INTRODUCTION

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Lysogenic phages regulate their developmental fate by encoding a product (termed CI in the case of phage ) that acts as a transcriptional regulator. CI binds to two operator regions on the phage genome, designated O_R and O_L, which are each further divided into three closely spaced binding sites (16 to 18 bp) termed operators O_R1/L1 to O_R3/L3 (reviewed in reference 27). Interspersed with the operators are two major promoters, P_R and P_L. In the P_R region, the binding of a CI dimer to its highest-affinity binding site (O_RI) turns off the P_R promoter, repressing the genes that mediate the lytic development of the phage. Furthermore, this interaction stabilizes the association of a second CI dimer to the O_R2 site. This activates the P_RM promoter, which is divergent from P_R, thereby activating the transcription of genes responsible for the maintenance of lysogeny (13, 17, 19, 21, 27). When the repressor concentration becomes high enough to cause occupancy of the O_R3 site, the transcription of cI is prevented. This leads to a reduction of repressor concentration which, in turn, provokes its unwinding from O_R3 and the resumption of cI expression. The repressor behaves then as an autogenous regulator of its own synthesis that functions positively at low concentrations and negatively at high concentrations (reviewed in reference 27). In other words, the commitment between lytic and lysogenic development is thus critically dependent on the ability of the repressor to discriminate between these different operators.

Very little is known about the commitment between lytic and lysogenic development from bacteriophages from gram-positive bacteria. Analysis of the genomes of different lactococcal phages reveals that rlt (24), BK5-T (4), and Tuc2009 (32) code for a product that shows significant homology with the CI repressor of lambdoid phages. Unlike them, the lactococcal phages seem to have only one early region. In the case of phage rlt, this may be subdivided into two regions (O₁ and O₂) with dyad symmetry (21 bp) separated by a 2-bp spacer, into which the 35 consensus regions of the two divergent promoters are embedded (24). In the case of phage BK5-T, there are three regions (O₁ to O₃) of dyad symmetry (18 bp) separated by 10- and 24-bp spacers. The putative O₁ and O₃ sites lie within the 35 and 10 consensus regions, whereas O₂ is placed between the two divergent promoters (4). In the case of the temperate bacteriophage A2, which infects Lactobacillus casei and Lactobacillus paracasei strains (16), the CI protein shows significant identity with the CI protein of lambdoid phages. The A2 cI gene encodes a 224-amino-acid polypeptide with a predicted molecular mass of 25,277 Da. It appears to be the central player in the maintenance of the lysogenic state because (i) it is synthesized in lysogenic cultures, presumably blocking the lytic development of superinfecting A2, and (ii) L. casei derivatives containing a chromosomal copy of cI are completely resistant to phage infection. In front of cI there are three regions (O₁ to O₃) of 20 bp, displaying partial symmetry, that are separated by 26- and 32-bp spacer stretches, respectively (20) (Fig. 1).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the A2 genome showing the location and organization of the development control region. (A) EcoRI physical map of the A2 genome. (B) Organization of its central region. The arrows refer to sequences functionally identified or similar to cI (repressor), orfX (unknown function), xis (putative excisionase), int (integrase), cro (putative homologue of  cro), ant (putative antirepressor), and attP (site of attachment to the bacterial chromosome). (C) Scheme of the genetic switch region, located in the intergenic stretch between cI and cro. The transcription start sites of the PL and PR promoters and the relative positions of the 10 and 35 hexamers with respect to the three operator sequences (O1 to O3) are indicated.

This paper deals with the molecular characterization of the CI repressor protein of bacteriophage A2 and how it acts to control gene expression.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and media. Escherichia coli XL-1 Blue (5) and DH10B (GIBCO BRL) were used as recipients for plasmid constructions. E. coli BL21(DE3)/pLys was used in expression studies of the genes cloned into plasmid pET11a (31). Plasmids pLys (31) and pUC18 (35) have been previously described.

DNA manipulations. Plasmid DNA was obtained by the alkaline lysis method (3) and purified through a CsCl gradient (29). Analytical and preparative gel electrophoresis of plasmid DNA and restriction fragments was carried out in 0.8% (wt/vol) agarose-Tris-borate horizontal slab gels or 8% (wt/vol) polyacrylamide-Tris-borate gels. The purification of DNA fragments was carried out by electroelution (29).

Protein purification. The overexpression of CI was achieved by the addition of 1 mM IPTG (isopropyl--D-thiogalactopyranoside) (Boehringer Mannheim) to exponentially growing cultures of E. coli BL21(DE3)/pLys carrying pET11a-cI. After 30 min, rifampin was added (to a final concentration of 200 µg/ml), and the cells were further incubated for 90 min. The cells were harvested, resuspended in 25 ml of buffer A (50 mM Tris-HCl [pH 7.5], 10 mM MgCl₂, 1 mM dithiothreitol [DTT], 0.1% Tween 20) per liter of culture containing 1 M NaCl, and broken by passage through a French press. During the purification procedure all steps were performed at 4°C. Crude extracts (total protein concentration of about 20 mg/ml) were centrifuged (12,000 × g for 30 min), the supernatant was discarded, and the pellet was washed twice with 25 ml of buffer A. The resulting pellet was thoroughly resuspended in the same volume of buffer A containing 1 M NaCl, until complete dissolution was obtained, and was then diluted four times in buffer A to achieve a final concentration of 250 mM NaCl. This sample was loaded onto a Q-Sepharose column (Pharmacia) equilibrated with the same buffer without DTT. The column was washed with 10 column volumes of buffer and eluted with a gradient of increasing salt concentrations (250 mM to 1 M NaCl). The fractions containing pure CI (as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) were pooled, diluted in glycerol (50% final concentration), and stored at 20°C. The protein was measured by using the Bio-Rad protein assay.

Filter binding assays. The formation of DNA-protein complexes was measured as described previously (11). In short, the standard reaction was performed for 30 min at 30°C, and the mixture contained 475 pg (81 pM) of the ³²P-labeled 183-bp DNA segment (see above) and 126 ng (100 nM) of CI in a total volume of 50 µl of buffer B (50 mM Tris-HCl [pH 7.5], 10 mM MgCl₂, 0.01% Tween 20, 100 mM NaCl, 5% glycerol), unless stated otherwise. Buffer B (1 ml) was added to stop the reaction, and the mixture was filtered and extensively washed with buffer B. Filters were dried, and the radioactivity bound to them was determined by scintillation counting. The DNA retained on the filter was corrected for the retention of radioactively labeled DNA in the absence of protein (1 to 3%). The specific activity of the labeled DNA was measured as trichloroacetic acid-precipitable material. All reactions were performed in duplicate.

Electrophoretic mobility shift assay (EMSA). The end-labeled 183-bp DNA fragment (240 pg or 81 pM) was incubated with increasing amounts of CI protein in 25 µl of buffer B at 30°C for 30 min, in the presence of poly(dI)-poly(dC) as an unspecific competitor (at a final concentration of 4 ng/µl). The reaction mixture was loaded onto a 5% (wt/vol) nondenaturing polyacrylamide gel. Similar reaction conditions were used with the Bacillus subtilis vegetative RNA polymerase (^A-RNAP) and in the competition experiments between ^A-RNAP and CI, except that the reaction mixture was loaded onto a 4.5% (wt/vol) nondenaturing polyacrylamide gel. Gels were run at 100 V in TAE buffer (40 mM Tris-acetate, 1 mM EDTA [pH 8]) at room temperature. Gels were vacuum dried and analyzed by autoradiography. Quantification of the repressor-operator complexes separated in these gels was performed by using an Instant Imager (Packard). The degree of DNA-protein hybrid formation was quantified by dividing the amount of complex formed by the amount of residual free DNA.

DNase I footprinting. The EcoRI-SphI or HindIII-KpnI DNA fragments obtained from pUO183 were end labeled at the EcoRI or HindIII sites with Klenow DNA polymerase and [-³²P]dATP (3,000 Ci/mmol; Amersham). End-labeled DNA (81 pM) was incubated for 30 min at 30°C in the absence or presence of CI and/or B. subtilis ^A-RNAP in 20 µl of buffer B plus poly(dI)-poly(dC) as an unspecific competitor (at a final concentration of 50 ng/µl), followed by the addition of freshly diluted DNase I (Boehringer Mannheim) (at a concentration to obtain, on average, one cut per molecule). The mixture was incubated at 30°C for 3 min, and the digestion was stopped by the addition of 1 µl of 0.5 M EDTA, pH 8. The DNA was precipitated, redissolved in a loading buffer, electrophoresed on a 6% denaturing polyacrylamide gel, and autoradiographed. Chemical sequencing reactions (22) for purines, obtained by an express protocol (2), were run in parallel to determine the sizes of the DNA fragments generated.

In vitro runoff transcription assays. Plasmid pUO183 digested with XmnI was used as a template for in vitro transcription assays. The reaction mixtures contained, in 25 µl of a solution consisting of 3 nM DNA, 0.2 mM each ATP, CTP, GTP, and UTP, 25 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 90 mM ammonium sulfate, 2 mM DTT, 7.5 U of RNasin (Promega), 30 nM B. subtilis ^A-RNAP, and different amounts of CI. After 30 min of incubation at 30°C, the reactions were stopped with 1 µl of 0.5 M EDTA, pH 8. The RNAs were precipitated and then analyzed by primer extension as follows. The pellet was dissolved in 10 µl of a solution containing 50 mM Tris-HCl (pH 7.5), 40 mM KCl, 7 mM magnesium acetate, 2 mM DTT, 200 µM each dCTP, dGTP, and dTTP, 100 µM [-³²P]dATP (3,000 Ci/mmol), 4 U of avian myeloblastosis virus reverse transcriptase (Promega), 10 U of RNasin, and 0.5 pmol of primers 5'-CGCCAGGGTTTTCCCAGTCACGA-3', for the P_R promoter, and 5'-GCGGATAACAATTTCACACAGG-3', for the P_L promoter. The reaction mixture was incubated at 42°C for 60 min, and the reaction was stopped by the addition of 0.5 µl of 0.5 M EDTA, pH 8, and 100 µl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8). Unincorporated nucleotides were removed by passing the sample through a Sephadex G-50 column. The resulting cDNAs obtained were precipitated, analyzed by denaturing PAGE (6%), and autoradiographed. Chemical sequencing reactions of purines (2, 22) were run in parallel to determine the sizes of the cDNAs obtained.

	RESULTS

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Purification of the bacteriophage A2 CI repressor. The CI protein was overexpressed as described in Materials and Methods. Under those conditions most of the CI, which accounted for about 2% of the total cell protein, was in the pellet of the crude extract centrifugations. This allowed the removal of soluble proteins and washing of the protein aggregates, resulting in the solubilization of most proteins coprecipitated with CI. The resulting pellet was solubilized in buffer A containing 1 M NaCl, and the solution was then diluted to lower the salt concentration to 0.25 M. The solubilized CI protein was further purified by a conventional chromatographic step (Q-Sepharose), rendering a product that was more than 95% pure, as judged by SDS-PAGE (data not shown).

CI specifically binds to the A2 cI-cro intergenic DNA segment. Analysis of the genomic organization of the early region of phage A2 (1, 20) (Fig. 1) revealed that the putative rightward promoter (P_R) would transcribe the cro and ant open reading frames, while the leftward promoter (P_L) would govern the expression of the genes coding for CI and the site-specific recombination machinery. This organization allowed us to postulate that the decision between lytic and lysogenic development would be critically dependent on the ability of CI to act as the A2 repressor, probably by discriminating among possible operators located between P_L and P_R. In support of this hypothesis was the finding of a putative helix-turn-helix domain in the NH₂-terminal half of CI that might mediate its DNA binding. To test whether the purified CI protein specifically binds to the early control region of the A2 genome, the 153-bp intergenic cI-cro region (obtained as a 183-bp DNA segment) was incubated with the CI protein and the mixture was subjected to filter binding assays.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Parameters involved in CI-DNA complex formation as detected with a filter binding assay. The CI binding parameter measured is indicated under each panel. Analytical binding reactions were performed in a 50-µl volume with 81 pM 183-bp [32P]DNA segment (475 pg) that comprises the cI-cro intergenic region and 100 nM CI (126 ng). The DNA retained on the filter was corrected for the retention of radiolabeled DNA in the absence of CI (about 2% of total DNA input).

Cooperative binding of CI to the cI-cro intergenic region. The rate of CI-DNA complex formation was determined as a function of CI concentration (Fig. 3A). The dependence of the DNA retention on protein concentration appeared to follow a sigmoidal curve. The apparent equilibrium constant (K_app) of the CI-cI-cro-DNA complex is estimated to be 80 nM at pH 7.5 and 30°C, whereas the K_app for nonspecific DNA is about 100 µM. The sigmoidal form of the curve suggested that the CI protein binds cooperatively to the cI-cro intergenic DNA segment.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Determination of the affinity between CI and the cI-cro intergenic DNA segment. The reaction mixtures were incubated for 30 min at 30°C. (A) Trapping of the 32P-labeled 183-bp DNA segment (81 pM) on membrane filters in the presence of increasing concentrations of CI. (B) Gel shift assay of the complexes formed by increasing concentrations of CI (0, 1, 2, 4, 6, 8, 10, and 50 nM) and the DNA in the presence of a 400-fold excess of nonspecific [poly(dI)-poly(dC)] competitor DNA. The unbound probe (Free) and the protein-DNA complexes (I and II) are indicated. (C) Fraction of DNA bound by increasing concentrations of CI obtained from quantification of the phosphorimage of the autoradiograph shown in panel B. (D) Binding of CI (7 nM) to increasing concentrations of DNA. [CI] is the total protein concentration, [DNAb] is the concentration of substrate bound to CI, and [DNAt] is the total substrate concentration.

CI binds to three discrete sites. For determination of the precise location of the sequences recognized by CI, the CI-DNA complexes formed with the cI-cro intergenic region were analyzed by DNase I footprints. The top strand contained three domains of CI protection of 22, 34, and 38 bp. These were separated from each other by 12- and 38-bp intervals (Fig. 4A; also see Fig. 8). Within this interspaced region, phosphodiester bonds hypersensitive to DNase I cleavage were identified. One of them was located between O₁ and O₂. The several hypersensitive sites between O₂ and O₃ are separated from each other by 10 ± 1 nucleotides, which is about one helical turn (assuming 10.5 bp per turn) in double-stranded DNA.

View larger version (81K): [in this window] [in a new window]   FIG. 4.   DNase I footprinting analysis of CI-DNA complexes. (A) The 172-bp EcoRI-SphI DNA fragment (top strand) from plasmid pUO183 containing the 153-bp cro-cI intergenic region from A2 was incubated with 3.5, 7, 14, and 28 nM CI. (B) The 166-bp HindIII-KpnI segment (bottom strand) was incubated with 3.5, 7, 14, 28, and 56 nM CI. The DNA fragments were end labeled at the HindIII or EcoRI restriction sites and partially digested with DNase I. The operator sites are indicated. A DNA sequencing ladder was used to determine the DNA fragment lengths. (C) The DNA half-sites of the inverted repeated sequences of the CI proposed operators are aligned. Conserved bases and their frequencies are indicated below the sequences.

Localization and characterization of the promoters of the genetic switch region. Northern blot hybridization analysis of the mRNAs produced from the genetic switch region, together with a visual inspection of its nucleotide sequence, suggested that the promoters of cI and cro were located in this intergenic region and were divergently orientated (20) (Fig. 1). To map the transcription start points of the two promoters, in vitro transcription runoff experiments were performed with ^A-RNAP and linearized pUO183, which contains the 153-bp segment of the genetic switch region, as a template DNA. After primer extension of the in vitro-transcribed products with the oligonucleotides described in Materials and Methods, we obtained two cDNA bands corresponding to segments of 84 and 100 nucleotides from the left- and right-oriented promoters, respectively (Fig. 5). These promoters were consequently named P_L and P_R. The cDNA bands seemed to indicate that the transcript from P_R would be about 10 times more abundant than the one from P_L. The 5' ends of transcription of these mRNA species map at guanosine nucleotides located at positions 12 and 130 (see Fig. 8). In both cases, 7 bp upstream from the transcription start sites, extended 10 consensus sequences (with TG dinucleotides at positions 14 and 15) were observed. These were separated by canonical 14-bp stretches from 35 consensus hexamers. Stretches of about 20 bp rich in A+T nucleotides could be predicted immediately upstream of the 35 hexamers (12). Consequently, all the crucial elements for RNAP promoter recognition in gram-positive bacteria, located near positions 59 to 38, 35, 14, 15, and 10 relative to the transcription start sites, were identified at both P_L and P_R (12, 15, 23, 33) (see Fig. 8). It is likely, therefore, that the data obtained from this work with the B. subtilis ^A-RNAP would correspond to those that might be obtained if RNAP from Lactobacillus was used.

View larger version (124K): [in this window] [in a new window]   FIG. 5.   Location of the transcription start sites in the cro-cI intergenic segment of phage A2 and role of CI in regulation of the PR promoter. B. subtilis A-RNAP (30 nM) and linearized pUO183 DNA (3 nM) were used for the in vitro generation of cI (lane 2) and cro (lane 3) runoff transcripts, and the resulting mRNAs were subjected to primer extension with reverse transcriptase in the presence of [-32P]dATP. Maxam and Gilbert (G+A) sequencing reactions were used as size standards (lane 1). The sizes of the cDNAs obtained are indicated. Successive lanes show the effects of increasing concentrations of CI (3.7, 7.4, 29.6, 59.2, 118.5, 177, 237, and 474 nM, respectively) on cro-specific transcript generation.

CI represses transcription from the P_R promoter. The CI repressor of lambdoid phages is known to repress the transcription of early lytic genes and stimulate its own transcription by binding to its operator sites (reviewed in reference 27). Consequently, a likely role for the A2 CI protein would be to repress the transcription of the P_R promoter. To address this question, we used B. subtilis ^A-RNAP and linearized pUO183 DNA (3 nM) to follow, by in vitro transcription assays, the expression of P_R in the presence of increasing amounts of CI. As shown in Fig. 5, CI reduced P_R utilization, in a concentration-dependent process, until no expression was detected. We estimated that 2.5 CI monomers (7.4 nM) per DNA molecule were enough to detect some repression from P_R, while about 60 polypeptides (180 nM) were needed to block it completely.

View larger version (55K): [in this window] [in a new window]   FIG. 6.   Binding of CI and A-RNAP to their cognate binding sites. The -32P-labeled 183-bp DNA segment (81 pM) was incubated with CI (6 nM) (lane 2) and increasing concentrations of A-RNAP (13, 17.5, 22, 26.5, 34, and 44 nM) (lanes 3 to 8) or with 22 nM of A-RNAP and increasing CI concentrations (2.7, 5.6, 11, and 22 nM). The unbound probe (Free), the CI-DNA complexes (I and II), A-RNAP-DNA (RPI and RPII), and the ternary complex CI-A-RNAP-DNA (II* and CI-RP) are indicated. Lane 1, unbound DNA probe.

CI relocates the RNA polymerase to the cI-cro intergenic region. The P_R and P_L divergent promoters are 81 bp apart and thus are located on the same face of the DNA helix. To investigate the type of complexes formed by CI, ^A-RNAP, or both proteins on the intergenic 153-bp cI-cro DNA fragment, DNase I footprinting experiments were performed. The binding of CI to DNA gives a characteristic DNase I protection pattern already shown in Fig. 4 (Fig. 7A), while ^A-RNAP shows an extended protection (about 40 bp), in the region where O₂ and the P_R promoter overlap, at concentrations of 15 nM and up (Fig. 7B).

View larger version (86K): [in this window] [in a new window]   FIG. 7.   Effect of CI on A-RNAP binding to the intergenic cI-cro segment of A2, as seen through DNase I footprinting analysis. The 172-bp EcoRI-SphI DNA fragment (top strand) from plasmid pUO183 was end labeled at the EcoRI restriction site and incubated with 3.5, 7, 14, or 28 nM CI (A), with 3.9, 7.8, 15.5, 31, or 62 nM A-RNAP (B), or with 31 nM A-RNAP plus 3.5, 7, 14, or 28 nM CI (C). In this last case the RNAP was incubated with the DNA for 15 min prior to the addition of CI.

View larger version (33K): [in this window] [in a new window]   FIG. 8.   Scheme of the stretches protected by CI (white bars) and A-RNAP (cross-hatched bars) in the cI-cro intergenic region of phage A2, based on the data shown in Fig. 4 and 7. In addition, the start points of transcription (*), the 10 and 35 regions of the PR and PL promoters (from the primer extension data shown in Fig. 5), and the UP-like sequence of PR (nucleotides in italics) are indicated. The inverted repeated sequences of the operators are shown by converging arrows, and hypersensitive sites are indicated by pointing arrows (filled for CI and open for A-RNAP). The black bar indicates the A-RNAP protected region in the presence of low CI concentrations.

	DISCUSSION

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The deleterious effect of bacteriophages on industrial processes that rely on the activity of lactic acid bacteria is fuelling the interest in their biology as a first step towards devising rational interference systems that abolish phage development. Furthermore, phages may be a source of genetic tools for these bacteria. The utility of the CI product in strain defense (reference 20 and unpublished data) has moved us to undertake an in-depth study of the regulation of the early processes that lead to either the lytic or lysogenic cycle of phage A2.

Northern blots from the A2 immunity region made in vivo (20) and in vitro transcription experiments showed that it is centered around a 153-bp segment comprised of the structural portions of the divergently transcribed cro and cI genes (Fig. 1). In front of these genes there appeared the corresponding promoters, P_R and P_L, respectively. Interspersed with these two promoters are three discrete, nonoverlapping, operator sites (O₁, O₂, and O₃) to which the CI protein was shown to bind specifically. Each of these operator sequences contains a 20-bp imperfect inverted repeated sequence with a dyad axis of symmetry. This organization suggests that CI, which is a monomer in solution, could dimerize on the DNA through recognition of each of the sides of the inverted repeats. The relative locations of promoters and operators are as follows: O₁ is centered 13 bp downstream from the transcription start point of P_R, while O₂ and O₃ have their axes of symmetry 33.5 and 86.5 bp upstream from that point and have embedded the 35 hexamers of P_R and P_L, respectively (Fig. 8). At low concentrations, CI formed two complexes with the intergenic cI-cro DNA segment, while only the most retarded one was observed at high CI concentrations (Fig. 3). The absence of a third intermediate complex suggests the strong cooperative binding of CI to two operator sequences, probably O₁ and O₂, based on its higher affinity to them than to O₃ (Fig. 4 and 7). This cooperative binding of CI probably provokes bending of the DNA, as judged by the pattern of increased sensitivity to DNase I cleavage with a periodicity of about 10 bp that occurs between the O₂ and O₃ sites (Fig. 4). However, the O₁ and O₂ sites are separated by a nonintegral number of turns in the DNA helix (the center-to-center distance is 46.5 bp, which means 4.4 turns), so we have to assume that the helix undergoes an unfavorable twisting process upon CI binding. At present the energetic cost of looping between these two high-affinity operator sites is unknown. On the other hand, O₂ and O₃ lay on the same face of the DNA, thus favoring DNA looping (the center-to-center distance is about 52.5 bp, i.e., 5 helix turns).

The overall organization of the genetic switch region appears to be significantly similar to the functionally homologous stretches of other bacteriophages, although in most of these the operators are uniformly spaced (14, 18, 26). However, the lambdoid phages HK022 and 80 also show asymmetric spacing (8, 25). Furthermore, A2 operators partially differ in their sequences, a feature previously thought to be important in phage  for the lysis-lysogeny decision, since they seem to be involved in the different affinities of CI and Cro for these sites.

The phage A2 CI protein represses P_R through binding to O₁ and O₂, which results in the displacement of the RNAP from its normal position, from nucleotide 2 to 50 upstream of the transcription start point of P_R, to place it on the P_L promoter, which presumably would result in the enhancement of cI transcription. However, at high concentrations of CI, even O₃ becomes occupied, which in turn provokes displacement of the RNAP from P_L, resulting in repression of cI.

The general arrangement of the operator sites relative to their promoters indicates that repression of the lytic cycle in phage A2 occurs by a principle similar to that proposed for the lambdoid phages (7, 27) but with clear mechanistic differences. First, the promoters of the lytic-lysogenic commitment of the A2 phage have four elements identified in bacterial promoters: the crucial 35 and 10 hexamers, the 14 to 15 TG dinucleotide which appears to be a basic third element found in a large portion of gram-positive bacterial promoters (15, 33), and a sequence rich in A+T called the UP element, located upstream from the 35 hexamer (6, 23, 28). The UP element, to which the C-terminal domain of the RNAP  subunit (-CTD) binds, has been located at positions 59 to 38 relative to the transcription start and has the consensus sequence 5'-nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn-3' (10). A poor match with an UP element could be predicted in the 59 to 38 interval of P_L (8 of 17 nucleotides), whereas an almost perfect match (15 of 17 nucleotides) was observed in the case of the P_R promoter (Fig. 8). These last two elements are not present in the promoters of the genetic switch region of bacteriophage .

Second, in vitro transcription of the cI gene from the P_L promoter of phage A2 takes place in the absence of CI, albeit about 10-fold less abundantly than cro expression which occurs from the P_R promoter (Fig. 5). This contrasts with the fate of the repressor synthesis from the P_RM promoter in phage  (13), which requires the presence of the repressor itself.

Third, in the case of phage A2 the spacer regions have 26 bp between O₁ and O₂ and 32 bp between O₂ and O₃ (Fig. 8), while in  these are reduced to 7 and 6 bp, respectively. The  CI protein activates transcription of cI when bound at O_R2 (which is centered at position 42 of promoter P_RM) (19, 21, 27). It has been recently shown, by using artificial constructs, that the  CI repressor bound to O_R2 at position 62 upstream of the promoter transcription start point does not activate RNA synthesis, because  CI cannot contact the  subunit of RNAP (9). In the case of the A2 phage, we have shown that the CI repressor bound to the O₁ and O₂ sites (centered at positions 131 and 84.5 of promoter P_L) helps in the binding of RNAP to the P_L promoter. This might be effected through contact between the  subunit of the B. subtilis RNAP and CI (our unpublished data) which, in the end, would enhance the expression from P_L. It is likely, therefore, that this situation is reproduced in vivo when an A2 genome enters a new host cell. As a result of early transcription, CI would bind the O₁ and O₂ sites, provoking repression of the transcription from the P_R promoter by covering part of the surface that must be occupied by ^A-RNAP, leading the phage into a lysogenic cycle. The CI dimers bound to O₁-O₂ help to position ^A-RNAP at the P_L promoter, further enhancing the synthesis of CI until its concentration allows the occupancy of O₃, which would result in a halt of transcription from P_L. In this way, as it occurs in , A2 CI would regulate its own synthesis and the maintenance of the lysogenic state of the cells.