Genetics of cosQ, the DNA-Packaging Termination Site of Phage : Local Suppressors and Methylation Effects

The cos site of the bacteriophage chromosome contains the sites required for DNA processing and packaging during virion assembly. cos is composed of three subsites, cosQ, cosN, and cosB. cosQ is required for the termination of chromosome packaging. Previous studies have shown cosQ mutations to be suppressed in three ways: by a local suppressor within cosQ; by an increase in the length of the chromosome; and by missense mutations affecting the prohead’s portal protein, gpB. In the first study reported here, revertants of a set of cosQ mutants were screened for suppressors, and cis-acting suppressors of cosQ mutations were studied; these included second-site cosQ point mutations, base-pair insertions within cosQ, and an additional genome-lengthening suppressor. The 7-bp-long cosQ, with the sequence 5 -GGGTCCT-3 , coincides exactly with the recognition site for the EcoO109I restriction/methylation system, which has the consensus sequence 5 -PuGGNCCPy-3 . In a second study, EcoO109I methylation was found to strongly interfere with the residual cosQ function of leaky cosQ mutants. cis-acting suppressors that overcome methylation-associated defects, including a methylation-dependent suppressor, were also isolated. Models of cosQ suppression are presented. MANY double-stranded DNA viruses have replication binds cosB to anchor gpA during cosN cutting. After a and recombination pathways that produce conconcatemer’s cosN is cut, terminase remains bound to catemers, i.e., end-to-end multimers of virus chromothe resulting cosB-containing DNA end, which is the left somes. For a subset of these viruses, including many end of the chromosome to be packaged. The terminasetailed bacteriophages and the herpes viruses, the concaDNA complex binds to the portal vertex of a prohead, temers are cut at specific sites to generate unit-length and translocation of the DNA into the shell ensues. virion chromosomes (Fujisawa and Hearing 1994). A Translocation moves the DNA-packaging complex along virally encoded enzyme, terminase, carries out the cutthe DNA until the next cos is encountered and the termiting reaction, which is coordinated with packaging of nase docked at the portal vertex recognizes and cuts the the DNA into an empty protein shell. downstream cos. Following cleavage, terminase undocks Phage chromosomes are 48.5-kb duplexes with 12from the newly filled head and remains bound to the base-long, single-stranded extensions at the 5 ends of left end of the next chromosome along the concatemer, the strands. These extensions, called cohesive ends, are sponsoring its packaging. cosQ, although not required for complementary and enable the chromosome to cyclize initiation of DNA packaging, is required for cleavage of in an infected cell. Late during infection, concatemers the downstream cos. Because cosQ mutants fail to cut produced by rolling circle replication and recombinathe downstream cos and fail to stop translocation at cos, tion are cut by terminase and packaged into empty shells the shell is filled to capacity, and because the protruding called proheads (Feiss 1986; Becker and Murialdo DNA prevents tail attachment, the cosQ defect is lethal. 1990; Catalano et al. 1995). The site at which terminase The bypassed downstream cos in cosQ mutants is properly introduces staggered nicks is cosN; cosN is located benicked on the top strand of cosN, but the bottom strand tween two other sites, cosQ and cosB. These cos subsites, is not nicked. The depolarization model proposes that which are located in a 200-bp segment, orchestrate cosQ acts in presenting a gpA subunit to the bottom the recognition, processing, and packaging of DNA strand of cosN by forming a bend in the region of DNA (Figure 1). In addition to cosN, the adjacent site cosB is between cosQ and cosN, forming a loop for cosQ and cosN required for cutting at cosN to initiate DNA packaging. to be aligned in the same orientation. A second version Terminase consists of a large subunit, gpA, which conof the model proposes that cosQ is needed for a pause tains the endonuclease, and a small subunit, gpNu1, which in the packaging process to recruit the second of two gpA subunits from solution to the cosN site for the nicking of the bottom strand of DNA (Cue and Feiss 1998; 1Corresponding author: Department of Microbiology, University of Wieczorek and Feiss 2001). Iowa, 3-315 BSB, Iowa City, IA 52242. E-mail: [email protected] Three classes of suppressors of cosQ mutations have Genetics 165: 11–21 (September 2003) 12 D. J. Wieczorek and M. Feiss Figure 1.—Structure of the cohesive end site and relative virus yields of the cosQ mutational analysis. The cos region of bacteriophage extends across the region of DNA from base pairs 48,473 to 166 and is composed of three subsites: cosN, cosB, and cosQ. cosN is the site at which staggered nicks are introduced by terminase to generate the cohesive ends of virion DNA and represents the junctions of individual chromosomes in the concatemer. cosN contains a 22-bp element, extending from base pairs 48,498 to 17, in which 10 of the base pairs show twofold rotational symmetry. cosN can be divided into two half-sites on the basis of this center of symmetry, denoted by the dot (•): the right half-site, cosNR, with a nicking site at N1, and the left half-site, cosNL, with a nicking site at N2. cosB is located between cosN and the gene Nu1 and is required for the initiation of chromosome packaging. It is composed of the three R sequences, R1, R2, and R3, which are bound by the gpNu1 subunit of terminase. Between R3 and R2 is I1, a binding site for E. coli integration host factor. cosQ is required for the termination of chromosome packaging. The cosQ region has been defined as the 7-bp sequence from base pairs 48,473 to 48,479, as denoted by the box. The genes near cos are also shown. The Nu1 and A genes encode the small and large subunits of terminase, W encodes a head-tail joining protein, and B encodes the portal protein. been identified (Cue and Feiss 1997; Wieczorek et al. article we report two studies dealing with how local changes affect cosQ function. First, we describe suppres2002). The first class consists of local suppressors affecting base pairs within cosQ. The second suppressor class sion of cosQ mutations by local mutations that alter cosQ function. Second, we describe how methylation of cosQ increases the length of the phage chromosome to near the capacity of the head. It has been proposed that the impacts cosQ function. The second study stems from the observation that the 7-bp-long cosQ segment coincides rate of translocation is dependent upon the length of DNA packaged and that the rate likely slows as more exactly with the recognition site for the EcoO109I restriction/methylation system, which has the consensus seDNA is packaged into the prohead. Such slowing of the rate of translocation has recently been documented for quence 5 -PuGGNCCPy-3 . The restriction enzyme cuts between the G residues, and the inner C residues are phage φ29 (Smith et al. 2001). Cue and Feiss (1997) proposed that the resulting increase in chromosome methylated by the methylase (Kita et al. 2001). We report experiments indicating that EcoO109I methylation length further slows the rate of translocation so that the translocation complex can more efficiently recognize has an impact on cosQ function. Methylation especially affects the residual cosQ function of leaky cosQ mutants. the mutant cosQ site, leading to more efficient termination. The third class of suppressors maps to the B gene, In addition, we describe cis-acting suppressors that overcome methylation-associated defects, including a methwhich encodes the prohead’s portal protein. It has been proposed that the portal protein acts as a sensing mechaylation-dependent suppressor. nism either to measure the rate of translocation or to identify the cosQ site (Cue and Feiss 1998; Wieczorek MATERIALS AND METHODS and Feiss 2001). These suppressors may slow the rate of translocation, allowing the mutant cosQ site to be Media: Media were prepared as described by Wieczorek more efficiently recognized by terminase or by the porand Feiss (2001) with the exception that chloramphenicol tal protein itself. was added at a final concentration of 30 g/ml for pACYC184based vectors. In a previous study, we used saturation mutagenesis Strains: Strains used in this study are described by Wieczto determine that cosQ is 7 bp long, composed of base orek and Feiss (2001). The standard bacterial host used was pairs 48,473–48,479 with the sequence 5 -GGGTCCT-3 MF1427, a galK derivative of the Escherichia coli C strain C1a (Wieczorek and Feiss 2001). The 7-bp sequence is (Six and Klug 1973). conserved in the related lambdoid phages 21, φ80, and General recombinant DNA techniques: General recombinant DNA techniques are described by Wieczorek et al. N15 (Smith and Feiss 1993; Ravin et al. 2000). In this 13 cis-acting cosQ Suppressors and EcoO109I Methylation (2002). Clones of the EcoO109I methylase were generously RESULTS provided by New England Biolabs (Beverly, MA). Phage DNA Local suppressors in pseudorevertants of cosQ was purified by CsCl centrifugation and phenol extraction as described by Arber et al. (1983). mutants Sequence designations: All references to sequence are Local suppressors in spontaneous pseudorevertants of based on the numbering convention described by Daniels et al. (1983). Numbering of the sequence begins with the first cosQmutants: cosQ is 7 bp long, so there are 21 possible base of the left cohesive end and continues along the top base pair changes in cosQ. Earlier, we constructed strand in a 5 to 3 direction. The position of each restriction phages with these 21 cosQ mutations and classified them cut site is given as the first nucleotide of the recognition by phenotypic severity (Wieczorek and Feiss 2001). Of sequence. the 21 point mutations in cosQ, 8 were severe lethals, Phage yield determinations: Phage yield determinations are described by Wieczorek et al. (2002). reducing the virus yield to 1.0 virion/induced lysogen, Identification and isolation of plaque-forming revertants: and 3 were moderate lethals with yields between 1.0 Lysogens of non-plaque-forming cosQ mutants were induced and 5.5 (see Wieczorek et al. 2002, Table 1). Recently and lysates titered on MF1427. Also, lysogens of cosQ T48,479C we reported on trans-acting suppressors of cosQ mutawere induced and lysates titered on MF1427 expressing the tions (Wieczorek et al. 2002). Here we report a characEcoO109I methylase. Plaque-forming revertants were selected and single plaque purified. Lysogens of the revertants were terization of the nature of local changes in pseudoreconstructed by infecting MF1427 with phages isolated from vertants of cosQ mutants. plaques. PCR amplification followed by restriction enzyme We first examined spontaneous revertants of cosQ analysis and DNA sequencing were performed to determine mutants. Efforts were taken to minimize siblings among the cosQ sequence. the revertants by using multiple induced lysates in the Introduction of B suppressor mutations into the genome: The introduction of B suppressor mutations into the genome screening process. We screened plaque-forming reis described by Wieczorek et al. (2002) with the exception vertants in unmutagenized lysates of phages with the that the mutations were introduced, by transformation, into eight severe lethal cosQ mutations: G48,473T, G48,473C, MF1427 lysogenic for cosQ G48,473A. G48,474A, G48,474T, G48,474C, C48,478A, C48,478T, and C48,478G (see E. coli mutD mutagenesis: The method of E. coli mutD mutaTable 1 in Wieczorek et al. 2002). Plaque-forming regenesis is described by Wieczorek et al. (2002). PCR mutagenesis: PCR mutagenesis of the B gene is devertants were screened by sequencing cos to identify true scribed by Wieczorek et al. (2002) with the exception that revertants and revertants with local, cis-acting suppresfor each lysogen, 4000 transformed colonies were scraped sors in cosQ. Of 423 revertants screened, 15 were pseudofrom the plates and resuspended in Luria broth. The pooled revertants carrying a local suppressor plus the original transformed lysogens were grown overnight, prophages were cosQ mutation. All 15 suppressors were suppressors of induced, and lysates were titered on MF1427 as described previously. only two cosQ mutations, both of which affected the first DNase protection assay: A 100l aliquot of each lysate was cosQ base pair, cosQ G48,473T and cosQ G48,473C. Furtherincubated with 5 units of DNase, 20 l of RNase A (500 g/ more, all 15 suppressors were insertions of either an ml), 6 mm MgCl2, and 10 mm CaCl2 at room temperature for A:T base pair or a T:A base pair between cosQ base pairs 30 min. Ten microliters of 0.5 m EDTA was added for 10 min 48,475 and 48,476 (see Wieczorek et al. 2002, Table 2). at room temperature to stop the reaction. Five nanograms of linearized pUC19 was included as a control for DNA recovery. That is, Rev12 of cosQ G48,473C (5 -CGGTCCT-3 ) conThe lysate was extracted twice with phenol-CHCl3-isoamyl alcotained an insertion of an A between base pairs 48,475 hol (25:24:1, v/v) and once with CHCl3. Samples included 30 and 48,476 to give the sequence 5 -CGGATCCT-3 . of 200 l (15%) of the extracted DNA. The DNA was subjected Rev28 and Rev15 of cosQ G48,473T contained insertions to electrophoresis on a 0.8% gel. To quantify the amount of DNA, the DNA was denatured and transferred to a Geneof A or T, respectively, also between base pairs 48,475 Screen Plus (New England Nuclear) membrane. DNA hybridand 48,476. These nucleotide insertions appeared to ization was performed using [ -P]dCTP-labeled (Amersham, suppress the original cosQ mutation by shifting the origiBuckinghamshire, UK) linearized pUC19 and whole-length nal mutation one position to the left, resulting in a DNA as probes. An autoradiogram was obtained by exposure new 7-bp cosQ site. The novel cosQ sites created by the of the membrane to a Fuji Super RX film for 8 hr at 70 . The recovery of pUC19 DNA and the amount of DNA packaged in insertion suppressors begin at base pair 48,474 instead the methylated and unmethylated lysates was determined by of 48,473, with the inserted base pair representing the phosphorimaging on a Packard Instantimager. The packaging only mutation in cosQ in the third base pair of cosQ. ratio is the yield of packaged phage DNA per induced lysogen Our previous work showed that mutants with an A in the presence of the methylase relative to the yield of packaged phage DNA per induced lysogen in the absence of the or T in the third cosQ base pair (5 -GGATCCT-3 or 5 methylase. For example, the effect of methylation on the packGGTTCCT-3 ) were viable (Wieczorek and Feiss 2001). aging of wild-type DNA was calculated by dividing the counts To test the proposal that the insertion mutations generper minute per induced lysogen from lane 8 by the counts ated new cosQ sites with sequences identical to those of per minute per induced lysogen from lane 3 (Figure 2). The cosQ G48,475A and cosQ G48,475T, we compared the yields of yields were further adjusted to account for the percentage recovery of pUC19 as an indicator of the overall recovery of the insertion-containing revertants with those of cosQ packaged DNA. More than 87% of the control pUC19 DNA G48,475A and cosQ G48,475T. First, we compared the yields was recovered for each sample. Portions of each culture were of Rev12 of cosQ G48,473C and Rev28 of cosQ G48,473T removed prior to induction, diluted 1:10,000 in 10 mm MgSO4, with that of cosQ G48,475A, which has the same 7-bp cosQ and plated on tryptone agar. Plates were incubated overnight at 31 to determine the number of viable lysogens. sequence. The yields of Rev12 and Rev28, at 15 and 14 D. J. Wieczorek and M. Feiss