VIROLOGY 188, 831-839 (1992) The Polarity Suppression Factor of Bacteriophage P4 Is also a Decoration Protein of the P4 Capsid MORTEN L. ISAKSEN,**t SVEIN T. RISHOVD,* RICHARD CALENDAR,t AND BJ0RN H. LINDQVIST*~’ *Institute of Biology and the Biotechnology Centre of Oslo, University of Oslo, Norway; and tDepartment of Molecular and Cell Biology, 40 1 Barker Hall, University of California, Berkeley, California 94720 Received December 13. 199 I; accepted March 9, 1992 We show that the product of the polarity suppression (psu) gene from bacteriophage P4 associates with P4 capsids. This association can occur when Psu is (i) provided in vivo from the P4 genome or from a plasmid or (ii) provided in vitro by mixing viable phage particles with Psu protein. Psu is unable to associate with the larger capsid of P4’s helper phage P2. Discrimination of the P4 and P2 capsids by Psu appears to be independent of the presence of the P4 genome in the capsid, since P2 size capsids filled with P4 DNA cannot accommodate Psu association. P4psu particles devoid of Psu are less stable than P4 particles carrying Psu. These results indicate that, in addition to its antitermination activity at Rho-dependent terminators, Psu is also a decoration protein that stabilizes the P4 capsids. o 1992 Academic PWSS, IN. INTRODUCTION Bacteriophage P4 is a satellite coliphage that uses all the morphological gene products of a helper phage such as P2 in order to make viable phage particles (Six, 1975; Bertani and Six, 1958). While P4 capsids contain mainly P2 proteins, P4 generates smaller capsids than P2. The reduction in volume of P4 capsids relative to P2 capsids reflects the difference in masses of the dou- ble-stranded genomes of P2 and P4 and is determined by the gene product of P4’s size determination (sid) gene (Shore et al., 1978; Agarwal et al., 1990). P4 cap- sids are also distinguished from P2 capsids by different amounts of minor capsid components and notably the presence of a 20-kDa protein, which is presumed to be the product of the P4 polarity suppression (QSU)gene (Barrett et al., 1976; Sauer et al., 1981). Psu protein relieves transcriptional polarity caused by certain amber mutations in P2 by acting as an antiterminator of Rho-dependent transcription termination (Sunshine and Six, 1976; Sauer et al., 1981; Lagos et al., 1986; Linderoth and Calendar, 1991). Three psu mutants have been isolated based on their inability to carry out polarity suppression (Sauer et a/., 1981). The psu gene has been sequenced, and this sequence predicts a protein of Mr = 21,314 (Dale et a/., 1986). All the psu mutants exhibit a two- to fivefold decrease in burst size when wild type P2 is used as a helper (Sauer et a/., 1981). It has been reported that P4 psul carries a double amber mutation in the psu gene, and that P4 psu3 car- ries a missense mutation in the carboxyl region of the ’ To whom reprint requests should be addressed. gene (Dale et al., 1986). The P4 psu 1 mutant is unable to make the 20-kDa protein in a host lacking an amber suppressor. In contrast, P4 mutants psu2 and psu3 both produce the 20-kDa protein (Sauer et a/., 1981). Here we confirm by microsequencing and by the use of Psu-specific polyclonal antibodies that the 20.kDa protein is coded for by the psu gene. Furthermore, we report that Psu helps to stabilize P4 capsids against certain kinds of environmental stress. We propose that the reduced capsid stability accounts for the lower burst size of P4 psu mutants. We also report the nu- cleotide change of psu2 and a revised sequence for psu+, psu 1, and psu3. MATERIALS AND METHODS Media and buffers Media and buffers used to grow and store phage have been described by Kahn eta/. (1991). P4 buffer is 80 mM MgCI,, 10 mMTris-Cl (pH 7.5), and 1% ammo- nium acetate. When necessary, antibiotics were added to the medium to a final concentration of 50 pg/ml for ampicillin, 25 pg/ml for kanamycin, and 200 @g/ml for streptomycin. Bacteria, phage, and plasmids Bacterial strains used were all derivatives of Esche- richia co/i strain C (Bertani and Weigle, 1953) and are listed in Table 1. The virl genotype of P4 and P4 psu mutants (Six and Klug, 1973) have been omitted from the text for simplic- ity. P4 ash8 sidlO1 was isolated by 0. Nilssen and carries an in-frame deletion in the sid gene (personal 831 0042.6822/92 $5.00 Copynght 0 1992 by Academic Press. Inc. All nghrs of reproduction in any form reserved.