J. Mol. Biol. (1976) 107, 549-569

Characterization of 2trp=transducing Bacteriophages made in Vitro A. S. HOPKINS,, NOR]SEN E. MURRAY AND W. J. BRAMiVZAR

Edinburgh University, Department of Molecular Biology King's Buildings, Mayfield Road Edinburgh EH9 3JR, Scotland (Received 4 .May 1976, and in revised form 5 August 1976) W h e n fragments resulting from the digestion of Escherichia cell D N A with endo R.HindIII were inserted into an a p p r o p r i a t e receptor chromosome three m a i n classes of plaque-forming Atrp phages were detected: AtrpA, ~trpC a n d ~trpB. Analyses of the D N A from these phages following digestion with endo R.HindIII show the inclusion of a single fragment within the central region of the chromosome left of the phage a t t a c h m e n t site. Targets for endo R.HindIII have been m a p p e d with respect to markers in the trpD and trpB genes of E. coll. F r a g m e n t s of bacterial D N A within these three classes of Atrp phages comprise the entire trp operon. The trpD gene was reconstituted b y in vitro recombination of D N A fragments from AtrpC and AtrpE phages; similarly, the trpB gene was reconstituted from the D N A of AtrpA a n d AtrpC phages. Ar and htrpABC recombinants were also isolated from a p p r o p r i a t e phage crosses. In rive recombination is m e d i a t e d via the homologous D N A of the host's trp operon a n d uses either the host (rccA) or phage (red) recombination p a t h w a y . E a c h class of Atrp phage has been subdivided according to the orientation of the inserted fragment ; in one subclass the trp gene is transcribed from t h e / - s t r a n d of the phage (e.g. ~trp(C)I) while in the other the trp gene is transcribed from the r - s t r a n d (e.g. trp(Cy). Transcription initiated a t PT., a n d measured b y expression of t r y p t o p h a n synthetase from ~trp(ABC)Zcro-, is in no w a y impaired b y the intervening phage a t t a c h m e n t site. Expression from the trp p r o m o t e r of ~$rp(GDEycro- is strongly inhibited b y convergent transcription from Pv.. The fragment of D N A containing the trpA gene does not include a trp p r o m o t e r : the trpA gene of ~trp(A) z can be expressed from Pv., t h a t of ~trp(Ay m a y be read from a weak constitutive p r o m o t e r in the b2 region of phage ~. The levels of anthranilate synthetase resulting from the trp p r o m o t e r of either Atrp(CDE)z ~trp(GDE)Tphages can be boosted to 25% of the t o t a l cell protein. 1. I n t r o d u c t i o n D e r i v a t i v e s o f b a c t e r i o p h a g e A c a r r y i n g Escherichia cell genes h a v e m u c h r e l e v a n c e to the understanding of both the organisation and expression of the incorporated b a c t e r i a l genes. T h e y also p r o v i d e for a m p l i f i c a t i o n of t h e p r o d u c t s o f t h e i r a c q u i r e d genes a n d in a n o t h e r p a p e r , Moir & B r a m m a r (1976) h a v e s h o w n h o w r e a d i l y trpt r a n s d u c i n g p h a g e s d e r i v e d in rive m a y be u s e d t o p e r s u a d e E . cell t o p r o d u c e g r e a t l y i n c r e a s e d levels o f t h e e n z y m e s e n c o d e d b y t h e trp operon. t Present address : Department of Molecular Biology, University of California, Berkeley, Calif., U.S.A.

549

500

A . S . HOPKINS, N. E. MURRAY AND W. J. BRAMMAR

Recent progress in nucleic acid biochemistry permits the extension of the advantages offered by the original plaque-forming transducing phages, such as ~trp (Franklin, 1974) and ~bio (Guha et a/., 1971 ; Cleary & Campbell, 1972) not only to many other E. coli genes but to other prokaryotic genes, since DNAs from any source may be fragmented by a restriction endonuclease and the resulting fragments recombined in vitro. The incipient ]in]~age between fragments depends on the use of a restriction endonuclease which generates fragments of DNA with mutually complementary single-stranded 5'-projections; hydrogen bonding between these cohesive ends is followed by treatment with polynucleotide ligase to provide covalent l~onding (Mertz & Davis, 1972). Plaque-forming transducing derivatives of phage ~ can be made in vitro by the insertion of fragments of donor DNA, produced by either endo R.EcoRI (Murray & Murray, 1974; Rambach & Tiollais, 1974; Thomas et al., 1974; Borck et al., 1976) or endo R.HindIII (Murray & Murray, 1975; Borck eta/., 1976), into appropriate phage receptor molecules. In the former case the donor fragment of DNA usually replaces that region of the phage chromosome concerned with integrative and generalised recombination. I f the bacterial genes within the acquired fragment are orientated so that they are transcribed from the same strand as the genes they have replaced, their expression is as described in detail for ~rp phages generated in vivo (Fran~lln, 1971,1974; Moir & Brammar, 1976). For those phages made via endo R . H i n d I I I the bacterial DNA is inserted left of the attachment region (Borck et al., 1976). These transducing phages remain integration proficient (Borck e~ al., 1976) and the relative position of the heterologous DNA differs from that in transducing phages derived in vivo (Campbell, 1962). In this paper we describe ~tri~ phages in which fragments of the trip operon of E. toll, resulting from digestion of E. coli DNA with endo R.HindIII, have been incorporated into the ~ genome to produce integration-proficient transducing phages. We have characterised these phages and studied the expression of their incorporated genes. The ~trT phages provide model systems for this approach because of the ease with which they can be analysed genetically and because the trp enzymes can be assayed readily. We find that bacterial genes with or without their own promoter can be expressed irrespective of their orientation within the phage genome, and we have taken advantage of the information gained from classical ~rp phages (Franl~]~n~ 1971) to maximise the expression of the inserted trp genes (Moir & Brammar, 1976). This information can now be applied to other bacterial genes, particularly those whose products are normally difficult to obtain in adequate amounts.

2. Materials and M e t h o d s (a) P ~ e s The nomenclature for targets for restriction enzymes uses the abbreviation for the enzyme (Smith & Nathans, 1973) followed by the genome concerned and the number of the site within the genome. Thus sites for endo R.EcoRI are described by still, stir2 etc., and sites for endo R.Hind~II should be described by shindIII~l, shindIII~2, etc. Since the latter description is particularly cumbersome, we have abbreviated it to 8hn~l, shn~2, etc., throughout this paper. The ~rp transducing phages were derived from a ~imm 2z receptor chromosome (Murray & Murray, 1975) which is deleted for approximately 21% of the wild-type complement of DNA (Fig. 1) and which retains only a single target for endo R.Hind_III (ahn~3). Fragments

2TRP-TRANSDUCING

[ srIX1-2]

art I

T

PHAGES

551

PL 0

0 P.

[ nin 5

0P~

shnX5

Fro. 1. The h receptor for fragments of DNA generated by endo R . H i n d I I I . Deletions arc symbolised by gaps and substitutions by the heavier lines. Two targets for endo R . H i n d I I I are removed by the deletion between 8rIhl and srI~2, a further 2 by the substitution of i m m 2i for i m m ~ and a 5th by a small substitution from ~80 in the region to the right of nin5 (Murray & Murray, 1975). The remaining target (shn~ 3) is located to the left of the attachment site. The combination of the 2 deletions and the substitution of i m m 21 for i m m ~ deletes the )~+ chromosome of approximately 21% of its DNA. Leftward transcription of the X chromosome is initiated at P,., rightward transcription at PR and late transcription from P'R. of D N A made by treating E . coli DNA with endo R . H i n d I I I were inserted at the site of breakage of the digested receptor chromosome. Phages were recovered from the ligase reaction (see section (i) below) b y transfection (see section (j) below) a n d the resultant plaques were harvested in the same way as plate lysates to provide 28 lysates each representing a sample of approximately 1000 plaques (Borck et al., 1976). Atrp-transducing phages were detected b y their ability to complement T r p - hosts and hence form "Trp + plaques" in the absence of exogenous t r y p t o p h a n (Franklin, 1971). The following auxotrophs were used to screen each of the 28 lysates for the 2trp phages : t r p A 8 8 ; t r p B 9 7 0 0 ; trpC10243, C9870; ~rpD159 and t r p E 5 9 7 2 . The htrp phages are described b y the fimetional genes incorporated; a ~trpC phage complements only gene G of the trp operon. Other phages used include the t r p - t r a n s d u e i n g phages ~trpBG2 (Murray & Brammar, 1973), r (Deeb et al., 1967) a n d the following donors of markers in the right arm of phage ~ : h r 8~ i m m ~ c I + ; h e 8~ immX ci857; h V8~ N a m T a m 5 3 c l A t 2 ; h r s~ c l A t 2 c r o l n i n 5 ; hO8~ ci857 n i n 5 Q a m 7 3 S a m T . Derivatives of the ~t/rp phages were isolated from crosses to the above hybrid phages b y selecting for h x a n d substitution of i m m 2z (turbid) b y i m m ~ (clear). (b) Bacterial strains These are listed in Table 1. (c) .Media The rich medium was L broth (Lemmx, 1955) containing (per 1): Difco Bacto Tryptone, 10 g; Difco Baeto yeast extract, 5 g; NaC1, 5 g; glucose, 1 g; adjusted to p H 7.2. Phage stocks for genetic analysis were prepared on L broth agar solidified b y Difco agar (10 g/l). Phage assays were made on Baltimore Biological Laboratories Trypticase agar, cont a i n i n g (per 1): Trypticase, 10 g; NaC1, 5 g; Difco agar, 10 g for plates a n d 6"5 g for top layers (Parkinson, 1968). EMBO agar contained (per 1): Difco Tryptone, 8 g; yeast extract, 1 g; NaC1, 5 g; Difco agar, 20 g. The p H was adjusted to 6"5 a n d 2 g K 2 H P O ; 0-4 g eosin and 0.065 g methylene blue added before sterilising. The minimal medium of Spizizen (1958) was used with glucose (0-2%, w/v) as carbon source and, for t r y p t o p h a n auxotrophs, Difco Bacto acid-hydi'olysed casein (0"05%, w/v) to provide all amino acids except tryptophan. Minimal plates contained New Zealand agar, 15 g/1. (d) General techniques The methods for preparing plating cells a n d phage stocks, assaying phage and carrying out phage crosses have been described (Murray et al., 1973). Stocks of htrp phages were sometimes made on t r p - d e l e t i o n hosts to prevent a n y illegitimate recombination events which might change the content of the trp operon carried by the htrp phages. The extent of the trp operon within the fragment of DI~A incorporated in a t r p - t r a n s d u c i n g phage was determined by transduction of a series of trp auxotrophs. A total of 109 ~trp phages 36

552

A. S. H O P K I N S , N. E. M U R R A Y AND W. J. B R A M M A R

TABLE 1 Bacterial strains Strain C60O W3110 Ymel ED8654 ED8149 KB8 QR47 QR48 KB3O trp strains

ED8520 ED8614 ED8740 ED8612 ED8801 ED8614

Relevant features

Source

supE, tonA F . W . Stahl sup ~ prototroph C. Yanofsky supS' A.D. Kaiser hsdR-, hsdM +, supE, supF trp( BE)V9, thyA endA, polA1, sup ~ J . P . Broekes supE E. Signer supE, recA E. Signer (trp-attCs~ C. Yanofsky trpA88, trpB9700 C. Yanofsky trpB51, trpB3 I. Crawford trpB21, trpB5 I. Crawford trpB15, trpB62 I. Crawford trpB12, trpB2 I. Crawford trpB18, trpB4 I. Crawford trpC10243, C9870 C. Yanofsky trpD159, trpD562 C. Yanofsky trpD1219, trpD876 C. Yanofsky trpD1538, trpE5972 C. Yanofsky trpE9914 C. Yanofsky (tonB-trpAC)V9 N. Franklin trp(DE)Vl02 C. Yanofsky trp(BE)V9trpRtrp(BE)V9trpR-, thyA trp(BE)V9trpR- (A) trp(BE)V9, hsdR-, supE, trpRtrp + derivative of ED8612 trp(OE)V1, supE

Reference Appleyard (1954)

Borck et al. (1976) Murray et al. (1973) Weft & Signer (1968) Weft & Signer (1968) Denney & Yanofsky (1974) Drapeau et al. (1968) Crawford et al. (1970) Crawford et al. (1970) Crawford et al. (1970) Crawford et al. (1970) Crawford et al. (1970) Yanofsky et al. (1971) Yanofsky et al. (1971) Yanofsky et al. (1971) Yanofsky et al. (1971) Yanofsky et al. (1971) Franklin (1971) Jackson & Yanofsky (1972b)

Murray & Brammar (1973)

Strains with ED numbers were constructed in this laboratory. were adsorbed to 0.2 ml of plating cells and after 20 min the infected cells were recovered b y centrifugation and resuspended in 1 ml 0.1 ~I-trisodium citrate (pH 7-0). Trp + transductants were selected by plating the infected cells on minimal medium supplemented with aeid-hydrolysed casein. (e) Phage lysates for D N A preparations To increase the titres obtained from lysates of the 2trp phages, derivatives giving clear plaques were isolated. Preparations of phage were made b y infection of exponentially growing cultures of E. coli C600 in L broth containing 10 -3 M-MgSO4. Growth was followed spectrophotometrically and when the adsorbance at 650 n m reached a m i n i m u m (usually about 2 h after infection) lysis was completed b y addition of CHCla (0"5 ml/1) a n d 15 rain later the lysate was clarified by centrffugation (10 rain at 10,000 g). Phages were recovered by eentrifugation, resuspended and treated with DNAase and RNAase (10 ~g/ml each, 2 h at room temperature), pelleted, resuspended and recovered by equilibrium centrifugation in 41"5~o (w/w) caesium chloride solution (Kaiser & Hogness, 1960): the caesium chloride step was repeated.

hTRP-TRANSDUCING

PHAGES

553

(f) D N A preparations Phage preparations were diluted to about 5 • 1011 to 5 x 1012 plaque-forming tmits/ml a n d dialysed against 10 rm~-Tris.HC1 (pH 8"0), 1 mM-EDTA. D N A was extracted b y gentle rolling with freshly distilled phenol (Kaiser & Hogness, 1960) followed b y dialysis against 10 mM-Tris.HC1 (pH 8.0), 1 m ~ - E D T A (4 changes in about 24 h). Bacterial DNA was prepared essentially as described by Marmur (1961), except t h a t the cells were lysed with lysozyme and Triton Xl00. Bacteria were gretna in 2 1 L-broth to a density of 2 • 108 cells/ml, a n d harvested b y centrifugation at 4~ The cells were resuspended in 48 ml of ice-cold 25% (w/v) sucrose in 0"05 M-Tris.HC1 (pH 8.0) a n d 6.9 ml of a freshly prepared solution of lysozyme (10 mg/ml in 0.25 ~-Tris.HC1, p H 8.0) were added. The suspension was shaken gently for 30 s at 37~ then placed on ice for 5 rain. Then 26 ml of ice-cold 0.25 ~ - E D T A (pH 8"0), were added, followed 5 min later by 54 ml of 2% (w/v) Triton Xl00, 0.05 ~-Tris.HC1 (pH 8.0), 0.063 M-EDTA to lyse the cells. Lysis was complete after 20 min on ice. The lysate was subsequently treated according to the Marmur procedure. (g) Enzymes and chemicals Pancreatic DNAase a n d RNAase were purchased from W o r t h i n g t o n Biochemical Corporation, Freehold, N.J., U.S.A. ; restriction endonuclease R . E c o R I was prepared essentially as described by Yoshimori (1971) and endo R . H i n d I I I was prepared as described b y I-I. O. Smith (Old et al., 1975) or Philippsen et al. (1974). Some of the endo R . H i n d I I I preparations used were generously provided b y Dr H. Cook or Mr R. Thompson. Lysozyme (Grade I egg-white enzyme) was obtained from Sigma Chemical Co., St. Louis, Me. 63178, U.S.A. T4 polynucleotide ligase was purchased from Miles Laboratories Inc., Elkhart, Indiana, U.S.A. Whenever possible chemicals used were of A R grade; caesium chloride and agarose were obtained from B.D.H. Ltd., Peele, Dorset, U.K. (h) Restriction endonuclease digestion and gel electrophoresis The q u a n t i t y of the restriction endonucleases required for complete digestion (37~ 30 min) of 1 ~g of h+DNA was determined in trial experiments with a series of digests, which were analysed b y electrophoresis in 1% (w/v) agarose gels (in 0.04 ~-Tris-aeetate, p H 8.0), containing 0.4 mg ethidium bromide/l; Sharp et al., 1973). For analysis of the products of digestion of the various I)NA preparations with the restriction endonucleases, samples of 1 to 2 tLg in about 20 td 0-01 ~-Tris.HC1 (pH 7.5), 0-01 M-MgC12, 0.01 M-2mercaptoethanol (and for R . E c o R I digests, 0"1 ~-NaC1) were incubated with the appropriate q u a n t i t y of enzyme at 37~ for 30 to 60 rain, heated at 70~ for 10 rain, cooled in ice, mixed with 5 ~l 50% (v/v) glycerol containing bromophenol blue (about 0.1%) concentrated to about 10 ~l in a v a c u u m desiccator a n d applied to weds in a n agarose slab gel (40 cm • 20 cm • 0"3 cm; Sharp et al., 1973) for electrophoresis, usually for about 18 h with a constant current of 40 mA. Gels were photographed under ultraviolet light on Ilford F P 4 film (4 • red filter), which was developed (9 rain at 18~ in Microphen (Ilford Ltd., Ilford, Essex, U.K.). The efficiency of restriction reactions was often assessed b y the decrease in plaqueforming ability on transfection of a suitable E. cell strain made competent b y starvation in CaC12 (Mendel & Higa, 1970) (see section (j) below). (i) Ligase reactions Equal quantities of restricted donor and receptor DNAs were mixed and incubated at 30~ for 15 rain to separate pre-annealed fragments. Reaction mixtures of 0.2 to 0-5 ml containing total DNA, 10 to 30 ~g ml, 66 mM-Tris.HC1 (pH 7.5), 1 mM-EDTA, 10 m_~-MgC12, 100 rm~-NaC1, 10 mM-dithiothreitol, 0.1 mM-ATP, 0.1 mg bovflm serum albumin/ml, T4 polynueleotide ligase (Miles Laboratories Ltd.), 0"5 units/ml, were incubated at 10~ for 6 h and then kept on ice for 2 to 10 days during sampling for transfectants. The m a x i m u m yield of plaques was usually obtained after 4 to 6 days.

554

A.S.

HOPKINS,

N. E. M U R R A Y A N D W. J. B R A M M A R

( j) Transfection of E. eoli to recover recombinant phages Cells (usually ED8654, an hsdRK-hsdMK+SUp-~ derivative of the M e t - strain 803) comp e t e n t for transfection were obtained b y growing in P m e d i u m (Kaiser, 1962) supplem e n t e d with glucose (1 mg/ml) a n d methionine (100 ~g/ml). The cells were starved in 0-1 M-CaC12 a t 0~ as described b y Mandel & Higa (1970). Cold D N A solutions (3 ~g/ml in 0"15 M-NaC1, 0.015 M-sodium citrate, p H 7"0) were m i x e d with the suspension of cells in CaC12 in the ratio 1 : 2 (v/v), placed in a w a t e r b a t h at 37~ for a b o u t 30 s and returned to all ice-bath for a t least 1 h before mixing with top layer agar and plating (A. Jacob a n d S. Hobbs, personal commtmication). (k) Electron microscopy D N A heteroduplex molecules were m a d e a n d observed in the electron microscope according to methods described b y Davis etal. (1971). The grids were visualized in a Siemens Ehnlskop 1A electron microscope a t an accelerating voltage of 80 kV and a magnification of 40,000 • (1) Enzyme synthesis during phage infections Infection of thy- strains b y htrp phages 1ruder conditions preventing replication of phage D N A were carried out as described b y F r a n k l i n (1971). Infected cells were incubated with vigorous aeration at 37~ W h e n replication of the phage D N A was desired, thy + hosts were used a n d the procednre was modified as described b y Moir & B r a m m a r (1976). Sampling and preparation of extracts of infected cells for enzyme assays were as described b y F r a n k l i n (1971). (m) Enzyme and protein assays Cell extracts were k e p t on ice a n d assayed as soon as possible for enzyme activities. Anthranilate synthetase a c t i v i t y was assayed as described b y I t o etal. (1969), using ~-glutamine as amino donor. Assays were carried out using a Locarte fluorimeter MKd, with an excitation wavelength of 313 n m and an emission wavelength of 386 ran. Reactions were carried out at 37~ in a water-heated cuvette holder. One tulit of anthranilate synthetase catalyses the formation of 0.1 t~ tool anthranilate in 20 rain at 37~ Specific activities are expressed as mlits of enzyane a c t i v i t y per mg protein. T r y p t o p h a n synthetase was assayed as the ~2fl2 complex b y the conversion of indole to t r y p t o p h a n as described b y Smith & Yanofsky (1962). One unit of t r y p t o p h a n synthetase causes the disappearance of 0.1 ~mol indole in 20 min a t 37~ Protein was measured b y the m e t h o d of Lowry etal. (1951), using bovine serum albumin as a standard.

3. Results (a) Isolation and classification of ~trp phages

~trp-transducing p h a g e s were i s o l a t e d from m o s t (24) o f t h e 28 l y s a t e s a n d classified according to t h e f u n c t i o n a l trp genes c o n t a i n e d ( B o r c k etal., 1976). T h e 40 ~trp p h a g e s i n c l u d e d 17 )~trpA phages, 13 ~trpC p h a g e s a n d 8 ~trpE phages. A few l y s a t e s y i e l d e d all t h r e e m a i n elasses o f ht~o phages. N o AtrpD p h a g e was d e t e c t e d b u t t w o p h a g e s earried t h r e e genes of t h e trp operon, trpA, trpB a n d trpC, a n d p r e s u m a b l y r e s u l t e d f r o m i n c o m p l e t e d i g e s t i o n of t h e d o n o r D N A b y endo R . H i n d l I I ; ,~trpABC19 is such a p h a g e where A , B a n d C refer t o t h e f u n c t i o n a l trp genes i n c o r p o r a t e d a n d 19 refers t o t h e l y s a t e f r o m which this p h a g e o r i g i n a t e d . T h e ,~trpA p h a g e s d i d n o t c o m p l e m e n t t h e tonB lesion o f t h e tonB-trp d e l e t i o n s t r a i n K B 3 0 , since lysogens of ~trpA in t h i s s t r a i n r e m a i n e d T o n B - . S i m i l a r l y , t h e htrpE p h a g e s d i d n o t i n c o r p o r a t e a f u n e t i o n a l eysB gene.

ATRP-TRANSDUCING

PHAGES

555

(b) Orientation of the trp genes within the transducing phages The fragments of E. coli DNA can be inserted into the receptor chromosome in either orientation to produce two classes of ,~trpE, ~trpC or AtrpA phages. The orientation of the trp gene within a ,~trp phage was deduced from crosses to a well-characterized (Deeb et al., 1967; Franklin, 1974) 4 8 0 t r p A B C D E phage (r trpl90). The rationale was t h a t from a cross between a lambda trp phage and a 480 trp phage, h a i m m q~e~ recombinants would originate from genetic exchanges within the inserted E. coli DNA. Recombination between the hr 6~

trpABCDE

480trp190

t

)~trp i m m 21 . . . . . . . . . . 9. . . . . . . . . . . . . h a (srI~l-2)V

i m m ~8~ r

,. . . . . . . . . trp

,

!

.

-

i m m 21

-

nin5

phage I)NAs would be infrequent, particularly so, because the known regions of homology (Fiandt et al., 1971) are deleted either in the )l receptor chromosome by the loss of DNA between srI,~l and srI,~2 or in the 480 trp phage by the substitution of E. coli DNA for the red genes of phage 480. The crosses (Table 2) clearly divided the htrp phages into two classes; those which provided homology as detected by recombination frequencies of 10-4 or greater, and those which apparently provided no more homology than the receptor phage itself, resulting in very low recombination frequencies (less than 10-6). TABLE 9,

Frequencies of ha immr 8o recombinants f r o m

crosses with 480trpABCDE

Phage

Recombination frequency

Phage

~trpA 13 ,~trpC1 herpC6 htrpO8 ,~trpC9 ~trpC]2 htrpC15 ,~trpC 17 MrpC20 MrpC27 ~trpE 1 )ttrpE3 htrpE5 ;ItrpE7 ,~trpE8 AtrpE l O h receptor

--t --t --t --t --t --r --t -- r --r --r --r --?

AtrpA1 ;ttrpA6 ~trpA8 AtrpA9 AtrpA17 )ltrpABC19 htrpC2 htrpClO )ltrpC19 ~trpE17

--r --~ --'~ --'~ --r

No recombinant detected among at least 10e progeny.

of htrp

imm 21 phages

Recombination frequency 1-9 • 1.7 x 2.6 • 1.5 • 3.8 • 2.6 • 6.1 • 1"6 • 1.3 • 3.9 •

10 -a 10-a 10-s 10 -s 10 -4 10-a l0 -4 10-a 10 -a 10 -~

666

A . S . H O P K I N S , N. E. MURRAY AND W. J. BRAMMAR

We suggest t h a t members of the first class of Atrp phages have incorporated E. coli trp genes in the same orientation as the r t~7~ phage, whereas those of the second class have theh' trp genes in the reverse orientation. An essential distinction is that, while in the first class the inserted trlo gene is transcribed from PL on t h e / - s t r a n d , as are the t~o genes in r ill the alternative class the trp gene must be transcribed from the r-strand. We use the abbreviations Atrlo(A) z and Atrp(A) r to distinguish /;hese two orientations. The AtrpA, AtrlJC and AtrpE phages were each subdivided into two classes of which Atrp(E)Zl7 and htrp(E)rlO; Atrlo(C)qO and Atrp(C)~8; and Atrp(A)Z9 and Atrp(A)rl3 were selected as representatives. The opposing orientations of the inserted fragments were confirmed b y electron microscopy of heteroduplex molecules. Where two Atrp phages of apparently identical complementation behaviour, but of opposite orientation, were used to m a k e heteroduplex molecules, a region of single-stranded non-homology was detected (see Fig. 2). The single-stranded loops were of the same lengths and originated at the appropriate position on the phage chromosomes. Since the isolation of the Atrp phages relied on complementation of T r p - strains of E. coli, the incorporated trp genes can be expressed, irrespective of their orientation within the phage genome.

Fro. 2. A heteroduplex molecule of Atrp(E)ll7 and Atrp(E)rlO. To the right of the photograph is a tracing of the molecule; single-stranded regions are indicated by dotted lines.

ATRP-TRANSDUCING

PHAGES

557

(c) Genetic mapping of the targetsfor endoR.HindlII in the trp operon The htrpE and ~trpC phages were presumed to contain part of the trpD gene, while the ~trpC and ~trpA phages were each expected to contain some of the trpB gene. The extent of the trpB and trpD genes carried by the ~trp phages was determined by their ability to transduce trpB and trpD auxotrophs to prototrophy (Table 3). The trpE phages recombined with only the closest trpD allele (trpD159), while the ~trpC phages recombined with all the trpD markers other than trpD159. At least one target for endo R.HindIII is defined between trpD159 and trpD562 (see Fig. 3). The ~trpG phages recombined with only the closest trpB marker (trpB4), while the ~trpA phages recombined with the remainder of the trpB alleles available. A second target for endo R.HindIII is thus defined in trpB between trpB4 and trpB18 (see Fig. 3). All available trp markers recombined with one of the three main classes of ~trp TABLE 3

Genetic mapping of the content of the 2trp phages Recipient bacteria Donor phage

trpA88

trpB18

trpB4

trpG/G

trpD562

trpD159

trpE5972

Receptor

O ~ 500 ~ 500 > 1000 0 0 0 O

0 106 224 > 1000 0 0 0 O

0 0 0 > 1000 25 36 0 0

0 0 0 > 1000 ~ 1000 > 1OO0 0 0

0 O 0 400 167 246 0 0

0 0 1 0 0 0 ~ 500 ~ 500

0 0 0 0 0 O > 500 ~ 500

trp(A)'9 trp(Ay13 trp(ABC)'19 trp(O)'lO trp(C)rl8 trp(E) z17 trp(E)rlO

T h e d a t a a r e e x p r e s s e d as t h e n u m b e r s o f T r p + colonies p e r 0.1-ml s a m p l e of i n f e c t e d cells. kfrpA9 or 13 k ?rl~4BCI9 i ~.t~8

(po) La

rrpE

I

I

trpD

T P~

Anf hrmilote synthetose

or I0

I

kfrpc"10 or 17

Phosphoribosyl ont hronilole tronsferose

t~pc 10:)43

Indole glycerol phosphate synthetose

L

~rpB i i

.r

t~pA

18 I:) 21 15 51 33/88

I 96

Tryptophon synthetose

Fro. 3. T h e g e n e t i c c o n t e n t o f t h e Atrp p h a g e s . T h e e x t e n t s o f t h e f r a g m e n t s i n c l u d e d in t h e Atrp p h a g e s a r e i n d i c a t e d a b o v e t h e g e n e t i c m a p o f t h e trp o p e r o n of E. cell ( Y a n o f s k y et al., 1971; I. Crawford, p e r s o n a l c o m m u n i c a t i o n ) . (po)La, left o f t h e trpE gene, a r e t h e r e g u l a t o r y e l e m e n t s o f t h e t r p operon, t h e p r o m o t e r (p), o p e r a t o r (o), t h e l e a d e r r e g i o n (L) a n d t h e a t t e n u a t o r (a) ( B e r t r a n d et al., 1976); P 2 w i t h i n t h e trpD gene, is a n i n t e r n a l c o n s t i t u t i v e p r o m o t e r ( J a c k s o n & Y a n o f s k y , 1972a). T h e s e d a t a (Table 3) l o c a t e o n e t a r g e t for e n d o R.HindIII in t h e trpD g e n e b e t w e e n m u t a t i o n s 159 a n d 562 a n d a s e c o n d t a r g e t in t h e o p e r a t o r - p r o x i m a l r e g i o n o f t h e trpB g e n e b e t w e e n m u t a t i o n s 4 a n d 18.

558

A. S. H O P K I N S ,

N. E . M U R R A Y

A N D W. J . B R A M M A R

phages : these data are consistent with the DNA of the E. coli trp operon containing only two targets for endo R.HindIII, one in each of the D and B genes. (d) Analysis of fragments resulting from digestion of DNA with endonucleases DNA from each of the six classes of ~trp phages was digested with endo R.HindIII, with endo R.EcoRI and with both of these enzymes. The resulting fragments of DNA were separated by electrophoresis on agarose gels (Fig. 4). The 2trp phages of like eomplementation behaviour, but of opposite orientation, gave identical patterns of fragments. The DNA from each of the htrp phages gave only three fragments when digested with endo R.HindIII, and those of differing complementation characteristics differed only in the size of the acquired fragment. From their mobilities the molecular weights of the inserted fragments are estimated to be 3.8 • 106 for ,~trpE, 1.9 • 106 for 2trpC and 2.0 • 106 for 2trpA. The DNAs from the receptor phage and the ~trp phages differ in only a single fragment following digestion with endo R.EcoRI. These variable fragments comprise the phage DNA between targets stir2 and srI,~3 plus any incorporated E. coli DNA (see bottom of Fig. 4). Digestion with endo R.HindIII in addition to endo R.EcoRI generates the inserted trp fragment from each of the 2trp phages. All of the phages yield a small Fragments

DNA

x~mz xtmc xtrp~

I[ II II

9Receptor

lI

XtrpA xtrpc. xtrpr

I I I

Receptor

I A

XtrpA

I

xtmc

I

XtrpE

I

Receptor

I A

EnzymeS

Hind Ill

i ! I I

B

I I

I I

I

I

I

I

C

I I I I

Eco RI

DE

I 1 I I

C k

III 11 II II

III

Hind]]I + Eco RI

P

DE

Receptor Torgets I| shnX srIX

3 i

FragmentsI

A' A

A

I

I

B' B

p

C

E

D

k C E D

Hind'fiT EcoRI

/~nd]I[+EcoRI

FIO. 4. Electrophoretic analysis of restriction enzyme digests of DNA from the Atrp phages and the receptor chromosome. Since Atrp phages of like complementation behaviour gave identical patterns, the data are presented for only 3 classes of htrp phages, AtrpA, ~trpC and AtrpE. Atrp phages digested with either endo R.HindIII, endo R.EcoRI or both of these enzymes, differed from the receptor phages with respect to only a single fragment; these fragments, indicated as dotted lines, m u s t contain the ~rp genes. The fragments of the receptor chromosome are identified by a letter immediately below each set of analyses. The origin of these fragments is indicated from the fragmentation patterns of the receptor chromosome presented at the foot of the diagram.

)~TRP-TRANSDUCING PHAGES

559

fragment (p) defined b y srI,~2 and shnA3 and a fragment (k) defined by shnA3 and srL~3 (Murray & Murray, 1975). The retention of these small fragments flanking shn)~3 is consistent with the origin of the )~trp phages b y the insertion of a discrete fragment of DNA at shn,~3 : the cohesive ends of the severed restriction target (shnA3) generating two new targets for endo R.HindIII. Since no target for endo R.EcoRI was detected in any of the three inserted fragments of DNA, the fragment of DNA containing the trp operon in an endo R.EcoRI digest of E. coli DNA must have a molecular weight greater than 7.7 • 106.

(e) The construction in vitro of AtrpCDE and AtrpABC phages DNAs from the phages ,~trp(E)q7 and ,~trp(C)q0 were digested with endo R.Hind I I I and the fragments recombined in vitro using polynucleotide ligase. Recombinant phages were recovered by transfection and phages able to complement a trp(ED)V host were selected. Four such phages were isolated, all of which complement lesions in trpE, trpD and trpC but not in trpB or trpA. One of the four phages was shown to have the trp genes in the same orientation as r (see section (b), above) and is designated ,~trp(CDE)~; the others had their trp genes in the reverse orientation and are designated Atrp(CDE) r. The restoration of the trpD gene confirms that the genetic content of this gene resides in the AtrpC and ~trpE phages. The simplest interpretation, t h a t the trpD gene contains a single target for endo R.HindIII, is strengthened by the recovery of both Atrp(CDE) I and AtrT(C.DE)r phages, showing t h a t the parental DNAs must have been well-fragmented. The construction of both classes of AtrpCDE phages facilitates study of the expression of the trpE gene, since the normal assay for the trpE gene product, anthranilate synthetase, is dependent on both the trpE and the trpD gene products (Ito & Yanofsky, 1966). DNAs from the phages Atrp(C)qO, ,~trp(C)T8 and trp,~(A)113 were digested with cndo R.HindIII and the fragments recombined in vitro using polynucleotide ligase. From each of the AtrpA q- AtrpC combinations, recombinant phages able to complement a trp(ABC)V host were recovered; endo R.HindIII probably splits the trpB gene into two fragments. (f) The construction in vivo of AtrpABC and AtrpCDE phages Crosses were made between htrp(A)19 and/ttrp(C)qO and a selection imposed for AtrpABC recombinants. Such recombinants were detected at a low frequency (10 -5 to 10 -6) as Trp + plaques on a trT(CBA)V host or as trpB + plaques on the same host using plates supplemented ~dth indole. Further crosses were made between other pairs of AtrpA and AtrpC phages and between pairs of AtrpC and AtrpE phages. Where the pairs of phages were of like orientation, the former crosses yielded ,~trpABC recombinants and the latter crosses yielded htrpD + recombinants, which were shown to be trpA-, B - , C +, D +, E +. In contrast, crosses between phages of opposing orientations failed to reconstitute the selected section of the trp operon. Our interpretation of the structure and origin of the Atrp phages implies the restitution of the trpB, or trpD, gene by recombination either within short homologous sequences provided b y the HindIII targets or, alternatively, mediated via the homologous DNA of the host's trp operon. The relevance of chromosomal homology was determined by repeating the crosses

560

A.S.

HOPKINS,

N. E. MURRAY

AND

W . J.

BRAMMAR

in a host deleted for the trp genes, trp(EDGB)V9, and in a Trp + derivative of this strain. AtrpABG and AtrpGDE recombinants were detected only from those crosses made in the Trp + host. AtrpABG, but not ~trpGJDE, recombinants were isolated from crosses made in a trp(ED#V host. The origin of these recombinants appears to be dependent on homology with the host chromosome and this implies a mechanism in which the restitution of the trpB, or trpD, gene is mediated b y recombination with the host's try operon. I f this is so, a ~trpB + recombinant should frequently acquire host DNA sequences from the region flan~iug the trpB gene. This prediction was tested b y making crosses between AtrTA and ~trpG phages in a host in which the trpC gene was marked b y two amber mutations. ~trpB + recombinants from this cross had frequently incorporated amber mutations into their trpG gene. We conclude t h a t the host chromosome can provide the homology to allow recombination between transducing phages carrying non-overlapping, closely linked sequences of the bacterial genome. AtrpB + recombinants were isolated from crosses between Red + phages in a recA(QR48) host or from crosses between R e d - phages in a Rec + (QR47) host but not in the absence of both the Rec and Red pathways. Attempts to reconstitute the complete trp operon from crosses between either AtrpA and AtryE, or AtrTA and ~tryGDE, failed, presumably because there is not enough space in the phage to accommodate all the bacterial DNA from the three ~ry phages. However, from a cross between ~trpA and ~trpE made in a trp(ED)V host, ~trTB + recombinants were isolated and shown to be trpA +, B +, G +, D - , E - . Bacterial homology, which in the case of the AtryE phage must be outside the structural genes of the try operon, permits the incorporation of the partially deleted trp operon. This isolation of tryB + recombinants from a cross between AtryA and AtryE phages demonstrates that the host chromosome can serve to restore the link between two non-contiguous segments of bacterial DNA carried within the ~trp phages. The implied mechanism of in vivo recombination should produce ~tryABG and AtrpGDE phages which comprise only the receptor chromosome and the appropriate fragments of the try operon. DNA was isolated from two such phages including the Atry(ABG) l phage used for studies of expression from the phage promoter, PL. The DNA was digested with endo R.HindIII and with both endo R.HindIII and endo R. EcoRI. The recombinants contained only those fragments present in the parental phages; the characteristic fragments p and k flanking the inserted DNA (see Figs 4 and 5), the latter of which contains the phage attachment site, were retained. The AtrpD+ recombinants from crosses between AtryG and AtryE unexpectedly included some tryG +, E - phages. These were found when the crosses were made in a host deleted for the try genes even though, as in all the crosses described, the phages were propagated in a trp(BE)V strain. Jackson & Yanofsky (1974) have shown t h a t the distal two-thirds of the tryD gene are sufficient to specify a polypeptide having phosphoribosyl anthranilate transferase (PRATase) activity. These authors selected deletions which fused the distal part of the tryD gene into the trpE gene b y demanding the restoration of PRATase activity to a tryD- strain t h a t lacked it. The anomalous ~trpG+D+E- phages require that the distal part of the tryD gene is translated, in phase, from a newly acquired initiation sequence. Either illegitimate recombination or mutation could generate such phages. Analyses of the fragments resulting from the digestion of DNA from AtrpG+D+E- phages, and preliminary genetic evidence: are consistent with both mechanisms contributing to the origin of these phages.

E

+

hi

I

i ~0 ,.o~

o

m ~

e.i

~ ~.~

!

'

I

E

~

o

~

o

o

~

~o~ 2~

Z 0

'If

9 ID

562

A.S.

HOPKINS,

N. E. MURRAY

AND

W. J. BRAMMAR

(g) Expression of the trp genes from the prophage

A lrlgE- host lysogenic for either Atrp(E)q7 op htrp(E)rlO is prototrophic, as expected if the trp promoter were present in the fragment of DNA that includes the trpE gene. Conclusive evidence for the trp promoter is presented later (section (h), below). The ~t~TC phages (see Fig. 3) must include the internal constitutive promoter located at the operator-distal end of the trpD gene (Jackson & Yanofsky, 1972a). The complementation of a trpC- host on lysogenisation by either htrp(C)q0 or ~trl~(C)r8 is readily explained by expression of the trpC gene from this promoter. The DNA fragment containing trpA does not include a known promoter for this gene. TrpA- bacteria lysogenic for 2trp(A)19 remained auxotrophic, although even in a recombination-deficient host they segregated prototrophie derivatives. Such prototrophs could result from base changes, deletions, or insertions that create a new promoter for transcription of the trpA gene. Since ~trp(A) l phages were very readily isolated as Trp + plaques, the expression of the trpA gene probably occurs from PL during the lytic cycle. TrpA- bacteria lysogenic for 2trp(Ay13 grew slowly in the absence of tryptophan, they very readily segregated prototrophic derivatives and, in contrast to the 2trp(A): lysogens, grew well when one drop of broth was added to the top layer of a minimal plate supplemented with aeid-hydrolysed casein. This very weak expression of the trpA gene may be from a constitutive promoter in the b2 region of the ~ genome. (h) The fragment containing trpE also carries the trp promoter The DNA fragment in the 2trpE phages is much larger than the 2000 base-pairs of the trpE gene and its control region. Formal proof that this fragment includes the t~T promoter and operator was derived from the tryptophan-dependent repressibility of trp gene expression during infection of a trpR + host. Nam7am53 derivatives of ~trp phages having the trpEDC genes in either orientation were used to infect a suppressorfree, trpR + host in the presence of low (2/~g/ml) or high (200 ~g/ml) concentrations of L-tryptophan. In these experiments, where trp gene expression from P~. was eliminated by the N mutations, the high rate of expression obtained in the presence of 2 /~g L-tryptophan/ml was severely repressed by high concentrations of the amino acid (Fig. 6). We conclude that the 2trpE phages, irrespective of the orientation of the DNA fragments containing the trpE gene, carry a functional trp promoter and operator region. (i) Transcription from PT. proceeds beyond art The availability of phages with trp genes inserted at shn23 allows one to determine whether N-dependent transcription initiated at P~. proceeds beyond the art region of the phage, cro- derivatives of htrp(CDE) l and 2trp(ABC) z were used to derepress transcription from P,., and expression of the trp genes was followed during infection of trp- hosts in the presence of repressing concentrations of tryptophan, trp gene expression was detected from both 2trp phages in a h-sensitive host (Fig. 7) but not in an immune host. The rate of expression of the trpA and B genes from ~trp(ABC) I was similar to that from the control phage, 2trpBG2 cro-, in which there is no att region between PT. and trp. The latter phage, unlike ,~trp(ABC) l, contains the trp attenuator (Squires et al., 1976), a transcription-termination signal located between the promoter and the structural genes of the trp operon (Bertrand et al., 1976). The

hTRP-TRANSDUCING PHAGES

563

1"6 A c

r

0,8

~

0-4

20

I0

50

Time offer infection (min)

Fro. 6. Anthranilate synthetase formation by ~trpCDE 1V- is repressed by tryptophan. htrp(CDE)ZlVam7am53cIAt2 and )ttrp(GDE)T1Vam7am53cIAt2 were used to infect the suppressorfree, trp-, thy- host strain, ED8149 at a multiplicity of 2 phage/cell, in the presence of 2 gg (solid lines) or 200 gg L-tryptophan/ml (dashed line). Phage DNA replication was prevented by thymine starvation. (O), ,~trp(CDE)tNam7am53cIAt2 ; ( 9 ~ttrp(CDE)rNamTam53cIAt2. .c =

16

0

2-0 --

A

== a

8

g

4 O

5

10

15

Time otter infection (rain) (o)

20

5

10

15

20

Time offer infection (min) (b)

FIG. 7. Trp gene expression from PL of ~trp(ODE)l and htrp(ABC) z. htrp(ABC)~clAt2crol, ~trp(ODE)lclAt2crol and AtrpBG2clAt2crol were used to infect the trp(BE)v, thy- host strain, ED8149, at a multiplicity of 2 phage/cell, in the presence of 200 /zg T.-tryptophan/ml to repress expression from the trp promoter. Replication of phage DNA was prevented by thymine starvation. (a) Tryptophan synthetase formation coded by the trpA and B genes of htrp(ABC)ZclAt2croI ( - - / k - - / k - - ) and htrpBG2clAt2crol ( - - 0 - - 0 - - ) . (b) Anthranilate synthetase formation coded by the trpD and E genes of Atrp(ODE)lclAt2crol ( - - / k - - / k - - ) and ~rpBG2cIAt2crol ( - - 0 - - 0 - - ) . s i m i l a r r a t e s of expression o f t h e trp genes f r o m t h e PL p r o m o t e r s o f t h e s e t w o p h a g e s suggests t h a t t r a n s c r i p t i o n a c t i v a t e d b y t h e l a m b d a N - p r o t e i n o v e r r i d e s this t e r m i n a t i o n signal. T h e s a m e conclusion h a s been r e a c h e d b y F r a n k l i n & Y a n o f s k y (1976). I n c o n t r a s t t o t h e a b o v e result, t h e r a t e o f expression o f t h e trpD a n d E genes f r o m htrp(CDE)' was m u c h lower t h a n t h a t f r o m t h e c o n t r o l phage. This difference can b e e x p l a i n e d b y t h e e x i s t e n c e o f a f u r t h e r t r a n s c r i p t i o n - t e r m i n a t i o n signal, p r e c e d i n g t h e trp operon, t h a t is o n l y p a r t i a l l y o v e r c o m e b y t h e )~ N p r o t e i n ( F r a n k l i n , 1974). This sequence, which we w o u l d suggest is p r e s e n t in t h e ,~trpE phage, w o u l d n o t be p r e s e n t

564

A. S. H O P K I N S , N. E. MURRAY AND W. ft. BRAMMAR

in AtrpBG2, which does not contain the t~o promoter (Davison et al., 1974; Squires et al., 1976). The int gene of phage lambda can be expressed from a weak promoter located in or near the xis gene (Shimada & Campbell, 1974), and it has recently been suggested t h a t the expression of int from this promoter m a y be activated b y lambda c I I and c l I I products (Oppenheim, 1976). Since this promoter is present in our Atrlo phages, we tested whether it contributed to the expression of the trp genes during phage infection. AtrIg(ABG) z was used to infect a lambda-lysogenic, tr~(EDCB)~" s ~ a i n in the presence and absence of 2imm 434 cI to provide the c I I and v i i i gene products. T r y p t o p h a n synthetase formation was not detected after 30 minutes of infection, with or without helper phage. The int promoter could not be making a significant contribution to the levels of t r y p t o p h a n synthetase obtained on infection of a sensitive host; the transcription detected is presumed to initiate at PT.. I t is clear from these data t h a t N-dependent transcription from P~, proceeds unabated beyond art into the central region of the lambda genome. (j) Convergent transcription of the t r p genes Since transcription from PT. can proceed beyond art into the trp genes located at shn23, it follows t h a t trp genes in the r orientation at this position can potentially be transcribed in either direction. We have investigated the outcome of this possibihty b y following the expression of the trpD and E genes, transcribed rightwards from the trp promoter, in the face of leftward transcription from PL. When ~trp(EDC) ~ cIAt2crol infected a )~-lysogenic host, trp expression from Pt~p

A r

~. 0"2

c m

0 o

o

r

io

o

~6

a)

c

0.1

I

5

I

I0

I

15

Time ofter infection (rain) (a)

I

20

5

I0

15

20

Time after infection (rain)

(b)

FIG. 8. Converging transcription of the ~rp genes of Xtrp(GDE)'. (a) Xtrp(CDE)rclAt2crol or (b) Xtrp(CDE)rclAt2cro+ were used to infect the h-sensitive host, ED8614 ( - - A - - A - - ) or the X-lysogenle host, ED8740 ( - - O - - 9 at multiplicities of 2 phage] cell, in the presence of 200 ~g T.-t.rvptophan]ml. Phage DNA replication was prevented in the ED8614 host by thymine starvation, and in the ED8740 host by the presence of the lambda repressor. Expression of the phages' trp genes was derepressed by the presence of the trpR mutation in both host strains.

ATRP-TRAI~SDUCING

PHAGES

565

proceeded at a high and constant rate (Fig. 8). During infection of a lambda-sensitive host, however, when transcription from PL can proceed, trl~ gene expression started normally, but was severely decreased after about five minutes. Infection using a cro +, ~trla phage showed a similar turn-off, with a suggestion that trl~ expression began to recover after about 15 minutes, when leftward transcription from PT. would be moderated by the action of the oro- product. Clearly, powerful leftward transcription from PT. interferes with productive expression of the trp genes in the opposing orientation. The mechanism of this inhibition remains to be investigated. (k) trp gene expression from replicating ~trp genomes l~Iethods have recently been described for optimising the production of enzymes coded by genes of transducing phages (Moir & Brammar, 1976). To demonstrate that these approaches are applicable to transducing phages constructed in vitro, we have monitored anthranilate synthetase formation coded by N - and Q-, S- derivatives of AtrpEDC phages. ~lrpEDC Nam7am53 and ,~trTEDG ci857 Qam73Sam7 were used to infect a suppressor-free, trp(EDCB)v, trpR- strain under conditions allowing replication of phage DNA. Despite the absence of active N-protein, N - derivatives of lambda can express their 0 and P genes and replicate their DNA, though at a reduced rate (Ogawa & Tomizawa, 1968). Anthranilate synthetase formation continued at a high rate for several hours after infection (Fig. 9) and very high yields of the enzyme were eventually obtained. Knowing the specific activity of pure anthranilate synthetase, we can calculate that after five hours of infection by klrpEDC Nam7am53, this enzyme represents approximately 25% of the total soluble protein of the infected cells. Parallel infections by ~trl~(EDC) l and 2~trp(EDC)" show no detectable difference in

c

60

~

4o

c

Q, o

=

20

I

2

3

4

5

Time ofler infecficn(h) FIe. 9. Formation of anthranilate synthetase during prolonged infection b y N - , a n d Q - , S derivatives of Atrp(CDE)L The t r p R - host strain, ED8520, was infected with Atrp(ODE)~Na~n 7am 53 clAt2 ( - - O - - O - - ) or Atrp(@DE)lcI857Qam73Sam7 ( - - A - - A - - ) at multiplicities of 2 phage/cell, under conditions allowing replication of phage DNA. During the first 1 to 2 h after infection the levels of anthranilate synthetase achieved from N - phages frequently lag behind those from ~r+Q- phages. This is expected, since N - phages replicate more slowly t h a n 2~r+ phages (Ogawa & Tomizawa, 1968) ; however, with some .N - phages (as above) no lag was detected.

566

A . S . HOPKINS, N. E. MURRAY AND W. J. BRAMMAR

anthranilate synthetase production. The orientation of an inserted fragment of DNA is clearly unimportant for successful expression of genes within that fragment from their own promoter in an N - phage.

4. Discussion Three main classes of ~t~ phages, each containing one functional gene of the trp operon, were defined by complementation tests. The formation of these htr~ phages by the insertion of a single fragment of DNA into the receptor chromosome is supported by gel analyses of the endo R.HindIII digests of their DNAs; these analyses would not, however, exclude additional fragments of less than 250 nucleotides. We propose that endo R.HindIII breaks the trp operon of E. coli into three fragments. The main evidence in support of this is the reconstitution of the trpD gene by recombination in vitro between fragments of DNA from )ttrpC and )ttrpE phages, and the restoration of the trpB gene from the DNA of ~trpA and htrTG phages. Alternative explanations require that deletions of small segments of DNA within the trpB and trpD genes are without phenotypic effect, or that the ~trp phages frequently comprise two fragments of donor DNA. The simplest interpretation is that the trpB and trpD genes each have a single target for endo R.HindIII, and that only two out of 40 phages isolated originate from incompletely digested DNA. The restitution of the trpB gene by recombination in vivo between ~trpA and ~trpC phages requires chromosomal homology. This implies an absence of overlapping DNA sequences in the bacterial components within these transducing phages. The host (Rec) or phage (Red) recombination systems are both effective in generating recombinant phages in which markers from two phages and the host chromosome can be included. This recombination in vivo provides a ready means of incorporating chromosomal markers into transducing phages and of extending the genetic content of the transducing phages to include non-overlapping, or even non-contiguous, closely linked regions of the E. coli chromosome. Each of the three classes of ,~trp phages was subdivided into those phages in which tile incorporated trp gene was transcribed from the/-strand and those in which .the trp gene was transcribed from the r-strand of the phage genome. Other workers (Bovre & Szybalski, 1969; Hendrix, 1971; Nijkamp et al., 1971) have shown that transcription from PL can traverse the phage attachment site and it was expected that any gene whose message is transcribed from the/-strand of the phage would be expressed from P~. unless transcription were stopped by a signal that impedes RNA polymerase even in the presence of the/V-gene product (Franklin, 1971,1974; Adhya et al., 1974). The expression of tryptophan synthetase from PL in a Atrp(ABC)Zcro phage paralleled that from the control phage 2trpBG2cro-. The quantitative agreement between these two phages permits the conclusion that transcription proceeds undiminished through the attachment region. These data also confirm the finding that the transcription stop within the trp regulatory elements, the trp attenuator (Bertrand et al., 1976), is not a barrier to transcription that is activated by the lambda N protein (Franklin & Yanofsky, 1976). Earlier data based on hybridisation of mRNA indicated a lowered rate of transcription left of att. As Szybalski (1971) has discussed, this may result from the preferential instability of the b2 message. The phage promoter P~. can be used for expression of genes in the b2 region just as readily as it can for those in the classical trp-transducing phages (Franklin, 1971,1974;

~ T R P - T R A N S D U C I N G PHAGES

567

]~Ioir & Brammar, 1976). The expression of the genes coding for anthranilate synthetase from P~. in ~rlo(GDE)lcro- phages was shown to be approximately tenfold lower than that from the control phage ~trpBG2cro-. This is consistent with the inclusion of a transcription termination signal in the DNA fragment containing trl~E which is only partially over-ridden by the action of the ~ N-gene product (Franklin, 1974). Our data show that the trp genes can be expressed from the phage promoter Pv. when they are inserted in the/-orientation. All three classes of phage were also found in which the fragment was in the alternative orientation. The fragment containing the trloE gene was shown to include the trio operator and promoter, and this gene could be expressed from its own promoter irrespective of its orientation. Similarly, the fragment containing the trloC gene includes the weak constitutive promoter in the trIoD gene (Jackson & Yanofsky, 1972a) and the trl~C gene can be expressed from this promoter in either orientation. In contrast, the trloA gene is separated from both of the known trl~ promoters and yet a ~trlo(A) r phage was isolated. Attempts to detect the trpA protein on infection of a TrpA- host with a ~trl~(A)rSam7 phage were unsuccessful, but this does not rule out a low level of transcription from P'R. Lysogens of 2trlg(A) r in a TrpA- strain do grow a little in the absence of tryptophan and expression from a weak constitutive promoter in the b2 region may suffice to provide enough rloA product to detect complementation of a TrpA- host. We have used the approaches described by Moir & Brammar (1976) to amplify the products of the trloD and E genes by reading from the trp promoter. When we block phage functions by defects in gene N, the resulting boost in anthranilate synthetase formation is irrespective of the orientation of the fragment within the phage. The availability of phages with trp genes in the r orientation has enabled us to investigate the consequences of converging transcription events. Expression of the trloE gene from the trlo promoter of ~trp(CDE)Tcro- phages was effectively eliminated by the N-dependent leftward transcription from PL. We do not know whether this effect reflects the relative efficiencies of the two opposing promoters or whether it is another feature of the action of the lambda N protein in overcoming barriers to transcription. Nor can we be sure that the rightward transcription of trl~ genes is prevented, since the effect could be due to an inhibition of translation of trp mRNA by the production of excess RNA of complementary sequence. Spiegelman e$ at. (1972) have suggested that the production of RNA complementary to the cro gene message during infection of a sensitive host by phage ~ could be important in inhibiting synthesis of the cro gene product and thereby enhancing the lysogenic response. I t is apparent that "termination by collision" (Szybalski, 1971) remains a possible mechanism for controlling transcription. An interesting feature of such a mechanism, illustrated by the result with the 2trI~(CDE) r phage, is that it can allow expression to proceed for a time that is accurately determined by the distance between the structural gene and the opposing promoter. We are grateful to Irving Crawford and Charles Yanofsky for providing bacterial strains and unpublished information; to Kenneth Murray for restriction enzymes and advice in their use; to Graham Brown and Susanna Winton for skilful assistance and to Anne Moir and Kathleen Borck for constructive criticism of the manuscript. One of the authors (A. S. H.) is indebted to Peter J. Highton for his guidance in the use of the electron microscope. 37

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This work was supported by grants from the Medical Research and Science Research Councils. One of the authors (A. S. H.) was the recipient of a Medical Research Council Training Grant. REFERENCES Adhya, S., Gottesman, M. & de Crombrugghe, B. (1974). Proe. Nat. Arid. Sei., U.S.A. 71, 2534-2538. Appleyard, R. K. (1954). Genetics, 89, 440-452. Bertrand, K., Squires, C. & Yanofsky, C. (1976). J. Mol. Biol. 103, 319-337. Borck, K., Beggs, J. D., Brammar, W. J., Hopkins, A. S. & Murray, N. E. (1976). Mol. Gen. Ger~t. 146, 199-207. Bovre, K. & Szybalski, ~r. (1969). Virology, 38, 614-626. Campbell, A. (1962). Advan. Genet. 11, 101-145. Cleary, P. & Campbell, A. (1972). J. Bacteriol. 112, 830-839. Crawford, I. P., Sikes, S., Belser, N. O. & Martinez, L. (1970). Genetics, 65, 201-211. Davis, R., Simon, M. & Davidson, N. (1971). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 21, pp. 413-428, Academic Press, New York. Davison, J. R., Brammar, W. J. & Brunel, F. (1974). Mol. Gen. Genet. 139, 9-20. Deeb, S. S., Okamoto, K. & Hall, B. D. (1967). Virology, 31,289-295. Denney, R. M. & Yanofsky, C. (1974). J. Bacteriol. 118, 505-513. Drapeau, G. R., Brammar, W. J. & Yanofsky, C. (1968). J. Mol. Biol. 85, 357-367. Fiandt, M., Hradecna, Z., Lozeron, H. A. & Szybalski, W. (1971). In The Bacteriophage Lambda (Hershey, A. D., ed.), pp. 83-96, Cold Spring Harbor Laboratories, New York. Franklin, lg. C. (1971). In The Bacteriophage Lambda (Hershey, A. D., ed.), pp. 621-638, Cold Spring Harbor Laboratories, New York. Franklin, N. C. (1974). J. Mol. Biol. 80, 33-48. Franklin, N. C. & Yanofsky, C. (1976). In R1VA Polymerase, Cold Spring Harbor Laboratories, New York. Guha, A., Saturen, Y. & Szybalski, W. (1971). J. Mol. Biol. 56, 53-62. Hendrix, R. W. (1971). In The Bacteriophage Lambda (Hershey, A. D, ed.), pp. 355-370, Cold Spring Harbor Laboratories, New York. Ito, J. & Yanofsky, C. (1966). J. Biol. Chem. 241, 4112-4114. Ito, J., Cox, E. C. & Yanofsky, C. (1969). J. Baeteriol. 97, 725-733. Jackson, E. N. & Yanofsky, C. (1972a). J. Mol. Biol. 69, 307-313. Jackson, E. N. & Yanofsky, C. (1972b}. J. Mol. Biol. 71, 149-161. Jackson, E. N. & Yanofsky, C. (1974). J. Bacteriol. 117, 502-508. Kaiser, A. D. (1962). J. Mol. Biol. 4, 275-287. Kaiser, A. D. & Hogness, D. S. (1960). J. Mol. Biol. 2, 392-415. Lennox, E. S. (1955}. Virology, 1, 190-206. Lowry, D. H., Rosebrough, N. H., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 198, 265-275. Mandel, M. & Higa, A. (1970). J. Mol. Biol. 58, 159-162. Marmur, J. (1961). J. ~Iol. Biol. 3, 208-218. Mertz, J. E. & Davis, R. W. (1972). Proe. Nat. Acad. Sei., U.S.A. 69, 3770-3774. Moir, A. & Brammar, W. J. (1976). Mol. Gen. Genet. In the press. Murray, lg. E. & Brammar, W. J. (1973). J. Mol. Biol. 77, 615-624. Murray, K. & Murray, N. E. (1975). J. Mol. Biol. 98, 551-564. Murray, N. F,. & Murray, K. (1974). Nature (London), 251, 476-481. Murray, N. E., Manduca de Ritis, P. & Foster, L. A. (1973). Mol. (~en. Genet. 120, 261-281. Nijkamp, H. J. E., Bovre, K. & Szybalski, W. (t971). Mol. Gen. Genet. 111, 22-34. Ogawa, T. & Tomizawa, J. (1968). J. Mol. Biol. 38, 217-225. Old, R., Murray, K. & Roizes, G. (1975}. J. Mol. Biol. 92, 331-339. Oppenheim, A. B. (1976). Nature (London), 261, 615-616. Parkinson, J. S. (1968). Genetics, 59, 311-325. Philippsen, P., Streeck, R. E. & Zachau, H. G. (1974). Nut. J. Bioehem. 45, 479-488. Rambach, A. & Tiollais, P. (1974}. Proc. Nat. Acad. Sei., U.S.A. 71, 3927-3930. Sharp, P. A., Sugden, B. & Sambrook, J. (1973). Bioehemis~y, 12, 3055-3063.

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Characterization of lambdatrp-transducing bacteriophages made in vitro.

J. Mol. Biol. (1976) 107, 549-569 Characterization of 2trp=transducing Bacteriophages made in Vitro A. S. HOPKINS,, NOR]SEN E. MURRAY AND W. J. BRAMi...
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