Article
Reference
Phage Mu transposase: deletion of the carboxy-terminal end does not abolish DNA-binding activity
BETERMIER, M., et al.
Abstract
We demonstrate that a specific site on the transposase protein, pA, of bacteriophage Mu is highly susceptible to proteolytic cleavage. Cleavage is observed in a minicell system on solubilisation with the non-ionic detergent Triton X-100 or following addition of a solubilised minicell preparation to pA synthesised in a cell-free coupled transcription/translation system.
Cleavage occurs at the carboxy-terminal end of the protein and generates a truncated polypeptide of 64 kDa, pA*, which retains some of the DNA-binding properties of pA. These results suggest that pA may be divided into functional domains for DNA binding and for interaction with the proteins involved in phage replication.
BETERMIER, M., et al . Phage Mu transposase: deletion of the carboxy-terminal end does not abolish DNA-binding activity. Molecular and General Genetics , 1987, vol. 210, no. 1, p.
77-85
DOI : 10.1007/BF00337761
Available at:
http://archive-ouverte.unige.ch/unige:149383
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1 / 1
© Springer-Verlag 1987
Phage Mu transposase: deletion of the carboxy-terminal end does not abolish DNA-binding activity
M. Betermier t, R. Alazard ~, F. Ragueh 1, E. Roulet 2, A. Toussaint 3, and M. Chandler 1 1 C.R.B.G.C. du C.N.R.S., 118 Route de Narbonne, F-31062 Toulouse Cedex, France
z Dept. de Biologie Moleculaire, University of Geneva, Geneva, Switzerland
3 Laboratoire de Genetique, Universit6 Libre de Bruxelles, B-1640 Rhode St. Genese, Belgium
Summary. We demonstrate that a specific site on the trans- posase protein, pA, of bacteriophage Mu is highly suscepti- ble to proteolytic cleavage. Cleavage is observed in a mini- cell system on solubilisation with the non-ionic detergent Triton X-100 or following addition of a solubilised minicell preparation to pA synthesised in a cell-free coupled tran- scription/translation system. Cleavage occurs at the car- boxy-terminal end of the protein and generates a truncated polypeptide of 64 kDa, pA*, which retains some of the DNA-binding properties of pA. These results suggest that pA may be divided into functional domains for DNA bind- ing and for interaction with the proteins involved in phage replication.
Key words: Phage Mu transposase Processing Protein domains - DNA binding
Introduction
The mutator phage Mu is perhaps one of the best character- ised of all bacterial transposable elements (for review see Toussaint and Resibois 1983). Unlike many temperate phages, insertion of the Mu genome into the bacterial chro- mosome occurs with little specificity for host DNA se- quences not only during lysogeny but also during lytic growth. The lytic cycle of Mu involves replicative transposi- tion of the phage from one site to another in the host ge- nome, while, as in the case of other temperate phages, lyso- geny is thought to occur by conservative (non-replicative) integration (Liebart et al. 1982; Harshey 1984; Akroyd and Symonds 1983).
Two Mu encoded proteins, the products of the A and B genes, are necessary for efficient transposition. The A protein (pA; mol.wt. 75 kDa), or transposase, is absolutely required for both lysogeny and lyric growth, and is used stoichiometrically during lytic growth of the phage (Pato and Reich 1982, 1984). The B protein (pB; mol.wt. 33 kDa) is also required for efficient integration of Mu (O'Day et al.
1978; Chaconas et al. 1985), for late transcription and for lyric replication. As might be expected for a transposase, pA binds specifically in vitro at sites located close to the ends of the phage (Craigie et al. 1984). In addition to its DNA-binding properties, it is likely that pA interacts in a specific way with pB and several host encoded proteins Offprint requests to." M. Chandler
to form the phage replication complex. In such multifunc- tional proteins, individual functions are generally located in distinct domains (Pabo and Sauer 1984).
We and others (van Leerdam et al. 1982; Roulet 1983;
Roulet et al. 1984; Chaconas et al. 1984; Craigie et al. 1985) have described the cloning of the Mu A gene, its conditional expression from the strong P1 promoter of bacteriophage lambda (Roulet 1983; Roulet et al. 1984), and some of the properties of pA. In the work presented here, we show that the 75 kDa pA can be partially chased into a species with an apparent molecular weight of 64 kDa (pA*). We demonstrate that pA* is a truncated form of pA, produced by post-translational cleavage of the carboxy-terminal re- gion. Moreover, pA* is shown to retain some of the proper- ties of pA, namely, the ability to bind DNA containing both ends of Mu. The data provide experimental support for the suggestion (Harshey et al. 1985) from an analysis of the amino acid sequence derived from the nucleotide sequence of the gene (Harshey et al. 1985; Preiss, personal communication) that the DNA-binding domain of pA is located at its amino-terminal end.
We present a model in which the stoichiometric require- ment for pA is proposed to result from its inactivation by proteolytic cleavage following a round of phage replica- tion and that, while the truncated form of the protein is able to bind specifically to Mu DNA, it has lost a domain necessary for interaction with other proteins involved in the phage replication apparatus.
Materials and methods
Bacterial strains andplasmids. The bacterial strains, bacteri- al and phage growth conditions, and the isolation of mini- cells from strain DS410 have been described in detail pre- viously (Roulet et al. 1984). Plasmid pPlc236 is a derivative of pBR322 carrying the P1 promoter of phage lambda (Re- maut et al. 1981); pPlc236:A is pP1c236 carrying the Mu A gene under control of PI (Roulet et al. 1983, 1984);
pLP117 is a pBR322 derivative carrying the left and right EcoRI fragments of phage Mu (van Leerdam et al. 1981);
pMK108 is a pBR322-based plasmid carrying a mini-Mu (Mizuuchi 1983); pBH857 carries the strong transcription/
translation terminator of gene 32 from phage T4 inserted between the HindIII and BamHI sites of plasmid pBR322 (see Prentki 1983; Prentki and Krisch 1984). Plasmid and phage DNA was purified by standard procedures (Maniatis et al. 1982).
78
Plasmid construction. The isolation of a series of pPlc236:A plasmids carrying "random" insertions of the transposon TnlO00 in the A gene is described elsewhere (Toussaint et al. unpublished). Briefly, the unique BamHI site located in the poly linker between the P1 promoter and the A gene carried by pPlc236:A was destroyed by cleavage with BamHI, the 5' extensions were rendered flush by treatment with the Klenow fragment of DNA polymerase I and the plasmid recircularised. The resulting plasmid was used as a target for TnlO00 and insertions were isolated by TnlOOO- mediated cointegrate formation with F-kan (Heffron et al.
1978), following conjugal transfer to a suitable recipient strain and resolution of the cointegrate.
Enzymes were employed according to the recommenda- tions of the supplier. Recombinant DNA techniques were as described in Maniatis et al. (1982).
Labelling and lysis procedures. Plasmid-carrying minicells (approximately 3 x 101°/ml) were resuspended in M9 mini- mal medium (Adams 1959) containing 0.4% w/v glucose and D-cycloserine to a final concentration of 20 gg/ml. A preincubation was carried out at 30 ° C for 15 min. Label- ling was for 1 h at 42 ° C following the addition of Difco methionine assay medium (0.5%) and 500 gCi/ml [L--$35] - methionine (970 Ci/mmol; 4.25 mCi/ml; Amersham). In the case of the pulse-chase experiment, incorporation was terminated by the addition of 100 gg/ml unlabelled methio- nine and samples were removed periodically for analysis.
Samples were centrifuged for 15 min at 15000 rpm, and the minicells washed and resuspended in SDS sample buffer (400 mM Tris, 40 mM DTT, 1% SDS, 10% glycerol, 0.02%
bromphenol blue).
Samples which were to be processed were washed and resuspended in a one-tenth volume of 10 mM Tris, pH 8.0, 5 mM EDTA, 10% sucrose and 0.3 M NaC1. Lysozyme was added at a final concentration of 200 gg/ml and the preparation incubated at 4°C for 1 h. The minicells were then frozen in a dry ice-ethanol bath and thawed (x 8).
Triton X-100 was added to a final concentration of 0.33%
v/v and incubation was carried out at 25 ° or 30°C (see figure legends). Aliquots taken at each step in the lysis pro- cedure and at various times after the addition of triton were diluted in SDS sample buffer.
Labelled polypeptides were separated by polyacryl- amide gel electrophoresis (PAGE) on 10% gels (Roulet et al. 1984) and visualised by fluorography. The $30 frac- tion used in the coupled transcription/translation reactions was obtained from Amersham and used according to the suppliers recommendations.
Densitometry tracing and normalisation of pA. The amount of protein in the relevant bands was estimated by densito- metry of autoradiographs using a Joyce Loebl or Vernon densitometer. The areas of the pA and pA* peaks in a given channel were divided by that obtained for vector spe- cified fl-lactamase in the same channel.
Isolation of proteins and limited proteolysis. Labelled protein bands obtained from minicells were cut directly from SDS gels and digested with Staphylococcus aureus protease V8 (Pierce) without prior elution, according to Cleveland et al.
(1977). Digestion was carried out within the stacking gel for between 5 and 6 h at room temperature. The products were analysed by electrophoresis through a linear
15%-22% SDS-polyacrylamide gradient gel, followed by fluorography.
Binding ofpA* to Mu DNA. Binding of pA* to DNA was performed according to the method of Kwoh and Zipser (1979) as modified by Roulet et al. (1985). Following the maturation procedure (see above), the preparations were enriched for pA* as follows: NaC1 was added to a final concentration of 1 M and the samples centrifuged at 12,000 rpm for 20 min at 4 ° C. The supernatant was then used as a source ofpA*. This procedure resulted in a signifi- cant enrichment of pA* (Fig. 1 C).
The binding of pA* to exogenous DNA was performed in the following way: 30 gl of partially purified pA* extract were mixed with 20 gg of DNA in a final volume of 300 gl (binding buffer: 10 mM Tris, pH 7.5, 1 mM EDTA, 50 ~tg/
ml bovine serum albumin, 0.1 mM DTT, 5 mM MgClz, 200 mM NaC1). After incubation at 30 ° C for 30 min, the preparation was sedimented through a 10%-25% sucrose gradient in binding buffer containing 50 mM NaC1. The position of protein and DNA in each gradient was moni- tored by gel electrophoresis and autoradiography.
Results
Stability and processing of plasmid encoded pA
Since it has been demonstrated that the activity of pA is unstable in Escherichia coli (Pato and Reich 1982, 1984), it was of interest to determine whether this reflects an insta- bility of the protein itself. We thus measured the stability of pA synthesised by the plasmid pPlc236:A in the minicell system by pulse chase experiments, as described in the Ma- terials and methods. The results (data not shown) indicate that no significant decrease in the amount of pA occurs over a 3 h chase period at 42 ° C.
These experiments were initially undertaken to deter- mine the DNA-binding properties of pA. Such studies in- volved the synthesis of pA in minicells, gentle lysis using a non-ionic detergent, and fractionation by centrifugation (Kwoh and Zipser 1979), as described in the Materials and methods. The results of the electrophoresis of samples taken at various stages of the procedure are shown in Fig. 1 A.
The untreated sample (lane 1) exhibits major protein species of 75, 33, 30 and 28 kDa, as previously described (Roulet et al. 1984). Treatment with lysozyme does not change this pattern (lane 2). Following freeze-thawing, however, a dis- tinct new band with an apparent molecular weight of 64 kDa appears (lane 3). This band increases in intensity following Triton X-100 treatment (lanes 4-8) and seems to reach a maximum after about 15 min incubation (lane 5), after which a decrease in the 64 kDa species is observed, presumably due to further degradation. Densitometer trac- ings of this autoradiograph show a correlation between the increase in intensity of the 64 kDa protein (pA*) and a decrease in intensity of the 75 kDa pA species (Fig. 1 B), suggesting that pA* is generated by post-translational cleavage of pA.
The distribution of protein species in the soluble and insoluble fractions of the "processed" minicell preparation is shown in Fig. I C. Under the high salt conditions we use (see the Materials and methods), the major fraction of uncleaved pA remains in the insoluble fraction together
1 2 3 4 5 6 78
i 2 3
O
B 1 2 3 4 5 6 78
A
C
Fig. 1A-C. Processing of 35S-labelled pA in minicells.
A 12.5% SDS-PAGE and autoradiography of pPlc236:A-directed proteins : lane 1, untreated; lane 2, after lysozyme treatment; lane 3, after freeze-thawing;
lanes 4-8, after addition of Triton X-t00 for 5, 15, 30, 60, and 180 rain, respectively. Incubation was at 25 ° C.
The size standards used were x4C-labelled albumin (69 kDa), ovalbumin (46 kDa), carbonic anhydrase (30 kDa), lactoglobulin (18 kDa), and cytochrome c (12 kDa).
B Densitometer tracing of autoradiograph shown in A:pA (e), pA* (+).
C Fractionation of pA* produced in minicells. After the addition of 1 M NaC1, 35S-labelled proteins were separated by 10% SDS-polyacrylamide gel
electrophoresis (PAGE) and visualised by
autoradiography: lane 1, unprocessed sample of pA synthesised in minicells (as in panel A, lane 1); lanes 2 and 3, lysozyme/freeze-thaw/Triton X-100 treated sample; lane 2, supernatant fraction obtained after the addition of 1 M NaC1 and centrifugation; lane 3, pellet fraction. Incubation following labelling was at
30 ° C
with membranes and other cell debris (lane 3), whereas the majority of pA* is soluble and remains in the supernatant (lane 2). This supernatant fraction was used as a source of pA* in all further studies.
Post-translational cleavage of pA synthesised in vitro To rule out the possibility that cleavage of pA to give pA*
is induced directly by Triton X-100, we have analysed the behaviour of pA synthesised in a coupled transcription/
translation system (Zubay 1973). Incorporation of S3S-me - thionine was terminated, in all cases, by addition of excess unlabelled methionine prior to addition of Triton X-100 or triton-extracts. The results of these experiments are shown in Fig. 2. Lane 1 shows the synthesis of pA directed by the plasmid pPlc236:A in the Zubay system. It will be noted that little apparent 64 kDa species can be detected under these conditions. Similar results are obtained when such a preparation is subjected to a regime identical to that used in the lysis of the minicell preparation, even in the presence of Triton X-100 (lane 2). If, however, the prep- aration is mixed with a preparation of plasmidless minicells (obtained by pretreatment with Triton X-100 following ly- sozyme treatment and freeze-thawing), the majority of pA
in the preparation is converted to pA* (lane 3). We note that certain minicell preparations generate a low level of the 64 kDa species when incubated with pA, even in the absence of the lysis pretreatment (data not shown). This is presumably due to a low level of lysis occurring during storage of the minicells.
Comparison of pA and pA* by 1/8 digestion
To provide further evidence that pA* is generated from pA by post-translational cleavage, we have compared pep- tides from both species generated by partial digestion with protease V8. The results are shown in Fig. 3. Both proteins were isolated from SDS-polyacrylamide gels; pA was ob- tained from a minicell preparation which had not been sub- jected to the maturation procedure, while pA* was isolated from fractionated preparations and treated as described in the Materials and methods. It will be noted that with in- creasing concentrations of V8, pA (Fig. 3A, lanes 1 3) is preferentially converted to a species exhibiting the same mobility as pA* (lanes 5 7) with little additional cleavage.
Increasing the concentration of V8 (Fig. 3 B) results in the appearance of several additional distinct fragments and the disappearance of the major 64 kDa species, The fragments
80
9Z4
1 2 3 4
68
43
25.7~
Fig. 2. Cleavage of pA synthesised in the Zubay coupled transcrip- tion/translation system and processing by the addition of lysed minicells: lane 1, pattern obtained with pPlc236:A; lane 2, follow- ing lysozyme/freeze-thaw and addition of Triton X-100 to the sam- ple shown in lane 1 and incubation for 20 rain at 30 ° C; lane 3, addition of Triton X-100 lysed minicells to the sample shown in lane 1 and incubation for 20 min at 30 ° C; lane 4, synthesis and processing of pA in minicells carrying pPlc236:A (as in Fig. 1, panel A, lane 5)
obtained from pA* (lane 2) are all present in pA. However, a single pA fragment of about 15 kDa (lane 1) is absent in the pA* sample. We therefore conclude from these results that pA* is generated by post-translational cleavage of pA.
Position of post-translational cleavage
To determine the point at which cleavage of pA occurs, we have constructed a set of deletions extending from the 3' towards the 5' end of the A gene. For this purpose we used a collection of pPlc236:A plasmids in which the un- ique BamHI site between the promoter and the A gene had been destroyed (see the Materials and methods), and which carry "random" insertions of the transposable ele- ment TnlO00 in the cloned A gene (Toussaint et al. unpub- lished). A sub-set of these plasmids in which TnlO00 had inserted in an orientation such that the BamHI site of the element is proximal to the 5' end of the gene (Fig. 4) were selected. The plasmids were cleaved with BamHI and ClaI, a procedure which results in deletion of the 3' extremity of the gene, all but 400 bp of TnlO00, and a region of phage lambda DNA carried by pPlc236:A (Fig. 4; Roulet et al. 1984). The remaining region of TnlO00 carries transla- tion termination signals in all three phases within the first 110 bp and thus the truncated A protein will carry a maxi- mum of 36 amino acids at its carboxy-terminal end encoded by the TnlO00 fragment. To ensure efficient transcription termination of the truncated A gene, a 124 bp BamHI-ClaI
200 ,'..- 97.4 =...- 68
1 2 3 4 5 6 7
200 97.4 68
1 2
43 le=.-- 43
25.7 "- 25.7 "-
18.4 14.3
A
18.4 14.3
B 6.2
Fig. 3A, B. Comparison of pA and pA* by partial V8 digestion.
A Preferential conversion of pA to a 64 kDa species: lanes 1 3, addition of 10, i or 0.1 ng V8, respectively, to gel slices containing an equal amount of 35S-labelled pA; lane 4, untreated labelled minicells carrying pPlc236:A; lanes 5-7, addition of 0.1, 1 or 10 ng V8, respectively, to gel slices containing similar quantities of pA*.
B V8 fingerprint of pA (lane 1) and pA* (lane 2) using 10 p.g of V8
EcoRI Hind III \ /
PL A1
I', A2:
... BamHl-Clal ... '
, 0.4 kb ...
Fig. 4. Physical map of pPlc236delBam:A::TnlO00. ~ vector sequences; E] A gene interrupted by insertion of TnlO00 (~]); | phage lambda sequences. Restriction sites are indicated: o, HindIII; v, BarnHI; ,, ClaI; o, EcoRI;., SalI
1 2 3 4 5 6 7 8 9 10 11 12
200
97.4 '-'- 68 "'-
43 ""
25.7 ""
Fig. 5. Labelling and processing of carboxy-terminal truncated proteins. Minicells carrying various deletion derivatives of pPlc236del- Barn:A::TnlO00 were labelled and processed as described in the Materials and methods. Lane 1: vector plasmid; lanes 2-4: untreated pPlc236:A (lane 2), treated supernatant fraction (lane 3), and treated pellet fraction (lane 4); lanes 5 and 6: pMi7, untreated and treated; lanes 7 and 8: pMil0, untreated and treated; lanes 9 and t0: pMil 1, untreated and treated; lanes 11 and 12: pMil2, untreated and treated. All incubations with Triton X-t00 were at 30 ° C. Black arrows indicate the position of the unprocessed protein. Open arrows indicate the position of the corresponding processed protein
fragment isolated from the plasmid pBH857 (see the Mate- rials and methods), and carrying strong transcription/trans- lation termination signals of T4 gene 32, was inserted be- tween the corresponding sites of the cleaved pPlc236:A plasmids. Four derivatives, pMil2, pMill, pMil0 , and pMi7, which carry deletions of increasing size extending towards the 5' extremity of the gene were analysed, to deter- mine the size of the truncated pA and whether post-transla- tional cleavage occurs in minicells. The size of the truncated proteins predicted from the nucleotide sequence of the trun- cated genes (data not shown) are 74 kDa (pMil2), 69 kDa (pMil 1), 68 kDa (pMil0) and 47 kDa (pMi7). The contri- bution of TnlO00 sequences to the truncated proteins is I l, 2, 1 I, 36 amino acid residues, respectively. The results obtained after expression in the minicell system are pre- sented in Fig. 5. The sizes observed correlate closely with those expected from the nucleotide sequence (data not shown): 74kDa (pMil2; lane 11), 68 kDa (pMill; lane 9), 65 kDa (pMil0; lane 7), and 41.5 kDa (pMi7; lane 5).
Moreover, post-translational cleavage of the pMil2 (lane 12), pMill (lane 10), and pMil0 (lane 8) polypeptides re- sults in the appearance of a species exhibiting a molecular weight indistinguishable from that of pA* (lane 3), whereas cleavage of the pMi7 polypeptide (lane 6) cannot be de- tected. This clearly demonstrates that cleavage occurs to- wards the carboxy-terminal end of pA.
Specific binding of pA* to Mu DNA
To determine whether pA* retains the DNA-binding prop- erties of pA, we analysed its behaviour in sucrose gradients in the presence and absence of Mu DNA, as described in the Materials and methods. A labelled minicell preparation enriched for pA* was mixed with various unlabelled DNA species in a standard binding reaction, and the mixture was subjected to centrifugation through a 10%-25% sucrose gradient and analysed as described in the legend to Fig. 6.
82
Fig. 6A-F. Binding of pA* to Mu DNA. A mixture of labelled pA* obtained as the supernatant fraction from processed minicells was mixed with various DNA species, incubated at 30 ° C for 30 min in 200 mM NaC1, and centrifuged through a 10% 25% sucrose gradient in the presence of 50 mM NaC1. Fraction 1 corresponds to the top of the gradient, fraction 18 to the bottom. Lane E (Panel A) shows an aliquot of unfractionated labelled minicells. Lane C (panels D-F) shows covalently-closed circular pLPI17 (23.5 Kb) DNA. Panels A-C Autoradiograph of 12.5% SDS-PAGE of the resulting fractions; A, no DNA; B, pLPI17 DNA; C, phage lambda DNA (48.5 Kb). D Ethidium bromide-stained, 0.7% agarose gel in acetate buffer showing fractions 7-18 of panel B. E Autoradiograph of the dried agarose gel shown in panel D. F Separation of pLPI17 closed circular and linear forms by extended electrophoresis of samples shown in panel D. In this gel system, the CCC form migrates more slowly than linearised plasmid DNA (data not shown).
Relative migration of these forms is known to be a function of plasmid size, gel concentration and field strength (Johnson and Grossman 1977)
In the absence of exogenous DNA, most of the labelled proteins remain at the top of the gradient (Fig. 6A). If DNA of the plasmid pLP117, which carries both ends of Mu, is included in the binding mixture, the labelled pA*
migrates down the gradient in two overlapping peaks
(Fig. 6B, fractions 7-11 and 13-17). When bacteriophage lambda DNA is substituted for that of pLPII7, a small amount of pA* is found to migrate with the DNA but the majority remains at the top of the gradient (Fig. 6C).
These results indicate that pA* retains the specificity of
. . . . . . . . Y T Y . . . . . . . . . . . . 7 Y V . . . .
,.~ dimer
-,~ oc
"~ linear CCC
..~ dimer oc
"~. linear
-,~ COC
A C
. . . Y v . . . ~ v v ~ p ~ .
B D
Fig. 7A-D. Binding of pA* to supercoiled plasmid DNA. Experimental conditions are identical to those described in Fig. 6. Panels A (pPlc236) and C (pMK108): ethidium bromide-stained, 0.7% agarose gel of fractions isolated from a sucrose gradient. Panels B (pPlc236) and D (pMK108): autoradiograph of gels shown in A and C, respectively. Electrophoresis conditions were as in Fig. 6.
Note the inversion of CCC and linear forms (lane C, panels A and C) compared with the larger pLP117 plasmid shown in Fig. 6
binding to bacteriophage Mu DNA exhibited by pA. More- over, autoradiography of the agarose gel shown in Fig. 6D reveals that fractions 13 17 have retained labelled protein (Fig. 6E). On the other hand, labelled protein is lost in fractions 7-11 when subjected to agarose gel electrophore- sis. Extended agarose gel electrophoresis of these samples (Fig. 6F) indicates that samples 7-11 and 13-17 contain a majority of linear and covalently-closed circular plasmid DNA, respectively. This indicates that not only does pA*
exhibit preferential binding to Mu DNA sequences but that it exhibits strong binding to a supercoiled substrate. This conclusion is confirmed by the data of Fig. 7 which show the results of binding experiments using the vector pPlc236 (panels A and B) and pMK108 (panels C and D), a small plasmid carrying both ends of Mu (see the Materials and methods). It is clear that strong binding, as judged by reten- tion in the agarose gel, is exhibited with CCC DNA of pMK108 (panel D) whereas no specific binding can be de- tected with pP1c236 DNA (panel B).
In vivo properties of the truncated protein
Since pA* lacks the carboxy-terminal domain of the trans- posase but retains the capacity to bind specifically to Mu DNA, it was of interest to investigate whether truncated pA, carrying the same domains as pA*, is active in transpo- sition. To investigate this possibility we have measured the plating efficiency of Aamao93 phage on strain K12delH1 car- rying pplc236, pPlc236:A, or pMil0. The latter plasmid was chosen since it specifies a truncated pA protein with approximately the same size as pA* and thus carries the same transposase domains as pA*. The results presented in Table I demonstrate that, in contrast to pPlc236:A, pMil0 is unable to complement the Aam mutant phage for growth at 37 ° C. Since these results indicate that the trt/n- cared protein is unable to promote replicative transposition, it was also of interest to determine whether it might influ- ence replicative transposition of a superinfecting wild-type phage, by for example interacting with and titrating sites
84
Table 1. In vivo effects of truncated pA
Plasmid Titre of superinfecting phage on plasmid-carrying strains a
Aamlo93 A +
30 ° C 37 ° C 42 ° C 30 ° C 37 ° C 42 ° C
pPlc236 < 103 < 103 < 10 3 5.0 X 10 9 1.5 X 10 i° 1.2 x 101°
pPlc236:A < 10 6 3 × 10 9 2 x 105 1.2 x 10 l° 6.0 X 10 9 1.5 X 1010
pMilO < 1 0 3 <10 3 <10 3 1.3 X 10 9 6.0 X 10 6 6.0 × 10 4
Infection and plating conditions have been described previously (Roulet et al. 1984). Strain Kl2delHl (Remaut et al. 1981) was used as an indicator. The values are based on three independent titrations
on the Mu genome recognised by intact pA. The results of superinfection of the plasmid-carrying strains with wild- type Mu are also included in Table 1. Truncated pA clearly exerts an inhibitory effect on growth of the A + phage. This effect is dependent on synthesis of the protein since little inhibition is observed at 30°C (conditions in which P1- directed synthesis is repressed in this strain). A second strik- ing observation resulting from these experiments is that while pPlc236:A is able to complement the MuAam phage efficiently when the cells are grown at 37 ° C, at 42°C a large reduction in plating efficiency occurs. This phenome- non does not occur when the superinfecting phage carries a functional copy of the A gene. Since the major difference between the two situations is that growth of M U A a m i 0 9 3
requires the supply of plasmid-specified pA in trans, while the MuA + phage can supply its own transposase (in cis), we suspect that the phenomenon is related to the source of pA. One possibility would be that pA has a tendency to aggregate when in sufficient concentration in the cell (and when not provided in cis). The level of synthesis of plasmid-specified pA at 42 ° C may be sufficient for aggrega- tion while synthesis at 37 ° may not.
Discussion
We have shown that the phage Mu transposase, pA, a pro- tein of 75 kDa synthesised from the plasmid pPlc236:A, is cleaved into a species, pA*, of 64 kDa presumably by a membrane-associated protease. Although pulse-chase ex- periments have demonstrated that pA is stable in the mini- cell system for a period of over 3 h, lysis of the minicell preparation using the non-ionic detergent Triton X-100 re- sults in the appearance of the 64 kDa species. Since no major protein species other than pA is observed with an apparent molecular weight greater than 64 kDa, it seemed likely that this species is derived from pA by post-transla- tional cleavage. We have confirmed this interpretation using a cell-free coupled transcription/translation system, primed with pPlc236: A DNA. The pA protein is stable in this sys- tem but can be chased into a 64 kDa form by addition of a preparation of minicells previously lysed with the deter- gent. This post-translational cleavage is not an artefact due to the presence of Triton X-100 since treatment of the cell- free system with the detergent alone does not result in cleav- age. Partial digestion of pA with the protease V8 confirms this interpretation: all partial digestion products exhibited
"by pA* are found in pA. Most pA is cleaved by V8 to
a form having the size of pA* before undergoing subsequent degradation. The region of pA which is cleaved to generate pA* in the minicell system is thus also preferentially cleaved by V8, Similar results using V8 and other proteases have recently been obtained by Nakayama et al. (1987).
Using a series of plasmids in which the A gene has been truncated as its 3' end, we have shown that post-transla- tional cleavage occurs at the carboxy-terminal end of pA.
Since pA* retains its specific DNA-binding properties, the carboxy-terminal end of the protein seems unlikely to con- tribute to the binding specificity of pA (see also Nakayama et al. 1987).
Our results may provide a physical explanation for the observed functional instability of pA in phage Mu develop- ment (Pato and Reich 1982, 1984). Cleavage of pA, to gen- erate pA*, could occur following a round of phage replica- tion and render the protein incapable of further participa- tion in replication, while permitting continued binding to its specific sites on the Mu genome. The observation (Ta- ble 1) that plasmid-specified pA deleted for it's carboxy- terminal domain not only fails to complement A,m phage for lytic growth but significantly inhibits growth of wild- type phage is consistent with this hypothesis. We emphasise, however, that cleavage has yet to be demonstrated in vivo during phage growth. It is interesting to note that pB, which acts in conjunction with pA during lyric transposition, is associated with the inner membrane (Schumann et al. 1984) and might serve to "deliver" pA to a membrane-associated protease.
We have suggested (Roulet 1983) that pA exhibits ana- logies with the O protein of phage lambda. O protein, which recognises and binds the origin of lambda replication, car- ries a DNA-specific binding domain within its amino-termi- nal region, and a domain at its carboxy-terminal end that enables it to interact with phage P protein and the host replication machinery (Furth and Wickner 1983). We have proposed (Roulet 1983) that pA has a similar type of archi- tecture; an amino-terminal domain exhibiting specific DNA-binding properties, and a carboxy-terminal domain involved in interaction with pB and/or the host proteins involved in Mu replication.
The collection of truncated A genes of which the four described here form part, will be useful in determining whether there is a strict correlation between the ability of a given protein to specifically bind Mu DNA and its ability to provoke low-level integration (leading to lysogeny) or high-level integration (which precedes lytic growth). Such truncated proteins will also be useful in determining 'the
various protein-protein interactions which occur in the transposition/replication complex.
Acknowledgements. We would like to thank E. Perret for help in the construction of the deletion mutants, B. Marty for help in minicell preparation, and M. Faelen for comments on the manu- script. This work was supported by a grant from the Ministere Francais de l'Industrie et de la Recherche to M.C. and in part by a grant from the European Community to A.T. and M.C.M.B.
was supported by a predoctoral fellowship (Ecole Polytechnique, France). We would also like to express our thanks to O. Fayet, D. Galas, P. Gamas, P. Ghelardini, P. Prentki, M.F. Prere and D. Zerbib for both intellectual and material support during the course of this work.
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