INTRODUCTION

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Only a few prophage genes are expressed in lysogens. Some, such as the genes encoding prophage repressors, prevent the expression of genes that lead to cell death and prophage loss. Others, such as phage  lom, bor, rexA, and rexB (2, 6), phage P22 sieA, sieB, and mnt (30, 31), phage P1 res and mod (35), phage P2 old and tin (9, 18), and prophage genes encoding virulence factors in a variety of pathogenic bacteria (see reference 32), have diverse functions. It is believed that the products of most of these genes confer a selective advantage on the lysogen, or, like repressors, prevent prophage curing and lytic phage growth. In some cases, for example  rexA (16), the expressed genes are cotranscribed with the repressor gene. In others, for example, P2 old (9), transcription initiates at dedicated promoters. In both cases downstream transcription terminators can prevent cotranscription of other prophage genes that might harm the host or destabilize the prophage. In phages of the  family, transcription antitermination mechanisms that function during lytic growth allow full expression of genes located downstream of the terminators only when they are needed for phage production (see reference 34).

Lysogens of temperate phage HK022 produce Nun, a protein that confers resistance to (or excludes) the related phage  (24). Although the nun gene is located immediately downstream of p_L, a major HK022 early promoter, this promoter can be inactivated by mutation without preventing nun expression (3). A second promoter, p_RM, is located upstream of p_L (Fig. 1). Transcripts initiating at p_RM in a lysogen direct the synthesis of prophage repressor, which is encoded by the cI gene. Previous results showed that at least some p_RM-directed transcripts extend through the repressed p_L promoter, which suggested that nun is cotranscribed with cI (3).

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Genetic map of the nun-cI region of HK022 (not to scale). Promoters and the direction of transcription are indicated by bent arrows. TIS903 is a transcription termination site within an insertion element found in our original isolate of HK022 (21). In a lysogen, pL is repressed and pRM is activated by the product of the cI gene. The map underneath is an expanded view of the region that contains two new promoters identified in this study (PNun and PNun'). DNA fragments from positions 2 to 72, positions 2 to 174, and positions 151 to 247, relative to the HK022 pL transcription start point, were fused to a promoterless lacZ gene. The corresponding -galactosidase (-gal) activities (numbers at left) were measured in exponentially growing cells carrying multicopy fusions. The relative strengths of PNun, PNun', and derepressed pL (but not pRM) are suggested by the thickness of the corresponding arrows. The binding site for oligonucleotide PM1, which was used to map the PNun and PNun' transcription start sites, is shown.

A potential problem with this idea is that nun transcripts initiating at p_RM will read through putL, a cis-acting transcription antitermination site that lies between p_RM and the beginning of nun (Fig. 1). Nascent putL transcripts modify elongating RNA polymerase molecules so that they read through downstream transcription termination sites (14). Indeed, the activity of putL is required for p_L-directed expression of phage genes located downstream of nun during lytic phage growth (20). Therefore, if nun were transcribed from p_RM in a lysogen, antitermination could lead to transcription of these downstream genes. This is likely to be harmful because some of the resulting proteins can reduce the fitness of the lysogen or destabilize the prophage (see Discussion). Accordingly, we looked for nun promoters located downstream of putL. In this article, we report that nun is transcribed from a dedicated promoter located between putL and the coding sequence, and that transcription from p_RM contributes little if anything to nun expression.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and bacteriophages. Bacterial strains, plasmids, and bacteriophages are listed in Table 1.

Phage and bacterial growth. Bacteria were grown in tryptone or Luria-Bertani (LB) broth and supplemented with ampicillin (100 µg/ml) or kanamycin (50 µg/ml) where appropriate. HK022 was grown and assayed as described in reference 21.

Cloning of fragments with promoter activity. Oligonucleotides were purchased from BioServe Biotechnologies (Laurel, Md.). Plasmid DNAs were isolated by using a Qiagen spin miniprep kit. Restriction enzymes and ligase were purchased from New England Biolabs (NEB). A 97-bp DNA fragment that immediately precedes the nun gene and contains the P_Nun promoter was amplified from plasmid pNGC25 by PCR with primers RK99 (5'-GGCGGATCCAATAAGCACCGTACGG-3') and RK100 (5'-CAGCGAATTCAAGTATTTATTGCAAAGATTC-3'). The amplified DNA was digested with EcoRI and BamHI (underlined sequences) and cloned into the promoterless lacZ vector pRS415.

Primer extension. Total RNA was isolated with an RNA isolation kit (Totally RNA; Ambion) from log-phase cultures of the following strains: SK37, SK38, and SK11 (Table 1). Strains SK37 and SK38 carry plasmids with inserts of HK022 DNA, and strain SK11 is an HK022 lysogen. Seven milliliters of culture was added directly to 10 ml of lysing buffer and then processed according to the Ambion protocol. RNA was suspended in diethyl pyrocarbonate-treated water supplemented with 1 mM EDTA. The purity and integrity of the RNA was analyzed on a 1.2% agarose gel, followed by staining with ethidium bromide. Primer extension reactions (20-µl volumes) were performed by using the Promega reverse transcription system. Oligonucleotide PM1 (5'-CTCGAGATGTAAGACCTC-3') is complementary to the nun mRNA and hybridizes approximately 90 bp downstream of the P_Nun start site. PM1 was 5' end-labeled in a 30-µl reaction mixture containing 50 µCi of [-³²P]ATP (Redivue [3,000 Ci/mmol]; Amersham) 10 U of T4 polynucleotide kinase (NEB) and 1× kinase buffer (NEB). Reaction mixtures were incubated for 30 min at 37°C. Primer extension reaction mixtures were incubated at 42°C for 1 h, and the reactions were stopped by adding 10 µl of Stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.02% xylene cyanol). Reaction mixtures were heated at 94°C for 3 min, chilled immediately on ice, and then loaded onto an 8% denaturing sequencing gel. Sequencing reactions were performed with the Perkin-Elmer Amplicycle sequencing kit. Sequence was obtained directly from purified pK1 plasmid and PCR-amplified template from wild-type P_Nun and P_Nun(10) mutant clones by using 5' end-labeled oligonucleotide PM1 as the primer.

In vitro transcription. Multiple round in vitro transcription reactions were performed on templates amplified from P_Nun⁺ and P_Nun(10) mutant clones. Reaction mixtures consisted of approximately 50 nM PCR template, 100 nM RNA polymerase holoenzyme (Epicentre, Madison, Wis.), 1 mM concentrations of each nucleoside triphosphate (NTP), 20 mM Tris-glutamate, 50 mM K-glutamate, 10 mM Mg-glutamate, and 0.001 µg of bovine serum albumin per ml. The reaction mixtures were incubated for approximately 1 h at 37°C. After ethanol precipitation and washing, the pelleted material was suspended in diethyl pyrocarbonate-treated water supplemented with 1 mM EDTA. The RNA products were then used directly in a primer extension reaction.

P_Nun and P_Nun' mutations. To inactivate the P_Nun promoter, plasmid pJO9.18 was digested with BsrGI, and the overhangs were filled in with Klenow fragment. The resulting blunt-ended plasmid was ligated and electroporated into MC1000 cells. Transformants were screened by PCR with primers PM1 and RK100, and the products were tested for their susceptibility to BsrGI digestion. The P_Nun' promoter was inactivated by replacing the sequence in the predicted 10 hexamer with a BamHI restriction site. The replacement was accomplished by overlap extension PCR with primers PM2 (5'-GCGGATAACAATTTCACACAGG-3'), PM3 (5'-CTTCTATTTTTTCGAACGACTTCTGGATCCAAAAGCTGCTTTGCTTTTTGTGAC-3'), PM4 (5'-CACAAAAAGCAAAGCAGCTTTTTGGATCCAGAAGTCGTTCGAAAAAATAG-3'), and PM6 (5'-CGCCAGGGTTTTCCCAGTCACGAC-3'). The BamHI site that replaces the predicted 10 sequence is underlined in the relevant oligonucleotide sequences. Amplifications were performed with Vent polymerase with pJO9.18 used as the template. The final amplified product was digested with HindIII and EcoRI and ligated into pJO9.18. The desired clones were identified by restriction analysis of purified plasmid DNA. The mutation was transferred onto HK022 by recombination, and the presence of the BamHI site was confirmed by PCR amplification with primers PM1 and PM5 (5'-CCTCATTAGGCAGTCAATCG-3') followed by digestion of the products with BamHI. Inactivation of both the P_Nun' and P_Nun promoters was accomplished by digesting the P_Nun' mutant construct with BamHI and BsrGI. The DNA ends were filled in by Klenow fragment, ligated, and electroporated into MC1000 cells. The desired deletion mutant was identified by analyzing the size of DNA amplified by PCR with primers PM1 and PM5.

Crossing P_Nun and P_Nun' mutations onto HK022 phage. Cells carrying pJO9.18 or various mutant derivatives were infected with HK022::Tn10(kan) cIts12 (phage O295), and the resulting lysate was used to infect MC1000 cells. This plasmid carries the wild-type HK022 cI gene but, for unknown reasons, does not prevent HK022 growth (21). The Tn10(kan) insertion is in the p_L operon, downstream from nun. Phage recombinants carrying the plasmid cI⁺ gene were selected by infection of a sensitive host and isolation of Km^r colonies at 42°C, the nonpermissive temperature for cIts12. The presence of the mutation on the prophage was confirmed by the size of PCR-amplified products and the ability to digest the products with the enzymes BsrGI or BamHI. Lysates of recombinant phage were made by induction of the lysogens with UV light. These lysates were used to lysogenize cells containing p_L-nutL-lacZ fusions (24).

Crossing lacZ fusions onto RS88. Promoter fusions in the lacZ reporter vector, pRS415, were transferred onto RS88 by recombination as described previously (14, 28). Lambda prophage copy number was determined as described previously (23).

Beta-galactosidase activities. Strains containing thermosensitive  repressor and a p_L()-lacZ fusion were grown overnight at 32°C in tryptone broth. The overnight cultures were diluted 1:50 in LB broth, grown to approximately 1 × 10⁸ to 2 × 10⁸ cells/ml, and shifted to 42°C for 60 min to allow derepression of the  p_L promoter. Beta-galactosidase activity was measured as described previously (17). It was important to use toluene to permeabilize the cells because -galactosidase produced by these fusions was destabilized by the alternative treatment with chloroform and detergent. Cells containing single-copy lacZ fusions were grown overnight at 37°C in tryptone broth. The overnight cultures were diluted 1:1,000 in LB broth and grown at 37°C to an optical density at 650 nm of approximately 0.5 × 10⁸ to 1 × 10⁸ cells/ml. Thereafter, aliquots were removed and placed on ice at various intervals and the -galactosidase activities were determined as described above.

Determination of HK022 prophage copy number. The copy number of HK022 prophages was determined essentially as described in reference 23 by using primers specific for the HK022 int and attP and the host attB sites, as follows: RK111 (int oligonucleotide [5'-AGCAATGCAGGGAGGCCAGC-3']), RK112 (attP oligonucleotide [5'-AATAATCTTGCGGTAGCCAGAC-3']), and RK113 (attB oligonucleotide [5'-GGAGATCTCCTGCTCCTGTTG-3']). Single-copy lysogens were identified by the amplification of an approximately 747-bp DNA generated from the attB and int primers. Polylysogens produced the 747-bp fragment in addition to an approximately 613-bp fragment.

Immunoblots (10). Cells were grown in LB broth at 37°C, chilled, concentrated, diluted into loading buffer, boiled for 5 min, and frozen at 70°C. Aliquots were loaded directly onto sodium dodecyl sulfate Tris-tricine-16.5% acrylamide gels (Bio-Rad), electrophoresed, and electroblotted to Immobilon polyvinylidene difluoride membranes. We found that Nun comigrated with prestained marker proteins (Bio-Rad) in the 14- to 18-kDa range. The membranes were blocked with 5% milk powder and allowed to react overnight with antiserum against partially purified Nun raised in rabbits (antiserum kindly supplied to us by David Friedman [11]). We treated the antiserum before use with an acetone powder prepared from strain MC1000 to remove antibodies to bacterial proteins (10). It was diluted 1:50 into a solution of 5% milk powder for use. Bands were visualized by treating the membranes with peroxidase-coupled anti-rabbit serum for 1 h, allowing them to react with ECL Western blotting detecting reagent (Amersham), and placing them in contact with X-ray film. To quantitatively estimate the amount of Nun, we mixed serial dilutions of purified Nun (a gift of Randy Watnick) with extracts of strain MC1000 and fractionated the mixtures in separate lanes of a gel (see Fig. 3).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Identification of promoters located between putL and nun. We have discovered two promoters located between putL and nun. These were found by extending an oligonucleotide primer that was hybridized to RNA isolated from cells that carry plasmid clones of HK022 DNA. Extension of the primer, which is complementary to the beginning of the nun coding sequence, produced a strong signal corresponding to a 5' RNA end close to the start of the nun coding region and a weak signal corresponding to a 5' RNA end further upstream, but still downstream of putL (Fig. 2). We also saw the strong primer extension signal with RNA isolated from an HK022 lysogen, although the amount of product was much less in this case (data not shown) (we did not see the weak signal, as would be expected if its intensity were correspondingly reduced). We call the putative promoter corresponding to the strong downstream signal P_Nun, and we called the putative promoter corresponding to the weak upstream signal P_Nun'.

View larger version (54K): [in this window] [in a new window]   FIG. 2.   Mapping of the transcription start sites of the PNun and PNun' promoters. Oligonucleotide PM1, complementary to the nun mRNA, was used for reverse transcriptase primer extension and sequencing. The DNA sequence of the promoter 10 regions is shown on the left. The arrows mark the position of the extension products (lane 1). The predominant initiation sites are marked by asterisks beside the DNA sequence (lanes G, A, T, and C).

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Sequence of predicted promoters and promoter mutants. The bent arrows indicate the observed 5' ends of PNun and PNun' transcripts (top and middle lines, respectively), as indicated by primer extension analysis (Fig. 2). The predicted 10 and 35 hexamers for these promoters are shown in bold type. The E. coli 70 consensus hexamers are TATAAT and TTGACA, respectively. The straight arrows indicate the sequence changes in the PNun(10) and PNun'(10) mutants (see Materials and Methods). The sequence [PNun-PNun'] is shown in the third line (see Materials and Methods). The slashes indicate the fusion points between PNun'(10) sequence (left) and PNun sequence (right). Possible alternative 10 and 35 hexamers are shown in bold type.

P_Nun and P_Nun' mutations. To see if these promoters contribute to Nun production in an HK022 lysogen, we mutated them individually or together and measured the activity and intracellular concentration of Nun. In mutant P_Nun(10), the predicted 10 hexamer of P_Nun was changed, in mutant P_Nun'(10), the predicted 10 hexamer of P_Nun' was changed, and in mutant [P_Nun-P_Nun'], the DNA segment extending from the (altered) 10 hexamer of P_Nun'(10) to the 10 hexamer of P_Nun was deleted (Fig. 3). We showed that P_Nun(10) had substantially reduced promoter activity: the steady-state level of -galactosidase activity in cells carrying a single copy of a P_Nun(10)-lacZ fusion was 1 to 3% of that of a comparable P_Nun-lacZ fusion (strains RK445 and RK444, respectively) (data not shown). The P_Nun'(10) and [P_Nun-P_Nun'] mutations were not checked in this way, and we have no experimental evidence that they inactivate the respective promoters. Indeed, inspection of the sequence revealed [P_Nun-P_Nun'] could have created a new promoter (Fig. 3). The three mutations were crossed from the plasmids in which they were constructed onto HK022 as described in Materials and Methods, and single lysogens of the mutant phages were isolated. The Nun phenotype of these lysogens was checked in several ways.

Plaque formation by  on lawns of HK022 lysogens (Table 2). A wild-type HK022 prophage reduced the number of plaques formed by superinfecting  by more than 8 orders of magnitude. No  mutants able to form plaques were found, probably because they cannot arise in a single step (25). However, the HK022 P_Nun(10) mutant prophage excluded  much less efficiently than did its parent. We observed a large number of tiny, uncountable plaques at a frequency of 10² plaques per added  phage. These are probably formed by limited growth of wild-type  on this host. We also found plaques of normal or near-normal size at a frequency of about 10² plaques per added phage. Most or all of these were formed by mutants that probably arose during limited  growth on the lawn (see below). The residual  exclusion by the P_Nun(10) lysogen is clearly due to Nun activity, since a lysogen of a nun::Tn10 insertion mutant plated  with unit efficiency, and the plaques were of near-normal size. The phenotype of a [P_Nun-P_Nun'] lysogen was similar to that of the P_Nun single mutant, although, surprisingly, the frequency of normal-sized (i.e., mutant; see below)  plaques was somewhat lower for the former (ca. 10³ plaques per added phage). These results suggest that P_Nun makes a major but not the only contribution to nun transcription in a lysogen. The quantitative difference in  exclusion between P_Nun(10) and [P_Nun-P_Nun'] can be accounted for by assuming that the deletion mutation creates a new promoter that is weaker than P_Nun but stronger than P_Nun(10) (see above). The P_Nun'(10) lysogen excluded  as efficiently as did its wild-type parent, but this result cannot be unambiguously interpreted because we have no experimental evidence that the mutation inactivated the promoter.

6

Effect of promoter mutations on the expression of a p_L()-nutL-lacZ fusion. Nun blocks  growth by binding to the boxB element of nascent nut transcripts and prematurely terminating early  transcription (5, 24) (see Discussion). Thus, measurement of -galactosidase production in a cell containing a nut site fused to a reporter gene gives a direct and quantitative estimate of Nun activity. As shown in Table 2, the wild-type Nun concentration greatly reduced -galactosidase accumulation by the p_L()-nutL-lacZ fusion but had no effect on a similar fusion that lacks a complete boxB. This agrees with previously published results (24). The P_Nun(10) mutation restored a high level of -galactosidase activity, similar to that observed in a nun insertion mutant. These results confirm that P_Nun makes a major contribution to nun expression. In contrast to measurement of phage exclusion, we saw no evidence of any residual Nun activity in this mutant. We propose an explanation for this difference in the Discussion. The [P_Nun-P_Nun'] mutation gave an intermediate level of lacZ expression compared to the wild-type and the P_Nun mutant lysogens, a result that is qualitatively consistent with its intermediate effect on  exclusion. The P_Nun'(10) mutation caused a small but reproducible increase in the expression of the nutL-lacZ fusion.

Effect of promoter mutations on the level of Nun antigen. We measured the effects of our promoter mutants on the intracellular concentration of Nun protein in HK022 lysogens by immunoblots. We found that the P_Nun(10) and the [P_Nun-P_Nun'] mutations reduced Nun protein concentrations to undetectable levels (Fig. 4). We estimate that we would have been able to see as little as 25% of the level of Nun found in a wild-type lysogen. By contrast, the P_Nun'(10) mutation had no detectable effect on Nun level. The simplest conclusion from these results is that P_Nun is the major promoter directing nun transcription in a lysogen. We consider the source of the residual phage exclusion activity by the P_Nun(10) prophage in the Discussion.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Detection of Nun in HK022 lysogens by immunoblotting. Cell extracts of strains lysogenic for wild-type or mutant HK022 prophages, of a nonlysogen, or of a strain carrying an induced PLac-nun fusion (as indicated below the gel image) were fractionated by electrophoresis in a denaturing gel. The Nun phenotype refers to the  exclusion data of Table 1. Nun was detected with antibody as described in Materials and Methods. The position of Nun is indicated by the arrow. Its mobility relative to protein standards is consistent with a previous report (21), and the identification is confirmed by the large amount of protein with identical mobility extracted from a Nun-overproducing strain (lane 7). Lanes 1 to 6 each contained 0.02 to 0.025 mg of protein, and lane 7 contained about half that amount. Lane 1, PNun'(10) (strain LM86); lane 2, PNun+ (strain LM87); lane 3, [PNun-PNun'] (strain LM89); lane 4, PNun(10) (strain RK422); lane 5, nun::Tn10 (strain JO350); lane 6, strain MC1000 (the nonlysogenic parent); lane 7, PLac-nun (strain RW4055). The first six strains were grown to 1 × 109 to 2 × 109 cells per ml and the seventh strain was grown to 4 × 108 to 6 × 108 cells per ml in LB broth at 37°C before extraction. Isopropyl--D-thiogalactopyranoside (IPTG) (1 mM) was added to the culture of RW4055, which contains a plasmid with a PLac-nun fusion, 50 min before the cells were harvested. The production of Nun by this strain was much reduced if IPTG was omitted (data not shown).

Further analysis of P_Nun. We estimated the amount of Nun per cell by comparing immunoblots of lysogens to those of known quantities of purified Nun protein (Fig. 3). There are 120 to 360 molecules of Nun per cell in a culture grown to 1 × 10⁹ to 2 × 10⁹ cells/ml in LB broth. This number is roughly consistent with the activity of -galactosidase per cell in comparable cultures of an HK022 lysogen containing a nun::lacZ translational fusion (strain RW4072) (the calculation assumes that the fusion protein is as active as native -galactosidase [26]). The specific activity in this strain and another strain with a P_Nun-lacZ transcriptional fusion (RK444) increased from three- to sixfold as the cultures proceeded from mid-log phase (0.5 × 10⁸ to 1.5 × 10⁸ cells/ml) into stationary phase (1 × 10⁹ to 2 × 10⁹ cells/ml). The mechanism and biological significance of this increase are unknown.

	DISCUSSION

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Our experiments show that P_Nun is the major promoter for expression of the nun gene in an HK022 lysogen. This promoter thus joins a fairly short list of identified promoters that are active in repressed prophages. We estimate that there are several hundred molecules of Nun per cell in early-stationary-phase cells, and perhaps one-sixth to one-third that number in exponentially growing cultures. The mechanism and biological significance of this growth phase regulation are unknown. Since Nun is stable in vivo (21), its low level probably reflects, at least in part, the weakness of P_Nun. Measurements of the activity of comparable promoter-lacZ fusions suggest that P_Nun is about 1% as strong as fully derepressed HK022 p_L (R. A. King and R. A. Weisberg, unpublished results). This result is qualitatively consistent with the relative strengths of the two promoters in vitro. The 70 consensus is not well conserved in P_Nun, and the transcript is unusual in that it starts with a U residue. These factors may contribute to the low activity of this promoter.

Nun acts by binding stoichiometrically to a stem-loop formed by the transcript of the boxB element in the nut sites of the nascent  p_L and p_R transcripts (5). It then interacts with and arrests elongating RNA polymerase (12, 13, 33). The arrested transcription elongation complexes are stable in vitro, but the arrested polymerase probably disassociates from the template in vivo (24, 25, 29). The efficiency of Nun action is remarkable: it completely blocks  lytic growth and cell killing and reduces to about 1% the expression of reporter genes that are fused to  p_L or p_R and preceded by either nutL or nutR, respectively. The reduction persists for more than 1 h after derepression (24) (Table 1).  p_L directs the synthesis of 10 to 20 transcripts/min in vivo (15), and the strength of  p_R is likely to be similar. It is unlikely that a few hundred molecules of Nun could arrest such a large number of transcription elongation complexes if Nun were consumed during the reaction. Therefore, we suggest that Nun bound to arrested elongation complexes is released in active form after termination and recycles to newly synthesized transcripts where it acts again.

RNA polymerase molecules that initiate at P_Nun do not transcribe the putL antitermination site (Fig. 1) and should therefore efficiently terminate at downstream transcription terminators. One of these terminators is within the IS903 insertion element located immediately downstream of nun (8, 21). Termination of nun transcripts is probably advantageous to HK022 and its lysogens because the prophage kil, cIII, xis, and int genes lie downstream of transcription terminators in the p_L operon, and the products of these genes are likely to destabilize the prophage or harm the lysogen. As expected, nun can also be expressed from the p_L promoter: we found that more than 10 times the amount of -galactosidase was produced by a p_L-P_Nun-nun::lacZ gene fusion in the absence than in the presence of repressor (unpublished results). However, lytic growth of HK022 does not require Nun (21).

Mutation of P_Nun depressed but did not completely prevent Nun production. Nun was undetectable by immunoblot (25% of wild type) and did not reduce expression of a p_L()-nutL-lacZ fusion in a P_Nun(10) lysogen. Nevertheless, it did reduce growth of superinfecting  (Table 2). Lambda growth could be more severely affected by low Nun levels than is expression of a reporter gene fused to nutL because the phage also has a nutR site, and this could amplify its sensitivity. The source of the residual Nun in the P_Nun mutant is unknown, but several possibilities are open. First, P_Nun(10) retains 1 to 2% of P_Nun⁺ promoter activity, as judged by expression of a reporter gene fused to the promoter, and this could be enough to impede the growth of superinfecting . Second, P_Nun' could be a source of nun transcripts. We have no independent evidence that the P_Nun'(10) mutation inactivates P_Nun', and we were surprised to find that the [P_Nun-P_Nun'] mutant retained considerable Nun activity. An inspection of the fused sequence suggests that this deletion creates a new promoter. Third, p_L could contribute to residual nun transcription if repression is incomplete. Finally, p_RM could contribute to residual nun transcription.

With respect to the last possibility, Cam et al. (3) detected low-intensity transcription initiating upstream of p_L and extending into putL in HK022 lysogens. They also found that a high level of HK022 repressor supplied in trans to a prophage by a plasmid reduced both Nun activity and the expression of a nun::lacZ gene fusion. Cam et al. (3) suggested that transcripts originating at p_RM are the principal source of nun expression in a lysogen and that excess repressor reduces nun expression by repressing p_RM. An alternative explanation of these results is that P_Nun responds to repressor. Although inspection of the DNA sequence revealed no obvious HK022 operator site in the vicinity of P_Nun, high repressor concentrations might relax the specificity of binding. Indeed, Carlson and Little (4) demonstrated binding of repressor to sequences adjacent to an isolated operator under such conditions.

Bacteriophage HK022 resembles other phages of the  family with respect to its genetic organization. However, in striking contrast to , the first gene in the HK022 p_L operon is expressed in the presence of the prophage repressor. Our identification of a dedicated, constitutive promoter for nun explains in a simple way the expression of the phage exclusion function of a repressed HK022 prophage. The advantage that Nun expression confers on HK022 is less clear. Nun excludes phages with nut sites of  specificity; the growth of several other phages, including some with related nut sites, is unaffected (15). Perhaps phage strains with nut sites of  specificity are important natural competitors of HK022. Alternatively, perhaps Nun has functions that are yet undiscovered.