The bun Operon of Bacteriophage P I LOCALIZATION OF THE PROMOTER CONTROLLED BY P1 REPRESSOR*

Repression of a strong promoter localized 5’ to the P1 ban gene is a prerequisite for cloning the ban operon in the multicopy plasmid pBR325. Repression is brought about by the binding of P1 repressor to the operator of the ban operon I., and Schuster, H. (1987) Mol. Gen. Genet. 206, 368-376). Binding of RNA polymerase in vitro overlaps with the operator and is inhibited by P1 repressor as shown by electron microscopy. The mu- tant P1 bac, which renders ban expression constitutive, contains a single base pair exchange within the operator. As a consequence, more repressor is required (i) for the inhibition of binding of RNA polymerase, and (ii) for the electrophoretic retardation of a P1 bac DNA fragment when compared to the corresponding bac’ fragment. A P1 ban recombinant plasmid containing a 4-base pair deletion close to the operator still allows binding of repressor but not of RNA polymerase. By that means, a repressible promoter is located at the P1 map position 72 in a distance of about 2.5 kilobase pairs to the beginning of the ban gene. Phage Expression of ban in the P1 prophage is repressed by P1 repressor

The bun Operon of Bacteriophage PI LOCALIZATION  Phage P1 codes for a dnaB analog (ban) protein (2,3). Expression of ban in the P1 prophage is repressed by P1 repressor and is constitutive in the regulatory mutant Pf bac (2). An additional P1 regulatory mutation crr leads to an overproduction of gpban in P1 bac crrin comparison to P1 bac lysogens (4,5). From genetic data the two regulatory mutations were found to be closely linked to the ban structural gene, the most probable order being bac-crr-ban in a hypothetical ban operon (4). The ban operon was found to be located on the P1 EcoRI fragment 3 (P1:3)' and the expression of ban in a h-P1:3 hybrid phage is repressible by P1 repressor (6,7). In order to obtain a more detailed insight into the regulation of ban, P1:3 and P1:3 subfragments from P1 wildtype and P1 ban regulatory mutant DNAs were cloned into multicopy plasmid vectors. Dissection of the ban operon by that means confirmed the order bac-crr-ban found genetically and a P1 repressor binding site was located 5' to the crr mutation (1). Cloning of the bun operon in multicopy plasmids requires the presence of P1 repressor in the cell, indicating the existence of a repressible strong promoter in the ban operon (1). A detailed analysis of that region now reveals an overlap of an RNA polymerase-and a PI repressor binding site. Two mutations within this site were analyzed. One of these is bac-1 (2) which is characterized as an operator-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The PI EcoRI DNA fragments 3 and 7 are abbreviated P1:3 and P1:7, respectively. constitutive mutation of the ban operon. The second mutation occurred during the cloning procedure in the absence of P1 repressor. It turned out to be a 4-bpZ deletion mutant unable to bind RNA polymerase. By that means, the structural requirements for RNA polymerase-and repressor binding appear in outline.
Binding of RNA Polymerase to DNA-Binding of E. coli RNA polymerase (Boehringer Mannheim) to plasmids or isolated DNA fragments for electron microscopic analysis was done essentially as described (11). Recombinant DNAs were linearized by restriction enzymes followed by incubation with RNA polymerase. In experiments testing the effect of P I repressor on the binding of RNA polymerase, DNA and repressor were first incubated in RNA polymerase binding buffer (30 mM triethanolamine hydrochloride (pH 7.9), 50 mM KCI, 10 mM MgCI,) for 15 min at 37 "C. RNA polymerase (0.1 units, approximately 200 ng) was added, and after 5-min incubation, the complexes were fixed for 15 min in 0.1% glutaraldehyde. Unbound repressor and RNA polymerase molecules were separated on Sepharose CL-4B (Pharmacia Biotechnology, Inc.) and the DNA-protein complexes prepared for electron microscopy by adsorption to mica as described (12). Between 100 and 130 DNA molecules were examined in each experiment by a computer program (11). In general, the results of three different restriction enzyme cuts of one DNA probe were compared in order to prove the reproducibility of the RNA polymerase binding sites. PI Repressor-Highly purified repressor protein (about 90% pure, 1-2 mg/ml) in RI buffer (20 mM Tris-HC1 (pH 7.6), 50 mM NaCI, 1 mM dithiothreitol, 0.1 mM EDTA, 10% (v/v) glycerol) was used. The cloning, overproduction, and purification of P1 repressor will be described el~ewhere.~ Ml3mp Cloning and DNA Sequencing-The 310-bp HincII fragment of P1:3 containing the P1 repressor binding site (see Fig. 3) was cloned in M13mp8/9 in two ways: (i) insertion into the Hind1 site of M13mp8 and M13mp9 directly (13); (ii) first, insertion into the SmaI site of the tac expression vector pJF118EH (14), followed by excision with EcoRI and Hind111 and insertion into the EcoRI-and HindIIItreated M13mp9. Cloning of the HincII fragment from plasmids of the pSS series (Table I)  in the recipient bacteria used for transformation, that of the pAJ series (Table I)   properties The cloning procedures are described in the text. The nomenclature of the recombinant plasmids has been described (1). Open (hatched) bars = Pl(pBR325) DNA. Relevant restriction enzyme sites are indicated by vertical lines (B = BssHII, E = EcoRI, P = PstI, S = SphI). bun marks the location and extent of the bun gene. The wavy line indicates transcription of ban starting from an unknown (question mark) or P1-specific (bold vertical line) promoter. The open circle marks the overlapping binding site of RNA polymerase and P1 repressor. Thermoresistance: (+), strain NY58 or NY58(pSS7) harboring the plasmid in question grows equally well at 30 and 40 "C; (-), strains grow only at 30 "C. Repressor requirement: (+), strain NY58 must harbor the plasmid pSS7 carrying the Plcl+ repressor gene. in order to stably maintain the plasmid in question (1); (-), no repressor requirement.
Methods-Cloning of P1:3 in pBR325, restriction enzyme treatments, transformation of bacteria by recombinant plasmids, and DNA fragment analysis by gel electrophoresis was done as described previously (1).

Cloning of the ban Operon of Phage PI
We have cloned P1:3 in pBR325 by two different approaches. In both cases the recombinant plasmids containing the ban gene were identified by the ability to suppress a cellular dnaBts mutation. The ban recombinant plasmids, which were used in the following experiments, all carry the inserted P1:3 DNA in the same relative orientation (pAJ1, pHS1, pSS1, or pSS1-12, Table I and Ref. 1). They were made smaller by SphI treatment which eliminates about 45% of P1:3 (the portion downstream of the ban gene) and about 30% of the pBR325 DNA (containing part of the tetracycline resistance genes). Religation by T 4 DNA ligase yields a truncated, tetracycline-sensitive recombinant plasmid (pAJ2, pHS2, pSS2, or pSS2-12, Table I and Ref. 1).
First Approach-P1:3 from PlCmclrlOO DNA was isolated and inserted into EcoRI-linearized pBR325. Upon transformation of NY58(pSS7) amp' + km' colonies were isolated at 30°C. The recombinant plasmid pSSl isolated from such transformants did not suppress the dnaBts mutation of its host (Table I).
However, thermoresistant mutants of NY58(pSS7) harboring pSSl were found at an appreciably higher frequency (about 50-fold) than with NY58(pSS7). In this respect, pSSl mimicks the property of prophage P1 in a P1 lysogenic E. coli dnaBts strain (2). Only strain NY58(pSS7), but not NY58, can be transformed at 30°C by plasmid pSSl indicating the requirement for P1 repressor in order to stably maintain pSSl (Table I). The same results as with pSSl were obtained with plasmid pSS2 (Table I) (1).
When the same cloning procedure was repeated using P1:3 from P1 bac crr DNA, the resulting plasmids pSS1-12 and pSS2-12 also required P1 repressor in the same cell in order to be stably maintained (Table I). But in contrast to pSSl and pSS2, these plasmids confer thermoresistance to NY58(pSS7) due to the bac-and/or crr mutation (Table I) (1). Second Approach-A X-P1:3 hybrid phage had been selected directly at 42 "C in an E. coli dnaBts(X) strain (6,7). P1:3 was isolated from X-P1:3 and inserted into the EcoRI-linearized pBR325. Upon transformation of NY58 amp' colonies were directly isolated at 40°C. Plasmid pAJl isolated from such transformants conferred thermoresistance to NY58 and did not require P1 repressor in the cell (Table I). Plasmid pAJ2, obtained from pAJ1 by SphI treatment and religation (see above), retained the properties of the latter (Table I). The ability to confer thermoresistance was not due to a bac-type mutation but rather to a promoter activity which arose by an (unknown) mutation in the vector. This was shown by replacing the vector DNA in pAJl by new, EcoRI-linearizedpBR325 DNA. The resulting plasmids pHs1 and pHS2, isolated at 30 "C, no longer conferred thermoresistance to NY58 (Table  I). However, thermoresistant mutants of NY58 (pHS1) again were found at an appreciably higher frequency than with the plasmid-free strain.
These results suggest that plasmids of the pSS series contain a PI-specific strong promoter which has to be repressed by P1 repressor. The same promoter has become inactive by mutation in plasmids of the pAJ-and pHS series. Confirmation comes from RNA polymerase-binding studies and DNA sequence analysis as described in the next sections.

Mapping of an RNA Polymerase Binding Site Controlled by PI Repressor
A strong, PI-specific RNA polymerase binding site was identified by electron microscopy when PstI-linearized pSS2 DNA was incubated with RNA polymerase (Fig. 1). Binding sites on the vector DNA belong to promoters of the bla and cat gene (left) and of the origin of replication (right, Fig. 1) (17,18). Binding of RNA polymerase to the P1-specific site is strongly and selectively inhibited when the DNA is preincubated with P1 repressor (Fig. 1). The DNA-repressor complex itself is not visible under the experimental conditions. In contrast to pSS2, the strong, P1-specific RNA polymerase  Table I. binding site does not exist in pAJ2 (Fig. 1).
When the same procedure is repeated using the EcoRIlinearized pSS2-12(bac err) DNA, a similar pattern of RNA polymerase binding sites emerges. Again a P1-and three vector-specific RNA polymerase binding sites are resolved (Fig. 2), and the relative strength of the different binding sites of pSS2-12 and pSS2 is nearly identical. It demonstrates that neither the bac-nor the err mutation changes the pattern of the RNA polymerase binding sites. However, in order to inhibit the binding of RNA polymerase to the P1-specific site in pSS2-12, about twice as much repressor is required than with pSS2 (compare Figs. 1 and 2). Similar results were obtained when the DNA was linearized with PstI (data not shown). In all plasmids tested, and irrespective of the presence or absence of P1 repressor, a minor RNA polymerase binding site about 2.6 kb downstream of the repressor-cont~lled binding site is observed (Figs. 1 and 2). It is not yet known  Table I. whether it is relevant for the transcription of bun.
Differences in the efficiency of binding of P1 repressor to pSS2 and pSS2-12(buc crr) were also observed by the retardation of DNA-repressor complexes during gel electrophoresis (19,20). When the plasmid DNAs were treated with Him11 and titrated with increasing amounts of repressor, a 310-bp H i n d fragment disappeared and one or two bands of decreasing mobility appeared instead. Conversion to these bands of the HincII fragment from pSS2-12 requires more repressor than that of pSS2 (Fig. 3).

~o r r e~t~o n of RNA Polymeraseand PI Repressor B i~i~
Site In order to localize the P1-specific RNA polymerase binding site more accurately, the 1.3-kb BssHII fragment of pSS2-12 was isolated and incubated with RNA polymerase. A strong binding site was identified (Fig. 4). As a control, the isolated BssHII fragment of pSS2 was incubated with repressor with and without subfragmentation by Him11 to verify binding of the repressor to the 260-bp BssHII-HincII fragment (Fig. 4,  inset). In parallel, the 310-bp HincII fragments ofpSS2, pSS2-12 (Fig. 3), and pAJ2 were cloned into M13mp8/9 and sequenced. (It was not possible to isolate stable M13mp8 clones containing the Hind1 fragment of pSS2-12.) Moreover, the  Table I. subjected to a DNase protection (footprinting) analysis in the presence of repressor as described (16,21).
The P1 repressor protects an AT-rich region in pAJ2 comprising at least 18 bp from DNase digestion (Fig. 4). The nucleotide sequence of this region coincides with a 17-bp consensus sequence found for different repressor binding sites in the genome of P1 (16, 24). The repressor-and RNA polymerase binding site are located approximately in the center of the 260-bp BssHII-HincII fragment. Both these sites and potential promoter sequences overlap (Fig. 4). The DNA sequences of pSS2, pSS2-12(bac crr), and pAJ2 are for the most part identical, but nevertheless have characteristic differences. pAJ2, which has lost the ability to bind RNA polymerase (Fig. 1) contains a 4-bp deletion (Fig. 4) within one of the two major contact regions of the RNA polymerase molecule (22) indicating again the importance of this sequence for binding of the enzyme. In pSS2-12(bac crr), a G + T base exchange in the sense strand (22) is located within the repressor binding site (Fig. 4), thus classifying P1 bac as an operator-constitutive mutation. Moreover, bac may be considered as a promoter-up mutation moving towards the consensus sequence of a potential -35 region ( Fig. 4 and  reference 22). (Transcription of the sense strand shown in Fig. 4 should proceed from left to right. The crr mutation and the ban gene are located downstream of the right HincII site as shown previously (I).)

DISCUSSION
The ban operon comprises, in clockwise direction on the P1 map, a P1 repressor binding site or operator overlapping with an RNA polymerase binding site, an unknown gene coding for a 14-kDa protein, and the ban gene (1). It is about 4.1 kb in length. The operator of the ban operon, abbreviated Op72 according to its map position (23), is one out of 11 operators so far identified in the P1 genome. The striking feature of these operators is the asymmetry of its 17-bp consensus sequence ATTGCTCTAATAAATTT (16, 24).4 In order to clone the ban operon in multicopy plasmids, the operator Op72 has to be occupied by P1 repressor. Obviously, the repressor-controlled promoter itself and/or the gene product of this promoter is deleterious to the plasmid and/or the bacterial cell. (It is not yet known whether the ban operon still contains a gene(s) in between Op72 and the gene coding for a 14-kDa protein.) Although P1 repressor still binds to the operator in pAJ2, its presence is dispensable because of the loss of the RNA polymerase binding site due to a 4-bp deletion mutation. The latter presumably arose by selecting the P1:3 recombinant plasmid in NY58 a t 40 "C in the absence of P1 repressor. On the other hand, the bac mutation weakens ' J. L. Eliason and N. Sternberg, personal communication.
the binding of repressor to the operator, Op72. This allows transcription of the ban gene, sufficient to suppress the dnaBts character of the host without impairing the recombinant plasmid itself. It is noteworthy that the bac mutation affects a highly conserved base pair within the operator (16), as it is known for numerous operator-constitutive mutants of phage X (25). The increase in the mutation rate to thermoresistance of NY58(pSS7) cells harboring ban recombinant plasmids allows the isolation of P1 bac-type mutants. A sequence analysis of these mutant plasmids may lead to a better understanding of the P1 repressor-operator interaction.