INTRODUCTION

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A temperate bacteriophage has a choice between two very different life cycles after infection of a sensitive host bacterium: it can enter either a lytic cycle or a silent state, transforming the host into a lysogenic bacterium. During the lytic cycle, phage genes are expressed in a temporal manner leading to production of new phages and cell lysis. In a lysogenic bacterium, the genome of the phage is present but only very few genes are expressed. The almost silent phage DNA is transferred to new daughter cells whether it is integrated in the genome, which seems to be the prevailing mechanism, or is present in the form of a plasmid, thus maintaining the lysogenic state of the host. The existence of these two life cycles argues for the presence of tightly regulated promoters and their cognate regulatory proteins in the phage genome. The paradigm for the early expressed promoters is represented by the p_L and the p_R promoters of bacteriophage  from Escherichia coli. They are recognized by the host polymerase, and repression is established through the cI repressor protein, while the Cro protein, by competing for binding to the same operator sites as cI, first represses synthesis of cI from the p_RM promoter and later in infection also represses transcription from the p_R and the p_L promoters. The cro gene is the first gene transcribed from the  p_R promoter, while the adjacent cI gene is divergently transcribed from the p_RM promoter (for a review, see reference 11).

TP901-1 is a temperate lactococcal phage belonging to the P335 group of phages infecting lactic acid bacteria, including lytic phage species (1). Analysis of the temporal transcriptional pattern during the lytic cycle resulted in localization of consecutive regions of early, middle, and late expressed genes on the phage genome (19). The early region was found to cover 13 kb of the 38.4-kb phage genome, and 6.4 kb of this region was sequenced and found to encode 11 ORFs as well as the phage attachment site attP (5, 19). The region was found to be divergently transcribed in accordance with the orientation of the ORFs, and two consensus promoters (p_L and p_R) were proposed from the sequence data. The start site for initiation of transcription for both promoters was identified by primer extension (19). The p_R promoter is oriented facing attP, and four ORFs were identified as being transcribed from p_R, the last of which (orf1) encodes the phage integrase (5). The lytic promoter p_L is located upstream of p_R, directing transcription in the opposite direction; the longest mRNA produced from this promoter reaches a size of 10 kb. The sequenced region covers ORF5 to ORF11 and part of ORF12 of the promoter-distal part of this mRNA. In this report, we present experimental evidence for the presence and regulation of these promoters obtained from promoter fusions and primer extension studies. The reported data identify ORF4 as a repressor protein for both promoters and furthermore show that ORF4 is sufficient for achievement of phage immunity. Finally, the data demonstrate that both ORF4 and ORF5 together with the divergent promoter region can function as a genetic switch, resulting in either repression or derepression of the lytic p_L promoter.

	MATERIALS AND METHODS

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DNA technology. Recombinant plasmid DNA from E. coli was isolated by the alkaline lysis technique. Plasmids were isolated from Lactococcus lactis subsp. cremoris by the alkaline lysis technique after incubation with lysozyme (20 mg/ml) in 20 min at 37°C. Preparative portions were further purified by passage through Qiagen columns as recommended by the supplier (Qiagen, Hilden, Germany). Pharmacia Biotech supplied restriction endonuclease enzymes, DNA polymerase Klenow fragment, T4 DNA ligase, and buffer systems. All enzymes were used as recommended by the supplier. PCRs were performed in a DNA thermal cycler (Perkin-Elmer Cetus) using Amplitaq polymerase and buffer supplied by Perkin-Elmer. DNA sequencing was performed by the method of Sanger and coworkers (24) as instructed by the protocol for the Sequenase version 2.0 DNA sequencing kit (U.S. Biochemical Corp., Cleveland, Ohio).

Construction of plasmids. The plasmids used in this study are listed in Table 1. Plasmid pPM92 was constructed by cloning a 2,787-bp XhoI fragment of pG5f4 (4) into the XhoI site of the E. coli vector pIC19R (20). This plasmid contains orf4 to orf9 of TP901-1. The following plasmids were constructed by cloning PCR products obtained by using pPM92 as a template, a vector primer (1211; see below), and primers located either in the region between orf4 and orf5 (24BamHI, P5A, and erm2ABamHI), within orf5 (pextPstI), or downstream of orf5 (orf4orf5PstI and porf7PstI). All clonings were performed in L. lactis subsp. cremoris MG1363. Plasmids pAJ93 and pAJ94 were constructed by cloning a BamHI-digested PCR product (primers 24BamHI and erm2ABamHI) into pAK80 (13) also digested with BamHI. The resulting plasmids contain p_L (pAJ93) and p_R (pAJ94) fused to the lacLM genes. PCR products obtained by using primer 1211 and pextPstI were cloned into pAK80 after digestion with PstI, giving rise to plasmids pPM126 and pPM137 carrying orf4 as well as p_L and p_R fused to the lacLM genes, respectively. Plasmids pPM127 and pPM132 were constructed by cloning a HindIII-digested PCR product (primers 1211 and porf7PstI) into pAK80. The plasmids thus contain orf4 and orf5 as well as p_L (pPM127) or p_R (pPM132) fused to the lacLM genes. A frameshift mutation was introduced in the beginning of the orf4 gene by filling in the SpeI site of pPM92 with Klenow polymerase. After ligation and redigestion with SpeI, two PCRs were performed on the ligation mixture. In the first PCR, primers 1211 and pextPstI were used, and a PCR product containing the mutated orf4 gene was cloned into pAK80 after digestion with PstI, giving rise to plasmids pPM138 and pPM136, carrying only the mutated orf4 gene as well as p_L and p_R fused to the lacLM genes, respectively. In the second PCR, primers 1211 and orf7PstI were used. After digestion with HindIII, a PCR product containing orf5 in addition to the mutated orf4 was cloned in pAK80, and plasmids pPM131 and pPM139 were constructed. Plasmids pPM131 and pPM139 thus contain p_L and p_R, respectively, fused to the lacLM genes. In all cases, several independent clones giving rise to the same -galactosidase specific activity were isolated. Furthermore, the inserts of plasmid pAJ93, pAJ94, pPM127, pPM126, and pPM132 were sequenced, as well as the mutation within the orf4 gene in plasmids pPM131, pPM136, pPM138, and pPM139. The PCR product generated by use of primer 1211 and erm2ABamHI was digested with BamHI and cloned into pCI372 (10) also digested with BamHI, resulting in plasmid pAJ80, carrying p_L, p_R, and orf4. The PCR product produced by using primer 1211 and P5A was cloned into pNZ8010 (7) after digestion with BamHI, giving rise to pAJ115 containing orf4 and p_L fused to the gusA gene. PCR products produced by using primer 1211 and orf4orf5PstI or Orf5A and Orf5B were cloned into pNZ8010 (7) after digestion with PstI and BamHI-PstI, respectively. Thereby plasmids pAJ98 and pAJ142 were constructed so as to contain orf4 and orf5 transcribed from p_R and p_L on a high-copy-number plasmid and orf5 under the control of the nisin promoter, respectively. The inserts of pAJ80, pAJ98, pAJ115, and pAJ142 were sequenced.

Primers used in this study. For construction of plasmids, the following primers were used: 1211 (5'-GTAAAACGACGGCCAGT-3'), 24BamHI (5'-GCGCGGATCCTATAGCGCATCTTGAAC-3'), P5A (5'-GCGCGGATCCTAATCATAACTCATTTATGT-3'), erm2ABamHI (5'-GCGCGGATCCCACGTTTCATGAACTTT-3'), orf4orf5PstI (5'-GCGCCTGCAGCCCAAGCTTCATCAGTTC-3'), Orf5A (5'-GCGCGGATCCAAGAAAGGAGAAACATAAAT-3'), Orf5B (5'-GCGCCTGCAGTTAATGAACTTTTGCATTAA-3'), pextPstI (5'-AAAACTGCAGAGACACAGTTCTCTCTG-3'), and porf7PstI (5'-AAAAAACTGCAGATCCCCCATGTGCTTTCC-3'). For primer extensions, primer PE (5'-AGACACAGTTCTCTCTGAAAGCCCC-3') was used for mapping of the p_L promoter, while primer 23 (5'-GGTCAGGAGATTGAACG-3') was used for p_R.

Transformation and selection in E. coli and L. lactis subsp. cremoris. E. coli XL1-Blue was made competent with CaCl₂ and was transformed as described by Sambrook et al. (23). Selection was performed with 100 µg of ampicillin per ml. L. lactis subsp. cremoris MG1363 was transformed by electroporation according to the method described by Holo and Nes (12), using 0.03 to 0.5 µg of DNA per electroporation. When L. lactis subsp. cremoris 3107 was made competent, the growth medium (12) contained only 0.2 M sucrose, and 25 µg DNA was used per transformation. Transformants were selected on plates containing 5 µg of erythromycin per ml. Screening on 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) plates was performed at a concentration of 90 µg/ml.

Bacteria and phages. Bacterial strains used in this study are listed in Table 2. The temperate bacteriophage TP901-1 was induced from L. lactis subsp. cremoris 901-1 (1) by the use of UV light as previously described (4).

Cell growth and enzyme assay. E. coli strains were grown with agitation at 37°C in Luria-Bertani broth (23) (Difco Laboratories, Detroit, Michigan). Bacto Agar (Difco Laboratories) was used at 1.5% (wt/vol) in solid media. L. lactis strains were propagated at 30°C without shaking in GM17 (M17 broth [Oxoid Limited, Basingstoke, Hampshire, United Kingdom] containing 0.5% [wt/vol] glucose) (26). For determination of -galactosidase activity, exponential growing cells were diluted in fresh medium to an optical density at 600 nm (OD₆₀₀) of 0.05. At an OD₆₀₀ of 0.8, 70 ml of culture was harvested and resuspended in 7 ml of Z-buffer, and cell extracts were obtained with a French press. The activity of -galactosidase was determined according to Miller (21), and specific activity was calculated in Miller units.

Induction of transcription from the nisin promoter. Exponentially growing cells of strains LKH169 and LKH206 were diluted in fresh medium (GM17 containing 5 µg of erythromycin per ml and 5 µg of chloroamphenicol per ml) to an OD₆₀₀ of 0.05. At an OD₆₀₀ of 0.2, nisin was added to the culture to a final concentration of 1 ng/ml. Samples were withdrawn before and after addition of nisin, diluted, and plated on plates containing X-Gal but no nisin. After overnight incubation at 30°C, blue and white colonies were counted.

Induction by mitomycin C. Strains were grown in GM17 containing 5 µg of erythromycin per ml at 28°C, due to the temperature sensitivity of the recA strain. Exponentially growing overnight cultures were diluted to an OD₆₀₀ of 0.05 in fresh medium, and growth was monitored. At an OD₆₀₀ of 0.4, each culture was diluted into two separate cultures to an OD₆₀₀ of 0.1. Subsequently, mitomycin C was added to a final concentration of 2.5 µg/ml to one of the cultures, while the other served as a control. Samples were removed at different time points, harvested, extracted, and assayed as described above for -galactosidase activity.

Plaque assay. TP901-1 phages were diluted in A buffer (0.8% [wt/vol] NaCl, 10 mM Tris-HCl [pH 7.5], 5 mM MgCl₂, 1 mM CaCl₂) and plated on lawns of L. lactis subsp. cremoris 3107 transformed with either pPM126, pPM138, pPM127, or pPM131. As a control, L. lactis subsp. cremoris 3107 was used as an indicator strain. After incubation at 30°C overnight, plaques were counted. For each strain, the efficiency of plaquing (EOP) was calculated as the number of plaques formed on a lawn of the strain of interest divided by the number of plaques formed on L. lactis subsp. cremoris 3107.

Extraction of RNA. RNA was isolated from strains PM93, PM94, and PM80 growing exponentially in defined medium SA (14) (1.0% glucose [wt/vol], 5 µg of erythromycin per ml), using the method described by Johnsen et al. (15) as modified by Madsen and Hammer (19).

Primer extensions. Primers PE and 23 were phosphorylated by using T4 polynucleotide kinase (Gibco BRL) and [³²P]ATP (Amersham) as described by Sambrook et al. (23); 25 µg of total RNA isolated from strain PM93, PM94, or PM80 was mixed with 1 pmol of phosphorylated primer in a volume of 10 µl and incubated at 70°C for 10 min. The mixture was allowed to cool to 37°C over a 1-h period. Then 1 µl of Superscript reverse transcriptase (Gibco), 4 µl of 5× first-strand buffer (Gibco), 2 µl of 0.1 M dithiothreitol (Gibco), and dATP, dGTP, dCTP, and dTTP to a final concentration of 500 µM were added, and the mixture was incubated at 37°C for 1 h.

	RESULTS

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ORF4 is a negative regulator of p_L and p_R. Previously, we identified two divergently orientated promoters (p_L and p_R) located between orf4 and orf5 of the temperate bacteriophage TP901-1 (19). To investigate the expression and regulation of the promoters, we cloned a phage DNA fragment of 837 bp, containing both promoters and orf4, in both orientations in the promoter probe vector pAK80. The lacLM genes contained in this plasmid give rise to -galactosidase activity when a functional promoter is inserted in the cloning region, and stop codons in all three reading frames ensure that a transcriptional fusion is constructed. When p_L (pPM126) and p_R (pPM137) are located upstream of the lacLM genes, low -galactosidase activities (1.0 and 0.5 Miller units, respectively) can be measured when the plasmids are present in L. lactic subsp. cremoris MG1363 (Fig. 1). These enzyme levels are, however, significantly different from the basal level of 0.02 Miller units measured in MG1363 containing the pAK80 vector, which is in agreement with the existence of the divergently orientated promoters p_L and p_R on this phage DNA fragment. The effect of the presence of orf4 on the activity of the promoters was investigated by introduction of an early frameshift mutation at codon position 6. The -galactosidase activities of the resulting plasmids, carrying the p_L fusion (pPM138) and the p_R fusion (pPM136) were measured as 62 and 15 Miller units, respectively, in MG1363. These results demonstrate the presence of divergently orientated promoter activities on the cloned phage DNA fragment and clearly show that the presence of a functional ORF4 protein represses the expression of both phage promoters. The levels of repression are 62- and 31-fold for p_L and p_R, respectively (Fig. 1).

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Effects of orf4 and orf5 on expression from pL (A) and pR (B). Fragments cloned upstream of the lacLM genes of the pAK80 vector are indicated by lines. The lacLM genes are located downstream of pL (A) or pR (B). Small vertical lines indicate a frameshift mutation in orf4. Plasmid numbers and specific activities of -galactosidase (-gal) of the corresponding strains are indicated. For details of cell growth and the -galactosidase assay, see Materials and Methods. For PM80 and PM95, only two independent experiments were performed; the remaining measurements are averages of three to five experiments.

Antirepressing activity of ORF5. In bacteriophage , the Cro and the cI repressors compete for the  operator sites and thereby regulate expression of the p_RM, p_R, and p_L promoters. To investigate the influence of both ORF4 and ORF5 on the p_L and p_R promoters of TP901-1, we cloned a 1,279-bp phage DNA fragment carrying both orf4 and orf5 in both orientations in pAK80, resulting in plasmids pPM127 (p_L fusion) and pPM132 (p_R fusion). The transformants arising from both plasmids exhibited clonal variation when screened on GM17 plates containing X-Gal. Of 200 transformants arising from pPM127 (p_L fusion), strong blue colonies were observed in 191 cases and the remaining 9 colonies were white, giving a frequency of 4.5% of white colonies. This frequency is comparable to the frequency of lysogenization of approximately 1 to 3% by TP901-1 (3). When plasmid pPM132 (p_R fusion) was used, the same type of clonal variation was observed but with opposite frequencies: 95% white colonies and only 5% light blue transformants were observed. The phenotype was stable for both plasmids when restreaked to single colonies more than three consecutive times and also after freezing and restreaking. When plasmid DNA was isolated from either blue or white transformants and used for retransformation of MG1363, segregation into white and blue transformants was observed again with the same frequencies as seen before. The phenotype of a transformant was thus independent of the phenotype of the strain from which the plasmid was prepared.

ORF4 and ORF5 are trans acting. The effect of ORF4 and ORF5 donated from a plasmid in trans to the promoter fusions contained on the compatible pAK80 plasmid was investigated. As observed in Table 3, the presence of orf4 on the donor plasmid results in repression of both a p_L fusion (pPM138) and a p_R fusion (pPM136) in the recipient strain, whereas the vector plasmid itself (pCI372) does not change the constitutive expression monitored as blue colonies. Thus, ORF4 is able to repress both p_L and p_R when given in trans. Subsequently, the effect of donation of a plasmid containing both orf4 and orf5 (pAB223) to either the p_R or the p_L fusion strain was investigated. The result clearly showed clonal variation, with the majority of the p_L fusions turning blue and the majority of the p_R fusions turning white. Thus, the same effect of ORF4 and ORF5 was observed whether orf4 and orf5 were donated in trans or were located on the same plasmid as the promoters (pPM127 and pPM132). Regulation of p_L and p_R thus seems to be dependent on ORF4 and ORF5, both being able to function in trans.

The state of p_L can be changed. The clonal variation mediated by the presence of both ORF4 and ORF5 was found to be stably inherited. Particularly, expression from p_L is affected by the choice of configuration, since an approximately 1,000-fold difference in the specific activities of -galactosidase of strains PM93 (p_L open) and PM94 (p_L closed) was found (Fig. 1). The ratio between the amount of ORF4 and ORF5 could determine the choice of configuration of p_L, and we therefore examined whether the state (repressed or open) of the p_L promoter could be changed by introduction of additional amounts of either ORF4 or ORF5. For this purpose, strain LKH148 carrying pPM127 (orf4 orf5), in which p_L is open, was transformed with plasmids expressing orf4 from the p_R promoter, and the color of the transformants was observed on plates containing X-Gal. In the presence of orf4 on a low-copy-number plasmid (pAJ80), only blue transformants were seen, whereas 92% white and 8% blue colonies were found when orf4 was present on a high-copy-number plasmid (pAJ115) (Table 3). It is thus possible to change an open state of the p_L promoter to a repressed state by introduction of a sufficient amount of additional ORF4.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Change of the pL promoter from the closed to the open configuration by a burst of induction of synthesis of ORF5 from the nisin promoter. Strains LKH169 and LKH206 both contain pPM127 carrying the pL promoter in the closed configuration. At time zero, nisin (1 ng/ml) was added to exponentially growing cultures. At the indicated times, samples were withdrawn, diluted, and plated on GM17 plates containing X-Gal. After incubation at 30°C, the number and color of the colonies were determined and the frequency of blue colonies was calculated. Circles, strain LKH206 containing pPM127 and pAJ142 (orf5 under the control of the nisin promoter); triangles, strain LKH169 containing pPM127 and pNZ8010 (the vector of pAJ142).

Study of the regulation of promoters by primer extension. In a previous study, locations of the divergent promoters p_L and p_R in the region between the divergent reading frames in the early expressed region of TP901-1 were determined by primer extension (19). The results from the transcriptional fusions presented in the present report have also demonstrated the presence as well as the regulation of divergent promoter activity. By primer extension analysis, we were furthermore able to simultaneously monitor expression from the p_R and p_L promoters in the same strain. Therefore, we decided to map the transcriptional start sites from both promoters by primer extension using mRNA prepared from three strains: PM93 and PM94, both containing pPM127 but carrying the p_L promoter in the derepressed state and the repressed state, respectively, as well as PM80, containing the corresponding plasmid with a mutation in orf4 (pPM131). In PM93 (orf4 and orf5 present), expression from the p_L promoter is high, as shown by the -galactosidase measurements. The mRNA prepared from this strain shows an mRNA start site from the p_L promoter but none from the p_R promoter (Fig. 3, lanes 1). On the other hand, when the p_L promoter is in the repressed state as in the clonal variant PM94 containing the same plasmid (pPM127) as PM93, transcription from p_R but not from p_L is observed (lanes 2). For comparison, we investigated the primer extension results from the strain containing the orf4 mutant plasmid, pPM131. Since the promoter fusions showed that ORF4 represses transcription from both promoters, we would expect to find transcriptional start sites from both promoters in this strain. As shown in lanes 3, this was also observed. The transcripts from p_R are 4 bp longer due to filling in of the SpeI site, which is located between the primer used and the promoter. Therefore, the same transcriptional start site is used for expression from p_R in strains PM94 and PM80, just as in the case of p_L expression in both PM93 and PM80. The results for strains containing pPM127 (orf4 and orf5 present) demonstrate that the two different phenotypes which can be obtained with this plasmid result from a strain where p_L is open and p_R is simultaneously closed, and vice versa: a strain where p_L is closed and p_R is open. However, as judged from the enzyme measurements in Fig. 1, p_R is only partially derepressed in this situation.

View larger version (73K): [in this window] [in a new window]   FIG. 3.   Primer extension analysis of regulation of pL and pR. Total RNA used in the analysis was isolated from the following strains, all derivatives of MG1363: lanes 1, PM93 containing pPM127 (pL open); lanes 2, PM94 containing pPM127 (pL closed); lanes 3, PM80 containing pPM131 (pL and pR open). Lanes G, A, T, and C contain the sequence reactions performed with the primers used for the analysis. (A) Mapping of the pL promoter; (B) mapping of the pR promoter. To the right of each lane, the sense strand sequence is given; the 10 and +1 positions of the promoters are also indicated.

Effect of ORF4 and ORF5 on phage immunity. The effect of ORF4 and ORF5 on the plating capacity of phage TP901-1 was investigated after transformation of the relevant plasmids into the phage indicator strain L. lactis subsp. cremoris 3107. We tested the presence of ORF4 alone by using pPM126 (p_L fusion) and, as a control, the corresponding orf4 mutant plasmid pPM138. A strain containing orf4 (LKH51) showed full resistance to TP901-1 and an EOP of less than 10⁶, compared to the EOP of 1 for both the orf4 mutant strain (PM97) and the indicator strain 3107 (Fig. 4). The effect of the presence of both orf4 and orf5 was tested with pPM127 (p_L fusion). Use of this plasmid also gave rise to clonal variation in the indicator strain 3107. LKH48 and LKH49 are representatives of clones containing repressed and derepressed p_L promoters, respectively. No plaques were detected with LKH48, while a few pinpoint plaques were obtained with LKH49. For comparison, we also used PM99 containing the orf4 mutant plasmid pPM131, which should produce ORF5 from p_L since orf5 is located downstream of p_L, which in this strain has a high activity (plasmid pPM131 in strain PM80 [Fig. 1]). As reported in Fig. 4, the presence of this plasmid is inhibitory to the plaque-forming capacity of TP901-1, giving an EOP of 10⁴. This is only about fivefold higher than the EOP on LKH49, indicating that the inhibitory effect in LKH49 is mainly due to the production of ORF5.

View larger version (11K): [in this window] [in a new window]   FIG. 4.   Effect of orf4 and orf5 on phage TP901-1 immunity. The lines indicate fragments cloned upstream of the lacLM genes of the pAK80 vector. In all cases, the lacLM genes are located downstream of the pL promoter, and the state of pL is indicated as either repressed or open, as judged from the color of the strains on plates containing X-Gal. A small horizontal line indicates a frameshift mutation in orf4. For each strain, the transformed pAK80 plasmid derivative and the EOP are noted. For details, see Materials and Methods.

Induction by mitomycin C is dependent on RecA and ORF5. For bacteriophage , it is known that during the maintenance of lysogeny the cI repressor is bound to the p_R promoter, preventing transcription of the genes involved in lytic development. In the presence of UV light or mitomycin C, which both induce the SOS response, the cI repressor is inactivated by autocleavage induced by the RecA protein (17). To investigate whether transcription can be induced from the clone containing the lytic p_L promoter of TP901-1 by treatment with mitomycin C, -galactosidase activity was monitored after addition of mitomycin C to strain PM94, carrying both orf4 and orf5. Strain PM94 is the clonal variant carrying the p_L promoter in the repressed state. As seen in Fig. 5, the -galactosidase activity was induced 28-fold by mitomycin C. Furthermore, mitomycin C induction of the p_L promoter fusion was found to be dependent on a functional recA allele, since no induction was seen in a recA mutant (LKH68 in Fig. 5). However when PM75, the recA⁺ strain containing the p_L fusion plasmid pPM126 with only orf4, was exposed to mitomycin C, no induction of -galactosidase activity was observed (data not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Induction of pL by mitomycin C in strains containing the pL promoter fused to the lacLM genes of pAK80. At time 5 min, mitomycin C (2.5 µg/ml) was added. Triangles, strain PM94 containing pPM127 (orf4 orf5 with repressed pL); circles, strain LKH68 recA derivative containing pPM127 (orf4 orf5 with repressed pL).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

By primer extension studies and use of transcriptional fusions, the early transcribed region of phage TP901-1 has been shown to contain two divergently orientated promoters. The untranscribed DNA between the mRNA start sites located at positions 3212 and 3314 has a length of 101 bp. Both promoters have close to consensus sequences with 17 bp between the 10 and 35 regions; furthermore, p_L has the TG motif 1 bp upstream of the 10 region. By deletion of the 10 region of the p_R, promoter it was shown that the efficiency of p_L is independent of the activity of p_R. Furthermore, it was shown that both promoters are active in the absence of expressed phage TP901-1-encoded genes.

In this study, we investigated the functions of ORF4 and ORF5 that are located topologically equivalent to the cI and Cro proteins of phage . The transcriptional fusions demonstrated that ORF4 is necessary for negative regulation of both p_R and p_L, and the primer extensions (Fig. 3) strongly indicate that the negative effect of ORF4 is at the level of transcription initiation. As shown by the phenotype of the strains containing plasmids pPM126 and pPM137, ORF4 alone is furthermore sufficient for repression of both promoters (Fig. 1). This repression is also seen when ORF4 is donated in trans to the promoter fusions (Table 3). The role of ORF4 as the putative phage repressor was further substantiated by proving that the orf4-containing plasmids conferred immunity to TP901-1 when transferred to the phage indicator strain 3107, while introduction of a frameshift mutation in orf4 resulted in abolishment of the immunity of strain 3107 (Fig. 4). The experiments furthermore showed that large amounts of ORF5 are inhibitory to formation of plaques.

We investigated the effect of the presence of both ORF4 and ORF5 on p_L and p_R promoter activities by analysis of plasmids pPM127 and pPM132 and found that both plasmids gave rise to clonal variation among the transformants. The presence of ORF5 was found to be necessary for the observed clonal variation, but ORF4 was still responsible for repression of the promoters. It was demonstrated that the p_L promoter was either open or closed in the obtained transformants and that the clonal variation was stably inherited. However, an open configuration of p_L could be changed to the closed configuration by introduction of additional amounts of ORF4 donated from a high-copy-number plasmid, while no change was seen when ORF4 was derived from a plasmid with moderate copy number (Table 3). Furthermore, a burst of ORF5 production from the nisin promoter was sufficient to alter the configuration of the p_L promoter from closed to open, and the phenotype was stably inherited when the cells were plated without nisin. These results show that the amount of the regulatory proteins ORF4 and ORF5 as well as the ratio determines the choice of the open or repressed state of the p_L promoter. We have also shown that ORF5 is able to inhibit the ORF4-mediated repression of p_L in the short fusion containing only 145 bp of the p_L transcript.

By primer extension analysis, we found that when p_L is open p_R is totally repressed, while p_R transcripts can be found when p_L is closed. However, the enzyme levels of the transcriptional p_R fusions demonstrated that p_R is only partially derepressed under these conditions, 0.5 Miller units in PM95 compared to 0.1 in PM81 (Fig. 1).

When the p_L promoter is open (43 Miller units in PM93), this results in a large production of p_L mRNA encoding ORF5 and therefore presumably rather high levels of ORF5 in the cell (Fig. 1). In these conditions p_R is repressed (0.1 Miller units), but some ORF4 must be produced since ORF4 is necessary for the repression of p_R, as verified by the orf4 mutation in strain PM92 (Fig. 1). The presence of the high level of ORF5 must somehow preferentially interfere with the binding of ORF4 to the p_L promoter but not with the binding of ORF4 to the p_R promoter. Thus, initiation of transcription from p_R is repressed by ORF4, whereas the presence of large amounts of ORF5 prevents repression of p_L by ORF4. This resembles the commitment to lytic growth where the p_L transcripts are needed (19).

In the situation where p_L is repressed, stimulating the lysogenic state, activity from the p_L promoter in PM94 is only 0.04 Miller units, and therefore only small amounts of ORF5 could be produced in these cells (Fig. 1). The p_R fusion in PM95 shows activity of 0.5 Miller units; thus, considerably more ORF4 is produced than when p_L is open. When both ORF4 and ORF5 are present in the cell, p_L is repressed 25-fold more than in the situation where only ORF4 is present, as seen by comparison of PM94 (0.04 Miller units) with PM75 (1.0 Miller units) in Fig. 1. This does not seem to be due to different levels of ORF4 produced in these cells since the output from p_R is the same under both conditions (compare PM86 [0.47 Miller units] with PM95 [0.48 Miller units] in Fig. 1). Therefore, we suggest that some ORF5 is produced when p_L is repressed and that ORF5 is able to enhance the repression of p_L in these cells. In summary, these results suggest that during commitment to lysogenic growth, sufficient ORF4 is produced from the p_R promoter to repress initiation of transcription from both p_R and p_L and that the repression of p_L is further enhanced by the presence of small amounts of ORF5.

We assume that the ORF4 protein binds somewhere in the +10 to 45 region of the promoters in order to repress initiation of transcription. ORF5 may also be a DNA binding protein since it contains a helix-turn-helix motif (from Gln19 to Asn38) (19). In bacteriophage , the regulatory proteins cI and Cro regulates expression of the p_R and p_RM promoters by competition for the same operator sites in the region of the two promoters. But a similar regulation is not likely for TP901-1 since ORF5, in the presence of ORF4, both enhances (large amounts) and inhibits (small amounts) the level of initiation of transcription of the p_L promoter. In addition to this, ORF5 alone is not able to repress transcription from the TP901-1 early promoters. Instead, ORF5 may exert its action by binding to the phage DNA in a complex with ORF4 or just by binding to ORF4 and thereby somehow prevent repression of p_L by ORF4. The latter case would thus resemble the situation for the antirepressor Ant from E. coli phage P22 (for a review, see reference 22). In the first case, the high amount of ORF5 present during commitment to lytic growth may result in formation of an ORF4-ORF5 protein complex which is able to repress initiation of transcription of p_R and not that of p_L. On the other hand, during commitment to lysogenic growth ORF4 is free to bind to the target sites in both promoters, since no inhibition occurs from small amounts of ORF5. This could explain why the amounts of the regulatory proteins ORF4 and ORF5 as well as their relative abundance have a significant effect on the choice of the open or repressed state of the p_L promoter.

The expression of the p_L fusion present in pPM127 can be induced from the repressed state to the derepressed state by addition of mitomycin C. This induction was found to be dependent on the presence of a functional RecA protein. When the shorter fusion pPM126 was used, no mitomycin C induction was observed. This could indicate that ORF5 is needed for mitomycin C induction; however, we cannot completely rule out the possibility that mitomycin C acts on a separate promoter present in the extra DNA found in pPM127 between bp 3458 and 3900. RecA has been shown to act by stimulating repressor self-cleavage in the investigated cases of LexA and  repressor and is therefore mechanistically a coprotease (17; for a review, see reference 18). A consensus AG autodigestion site has not been found in ORF4 (19), but ORF4 could belong to a different protein family than the repressors analyzed so far. Furthermore, the possibility exists that RecA acts on a protein complex consisting of both ORF4 and ORF5.

A homology search between ORF4 and ORF5 of TP901-1 identified a region of ORF4 (from Tyr59 to Asp76) that is similar to the proposed helix-turn-helix motif in ORF5 and thus might constitute a DNA binding motif. This notion is further supported by the very high similarity of this region of ORF4 to the suggested helix-turn-helix motif of ORF4 (from Leu64 to Asp82) of Streptococcus thermophilus phage O1205 (25). A high degree of similarity on the level of protein sequences is found between ORF4 and ORF5 of TP901-1 and O1205, the degrees of identity being 48% in 122 amino acids for ORF4 and 47% in 67 amino acids for ORF5. Interestingly, ORF4 of O1205 is considerably shorter than ORF4 from TP901-1 (134 versus 180 amino acids) and also does not contain a suggested RecA binding site. Furthermore, ORF4 and ORF5 show extensive homology to the repressor (50% identity in 168 amino acids) and protein 31a (75% identity in 44 amino acids), respectively, of Staphylococcus aureus phage phiPVL (16). In addition to this, the intergenic region located between orf4 and orf5 of TP901-1 and the repressor and 31a genes of phiPVL show 62% identity at the nucleotide level in a 66-bp region. The similarity between the TP901-1, O1205, and phiPVL phages in this region indicates that they may share a common mechanism for the switch determining lysis or lysogeny, but this has to be proven experimentally. Future studies may enable us to determine whether a protein complex consisting of ORF4 and ORF5 is responsible for the features of this new type of phage switch exemplified by TP901-1.