Roles of pIII in filamentous phage assembly1

Article No. mb982006

J. Mol. Biol. (1998) 282, 25±41

Roles of pIII in Filamentous Phage Assembly Jasna Rakonjac and Peter Model The Rockefeller University 1230 York Avenue, New York NY 10021, USA

Filamentous phage protein III (pIII), located at one end of the phage, is required for infectivity and stability of the particle. Cells infected with phage from which gene III has been completely deleted produce particles that are not released into the medium but stay associated at the surface. These particles are much longer than normal phage. They can be released by subsequent expression of pIII. Viewed with the electron microscope, cells infected with gene III deletion phage are decorated with structures that resemble extremely long pili. Surprisingly, such cells are viable and can form colonies. The pIII de®ciency can be complemented in trans, but there is a threshold concentration below which assembly does not occur. Above this threshold, pIII is used very ef®ciently and is incorporated into infectious but longer than unit length phage. As the concentration of pIII is increased, the number of infectious particles increases, and their average length decreases. pIII stabilizes pVI, a second phage protein found at the pIII end of the particle. In the absence of pIII, degradation of pVI is very rapid. pIII is thus not only required for infectivity and particle stability, but to terminate assembly and release the phage from its assembly site. # 1998 Academic Press

*Corresponding author

Keywords: ®lamentous phage; gene III encoded protein; gene VI encoded protein; phage assembly

Introduction The f1 (fd, or M13) phage is a ®lament, 880 nm long and 6 to 7 nm in diameter (Model & Russel, 1988). The virion is comprised of a circular singlestranded DNA genome, wrapped in a tube composed of around 2700 copies of the major coat protein, pVIII. The two ends of the ®lament bear two different pairs of proteins, called minor coat proteins, each present in three to ®ve copies per parAbbreviations used: Amp, ampicillin; Ab, antibody(ies); BPB, bromophenol blue; Cm, chloramphenicol; DNase, deoxyribonuclease; EtBr, ethidium bromide; gI, gene I ; gIII, gene III ; gVI, gene VI ; IPTG, isopropyl-b-D-thiogalactopyranoside; lac, lactose operon; NP40, Nonidet P40; PCR, polymerase chain reaction; pIII, gene III protein; psp, phage shock protein operon; RNase A, ribonuclease A; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; wt, wild type; (..), denotes plasmidcarrier state; SS, single-stranded; PEG, polyethylene glycol; m.o.i., multiplicity of infection; HRP, horseradish peroxidase; TCA, trichloroacetic acid; PBS, phosphate buffered saline. E-mail address of the correspondng author: [email protected] 0022±2836/98/360025±17 $30.00/0

ticle (Goldsmith & Konigsberg, 1977; Woolford et al., 1977; Grant et al., 1980; Lin et al., 1980). One pair (pVII and pIX) is at the end at which assembly initiates (Lopez & Webster, 1983; Russel & Model, 1989), while the other pair (pIII and pVI) is at the end of the phage at which assembly terminates (Lopez & Webster, 1983). Most of the structural proteins of the f1 virion are very small and hydrophobic. The exception is pIII, which is 406 amino acid residues long, and contains a large hydrophilic portion (Beck et al., 1978). pIII consists of three domains divided by two glycine-rich stretches. Two N-terminal domains, N1 and N2, mediate infection of the host cells (Armstrong et al., 1981; Jakes et al., 1988; Stengele et al., 1990; Riechmann & Holliger, 1997), while the third domain, CT, is suf®cient for the formation of stable, but non-infectious phage (Nelson et al., 1981; Crissman & Smith, 1984). Upon entry of the infecting phage into the host cell, the major coat protein (pVIII) integrates into the host inner membrane and can be incorporated into progeny phage (Trenker et al., 1967; Smilowitz, 1974; Armstrong et al., 1983). Indirect evidence suggests that minor proteins pVII and pIX may be recycled and reused in the assembly of # 1998 Academic Press

26 the progeny phage (Lopez & Webster, 1983). Marco et al. (1974) showed that truncated pIII from the infecting phage could be detected in the host cell. No studies on the fate of pVI have been carried out. The phage structural proteins are anchored in the inner membrane prior to incorporation into the virion (Endemann & Model, 1995), and the phage is assembled at the membrane. The sites of assembly are points in the cell envelope where the inner and outer membranes are in close contact (Lopez & Webster, 1985). The particle extrudes from the cell as it assembles, without killing the host (Marvin & Hohn, 1969). The ssDNA genome is sequestered before assembly in a rod-shaped complex with the phage-coded ssDNA binding protein pV (Salstrom & Pratt, 1971; Gray, 1989). Genetic studies suggest that assembly is initiated when a sequence, called the packaging signal (Dotto & Zinder, 1983), interacts with structural proteins pVII and pIX and morphogenetic protein pI (Russel & Model, 1989), the event that results in the formation of the leading end of the phage (Lopez & Webster, 1983). The particle is elongated by simultaneous removal of pV from DNA and incorporation of the major coat protein pVIII as the phage particle is extruded from the cell. Morphogenetic protein pIV, essential for the assembly of the phage, most probably forms a channel in the outer membrane through which the phage is extruded (Kazmierczak et al., 1994; Linderoth et al., 1997). When all of the DNA is wrapped in pVIII, minor proteins pIII and pVI are added, and the particle detaches from the host cell (Lopez & Webster, 1983). Any DNA that contains the f1 origin of replication and the packaging signal can be assembled into a virion-like particle in the presence of helper phage, and the length of the assembled particle depends on the size of the DNA molecule (Dotto et al., 1981). Virions can contain more than one ssDNA molecule: in a population of f1 wt virions, there are about 5% double-length particles that contain two genomes (Scott & Zinder, 1967). Studies of phage mutants defective in the genes that form the ends of the phage (gVII, gIX, gIII and gVI) showed that, under non-permissive conditions, extremely long virions (called polyphage) are released into the medium (Pratt et al., 1969; Lopez & Webster, 1983). This suggests that assembly need not stop when the end of the ssDNA substrate meets the assembly machinery, and that an excess of the substrate for packaging and/or major coat protein can stimulate the elongation of the phage particles at the expense of termination. A role for pIII and pVI in the termination of assembly was suggested by the fact that they are added last to the assembling phage particle (Lopez & Webster, 1983). This hypothesis was tested by studies of amber mutants of gIII or gVI, or of a strain with an incomplete deletion of gIII (Lopez & Webster, 1983; Crissman & Smith, 1984; Gailus

Roles of pIII in Filamentous Phage Assembly

et al., 1994). In the analyses of gIII mutants, despite the fact that some of the samples reportedly contained a high background of revertants, it was found that most of the released virions were noninfectious, very long, and unstable to heat and detergents (Pratt et al., 1969; Lopez & Webster, 1983; Crissman & Smith, 1984). The abundance of released phage suggested that termination of assembly was rather ef®cient in the absence of pIII. pVI was an obvious candidate as the secondary terminator of assembly, and this was supported by the ®nding that gVI amber mutant phage grown under the non-permissive conditions generated very few phage, and that these are extremely unstable (Lopez & Webster, 1983). However, pVI could not be detected in the gIII-negative polyphage (Gailus et al., 1994) so the question of termination of pIII-de®cient phage remained unresolved. Studies of interactions of pIII and pVI in the phage particles by cross-linking or co-immunoprecipitation detected a very stable multimolecular complex, termed ``the adsorption complex'', which was resistant to agents that dissociate the other components of phage particles (Gailus et al., 1994; Endemann & Model, 1995). This suggested that pIII and pVI might terminate phage assembly by forming a tight complex. However, studies of the interaction of these two proteins in the host cell, prior to assembly into the phage particles, did not offer a clear answer. pIII was found in a large complex by gel ®ltration, but the presence of pVI in it was not directly examined (Gailus et al., 1994). On the other hand, co-immunoprecipitation of cell extracts failed to detect a stable interaction of pIII with pVI (Endemann & Model, 1995). Another inconsistency related to phage assembly in the absence of pIII came from the effect of phage infection on the viability of the host. Although f1 infection does not usually kill the host cells, the infection can be lethal if assembly is impaired, and amber mutants in all genes except gII kill the host (Pratt et al., 1966). The killing effect is relieved by mutations that decrease the ef®ciency of replication, and presumably decrease the otherwise intensive production of the building blocks of the phage (Horiuchi et al., 1978; Model & Russel, 1988; Smith, 1988). Therefore, the lethality of most of the phage mutants is caused by overproduction of any of a number of the phage components in the absence of assembly (Schwartz & Zinder, 1968; Model & Russel, 1988). The killing effect reported for gIII mutants is hard to reconcile with abundant phage production, compared to extremely low phage production by the other killer mutants (Pratt et al., 1969; Lopez & Webster, 1983; Crissman & Smith, 1984). Here, the question of the role of pIII in the phage assembly is revisited. A genetically stable system was used, which consists of phage with a complete deletion of gIII (Rakonjac et al., 1997), and either of two complementing plasmids that do not recombine with the phage. Phage assembly at

Roles of pIII in Filamentous Phage Assembly

various intracellular concentrations of pIII was followed by measuring phage release, length, infectivity, composition and association with the infected cells. In parallel, the steady-state amount, stability and interaction of pIII and pVI in the cells was measured.

27 to 0.198 phage/molecule as the pIII concentration increases from 161 to 1350 molecules per cell (Table 1).

Results Phage release is extremely inefficient in the absence of pIII Phage release was measured after infection with f1d3 in the presence or absence of pIII produced from a lac-gIII fusion on a plasmid (Figure 1A and B). Phage release after f1d3 infection was very ef®cient in the presence of complementing pIII, but very poor in the absence of pIII. The lengths of phage particles released after infection of f1d3 in the presence or absence of pIII were very different. Hence, the amount of released phage shown in Figure 1A is expressed as a number of monophage equivalents. A single monophage equivalent corresponds to one ssDNA genome wrapped in coat protein, and for f1 wt corresponds to one phage particle. The number of monophage equivalents was calculated from the amount of encapsidated phage ssDNA, which in turn was quanti®ed by densitometric analysis of EtBr-stained bands after agarose gel electrophoresis of SDS-disassembled virions (Figure 1B). In an uncomplemented f1d3 infection, released phage particles were also observed by electron microscopy (not shown). The average length of particles (n ˆ 200) was ten times that of monomer. The amount of phage released into the supernatant in this experiment was 1  1010 monophage equivalents/ml, which is 1/100th of the amount released after infection with wt phage. Since the estimated average length of f1d3 phage was a 10-mer, the ef®ciency of the termination of phage assembly was about 1/1000th of that of f1 wt phage. Intracellular concentration of pIII and assembly of infectious phage The dependence of phage release on the amount of pIII was measured after a strain containing a lac-gIII fusion was infected with f1d3, at several concentrations of IPTG (Table 1). The steady-state amount of pIII in this experiment was determined by Western blotting (Figure 2A and E; see Materials and Methods for the method of calculation). Release of infectious phage (Table 1, Figure 3) was extremely inef®cient (less than one per cell) below a threshold concentration of pIII, calculated to be 160 molecules per cell (Table 1). As the concentration of pIII increases further, intracellular pIII is assembled into phage particles more ef®ciently. This is re¯ected by an increase in the ratio of infectious phage to intracellular pIII from 0.013

Figure 1. Time course of phage release in complemented and non-complemented f1d3 infection. The host strain was K561 containing gIII under the control of lac promoter on plasmid pJARA200. A, Released virions expressed as the number of monophage equivalents per ml of culture. (^) f1 wt; ( & ) f1d3 grown in the presence of 1 mM IPTG; (~) f1d3 grown in the absence of IPTG. Data in the graph were derived from the phage samples shown in B. The amount of phage ssDNA was calculated as described in Materials and Methods. B, Agarose gel electrophoresis of ssDNA from SDS-disassembled virions from unconcentrated culture supernatants. wt f1 ssDNA migrates slower because it is longer by 1277 nucleotides than f1d3. 1 to 5, f1 wt: 1, time point 0; 2, 20 minutes; 3, 40 min; 4, 60 minutes; 5, 80 minutes; 6, f1d3, time point 0; 7 to 10, f1d3, in the presence of IPTG: 7, time point 20 minutes; 8, 40 minutes; 9, 60 minutes; 10, 80 minutes; 11 to 14, f1d3, no IPTG: 11, time point 20 minutes; 12, 40 minutes; 13, 60 min; 14, 80 minutes. An exponentially growing culture was divided and infected with either f1 or f1d3. After 15 minutes of infection, the excess infecting phage were removed by centrifugation, and the cells were resuspended in fresh medium. f1d3-infected cells were divided into two ¯asks, and 1 mM IPTG was added to one. Aliquots were collected at 20 minute intervals. Cells were removed by centrifugation, and phage in the supernatant detected by agarose gel electrophoresis of SDSdisassembled virions (Nelson et al., 1981).

28

Roles of pIII in Filamentous Phage Assembly

Table 1. Intracellular concentration of pIII and release of infectious phage

a

Sample

K561/f1wt lac-gIII/f1d3: 0.04 mM IPTG 0.01 mM IPTG 0.004 mM IPTG 0.002 mM IPTG No IPTG

Molecules of pIII per cellb 15483 1350 409 284 161 83

Infectious phage per cell 1644 267 19 7 2.1 0.07c

Phage per pIIId 0.106 0.198 0.047 0.023 0.013 0.0008

a Top row: strain K561 infected with f1 wt. Rows 2 to 6: K561 containing lac-gIII fusion, infected with f1d3 and incubated at several concentrations of IPTG for 90 minutes. b The amount of pIII was calculated from densitometric analysis of the western blots shown in Figure 2A, as described in Materials and Methods. c The titer of infectious phage in this sample was equal to the background titer of the residual phage from the stock of f1d3 used to infect the cells, immediately after removing the excess infecting phage. d The numbers in this column were obtained as a ratio of values in column Infectious phage per cell to Molecules of pIII per cell.

Infection by wt f1 leads to the synthesis of much more pIII than can be obtained from the complementing plasmid, even at maximal induction of pIII production. However, a smaller proportion of the intracellular pIII is incorporated into phage particles. Hence, the ratio of released infectious phage to intracellular pIII (0.106) is lower than in f1d3 at maximal induction of pIII production (0.198). Cells containing the lac-gIII fusion made only about 1/10th as much pIII (even when fully induced) than is made in a wt infection, and pIII may be the limiting component for assembly of f1d3, but not wt f1. lac-gIII fusions that can be induced to a higher level have a basal expression of pIII that renders cells resistant to f1 infection (J.R. & P.M., unpublished results). Control of the length of the virions in f1 assembly The amount of released phage, measured as the number of monophage equivalents, and the titer of infectious phage (measured by plating) rose as the amount of pIII went up (Table 1, Figure 3A). The increase in the number of infectious particles was more dramatic than the increase in the number of monophage equivalents, because more of the released particles were infectious, and possibly shorter (Figure 3A). To examine the lengths of the virions produced at different concentrations of pIII, virions were separated by size on agarose gels (Figure 4B, lanes 2 to 7), by a method modi®ed from that of Nelson et al. (1981). All samples contained a preponderance of virions that were longer than monomer, even those released at the highest pIII concentration attainable from this plasmid. At 0.01 mM IPTG and below, very long phage were present,

Figure 2. Steady-state amounts of pIII, pVI, pI and pIV in the f1d3-infected cells that produce increasing amounts of pIII. A to D, Western blots of cell lysates, from the experiment depicted in Figures 3 and 4: A, pIII; B, pVI; C, pI; D, pIV. Lanes: 1, K561, uninfected; 2 to 7, K561 containing plasmid with the lac-gIII fusion (pJARA200): 2, no IPTG; 3, 0.002 mM; 4, 0.004 mM; 5, 0.01 mM; 6, 0.04 mM IPTG; 7, K561, infected with f1 wt. Each lane was loaded with the total cell lysate of an equivalent of 0.15 A660 units of cells. E, Lanes 1 to 4, Western blot of a gel loaded with 2.3 times as much lysate per lane as the gels in A to D. Lanes: 1, K561(pJARA200) infected with f1d3, no IPTG; 2, K561 infected with f1d3; 3, K561(no plasmid), uninfected; 4, K561(pJARA200), uninfected, no IPTG. Lanes 5 to 8, standard used in quantitative analysis, containing the following amounts of pIII per lane: 5, 0.2 ng (2.8  109 molecules); 6, 0.4 ng (5.7  109 molecules); 7, 1.6 ng (2.3  1010); 8, 6.3 (9.1  1010 molecules). The amount of pIII was calculated as described in Materials and Methods. Cells were collected by centrifugation and lysed by heating in SDS to 100 C. Proteins from the total cell lysates were separated by SDS-PAGE in either Laemmli (A, C and E), or tricine (B and D) gel system.

many of which remain in the well (Figure 4B, lanes 1 to 5). In the absence of IPTG or in cells that did not contain a complementing plasmid, most of the virions were too long to enter the gel at all (Figure 4B, lanes 1 and 2). Crissman & Smith (1984) found that virions lacking pIII were extremely sensitive to incubation with the detergent sarcosyl. Sarcosyl treatment

Roles of pIII in Filamentous Phage Assembly

Figure 3. Dependence of the release of f1d3 phage on the amount of pIII in the cell. Host strain was K561 containing gIII under the control of lac promoter on plasmid pJARA200. A, Phage release at different IPTG concentrations. (}) monophage equivalents of phage released into the supernatant; ( & ) infectious phage titer. Data in the graph were derived from the phage samples shown in B. The amount of the phage ssDNA was calculated as described in Materials and Methods. B, Agarose gel electrophoresis of phage ssDNA from SDSdisassembled virions from unconcentrated culture supernatants. Lanes: 1, no IPTG; 2, 0.004 mM; 3, 0.01 mM; 4, 0.04 mM; 5, 0.1 mM IPTG. Cells were infected with f1d3, and the excess infecting phage removed as in the legend to Figure 1. IPTG was added at increasing concentrations at 37 C for a 90 minute period.

resulted in the disassembly of virions and the release of phage ssDNA into the medium. pIII-containing virions stayed intact under the same conditions. Hence, f1d3 phage were incubated with sarcosyl, and subjected to native virion electrophoresis to assay resistant (infectious) and sensitive (non-infectious) virions. Free ssDNA released from sarcosyl-sensitive virions was detected by staining the gel with EtBr immediately after electrophoresis (Figure 4C). The position of the bands of the intact (sarcosyl-resistant) virions was revealed by treating the gels with NaOH to disassemble the sarcosylresistant virions, and then staining the newly exposed phage ssDNA with EtBr (Figure 4D). At the lowest pIII concentrations, all the phage were sensitive to sarcosyl (Figure 4C, lanes 1 and 2), while phage grown at the highest concentrations of pIII were resistant (Figure 4C, lanes 6 and 7). Phage released at intermediate concentrations of

29 pIII contained a mixture of sarcosyl-sensitive and -resistant phage (Figure 4C, lanes 3 to 5). The amount of phage ssDNA in the bands of sarcosyl-resistant virions of various lengths was estimated from a densitometric scan of the gel. At 0.002 mM IPTG (Figure 4D, lane 3), the ratio of ssDNA in longer (6-mer and up) to shorter (1 to 5mer) phage was 4.2 to 1, while at 0.04 mM IPTG (Figure 4D, lane 6), it was 0.7 to 1. Hence, within the population of sarcosyl-resistant phage, the length distribution changed in favor of shorter phage as the IPTG concentration increased. Even at the highest pIII levels we were able to achieve, 85% of the f1d3 were longer than monophage, while the wt f1 were mostly monomers. Since the concentration of pIII in a complemented f1d3 infection was about tenfold lower than in a wt f1 infection (Table 1), it seems likely that the predominance of longer phage is due to the lower concentration of pIII. To examine the dependence of the length of virions on pIII concentration, gIII amber mutant R171 grown on the suppressor strain K37 were analyzed by native virion electrophoresis (Figure 5, lane 2). Suppression of the gIII amber mutations is not complete; Pratt et al. (1966) and Horiuchi et al. (1978) found that the virions released after infection of suppressor strains were longer than wt. The population of R171 virions (Figure 5, lane 2) contained more long phage than did f1 wt (Figure 5, lane 7), but fewer than complemented f1d3 (Figure 4B, lanes 6 and 7). 43% of phage R171 grown on suppressor strain K37 were monomers. When K37 also carried the complementing plasmid, which added more pIII, the fraction of R171 monomers increased to 67% (Figure 5, lane 1). This supports the proposal that the length of the phage is inversely related to the amount of pIII in the cells. amber mutants of gVI, which encodes the other protein added to the virion during termination of assembly, also give rise to longer particles (Horiuchi et al., 1978). gIII, gVI and gI amber mutants grown in the suppressor strain produced particles longer than wt particles, and this could be partially corrected by addition of a plasmid that produced only the affected protein (Figure 5). That a shortage of a termination protein should lead to a larger population of phage particles longer than monomers is readily understandable. However, pI (Figure 5, lanes 5 and 6) takes part in the formation of the assembly machinery and the initiation of phage assembly, and is also involved in elongation (Russel, 1995). Moreover, mutations that lead to reduced expression of pVII and pIX (but not pVIII) also give rise to longer phage (data not shown). These results can all be rationalized in terms of competition between further elongation and termination, but not as simply as for limiting amounts of pIII and pVI.

30

Roles of pIII in Filamentous Phage Assembly

Figure 4. Native virion agarose electrophoresis of released f1d3 showing the length and stability in sarcosyl at several intracellular concentrations of pIII. A and B, untreated virions; C and D, virions incubated in the detergent sarcosyl (0.1%, RT, ten minutes) prior to loading. A and C, Free phage ssDNA; B and D, free phage ssDNA and virions. Lanes: 1, K561 (no plasmids); 2 to 7, K561 containing plasmid with the lac-gIII fusion (pJARA200): 2, no IPTG; 3, 0.002 mM; 4, 0.004 mM; 5, 0.01 mM; 6, 0.04 mM; 7, 0.1 mM IPTG. Free ssDNA, free phage ssDNA in the phage samples; Monomer, virion with one copy of the genome; Dimer, Trimer, Tetramer, Pentamer, Hexamer and longer; virions that contain to, three, four, ®ve, or six or more copies of the genome, respectively. Phage in wells, long phage that did not migrate from the gel wells during electrophoresis. Samples contained 7.6  1010 to 1.5  1011 monophage equivalents of the phage (200 to 400 ng of phage ssDNA) per lane. Phage samples in lanes 2 to 7 are from the experiment depicted in Figure 3, and were concentrated 25 to 400 times prior to incubation with sarcosyl and loading. Methods of phage concentration, electrophoresis and detection are described in Materials and Methods.

Initiation and elongation proceed with high efficiency in the absence of pIII Cells infected with f1d3 in the absence of pIII release about 100-fold fewer monophage equivalents than do f1 wt infected cells. To ask whether, in the absence of pIII, elongation can proceed without termination, and whether the assembling phage stay associated with the cells, K561 cells (no gIII) infected with f1d3 were examined by electron microscopy. Many long phage ®laments that form a dense network around the cells were seen to emerge from the cell surface (Figure 6A). Cells that contained the lac-gIII fusion infected with f1d3, but not induced (no IPTG), looked similar (Figure 6E and G). In contrast, very few ®laments were associated with cells infected with f1 wt under the same conditions (Figure 6D). The cell-associated ®laments labeled intensively in an immunogold assay when anti-f1 was used as the primary Ab (Figure 7A). To detach the cell-associated ®laments, f1d3infected cells were subjected to mechanical shearing, and the cultures were observed by electron microscopy (Figure 6B and C). After mechanical shearing, ®laments appeared uniformly distributed in the background (Figure 6C) and they could be separated from the cells by centrifugation (Figure 6B). The material detached from cells by mechanical shearing was heated in SDS-containing

buffer to disassemble the virions, and analyzed by agarose gel electrophoresis. Much ssDNA was detected in the supernatant after shearing, in comparison to the culture prior to shearing (Figure 8, lanes 2 and 4). To examine whether the ssDNA detected in these samples was free or encapsulated into the phage-like ®laments (seen by electron microscopy, Figure 6C), native, unconcentrated supernatants (not treated with SDS) were subjected to agarose electrophoresis and the gels were stained with EtBr. No free phage ssDNA or other DNA species were detected, indicating that all ssDNA was encapsidated into the phage-like ®laments (not shown). Shearing did not lyse the cells, as determined by counting under the light microscope. The dependence of the number of monophage equivalents of cell-associated phage on the amount of pIII in the cells was estimated (Figure 8, Table 2). Spontaneously released and mechanically sheared (cell-associated) particles were collected separately, and the number of monophage equivalents of phage estimated from the intensities of ssDNA bands after electrophoresis of SDS-disassembled virions. The number of phage particles (as distinct from the monophage equivalents) was hard to estimate, since samples contained virions of variable length. When IPTG was not added, almost no DNA was detected in the supernatant of the infected cells, while much could be recovered after

31

Roles of pIII in Filamentous Phage Assembly

Figure 5. Native virion electrophoresis showing the length of the phage in suppressed and supercomplemented gIII, gVI or gI amber mutants. Lanes: 1, phage R171 (gIIIam) grown on supD strain that also contained a plasmid with phage infection-inducible psp-gIII fusion (pJARA112); 2, R171 (gIIIam) grown on supD; 3, phage R5 (gVIam) grown on supD in the presence of a plasmid with tac-gVI fusion (pJARA300), induced with 1 mM IPTG; 4, R5 (gVIam) grown on supD; 5, R2 (gIam) grown on supD in the presence of the plasmid that produces pI (pJIH1; Horabin & Webster (1986); 10, R2 (gIam) grown on supD; 7, supD infected with the f1 wt. ‡‡, phage grown in the presence; and ‡, in the absence of a supercomplementing plasmid. Monomer, virion with one copy of the genome; Dimer, Trimer, Tetramer, virions that contain two, three or four copies of the genome, respectively. A supD strain K37, either with or without appropriate supercomplementing plasmids, was infected with phage containing an amber mutation in gIII, gVI or gI or by wt phage; 120 minutes later phage were harvested, concentrated by PEG precipitation and subjected to agarose electrophoresis. Each lane contained 1.2  1011 monophage equivalents of phage.

shearing. Release of phage increased with increasing amounts of pIII: in an f1 wt infection, more than 99% of the phage were released, in comparison to 3% for f1d3 in the absence of pIII (Table 2). However, the total number of monophage equivalents of extruded phage-like ®laments (cellattached and released), was only threefold lower after f1d3 than after f1 wt infection, suggesting that initiation and elongation proceed ef®ciently in the absence of termination. A remaining question was whether cell-associated phage, which have started to assemble in the absence of pIII, could be terminated and released if

pIII were supplied later. To test this, pIII production in cells that contained the lac-gIII fusion was induced 30 minutes after infection with f1d3. Electron microscopy indicated that the cell-associated ®laments decreased in number and length after the onset of pIII production, while in the uninduced culture they increased and lengthened (Figure 6E, F and G). Agarose gel electrophoresis and quanti®cation of cell-associated and released phage also suggested that cell-associated phage were released after induction (Figure 8, Table 2). The absolute number of monophage equivalents of cell-associated phage decreased, suggesting that a fraction were released into the supernatant upon induction, while the sum (released plus cell-associated) of monophage equivalents of phage increased. Thus there was both release and new synthesis of phage. Cells from the late-induction culture nevertheless contained more attached phage than did f1 wt infected cells. Cells containing a psp-gIII fusion (pJARA112), which is induced by phage infection, also retain more phage on their surface than do f1 wt infected cells (Figure 8, Table 2), even though they make more pIII than is made by lac-gIII fusion (Rakonjac et al., 1997; and unpublished results). When observed by electron microscopy, these cells looked like those from late induction of the lac-gIII, shown in Figure 6F. Thus it appears that the delay in supplying pIII is not responsible for the residual numbers of attached phage. (A lac-gIII fusion induced at the time of phage infection cannot be used as a control, because pIII production blocks infection (Boeke et al., 1982).) pIII stabilizes pVI The low ef®ciency of phage release in the absence of pIII suggested that pVI, which is the other protein at the pIII end of the phage particle, cannot, by itself, ef®ciently terminate assembly. Western blotting was used to measure the amount of pVI in f1d3 infected cells (Figure 2B). The amount of pVI in the absence of pIII was very low. When pIII was supplied, the abundance of pVI depended on the amount of pIII produced (Figure 2B). This suggests that pIII may stabilize pVI. The amount of two other phage proteins, pI and pIV, was not affected by the level of pIII (Figure 2C and D). gIII and gVI were expressed from compatible plasmids, pJARA200 and pJARA300, respectively, and the stability of each of the proteins, when expressed together or separately, was measured in a pulse ± chase experiment. The amount of pVI detected after labeling suggested that its rate of synthesis was not affected by pIII. Degradation of pVI was very rapid in the absence of pIII, but markedly slowed in its presence (Figure 9). The half-life for pVI was one minute in the absence of pIII, and nine minutes in its presence. The stability of pIII was not affected by pVI: the amount of protein labeled in the pulse was the same whether

32

Roles of pIII in Filamentous Phage Assembly

Figure 6. Electron micrographs of cells and cell-associated phage. A, f1d3-infected strain K561, 100 minute infection. B, An aliquot of the sample in A, mechanically sheared, ®xed, washed and stained. C, Same as B, except the cells were not washed after ®xation and prior to staining. D, f1 wt-infected strain K561, 100 minute infection. E, K561 strain containing lac-gIII fusion, infected with f1d3, incubated for 30 minutes in the absence of IPTG, and ®xed. F, Culture in E, induced with 1 mM IPTG for another 45 minutes, and ®xed. G, Culture as in E, incubated without IPTG for another 45 minutes and then ®xed. H, uninfected culture of K561. Magni®cation: 11,250. The bar represents 1 mm.

33

Roles of pIII in Filamentous Phage Assembly

Figure 7. Cell-associated ®laments in f1d3-infected cells are made of f1 coat protein. Cells were incubated with: A, anti-f1 (directed against major coat protein, pVIII); or B, a non-speci®c IgG. The bound antibody was detected after incubation with gold-conjugated secondary Ab and electron microscopy. Magni®cation: 67,000. f marks a ¯agellum. The bar represents 100 nm.

or not pVI was present, decreased to 50 to 60% during ®rst 20 minutes of chase, and stayed at the same level for two hours (not shown). The dependence of pVI stability on pIII suggested that they might interact directly. Coimmunoprecipitation experiments using a Triton X-100-solubilized membrane fraction of the cells that contained gIII and gVI on plasmids or phage did not support this hypothesis (Figure 10). Neither anti-pVI nor anti-pIII serum co-immunoprecipitated the interaction candidate (Figure 10, compare lanes 5, 6, 7 with 10, 11, 12). The absence of detectable co-immunoprecipitation cannot be explained by interference of the antibody with a putative protein ±protein interaction, since these very sera can detect a pIII ± pVI complex from virions (Endemann & Model, 1995). Quantitative analysis of pVI in Western blots of cells that contained an increasing amount of pIII (Figure 2A and B), showed that the steady-state amount of pVI was proportional to the amount of pIII over a range of concentrations from 83 to 1350 molecules per cell. The maintenance of a stable ratio of pIII:pVI of 1:5 (determined from the Western blots; Figure 2A and B) suggests that pVI might be stabilized by a stoichiometric interaction with pIII. However, the interaction may be either transient, or unstable, under the conditions used in the co-immunoprecipitations. Similarly, kinetic analyses of pIII and pVI accumulation in f1d3 infected cells after lac-gIII

induction showed that the amount of pVI increased in parallel with pIII within a two minute time span (Figure 11), suggesting that stabilization could be a consequence of a direct protective effect of pIII protein. Can pVI terminate phage assembly in the absence of pIII? The intracellular concentration of pVI in infected cells in the absence of pIII estimated from the Western blots was 400 molecules per cell, about 40fold lower than in f1 wt infection (Figure 2B). Nevertheless, the small amount of pVI could have terminated the assembly of the few virions that were released. The incorporation of pVI into those virions was examined by monitoring the amount of pVI in the released phage over time (Figure 12). pVI in the virions increased twofold, while there was a 13-fold increase in the number of monophage equivalents of the released phage. The amount of pIII did not change (Figure 12A). The increase in pVI was too small to suggest that pVI is associated with every monophage equivalent, a conclusion that agrees with reports from Gailus et al. (1994) The average length (in monophage equivalents) of virions released in the absence of pIII is about 10. Hence the increase in released pVI might be attributed to the presence of pVI at the end of the released particles.

34

Roles of pIII in Filamentous Phage Assembly

Figure 8. Comparison of cell-associated and spontaneously released phage after f1d3 infection. Agarose gel electrophoresis of phage ssDNA from SDS-disassembled virions of: R, spontaneously released phage (odd number lanes); A, cell-associated phage, detached by mechanical shearing (even number lanes). Lanes 1 to 6, K561(pJARA200) infected with f1d3: 1, 30 minutes, no IPTG, spontaneously released phage; 2, 30 minutes, no IPTG, cell-associated phage; 3, 75 minutes, no IPTG, spontaneously released phage; 4, 75 minutes, no IPTG cell-associated phage; 5, 30 minutes without and 45 minutes with 1 mM IPTG, spontaneously released phage; 6, 30 minutes without and 45 minutes with 1 mM IPTG, cell-associated phage; lanes 7 and 8, K561 containing plasmid with phage infection-inducible psp-gIII fusion (pJARA112), infected with f1d3 for 75 minutes; 7, spontaneously released phage; 8, cell-associated phage; 9 and 10, K561(pJARA200) infected with f1 wt for 75 minutes: 9, spontaneously released phage; 10, cell-associated phage. wt f1 ssDNA migrates more slowly because it is longer by 1277 nucleotides than f1d3. Each lane contains 30 ml of unconcentrated sample.

gIII and gVI deficiency is not lethal to the host K561 infected with f1d3 phage showed the same ef®ciency of colony formation as uninfected cells. At an m.o.i. of 50, the majority of the cells became infected, and gave rise to small, transparent colo-

Figure 9. Stability of pVI in the presence or absence of pIII. The time course of degradation of labeled pVI was monitored in a pulse ± chase experiment as described in Materials and Methods. Lanes 1 to 5, gVI only, K561 containing a plasmid with a tac-gVI fusion (pJARA300) and a vector with lac promoter (pGL101); 6 to 10, gVI ‡ gIII K561 containing tac-gVI fusion on plasmid pJARA300 and lac-gIII fusion on plasmid pJARA200 (derivative of pGL101). Lanes 1 and 6: P, samples at the end of pulse; 2 and 7, two minutes (20 ); 3 and 8, ten minutes (100 ); 4 and 9, 20 minutes (200 ); 5 and 10, 60 minutes (600 ) after addition of cold methionine.

nies. This was surprising, since infection with amber mutants in gIII or gVI is deleterious to colony formation and is often lethal (Pratt et al., 1966). Non-suppressing cells, infected with gVI amber mutant phage R5, did not form colonies. Because cells infected with f1d3 contain very little pVI, and yet are viable, and gVI is probably involved only in the termination of phage assembly, gVI amber mutants could have killed their host due to a polar

Table 2. pIII and release of the assembling phage from the infected cells

Samplea

Shearingb

Lane in Figure 8

Phage (monophage equivalents per ml)c 8

Total phage (monophage equivalents per ml)d 11

Attached (%)

Released (%)

99.6

0.4

f1d3, ÿIPTG, 30 minutes

R A

1 2

6.0  10 1.6  1011

1.6  10

f1d3, ÿIPTG, 75 minutes

R A

3 4

7.3  109 2.5  1011

2.6  1011

97

3

f1d3, ÿIPTG, 30 minutes ‡ IPTG, 45 minutes

R A

5 6

4.5  1011 7.4  1010

5.3  1011

14

86

f1d3, psp-gIII, 75 minutes

R A

7 8

4.2  1011 5.3  1010

4.8  1011

11

89

f1 wt, 75 minutes

R A

9 10

7.3  1011 4.0  109

7.4  1011

a

0.5

99.5

Samples were prepared as described in the text and the legend to Figure 8. Rows labeled with: R, spontaneously released phage; A, cell-associated phage, detached by mechanical shearing. c Calculated form the amount of phage ssDNA in the agarose gel electrophoresis of SDS-disassembled virions, as described in Materials and Methods. Data were derived form samples shown in Figure 8. d The sum of the number of the monophage equivalents of the phage before and after shearing. b

35

Roles of pIII in Filamentous Phage Assembly

Figure 10. Co-immunoprecipitations of pVI and pIII. The Triton extract of the inner membrane fraction of [35S]Met-labeled cells was subjected to immunoprecipitation with: lanes 1 to 4, anti f1 antiserum; lanes 5 to 9, anti-pVI antiserum; lanes 10 to 14, anti-pIII antiserum (Gailus et al., 1994). Upper panel: immunoprecipitated proteins separated on tricine gel, 16%; lower panel: same set of samples, run on Laemmli gel, 10%. Lanes 1, 5 and 10, strain K561, infected with f1 wt; lanes 2, 6 and 11, K561, infected with gVIIIam mutant phage (R240); 7 and 12, K561 containing lac-gIII and tacgVI fusions on two plasmids (pJARA200 and pJARA300, respectively); 3, 8 and 13, K561 that contains a lac-gIII fusion on pJARA200; 4, 9 and 14, K561 that contains a tac-gVI fusion on pJARA300. The positions of pIII, pVI and pVIII on the gels are indicated. The vertical bar along the left side of the lower panel (Laemmli gel) corresponds to the region indicated by a shorter vertical bar along left side of the upper panel (tricine gel).

effect on gI, which is in the same operon and downstream from gVI (Pratt et al., 1966). Killing by R5 was relieved in non-suppressing cells that expressed pI from a plasmid. The polarity of gVI amber mutation was con®rmed by Western blots, which showed that amount of pI in R5-infected

cells was signi®cantly lower than in f1 wt or f1d3infected cells, and did not increase when pVI was overexpressed in trans from a plasmid (not shown). Interestingly, the killing effect of R5 was also relieved by expression of pVI alone from this plasmid, which at the same time partially complemented the gVI mutation for production of infectious phage. Presumably the combined effect of somewhat lower phage production (due to the absence of termination) and a reduced number of assembly sites (due to lower pI levels) was suf®cient to unbalance the host more severely than either defect alone.

Discussion

Figure 11. pVI accumulation in f1d3-infected cells upon induction of gIII expression from lac-gIII fusion. pIII and pVI were detected by Western blots in the total cell lysates at several time points after addition of IPTG to the culture of K561 that contained plasmid pJARA200 (lanes 4 to 9). For comparison, total cell lysates of: uninfected K561 (lane 1), K561 infected with f1d3 (lane 2), or with f1 wt (lane 10) for 90 minutes were run on the same gel. As a difference from Figure 2E, lane 1, pIII band is not detectable in the lysate of K561(pJARA200) in the absence of IPTG (lane 3), because the antiserum used to detect pIII in this experiment, R164, is less sensitive than 1F8, used in Figure 2.

Although previous work with amber mutants of gIII demonstated that pIII was involved in the termination of phage assembly, the abundance of released virions from cells infected with gIII mutant phage argued that pIII was not needed for the formation and release of non-infectious phage particles (Pratt et al., 1969; Lopez & Webster, 1983; Crissman & Smith, 1984). We measured phage release in the absence, and at several intracellular concentrations, of pIII. The release of phage particles from infected cells in the absence of pIII was extremely low. Instead, the assembling ®laments stayed attached to the host cell. pIII supplied in trans stimulated phage release, suggesting that it is necessary for the termination of assembly.

36

Figure 12. Accumulation of pVI in released phage particles during the time course of non-complemented f1d3 infection. A, Relative amount of pIII, pVI and phage ssDNA. The amounts at the time point 0 were taken as 1. (~) pIII; ( & ) pVI; (^) phage ssDNA. B, pIII, pVI and ssDNA from concentrated phage samples. The left and the middle panel, Western blots. Each lane was loaded with 10 ml of 1000-fold concentrated phage sample. Right panel, ssDNA from the SDS-disassembled phage. Each lane was loaded with 20 ml of a 100-fold concentrated sample. Lanes: 0, time point 0; 20, 20 minutes; 40, 40 minutes; 60, 60 minutes; 80, 80 minutes. Phage samples were from the experiment depicted in Figure 1, in which strain K561 containing plasmid with lac-gIII fusion, pJARA200, was infected with f1d3 in the absence of IPTG (Figure 1B, lanes 6 and 11 to 14).

By counting the emerging ®laments from highmagni®cation electron micrographs of f1d3-infected cells in the absence of pIII, it was possible to estimate that the number of assembly sites is between 150 and 300. This is in agreement with the estimated increase of the number of adhesion zones upon phage infection (Lopez & Webster, 1985). Between three and ®ve molecules of pIII are located at the end of the phage particle, and it has been proposed that they function as a multimer (Gailus et al., 1994). Therefore, at the lower concentrations of pIII, the probability that there will be three to ®ve molecules at any one assembly site will be small, in agreement with our ®nding that a threshold pIII concentration (160 molecules per cell) is needed for termination of phage assembly and formation of infectious particles. Based on the number of infectious phage released at very low concentrations of pIII (up to 409 molecules/cell), a Poisson calculation that assumes 200 assembly sites suggests that between four and ®ve molecules of pIII per assembly site are required for the formation of the infectious particle.

Roles of pIII in Filamentous Phage Assembly

When pIII is above the threshold concentration, but is still the limiting component of phage assembly (161 to 1350 molecules/cell), the number of released infectious phage is proportional to the square of the pIII concentration. The physical interpretation of this relationship is complex, but is in agreement that more than one pIII is necessary for assembly of one infectious phage. pIII protects pVI from rapid degradation. In the absence of pIII, the steady state amount of pVI in the cells infected with f1d3 was at least 40-fold lower than in an f1 wt infection. A few long, phage-like ®laments released from these cells most likely contain pVI at the end of the particle, suggesting that pVI may be capable of terminating the assembly. Therefore, the requirement for pIII in the termination of assembly could be indirect, in that it might stabilize pVI, which would terminate the assembly if present in suf®cient quantity. Since pIII protects pVI from degradation, the two proteins might interact in cells prior to their incorporation into phage, as proposed by Gailus et al. (1994). We were unable to demonstrate a pIII ±pVI interaction by co-immunoprecipitation, but the existence of such a complex is suggested by the maintenance of 1 to 5 ratio of pIII to pVI over a range of intercellular pIII concentrations from 83 to 1350 molecules per cell (Figure 2A and B). Detection of a pIII-pVI interaction by co-immunoprecipitation from phage disrupted by deoxycholate in the presence of chloroform (Endemann & Model, 1995), but not from cell membranes solubilized by deoxycholate/chloroform or Triton X-100, suggests that pIII and pVI may undergo a conformational change and form a stable complex upon the transition from the inner membrane into to the virion. Since both proteins interact with pVIII prior to assembly (Endemann & Model, 1995), it is possible that pVIII might dock them (separately or together) to the assembling virion, when the whole ssDNA genome has been incorporated into the ®lament. A conformational change in pVIII upon incorporation into the ®lament (Nambudripad et al., 1991) could trigger the change in pIII and pVI that enables them to form a stable complex, and to terminate phage assembly. Formation of the complex could terminate assembly by disrupting the connection of the growing ®lament with the cell envelope and at the same time capping the virion. Although initiation and elongation of phage assembly proceeds quite ef®ciently in the absence of termination, the sum of cell-associated and released phage (expressed as the number of monophage equivalents) is threefold lower than in a wt infection, suggesting that the addition of the new ssDNA ± pV complexes to the growing phage ®lament is somewhat less ef®cient then the initiation of assembly of new particles. The virions released by pIII or pVI amber mutants grown on a suppressor strain were longer than f1 wt. When yet more pIII or pVI was supplied from plasmids, the ratio of monomeric to multimeric

37

Roles of pIII in Filamentous Phage Assembly

phage increases markedly. This suggests that the high percentage (>95%) production of normal length (monomeric) particles in an f1 wt infection is based on the relative abundance of the components that take part in elongation to the proteins that carry out termination. In assembly-incompetent mutant phage, the accumulation of the components of assembly is what presumably leads to the killing of the host (Pratt et al., 1966; Hohn et al., 1971; Horiuchi et al., 1978). It has been observed that gIII and gVI amber mutant phage do not kill the cell as fast as other mutants (Model & Russel, 1988). We found that neither the gIII deletion mutant f1d3 nor the gVI amber mutant R5 (when compensated for the partial polarity on gI) kill host cells, and that the infected cells are able to form colonies, albeit of smaller size than f1 wt infected cells. In the infection with gVI amber mutant R5, each of the de®ciencies that lowers the ef®ciency of assembly (no pVI or lower amount of pI) by itself did not cause the killing of the host. However, the combination was lethal. Therefore, it seems that the cells are tolerant to only a minor increase in the concentration of the precursors of phage assembly over the level present in an f1 wt infection. Early experiments that followed the fate of pIII after the entry of the phage into the host cell by tracing radioactively labeled pIII, detected a cleaved product in the infected cell (Marco et al., 1974). However, Western blots of f1d3-infected cells that do not contain any gIII detected a very faint band of the full-length pIII (at the border of detectability; Figure 2E, lane 2), but no truncated product (not shown), arguing in favor of preservation of at least a fraction of pIII after entry into the cells. The existence of a threshold concentration of pIII in the cell necessary for ef®cient release of infectious progeny suggested that pIII from the infecting phage is not re-used at a 100% ef®ciency for the assembly of infectious progeny. Some infectious phage (0.01 to 0.1/cell) were detected after the infection with f1d3 in the absence of gIII, but it could not be determined whether those were progeny particles or residual f1d3 phage from the stock used to infect the cells. A very sensitive assay, which detected only phagemid-containing particles released after the infection of phagemidcontaining cells with the gIII deleted helper phage R408d3 (Rakonjac et al., 1997) showed that production of infectious particles in the absence of gIII was extremely inef®cient (2.5  10ÿ7/cell, as opposed to 700/cell with wt helper phage). In that respect, pIII seems to be different from the minor proteins pVII and pIX, which were found to be reutilized rather ef®ciently under similar experimental conditions (Lopez & Webster, 1983). It may be worth pointing out that the ®laments made in the absence of pIII morphologically resemble conjugative pili, reinforcing the intuitive idea that the two are likely to be evolutionarily related.

Materials and Methods Bacterial and phage strains The bacterial strains used are all derivatives of E. coli K12, strain K38 [HfrC l‡ relA1 spoT1 T2R(OmpF627 fadL701)], from our laboratory collection. K37 ˆ K38 (supD); K561 ˆ K38 (lacIq); K1653 ˆ K561 (pJARA200); K1762 ˆ K561 (pJARA112, pJARA131). All phage used are from our laboratory collection: f1 wt, and a set of amber mutants, R171 (gIII), R5 (gVI), R2 (gI), R240 (gVIII). The construction of the gIII deletion mutants, f1d3 and R408d3, has been described (Rakonjac et al., 1997). These two phage have the same deletion, which removes the complete gIII coding sequence including the start (GTG) codon. In this phage gVI is translated from the gIII ribosome binding site. Plasmid construction pJARA200 contains a lac-gIII promoter fusion that was constructed by Davis et al. (1985) in plasmid pGL101 (a derivative of pBR322 (Lauer et al., 1981)), which contains the lacUV5 promoter, and has a very low background expression in the absence of IPTG (Davis (1985); this paper). pJARA200 was constructed by inserting the 30 portion of gIII from the BamHI site to the stop codon into plasmid pND372 (Davis et al., 1985), which contains the 50 portion of gIII fused to the lac promoter. The 30 portion of gIII was ampli®ed by PCR using f1 wt RF as a template and primers 33 (f1 gIII 2210 to 2225; 50 -GCTTTAATGAGGATCC-30 , including the resident BamHI site at position 2220), and 32 (f1 gIII 2853 to 2837 HindIII; 50 -CCCAAGCTTCTATTAAGACTCCTTATTAC30 ). The ampli®ed fragment was inserted into BamHIHindIII-digested pND372. Plasmid pJARA300 contains a tac-gVI promoter fusion in the vector pGZ119EH, which has a ColD origin of replication and a chloramphenicol resistance gene (Lessl et al., 1992). The pVI sequence (from the ribosome binding site to the stop codon) was ampli®ed by PCR, using f1 RF DNA as a template, and primers 46 (f1 gVI 28392857 BamHI; 50 -CGGGATCCAATAAGGAGTCTTAATCAT-30 ) and 47 (f1 gVI 3195 to 3179 HindIII; CCCAAGCTTTATTATTTATCCCAATCC-30 ), and then cloned into the BamHI-HindIII-digested vector pGZ119EH. pJARA250 is a phagemid that contains a tac promoter fused to the pelB signal sequence, followed by a multiple cloning site. It was made by inserting the HpaI-XbaI fragment of pGZ119EH into HpaI-XbaI-cleaved pET25b (Novagen). pJARA112 is a pBR322 derivative that contains a pspgIII fusion. pJARA131 is a derivative of pGZ119EH that contains the pspFABCD operon. Construction of these plasmids has been described (Rakonjac et al., 1997). Plasmid pJIH1 contains a pL-gI fusion (Horabin & Webster, 1986), and plasmid pJF4 an araC-gI fusion (Jian-nong Feng, unpublished results). Bacterial strains containing pBR322 or pGZ119EHderived plasmids were selected on Amp (60 mg/ml) or Cm (25 mg/ml), respectively. Phage infection and growth In all experiments in which phage release from infected cells was measured, a logarithmic phase culture (around 108 cells/ml) of the appropriate strain in TB

38 medium (10 g Bacto tryptone, 1 g yeast extract, 4 g NaCl and 1 g glucose per liter) was infected with phage at m.o.i. 50, for 15 minutes at 37 C. Infected cells were separated from unabsorbed phage by centrifugation and resuspended in fresh medium. The cultures were then incubated under the conditions described in the Figure legends. At the end of the incubation, cells were chilled, titered, and then separated from the phage by centrifugation. Bacterial pellets were resuspended in TE (10 mM Tris-HCl, and 2 mM EDTA, pH 7.6) at 50 A660 units per ml, frozen on dry ice, stored at ÿ70 C and later used for the analysis of the proteins by SDS-PAGE and Western blotting. For the shearing experiment, cells were resuspended in chilled medium, exposed to mechanical shearing in a Sorvall Omnimixer (one minute at 10 and six minutes at 5), and the detached phage were separated from the cells by centrifugation. Cells were titered prior to infection, after infection, after washing away the unabsorbed phage and resuspension in the fresh medium, or at the end of incubation. In the experiments that assessed the killing effect of mutant phage, cells were plated 15 minutes after infection, and their titer was compared with that of a parallel uninfected culture. Infected cells make small, transparent colonies. The absence of small colonies and a smaller number of large colonies indicates that the phage infection has killed the infected cells. The number of infectious phage was determined by titering. Strain K1762, which is K561 with a psp-gIII fusion on pJARA112 and pspFABCDE on pJARA131, allows plaque formation by the gIII deletion phage (Rakonjac et al., 1997), and thus was used for titering gIII deletion phage f1d3 and R408d3 and generation of stocks (from a single plaque). f1 wt was titered on K561, and all the amber mutant phage on K37 and K561. In the experiments that followed phage release in noncomplemented infections with f1d3 phage, the background of infecting phage that remained after washing and resuspension of the cells in fresh medium was determined by titering. Cultures were incubated at 37 C, except for growth of gI amber mutant phage (Figure 5), which was incubated at 34 C. This was necessary to prevent killing of the cells by overexpression of heat-inducible pL-gI fusion from the plasmid pJIH1 (Horabin & Webster, 1986). Phage purification and concentration Phage released by the f1d3-infected cells in the absence or at very low concentration of intracellular pIII are fragile, so they were pipetted as little as possible, and then with cut off pipette tips, and kept at 0 to 4 C, except when indicated. They were concentrated by ®ltration in an Amicon stirred cell (200 ml volume), using membrane XM300 (cutoff molecular mass 300 kDa) at the recommended pressure. After the medium was ®ltered out, the membrane with the phage was taken out of the unit, and placed in a sterile Petri dish. To elute the phage, 2 ml of TE buffer (10 mM Tris-HCl and 2 mM EDTA, pH 7.6) was pipetted onto the membrane and incubated overnight. Phage were collected and separated from the residual cells by centrifugation. If necessary, phage were further concentrated in Centricon 100 units (2 ml), and again allowed to elute overnight. This procedure was used to concentrate f1d3 phage released in the following experiments: time course, grown in the absence of IPTG (Figures 1 and 11), phage grown in

Roles of pIII in Filamentous Phage Assembly increasing concentrations of IPTG (Figures 3 and 4), and infection of strain K561. No free single-stranded phage DNA was detected in the concentrated samples (see Figure 4), suggesting that this procedure did not disrupt the virions. The absence of free ssDNA was not a result of presence of DNases in the culture medium: in a control experiment, free f1 phage single-stranded DNA was put into f1d3-infected culture at the time of the infection and it stayed intact during a typical incubation period (90 minutes). Alternatively, phage were concentrated by a short PEG precipitation in 5% PEG 8000 and 0.5 M NaCl (ten minutes), and the pellet was allowed to dissolve in TE buffer overnight (without pipetting). This procedure was used to concentrate the samples from the experiment in which phage were mechanically sheared from the cells (Figure 8). About 5% of the sheared off phage were disrupted during this procedure; this was detected as free phage ssDNA on native virion electrophoresis (not shown). Phage from infection with f1 wt or suppressed amber mutants were puri®ed by PEG precipitation, for one hour on ice. Precipitated phage were resuspended in the TE buffer.

Agarose electrophoresis of the phage and quantification A monophage equivalent is a measure of particle mass, and is de®ned as a particle (or its portion) containing one encapsidated genome. Thus, a particle containing ten genomes represents ten monophage equivalents, as do ten particles containing one genome each. In all experiments, the number of monophage equivalents was determined from agarose electrophoresis of phage ssDNA, released from the SDS-disassembled virions (Nelson et al., 1981). Prior to electrophoresis, chromosomal DNA and RNA were removed by digestion with DpnI and RNase A, respectively, for two hours at 37 C. Virions were then disassembled by incubation in SDScontaining buffer (1% SDS, 1  TAE, 5% glycerol, 0.25% BPB) at 70 C for 20 minutes, and then subjected to agarose electrophoresis in 1  TAE buffer. After electrophoresis, phage ssDNA was stained with the EtBr and quanti®ed densitometrically. Since the amount of ssDNA in a band is not linearly proportional to the intensity of the ¯uorescence, every gel contained a set of twofold dilutions of a standard, typically from 1280 to 2.5 ng per lane, used for calibration. Puri®ed phage ssDNA, the concentration of which was determined spectrophotometrically, was used as a standard. The gel was photographed with a CCD camera, and quantitative analysis performed using software packages IS 1000 (Alpha Innotech Corp.), NIH Image (Wayne Rasband, National Institutes of Health), Cricket Graph (Computer Associates International, Inc.) and Excel (Microsoft). A third order polynomial function was used to ®t the standard curve over a range of about 500-fold. Conversion of the calculated amount of ssDNA in the samples into the amount of phage was done based on the molecular mass of ssDNA genome of a particular phage strain, which was again calculated from the base composition and length. A 1 ng sample of single-stranded phage DNA equals 3.06  108 genome equivalents or monophage equivalents for f1 wt, and 3.82  108 f1d3, which is shorter. In f1 wt samples, the calculated number of monophage equivalents matched the titer, as would be expected from the prevalence of monomeric virions (90 to 95%).

39

Roles of pIII in Filamentous Phage Assembly

Native virion agarose electrophoresis was used to separate virions of various lengths and to detect free phage DNA when the stability of phage was analyzed (Nelson et al., 1981). The method was modi®ed by making very low density agarose gels (0.4%) and loading the samples in buffer containing molten agarose (1  TAE, 0.05% BPB, 0.4% agarose), preheated to 50 C. Electrophoresis was performed at 2 V/cm for ten hours. After electrophoresis, free phage single-stranded DNA was ®rst detected by staining the gel in EtBr. To detect the position of the particles in the gel, virions were disassembled by soaking gel in the alkaline buffer, and then the phage DNA was stained in EtBr. Protein electrophoresis, Western blots and quantification of pIII and pVI Proteins from the cell lysates and phage samples were separated by SDS-PAGE, using either (Laemmli, 1970) or tricine (SchaÈgger & von Jagow, 1987) gel systems, transferred to nitrocellulose ®lters, and then detected using appropriate antibodies. Protocols varied slightly, depending on the antibody used. With antibodies R164 (anti-pIII; Rakonjac et al., 1997), 19 to 38 (anti-pVI; Endemann & Model, 1995), anti-pI (M. Russel, unpublished results), or 19 to 87 (anti-pIV; Kazmierczak et al., 1994), an anti-rabbit antiserum conjugated to HRP (Sigma) and the ECL detection system (Amersham Corp., Arlington Heights, IL) were used. With the monoclonal antibody 1F8 (anti-pIII; Tesar et al., 1995), an antimouse antibody conjugated to AP, and immunostaining detection (Blake et al., 1984) was used. The basic buffer was TBS (30 mM Tris, 150 mM NaCl, pH 8.0) with 0.05% Tween 20. Blocking and antibody binding buffers also contained 5% non-fat dry milk. With anti pI antibody, the buffer included 0.02% SDS, and with antibodies R164 and 19 to 38, 0.5% deoxycholate, 0.2% NP40 and 0.02% SDS. Antibody 1F8 was used in Figures 2 and 11, and R164 in Figure 12. The amount of pIII or pVI in various samples was determined from densitometric analysis of the protein bands. A series of twofold or fourfold dilutions of puri®ed and concentrated f1 wt phage sample was used as a standard for the calibration. Every gel used in quantitative analysis contained the calibration standard. The concentration of the f1 phage used as a standard was determined from the amount of ssDNA on the disassembled gel electrophoresis, as described above. The amount of pIII and pVI in the standard were derived from the amount of ssDNA, based on the following assumptions: DNA represents 12% of the mass of the phage; there are four molecules of pIII or pVI in each virion (Goldsmith & Konigsberg, 1977; Woolford et al., 1977), and they represent 1.2% or 0.34% of the mass of the phage proteins, respectively. When the amount of pIII or pVI in the cell lysates was analyzed, the standard was mixed with a lysate of non-infected cells, to equalize the background. The Western blots of the gels were scanned on the Hewlett Packard ScanJet IIp scanner and analyzed using software NIH Image, Cricket Graph, and Excel. A third order polynomial function was used to ®t the standard curve over a 256-fold range of protein amounts. In the experiment depicted in the Figure 2A and E, and Table 1, the number of pIII molecules per cell was calculated as follows: the amount of protein per lane (in ng) was ®rst converted into the number of molecules per lane. Subsequently, from the loading volume and the concentration factor, the number of molecules per ml of

unconcentrated culture was determined, and then divided by the titer of the cells per ml in the culture at the time of harvest. In vivo labeling, cell fractionation and immunoprecipitation Exponential cultures of bacteria were grown in the DO minimal medium (Vogel & Bonner, 1956) supplemented with 0.4% (w/v) glucose, 5 mg/ml thiamin and 18 amino acids (0.2 mg/ml, no methionine or cysteine). When plasmid-encoded proteins were analyzed, the culture was induced with IPTG for 15 minutes, and for analysis of the phage-encoded proteins, cells were infected for 30 minutes. Samples (400 ml) were then labeled for 120 or 240 seconds with 40 mCi [35S]methionine. In the pulse ± chase experiments, nonradioactive methionine (2 mg/ml) was then added, and samples precipitated in cold TCA (equal volume of 10% solution) at the indicated times. Precipitated proteins were resuspended in 20 ml of 2% SDS and heated to 100 C. To immunoprecipitate the proteins, lysates were diluted 50-fold into buffer containing the appropriate antibody, as described (Davis et al., 1985). In the coimmunoprecipitation experiments, cells were labeled for 240 seconds, subjected to fractionation as described by Russel & Kazmierczak (1993), and the inner membrane proteins immunoprecipitated with the appropriate antibodies. The immunoprecipitations were performed using the Staphylococcus aureus Protein A (Staph A) beads (Protein A ± Sepharose, CL-4B, Pharmacia), as described by Davis et al. (1985). Bound proteins were released by boiling in SDS, and then separated by PAGE-SDS electrophoresis. Labeled proteins were detected by a phosphoimager (Molecular Dynamics). The signal from the pVI band in the pulse ± chase experiment was quanti®ed using the software packages ImageQuant (Molecular Dynamics) and Excel. Anti-pIII Ab used in immunoprecipitations (Gailus et al., 1994) was a gift from M. Erdmann. Preparation of cells for electron microscopy and mechanical shearing Infected or uninfected cells were chilled on ice, pelleted by centrifugation and resuspended in fresh chilled medium. Samples were then divided, and one aliquot was exposed to mechanical shearing in a Sorvall Omnimixer (one minute at 10 and six minutes at 5). Samples were prepared for electron microscopy by a modi®cation of the procedure of Lopez & Webster (1983). Brie¯y, cells were ®xed by adding 1/4 volume of 0.5 M glutaraldehyde in 0.05 M sodium phosphate (pH 7.5), 750 M NaCl (5  concentrated PBS) for 20 minutes, and then treated with NaBH4 at a ®nal concentration of 25 mM. Bacteria were then washed twice in PBS by centrifugation (except when indicated). The immuno-gold labeling experiment (Figure 7) was carried out according to Lopez & Webster (1983). Brie¯y, the ®xed cells were ®rst incubated with anti-f1 (or fd; Sigma) or a non-speci®c rabbit IgG (30 mg per 0.25 ml cells) in PBS 7.5 with 2 mg/ml BSA at RT for 30 minutes. Ab was removed by centrifugation, and cells were resuspended in PBS 7.5 with 2 mg/ml BSA. Goldconjugated anti-rabbit IgG Ab (10 nm particles; Sigma) was added at dilution 1:10 and incubated at RT for 30 minutes. Cells were washed twice in PBS by centrifugation. Throughout the procedure, cells were handled carefully and pipetted as little as possible to prevent

40 breakage of the long, cell-associated, phage. Samples were adsorbed onto glow-discharged carbon-coated grids, negatively stained with uranyl acetate and examined on a JOEL 100 CX electron microscope.

Acknowledgments We thank Marjorie Russel, Jian-nong Feng and Simon Dae¯er for critical reading of the manuscript, J.-n. Feng for plasmid pJF4, Robert Webster for plasmid pJIH1, Michael Tesar for the mAb 1F8 and Mark Erdmann for anti-pIII Ab. We specially thank Eleana Sphicas for her excellent work on the electron microscopy of phage and infected cells. This work was supported by NSF grant MCB 93-16625.

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Edited by M. Gottesman (Received 20 March 1998; received in revised form 28 May 1998; accepted 18 June 1998)