Amber and deletion mutants were used to assign functions in cell lysis to three late genes of bacteriophage P1. Two of these genes, lydA and lydB of the dar operon, are 330 and 444 bp in length, respectively, with the stop codon of lydA overlapping the start codon of lydB. The third, gene 17, is 558 bp in length and is located in an otherwise uncharacterized operon. A search with the predicted amino acid sequence of LydA for secondary motifs revealed a holin protein-like structure. Comparison of the deduced amino acid sequence of gene 17 with sequences of proteins in the SwissProt database revealed homologies with the proteins of the T4 lysozyme family. The sequence of lydB is novel and exhibited no known extended homology. To study the effect of gp17, LydA, and LydB in vivo, their genes were cloned in a single operon under the control of the inducible T7 promoter, resulting in plasmid pAW1440. A second plasmid, pAW1442, is identical to pAW1440 but has lydB deleted. Induction of the T7 promoter resulted in a rapid lysis of cells harboring pAW1442. In contrast, cells harboring pAW1440 revealed only a small decrease in optical density at 600 nm compared with cells harboring vector alone. The rapid lysis phenotype in the absence of active LydB suggests that this novel protein might be an antagonist of the holin LydA.

Prophages P1 and P7 exist as unit copy DNA plasmids in the bacterial cell. Maintenance of the prophage state requires the continuous expression of two repressors: (i) C1 is a protein which negatively regulates the expression of lytic genes including the C1 inactivator gene coi, and (ii) C4 is an antisense RNA which specifically inhibits the synthesis of an anti-repressor Ant. In addition, C1 repression is strengthened by lxc encoding an auxiliary repressor protein. The repressors C1, C4 and Lxc are components of a tripartite immunity system of the two phages. Here, the mode of action of these regulatory components including their antagonists Coi and Ant is described.

Gene 10 of bacteriophage P1 encodes a regulatory function required for the activation of P1 late promoter sequences. In this report cis and trans regulatory functions involved in the transcriptional control of gene 10 are identified. Plasmid-borne fusions of gene 10 to the indicator gene lacZ were constructed to monitor expression from the gene 10 promoter. Production of gplO-LacZ fusion protein became measurable at about 15 min after prophage induction, whereas no expression was observed during lysogenic growth. The activity of an Escherichia coli-like promoter, Pr94, upstream of gene 10, was confirmed by mapping the initiation site of transcription in primer extension reactions. Two phage-encoded proteins cooperate in the trans regulation of transcription from Pr94: Cl repressor and Bof modulator. Both proteins are necessary for complete repression of gene 10 expression during lysogeny. Under conditions that did not ensure repression by Cl and Bof, the expression of gplO-LacZ fusion proteins from Pr94 interfered with transformation efficiency and cell viability. Results of in vitro DNA-binding studies confirmed that Cl binds specifically to an operator sequence, Op94, which overlaps the -35 region of Pr94. Although Bof alone does not bind to DNA, together with Cl it increases the efficiency of the repressor-operator interaction. These results are in line with the idea that gplO plays the role of mediator between early and late gene transcription during lytic growth of bacteriophage P1.The maintenance of the lysogenic state of the temperate bacteriophage P1 requires at least three regulatory functions, which are components of a complex tripartite immunity system (for a review, see reference 54). First, the major repressor protein Cl (1,8,15) is encoded by the cl gene (35,38). It binds to at least 15 operator sequences, scattered widely over the P1 chromosome (55). The analysis of the operator sequences reveals an asymmetrical, 17-bp consensus sequence ATIGCTCTAATAAATlTT (5,9,17,45). The operator sequences are numbered according to their positions on the P1 genetic map (55). Second, the transcript of the c4 region confers phage strain-specific immunity, acting as a special type of antisense RNA (4). Third, the bof gene (7,43) was recently shown to code for an auxiliary repressor protein, which controls the efficiency of repression exerted by Cl (33,34,46,47).The number of genes and operons known to be transcriptionally controlled by Cl and Cl/Bof is steadily increasing. They can be classified into two groups: (i) functions regulating the switch from the lytic to the lysogenic pathway such as cl/Op99a,b (46), coi/Op99d (17, 18), and c4, antIOpSl (2,5,19); and (ii) functions associated with viral DNA replication such as kilA, repL/Op53 (6,13,39), ban/Op72a,b (16,20)

After digestion of bacteriophage P1 DNA with EcoRI in the presence of P1 repressor, 6 repressor binding sites were identified in 5 of 26 EcoRI fragments. Binding sites were localized by the decreased mobility of DNA fragment-repressor complexes during electrophoresis and by DNase protection ("footprinting") analysis. The repressor binding sites, or operators, comprise a 17-base-pair-long consensus sequence lacking symmetrical elements. Three operators can be related to known genes, whereas the function of the others is still unknown. The mutant P1 bac, rendering ban expression constitutive, is identified as an operator-constitutive mutation of the ban operon.P1 is a temperate bacteriophage with a genome size of about 90 kilobases (kb). In the prophage state the proviral DNA is maintained as a plasmid, and the vegetative P1 functions are repressed. Repression is accomplished by a phage-specific repressor, the product of the cl gene, which is located at the far right side of the P1 genetic map in EcoRI restriction fragment 7 (P1:7) (1, 2). Partially purified P1 repressor binds in vitro to at least two regions near cl within BamHI fragment 9 that itself is located within P1:7 (3, 4).The binding sites close to the cl gene, however, are not the only region at which the P1 repressor acts. The latter can also repress in vivo the expression of the P1 ban gene, which is located in P1:3 (5, 6). Furthermore, P1:14 also contains a promoter repressible by the product of cl (7). Together these results reveal that the P1 cl repression system must differ from that of other temperate phages such as A, P2, and P22, in which only promoters adjacent to the repressor gene are repressed (7).During our studies on the regulation of phage P1 ban expression we have localized by indirect methods a region 5' upstream of the ban gene within P1:3 at which the P1 repressor acts (8). Highly purified P1 repressor protein binds to this region in vitro (H.S., unpublished data). Now in a systematic search for other repressor binding sites in the phage genome, EcoRI-digested P1 DNA is incubated-with repressor, and binding regions are identified by the decreased mobility of EcoRI fragment-repressor complexes during electrophoresis (9,10). Thus binding regions were detected in P1:7 and P1:14, as expected and, in addition, in P1:9 and P1:11. Moreover, the results of DNA sequence and DNase protection analyses reveal an asymmetric 17-base-pair (bp)-long consensus sequence for the P1 repressor binding site. MATERIALS AND METHODSBacteria, Phage, and Plasmids. Escherichia coli K-12 strains used included the following: C600, HB101 (recA13) (11), NY58 (dnaBI07, recA56) (12), JM101 (13), N100 (galK, recA) (14), DW101 (sup'), and DW103 (supD) (15). For the cloning of P1 DNA fragments containing the ban operon (8) the recipient bacteria C600, HB101, NY58, and JM101 carried the plasmid pKT101-P1:7 harboring the P1 repressor gene (8). Strains DW101 and DW103 were transformed by plasmid pBR325-P1:11 for marker rescue tests (2).Phage used were M13mp8/9 (16)...

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