J. MOT. Biol. (1978) 119, 167-178

Isolation of Bacteriophage MX4, a Generalized Transducing Phage for Myxococcus xanthus

JOSEPH M. CAMPOS~,JANET GEISSELSODER$ AND DAVID R.ZUSMANQ:

Department of Bacteriology and Immunology University of Calafornia

Berkeley, Calif. 94720, U.S.A.

(Received 17 June 1977, and in revised form 11 October 1977)

A new bacteriophage, MX4, was isolated from nature and tested for transduction in strains of Myxococcus xanthus. The phage shows a lytic cycle of growth in strain DZl with a latent period of 180 minutes at 35°C and an average burst size of 75 phage per infected cell. The phage was found to adsorb to but not form plaques on wild-type strains of M. xanthus; it could form plaques on a subclass of host mutants which were isolated as spectinomycin-resistant clones. Auxo- trophs were isolated in one of these strains, DZFB, using a new growth medium suitable for nutritional selections. The auxotrophs were transduced to proto- t,rophy at a frequency of about 2 x 10m6 to 4 x 10e5 per plaque-forming unit. Transductions were also performed with several antibiotic resistance markers using a temperature-sensitive phage mutant and maintaining all incubations at 35”C, the restrictive temperature. In this manner, rifampicin and streptolydigin resistances were co-transduced at frequencies of 88 to 100%. A mutant of phage MX4 was isolated which shows high transduction frequencies. At high multi- plicities of infection, this mutant should permit generalized transduction of many unselected markers to recipient strains. For test markers, this frequency was as high as 1 in 200 to 1 in 1200 viable recipient cells. Further, a host range mutation (hrm-I) has been introduced into thr phage mutant. Thus genetic anal;ysis of developmental genes in wild-type strains of M. zanthus is now possible.

1. Introduction Myxococcus xanthus is an ideal prokaryote for the study of the regulation of develop- ment’. The complex life cycle involves a temporal sequence of cellular aggregation, mound formation, and myxosporulation (Dworkin, 1966). Unfortunately, many studies with these organisms are limited by the lack of a genetic transfer system. While several phages have previously been isolated in species of myxobacteria (Burchard & Dworkin, 1966; Brown et al., 1976: Anacker & Ordal, 1955), none of these was found to be capable of genetic transduction. Recently, Kaiser & Dworkin (1975) reported genetic transfer of chloramphenicol resistance between Escherichia co& a.nd M. za,nthus using coliphage PICM. In addition. Parish (1975) reported transfer

t Present address: Department of Microbiology and Immunology, Temple University Medical School, Philadelphia, Pa 19140, U.S.A.

$ Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Mass. 02116, U.S.A.

§ To whom reprint requests should be sent.

l(i7

I ti?i J. M. C’AMPOS. .I. GElSSELSO1)El-i ANI) 1). R. ZUSMAN

of chloramphenicol, kanamycin and neomycin resistance from an R factor-carrying strain of E. coli to either M. xanthus or Myxocmcus fulvus. In both cases, the anti-

biotic resistance phenotypes were unstable in the recipients, suggesting that the transferred genetic material failed to integrate into the recipient chromosomes.

In this paper, we describe the isolation from nature of phage MX4, an efficient

generalized transducing phage for M. xanthus. Procedures for stable transduction of auxotrophic and antibiotic resistance markers were established. In addition, a phage mutant was isolated which shows high transduction frequencies. With this mutant at high multiplicities of infection, the frequency of transduction among survivors is

sufficient to permit the direct screening of unselected markers among viable recipient

cells.

2. Materials and Methods (a) Bacterial and bacteriophage strains

A description of the strains used is presented in Table 1. Mutants were isolated as described below.

(b) Media and cultural conditions

Bacteria were routinely grown in CT broth (1% Casitone, 0.1% MgSO,. i’H,O) or CYE broth (1% Casitone, 0.5% yeast extract, 0.1% MgSO,. 7HzO; pH adjusted to 7.0 with NaOH). Phage MX4 was usually grown as a liquid lysate on exponential phase cultures. Early log phase cultures (2 x IO8 cells/ml) completely lyse when infected at a multiplicity of about 0.01. Stationary phase cultures appear to be resistant to adsorption. Phage were also grown as plate lysates.

For many transduction experiments we used a new growth medium, 17P. 17P supports the growth of wild-type strains of M. xanthus with good plating efficiency using the soft agar overlay technique. The medium does not support the growth of liquid cultures. The requirement for pyruvate was determined by Bretscher & Kaiser (unpublished results).

17P bottom agar contains 17 amino acids: L-&nine, n-arginine, L-asparagine, L-cysteine, r,-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tyrosine, L-valine each at 50 pg/ml, L-proline (1 mg/ml), sodium pyruvate (1 mg/ml), tris-hydroxymethylaminomethane (1.2 mg/ml), NaCl (0.2 mg/ml), KHzPO, (O-14 mg/ml), MgS0,.7H,O (0.1 mg/ml), (NH&SO4 (0.1 mg/ml), C&l, (0.002 mg/ml), and Ionagar no. 2 S (0.8%) (Colab). The pH was adjusted to 7.5 with HCl.

17P top agar is identical to the bottom agar except that it contains 0.4% Ionagar. For some experiments, we used a minimal version of 17P, 4P, which was identical to 17P except that it contained only 4 amino acids : L-asparagine, L-phenylalanine, L-tyrosine, and L-proline, each at 50 pg/ml except proline (1 mg/ml).

(c) Isolation of phage MX4

Bacteriophages were isolated from soil and manure collected at the Tel Aviv University research zoo (J&a, Israel) and from material taken from a farmyard manure pile near Firebaugh, Calif. U.S.A. A small amount of the material was suspended in 0.1% MgSO, * 7H,O. The suspension was passed through a sterile Millipore filter apparatus (0.45 pm pore size) and added to exponentially growing cells of M. zanthus DZl. After overnight incubation at 3O”C, the culture was treated with chloroform and centrifuged to remove cellular debris. Dilutions of the supernatant were added to DZl indicator cells and plated in CT agar. Plates were examined for phage plaques after 48 h at 30°C.

(d) Auxotroph isolation

Cultures were mutagenized with N-methyl-N’-nitro-N-nitrosoguanidine by adding 4 pg/ml directly to an exponential phase culture and incubating overnight with shaking at 30°C. Since many of the mutants isolated from these cultures had high reversion rates (about 10e5 to 10e6), we also used the frameshift mutagen ICR-191 (gift of Dr H. Creech).

TABL

E 1

Bact

eria

an

.d B

acte

rioph

age

.dra

ivL.

r

S t,ra

in Pa

rent

LMuta

gen

Phen

otype

? So

urcP

M.

rnnt

hus

DZl

DZ

l-59

DZl-1

01

DZl-1

02

DZ2

FB

DZF6

DZ

F25

DZF3

3 DZ

F37

DZF3

8 DZ

F41

DZF4

5 DZ

F66

DZF8

9 DZ

F91

DZF9

9 DZ

F121

DZ

F140

Hacte

rioph

aga

MX4

MX4

ts18

MX4

ts18t

s27

1MX4

tsl

8ts2

7htf-

-1

MX4

hrm

-1

MX4

ts27h

tf-lhr

m-I

ERl

spon

taneo

us

DZl

Nitro

sogu

anidin

r DZ

l sp

ontan

eous

DZ

l sp

ontan

eous

- FB

DZ

F6

DZF6

DZ

F6

DZF6

DZ

F6

DZF6

PB

DZ

F38

DZF3

8 DZ

F38

DZF3

8 DZ

F26

Nitro

sogu

anidin

r IC

R-19

1 IC

R-19

1 IC

R-19

1 IC

R-19

1 IC

R-19

1 IC

R-19

1 Ni

troso

guan

idine

ICR-

191

ICR-

191

ICR-

191

ICR-

191

Spon

taneo

us

MX4

.MX4

ts

l8

- Nitro

sogu

anidin

e Ni

troso

guen

idine

Nitro

sogu

enidin

o

11x4

No

m

St+,

Fr

u -

, Mo

t -

Zusm

an

et nl.

(19

78)

Stra

, Fr

u ~,

Mo

t -,

Bio

- St

ra,

Fru

- ,

Mot

- ,

Spca

, Er

ya

St+,

Fr

u-,

Mot

-. St

da,

Rifa

Wild

type

Camp

os

c Zu

sman

(19

75)

Wild

type

Dwork

in (19

62)

Spca

, MX

4s

MX4*,

Tr

p -

MX4s

, Pa

n- MX

4s,

Dal-

MX4s

, Ad

e -

MX4=

, Tr

p MX

4s,

Ad?

Trp

- MX

4S,

Ado-,

Me

t MX

4s,

Ade

~,

Lys

- MX

4S,

Ade-,

His

MX

4s,

Ade-,

‘I%

- MX

4s,

Trp-

, Ri

fa

Wild

type

Soil

and

co\%

dunp

Te

mpera

ture-s

ensit

ive

Temp

eratur

e-sen

sitive

Te

mpera

ture-s

ensit

ive

high

freyu

ency

ge

ncral

izecl

trans

ducti

on

Host

range

m

utant

1).

Kaise

r Te

mpe

ratur

e-se

nsitiv

e. hig

h tra

nsdu

ction

fre

quen

cy.

- ho

st ran

ge

muta

nt

t Sy

mbols

for

ph

enoty

pes

art’:

ability

t,o

fo

rm

wild-

type

fruitin

g bo

dies,

Fru+

; m

otile,

Mo

t +

; ab

ility

to pla

que

phag

e MX

4, MX

4S

; oryt

hromy

cin-re

sistan

t (50

pg

/ml),

Er

yR:

rifamp

icin-re

sistan

t (50

pg

/ml),

Ri

fa;

sprct

inrm

~ycin

-resis

tant

(500

pg/m

l),

SpcR

; str

eptol

ydigi

n-res

istan

t, St

da.

, stre

ptom

ycin-

resis

tant,

(1 m

g/m

l),

StrR

: ad

enos

ine,

.idr;

n-alan

ine.

Dal:

biotin

, Ri

o; his

tidine

, Hi

s; lys

ine,

lys;

methi

onine

, M

et;

panto

thenic

ac

id,

Pan:

thiam

ine.

Thi;

trypto

phan

. Tr

p.

170 .J. ill. CAMPOS. .I. GEISSELSODER ANI) I). K. %l;S>I.AS

ICR-191 (4 to 8 pa/ml) was added to mrly log phase cultures a~td i~~cwt~atwl f’or 30 II ii~ 30°C wit)11 shaking. Mutagenized criltjurcs were plated on CYE agrr. For arixotropl~ isolations, about 10,000 colonies were picked ont,o master plates. ITsing a rnnltiprongc~ti inoculator (consisting of 48 metal pins arranged in 8 rows of 6 pills) thr coloiiit*s \v(~I’(’ replica picked onto CYE agar. 17P agar and 4P agar. The mutants woe charact,crizrti h>. t,esting colonies for growth on mediacorlt,ainillg appropriate supplrmc~nts.

(e) Isolation of temperature-sensitive yhage mutants

Exponential phase cultures of DZl growing in CYE were infected witti phage MX4 at a multiplicity of infection of 10. After 10 min of incubation at 28”C, the cells were collected by centrifugation, washed with TM buffer (tris-hydroxymethylaminomethane, sodium maleate, and ammonium sulfate, each at. 0.05 fir, pH 6) and resuspended in TM buffei containing 100 pg nitrosoguanidine/ml. After 50 min incubation in t,he dark at 28”C, the cells were centrifuged, washed, and resuspended in fresh CYE broth. The cells were then incubated at 28°C with shaking until visible lysis occurred. The lysates were treated with chloroform, centrifuged to remove cell debris, and st,ored over chloroform at 4°C. Muta- genized plaques were picked onto mast)er plates (CT medium) seeded with DZl and con- taining prong marks which matched our multipronged inoculator. After incubation for 2 days at 28”C, the plaques were replica picked with the inoculator onto duplicate plates seeded with DZl. The replica plates were then incubated at 28°C or 35°C. Several t,c,rn- perature-sensitive phage mutants were isolated in this manner.

(f ) Transduction experiments

Auxotrophs were generally transduced to prototrophy as follows. Phage (0.1 ml), propagated on the appropriate donor strain, were added to O-2 ml of recipient cells at a multiplicity of infection of 0.1 to 0.3. Recipient cells were usually prepared by centrifuging exponential phase cells (5 x 10s to 10 x 10s cells/ml) and resuspending them in 0.01 M-Tris buffer (pH 7.5), containing the salts mixture used in the 17P medium. Selective top agar (2.5 ml), consisting of 17P or 17P minus the required amino acid, was added to the infected cells and the mixture poured onto selective bottom agar. Plates were incubated for 5 to 10 days at 35°C before scoring for transductants. Uninfected cells were also plated on the selective media as controls.

Antibiotic resistance markers were transduced as follows. Phage (MX4 tslSts27),

propagated on the appropriate donor strain, were added to exponential phase recipient cells at a multiplicity of infection of 0.5. The infected cells were incubated for 8 to 12 h with aeration at the restrictive temperature for the phage, 35°C. Samples (0.1 ml) were plated in 2.5 ml CT top agar on CT bottom agar containing the appropriate antibiotic. Uninfected cells were also plated in the antibiotic media as controls. Plates were incubated for 5 to 10 days at 35°C before scoring transductants. Transductants resistant to one antibiotic were sometimes screened for resistance to a second antibiotic to determine possible cotransduction of the markers.

(g) Isolation of a high frequency transdwtion mutant

An exponential phase culture of DZI growing in CYE broth was infected with phage MX4 tslSts27 at a multiplicity of infection of 3. After 5 min at 28”C, nitrosoguanidine (100 rg/ml) was added to the culture ; the culture was incubated until lysis was observed. The lysate was treated with chloroform, centrifuged to remove debris, and stored at 0°C.

The titered lysate (l-2 x log phage/ml) was diluted and plated in soft CT agar containing DZl indicator cells. After 3 days incubation at 28”C, approximately 5000 plaques were picked onto master plates containing CT agar seeded with DZl indicator cells. After incubation at 28”C, the master plates were replicated with the inoculator onto test plates containing 17P medium (which lacks tryptophan), rifampicin (25 pgglml), and indicator cells (DZF140; Trp-, Rifa). The test plates were incubated for 7 days at 35°C to prevent phage killing of transductants. Transductants were found in or near most prong marks. Control plates gave only 2 or 3 Trp+ revertants per plate. Plaques which corresponded

GENERALIZED TR.4NSDUC’l’IO~ IS 31. S=1~VTHCS 151

to clustrIs of t,ransductants on the tjest plates (5 or I~O~P) were picked w-ith Pasteur pipets and resuspended in 1 ml of 0.02 AI-N&l. TIw phape snspensions LWTP t,hen tit,pred and testfd for tmnsd~~ction frequent-y at several rmlltiplicities of infectioll.

3. Results

Genetic studies of development in M. xmthus have not been possible because of the absence of any genetic transfer system. We t’herefore isolated six new phages from soil and dung, the natural habitat’ of the myxobacteria (see Materials and Methods). Preliminary experiments wit’h our phages indicated that three of the isolat#es (one isolated in Israel. two in California) were very similar and were capable of t)ransducing an auxotrophic mutant of DZ1 to protot)rophy. One of the California isolates, phape MX4. wa,s chosen for further examination. Physical characteriza,tion of the phage is provided in the accompa.nying paper (Geisselsoder nt al.. 1978).

Phage MX4 forms large clear plaques on DZi and completely lyses liquid cultures of this st’rain, yielding phage titers in thr range of 10” to 101’ phage/ml. A one-step growth cycle of MX4 shows a latent period of 180 minutes and an average burst, size of 75 progeny/infected cell. The host showed a generat,ion time of approximately 270 minutes at 35°C.

(b) The problew of hoot mlqr

A’. xnnthus strain DZl is an excellent host for phage MX4 but is non-motile and unable to participate in the developmental cycle leading to fruiting body formation. If transductional analysis of myxobacterial development were to be possible: a solution had to be found providing for phage progeny production in a fruiting host strain. We first explored the nature of the phage restriction in the “wild-type” host strain FB. A one-step growth experiment was performed in which phage-infected strain FB was plated in soft agar containing indicator cells of strain DZl (Fig. l(a)). The experiment showed that, phage adsorb to t’he \+ild-type host, since an eclipse period is observed. About 1 vi0 of the cells actually showed a small burst. However. the net result of one cycle of infection was about a tenfold decrease in viable phage per ml. Host modification and rest’riction of the DNA was considered unlikely since the progeny phage never form plaques on a lawn of FB.

The possibility that phage development was blocked at the transcriptional or translational level was explored by isolating mutants of strain FB resistant to rifampicin (50 pg/ml, 1000 mutants), streptomycin (1 mg/ml, 131 mutants), spectino- mycin (0.5 mg/ml, 200 mutants), or erythromycin (50 pg/ml, 52 mutants) and then screening each colony as hosts for plaque formation. Analysis of the mutants showed that only some of the spectinomycin-resistant mutanbs (about 7%) were able to plaque phage MX4. A one-step growth experiment with one of these mutants, DZF6, is presented in Figure l(b). Here, the net result, of one cycle of infection is about a tenfold increase in phage per ml. The one-step growth curve also shows that only 20 to 250/ of the phage-infected cells give rise t,o progeny phage and form a plaque. This is consistent with the 7574 lower plating efficiency one observes with these strains when compared to strain DZl. Adsorption of phage MX4 on FB or DZF6 is about 95% in 10 minutes. DZF6 is unaltered in development and forms normal fruiting bodies. In contrast, some of the spe&inompcin-resista,nt mutants were defeot.ive in development.

10’0 I-- Wild type

100 ZOO (a) (b)

Time after infection (min)

Fxa. 1. Growth of phage MX4 on wild-type M. xanthus strain FB and on a speotinomyoin- resistant mutant DZFG. M. xanthw FB (a) or DZF6 (b) were infected with phage MX4 at multi- plicities of infection of about 3. Thirty min after infection, antisera to phage MX4 was added to inactivate mu&orbed phage. Samples (0.1 ml) were removed at intervals and either assayed directly for plaque-forming units (--O-O-), or treated with chloroform prior t,o titering (-O-O-). All plaque assays were made with strain DZl as indicator.

(c) Generalized transduction with phage MXd

Since M. xanthus DZF6 was able to plaque phage MX4, we chose this strain for mutagenesis and auxotroph isolations (see Materials and Methods). Table 2 contains a list of the auxotrophic mutants which we successfully transduced to prototrophy. Preliminary experiments indicated that many of our transductants were being killed by viable lytic phage. We therefore isolated a temperature-sensitive phage mutant, ts18, for these experiments and carried out all incubations at the restrictive tem- perature, 35°C. The frequency of transduction per viable pha,ge was between 2 x 1O-s and 2 x 10e5 for most markers tested.

Trp + transductants of strain DZF41 were tested for stability and phage sensitivity. The transductants maintained their Trp+ phenotype after several generations of growth in rich medium. Liquid cultures derived from transductant colonies did not release phage progeny during growth. When used as indicator cells, transductants supported phage MX4 plaque formation at 100% efficiency. Donor phage propagated on strain DZF41 failed to transduce strain DZF41 to prototrophy, but did transduce other auxotrophs to prototrophy. These properties, taken together with the diversity of recipients suocessfully transduced, are consistent with a generalized mode of transduction.

GENERALIZEI) TRANSDUCTIOh’ IK M. Xd-VY’HIIS

TABLE 2

Transduction of auxotrophu to prototrophy by bacteriophage MX4t

I73

Recipient Phenotype transduced Transductants/plaque forming

to prototrophy unit

DZF25 DZF33 DZF37 DZF41 DZF4b DZF66 DZF89 DZF91 DZF99 DZFlZl DZl-59

Trp pan Dal ‘lhp Ade - Trp Met - Lys - His Thi- Rio -

1.5x 10-S 1.9x 10~~” 3.6x 10-V 2.7 x 10 5 1.4x 10-s 2.8 x lo-” 3.8~ IO-” 4.1 x 10-E 4.1 x 10-h “.9x 10-S 2.6 x 10-Z

t Phage MX4 ts18, propagated on strain DZl, was mixed with cells of the specified strain at low multiplicities of infection. Tho mixtures were plated in soft agar (in quintuplicate) on the appro- priate selective bottom agars. Plates were incubated for 5 to 10 days at 35°C and then scored for transductants. The data represent t,he best transduction frequency normally observed with each recipient.

The transduction of antibiotic markers is more difficult than auxotrophic markers since outgrowth under non-selective conditions is required to permit expression of the newly acquired phenotype. Superinfection and killing of potential transductants during outgrowth was observed, even with the temperature-sensitive phage, ts18, at the restrictive temperature, 35°C. We therefore isolated a double temperature- sensitive mutant, ts18ts27, for these studies. The plaque-forming efficiency of this double mutant was about five logs lower at 35°C than at the permissive tempera,ture, 28°C. Table 3 lists the results of several transductions to antibiotic resistance. The frequencies of transduction, after correcting for the appearance of spontaneous mutants and the increase in cell numbers during the eight hours of outgrowth ranged between 6.7 x 10e7 and 4.6 x low6 transductantslviable phage. Cotransduction was

TABLE 3

Transduction of antibiotic resistance

Bacterial donor and phenotype

Antibiotic-resistant Recipient Selected antibiotic resistance colonies per plate

--- Phage + Phage

DZl-101 (Spcs, Erys) DZF25 DZI-102 (StdR, Rifa) DZl DZl-102 (Stda, RifR) DZF26 DZl-102 (Std*, RifR) DZF26 DZl-102 (StdR, RifR) DZFPS

Erythromycin 4 28 Rifampicin 6 42 Rifampicin 10 143 Streptolydigin 11 32 Streptolydigin and rifampicin 0 14

Phage MX4 ta18t.927 was added t,o recipient cultures at a multiplicity of infection of 0.5. The cultures were incubated for 8 h at 36°C before plating on selective bottom agar. Plates were incubated for 7 days at 36°C.

154 J. M. CAMPUS, J. UEIS8El~HOI)EH ANI) I). It. ZUSMA?;

TABLE 4

Cotran.sdwtion jrequewy of streptolydigin and sifantpicipz resistancest

Selected drug Colonies Rifampicin-resistant Streptolydigin-resistant Cotransduction resistance tested colonies colonies (%I)

1. Streptolydigin + phage 59 59 100 - phage 65 0 0

2. Rifampicin + phage 54 48 88 - phage 20 0 0

t Streptolydigin and rifampicin-resistant colonies which arose in a transduction experiment using phage MX4 ta18ts27 grown on DZl-102 were tested for resistance to both antibiotics. The data are corrected for the number of spontaneous mutants to antibiotic resistance observed in control experiments.

observed between rifampicin resistance and streptolydigin resistance (Table 3). The frequency of cotransduction (Table 4) was 88 to lOOo/,.

(d) High jreyuency generalized transduction

Since developmental functions may not always permit a positive selection for transductants, we searched for a mutant of phage MX4 which shows a high trans- duction frequency (htj) (see Materials and Methods). Testing of the most promising mutant plaques showed that one of these, htj-I, had a tenfold increase in transduction frequency per plaque-forming unit for the Trp marker. This increased frequency of transduction was maintained even at high multiplicities of infection (Fig. 2). Thus, at a multiplicity of about 8, the frequency of Trp + transductants among viable colonies was about 8 x 10m4 or 1 in 1200 colonies. Similarly high transduction fre- quencies were obtained for other markers tested including His, Lys, and Met (Table 5). For example, at a multiplicity of 20, the frequency of His+ transductants among viable colonies was very high, about 4.7 x 10e3 or about 1 in 200 colonies. However, transduction of the rij” locus was never higher than 10e5 transductants/recipient (about 50-fold higher than the parental phage). The htj-2 mutant is therefore useful for high frequency generalized transduction.

(e) Host range high transduction frequency mutants

While this work was in progress, Marianne Wolfner, working in Dale Kaiser’s laboratory, isolated a mutant of phage MX4 which is able to form plaques on wild- type strains of M. xanthus. The mutant, originally named MX4*, will be referred to here as MX4 hrm-1 (host range mutant). A phage cross was performed with MX4 hrm-1 and MX4 tsl8tsZTh[f-1 by infecting a culture of DZl with both mutants at multiplicities of 2 and 8, respect’ively. The progeny phage were titered on strains FB and DZl and 35°C and 28°C. A total of 26% were found to be of the hwn-l- genotype and 76% of the hrm-l+ genotype. The hrm-l- progeny were then screened for temperature-sensitive recombinants : 4.3 y. contained the ts27 genotype (the ts18 mutation is too leaky to follow). A total of 45 hrm-lts27 recombinants were then screened for the htj-1 locus by picking the plaques and examining the frequency of

I I 1 I 0 4 8 12 16

Multlphty of mfectmn

FIG. 2. Effect of multiplicity of infection on transductants per viable colony. Phage MX4 la18 ts27 (-o-o-) or tsl&s27hfl-1 (-O-e-) was added at various multiplicities of infection to exponential phase DZF140 (Trp-, Rifa). After 6 min, the cells were diluted and plated on a selective medium consisting of 17P plus rifampicin (25 pg/ml) or on a supplemented medium which also contained tryptophan. The Trp + transductants on the selective medium and the viable counts on t,he supplemented medium were determined aft,er 7 days growth at BFi”C.

transduction of a Trp- auxotroph (DZF140) to prototrophy. Three such recombinants were found.

The MX4 ts27htflhms-1 recombinants form plaques with equal efficiency on strains DZl and FR. The high transduction frequency characteristics of recombinants using the Trp marker are similar to those of the parental htf-1 phage. The recom- binant phage has also been used for transduction of unselected markers. For example, yellow pigment was transduced into a red pigmented strain of FB. About 1 in 2000 colonies showed the yellow (wild-type) pigment. A motility gene was also transduced in a similar manner by direct screening of surviving colonies.

4. Discussion 111 this paper, we report the isolation of a new bacteriophage, MX4, which is

capable of generalized transduction in M. xanthus. The average transduction fre- quency for most markers tested is high, about 0.5 x lo- 5 to 5 x 10e5 transductantsj plaque-forming unit. This frequency is similar in magnitude to the frequencies observed with Pl transduction in Escherichia co& (Wall & Harriman, 1974) or P22 transduction in Salmonella typhimurium (Schmieger, 1972). The frequency is especially high considering the much larger size of the M. xanthus chromosome (3 to 4 times

TABL

E 5

Tran

sduc

tion

frequ

encie

s at

high

m

ultip

licitie

s of

inj

ectio

n at

35°

C

Recip

ient

Selec

ted

strain

m

arke

r m

.o.i.+

td8t

s27h

tf-1

Survi

val

Tran

sduc

tant

s/viab

le co

lony

(%)

m.0

.i.

tal8t

s27

Survi

val

(%)

Tran

sduc

tttnt

s/viab

le co

lony

-.

DZF1

40

TOP

I.5

14

7.6

x 1O

-4 6.

8 16

i.9

,r:

1O-

6

DZF9

9 Hi

s 10

35

2.

1 x

10-a

20

18

4.

7 x

10-a

20

19

“.0

x 10

-S

DZF9

1 Ly

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GENERALIZED TRANSDUCTION IN M. XALVTHUS 177

larger than E. co&; Zusman et al., 1978) and the size of the phage genome (M, 39 x lo6 ; Geisselsoder et al., 1978). I f one assumes that transducing particles package all genes with equal frequency (1 x 10m5) and that recombination occurs in about 5076 of all infected cells, we can estimate the frequency of transducing particles present in a phage lysate at about one transducing particle/250 phages. The high frequency t’ransducing phage mutant, htf-1. may produce about ten times that number or one per 25 phages. The combination of the htf mutation and double ts mutations permit* us to use high multiplicities of infection without, extensive host killing at 35°C. Under these conditions, we can increase the number of transductants per viabl- host cell 50 to loo-fold (Fig. 2 ; Table 5). This frequency is sufficiently high to permit the routine screening of unselected markers among transductants. Genetic analysis of many developmental mutants is now feasible.

The problem of host restriction of phage MX4 in wild-type strains of M. xanthus presented a significant barrier for further work with this phage. We were able t,o isolate host mutants which plaque the phage and remain capable of fruiting body formation. These mutants were found as a subclass among spectinomycin-resistant colonies. Spectinomycin-resistant mutants were found to be heterogeneous in pheno- type. Some of these mutants showed altered growth rates, colony morphology, phage growth, and ability to form fruiting bodies. These results with M. xanthus are analogous to those of Chakrabarti & Gorini (1975) who found that 24 of 26 spon- taneously isolated independent streptomycin-resistant mutant’s of male E. coli were

pleiotropically able to plaque phage T7. The problem of host restriction of phage MX4 was better resolved with the isolation

of t,he host range mutant, hrm-1. This mutant permits phage growth on all strains of M. xanthus tested. We introduced the hrm-1 locus into the ts27htf phage mutant, without significantly changing its transduction frequencies. Thus, the developmental mutants already isolated in several laboratories can now be used as hosts for the phage. The genetic control of cell-cell interactions during aggregation, mound formation and myxosporulation is clearly an important area for future work.

This research was supported by grants GM 20509-04 from the National Institute of General Medical Sciences and PCM 75-09231 A01 from the National Science Foundation. This project was started while one of us (D. Z.) was visiting the laboratory of Dr Eugene Rosenberg at the Department of Microbiology, Tel Aviv University, Tel Aviv, Israel. The authors thank Joanne Uomini for performing the experiments described in Figure 1 and for wonderful assistance in the laboratory. The authors also thank Dr Dale Kaiser and his laborat’ory for sharing their unpublished findings with us and for reading the manuscript.

ADDENDUM

While this work was in progress, a second generalized transducing phage has been isolat>ed for M. zanthus called MX8 (S. Martin and D. Kaiser, personal communication). This phage has recently been used for studying t,he genet,ics of motility (Hodgkin & Kaiser, 1977).

REFERENCES

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(1976). Arch. 1MicrobioZ. 108, 271-279. Burchard, R. P. & Dworkin, M. (1966). J. Bacterial. 91, 1305-1313. Campos, J. M. & Zusman, D. R. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 518-522. Chakrabarti, 8. & Gorini, L. (1975). J. Bacterial. 121. 670.674. Dworlrin, M. (1962). J. Racterbl. 84, 360 2.57.

178 J. M. CAMPOS, J. GEISSELSODER AXII) 1-j. R. ZUSMAX

Dworkin, M. (1966). Anlzu. Rev. Microbial. 20, 75--106. Geisselsoder, J., Campos, J. M. & Zusman, D. R. (1978). ,I. Mol. Biol. 119, 179-189. Hodgkin, J. & Kaiser, D. (1977). P rot. Nat. Acad. Ski., V.S.A. 74, 2X38- 2942. Kaiser, D. & Dworkin, M. (1975). Science, 187, 653-655. Parish, J. H. (1975). J. Gen. Microbial. 87, 198-210. Schmieger, H. (1972). Mol. Gevz. Belzet. 119, 75-88. Wall, J. D. & Harriman, I’. D. (1974). Virology, 59, 532-544. Zusman, D. R., Krotoski, D. M. & (‘urnsky, M. (1978). J. Bacterial., 133, in t,hf: press.