Low-frequency specialized transduction with Bacillus subtilis bacteriophage ∅105

VIROLOGY

62, 393-403 (1974)

Low-Frequency

Specialized

Bacillus subtilis JAMES

A. SHAPIR0,2 Rosenstiel

DONALD

Transduction

Bacteriophage H. DEAN,

AND

with

$105’ HARLYN

Basic Medical Sciences Research Center, Brundeis Waltham, Massachusetts 02154

0. HALVORSON3 University,

Accepted July 31, 1974 Low-frequency specialized transduction of phe A, leu B, leu C, and ilu B by Bacillus subtilis bacteriophage $105 has been found. All of these markers are near the attachment site. Lysates of $105, prepared by mitomycin C induction of lysogenic B. subtilis 168 (trp-) or 168 trp+, were used to infect lysogens auxotrophic for the selected marker. The frequency of transduction of these markers is two to three orders of magnitude higher than other markers and is 6.5 x 10m7per plaque-forming unit for leu B. Effective transduction occurs only with lysogenic mutants, and the absence of a prophage in the recipient leads to a drop in transduction frequencies of more than two orders of magnitude. Transformation and generalized transduction have been eliminated as possibilities because of DNAase treatment of the lysates, the selectivity of the transduced markers, the requirement for lysogenicity, and density gradient centrifugation of transducing particles. High-frequency transducing phages have not been detected. Because 4105 does not excise from the chromosome according to the Campbell model, we suggest that the low-frequency transduction is due to infrequent errors in excision which may be corrected on subsequent infection and excision. INTRODUCTION

Bacillus subtilis has several properties which make it a model organism for the study of sporulation. It has systems of recombinational genetic analysis by transformation and generalized transduction. However, studies of regulation at the molecular level in B. subtilis have one very important obstacle when compared to similar studies in the enteric bacteria: episomes in general, and specialized transducing phages in particular, are so far unknown in this species. Specialized transducing phages have been of great importance. in regulation studies with E. coli for three reasons: (1) they provide a system of 1 Supported by Grant GM 18904-98 from United States Public Health Service. *Present address: Department of Microbiology, University of Chicago, 920 East 58th Street, Chicago, Illinois 60637. 3 To whom reprint requests should be addressed.

functional genetic analysis for dominance and complementation studies (Morse et al., 195613); (2) they have made it possible to rearrange the bacterial chromosome and thereby facilitated genetic analysis of regulatory genes and controlling elements (Beckwith et al., 1966; Beckwith and Zipser, 1970); and (3) the DNA of specialized transducing phages has served as an indispensable substrate for in vitro studies of gene expression and regulation (Gilbert and Muller-Hill, 1966; Zubay and Chambers, 1969; Zubay et al., 1971 and 1972). If specialized transducing phages for B. subtilis carrying vegetative or sporulation markers can be isolated, their DNA will provide a template approximately loo-fold enriched for those loci. This DNA could be used both for assaying specific messenger RNA molecules (DiCioccio and Strauss, 1973) and for directing in vitro messenger RNA synthesis with RNA polymerase from vegetative and sporulating cells (Losick, 393

Copyright All rights

0 1974 by Academic Press, Inc. of reproduction in any form reserved.

394

SHAPIRO.

DEAN

AKD HALVORSON

1970). Since the recessiveness of antibiotic resistand Sonenshein, Shorenstein, to Hence, the object of the present study is to ance markers would make it difficult isolate a specialized transducing phage in detect specialized transducing particles deB. subtilis to facilitate the study of’ the rived from SPO2. we chose to study @lOFi. If’ control of bacterial sporulation. this phage produces specialized transducIn addition to their utility as tools for the ing particles which incorporate the phe, study of regulation in B. subtilis, specialleu, or ilu markers, they should be readily ized transducing phages are also of interest identified by transducing auxotrophic mufor the information they provide on the tants with lesions in those loci. mechanisms of lysogenization by temperMATERIALS AND METHODS ate phages in species outside the enteric bacteria. As will be seen from the DiscusBacteria and Bacteri0phnge.s sion, at least one B. subtilis temperate The bacterial strains used in this work phage appears to differ markedly from are all derivatives of’ B. subtilis 168 trp better-characterized phages (such as X) in and are listed in Table 1. Bacteriophage its interaction with the host chromosome. $105 was isolated from strain JAS-15 iniSpecialized transducing phages are iso- tially and used to lysogenize strain ANS-1. lated as rare variants of temperate phages Phage lysates were induced by a lo-min which have substituted a portion of the exposure of broth cultures of lysogenic bacterial chromosome for part of the viral strains to 1.5 pg/ml mitomvcin C at 37 C genome (Campbell, 1962). They are gener- and sterilized by passage through a Milally selected from induced lysates of a lipore filter. Phage lysates tested for transtemperate phage by searching for phage duction were induced from strain ANS-1 particles which will transduce bacterial (4105). Lysates were routinelv incubated markers located close to the prophage at- with 5 pglml DNAase I (Worthington). 30”, tachment site (Signer, 1966). Specialized 30 min but omission of this step in early transducing phages have classically been work did not alter the results. distinguished from generalized transducing phages by, their marker specificity and by their ability to reproduce themselves Pur a and, upon induction, produce a phage lysate (HFT lysate) which contains a high titer of transducing particles (Morse ct al., 1956a). B. subtilis has two temperate phages which have been shown to lysogenize by integration into the bacterial chromosome and whose attachment sites have been mapped: SP02, whose prophage maps between the spcA and lin antibiotic resistance markers (Smith and Smith, 1973). and 4105, whose prophage maps between the phe A and SpoO31 markers on one side and the leu-ill: gene cluster on the other side (Rutberg, 1969; Young and Wilson, 1972). These are shown in Fig. 1. In specialized transduction with X phage. the inheritance of donor markers occurs by the formation of a partial diploid. or heteroFIG. 1. Partial map of B. subtilis 168 showing genote, and the presence of the donor allele attachment sites for temperate phages @lo5 and is detected by complementation rather SP02. Compiled from Hara and Yoshikawa. 1973 and than recombination (Morse et al., 1956b). Young and Wilson, 1972.

SPECIALIZED TABLE

1

BACTERIAL STRAINS (B. .subtilis Strains JAS-8 JAS-9 JAS-11 JAS-12 JAS-15 JAS-19 ANS-1 BCY37 BC-60 BC-52 BR-63

Isolation

Genotype

(‘iear-Plaque

168) Source

leu B, trp C leu C 7, trp C 2 iluB3,trpC% ILL) A :i, trp C ‘2 168 (410.5) phe A, Spa 031 Protot rophic (168 trp ’ ) pur A16, met B5. /ys A8. phe Al2, arg A3 pw AL6. leu A8, met B5, nit pur A16, leu A8. met B5. ilc A6:i phe A of

TRANSDUCTION

S. Zahler S. Zahler S. Zahler S. Zahler L. Rutberg I. Takahashi A. Schwartz J. C. Copeland d. C. Copeland J. C. Copeland A. L. Sonenshein

Mutants

of

4105

Strain ANS-1 (4105) was grown at 30” and mutagenized with nitrosoguanidine. After recovery at 30”, exponential cultures were given a 20-min heat shock at 45” and then incubated at 37” for 1 hr. The cultures were then centrifuged, and the supernatant was filter sterilized and plated for isolated plaques on ANS-1 at 45 C. Clear plaques were picked, purified, and then retested on ANS-1 at 30” and 45”. Two plaques were found to yield phages that gave clear plaques at 45” and turbid plaques at 30”; these were named @lOFiclOS and @105clO5. One plaque yielded clear plaques at both temperatures and was named @105c30. Isolation

of

Lysogens Containing

a Defec-

Strain JAS-9 (leu C) was lysogenized with temperature-sensitive clear plaque phage @105c103 and mutagenized with NTG. NTG-mutagenized lysogens yielded mutants capable of forming colonies at 45” with a frequency of about 10m4. These mutants should contain a second mutation in the prophage which either makes its repressor temperature stable or renders the prophage nonlethal. Five hundred and eight thermoresistant mutants were repurified and tested for nutritional requirements, their ability to produce phage. and whether the phage produced gave clear or

WITH

395

$105

turbid lysis at 45”. Of these 508. one of the prophage mutants (JAS-50) contains at least two additional mutations, one rendering the prophage defective (does not induce with UV) and the other creating a cold-sensitive immunity (immune at 45” but not at 30”). JAS-50 remains auxotrophic for leutine but has an additional auxotrophic requirement which was determined to be isoleucine-valine. The ilu requirement in this strain confirms the close linkage of the ilu gene cluster to the 4105 prophage. Isolation of a defective prophage was important for mapping. Determination of linkage of nutritional markers to the 4105 attachment site was only possible with immune recipients which cannot produce active phage. The reason for this is that free phage on the selection plate infect the nonlysogenic transformants and alter the linkage results in two ways: (a) lysing some cells, and (h) lysogenizing other cells. Both of these effects will reduce the apparent number of nonlysogenic transformants. Other Methods General phage and genetic experiments were performd as described by Rutberg (1969). DNA was extracted from strain ANSI for transformation by resuspending cells from 100 ml of an overnight broth culture in 10 ml EDTA-saline (pH 8.(l), treating with 4 mg lysozyme for 20 min at <‘3r1o I adding 1 ml 2078 SDS, and then extracting with an equal volume of 90’7 phenol at 4”. The aqueous phase was precipitated with 20 ml 95% ethanol, and then resuspended in 2 ml SSC. Nitrosoguanidine mutagenesis was by the method of Cerda-Olmeda, Hanawalt and Guerola (1968). RESULTS

Proximity of Prophage

SeLleral

Markers

to

the

Signer (1966) demonstrated that, for coliphage 480, the probability of formation of a specialized transducing phage for a given marker decreases with increasing distance from the prophage. We wished to determine whether markers near the att 4105

396

SHAPIRO.

DEAN

AND HALVORSON

were close enough to be incorporated into the phage head (i.e., not further than the length of the prophage) Transformation mapping of leu, ill), and att 4105 was done and the results are shown in Table 2. Competent cells of JAS-50 were incubated with DNA extracted from ANS-1 and then plated on selective medium. After 48 hr at 45” transformant clones were purified onto the same medium and replica plated onto appropriate selective medium to score the inheritance of nonselected markers. These results confirm the order of loci as att $105leu C-ilu, and show that both the leu C and ilc mutations are very close to the prophage attachment site (40% cotransformation, 0.6 recombination distance). The size of the transforming DNA extracted by the procedure we use is not greater than 20 x lo6 daltons. (A. L. Sonenshein, personal communication). Thus, from the recombination distances, we can estimate that the distance between the $1105attachment site and the leu C markers is not greater than 12 x lo6 daltons of DNA. Table 3 shows the recombinant classes of a two-factor cross linking phe and att 4105. There is a 40%) cotransformation ofphe+ and 4105d50 prophage from the donor. This is equivalent to a recombination distance of 0.6 or a physical distance of approximately 12 x lo6 daltons. In this we show a closer linkage than previously reported by Rutberg (1969) who reported lo-15Y~ cotransformation of phe to a prophage marker. The position of a prophage marker with respect to the end of the prophage nearest to phe would account for these differences. The size of the $105 genome is about 25 x lo6 daltons (Birdsell, Hathaway, and Rutberg, 1969). Hence, the DNA between the 4105 pro-

phage and the leu C and i/c markers or phc A could be incorporated into a phage head together with part of the viral genome. The Effect of Lysogenic Immunity sible Transduction b>, @lOi

Our initial approach to isolation of a specialized transducing phage was to perform the simplest experiment of inducing $105 from a prototroph, absorbing the phage to an auxotrophic recipient, and plating on selective medium for transductants. When this experiment was performed with strains carrying the phe A, leu C, and ilu A markers, a few very small colonies were seen on the selective medium. Since the strains were sensitive to 4105, it was possible that free phage on the selective plates were infecting and killing protential transductants. Thus, the experiment was repeated with the same recipients lysogenized with $105. It was also possible that the resident prophage might have a “helper” effect on transduction. More colonies were detected on selective media, and they were of normal size. Table 4 summarizes these results, showing that the appearance of leu+, ilu+, and phe+ colonies after 4105 infection was at least TABLE LINKAGE

-

Selected marker leu * ilv+

leu+

i/a.

35 29

4’

OF phe

35 81

leu

:1

$105

BY TRANSFORMATION”

Recombinant

classes

phe’ $105 d50

phe’ @lo5

45

75

phe (120)

UJAS-50 DNA was used to transform BR63 ($105). Primary selection was for phenylalanine. The type of prophage was assessed by replica plating onto B. subtilis 3610 cells and scoring spontaneous induction of wild-type phage. 2

CLASSES FROM TRANSFORMATION

leu i ilv * 4

TO att

Selected marker

TABLE RECOMBINANT

on Pos-

ilc 28

4’

OF JAS-50”

leu. ilc

4

leu ilc’ +-

leu

ilu’ 4

61 45

5

DDonor DNA from ANS-1 was used to transform JAS-50. The presence of $105 prophage was determined by replicating onto a lawn of $105 ~30 on nutrient agar plates and scoring for growth at 45”. No growth indicated inheritance of the donor att site C&l; growth indicated inheritance of the recipient prophage (4’ ).

SPECIALIZED

TRANSDUCTION

lo- to loo-fold higher with lysogenic than nonlysogenic recipients. Evidence That the Formation of Transductanks after $105 Infection Is Not Due to Transformation or Generalized Transduction The appearance of colonies selected for the leu-, ilv+, ot phe+ phenotype after infection by 4105 may have been due to transformation by DNA in the phage lysates (which, in the initial experiments, were not treated with DNAase) or to generalized transduction. Transformation was unlikely since $1105lysogens are very poorly competent (Peterson and Rutberg, 1969; Yasbin, Wilson, and Young, 1973). $105 lysogens yield less than loo-fold fewer transformants than nonlysogens. Thus, if transformation were occurring, we would expect lysogenic recipients to have yielded

WITH

397

$105

fewer recombinants than nonlysogenic recipients, and Table 4 shows that we obtained the opposite result. To eliminate the possibility of transformation, lysates were subsequently treated with DNAase (*5 pg/ ml), and an experiment was carried out with an untreated lysate to determine whether it was capable of transducing a number of other markers as well. If transformation or generalized transduction were occurring, it should also occur for the other markers in the recipient strains which are completely unlinked to the $105 prophage (Fig. 1). The results in Table 5 and Fig. 2 indicate that the transduction effect was specific for markers closely linked to the 4105 prophage. Only those markers which are closely linked to att4105 are transduced at levels above 10m9. These results agree with those of Birdsell et al. (19691 who had previously found no evidence for

TABLE

4

EFFECT OF LYSOCENK IMMUNITY ON POSSIBLE TRANSDUCTION BY $105” Lysate

PFU!ml

Colonies of possible transductants Lysogenic recipients leu +

ilu +

per infecting

phage

Nonlysogenic phe+ h

leu +

recipients

ilu *

phe’ fi

1

8.9 x lo9 1.5 X 10-T 3.0 x 10 7 3.4 )/ 10 3 1.1 X 10 I0 6.7 x 10 I” 2.2 * 10 I” 8.1 x lo9 1.5 x 10 7 1.7 x 10 7 1.2 x 10 q <1.2x10 ‘0 5.4 Y 10 1” <1.2 b 10 lo ~~~ ~__ 0 Exponential cultures (-5 x 10B/ml) of strains JAS-9, JAS-12, JAS-19, JAS 9 (@105), JAS-11 (&lO.i), and JAS-19 (4105) were infected with lysates of 4105 induced from ANS-1 (4105) at a multiplicity of approximately 5-10, allowed to absorb phage for 20 min at 37”, and then plated on appropriate selective medium. Colonies were counted after 72.hr incubation at 37”. Uninfected cultures were plated as controls for reversion and contamination. h phe recipient in this experiment was JAS-19 which may be a deletion mutant for phe A and Spo (131, see text. 2

TABLE

5

4105 TRANSDUCTION IS SPECIFIC FOR MARKERS NEAR THE PHACE A~ACHMENT SITE” Transductants/PFU

(x 10 l”)h

leu C

leu B

phe A

nit

1400

1500

(ND)

(ND)

6500 (0.25)

200 (0.06)

(0.15)

pur A’

arg A

ilv B

2.9 (1.5)

0.3 (0.3)

185

1YS

trp c

met B

ilu A

8.8

0.1 (0.01)

0.1 (0.25)

8 (0.01)

(0.09)

0 JAS-9 (&105), JAS-8 (+105), JAS-11 ($105), BC-37 ($105). BC-50 (1$105), and BC-53 (6105) were infected with 4105 induced from ANS-1 (+105) and plated as described in the legend to Table 3. The att $105 site is between leu B and phe A. The numbers in parentheses are the reversion rates (revertants per colony-forming unit x 10-r). h Results from a typical set of experiments corrected for reversion rates. ( Markers are arranged according to Hara and Yoshikawa, 1973. and Young and Wilson. 1972.

398

SHAPIRO,

DEAN

generalized transduction by 4105. (Their experiments, however. were performed with nonlysogenic recipients.) In order to demonstrate that we are in fact observing bacterial DNA carried by $105 particles, we performed the CsCl density-gradient experiment shown in Fig. 3. Transducing activity clearly bands at the same buoyant density as infectious 4105 and is not due to DNA. Our original observation that JAS-19 was transduced at especially low frequency (Table 4) led us at first to incorrectly assume that phe A was not transducible. The subsequent finding that the wild-type phe A marker could be transduced into other auxotrophs (Table 5) as well as our difficulty in mapping the phe A-SpoOSl-utt 4105 region by transformation with JAS-19 have led us to suspect that phe and Spo are deleted in this mutant. Attempts

to Obtain an HFT Lysate

As noted in the introduction, one of the methods for proving the existence of a specialized transducing phage is to obtain an HFT lysate. This was especially important for 4105 since transduction results are

AND HALVORSON

FIG. 3. CsCl density gradient of induced $105. Phage were induced from JAS-15, and the concentrated lysate had a titer of 10” PFU/ml. A 12-ml gradient with an initial density of 1.5 g/ml was centrifuged 40 hr at 30,000 rpm in the 5OTi rotor at 21”. Sixty fractions were collected into l-ml phage buffer and assayed for PFU (--O--J on ANS-1 and for leuB+ transducing particles (- -A- -1 on eJAS-8.

of low frequency. To try to obtain HFT lysates, we used the following technique: all potentially transduced clones from an infection were resuspended in broth, induced, and the resulting lysate was used to infect mutant recipients. If any of these clones were lysogenic for a specialized transducing phage, we should obtain a higher frequency of transduction after infection with the lysate as is found with phage lambda transduction for the E. coli gal or aro G markers (Shapiro. 1969). The results in Table 6 show that we were not able to obtain any HFT pooled lysates. DISCUSSION

FIG. 2. Transduction of markers plotted with respect to their location on the chromosome. 0 = origin of replication which proceeds to the left and right; T = terminus.

A low-frequency specialized transduction of leu B, C, ilu B, phe A, and nit has been observed using B. subtilis bacteriophage 4105. The frequency of transduction of the leu B marker is 6.5 x 10m7/PFU and transduction occurs only for those markers which are closely linked to the phage attachment site (Fig. 2). Several lines of evidence eliminate transformation or generalized transduction as the basis of the effect. Only a few markers are transduced at a significant frequency. Transduced markers are closely linked on both sides to the attachment site. Markers

SPECIALIZED TABLE TRANSDUCTION

Selected marker

leu *

ilc * phe’

TRANSDUCTION

6

WITH LYSATES FROM POOLED TRANSDUCTANTS”

Possible transductant clones resuspended and inducted 136 132 - 7000 “72 1 - 1000 4 45

Pooled Ivsate phage titer (PFUlmll

3.5 2.0 6.2 5.0 7.3

x x x x x

10” 107 1O’O 107 109 108 6.9 x lo9

Transductants per infecting phase

1.7 X 10 ’ 4 x 10-7 6x10’ 6.8 x 10 7 2.7 x 10~ ‘” 10-s 3.2 x 10 y

“Transductant colonies of JAS-8 ($105). JAS-9 ($1051, JAS-11 ($11051, and JAS-19 ($1105) were selected on appropriate media after infection with $105 as described in the legend to Table 2. These colonies were patched on the same selective medium with sterile toothpicks, incubated 24 hr at 30”, and then resuspended en masse into broth. When -7000 possible transductants were used. the cells were pooled grown in minimal medium containing only tryptophane hefore reinoculating into broth. When they had reached exponential phase at 35”, they were induced with mitomycin C. and the resulting lysates were filter sterilized. l&ese were then used to infect the original recipient strain, and the infected cultures plated on selective medium. Colonies were counted after 72 hr at :30”.

which are closest to att 6105 are transduced at greatest frequency. Both of these are characteristic of specialized transduction. Moreover, the transducing activity is resistant to DNAase treatment, has the same buoyant density as infectious 4105 (Fig. 3), and is greatly increased with lysogenic recipients (Table 4). All of these results clearly distinguish the effect we observe from transformation. It may be argued that $105 is capable of a low level of generalized transduction and that the specificity we observe for prophage-linked markers is due to their selective replication after induction (Rutberg, 1973). This explanation is extremely unlikely for the following reason. The enrichment of DNA carrying prophage-linked markers after induction is at most 7- to lo-fold (Rutberg, 1973). and we observe a specificity of more

WITH

$105

399

than two orders of magnitude (Table 5 and Fig. 2). Our inability to obtain an HFT for 4105 has been a cause of concern. We have shown that massive lysates of as many as 7000 transductants did not yield higher transduction frequencies (Table 6). Transformation mapping showed that this failure is probably not due to the distance of at least the leu and phe markers (Tables 2 and 3) from the prophage. It is possible that we have not looked hard enough for a 4105 HFT. It is known that the lambda prophage integrated at sites on the bacterial chromosome distinct from its normal attachment site is capable of specialized transduction of nearby markers (Shimada et al., 1972). However, in some cases it is difficult to obtain an HFT lysate for these markers. When the lambda prophage is integrated near the arabinose operon, it is necessary to go through two cycles of pooled lysates starting with thousands of potentially transduced clones before an HFT lysate can be detected for aru- mutants (Lis and Schleif. 1973). Hence, it is possible that our failure to find a high-frequency specialized transducing phage for markers near the $1105 prophage was due to using too few initial transductants and only going through a single cycle of pooled lysates. There is, however, a biological reason that $105 may not yield specialized transducing phages at high frequencies. Phages such as lambda and $80 in E. cd which produce specialized transducing phages are circularly permuted when they integrate into the bacterial chromosome (Campbell, 1962). This means that the part of the viral genome which corresponds to the cohesive ends of the DNA molecule packaged into the phage particle is in the middle of the prophage, and therefore one end of the prophage can be substituted with bacterial DNA in the formation of a specialized transducing phage without losing the sequences necessary to generate cohesive ends and package the DNA into a phage head. Kayajanian (1970) has demonstrated that the presence of the cohesive end sequences is necessary to form a specialized transducing phage for X. 4105, on

400

SHAPIRO.

DEAN

the other hand, does not circularly permute when it integrates into the bacterial chromosome (Armentrout and Rutberg, 1970; Chow and Davidson, 1973a); so the attachment site of 4105 must be located at or near the ends of the linear phage chromosome. The physical studies of Chow and Davidson (1973a) show that the 4105 attachment site is located within 20 nucleotides of the end of the viral genome. Thus, it is possible that integration of $105 into the bacterial chromosome does not occur by the model proposed by Campbell (1962), but rather by recombination at both ends of the phage to sites on the host chromosome (Fig. 3). This possibility has been suggested in the studies of Garro (1973). The fact that circularity of the phage genome has not been observed either in sucrose gradients (Armentrout, Skoog, and Rutberg. 1971), or in electron micrographs (Chow and Davidson, 1973b) makes this possibility more likely. The excision of d105 from the bacterial chromosome after induction also appears to differ from the Campbell model. In their early work, Peterson and Rutberg (1969) observed that prophage excision follows its replication. It was observed that newly replicated prophage DNA remains with the rapidly sedimenting bacterial DNA. and that prophage markers are linked genetically with markers such as nit (nia), phe A, and ilv during early induction. The genetic linkage between these markers and prophage markers decreases during induction but well after phage DNA has begun replicating (Armentrout and Rutberg, 1971). More recently Rutberg (1973) has shown that replication of the 4105 prophage is bidirectional and bacterial markers on both sides of the prophage are replicated as much as 30% during induction. We have constructed a model (Fig. 4) which accounts for the low frequency of transduction with 4105, for our present failure to obtain an HFT lysate, and for what is known about integration and excision of the phage. After infection the phage genome remains linear (Armentrout and Rutberg, 1970). Integration of the phage genome into the chromosome is presumably by recombination at both ends of this

AND HALVORSON

linear structure. Two possible routes of’ recombination are indicated in Fig. 1. One involves addition of the viral genome to the host chromosome without loss of genetic material, and the other involves substitution of the viral genome for a nonessential portion of the chromosome. PBS1 transduction indicates that 4105 integration increases the physical distance between pheA and 1euB by approximately 32-50 x’ lo6 daltons (Dean et al., unpublished observations). These results, therefore, favor the addition model. The first step of induction (Fig. 4) involves bidirectional replication which continues onto adjoining regions of the bacterial chromosome (Rutberg, 1973). The next step, excision, involves breaking the physical and genetic linkage between the prophage DNA and bacterial DNA. This is probably via a cutting enzyme (see below). Armentrout. Skoog. and Rutberg (1971). have indicated by studies with exonucleases I and III that the ends of $105 are probably double stranded. If a rare excision event occurred, such that an adjacent marker remained with the excised prophage, this might be packaged in a phage. The possible fate of this hypothetical @105-mediated DNA is traced in Fig. 5. Upon infection the phage DNA carrying a bacterial marker could recombine with the lysogenic host’s chromosome at new recombination sites, transducing the auxotrophic lysogen to prototrophy. Subsequent induction, DNA replication and excision at the normal excision sites would not result in an increase in erroneous excisions or in the frequency of transduction of the prototrophic marker. If this model is correct, it would also account for the verv low transduction frequencies obtained with nonlysogenie recipients (Table 4). The reduction in transduction frequencies for the leu and ilu markers caused by the absence of a resident prophage is on the order of 50. to 100-fold. This is much greater than the killing of nonlysogens by 4105 infection at multiplicities of 5-10 (150% killing, our observations). The model presented in Fig. 5 predicts that the absence of prophage sequences in the recipient genome will reduce recombination due to lack of homol-

SPECIALIZED

TRANSDUCTION

WITH

401

4105

Infectinn Viral

gcnomr

I

Recombination

\

--

A nit *

phr *

AB

Spa .

-KL

;

) ku+ilv

--

non-essential bacterial

region

”

Induction DNA

I Replication

A0

..KL.,.

I,”

+

I AB ror.

FIG. 4. Hypothetical

model for integration,

replication,

ogy at one end of the transducing fragment. The observation that transducing activity for leuB+ shows a sharp peak in equilibrium CsCl gradients (Fig. 3) is very different from LFT lysates of X (Adler and Templeton, 1963). This suggests that maturation of $105 after excision involves a mechanism for measuring the amount of DNA that is packaged in each phage particle-even when the ends of the encapsulated DNA molecule do not correspond to the normal ends of the viral genome. If this

tronrducsng

KLy.lru*

phage

and excision of temperate

gmom.

phage $105.

is so, and if excision of $105 follows replication in the integrated state (Rutberg, 1973), then we predict that normal excision and maturation of induced 4105 requires two separate functions: a cutting function that separates the prophage DNA from chromosomal DNA at a specific site at the end of the prophage, and a measuring function that packages exactly one viral equivalent of DNA in the phage particle. Formation of transducing particles would involve an error in the cutting function.

402

SHAPIRO.

DEA&

AB

AND

HALVORSON

KL../eu+

tronsducfng

Phogo

DNA

”

Recomblnotton

AB

..KL.,

Icu+

I lysogcnic

rccombinont

recipient

transductont

”

lnductlon

c DNA

Repllcatlon ”

Excision

AE-

.KL.;/&

A0 ~..~KL../eu+

; ,,B..

..,KL...

rare

FIG. 5. Hypothetical

fate of $105 transducing

In conclusion, we wish to observe that although we were unsuccessful in obtaining HFT lysates of 4105 transducing phages for genetic and biochemical studies, the apparently very novel mechanisms for 4105 prophage intregration and excision may prove relevant to other systems of viralhost chromosome interaction. Note added in proof. Birdsell et al. (1969) and Chow, Boice and Davidson (J. Mol. Biol. 68, 391 [1972]) have observed circular molecules in electron micrographs of DNA extracted from $105 particles. Thus, it remains possible that 4105 does lysogenize by reciprocal recombination followin:: circularization.

tronsducmg

phoge

phage during integration

genome

and excision

Even if this were the case. transducing phage genomes (formed by the Campbell model or by the mechanism outlined in Fig. 4) would very likely not have the proper terminal sequences for subsequent circularization. This would prevent transduction by lysogenization and lead to the result depicted in Fig. 5. ACKNOWLEDGMENTS We are grateful for the advice and encouragement of A. L. Sonenshein and R. Schleif. We thank Maryvonne Arnaud for her excellent technical assistance. REFERENCES ADLER, J., and TEMPLETON,

galactose

genetic

material

B. (1963). The amount of in hdg bacteriophage

SPECIALIZED

TRANS ;DUCTION

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