 6.
Role of E. coli NAPs and their binding sites on the Mu genome, in Mu domain configuration.
(A) Schematic showing binding sites for IHF, Fis and HU on the Mu genome. O/E, operator/enhancer; G, invertible G segment; sis, Fis-binding enhancer site for G inversion; Pmom, promoter for the mom gene. (B) RE of loxP sites in strains either deleted for the indicated NAPs or for their binding sites on Mu. Strains: MU (ZL524), ΔH-NS (ZL624), ΔHU (ZL634), ΔFis (ZL614), ΔIHF (ZL604), ΔIHF site (ZL656), ΔHU site (ZL652), Δsis (ZL660), ΔPmom (ZL670), Δsis ΔattR (ZL662), Δsis ΔPmom ΔattR (ZL672).
Figure 7.
Mu domain at an NS chromosomal location, and probing for a domain configuration for prophage λ.
(A) E. coli chromosome macro-domains and position of prophages at the loci examined in this study. (B) RE of loxP sites flanking Muc+ prophage (Mu) located in lacZ, and its isogenic mutants. Mu (ZL911), ΔSGS (ZL921), ΔH-NS (ZL931), ΔIHF (ZL936). (C) RE of loxP sites flanking prophage λ (ZL808) compared to Mu (ZL911).
Figure 8.
Model of the Mu prophage domain.
See text for description. The single supercoiled Mu loop shown is not intended to imply absence of branching. NAPs not tested in this study, such as SMC-like proteins, may also be involved in domain maintenance. A variation of this model was proposed earlier for replicating Mu, to account for strong MuB binding only within Mu [47].
Table 1.
Strains.
Table 2.
Plasmids.