Analysis of bacteriophage P 1 immunity by using XP 1 recombinants constructed in vitro ( P 1 repressor genes / PI antirepressor gene / PI dnaB analog gene / DNA cloning )

We describe the dissection and reconstruction of a complex control circuit, the P1 immunity system, by a method that involves inserting EcoRI-generated fragments of P1 DNA into X vectors that can then be sequentially inserted into a bacterial cell. Using these techniques we have isolated X-P1 hybrid phages that express the products of P1 genes cl, c4, ant, and ban and, in appropriately constructed lysogens, confirmed the roles played by the first three of these products in phage immunity. In addition we have'localized to particular P1 fragments the sites requisite for expression and repression of these gene products. The analysis leads to the conclusion that gpant acts in trans to antagonize repression mediated by gpcl, in support of' one of two proposed models for gpant action. Moreover, two features of the, immunity system are revealed: (f) a hitherto unknown component that effects gpcl repression; and (if) an unexpected ability of gpc4 to channel a superinfecting cl'+ phage into the lysogenic state, which suggests that gpc4 activity regulates the establishment phase of lysogeny. Analysis of complex genetic'controls requires methods that permit parts of the control circuits to be isolated, characterized, and variously reassembled. We show here how the insertion of defined DNA segments into X vectors that can be successively integrated into the chromosome of a suitable bacterium permits the analysis of several interacting genes by reconstruction as well as dissection experiments. Because of the phenomenon of incompatibility, this type of analysis would not be possible if the genes to be studied were located in a plasmid. The subject of our analysis is the immunity to superinfection conferred by the plasmid prophage of coliphage Pl. This immunity is thought to' require the presence of two repressor proteins, the products of genes ci (1) and c4 (2) (gpcl and gpc4) that are located in the separate immC and immI regions of the phage genetic map (Fig. 1). Gpcl represses the viral lytic functions. Gpc4 is thought to repress a second immI' gene, ant (or reb). The product of the ant gene has been postulated to act either in trans to antagonize gpcl-mediated repression (9) or in cis to replicate DNA of the superinfecting phage even in the presence of gpcl (i.e., it acts as a repressor bypass) (10). Phage P7, which has a ci gene functionally identical to the ci gene of P1 but differs from P1 in the specificity of its c4 repressor (9), is able to grow in a P1 lysogen because gpant of the infecting P7 phage is not repressed by gpc4 of the prophage. We describe here the isolation, characterization, and interactions of individual X-P1 hybrids bearing EcoRI-generated fragments of P1 (11) that express each of the known components of the P1 immunity system. An additional hybrid that'expresses the P1 ban gene (12), known to be directly under cl control (13) (Fig. 1), provides'an indicator of cl repression. MATERIALS AND METHODS Bacterial and Phage Strains. The Escherichia coli strains used were NS985 (HfrH, sup+), RW842 [HfrH XA(int-FII)gajT] (14), YMC (supF) (15), K175 (supD, Xr) (10), Q1508 [dnaB70(ts), thyAl (12), NS91 (C600 groPA15) (16), and N3072 [HfrH, sup+, A (pro-lac) Xlii] (6). The X vector X Dam15b538sr1X3c1857(ts)nin5 contains a single EcoRI site for inserting EcoRI fragments of P1 DNA (11).' Hybrid phages obtained by inserting P1 fragments (11) are designated X-Pl:c4, X-Pl:c1, etc.; :c4 or :cl refers to pertinent genes located on the inserted P1 fragment. Other phages used were PlCm, PlCmcl.100(ts), PlCmcl.55(am), PlCmc4.32, PlCmelrs, and Plvdrsban-l. Unless otherwise indicated, all of the P1 phages carried the Tn9 transposon (Cm). PlCmvirsant-10 and PlCmc4.32ant-17 were isolated from P1 lysogens made by infecting strain NS985 with either PlCmvdrs or PlCmc4.32. The presence of the antmutation in these phages was confirmed by their inability to make plaques on strain.K175(Plcry) (5). Mutational defects are indicated here by a minus sign superscript unless an allele numbei is specified. Media and Growth Conditions. Media and commonly used microbiological procedures for P1 and N were as described (13, 17). Because the attX-int region of the X vector has been removed' by the b538 deletion (18), homologous recombination with the cryptic X prophage of 'strain RW842 was used to lysogenize with primary X-P1 hybrids. The experiments described in this report were carried out at an EKi-Pi containment level in accordance with the'stipulations specified in FCRC MUA# 1. Construction of Xatt+imm21-P1 and Xatt+imm434-P1 Hybrid Phages. The Xb538cI857(ts)nin5-P1 hybrids (X-P1 hybrids) -were restructured first by recombination with Xfmm21nin5 or Ximm434nin5 to replace the immX' region with'imm2l or imm434 and then by recombination with Xb515b519Nam7cI857(ts) to replace the b538 deletion with the two smaller deletions, b515 and b519, which leave attX and the X int gene intact (18). The restructured hybrids (designated Ximm2l-Pl or Ximm434-Pl) can lysogenize standard strains at 32 or 42°C. Isolation of a X-P1:ban Hybrid. We took advantage of the ability'of the P1 ban gene product to substitute for the dnaB protein of the host (12) in order to isolate a X-Pl:ban hybrid. Strain Q1508(X) was infected with a pool of \-P1 hybrids (derived from 1000 plaques). The infected cells were spread on thymine-supplemented'Ll plates and incubated for 36 hr at 42°C. The frequency of infected cells that survived to form colonies (3 X 10-6) was about 30-fold greater than the dnaB70(ts) reversion frequency. Phage that formed turbid plaques at 320C and clear plaques at 42°C [phage with the Abbreviations: Cm, chloramphenicol resistance transposon Tn9; am, amber, ts, temperature sensitive; gpc4, gpcl, etc., gene product of gene c4, cl, etc. 5594 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 75 (1978) 5595