a novel p22 prophage in salmonella typhimurium · copyright 0 1987 by the genetics society of...

Copyright 0 1987 by the Genetics Society of America

A Novel P22 Prophage in Salmonella typhimurium

Diana M. Downs and John R. Roth Department of Biology, University of Utah, Salt Lake City, Utah 84112

Manuscript received July 20, 1987 Accepted August 3, 1987

ABSTRACT Under several sets of conditions, all of which seem to perturb purine metabolism, Salmonella

typhimurium releases a variety of phages which were not known to be present in the strain. These cryptic phages are not induced by UV irradiation. Furthermore, the induction process does not require a functional recA gene product. While phages of several phenotypic classes have been recovered, including both turbid and clear plaque formers, all appear to be variants of P22 because all show DNA restriction patterns indistinguishable from that of P22. The variety of types suggests that the cryptic prophage is mutagenized as a consequence of the induction process. All the temperate phages tested are capable of transducing a variety of chromosomal markers with high efficiency. The phages induced in this novel way are capable of forming plaques on the strains that gave rise to them. Since the strains releasing phage are not immune to P22, the parental lysogens must not express immunity and the phage must be held in a cryptic state by a novel mechanism. The released phage possess an intact P22 immunity system because many can form standard immune lysogens after reinfection of Salmonella. These results raise the possibility that Salmonella typhimurium harbors cryptic phages that are subject to a novel system of global control related to purine metabolism. Preliminary evidence suggests that the regulation system may involve DNA modification.

OST temperate bacteriophages are able to in- M tegrate into the bacterial chromosome as prophages and express a repressor that renders the lysogenic cell immune to superinfection. Bacteria carrying such functional phage genomes are identified by occasional release of phage particles which can be detected by plaque assay on a sensitive host. If a nonly- sogenic host is not available, it can be difficult to determine whether a particular bacterial strain is lysogenic because released phage would not be expected to plaque on the immune lysogenic strain (LWOFF 1953). Detecting a defective prophage is even more difficult since no plaque-forming virus particles are made. Many commonly used bacterial strains are known to carry cryptic prophages (BERTANI 1951; BOYD and BIDWELL 1957, 1959; COHEN 1959; RED- FIELD and CAMPBELL 1984; WEIGLE and DELBRUCK 1951; ZINDER 1958).

Ultraviolet light and other DNA damaging agents that induce the SOS response can serve to induce some functional prophages. Many phage repressors are sensitive to proteolysis by RecA in its activated form (IRBE, MORIN and OISHI 1981; ROBERTS and ROBERTS 1975; ROBERTS, ROBERTS and CRAIG 1978). Cleavage of the repressor induces the lytic growth cycle of the phage, with concomitant lysis of the host (ROBERTS and ROBERTS 1975). Even when viable phage or virus particles cannot be detected, lysis of a strain following SOS induction suggests the presence of an inducible prophage. Some of the well-character-

Genetics 117: 367-380 (November, 1987)

ized UV-inducible phages are A, P1, P22, 980, 434, 186 and 21 (LEVINE 1961; MOUNT 1977; OISHI and SMITH 1978; WOODS and EGAN 1974). These phage are also induced by a number of other treatments such as nalidixic acid, mitomycin C and amino acid starvation (IRBE, MORIN and OISHI 1981; LEVINE 1961; MELECHEN and Go 1980). Because of their dependence on a functional RecA protein, all of these treatments are thought to act through induction of the SOS system (WALKER 1985). In several cases, the repressor proteins of these phage have been shown to be subject to RecA-dependent cleavage (PHIZICKY and ROBERTS 1980; ROBERTS, ROBERTS and CRAIG 1978). There remain a number of temperate phage which are not inducible by UV. These phage, which include P2 (BERTAINI 1968) and Mu (HOWE 1973), probably have repressors which are not sensitive to the recA function. These phage are not induced by any of the treatments which induce the UV-inducible phage. No other global regulation system has been found to induce phage that are not UV-inducible. It is possible, however, that these phage are subject to induction by yet undiscovered global regulatory systems.

There have been scattered reports in the literature of novel phage or variants arising in cells after viral infection (BENZINGER 1962; COHEN 1959; FREIDMAN et al. 1981; NORTHROP 1965; YOUNG, HARTMAN and MOUDREANAKIS 1966). These variants cannot generally be explained by simple phage mutation. Often the variants differ significantly from the original infecting

368 D. M. Downs and J. R. Roth

phage in morphology, immunity and host range. In several cases, the immunity acquired by the incoming phage was not being expressed by the host cell (FREID- MAN et al. 1981; REDFIELD and CAMPBELL 1984). These novel phage have generally been attributed to recombination between the known phage and cryptic, possibly defective, prophages in the host chromosome. In none of the reported cases was the novel phage released without prior infection by a functional phage. In addition there are a few reports that phage growth in a particular strain alters the phage’s host range (BERTANI and WEIGLE 1953; LURIA and HUMAN 1952). This phenomenon may reflect the existence of defective phage elements in the chromosome capable of recombining with incoming phages. The dependence of these phenomena on recA was not tested.

There are several reports of an autoplaquing phenomenon occurring in a variety of organisms (BREYEN and DWORKIN 1984; CAMPBELL et a l . 1985). In all cases this phenomenon appears to be due to something other than virus infection: the “plaques” could not be propagated as a lysate. In these instances, it has been suggested that the apparent plaques were caused by a toxic substance released by some cells causing autolysis of neighboring cells.

The Salmonella LT2 strain generally used for bacterial genetics is known to carry several functional prophages (Felsl , Fels2) (BOYD and BIDWELL 1959; ZINDER 1958). Phage P22 can recombine rarely with these prophages to generate hybrid phage with different morphology (P221) (YAMAMOTO 1964). Strain LT2 has been cured of one of these prophages to generate the strains that are standardly used for genetic analysis of phage P22 (e.g., DB21, DB9071) (BOTSTEIN, CHAN and WADDELL 1972). These cured strains carry no UV-inducible prophage and do not lyse following UV irradiation. Moreover, P22 does not appear able to rescue any markers from these phages (BOTSTEIN, CHAN and WADDELL 1972). A natural isolate of S. tjphimurium, Ql, is thought to be free of cryptic prophages able to grow on S. typhimurium strains (BOYD and BIDWELL 1959).

We report here conditions which cause induction of cryptic P22 phage from all of the above S. typhimurium strains, none of which were thought to be lysogenic for P22. The induction conditions appear to require interference with purine metabolism. The cryptic phage sequences appear to be held inactive by some sort of DNA modification.

MATERIALS AND METHODS

Bacterial strains: All strains used in this study are listed with their sources in Table 1. Strains carrying the musA mutation, which confers sensitivity to phage Mu, were constructed using mutants obtained through the Salmonella Genetic Stock Center. The origin of the mus mutation is described by FAELEN et al. (1 98 1).

TABLE 1

Strain list

Strain Genotype Source

LT2 DB9071 Q1 TR6770 TR6924 TT10885 TTI 0886 TT10882 TT10883 TT521 TT287 TT289 TTll TT273 T T 3 17 T T 3 15 TR6583 TR6761 TR6762 TR6763 TR6764 TR6765 TR6766 TR6767 TR6768 TR6769 TR2311 TR2228 TR52 1 TT10919 TT10920 TT10921 TT10922 TTI 0923 TT10924 TT10925 TT10926 TT8375 TR6771 T T l l l O 6

T T l l l O 7 TR6912 TR6913 TR6908 TR6909 TR6910 TR6911 TR6915 TR6773

Wild-type Wild-type (derived from LT2) Wild-type purF145 pur+ (isogenic to TR6770) purF145 srl-202::TnlO recAl purF145 srl-202::Tn 10 purF2058 purF2058 zeh-1841::TnlO srl-202: :Tn 10 recAl purC882::TnlO purE884: :Tn 10 pur11757::TnlO purA874::Tn 10 purFl74 1: :Tn IO purGl739::TnlO ara9 metE205 ilv589 pyrB85 pyrF146 hisF440 his640 leu447 trp 130 trpDl8 argD 2 1 pyrB64 (P22 saeA27) pyrDl3 hut+ galE542 hisC527,, cysA I348 supD5Ol (Ql) srl-202:TnlO recAl (Ql) srl-202::TnlO (LTZ) srl-202::TnlO recAl (LT2) srl-202:TnIO (DB907 1) srl-202: :TnlO recAl (DB9071) srl-202::TnlO

Lab collection D. BOTSTEIN B. STOCKER Lab collection This work This work This work This work This work Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection Lab collection This work This work This work This work This work This work

(DB9071) purF2058 zeh-1841::TnlO This work (Ql) purF2058 zeh-1841::TnlO This work musAl Lab collection LT2 (P22 sieA44 sieBl m44) This work pyrB64 srl-202::TnlO recAl This work

pyrB64 srl-202::TnIO (P22 sieA27) This work leu485 Lab collection his203 Lab collection DB907 1 (DD37) This work

This work DB9071 (DD36) DB9071 (DD38A) This work DB907 1 (DD3 1) This work purDH343 J. GOTS leu1051 cysC1175 malB479 Lab collection

(P22 sieA27)

Arphs-rfii - _ -

Media and biochemicals: The E medium of VOCEL and BONNER (1956), supplemented with 0.2% glucose, was used as minimal medium. Alternative carbon sources were added at 0.2% to E medium lacking citrate (RATZKIN and ROTH 19%). Difco nutrient broth (8 g/liter), with NaCl (5 g/liter) added, was used as rich medium. Difco Bactoagar was added to a final concentration of 1.5% for solid medium. The following additives were included in media as needed (final concentrations given): tetracycline (1 5 pg/ml in rich media), thiamine (0.05 miw). Purine concentrations are described in

Novel P22 Prophage 369

the text. Mitomycin C (Sigma Chemical Co.) was stored frozen as a 1000 pg/ml stock solution and added to desired concentration. Lambda top agar (DAVIS, BOTSTEIN and ROTH 1980) was used for phage purification and assay. Reagents were purchased from the following commercial sources: diethyl sulfoxide (DES), Eastman Kodak Co.; N- methyl-N'-nitro-N-nitroso-guanidine (NG), Aldrich Chemi- cal Co.; agarose, FMC Corp.; nitrocellulose filter paper, Schleicher & Schuell; proteinase K, Boeinger Mannheim. All other chemicals were purchased from Sigma. [CY-~~P]- dCTP (2000 Ci/mmol) was purchased from New England Nuclear Corp.

Transductional methods: The high frequency generalized transducing bacteriophage P22 mutant (HT 105/1, int201) (SCHMIEGER 1972) was used for all transductional crosses. Recipient cells (1 0') and transducing phage [ 10' to 1 O9 plaque-forming units (pfu)] were spread directly on selective plates. Transductants were purified by streaking on nonselective green indicator plates and putative phage- free clones were identified by their light colonies (CHAN et al. 1972). Possible phage-free colonies were checked for phage sensitivity by cross-streaking with phage P22.

Construction of strains carrying the recA mutation: The recA mutation was introduced into strains using its 50% cotransductional linkage to srl (SANDERSON and ROTH 1983). The strain into which a recA mutation was to be introduced was transduced to tetracycline resistance (Tet') with a P22 lysate grown on a srl::TnlO recAl double mutant (TT521). Greater than 50% of the Tet' transductants from this cross are sensitive to ultraviolet radiation, indicating that they have inherited the donor's recAl allele. The recAl allele used is probably a missense mutation. This is based on the fact that this allele does not detectably alter the mobility of the RecA protein on an SDS-polyacrylamide gel. This recAl allele is completely recombination-deficient and shows no UV induction of P22 or of a umuC::lac fusion (WING, LEVINE and SMITH 1968; D. M. DOWNS and J. R. ROTH, unpublished results).

Construction of strains containing a purF deletion: The purF2058 allele used was obtained as a TnlO-generated deletion starting with a strain carrying a TnlO in the purF locus. The deletion fails to recombine with 9 of 10 purF point mutations tested; it can be transduced to prototrophy by P22 grown on a wild-type donor strain. A TnlO insertion near the purF2058 deletion was obtained as previously described (KLECKNER, ROTH and BOTSTEIN 1977) and a double mutant carrying both purF2058 and the linked TnlO was constructed (TT10883). P22 lysates grown on TT10883 were used to transduce a recipient strain to Tet'. Of the Tet' transductants, more than 95% coinherit the purF deletion mutation.

Mitomycin C treatment: Cells were grown in nutrient broth, with shaking, at 37" to a density of about 10' cells/ ml. Mitomycin C was added to the appropriate concentration (see RESULTS) and incubation was continued for 10 min. Cultures were diluted 10-fold into fresh medium and incubated for up to 4 hr. Cells were harvested and the supernatants were tested for phage.

Phage appearance during bacterial nutritional tests Overnight cultures grown in nutrient broth were centrifuged 15 min at 3500 rpm to pellet the cells. The supernatant was discarded and the pellet resuspended in an equal volume of 0.85% w/v NaCl. The resuspended cells were pelleted a second time and again resuspended in 0.85% w/ v NaCI. A 0.1-ml aliquot of the washed cell suspension was then spread on minimal glucose plates supplemented with thiamine. A crystal of adenine, adenosine, or alternative

supplement was placed near one edge of the plate. Plates were incubated 1-2 days at 37".

Liquid assay of phage induction: Overnight cultures were grown in minimal glucose medium with the appropriate supplements. These cultures were washed twice as described in the previous section. The cells were then pelleted a third time and resuspended in fresh medium of the same composition as that in which they were to be grown. The resulting suspension was diluted 30-fold into the same medium and grown with shaking at 37".

At various times turbidity was measured and a sample was taken. The samples were centrifuged (15 min at 3500 rpm) to pellet the cells. The supernatant was removed and sterilized by vortex mixing with chloroform. (All batches of chloroform were free of phage contamination.) The sterile phage suspension was assayed for plaque formation using the respective parent strain as indicator host.

Filter washing: Overnight cultures (2 ml) were deposited on sterile Nalgene filter units (0.45 pm pore size). The cells were then washed by passing 100 ml E medium containing 0.2% glucose over them. Cells were resuspended on the filter (with no suction), in 2 mi sterile NaCl (0.85% w/v). These resuspended cells were used to inoculate growth media (minimal media containing 0.1 % adenosine). The cells were grown to full density and the supernatant assayed for phage accumulation.

Anaerobic growth of Salmonella: All manipulations of anaerobic cultures were performed inside an anaerobic chamber (Forma Scientific). Anaerobic growth of cells was achieved by allowing culture media to become low in dis- solved oxygen by overnight exposure to a low oxygen at- mosphere inside an anaerobic chamber. Large volumes of anaerobic cultures were obtained by inoculating medium in 125-ml culture flasks fitted with rubber stoppers to maintain anaerobic conditions.

Mutagenesis: DES mutagenesis was performed as follows: 0.1 ml of an overnight culture was incubated in E medium saturated with DES for 30 min at 37". Cultures were started from a 0.1-ml inoculum from these tubes. The cultures were grown to full density. Mutagenesis by NG was performed as follows. An 0.1-ml aliquot of an overnight culture was spread on a nutrient broth plate. A crystal of NG was placed in the center of the plate. Plates were incubated 1 day at 37". The number of plaques visible on the lawn were observed.

Purification of released phage: Plaques were picked from plates containing washed cells and streaked on a layer of top agar containing a lawn of strain DB907 1. Individual plaques from these streaks were picked and added to a log- phase (10' cells/ml) nutrient-broth culture of strain DB9071. The cells and phage were incubated for 8-14 hr at 37" with shaking. The lysate was then centrifuged to pellet the cells. Chloroform was added to the supernatant and the tube vortexed to kill any remaining cells. These lysates were titered on DB907 1.

Testing phage growth and ability to transduce: A 0.1- ml aliquot of high titer lysates of the appropriate phages were spotted on a top-agar lawn of the desired cells. The spots were streaked out so that single plaques could be seen if phage growth was significant. Growth was scored as positive if clearing and/or individual plaques were visible. Phage lysates grown on DB9071 were used to test transduction ability of the phage. The lysates were combined with leu485 or his203 recipient strains on separate minimal glucose plates. The frequency of prototrophic transductants was scored.

Lysogen isolation: High-titer (1 0') lysates of the temperature phage were spotted on a top-agar lawn of strain

370 D. M . Downs and J. R. Roth

DB907 1. Cells from the center of the resulting cleared area were streaked for single colonies on green indicator plates (CHAN et al. 1972). Light colored colonies from each donor were tested for sensitivity to their parent phage. Putative lysogens (those resistant to their parent phage) were grown overnight in nutrient broth to test for release of phage. These cultures were centrifuged to pellet the cells. The supernatants were then chloroformed. Equal volumes (0.1 ml) of the supernatant and LT2 were combined in top agar and spread on nutrient plates. Plaques were scored. Those cells meeting the above criteria ( L e . , release of phage able to plaque on LT2 and resistance to the phage they release) were concluded to be lysogenic.

Phage DNA isolation: Phage DNA was isolated by lysing a concentrated phage suspension with 0.5% SDS and 50 pg/ ml proteinase K at 65" for 15 min followed by several phenol extractions. NaCl was added to a final concentration of 0.4 M in the aqueous phase. T w o volumes of cold ethanol were added to precipitate the DNA. The DNA was pelleted and resuspended in TE (1 0 mM Tris, 1 mM EDTA, pH 7.5) buffer.

Chromosomal DNA isolation: Overnight cultures (30 ml) were pelleted, resuspended in 5 ml SET buffer (20% sucrose, 10 mM Tris-HCI, 1 mM EDTA), pelleted and finally resuspended in 5 ml SET buffer. Lysozyme was added to a final concentration of 75 pg/ml and the cell suspensions sat at room temperature for 15 min. SDS was added to a final concentration of 0.1 % and the suspension was incubated at 65 O for 20 min. The mass of lysed cells was pushed through an 18-gauge needle. Proteinase K was added to a final concentration of 50 pg/ml and the suspension was incubated 30 min at 37". The mass was then phenol-extracted repeatedly, adding more buffer to reextract the interface if nec- essary. DNA in the final aqueous phase was ethanol precipitated by adding NaCl to a final concentration of 0.4 M and 2 volumes of cold ethanol. DNA was pelleted by centrifugation. DNA was then resuspended in 2 ml T E buffer (1 0 mM Tris-HCI, 1 mM EDTA) and RNase was added to a final concentration of 100 pg/ml. The suspension was incubated at 65" for 30 min, or until the DNA was resuspended. The resulting solution was ethanol precipitated. The DNA pellet was resuspended in a small volume (500 pl) of T E buffer.

Agarose gel electrophoresis: Restriction endonuclease digests were electrophoresed in 0.8% agarose submerged gels. Gels were cast and electrophoresed in 40 mM Tris- acetate 1 mM EDTA buffer (pH 8.0). Electrophoresis was at 4.4 V/cm for 5 hr. Southern blot hybridization was preformed as described by MANIATIS, FRITSCH and SAM- BROOK (1982).

RESULTS

Initial Observations The first evidence of phage induction was encoun-

tered during routine characterization of a PurF mutation. It was observed that plaques frequently appeared on the lawn of a purF mutant if the cells had been washed before spreading on a minimal glucose plate containing thiamine. The purF gene encodes the first enzyme of purine biosynthesis. Mutants in purF require adenine and thiamine for growth, because thiamine is derived in part from an intermediate in purine biosynthesis (NEWELL and TUCKER 1968). Cells of a purF mutant (purF145) were washed and then plated on minimal medium containing thiamine and

a crystal of adenine was added to the plate to permit growth. When this is done, a lawn of cells grows up around the adenine crystal. Phage plaques appear in this lawn. Often the plaques appear most densely at the outer border of the growth, suggesting that phage release depends on purine starvation. Figure 1 shows two typical patterns of plaque formation. In Figure 1 A the plaques are primarily at the border of the zone of growth. Figure 1B shows another pattern in which plaque formation is not limited to the cells which would be starving for purine; however, these cells were subject to starvation immediately after plating, before purine had diffused. Although it is difficult to see in the photograph, the plaques become more numerous and closely packed as cell growth decreases. A few plaques are occasionally observed when washed cells of purF145 are spread on plates containing a standard concentration (5 mM) of adenine. The autoplaquing phenomenon does not occur every time washed cells are plated. The reason for this inconsist- ency is not clear.

Thus, by starving washed cells of a purine auxotroph, we occasionally cause these cells to release phage. Washing is critical for this phage release. The striking thing about the phage which are released is that they form plaques on the strains from which they came. We never observe this phenomenon with un- treated cultures of LT2. When the known prophages of LT2 are induced by UV irradiation, the released phage do not form plaques on LT2. We realize that contamination is a prime suspect in accounting for these observations. We believe we have checked the sterility of every component of our media and equip- ment. Below are several additional lines of evidence that we feel argue against contamination.

Phage release by other strains of S. typhimurium: Wild-type S. typhimurium strain LT2 is known to carry several cryptic prophages inducible by UV irradiation (BOYD and BIDWELL 1959; ZINDER 1958), but these phages do not plaque on LT2. This observation suggested the possibility that the known phage might be released under particular growth conditions and either undergo mutation or recombination with cryptic prophages to generate new phages capable of forming the observed plaques. To test whether or not phage release depends on the phage carried by our L T 2 strain, we tested two S. typhimurium strains known to be free of UV-inducible cryptic phage. These strains are Q1 and DB9071. Strain Q1 is an independent isolate of S. typhimurium that is used as an indicator for Salmonella phage. It appears not to be lysogenic for any known S. typhimurium phage. Strain Q1 contains only cryptic prophage B5d. Phage B5d is weakly UV-inducible and only known to plaque on Salmonella gallinarum (BOYD and BIDWELL 1959). Strain DB9071 is a derivative of LT2 that has been

Novel P22 Prophage 37 1

FrcmE I.-Variability of aut* plaquing. TR6770 (purF145) was grown as described in MATER- AND METHODS. An 0.1-ml aliquot of the washed culture was added to 4 ml of 0.7% agar and poured on a minimal glucose plate containing thiamine. A sterile filter disk was placed in the center of the plate with 10 pl of a 2% solution of adenine. The white zone around the center disk indicates growth of the cells. A and B are separate cultures put through identical procedures.

cured of all known UV inducible cryptic prophages and also lacks the cryptic plasmid carried by LT2 (BOTSTEIN, CHAN and WADDEU 1972; D. BOTSTFCIN personal communication). Strain DB9071 sponta- neously releases a phage which will plaque on Ql; release of this phage does not depend on a functional RecA protein, and the phage is not UV-inducible (see Table 5). A purF deletion has been introduced into each of these wild-type strains. For each derivative, phage plaques'appear following washing and starvation.

Phage induction is reCA independent: To test whether induction requires recA function, a strain containing purFZ45 and recAZ (TT10885) was constructed by transduction. Figure 2 shows that the absence of a functional recA gene product does not prevent phage induction. Figures 2A and 2C show unwashed cultures of strains TT10886 (recA+) and TTlO885 (recAZ), respectively, supplemented with adenine on the disk in the center of the agar plate. Figures 2B and 2D show washed cultures of the same strains plated under identical conditions. Phage was released by washed cells regardless of whether or not a functional RecA protein was present. Neither strain released any phage without prior washing of the CUI- tures.

The recAZ allele is known to prevent induction of phage P22 (WING 1968). We have confirmed this observation by treating isogenic recA+ (TT 1 1 107) and recAZ (TTl 1 106) P22 lysogens with Mitomycin C. As shown in Table 2, we saw induction of P22 only in the rec+ strain.

Purine mutants that show phage induction: Since the initial observation of autoplaquing was made during experiments involving a purine auxotroph (PurF),

we explored the possible relationship between purine metabolism and phage induction. Mutants blocked at specific steps of the purine pathway were tested for their ability to release phage. The results in Table 3 show that phage release was not limited to purF mutants, but occurs when cells of any adenine-requiring mutant were washed and plated in this way. Washing the cells in solutions of thiamine, adenine, histidine, guanosine+adenosine+inosine, or nutrient broth did not prevent the release of phage. It should be noted that guanine-requiring auxotrophs repeatedly failed to show phage release under these conditions. As shown in Table 3, repeated tests gave no evidence of phage induction for auxotrophs other than adenine- requiring mutants. It should be stressed that, because this procedure remains intrinsically variable, a negative result should be interpreted with caution. Exper- iments giving such negative results were repeated several times.

Exogenous purines can cause phage rel- In an effort to cause phage release without blocking the purine pathway, we washed cultures of wild-type Sal- monella (strain LT2) and grew them in a number of purine-supplemented minimal media. We noticed that some cultures of LT2, when washed and transferred to liquid E glucose medium with adenosine, appeared to be lysing. The cell density increased for a short time after transfer, then dropped before slowly increasing to full density. Cells removed from these cultures after full growth were often resistant to P22 clear-plaque mutants, suggesting that the second stage of growth was due to phage-resistant mutants or a few cells surviving after lysis of the culture. Cultures of LT2, when washed and grown in E glucose plus adenosine, frequently accumulate between 1 O6 and


FIGURE 2.-Autoplaquing on rccA. Washed cells were prepared as described in MATERIALS AND METH- ODS. Unwashed cells to be plated were taken from a growing nutrient broth culture. Both washed and unwashed cultures were plated in the same way, 0. I ml of the culture being added to 4 ml of 0.7% agar and spread on a minimal glucose plate containing thiamine. A sterile filter disk was placed in the middle of the plate with 10 pl of a 2% solution of adenine. The white zone around the disk indicates growth of the cells. (A) TT10886 (rccA+ purFI45)-unwashed. (B) TT10886 (rccA+ pur- FI45bwashed. (C) TT10885 (rccAI purFI45)-unwashed. (D) TT10886 (rccA I purF145)washed.

TABLE 2

Prevention of P22 induction by redl parent strain. Adenosine was the purine source which caused phage release most frequently in all strains (20-80% of identically treated cultures). Guanine, guanosine, adenine, inosine, hypoxanthine, and xanthine all failed to cause significant phage release in

TT11107 rccA* (P22) None 7 x 1 0 4 any of the strains tested. Guanosine, inosine, adenosine and adenine were used at a concentration of TTI 1106 rccAl (P22) None 1 x 10'

TTl1107 rccA+ (P22) 4 pg/ml 5 x 108 TT11106 rccAl (P22) 4 &ml 1 x 105 0.05% w/v, hypoxanthine and xanthine at 0.02% w/ TT11107 rccA+ (P22) 8 rg/ml 7 x IO8 v and guanine at 0.01% w/v due to its low solubility. T T I 1106 rccAl (P22) 8 r g h l 7 x IO5 With purines other than adenosine, the success rate

(tubes containing phage/tubeS treated) was less than 5% and, whenever phage was released, lysates showed titers less than lo3 pfu/ml. In all instances, however, phage release from wild-type cells depended on washing the cells prior to growth and on the presence of some purine source in the growth media.

In the case of purF mutants, it is possible to induce phage without cell washing. As discussed earlier, starvation of washed PurF mutants causes phage indue- tion* In addition, growing PurF mutantS in a great excess of purines (0.1% guanosine + 0.1 % adenosine + O s 1 % inosine) occasionally causes Phage release without Prior washing. In this case, Phage induction does not depend on the most contamination-prone aspect of the treatment described above.

Phage release by phage resistant strain: In an effort to further show that we were detecting phage released from within cells, we used a phage-resistant mutant in the induction procedure. A mutation in rfb makes cells resistant to phage infection by most phages, including P22, due to the loss of the 0 antigen which acts as a phage receptor. A strain containing an

Mitomycin C Strain Genotype concentration Phage/ml

cells were grown in nutrient broth to a density of M. io9 cells/ ml and mitomycin C was added to the indicated concentration. Incubation was continued for up to 4 hr and cultures were assayed for phage titer.

10'' phage/ml by the time they reach a density of 2 X lo9 cells/ml. The phage were assayed as plaque- forming units on LT2 host cells. Figure 3 shows a typical growth and phage release patte,.,, for LT2 grown in adenosine with and without prior washing. The same results were obtained for the other two strains of S. t)lphjmurium (Table 4). It should be noted that phage accumulation in these cultures could rep resent increasing phage release or it could reflect the multiplication of a small number of phage released early in the culture.

Table 4 shows the number of phage released after a typical successful adenosine treatment for the three wild-type strains and their recAl derivatives. The three previously described wild-type strains of S. typhimurium were tested for phage release after washing and outgrowth in different purine sources. Phage titer was assayed by plaque formation on the respective


TABLE 3

Phage release by starvation of auxotrophic strains

Phage Strain Genotype Supplement released

TT287 purC882:TnlO Adenine TT289 purE884::TnlO Adenine TT11" purll757::TnlO Adenine TT273 purA874::TnlO Adenine T T 3 17" purFl741 ::TnlO Adenine TT315" purG1739::TnlO Adenine TR69 15" purDH343 Adenine TR6770" purF145 Adenine TT 10882" purF2058 Adenine TT10885" purF145 srl-202:TnlO Adenine

TT10886" purF145 srl-202::TnlO Adenine TT275 guaB554::TnlO Guanosine TT278 pA554::TnlO Guanosine TR6761 ilv589 Isoleucine and

TR6762 gyrB85 Uracil TR6763 pyrF146 Uracil TR6583 ara9 metE205 Methionine TR6764 hisF440 Histidine TR6765 hisEl640 Histidine TR6766 leu447 Leucine TR6767 trpl30 Tryptophan TR6768 trPD18 Tryptophan TR6769 arpD21 Arginine

recA 1

valine

- - Cells were grown overnight in nutrient broth, washed as de-

scribed in Materials and Methods, and plated on minimal glucose medium. Crystals of the indicated supplement were put on one side of the plate. Adenosine crystals gave the same results as adenine. Phage release was scored as positive if any plaques were visible on the lawn of cells.

Cells plated on minimal glucose plates supplemented with thiamine (0.05 mM).

r - mutation (e.g., TR67'73) releases phage when treated by washing and adenosine outgrowth. (This strain is described in detail in a subsequent section.) The titer of these lysates is between 10' and lo4 pfu/ ml. The released phage cannot grow on this strain and thus must be assayed on a phage-sensitive strain (e.g., LT2). This result supports the idea that phage release is from within the cells, because contaminating phage would be unable to infect these strains. If contamination is occurring, it must be with significant titers of phage. The lower titers seen with such phage- resistant strains are expected, because released phage cannot propagate in these cultures. In addition, the phage released from these cultures form smaller plaques than those released in phage-sensitive cultures (see DISCUSSION).

Phage release by LT2 in liquid medium is independent of the SOS system: As seen for auxotrophs starved on plates, adenosine-induced release of phage in liquid cultures of LT2 is independent of recA (Table 4). All three wild-type strains released phage at a similar frequency, regardless of the presence or absence of the recA1 mutation. These results indicate

"I

- TIME (MINUTES)

FIGURE 3.-Phage induction by adenosine in washed and unwashed cultures. Overnight cultures of LT2 were treated in one of two ways: (U) cells were diluted 30-fold into fresh minimal medium containing 0.1 % adenosine, or Q cells were washed as described in MATERIALS AND METHODS and then diluted 30-fold into fresh minimal medium containing 0.1 % adenosine. Dilution was made at time equals zero. Numbers to the right of the washed-cell curve 0 represent pfu/ml found at that time. Phage were titered on LT2. No pfu were found at any time in the unwashed (U) culture. Cell density was monitored with a Klett-Summerson photoelectric col- orimeter.

TABLE 4

Phage release in teeAI strains after growth in adenosine

Phage released (pfu/ml)

Wild-type Relevant Strain background genotype Unwashed Washed

TT10922 LT2 recA+ 0 2 x 10'0 TT10921 LT2 recA 1 0 7 x 109 TT10920 Q1 recA+ 0 1 x 10'0 TT10919 Q1 recA 1 0 3 x 10'0 TT10924 DB9071 recA+ 0 4 x 109 TT10923 DB9071 recAl 0 3 x 109

Cells were grown in minimal glucose medium with 0.05% adenosine, washed as described in MATERIALS AND METHODS and inoculated into fresh medium. Newly inoculated cultures were grown to a density of 2 X lo9 cells/ml and assayed for plaque forming units on the corresponding parent strain. The plaques exhibited a variety of phage morphologies.

that the induction of these cryptic prophages is not mediated through the SOS system.

T o further test this conclusion, we tried to induce these phage by treatment with mitomycin C; we also used the liquid adenosine shock method to test induction of phages from recA- cells. Table 5 shows the phage induced from wild-type strains after various treatments. No phage able to plaque on their parent strains were released under conditions (mitomycin C) that induce a P22 prophage. It should be stressed that


TABLE 5

Effect of various treatments on phage release

Treatment"

Mitomycin c" Adenosine"

Q1 DB9071 LT2 Q1 DB9071 LT2

NT NT + + + N T S S NT NT S N T s' S + + +

+ + + + + + + + + + + + + + +

- - - - - - -

- - - - - S

S - -

Strain Wild-type

background Relevant genotype

Nutrient broth'

Q1 DB9071 LT2

T T l l l 0 7 TTl1106 TT 10922 TT10921 TT10920 TT10919 TT 10924 TT10923

recA+ (P22) recAl (P22) recA +

recAl recA +

recA I recA +

recA I

S indicates spontaneous release of phage (less than lo4 pfu/ml). NT = not tested. + indicates greater than loR pfu/ml. ' Cultures were grown overnight in nutrient broth and assayed for phage accumulation by plating on each of three host strains. ' Cultures were treated with 4 pg/ml mitomycin C as described in MATERIALS AND METHODS.

Cultures were washed and grown in adenosine as described in MATERIALS AND METHODS. Release of phage from LT2 is slightly increased (Xl00) after treatment with mitomycin C.

all of the adenosine-induced phage tested were capable of plaquing on all three wild-type S. typhimurium strains.

Phage is not released by mutagenesis: The possibility was entertained that our "inducing" conditions might be mutagenic and thereby cause alteration of a cryptic prophage. We therefore directly tested whether mutagenesis by standard procedures causes phage release. Mutagenesis by DES and NG does not cause phage release in LT2. Cultures of LT2 were mutagenized as described in MATERIALS AND METH- ODS. More than 10'' cells were mutagenized by DES and no phage were found which plaqued on LT2. More than IO' cells were mutagenized with NG and no plaques were detected. Mutagenesis was successful in both cases as judged by the high frequency (1 0-') of cells which acquired resistance to streptomycin (2 mg/ml). Background reversion to this level of streptomycin resistance is less than IO-'. Conversely, the induction treatment (washing and outgrowth in adenosine) is not itself mutagenic. This was concluded from the observation that no streptomycin-resistant mutants were found in three independently treated cultures (6 X IO' cells), all of which released phage. It should be noted that, if our inducing treatment were causing only frameshift mutations, we would not have detected them by scoring streptomycin resistance.

Success rate in cryptic prophage induction: The method described above for causing phage release in liquid medium is variable; frequently, only a fraction of cultures treated in parallel will produce phage. The success rate (tubes releasing phage/tubes treated) var- ies between 20 and 80%.

In an effort to allow more precise reproduction of the phenomenon, filtration was substituted for washing by centrifugation. Described in detail under MA- TERIALS AND METHODS, this modification involves col-

lecting 2 ml of an overnight culture on a sterile filter and washing with 100 ml of sterile media. These washed cells were then used to inoculate E medium containing adenosine. This modified procedure re- sulted in about the same success rate (10-60%) for most strains. However, because this modification results in a more easily controlled procedure, we used it in most of the experiments below in which parameters of the procedure were varied.

Extensive efforts to determine the cause(s) of the variability of the success rate by independently alter- ing various parameters of the protocol have been unsuccessful. The following parameters were altered to determine their effect on phage release. Cultures were grown and washed anaerobically. Cultures in various growth phases, from mid-log phase to cultures that had been in stationary phase for up to 40 hr, were washed. Inocula ranging from 10' to 4 x IO' cells were used for the outgrowth phase. Temperature of washing was varied. The effect of detergent in the glassware was tested. Washing was conducted in combination with UV irradiation. Incubation of cells (at 37 ") in saline between centrifugations was tested. Metabolism was inhibited with cyanide and/or chlor- amphenicol during the wash. DES mutagenesis was tried in combination with washing. The pH of the wash medium was varied. Washes were done with proteinase K present. Washes were done with hydro- gen peroxide present. Outgrowth was done in medium limiting for phosphate. No correlation was observed between any of these parameters and the frequency of phage release.

Initial characterization of released phages Co-immunity with P22: A series of phage arising

from various strains were purified from independent plaques. Both turbid and clear plaques were chosen. Several properties of these phages are shown in Table

Novel P22 Prophage

TABLE 6

Characterization of released phage

375

~ ~ ~~~ ~~ ~

Wild-type background Parent strain Morphology Growth on Forms stable Capable of

Phage of origin of origin on LT2 nausAl lysogen transduction

P22 c2 + - clr +/- DD1 LT2 TT10885 1 DD15, DD21 LT2 DD27 Q1 TR6770 TT10925 1 ch. DD38A" DB907 1 TT10926 J

+/- NT NT

+ NT N T TT10885

+/- + +

DDBB, DD9 LT2 DD22b LT2

P22 t

DD2, DD3 DD4-7*

DD16, DD18 DD19 DD25, DD30 DD34, DD35 DD39"

DD10-14

LT2 LT2 LT2 LT2 LT2 Q1 DB907 1 DB907 1

TT10885 TR6770 TR6770 TR6770 TT10885 TT10925 TT10926 TT10926

c + - +/-

+ - DDSA LT2 TR6770 t +

+/- + + I t TT 10925 TTl0926 DD36, DD37 DB907 1

DD38B" DB907 1 TT10926

DD3 1 Q1

Growth on various strains was tested as described in MATERIALS AND METHODS. Morphology on LT2; clr = clear plaques; t = centered

a Indicates plaque formation on E. coli K12. plaques. Transducing ability was tested as described in MATERIALS AND METHODS. NT = not tested.

Indicates growth on rbfB strains of S. typhimurium.

6. All of the phage cause only slight clearing on a lawn of an LT2 (P22 sieA sieB) lysogen and show plaques on this host at a frequency of IO-' per phage. This frequency is similar to that at which virulent P22 Vy mutants occur (SUSSKIND and BOTSTEIN 1978). The sieA and sieB genes encode the two main superinfection exclusion systems of P22 (SUSSKIND and BOTSTEIN 1978). These systems act to prevent superinfection of a lysogenic cell and must be removed before the effect of the immunity system of the lysogens can be tested. The above result suggests that the new phage are subject to P22 immunity.

High frequency generalized transducing phages: All the temperate phage were able to mediate general transduction with an efficiency similar to that of P22 HT105. This is significantly higher than for wild-type P22 for most markers.

Growth on P22-resistant Salmonella strains: A galE mutation prevents Salmonella from making the galactose required for the 0 antigen. These mutants can make the 0 antigen if exogenous galactose is provided. All of the new phage will grow on galE strains of S. typhimurium only when both glucose and galactose are present (galactose alone kills these hosts), suggesting that, like P22, they require a complete 0 antigen for adsorption. Strain TR6773 was isolated as a his deletion (J. CASADESUS, personal communica-

tion). It was found to be resistant to P22, and was assumed to be a deletion extending from his into the nearby rfa operon. Based on this criterion, this strain was used as a rfa- strain to test the growth of the new phage on rough strains, on which P22 and most of the new phage cannot grow. Wild-type P22 does not form plaques on this strain (ClO-'I). Phage DD7, DD 12 and DD22, while not capable of growth on this rfa- strain, acquire mutations at frequencies of 1 P , lo-' and lo-', respectively, that allow them to form plaques on this strain. These phage mutants retain the plaque morphology of their parent phage, i.e., clear or turbid.

The musA mutation of Salmonella has been described in detail by FAELEN et al. (1981). It allows phage Mu to infect Salmonella. While P22 and most of the new phage form poor, hazy plaques on S. typhimurium carrying a mus mutation, phage, DD9, DD22, DD8A and DD8B plaque well on these strains.

Growth on P1-lysogenic Salmonella strains: All of the new phage are capable of plaquing on a S. typhimurium strain lysogenic for coliphage P 1. These tests were done in a galE strain of Salmonella in which both P1 and P22 can grow. If glucose alone is present in the medium, the strains are P1-sensitive, if both glucose and galactose are present, the strains are sensitive to P22 (and to the new phage). Such a strain was made


lysogenic for P1 (when grown on glucose alone). All of the new phage plaque on this P 1 lysogen when both galactose and glucose are present.

Lysogen formation: Temperate phage (those forming turbid plaques) were tested for the ability to establish stable lysogens (see MATERIALS AND METH- ODS). Of 24 tested, only four form stable lysogens as determined by our procedures (Table 6). P22 does not grow on these lysogens. This supports the earlier data suggesting that the phage are co-immune with P22, but does not exclude the possibility that growth of P22 is being prevented by superinfection exclusion systems of the new phage. T w o of these lysogens (TR69 1 1, TR6908) are easily transducible using P22 at a low multiplicity of infection (0.3 phage/cell). This suggests that these prophages are not expressing a &A superinfection exclusion system which is known to exclude both P22 and its transducing fragments (SUSSKIND and BOTSTEIN 1978).

The variety of phenotypic phage types which were found after induction suggests that mutations were either selected or induced by the induction treatment. We looked for conditional lethal phage mutations to support this idea. Phage were induced under permis sive conditions, in this case low temperature in an amber suppressor background. Ten independent cultures of TR251 (su,,) were washed and grown in adenosine at 30" (see MATERIALS AND METHODS). Three of the ten cultures produced high-titer lysates. These lysates were screened for temperature-sensitive as well as amber phage mutants. N o amber mutants were found. Temperature-sensitive mutants were found in two of the three lysates. Since the independ- ence of the mutants within a lysate could not be determined, only one mutant from each lysate was considered. Phage DDJ 4 has a significantly reduced burst size at 42" compared to 30" as judged by the pinpoint plaques made at 42" vs. the large clear plaques made at 30". Phage DD,l does not make visible plaques at 42" compared to large plaques at 30". Phage P22 makes plaques of equal size and number at 30" and 42".

Southem blot hybridization analysis Since the phage show extensive similarities with

P22, we compared their DNA to that of P22. The DNAs of phage DD22, DD25, DD3 1 and DD39 were digested with several restriction enzymes and analyzed by agarose gel electrophoresis (data not shown). Al- though these phages are phenotypically distinct (Table 6), the restriction digest patterns of their DNAs are indistinguishable from each other and from wild-type P22. We have tested more than 40 restriction sites. S . CASJENS (personal communication) has tested more than 50 additional restriction sites. All phage bands hybridize to P22 DNA used as probe in a Southern hybridization. This suggests that their phenotypic dif-

2.5-

1.0-

-2dkb 1 @ -1.0

FIGURE 4.--Southem blot hybridi~ations. DNA was isolated from the listed bacteria, digested with EcoRI and analyzed by agarose gel electrophoresis. Lanes: 1 ) A P22 lysogen (TR2311) grown in NB (0.5 rg) LT2 grown in NB (0.5 rg); 3) Q1 grown in NB (0.5 rg); 4) A purF+ transductant out of purF145 (TR6924) grown in NB (0.5 pg); 5) purF145 (TR6770) grown in NB (0.5 rg); 6) TR6770 grown in NB, treated with DNase prior to loading (0.5 rg); 7) TR6770 grown in E medium containing 0.2% glucose, 0.1 % adenosine, 0.1% guanosine, 0.1% inosine (0.6 rg); 8) TR6770 grown as for lane 7 (0.25 pg). The DNA fragments in the agarose gel were denatured and transferred to nitrocellulose paper as described (MANIATIS, FRITSCH and SAMBROOK 1982). "P-labeled DNA of phage DD39 (7 ng. 4 X loR cpm/ml) was used as a hybridizationprobe to DNA bound to the filter. The hybridization solution contained 0.75 M NaCI, 0.75 mM sodium citrate, 10% dextran sulfate, 0.1% sodium pyrophosphate, 0.1% SDS, 0.2% Ficoll. bovine serum albumin, and polyvinylpyrrolidone. After 12 hr at 65O, the filter was washed at 65' in 40 mM NaCI. 3.7 mM sodium citrate. 0.1 % SDS.

ferences are due to subtle genetic changes and the induced phage are in fact P22.

We wanted to physically demonstrate the presence of prophage DNA in the strains from which these phages were released. To do this, we carried out Southern blot hybridization analyses to look for phage homology in the chromosomes of these strains. Phage DD39 was chosen as the probe because it is the most phenotypically different from P22, although its DNA cross-hybridizes with all restriction bands of P22 DNA. Nick-translated DNA of phage DD39 was then used in hybridization analysis with EcoRIdigested chromosomal DNA from various strains. Figure 4 shows the results of these hybridization analyses. Lane 1 shows the strong bands of hybridization in a strain carrying a standard lysogen of P22. Lane 2 and 3 contain DNA from wild-type Salmonella strains LT2 and Q1, respectively. DNA from LT7 (an independent Salmonella wild-type) shows the same pattern (data


not shown). The hybridization signal we see in these strains is very weak and involves a single sharp band. However, the homology in this band is good as judged by the fact that conditions of high stringency (65’, 10 mM NaC1, 0.9 mM sodium citrate, 0.1% SDS) do not prevent the hybridization. Three things are apparent about these results. The mobility of the single band does not correspond to a size sufficient to include an entire standard P22 genome (44 kb). The band does not correspond in mobility to any internal P22 EcoRI fragment. Hybridization is weaker than we would expect if one copy of P22 were present per genome.

Needless to say, these results were contrary to our expectations. Since we are convinced that the induced phage are coming from these strains, we have pursued the possibility that the unexpected behavior of the P22 sequences could be due to some kind of modification of these specific DNA sequences. Such hypo- thetical modification might be responsible for phage encryptification and might also prevent normal DNA digestion and/or hybridization. We thought it was possible that this DNA would only be uncovered during inducing conditions. Therefore, we examined DNA of strain purF145 which releases phage in response to the widest variety of growth conditions and with the least number of manipulations. We isolated DNA from purF145 cells grown under different conditions and checked hybridization to phage DNA. Lane 5 (in Figure 4) contains DNA from strain pur- F145 grown in nutrient broth. The weak band seen for wild-type strains is present but, in addition, a very strong hybridization signal was noticed running as a smear at an apparent size of less than 1 kb. This apparent homology with DNA of low molecular weight is present in purF145 with or without restriction digestion and hybridization is not prevented by highly stringent conditions. Lane 4 contains DNA of a pur+ transductant isogenic with pur145 and grown under the same conditions. Note that this strain does not show the homology with DNA of low molecular weight. It was important to eliminate the possibility that the material of low molecular weight reflects something other than DNA hybridization. Lane 6 shows that the material seen in Lane 5 is absent when the sample is pretreated with DNAse. Lane 7 and 8 illustrate the effect of growth conditions on the avail- ability of the homologous sequences for hybridization. T w o cultures of strain purF145 were grown in medium containing a great excess of purines (0.1 % adenosine, 0.1 % guanosine and 0.1 % inosine) which occasionally causes release of phage. Both cultures were handled in parallel and treated identically. One of the two cultures released phage. DNA from cells of these two cultures was used in hybridization analysis. Lane 7 shows DNA from the culture that did not release any phage. In this case, the material of apparently low

molecular weight is no longer present: only the single weak band, present in all LT2 strains, is seen. Lane 8 shows the hybridization pattern of DNA from the parallel culture of purF145 that released phage. In this case, the material of apparently low molecular weight is absent and several strong bands are apparent. Some of these bands correspond in size to bands seen for the P22 lysogen (Lane 1). However, other bands are new and may be due to end fragments or to packaged genomes present in the culture.

DISCUSSION

The results presented in this paper describe the appearance of previously undetected P22 phage from several standard strains of S. typhimurzum. The striking aspects of this phenomenon are:

1) Phage are induced by perturbing purine metabolism.

2) The induction process is independent of the SOS control system.

3) The phage plaque on the strains from which they emerge. Thus, the original “lysogenic” strains do not exhibit immunity to the released phage or to P22 (with which the released phage are co-immune), suggesting that the cryptic prophage is held inactive by a cis-acting mechanism that does not prevent expression of superinfecting phage genomes. 4) Sequences of the cryptic prophage are detecta-

ble by a standard Southern blot procedure. However, the sequences behave anomalously in that hybridization is weak and fragments characteristic of P22 are not seen.

An obviously mundane alternative to the provoca- tive results outlined above is that we have contami- nated cells and DNA with exogenous P22 material. We believe we have eliminated this possibility for the following reasons.

1) Procedures have been rigorously checked for introduction of contamination, yet the phenomenon can be observed routinely.

2) For wild-type cells, both washing and growth in purine (specifically adenosine) are essential for phage induction. Neither condition alone induces. Thus, neither washing nor purine growth introduces contamination.

3) Strains of PurF mutants will sometimes release phage during growth in high concentrations of purines. This eliminates the manipulation which is most likely to introduce contamination.

4) We see a variety of phage types emerging from these strains. The possibility of contamination occurring simultaneously with several phage types is un- likely.

5 ) Phage are released by phage-resistant strains, indicating that the observed phage could not be due


to a few contaminating phage propagating within the cultures.

6) Although the behavior of the sequences in the Southern hybridizations is abnormal, homology with the released phage is observed. The DNA manipulation methods do not seem to introduce contamination by P22 DNA, because Bacillus subtilus DNA, prepared and treated in parallel, shows no P22 homology.

Essential to our method of phage induction for wild- type cells is extensive washing by either centrifugation or filtration. Outgrowth in the presence of excess adenosine greatly enhances the effect of washing the cells. We have never seen phage release from wild- type strains that have not been extensively washed (less than 1 phage/l0I3 cells). At present, we don’t know what the washing does to the cell. However, washing the cells in nutrient broth and other nutrient- rich solutions (tryptone or peptone broths) still causes phage release; thus, if metabolite pools are being depleted, the critical substance is not one that resting cells can quickly recover from rich media.

Various aspects of adenosine uptake and/or metabolism could be responsible for causing the phage induction after wild-type cells have been washed. It is known that both resting and growing cells take up quantities of adenosine greatly in excess of their growth requirements (HOFFMEYER and NEUHARD 1971; PETERSON and KOCH 1966). The adenosine they take up is deaminated to inosine, much of which later appears extracellularly. Some inosine is con- verted to hypoxanthine which, like inosine, accumu- lates in the medium, but at a slower rate (MANS and KOCH 1960). Therefore, it is likely that growth on adenosine causes large intracellular pools of inosine and hypoxanthine to accumulate. HOULBERG andJEN- SEN (1 983) have shown that hypoxanthine is the purine primarily responsible for repression of pur gene expression. We suspect that the release of phage caused by shocking washed cells with adenosine is due to an imbalance in internal purine pools. If the rate of adenosine uptake is significantly greater than that of hypoxanthine release into the medium, an accumulation of internal hypoxanthine could result in transitory repression of de novo purine synthesis. This repression, at the critical time when the cells are trying to restore the balance of their pools after washing, may trigger events that ultimately result in the release of phage. The fact that external inosine and hypoxanthine do not cause phage release may simply be a consequence of their slower uptake rates.

Induction of phage from strains containing a purF mutation occurs after a variety of treatments. All of these probably alter the purine metabolism of the cells. Purine starvation, washing, and growth in a great excess of purine can all cause phage induction in purF strains. The purF gene encodes the feedback-

sensitive enzyme in de novo purine synthesis and is thought to be a primary regulatory point in the pathway (HENDERSON 1972). It is therefore plausible that a strain deleted for this gene may be unable to prop- erly regulate its internal purine pools. If this is the case, strains carrying a purF deletion might have internal purine pools that are affected by exogenous purine sources. According to this hypothesis, internal purine pools in LT2 (purF+) would be less perturbed by variation in exogenous purine concentrations. Our findings are consistent with this hypothesis.

Purines are known to influence various aspects of the SOS system. E. coli strains carrying the recA440(t$) mutation are sensitive to adenine due to its apparent stimulation of the proteolytic activity of the mutant RecA protein in vivo (TESSMAN and PETERSON 1980). dATP is the most effective nucleotide triphosphate in promoting proteolytic action of the RecA protease in vitro (PHIZICKY and ROBERTS 198 1). These involve- ments of adenine in the well described SOS response leave open the possibility that we are simply looking at another aspect of that system. However, induction of the SOS system, as it is currently understood, requires a functional RecA protein (WALKER 1985). Nucleotide effects of the SOS system are mediated by the RecA protein. The induction of the cryptic prophages we have described seems to be independent of recA function. Induction occurs just as well in strains carrying a recA mutation as in those which are recA+. This result suggests that phage induction caused by shocking washed cells with adenosine is not mediated through the SOS system, at least in the classical sense. Efforts to induce these phages with mitomycin C also were unsuccessful. We conclude that the standard SOS response is not capable of inducing these phages.

The released phage are able to grow on the strains from which they emerged. This suggests that the strains of origin are not immune. Alternatively, the released phage could be mutants that escape immunity. Since the released phage are subject to P22 immunity and some are able to establish and maintain immunity to P22, we think that the released phage have not escaped a standard immunity system. This is supported by the fact that wild-type P22 phage can grow on strains able to release these P22-like phages. It seems that the parent strains must carry P22 in a new sort of lysogenic state which we called archived. The absence of superinfection immunity suggests that an archived prophage is kept inactive by a cis-acting mechanism that does not interfere with expression of a superinfecting phage genome. (A system of heritable DNA modification would provide such cis-acting inactivation.)

The phage released after induction display a wide variety of phenotypes. This result may be explained if the “dearchiving” process is mutagenic to the phage


sequences or if some modifications are present in the released phage. As mentioned above, cultures of phage-resistant cells also release phage when washed and grown in adenosine. The phage released by these cultures make very small plaques compared to those accumulated in phage-sensitive cultures. This would be consistent with the idea that the released phage are partially defective, resulting in a decreased burst size. The defects may be due to the postulated mutagenic properties of the “dearchiving” process. We speculate that the large plaques produced by phage released from phage-sensitive hosts is due to recombination among the released defective phage or among these phage and the partially “dearchived” prophages in the host cells. Such recombination events could only occur in cultures of phage-sensitive cells that permit multiple rounds of infection. We suggest that, while our strains cannot normally donate markers to incoming P22 phage, cells in the induced state (i.e., washed and growing in adenosine), expose these sequences to recombination. This predicts that the few P22 phage initially released during an induction are partially defective and the phages which are selected during outgrowth are recombinants between these and the remaining prophages. Such recombination could yield the variety of phenotypic phage types we regularly see after inducing a culture of phage-sensitive cells.

The Southern hybridization patterns are not yet understood. The single band of P22 homology appears to contain an entire P22 genome with an abnormal mobility (D. M. DOWNS and J. R. ROTH, unpublished results). Our working hypothesis is that the DNA of “archived” prophages is modified so that internal restriction sites are hidden and a single band is generated by the first available pair of flanking restriction sites. We propose that DNA modification causes the unusually high mobility of these sequences. According to this hypothesis, stress in the form of purine imbalance causes these modifications to be lifted and causes occasional appearance of the phage.

In light of these findings, we propose that several S . typhimurium strains harbor an archived cryptic P22 prophage that is kept inactive by a system of localized DNA modification. The “archived” state can occasionally be lifted by conditions that perturb purine synthesis.

We thank ROBERT ROWAN for valuable assistance with the South- ern hybridization analyses. We thank MIMI SUSSKIND, PHIL YOUD- ERIAN and BRUCE STOCKER for helpful discussions. This work was supported by National Institutes of Health grant GM23408.

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a novel p22 prophage in salmonella typhimurium · copyright 0 1987 by the genetics society of...

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