Efficient Mu transposition requires interaction of transposase with a DNA sequence at the Mu operator: implications for regulation

Phage Mu transposition is initiated by the Mu DNA strand-transfer reaction, which generates a branched DNA structure that acts as a transposition intermediate. A critical step in this reaction is formation of a special synaptic DNA-protein complex called a plectosome. We find that formation of this complex involves, in addition to a pair of Mu end sequences, a third cis-acting sequence element, the internal activation sequence (IAS). The IAS is specifically recognized by the N-terminal domain of Mu transposase (MuA protein). Neither the N-terminal domain of MuA protein nor the IAS is required for later reaction steps. The IAS overlaps with the sequences to which Mu repressor protein binds in the Mu operator region; the Mu repressor directly inhibits the Mu DNA strand-transfer reaction by interfering with the interaction between MuA protein and the IAS, providing an additional mode of regulation by the repressor.     

Conformational isomerization in phage Mu transpososome assembly: effects of the transpositional enhancer and of MuB.

Initiation of phage Mu DNA transposition requires assembly of higher order protein-DNA complexes called Mu transpososomes containing the two Mu DNA ends and MuA transposase tetramer. Mu transpososome assembly is highly regulated and involves multiple DNA sites for transposase binding, including a transpositional enhancer called the internal activation sequence (IAS). In addition, a number of protein cofactors participate, including the target DNA activator MuB ATPase. We investigated the impact of the assembly cofactors on the kinetics of transpososome assembly with the aim of deciphering the reaction steps that are influenced by the cofactors. The transpositional enhancer IAS appears to have little impact on the initial pairing of the two Mu end segments bound by MuA. Instead, it accelerates the post-synaptic conformational step(s) that converts the reversible complex to the stable transpososome. The transpososome assembly stimulation by MuB does not require its stable DNA binding activity, which appears critical for directing transposition to sites distant from the donor transposon.     

The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth

We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site-specific recombinational switches. Furthermore, we show that induction of a Mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucts62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage lambda.     

The phage Mu repressor c and IS30 transposase proteins are significantly related

The IS30 transposase exhibits significant amino acid sequence homology to the phage Mu repressor c in the amino- and carboxy-terminal regions of the proteins. The conserved sequences include the proposed Mu repressor DNA binding site, which is also related to the proposed Mu and D108 transposase DNA binding sites. The carboxy-terminal homologies are characterised by two almost complete, and one partial, somewhat diverged amino acid sequence repeats. Only weak homologies to this domain are present in the Mu transposase (Mu A). Nevertheless, a clear link between an insertion sequence and a bacteriophage has been established.     

Transposase A binding sites in the attachment sites of bacteriophage Mu that are essential for the activity of the enhancer and A binding sites that promote transposition towards Fpro-lac.

In this paper we determine which of the A binding sites in the attachment sites of phage Mu are required for the stimulatory activity of the transpositional enhancer (IAS). For this purpose the transposition frequencies of mini-Mu's with different truncated attachment sites to an Ftet target were measured both in the presence and the absence of the IAS. The results show that in our in vivo assay the L3 and R3 sites are dispensable for functioning of the IAS. An additional deletion of L2 or R2 however abolishes the stimulating activity of the enhancer suggesting an interaction between A molecules bound to these sites and the IAS. The residual transposition activity of a IAS-containing mini Mu in which R2 (and R3) are deleted is much lower than the activity of the comparable construct without the IAS. This means that in the absence of R2 the IAS is inhibiting transposition. Such an inhibition is not observed when L2 (and L3) are deleted. This suggests that the IAS interacts with the attachment sites in an ordered fashion, first with attL and then with attR. Furthermore we show that mini-Mu transposition is enhanced when Fpro-lac is used as a target instead of Ftet. We show that this elevated transposition is dependent on the Mu A binding sites L2,L3 and R2. These sequences could possibly mediate an interaction between the mini-Mu plasmid and sequences present on Fpro-lac.     

Preferential binding of bacteriophage Mu repressor to supercoiled Mu DNA

It was shown, using a relatively simple assay, that Mu repressor, cI, binds specifically to a region which spans the leftmost HindIII cleavage site on the phage genome. This extends the observations of Kwoh and Zipser [Nature (London) 277, 489-491 (1979)], who were able to define a binding region to the left of this site. These results provide support for the idea that the eight blocks of repeated DNA sequences, which also span the HindIII cleavage site, are involved in repressor binding. These results also indicate that cI repressor has a marked preference for supercoiled DNA.     

Replacement of the Bacteriophage Mu Strong Gyrase Site and Effect on Mu DNA Replication

The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the Escherichia coli chromosome which might be involved in organizing nucleoid structure, fragments of E. coli chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.     

Mapping of binding sites for Mu repressor and ner product within the left-end EcoRI. C fragment of the Mu genome

Bacterial cells containing the ner gene of phage Mu inserted into pBR322 express a binding activity with specificity for the left-end EcoRI.C fragment of Mu DNA. Crude extracts containing either Mu repressor or ner protein have been used to localize binding sites on TaqI subfragments of the EcoRI.C fragment. There are at least 3 distinct binding sites for the Mu repressor and 1 binding site for the ner protein on the EcoRI. C fragment. The possible biological function of these binding sites is discussed.     

Reversal of Mu gem2ts-induced mutations

Abstract: Mutations induced by the integration of a Mu gem 2ts mutant prophage can revert at frequencies around 1 × 10⁻⁶, more than 10⁴-fold higher than that obtained with Mu wild-type. Several aspects characterize Mu gem 2ts precise excision: (i) the phage transposase is not involved; (ii) the RecA protein is not necessary; and (iii) revertants remain lysogenic with the prophage inserted elsewhere in the host genome. In addition, prophage re-integration seems to be non-randomly distributed, whereas Mu insertion into the host genome is a transposition event without any sequence specificity. In this paper, we describe that the site of re-integration somehow depends on the original site of insertion. Two alternative models are proposed to explain the strong correlation between donor and receptor sites.     

Fis plays a role in Tn5 and IS50 transposition.

The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition. Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria. This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins. We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited. The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate. Using a mobility shift assay, we also identified another potential Fis site within IS50. Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition.     

Simultaneous expression of a bacteriophage Mu transposase and repressor: a way of preventing killing due to mini-Mu replication

In vitro studies of bacteriophage Mu transposition have shown that the phage-encoded transposase and repressor bind the same sequences on the phage genome. We attempted to test that prediction in vivo and found that Mu repressor directly inhibits transposition. We also found that, in the absence of repressor, constitutive expression of Mu transposition functions pA and pB is lethal in Escherichia coli strains lysogenic for a mini-Mu and that this is the result of intensive replication of the mini-Mu. These findings have important consequences where such mini-Mus are used as genetic tools. We also tested whether in Erwinia chrysanthemi the effect of transposition functions on a resident mini-Mu was the same as in E. coli. We observed that expression of pA alone was lethal in E. chrysanthemi and that a large fraction of the survivors underwent precise excision of the mini-Mu.     

Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain.

Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42 degrees C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47----Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of beta-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.     

Effect of mutations in the C-terminal domain of Mu B on DNA binding and interactions with Mu A transposase

Bacteriophage Mu transposition requires two phage-encoded proteins, the transposase, Mu A, and an accessory protein, Mu B. Mu B is an ATP-dependent DNA-binding protein that is required for target capture and target immunity and is an allosteric activator of transpososome function. The recent NMR structure of the C-terminal domain of Mu B (Mu B223-312) revealed that there is a patch of positively charged residues on the solvent-exposed surface. This patch may be responsible for the nonspecific DNA binding activity displayed by the purified Mu B223-312 peptide. We show that mutations of three lysine residues within this patch completely abolish nonspecific DNA binding of the C-terminal peptide (Mu B223- 312). To determine how this DNA binding activity affects transposition we mutated these lysine residues in the full-length protein. The full-length protein carrying all three mutations was deficient in both strand transfer and allosteric activation of transpososome function but retained ATPase activity. Peptide binding studies also revealed that this patch of basic residues within the C-terminal domain of Mu B is within a region of the protein that interacts directly with Mu A. Thus, we conclude that this protein segment contributes to both DNA binding and protein-protein contacts with the Mu transposase.     

Information analysis of Fis binding sites.

Originally discovered in the bacteriophage Mu DNA inversion system gin, Fis (Factor for Inversion Stimulation) regulates many genetic systems. To determine the base frequency conservation required for Fis to locate its binding sites, we collected a set of 60 experimentally defined wild-type Fis DNA binding sequences. The sequence logo for Fis binding sites showed the significance and likely kinds of base contacts, and these are consistent with available experimental data. Scanning with an information theory based weight matrix within fis, nrd, tgt/sec and gin revealed Fis sites not previously identified, but for which there are published footprinting and biochemical data. DNA mobility shift experiments showed that a site predicted to be 11 bases from the proximal Salmonella typhimurium hin site and a site predicted to be 7 bases from the proximal P1 cin site are bound by Fis in vitro. Two predicted sites separated by 11 bp found within the nrd promoter region, and one in the tgt/sec promoter, were also confirmed by gel shift analysis. A sequence in aldB previously reported to be a Fis site, for which information theory predicts no site, did not shift. These results demonstrate that information analysis is useful for predicting Fis DNA binding.