Sites of P22 and P221 prophage integration in Salmonella typhimurium

VIItOLOGY

28, 265-270 (1966)

Sites of P22 and P221 Prophage

Integration

in Salmonella

typhimorium’

B. G. YOUNG2 The Johns

Hopkins Cancer

University, Institute,

Department National AND

The Johns

Hopkins

of Biology, Baltimore, Maryland, and National of Health, Bethesda, Maryland

Institutes

PHILIP

C’nioersit;y,

E. HARTMAN

Department

of Biology,

Baltimore,

Maryland

Accepted October 15, 1965 The sites of P22 and P221 prophage integration in the linkage map of Salmonella typhimurium have been determined by means of colicine factor-mediated conjugat,ion experiments. The P22 prophage is integrated near a cluster of proline genes, and P221 prophage is integrated between a cluster of tryptophan and a cluster of galactose genes. A common c region is shared by P22 and P221 phage particles. We conclude that the c region plays no determinative role in prophage location. INTRODUCTION

A number of different bacteriophages are released from Salmonella typhimurium, strain LT-2, infected with I’22 and P22-related bacteriophages (Yamamoto and Anderson, 1961; Yamamoto and Weir, 1966a; Young, et al., 1966; Yamamoto, personal communication). Among the phages released is a class of phages which has been designated P221 (Yamamoto and Anderson, 1961; Yamamoto and Weir, 1966b). P221 phages possess the c and h21 markers, but not the m3 marker (Levine, 1957; Levine and Curtiss, 1961) of phage P22 and are serologically unrelated (Yamamoto and Anderson, 1961; Yamamoto and Weir, 196613) and partially dismune t’o I’22 phage (Yamamoto and Weir, 1966b; R. G. Wilkinson and B. A. D. Stocker, personal communication). The acquisition of colicinogeny confers upon X. typhimurium the ability to mate and to transfer chromosomal markers at a 1 This work was supported, in part, by grant Al 01650 of the National Institute of Allergy and Infectious Diseases, Public Health Service. p Present address: Laboratory of Viral Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. 265

low frequency (Ozeki and Howarth, 1961; Ozeki et al., 1962). This acquired ability has been used to determine the sites of prophage location both for P22 and for one of t,he genetically related P221 phages. P22 phage is integrated very close to a clust’er of proline genes while P221 prophage exhibits an entirely different location on the bacterial chromosome. We conclude that the c region plays no determinative role in the prophage locations of these t’wo phages. MATERIALS

AND

METHODS

Media. Media used for growth of bacterial cultures and for assay of phage and bacteria are described by Young et al. (1966). Select’ive media for the detection of recombinants in conjugation experiments consisted of E minimal medium (Vogel and Bonner, 1956) supplemented by the appropriate amino acids at final concentrations of 20 rg L-amino acid per milliliter and 0.6% dextrose. When required, streptomycin sulfate (Parke, Davis and Co.) was added at a final concentration of 1.0 mg/ml. Recombinants were examined for ability to utilize galactose or arabinose on a minimal medium containing low citrate (Demerec et al., 1958) and supplemented

266

YOUNG

AND TABLE

BACTERIAL

Strain Salmonella typhimurium LT.2 derivatives stc St/22 SB133

SB135

SL689

SL894

Escherichia CL136

CL150

HARTMAN I STRAINS

Description

and comments

Strain LT-2, cured of a B phage (Zinder, 1958). Sensitive to P22 phage; resistant to P221 phage. Obtained from IIr. H. H. Plough P22-resistant mutant of strain LT-2 (Yamamoto and Anderson, 1961). Sensitive to P221 phage. Gift of Dr. T. F. Anderson atr-r hisH32 tryB2 gal-101 (P22+). A st,reptomyciri-resistant, histidilleand tryptophan-requiring mrltant (SL800 of Stocker) was t,reated with diethylsulfate and a galactose mrltant selected (SB98) and lysogenized with wild-type P22 phage. Adsorbs P22 phage, rmable to adsorb P221 p&e Doubly lysogenic derivative of St/22 carrying the phages (P22 mS cd h21) (P221 c+ h21f) = SB134. Obtained from Dr. N. Yamamoto (Yamamoto and Weir, 1966b) and infected wit,h (col El) from CL136 met&$ H2enx Hf b tryB2 (col I). Donor of colicinogenic factor (co1 I) and carrying requirements for methionine and tryptophan as well as flagellar markers. Obtained from Dr. B. A. D. Stocker str-r adeC7 row401 iM-i0 $a-66 gal-422 pro;146 ara-430 ile-405 (P22+). A streptomycin-resistant mutant with nlltritional requirements for purines, proline, and isoleucine, inability to ferment galactose and arabinose, and flagellar markers. Can adsorb P221 phage; unable to adsorb P22 phage due to rough phenotype. Obtained from Dr. B. A. I). Stocker

coli derivatives str-r gal- tit- (col El). A streptomycin-resistant, galactose- and citrat,enegative strain carrying colicinogenic factor co1 El (Ozeki et al, 1962). Obtained from Dr. Stocker str-r. A streptomycin-resistant mutant sensitive to colicine El (Ozeki et al., 1962). Obtained from Dr. Stocker

with the appropriate sugar at 0.3 % concentration. Difco eosin methylene blue (EMB) supplemented with 2% galactose or arabinose was used to verify sugar fermentation. Bacterial strains. Bacterial strains are listed in Table 1. Strains with SL or CL stock numbers were obtained from Dr. B. A. D. Stocker, Lister Institut,e for Preventive Medicine, London. A strain carrying colicinogenic factor (co1 El) was obtained by mixed culture of a doubly lysogenic Salmonella strain with Escherichia coli strain CL136 and selection of citrate-positive, colicinogenic derivatives by methods analogous to those utilized by Ozeki et al. (1962). Strain CL150 served as an indicator for the presence of co1El. A total of 1s colicinogenic, citrate-positive colonies of SB135 were test)ed. All retained P22 prophage but two failed t,o release P221 phage. One of these

colicinogenic, doubly lysogenic strains (SB135) was used as male parent in the conjugation experiments. Colicine factor-me&fated crosses. Conjugation experiment’s were performed by a procedure modified after one utilized in the laboratory of Dr. B. A. D. Stocker (Ozeki and Howarth, 1961; Oeeki et al., 1962; Smith and Stocker, 1962; Stocker, personal communication). A 0.1 ml sample of a 1: 200 dilution of an aerated overnight broth cult’ure of SLGS9 and 0.1 ml of a 1:lO dilution of SB13.5 were inoculated together into 10.0 ml nutrient broth and incubated overnight at 37” without aeration to allow infection of SB135 with (co1 1). The mixed culture was centrifuged to remove contaminating phage, and the cells were resuspended at a 1:lO dilution in broth. An unaerated overnight broth culbure of recipient bacteria (SLS94

1’2‘2 AK11 1’221 PROPHAGE

or SB133) was diluted 1: 10 in broth, and equal volumes of the donor mixture and the recil)ient culture were mixed and incubated at 37” for 2 hours. Readily discernible clumping of the bacteria occurred. The culture was centrifuged and t,he bacteria were resusljcnded in one-fifteenth of the original volume. Aliquot)s (0.1 ml) were spread on minimal plus amino acid selection plates for the detect,ion of recombinant clones. In the first cross (Table 2), a/e+, PM+, and ile+ rec~ombimtnts were selected 011 media from whicbh growth factors were individually omitted. In the second cross (Table 3), his+ TABLE

a&

I

pro+* p,.o+gu1+ pro+ara+ pro+nra+gal+ ut1e+f’ ade+ile+ ileib i/e+WU+pR7+

I

str gal

his

Recombinant selected (averagenumber of colonies/plate)

lhr~or mixture alone llecipient alone I)onor mixture atid recipieiitS I~ecombinar~ts/pl:~te I(econlbinarlts/plate (‘)L, of colonies)

ora

2

C:ROSS 1 : COLICINE ~AcT~R-~ED~.~‘~‘EI~ CROSS ‘TO Pos~~ros ‘I~HE P22 AND P221 I?ROPHAOES (SB135 X SL894)($

Bacteria spread

267

SITES

22.7 0.0 33.9

8.7 1.2 25.9

31.2 1.7 91.1

Phagereleased

0

4

16 16 16 0 0 0 4 -

1 4 2 10 5 10 0 -

58

3G

y The bact,erial straitts used and the mating procedures are described in the text. The frequencies of recombination found per input male bacterium were: adenine+, 1.8 X lo-‘; proline+, 1.2 X 10-T; isoleucine+, 3.4 X lo-“. b Colonies examined included spontaueous revertants.

FIG. 1. Linkage

map of Salmonella

typhinrrc~i/rm.

and try+ recombinants were similarly selected. In both crosses the media contained streptomycin to contraselect, against the streInomycin-sensitive donor prototrophic, strains; SL689 also was eliminated by the absence of methionine in the medium. Spontaneous revertants of the individual recipient markers were estimat’ed by comparison with control plates of the same media spread separately either with the donor mixture of bacteria or with the recipient bacteria. After 48 hours’ incubation at 37”, clones were picked from the selection plates and streaked on identical selective media for single-cell isolation. Isolated recombinant clones were further tested by replica plating t’o determine cotransfer of unselected markers. The clones were also examined for the spontaneous release of phages. Overnight aerated broth cultures were centrifuged, and the supernatants were shaken with chloroform, diluted, and plated with St,c and with St/22 bact,eria on modified indicator agar (Young et al., 1966) for analysis of phage content,. The VZSplaque morphology marker (Levine, 1957) was cont’ained in the donor strain I’22 prophage while the recipient carried wild I’22 prophage. 1’221 phage was det’ected by its ability to plate on St,/22 (Yamamoto and Anderson, 1961). The presence of one or both donor strain phages and the concomit~aru abseme of the phage originally present in the recipient, bact,eria were

268

YOUNG TABLE

CROSS

[I : COLICINE POSITION

Bacteria

3 CROSS TO

FACTOR-PL’IEUIATED

THE I’221 PROPHAGE (SB135 x SB133) Recombinant selected (Average number of colonies/plate)

spread

-.iris+

__.

I

I

~.

try+gal+ try+his+gaP

IV+

..~

Donor mixture alone Recipient alone Donor mixture and recipien t Recombinants/plate Recombinants/plate (‘/;L of colonies)

try+ try+his+

AND

0.0 1.0 208.0

206 0 (96.2)

207.0 (99.5)

Phage DOnOr P221

11 8

1 0

reieased

DOllOr P22(m3)

Recipient P22 cm+)

-

11 8 1 4 -

23

24

9

9

15 43

13

i

0.0 8.0 214.0

QThe bacterial strains used and the mating procedures are described in the text. The frequencies of recombination found per input male bacterium were: histidine+, 1.0 X 10-S; tryptophan+, 8.0 X 1O-5. b Colonies examined included donor strain (if any) that survived strept,omycin.

used as indications of cotransfer of the prophage and the selected marker. As described in Results, adsorption of phage by recipient bacteria exerted no determinative influence on the crosses. RESULTS

cross I

The first cross involved SBl35 as donor and SL894 as recipient. The data (see Table 2) show that the recombinant classes tie+, adef de+, and ilef release no P22 phage derived from the donor parent. Thus, it is assumed that P22 prophage is not located in these two chromosomal regions. This result also indicates that no significant proportion of recipient bacteria is infected by P22 phage

HARTMAN

released from the donor bacteria during the course of the experiment. Sixty per cent of pro+ clones release donor P22 phage. Since it is expected that approximately 40 7%of these clones are derived from sponbaneous mutation of recipient bacteria to pro+, close linkage of P22 prophage and pro+ is indicated. All pro+ ara+ gal+, 80 %Bof t,he pro+ ara+, and 94 % of the pro+ gal+ recombinants release 1’22 donor type phage. From these data it is concluded t)hat the site of P22 prophage is very near the proline loci (Miyake and Demerec, 1960) in the fi,\‘. typhimwiwm linkage map, possibly very slightly clockwise from the proline loci as the map is usually drawn (Sanderson and Demcrec, 196.5) (Fig. 1). Two of t’he 16 pro+ gulf ara+ recombinant clones release approximately equal numbers of both donor-type and recipient-type P22 phage. These presumed double lysogens have not been invest’igated further. The only classes of recombinant t,hat rclease P221 phage are the pro+ gal+ and t,he pro+ gal+ ara+ classes (Table 2). Only 28 % of these clor~es release P221 phage. This frequency is smaller than might be expected were the P221 prophage located between the pro and gal loci but is consistent with a locat’ion nearby, but dist)al, to gal (Fig. 1). cross II

A second bacterial cross was devised to define more closely the site of P221 prophage int,egration. Cross II was made between the same donor as used in the first cross (SB13.5) and SBl33 as recipient (Table 1). The data in Table 3 eliminate the presence of P221 prophage in the try-his region of the chromosome since t,he try+ his+ recombinant class is devoid of P221. In contrast, all the try+ gal+ recombinant clones release P221 phage. This result, coupled with the results of the first cross, indicates that prophage P221 is located between these two loci. Since only one of eleven tyy+ clones released P221 phage, the P221 prophage may be located closer to the gal locus than to the try locus. The prevalence of donor type P22 phage in try+ gal+ clones

remains

unexplained.

HOW-

ever, in this cross, unlike the first cross, the

P22 ANl)

P221 PKOPHAGE

recilkmt~ was able t,o adsorb I’22 l>hage and, thus, a high rate of infection may have occsurred.

wit,h 1’22 phage

I)ISCUSSION

The posit,ions assigned for the 1’22 and 1’221 I)rol)hages, as well as the locations of other chromosomal markers used in t)hc crosses, are deI)ict’ed in Fig. 1 (cf. Smit,h and Stocker, 1962; Sanderson and Demereca, 19A.i). Zindcr (1963), Smith and Stochker (19(X), and Smith and I,evinc (196.5) have obtained results on the I)rol)hage location of 1’22 in agreement with ours. At least seven indq)cndent lysogcnizations wit,h 1’22 indicate :I unique l,rol,hage sit,e for this ljhagc. I)xt,a presented by lVan~an~oto and Anderson (lY(il), Ynmamoto and Weir (1966b), and Young et al. (1966) show that 1’221 l)hages obtain their c regions and h+?ZImarkers, but not their a (Young et al. 1966) and ,/r/3 markers from 1’22 I)hage. In addition, as shown hero, 1’2: and 1’221 l)rophages arc intcgratcd at different sites in the Sallrzonella linkage ma],. These observations indicate that, the c region alone cannot control l,ro$l:~gc location. WC conc*lude that I)rol)hage location is determined by some other portion of the llhage genomc. The c region in the 1’22 I)hage genomc consists of three closely linked, but discrete, regions termed ci, cd, and c3 (Levine, 19.57). Levine and Smith have shown that the ~1 locus c*ontrols early inhibition of l)hage DKA synthesis, and that the c,%?region functions in maintaining inhibition of I)hagc DKA syrlthesis when csellular I)?;A synthesis is rcsumcd. Thus, cool)eration among Ihe c loci is required for the sucorcssful establishment1 of lysogeny (1,evine and Smit,h, 1964; Smith and J,evinc, 1964) rat’her than determining where in the host genome the I,rophage becomes integrated. In coliphagc X t,hree closely linked c loci also have been described (Kaiser, 1957). The ~1 locus of X, assumed to be analogous to the & locus of 1’22 Ijhage, has been rq)ort,ed as rq)onsible for determinating the prophage sit c of X (Kaiser and Jacob, 19.57). *Jacob and Wollman (19.54) report,ed that, loci other than the r loci l)layed roles in the stability of the I)rol)hwgc condition. In view of the sim-

269

SITES

ilarit’y in t(he genetic structures of 1’22 and X phages, it would seem of interest to examine more closely t’he roles of t,hese “stabilizing” loci. Ik%hermore, t)he X-434 hybrid l>hage (Kaiser and Jacob, 1957), which has been used in so many studies, should be tested t’o see whether it is as truly isogenic with X as believed. The data of Tamamoto and Weir (1966 a, b) indicate t’hat’ rccombinations obtained between more distantly related ljhages may I)redominant~ly, if not exclusively, involve double crossovers. A variant of 1’2 l,hagc, 1’2 Hy L&s, has been described which has the same serological q)ecificit’y as 1’2 but is dismune wit,h 1’2 l)hage and has an altered host) range, heat sensit,ivity, and I)laque moq,hology (Cohen, 19.59). This variant was considered to have originat’cd by recombination between phage 1’2 and a defective I)rophage in the host. In contrast to findings rel)ortcd in this l)aI,er for 1’22 and I’221 ljhage, 1’2 Hy Dis was located at the same c~hromosomal sit,c as 1’2 (Bertani and Six, 195s). I:EFERE?;CES B~wrasr,

CT., and

SIX,

Ii:. (1958). Inheritance crosses. Virology

prophsge P2 in bacterial

of 6,

357-381. I). (1959). A variant of phage P2 origillating in Exherichia coli, strain B. Virolog!/ 7, 112-126.

COHEN,

IhlIGRlX,

M.,

(:OId,MAS,

I.,

alId

LAHR,

E.

1,.

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270

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AN11 HARTMAN

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in

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YAMAMOTO, N., and WEIR, M. L. (1966a). (ielrctic relatiotlships between serologically ullrelatetl bacteriophages P22 and P221b. TTi/,o/og!/ ill press. Y.~M~Mow, N., and WEIR, M. I,. (19Mb). Bou~ldary struct we bet,weerr homologous :uld 11011~ homologous regions in serologically utlrelnted bacteriophages P22 and P221. I. Alapping by recombination found ill the cultures of dol~ble Iysogetlic struills for P22h :trld P221. Virolog!/ 28, 324-329 YOCNG, B. G., HAWMAS, 1'. E., :t~l R~oL-DRIANAKIS, 15. S. (196G). Some phages released from P22-infected Salmonella. Virology 28, 249+264. ZINDER, N. I>. (1963). I)iscussion. In “Rletl~odology ilr Basic (;enetics” (W. J. Burdette, ed.), pp. 124-12(i. HoldewDny, San Francisco.