Characterization of the gene transfer agent made by an overproducer mutant of Rhodopseudomonas capsulata

J. Mol. Bid.

(1979) 131, 157-168

Characterization of the Gene Transfer Agent Made by an Overproducer Mutant of Rhodopseudomonas capsulata H. c. YEK,

N. T. Hu AND B. L. &RR5

E. A. Daisy Department of Biochenristry ASaint Louis University A’aint Louis, MO. 63204, TJ.8.A. (Received

7 July

1.978, and in revised form, 79 December 1978)

Most wild-type isolates of the photosynthetic bacterium Rhodop8eudomon.as capsulata spontaneously produce nucleoprotein particles that act as vectors of genetic exchange in this species. The low yield of these particles, termed gene transfer agents, has made their characterization difficult. We have utilized a new screening technique to select a mutant, Y262, which produces about three orders of magnitude more GTAt per culture. The GTA produced by Y262 seem to bc identical to those of wild type by immunological, genetic and sedimentation analyses, and they have been further characterized by a variety of techniques. GTA resemble tailed bact,eriophage, although GTA with a head diameter of 30 nm are smaller than most morphologically similar viruses. DN.4 isolated from GT;Z weight. There, is in the form of linear duplex molecules of about 3 x lo6 molecular is no phage-like DNA in GTA particles as judged by C,t and restriction endonuclease analyses. Instead, GTA DNA shows the same kinetic complexity and restriction endonuclease digestion pattern as the R. capsulatu genome. The GTA of ten independently isolated wild-type strains of R. capsdata show a high degree of immunological cross-reactivity; thus this genetic exchange system, while not a species-wide trait, does seem to bc the produrt of a conservative rvolut~ionar> process.

1. Introduction An unusual mechanism of genetic exchange has been demonstrat’ed in Rhodopseudomenus capsuluta, a non-sulfur purple photosynthet’ic bacterium (Marrs, 1974). A majority of the wild-type strains of this species spontaneously release DNA-containing particles, termed gene transfer agents, which serve as vectors for transmission of genetic information to other cells in the culture (Wall et al., 1975; Solioz et al., 1975: Solioz & Marrs, 1977). Genetic transfer has been demonstrated for a wide variety of markers, and it appears that any region of the chromosomes may be transferred. A map of the region of the chromosome coding for carotenoid and bacteriochlorophpll synthesis has been constructed using this genetic system (Yen & Marrs, 1976). Although GTAT-mediated genetic exchange clearly resembles generalized transduction, several features of the GTA system are not typical of genetic exchanges mediat)ed by bacterial viruses. No viral activities are found associated with gem i Abbreviations used: GTA, gene transfer agents; Cot, moles of nucleotides the key parameter in renaturation kinetics analysis.

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transfer activity; i.e. no plaque-forming or killing activit(irs have been tlet.ectetl in active, purified GTA preparations, and GTA preparat,ions do not bestow t,he abilit,y to produce GTA upon naturally occurring non-producing strains, even though such strains can receive genetic information from GTA (Wall et nb., 1975; Yen & Maws. unpublished data). The kinetics of GTA release by growing cultures are unlike t-hi kinetics of phage release by lysogenic cultures (Solioz et al., 1975). The GTA parti+ has a slower sedimentation rate and carries a smaller piece of DNA than any knouw transducing phage (Marrs, 1974; Solioz & Marrs, 1977). Naturally occurring strains of’ R. mps’ulata produce quantIit’ies of GTA that, arti easy t,o detect by bioassay yet are insufficient for direct, chemical or physical charact.erization. In this paper we describe t,he isolation of a mutant. strain of N. eapsuhttt that produces approximately 1000 times as much GTA as the wild-type. We domonstrate that thr GTA produced by t.his mutant is essentially the same as t,he wild-typci product,, and we further characterize t,he particle and its nucleic acid. It is demonstrated that there is no detectable virus-like DNA in GTA particles, and that CXA isolated from diverse geographic: produced by wild-type strains of R. capsdata locations shorn a high degree of immunological cross-reactivity. These results aw int’erpretated as evidence that this genetic exchange s>rstem has evolved in fi’. capsdata as a means of promoting recombination.

2. Materials and Methods (a) Bacterial The strains

of K. capsulata

used in ttlis work

strailts are listed

and descriIWd

in Table

I.

Peptone-yeast extmct (PYE), HCV and 7Y media Ilave been described elsewhere (Y(~II & Marrs, 1977), as has G-buffer (Solioz & Marrs, 1977). Media for t.hc growth of YBlO23 were supplemented with 10 pg L-tryptophan/ml. Solidified media were ma.de with 1*20/b ngar, except for soft, agar, which was 0.6% agar. Anaerobic incubation of plates was Ttontitw growth procedures have bwt achieved with the Gas Pak system (Bioquest). described (Yen & Marrs, 19’76).

From 200 to 400 colony-forming units of nitrosog\lallidirle-lnlltagerrizecl (IMarrs et
The following procedure is suitable for purifying GTA from cultures of Y262 or ot,h+r overproducer strains, and is considerably simpler than t,hat necessary to purify GTB beginlliny with t,llr lower tibers present in t,hc wild-type rrdtures (Solioz 6%Marrs. 1977). Ttrcb

GENE

TRANSFER

AGENT

OVERPRODUCER

I .Y!)

cell-free supernatant from a 30-min, 8000 g centrifugation is passed through all dmicotl thin-channel filtration system TCF-IO fitted with a DP045 membrane. The clear filtrat,ct is concentrated 20-fold on a Millipore Pellicon cassette system, using membrane typo PTHK 000 01, and then precipitated with 10% (w/v) polyethylene glycol (carbowax 6000) at pH 7 in the presence of 0.5 M-Nacl. The precipitate is resuspended in 5 to 10 ml of G-buffer and layered on 15% to 30% sucrose gradients and centrifuged at 15,000 revs/mill for 16 h in a Beckman SW25 rotor. The fractions containing GTA activity are determined by bioassay (Solioz & Marrs, 1977), pooled, and concentrated on an Amicon model 52 stirred cell fitted with a PM10 membrane. Then 3 ml of concentrate are layered on 9 ml of saturated RbCl and centrifuged in a Ti50 rotor at 47,000 revs/min for 24 h. The GTA band at, p : 1.33 can be recognized by it,s turbidity or by bioassay for gene transfer activity. (H) Gel electrophoresis GTA proteins were sized by electrophoresis of a 75-rg sample on a 7.5% to 15:’ polgacrylamide gradient gel, essentially as described by Chua & Bennoun (1975). Bgarose gel electrophoresis and restriction enzyme digestion of GTA DNA were performed essentially as described by Murray & Murray (1975). Polyacrylamide gel electrophoresis of DNA was performed according to the method of ,Jeppesen (1974). (f)

C,t analy&s

Purified GTS were dialyzed against 0.01 M-Tris.HCl (pH 7.5), 0.01 M-MgSO, and the11 extracted with Tris-saturated phenol until no precipitate appeared at the water/phenol interface. The aqueous phase was then dialyzed against 0.015 M-sodium citrate, 0.15 XIsodium cllloride, pH 7.0. DNA concentration was determined by the diphenylamine reactiolt (Burton, 1956). This DNA was then fragmented by 2 passages through a French pressure’ cell (American Instrument Company) at 12,000 lb/in’. The fragmented DNA was denatured into separate strands by boiling in 0.18 M-NaOH for 5 min, followed by immediate cooling on ice. The solution was then neutralized with 0.9 M-HCl in the presence of O-01 m-PIPES. The reassociation reaction was carried out in micro reaction vessels (Supelco) at 67°C. Thrl Pxt,ent’ of reassociation was determined by S1 nuclease digestion (Britten et al., 1974). (g) Antibody

preparation

awl assays

Antibodies were raised in rabbits by direct injection of GTA (purified as described abovcs) into the ear vein. An initial injection (2 mg GTA protein/rabbit) was followed by 2 boosters ( 1 mg each) at 2 week intervals. High anti-GTA titers were observed about 6 weeks after the initial injection. The serum for the experiments described here was collected from one rabbit, which was sacrificed about 10 weeks after the initial injection. The immunoglobitl G fraction was prepared from that serum by precipitation with (NH,),SO, and DEAEchromatography. Control serum was taken from each animal before the first injection of GTA. Antibody inactivation of gene transfer activities from various strains of h?. capsdattr was measured by the following procedure. A cell-free filtrate was prepared from a PYEgrown culture of each strain ; 0.2 ml of filtrat,e was mixed with 0.4 ml of G-buffer and 0.4 ml of each of a series of dilutions of antibody, and incubated at 35°C for 20 min. A 0.2-ml suspension of recipient cells (YB1023) in G-buffer was then added to each and incubation continued for 1 11 at 35°C. Cells were then plated in minimal medium (RCV) to quantitntcb the number of t,ryptophan-independent recombinants resulting from surviving GTA.

3. Results (a) Isolation

of GTA-overproducing

m,utan.t

A procedure was designed to screen for clones of R. capsubta that produce more GTA than wild type (see Materials and Methods). The method relies on visualizing gene transfer occurring in solidified medium between photosynthetically competent, donors seeded in the agar and an overlying lawn of non-photos@hetic mutants.

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Since most growth media for K. capsdata inhibit GTA-mediat’etl genetic exchange at one point or another (Solioz et aZ., 1975), a new medium? iv. was dcvisetl thal allowed GTA production aud utilization of GTA-derived genetic information. a:: u (,]I as colony formation. Figure 1 shows bhe resuhs of a reconstruction of this OL-Nproducer-screening procedure using a mixture of wild-type (SB1003) and mutant (Y262) cells. Y262 is a GTA-overproducing st’rain that was isolated by ,scrc:ening mutagenized cells as described in Materials and Methods. Y262 forms smaller color& t,han SB1003, but both produce wild-t,ype pigmentation, Each Y262 colony is toppta(i transferants which RI'Oht by a disc of photosynthet8ically competent microcolonies. from the interact,ion between GTA from the YZfk? tiottw ~lony and 1.1 (i;S (photo svnthetically incompetent) recipient cells in t,he overlying Iall 11. The ~a~c)tt~rloic-ll(,~~ phenotype of the microcolonies serves as a marker, tlcmonstratin, cr that t~llexo colonicx\ are indeed derived from the Yl6Ei parent. (Bacteriochloro~)~~~l~ has only a minor absorption peak in the visible region c.)f the spectrum. thus thr F)hotosyntllr:t,icall? competent recombina& appear nearly colorless.) A rare pigmcntcd colony- ma>’ 1~ seen among the carotenoidless microcolonies of one: disc i ti t.hcx rlpp<‘r 1ayc.r.. Si~c,h colonies are observed with a frequenc;v consistent with the hypothesis that they ari~t~ from two independent genetic exchanges occurring in the satnfl ~11. ‘l’h(k t*wo pigmon1 mutat’ions in Yl 65 are unlinked in GTA-mediated CIWSW (Yen & Marrs. 1976).

We attempted to transfer the ability to produce high titers of GTd from st’rain Y262 to SB1003 uia GTA. By using a cell-free filtrate of stra,in Y262 as a source of GTA, the observed frequency of recombinants for a single marker per recipient ccl1 can be as high as 10e3. This value is somewhat greater t#han the previously observt~tl value of 4~ 10e4 recombinant,s per recipient’ (Solioz uf r/l.. 1975) obt8ained with lo\\.clt, titer GTA preparations. SB1003 cells were treated with Y262 filt,rat’e and plated as described in &Iatorials and Methods to screen for overproduction of GTA. Several clones were isolat,ctl (frequency w.5 x 10 -‘) that produced between 10 and 30 times as much GTA as tloc~s SB1003, but none that’ produced as high a titer as Y262. Onr of t#hcse clones wah hreated a second t,imc with filtrate from st,rain Y262 and screened for a further increase in GTA production. Strain Y402, which produces GTA t,iters as higIl as YP62: was isolated in this way. No spont,aneous overproducer was observed in untrratetl controls. Both Y262 and Y402 grow poorly on PYE metliutn~ probably tlnc to lyxix accotnpanying GTA production. Since RCV medium had been shown to greatly inhibit, GTA production by wild-hype strains, we adopted the procedure of maintaining cultures of these strains in RCV medium: and using an RCV-grown inoculum in PYE medium when GTA production is desired. When such :L transfer is performed, thtl culture in PYE medium undergoes several divisions. att(I t,hctl lysis of lo’>;, to ~O’~,, of the cultjurc usually occurs, accompanied by tjlrc>appcaranw ot’ high t#itcbrs of G’I’X. (c) Contparison

of uild-ttjpt

und mwrprotlucer

(:1',-1

In cont’rast to the difficulties experienced during the purification of G?‘A f’WJt1 wild-type strains (Solioz & Marrs. 1977), t>hp material isolat)ed from overprodncrt, \1’1* st,rains could be purified by techniqncs commonly r~setl fat, baotcria,l I-irusw.

FIG. 1. (a) Photograph of a culture plate illustrating the assay used to scrwn for mutants of R. eapsuhtrr producing more GTA than do wild-typo CICIIIW. ,I mixture of about 20 colony-forming units each of strains SB1003 and Y262 were plated as described in Materials and Methods. See the t,ext for discussion. (b) Au enlargement of 2 colonies of the plat,o shown in (a) illustrating the photosynthetically competent, carotenoidless microcolonies lying nbovcb a \i262 colony in numbers greatly cscoocling those ahovc an SRI003 colony.

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attribute our previous inability to purify GTA from wild-type by these techniques to the very small amounts of material present and the resulting sensit,ivity to nonspecific inactivating processes such as adsorption. We performed a series of control experiments to determine whether GTA produced by overproducer strains is the sam(’ as that produced by wild-type. By mixing crude, cell-free filtrate from a rifampicinresistant (rifR) strain (SB1003) that produced GTA at typical titers, with a filtrate from a streptomycin-resistant (strR) overproducer (Y262)> we established that the GTA from these two strains copurified through the techniques described in Materials and Methods, by measuring the ratio of strR and rifR gene transfer activities. We also observed that GTA from those two sources cosediment during isokinetic sucrose density-gradient centrifugation. In addition, cotransfer frequencies determined by genet.ic crosses using either strain as donor are identical. Cotransfer frequency should be very sensitive to the length of DNA carried by GTA, since the map function derived for this system suggests that cotransfer frequency = (1 - Distance between two markers/Length of GTA DNA)’ (Yen & Marrs, 1976). These results and others described below all support the hypothesis that the GTA overproduced by strain Y262 is undistinguishable from wild-type GTA. (d) BTA

morphology

Figure 2 is an electron micrograph of GTA purified from strain Y262 and negatively stained with phosphotungstic acid. GTA particles look like tiny tailed bacteriophages. The 30 nm diameter head appears icosahedral with short apical spikes and is joined to a tail of variable length by a stain-excluding collar. The tail is 5 to 6 nm in diameter with a pattern of cross-striations repeating at) about’ 3 to 4 nm intervals. Suggestiou:: of tail fibers are occasionally seen. When concentrated GTA preparations from wildtype cells were examined by electron microscopy, a variety of particulate materials was observed, including particles identical to GTA purified from strain Y262. (e) Macromolecular

composition

of GTA

The DNA isolated from wild-type GTA has been determined to be linear duplex weight by sucrose density-gradient sedimolecules of about 3.6 x lo6 molecular mentation and enzymatic digestion studies of small amounts of material labeled to high specific activity with [3H]thymidine (Solioz & Marrs, 1977). DNA extracted from GTA produced by strain Y262 sedimented with an s20,W value between 14 and 15 S, as does DNA from GTA produced by wild-type cells. Electron micrographs confirmed the linear duplex nature of the DNA. DNA isolated from GTA particles was sized by agarose gel electrophoresis. .Using Hind111 restriction endonuclease-generated fragments of bacteriophage h DNA an molecular weight standards (Fig. 3(a)), GTA DNA shows the mobility of 2.5 x IO” molecular weight DNA. The GTA DNA band appears broader than restriction fragment bands containing comparable amounts of X DNA. indicating a slight heterogeneity in mobility. This may reflect either size or compositional heterogeneity. The most purified preparations of GTA show about 2.5 pg of protein per pg of DN$, and therefore the t,otal protein component of each GTA particle should have a mass of M, = 7.5 x 106. The proteins of the particle have been sized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis as shown in Figure 4. The tive largest polypeptides (bands 1 to 5) appear to stain with the same relative intensities in clifferent preparations. Il’he weak band bet,ween bands 2 and 3 is a residual coutarnimant

Y

I

IOOnm Kc. 2. Electron rlngatively st.aincd

micrograph of GTA, with phosphot.ungstic

purified acid.

as tlcncribctl

in Milatrrials

mtl

Methods.

antI

Three lower molecular weight polypeptides (bands 6 to 8), although always present. vary in intensity relative to the others. Since electron microscopy shows a variable fraction of particles that appear empty, the small polypeptides could represent internal proteins or proteins bound non-specifically to GTA. Coomassie blue-stained bands 1 and 6 fluoresce violet in strong white light. Although fluorescence of this typcl has been described elsewhere (Clayton & Haselkorn, 1972), why some proteins givca rise to fluorescent bands while others do not remains unexplained. It can provide a useful way of identifying particular protein bands in gel separations with complex banding patterns. (f) The sequence cow$exity

of GTA DNA

Two types of experiments were performed to determine any DNA sequences that were repeated more frequently

if GTA particles carried in GTA DNA t.han in

-

(b) 3. (a) Sizing of GTA DNA by agarose gel electrophoreais. DNA was extracted from purifier1 GTA and electrophoresed on a 0.8% agarose gel next. to a series of molecular weight standartls created by restriction endonuolease Hind111 digestion of bacteriophage h DNA. (b) Restriction endonuclease analysis of GTA DNA. Various DNA samples were treated with restrict,ion endonucleases and electrophoresed on a 2.~r y/, to 7.5% acrylamidc gradient, gr>l a~ ‘T digest~ed with e~cvs~ described in Materials and Methods. The leftmost slot contains GTA D?\A HindIII. Note the large amount of uncut DNA that barely entered this retentive separating gel, and a faint smear of smaller fragments. The 2nd slot from the left contained R. cqmdata DNA. unfractionated, digested with H&U. The 3rd slot contained 10 times as much GTA. DNA trea.tetl with HpafI. The distribution of HpnII-cut> total R. eopt~latrz and GT.4 DX’;zs appear the SUTIX~. Discrete bands are not, detectable in ekher sample. Thr 4th slot shows the results of H/X/~ I digest,ion of Xezrrosporn CTOSSQ mitochondrial DNA bbf thn H/xLII v~zym~, tlcmonst,rating bo18hthv resolving power of the gel and the lack of generalized nncl~tas~~actwlt?; in the rnzymc. FIG.

R. cupsdata cellular DNA, Restriction endonucleases \verc nsc:ti to warch for cordant size cleavage products, and haybridization kinetics (Cot) analysis ww performed to search for rapidly reannealing species. The absence of discrct.c bands in tShe Hpc1.11 digest of GTA DNA (Fig. 3(b)) sh ows that no sequence is cut’ from this DNA more frequently than predicted by the distribution of sizes cut’ from whole cell DNA. (‘,,I analysis (Fig. 5) shows no detectable rapidly reannealing component in GTA DNA. but instead all GTA DNA shows a kinetic complexity identical t’o whole-ccl1 DNA. The size of the R. mpsulata genome is estimated to be JJr =- 2.9 x 10’ by standardizing our Cot system with Escherichia c&i DNA (molecular weight taken as 2.5 x 10’ : Wetmur & Davidson. 1962). (g) Serological

stuch

Antibodies raised against GTA from strain Y262 were isolated and used to comparcx all of the gents phage and GTA produced by various strains of R. capsdata.

GENE

TRANSFER

ACENT

z-3 L c 0) E o,.E .E 0 ‘; 4 .z i -

‘OVERPRODUCER

6’8

I &

6

2

migrated

4 5 1 14

IO

Distance

165

1

(cm)

@‘IO. 4. Absorbancg scan of Coomessie brilliant blue-stained polyacrylamide gel after electrophoretic separation of the proteins of GTA. Molecular weights are calculated assuming a linear relation between the logarit,hm of molecular weight and the distance migrated, as compared to Fi st~andard proteins electrophoresed in an adjacent slot. Band 1, 40 x 103; band 2, 39 Y 10s; band 3, 2i x 103; band 4, 23 x IV; hand 5, 20 x 10s; band 6, 15 x 103; band 7, 14 x 103; band 8, 13 x 10s.

IO4 t ’

105 ,

Nucleotide

pairs

IO6 T

107

Cot

t

(mol

’

108 1

IO9 /

10’0 I

s C-‘)

Fro. 5. Kinetic analysis of renatur&ion of GTA DNA compared to chromosomal DNA of R. ,-crps&tn. Cot analysis performed as described in Materials and Methods. The nucleotide pairs scale was positioned by C,t analysis of E. co& DNA under our conditions (data not shown). Filled s.ymbols are separate determinations of GTA DNA renaturation; open symbols, chromosomal. No DNA sample was observed to be more than 96% solubihzed by is1 nuclease in our procedure (including E. coli DNA), even at the lowest Cot values.

transferring aot,ivity of the cell-free filtrates tested could be inactivated by these pllrified antibodies (Table 2). Preimmune control serum had no effect on GTA activity at the dilutions used here. Since the same antibody preparation was used to test each GTA preparation, the K values given in Table 1 are proportional to the relative rates of antibody-antigen interaction for each t,ype of GTA. For this particular antibody 1J

H.

166

C. YEN,

N.

T.

HU

ANY

TABLE

1

Rhodopseudomonas strain St. Louis BlO H9 JHl JH2 C3 LB2 LB4 EY3 SP108 HP3 BB103 SB1003 Y262 Y165 Y401 YB1023

B.

capsulata

Reference

L.

MAIIERS

strains

or remarks

Wild type American Type Culture Collection uo. 23782; Wall et rrl. (1975) Wild type Marrs (1974) ; Wall et (~1. (1975) Wild type Marrs (1974) Wild type Wall et al. (1975) Wild type Wall et al. (1975) Wild type Wall et al. (1975) Wild type Wall et al. (1975) Wild type Wall et d. (1976) Wild type Wall et al. (1975) Wild type Wall et al. (1975) FVild t)ypo Wall et ctl. (1975) Spontaneous strcptomycirl-resistant; Marrs (1978) Spont,aneous rifampicin-resistant; Yen & Marrs (1!176) GTA overproducer derived from BB103 by nitrosoguanidine mutagens+ ; this paper Double mutant blocked in chlorophyll and carotenoid synthetic pathways: Yen & Marrs (1976) GTA overproducer from cross of Y262 GTA x SBlOO:% Tryptophan auxotroph; Maws (1978)

TABLE

Antibody

inactivation various

2

of gene trunsfer strains

activities

from,

of R. capsulata

Strain

K

(min-‘)

Y262 BlO St. Louis SP3 LB2 JH2 .JHl

97 1Oi 9K 07 77

71

70 6X ;i4 48

c3 EY3 H9 SP108 LB4 c-(KtID), K is defined by the equation H/R, activity after exposure to antibody diluted D times

41 ‘3‘) 11 where R/R, is the surviving for t minutes at 35°C.

gsrn~ t,ransf&l

preparation, reaction rates for all strains tested fall within a factor of three of ant another. This indicates a degree of cross-reactivity comparable t’o that within t,hc T-even phage group (Delbriick, 1946). The same antibody preparat’ion showed no inactivation of the phages carried by strain BlO (see Wall et al., 1975).

4. Discussion Many wild-type strains of R. capdata spontaneously release into the medium small (70 t,o 100 S) particles containing short (M, 3~ 108) pieces of linear, doublrstranded DNA. These gene transfer agents may he adsorbed by recipient cells, and

GENE

TRANSFER

AGENT

OVERPRODUCER

165

the DNA utilized to effect genetic exchanges from donor ho recipient. The titer of GTA, measured by bioassay for gene transfer, varies in waves in exponent8iallJ growing cultures, reaching peak activities of about lo5 gene transfer units per ml for a particular marker as the culture enters stationary phase (Solioz et al., 1975). While this level of activity is sufficient for genetic analyses, the GTA particles themselves are virtually undetectable by physical or chemical means at, that concentrat’ion, and purification from such dilute starting material was difficult (Solioz & Marrs, 1977). The isolation of a mutant strain of R. capsulata that produces GTA at titers about. three orders of magnitude greater than wild-t,ype strains has greatly facilitated thck characterizat’ion of this unusual genetic vector. The general appearance of GTA in the electron microscope is unmistakably phagelike, and the genetic exchange activity mediated by GTA particles is like generalized t,ransduction. However, the putative GTA-associated virus must be considered ho he a defective phage of a most interesting sort. Although virtually all known wild-type isolates of R. capsulata have been tested, none acts as an indicator for plaque-forming activity arising in GTA-producing strains (Wall et al., 1975). (GTA served as a genetic: v&or for approximately two-thirds of the strains tested.) The small GTA hcacl volume and small (42, = 3x 106) DNA molecules carried also suggest the defective: nature of this structurally complex putative virus. Any defect,ive viral genomtt coding for GTA would not seem to be amplified during GTA production, since neither Cot nor restriction endonuclease analyses detected any GTA-borne DNA less complex than that entire R. capsulata genome. The limit,s of detection afforded by Dhesc analyses allow us to conclude that no more than a small proportion of the GTA particles could contain phage-like DNA. GTA can be demonstrated to cont,ain samples of all known replicons in donor cells, including cryptic and heterologous plasmids as well as the chromosome (Hu, Jasper & Marrs, manuscript in preparation) : thus selectivity in packaging does not seem a probable cause for the absence of phagelike DNA in GTA particles. Antibodies prepared against GTA do not inactivate t,hct phages carried by one GTA-producing wild-type strain, BIO. GTA-like genetic exchange activity is widespread among wild-type strains of’ R. capsubata (Wall et al., 1975), and the immunological studies described in this work indicate that that activity is carried out by a single class of closely related vectors. all cross-reacting with antibodies prepared against GTA. This suggests that t,htb putative GTA-associated virus became defective early in the evolutionary history of t,his species, since the alternative hypothesis, i.e. a widespread virus that infects this species and frequently becomes defective, would seem unlikely because of absence of evidence for the existence of such a virus in a non-defective form. The defective phagc functions may have been maintained through t)he evolutionary history of the species because of the genetic function they perform. It is not’ likely t,hat GTA-production abilit,y spread through the population by promoting its own transfer, because (1) strains that do not naturally produce GTA cannot be induced t.o do so by a sing10 round of GTA treatment and (2) incorporation of genetic information via GTA requires DNA-DNA homology (Jasper & Marrs, unpublished observations). ‘l’h~~ observation that two rounds of treat’ment with GlTA from strain Y262 are necessarjp t,o fully elevate the production of GTA by a strain that initially produced moderattl Ievels, strongly suggests that two mutations separat.e the overproducer strain from wild type. Control elements may be defined by t,hese mutations. but their nature 01’ mode of action remain completely unknown.

168

H.

C. YEN,

N. T. HU

AND

H. L. XARRR

An intriguing alternative evolutionary scenario for GTA can be entertained. The existing data are consistent with the hypothesis that, the genes coding for CX’A production did not arise from an ancestral virus that became defective, but evolved from bacterial genes concerned with genetic exchange. Since the basic biological function needed to effect genetic exchange between bacteria is quite similar t,o the function of a phage virion, it might, not be surprising that the vectors resemble one another. Indeed, it might be further hypothesized that bacterial viruses evolved from just such a bacterial precursor, rather than the ot,her way around. We are deeply indebted to Drs Carmen Mannella and Alan Lambowitz for suggesting and performing the restriction endonuclease assays. We thank Sandra Bilyeu for excellent technical assistance, Ray Narconis and Jesse Urhahn for the electroth microscopy. and Chris Poon for the immunological assays and technical assistance. This work was supported by Public Health Services grant GM-20173, by Research Career Development Award GM00098 from the National Institute of General Medical Sciences and hy grant PCM75- 19843 from the National Science Foundation.

REFERENCES Britten, R. J., Graham, D. E. & Neufeld, B. K. (1974). In Methods %n Enzymology ((:rossman, L. & Moldave, K., eds), vol 29E, p. 364, Academic Press, Now York. Burton, K. (1956). Biochem. J. 62, 315323. Chua, N.-H. & Bennoun, P. (1975). Proc. Xat. dead. Sci., IJ.8.A. 72, 2175 6179. Clayton, R. C. & Haselkorn, R. (1972). J. Mol. Riol. 68, 97-105. Delbriick, M. (1946). Biol. Rev. 21, 30-40. Jeppesen, P. G. M. (1974). Anal. Biochem. 58, 195-207. Marrs, B., Lien, S., Stahl, C. & Gest, H. (1972). Proc. Nat. Acarl. Aci., I:.S.,4. 69, 916.-920. Marrs, B. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 971.-973. Marrs, B. L. (1978). Current Topics Bioenerget. 8, 261.-294. Murray, K. & Murray, N. E. (1975). J. Mol. Biol. 98, 551--564. Solioz, M. & Marrs, B. (1977). Arch. Biochem. Biophys. 181, 300-307. 123, 651mm657. Solioz, M., Yen, H. C. & Marrs, B. (1975). J. Racteriol. Wall, J. D., Weaver, P. F. & Gest,, H. (1975). Arch. Microbial. 105, 217- 224. Wetmur, J. G. & Davidson, N. (1968). .I. Mol. Biol. 31, 349. Yen, H. C. & Marrs, B. (1976). J. Bacterial. 126, 619--629. Yen, H. C. &, Marrs, B. (1977). Arch. B&hem. Biophys. 181, 411-418.