R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata

J. Mol. Biol.

(1982) 162, 17-41

R-prime Site-directed Transposon Tn7 Mutagenesis of the Photosynthetic Apparatus in Rhodopseudomonas capsulata JAMES T. ELDERS, DIANE E. SANI>LIS”~ C. YOVVAN~‘, ZSEBO’.~, DANIEL P. ALDER’, NICKOI,AS J. PAXOPOVLOS~ BARRY L. MARRS’ ANI) JOHN E. HEARST”~

Dorw~s KRISZTINA

1Melvin

Calvin Laboratory, Lawrence Berkeley Laboratory ‘Department of Chemical Engineering 3Group in Comparative Biochemistry 4Department of Plant Pathology 6Department of Chemistry University of California, Berkeley, Calif. 94720, U.S.A. and ‘Department of Biochemistry Medical Center, St. Louis University St. Louis, MO. 63104, U.S.A.

(Received

Site-directed

4 May 1982, and in revised form 26 July

mutagenesis

of

1982)

the

photosynthetic apparatus (HA) genes in utilizing a transposon Tn7 mutagenized R-prime. The R-prime, pRPS404, bears most of the genes necessary for the differentiation of the photosynthetic apparatus. Mutagenesis of the R-prime with conjugation into R. capsulata, and homologous Tn7 in Escherichia coli, recombination with the wild-type alleles efficiently generates photosynthetic apparatus lesions. Wild-type alleles are lost spontaneously and the Tn7-induced lesions are revealed by subsequent intramolecular recombination between IS21 insertion elements that bracket the prime sequences in direct repeat. The molecular nature of the intermediates involved in the transposition, recombination and deletion have been investigated by Southern hybridization analysis. The spontaneous loss of wild-type alleles after homologous recombination with the chromosome may be of general use to other prokaryotic site-directed transposon mutagenesis schemes. The IS21 -mediated deletion of the prime DNA is dependent on the RecA protein in E. coli, generating the parental R-factor bearing one IS21 element. A genetic-physical map exists for a portion of the prime photosynthetic apparatus DNA. When Tn7 is inserted into a bacteriochlorophyll gene in the Rprime and then crossed into R. cupsulata. mutants are produced that accumulate a bacteriochlorophyll precursor. which is in excellent agreement with the existing genetic-physical map. This corroborates our mutagenesis scheme. Mutants arising from Tn7 insertions outside of the genetic map have been isolated. Light harvesting IT mutations have been isolated ; one mutant lacks only the 14,000 M, polypeptide.

Rhodopseudomonas capsulata is presented

t Author to whom reprint

requests shauld be addressed 17

0022-2836/82/330017-25

$03.00/O

‘6 198%Academia Press Inc. (London)

Ltd

IX

1. Introduction Rhodopsv/cdomorins cn~~sr~lntu is a model organism for the study of anoxygcnic~ photosyntjhesis because t,he structure and photochemist,ry of the phot~osynt~hetic~ apparatus have been investigated extensively (for a review. see Drews. 1978) and because genetic techniques have been developed for this organism (for reviews, see Marrs. 197Xn.b: Marrs et 01.. 1980). Hacteriochlorophyll and carotenoid 1976: biosynthetic pathways and genes have been characterized (Marrs & Ytw. Scolnik et al.. 1980a.h). Reaction center and light harvesting polypeptides can be separated and identified b,v sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Schumacher & Drew. 1978; Shiozaua rt rrl.. 1980: Touvan rt rrl.. 1982). A gene transfer agent similar to a generalized transducing phage has IEYW used for rnapping closely linked markers (Solioz rf al.. 1975: Yen rd al.. 1979). including those encoding bacteriochlorophyll and c*arotenoid biospnthesis. Large clusters of genes can be mobilized with an R-factor (Marrs. 1981). I-sing t,he Rfactor mobilization technique, Marrs and co-workers ha.ve recently isolated it11 Kprime, pRPS404, bearing 50 kbt of chromosomal DSA that can recombine and correct most’ lesions in mutant)s blocked in t,he differentiation of the PSA4. This implies that’ rnost or all of the photosynthetic genes are c+lustered in one 50 kl) region of the bacterial chromosome. A genetic map has been detjerminetl for approximately 12 genes of the PSA using the gene transfer agent. This map has been correlated lvith a physical restriction map of the R-prirne by returning subclones to rnutants and checking for I,et~ornbinat,ioil (Taylor rt (11.. 19X2: (‘lark rt nl.. 198%). Many PSA genes on the R,-prime remain t’o be mapped. presumabl? including t)he struct,ural genes for the R(’ and LH polypeptides. Very rec*ent,ly we have show~l (Yonvan et al., 19X2) by c,omplc~nlentatiou of enhanced Huoreswn(*e mutants that most or all of the R(‘ and LH genes are csarried by the Kprime and that these genes are actively transcribed in trans in the homologous organism. Our approach is to use site-directed transposon mutagenesis to determine the coding identity of the D;l;A outside of the genetically mapped region within the ttprime. The R-prime is mutagenized in Eschuichiu coli with Tn7 and then ret,urned t’o wild-type R. capsrdata (Ret+ ). Reciprocal homologous recombination trades the mutagenized copy of the WA genes for the wild-type chromosomal copy. The wilcltype alleles, now on the R-prime. delete 7Ga intrarnolewlar rec~ombinat~ion of insert,ion elements bracketing t,he prime I)SA in direct) repeat. This deletion reveals the mutagenized copy of the PSA genes on the chromosotne, The I’S;\ composit,ion of t’hese mutants is screened using a rapid microprocedrrrr (Youvan d (I/.. 1982). F’SA lesions are cwrrelatcd with the position of t)he t,ransposon insertion in thcl K-prime ; a (alone of the corresponding tnutagenized R-prime has been maintained ill a Ree- E. coli library. where it is stable. The target, 1)SA scyuer~w may be subcloned by direct selection for the ‘1‘117 markers (streptomycin, sp~~c:t,iliom!.c:irl and trimethoprim). Transpositional properties of Tn7 hare been csharacterized using l
TRANSI’OSON

MUTAGENESIS

OF THE

PHOTOSYNTHETIC

APPARATI’S

19

the terminal sequences of Tn7 have been determined (Lichtenstein & Brenner, 1981,1982). Twenty-four different transposon mutagenized R-primes have been conjugated repurified transconjugants were isolated into R. capsulata. Four independently from each cross, Each of these four transconjugants, “cousins”, arises from a single clone of donors bearing identical Tn7 mutagenized R-primes. If cousins arising from a single donor show different phenotypes, this is due to the recombinational pathway. The R-prime, pRPS404, bears a crtD lesion that results in a green colony color that is recessive to the normal red colony color. We find that the cousins from a single donor may differ in the segregation of the green lesion and the transposon lesion, but the PSA lesion, caused by the insertion of the transposon into the chromosome, is identical among cousins that have undergone recombination. For example, when an R-prime bearing a Tn7 insertion in what is known to be the bchC or bchA gene is crossed into R. capsulata, mutants accumulate a bacteriochlorophyll precursor characteristic of a bchA lesion. Mutants may be either green or red, depending on whether or not the crtD lesion was transferred to the

chromosome. Two other transposon

mutagenized

R-primes cause LH II lesions.

One may have an insertion near the 14,000 M, LH II structural gene, since only this protein is missing from the PSA. This paper focuses more on the description and characterization of the R-prime site-directed transposon mutagenesis scheme than the isolation of new PSA mutant’s, All steps in the scheme have been investigated by Southern hybridizations using R-factor, R-prime and Tn7 probes. A mutagenesis model is presented that incorporates both the physical hybridization data and the genetic data.

2. Materials and Methods (a) Media and cu~ltivntim E. coli strains were grown on L medium or M9 minimal medium. R. capsulata strains were grown chemoheterotrophically on PYE medium or minimal RCV medium (Yen & Marrs. 1977) at 32°C. Agar plates were sealed with Parafilm to maintain humidity and enhance the growth of R. capsulata strains. Liquid RCV cultures were grown without shaking and supplemented with 50 mM-dimethyl sulfoxide. @S% (w/v) dextrose, and @5% (w/v) sodium pyruvate (Yen & Marrs, 1977). Photosynthetic growth defective (PSG-) mutants were screened by making arrayed spots of late-log inocula on duplicate PYE plates. One plate was incubated incubated

aerobically in the under photosynthetic

temperature-controlled

dark (permissive for PSG-) conditions in an anaerobic

light-box

and the duplicate plate was jar (BBL GasPak) within a

(3 mW/cm’). (b) Conjugal

matin.gs

R-factors and R-primes were conjugated in spots on well-cured agar plates permissive for the growth of both the donor and the recipient. L agar plates (Marrs, 1981) were used for E. coli matings (37°C) and PYE plates were used for matings involving R. capsulnta donors or recipients (32°C). Equal volumes of late-log cultures were mixed and 50 ~1 was spotted and dried. Most matings were incubated for 4 h. but those involving transposition were incubated longer if the recipient. was R. capsuluta. After mating, bacteria from the spots were resuspended in minimal medium and serially diluted for viable counts and selection of transconjugants. Antibiotics were obtained from Sigma and used at the following levels

10

1). (‘.

YOUVAS

(pg/ml) : kanamycin. 30: streptomycin. cycline. 20: and trimethoprim, 500.

ET’

.-lf,

I.5 ; naladixic

(c) Chromntophorr

arid,

50:

rifampiein.

75:

trt’ra-

artnlysis

Absorbtion spectroscopy and gel electrophoresis of reaction center and light-harvesting polypeptides were performed as described (Youvan et n/., 1982). This micromethod utilizes lysozyme and sonication, rather than a French pressure cell. to disrupt the bacteria and release chromatophores. SDS/polyacrylamide minigels can he used to screen large numbers of mutants. (d) Suclric

nxd nrrnlysis

rrcid isolatiorr

Plasmid DNA was isolated essentially as described in the rapid alkali/SDS procedure (Birnboim & Daly, 1979) and total cellular DNA was isolated from R. capsuluta and E. coli strains essentially by an existing procedure (Marmur. 1961). DNA samples were digested with restriction enzymes purchased from BRL as specified by the company. Heat’inactivated RNAase was added to 1 pg/ml during the digestion. Typically. 07% (w/r) agarose gels were run in a horizontal submarine apparatus (BRI, model H4) in E buffer (40 mM-Tris base. 20 mw-sodium acetate. 1 mM-EDTA. adjusted to pH 7% with glacial acetic acid). Analytic gels were 150 ml in volume and Southern gels were 300 ml. Samples were loaded in E buffer, 00.50/, (w/v) bromophenol blue. PO (w/v) sucrose. Gels were electrophoresed for 16 h at 2 V/cm. Nick-translation of DNA probes was performed as described (Maniatis rt al.. 1975). Bidirectional transfer of DNA to nitrocellulose and hybridization was performed as described (Smith & Summers. 1980). Each lane blotted contained approx. 2 pg of DNA. and each filter was probed with approx. 0.5 pg of DNA. (e) AYrrfi~~ constructitn~ Antibiotic-resistant mutants of the multiple auxotrophic RecA- E. co/i strain &IV12 were selected to serve as recombination-free donors and recipients. The rifampicin-resistant derivative is denoted NECOlOO and the naladixic acid-resistant derivative is denoted NECO125. A strain bearing a chromosomal copy of Tni. NECOlOO : Tn7. was prepared 1~~ introduction of RPl,, : : Tn? into NECOlOO from SK1593 fin conjugation followed by heat curing of the plasmid in liquid medium at 42°C. Survivors resistant, t,o rifampicin and streptomycin were screened for the loss of R-factor markers (kanamycin and tetracycline) and expression of trimethoprim resistance (a Tn7 marker). Further confirmation of the loss of the R-factor and transposition of Tn7 to the chromosome was indicated b:y the inability of NECOlOO : : Tn7 to transfer transposon markers to SBlOO3 vin conjugation m the absence of a conjugative R-factor. Physical evidence that the transposon is present, only on the chromosome and that the temperature-sensitive R-factor has been cured includes the loss of detectable plasmid bands upon agarose gel electrophoresis of preparations that should yield supercoiled DNA, and the hybridization of Tn7-specific probes to 2 bands indicative of insertion of Tn7 into the chromosomal preferred insertion site described below. was constructed by conjugal transfer of the R-prime. pRPS404. into us404 NECOIOO : : Tn7 from HBlOl When IX404 is crossed with either ti. coli or R. capsulntcx recipients, pRPS404 rescues Tni at an efficiency of about IV4 and transfers t,o recipients with a conjugation efficiency of lo-’ per recipient, which yields an overall frequency of 10m6 per recipient. The NECOlOO : : Tn7 background is counterselected by auxotrophy. and the recipients are selected on plates containing 15 pg st,reptomycin/ml (supplemented MO agar for E. co/i recipients such as SK2267 or RCV agar for R. cnpsubta recipients such as SB1003).

3. Results (a) Insertion We

have

expressed

found

that

the

in R. capsulata.

of Tn7 in the R-prime

streptomycin For

the

purpose

resistance

pRPB404 marker

of mutagenizing

of’ transposon the

conjugative

Tn7

is It-

TRANSPOSON

MUTAGENESIS

OF THE

PHOTOH\~STHETIC

APPARATUS

“1

prime, a recA - strain of E. coli was constructed (NECOlOO: : Tn7) bearing a chromosomal Tn7 insertion that is not conjugative. The R-prime was conjugated into this strain to make US404, which is NECOlOO: : Tn7, pRPS404. When this strain is used as a donor and selection is for streptomycin and kanamycin in the recipient background, then the Tn7 is transferred by insertion into the R-prime. Construction of US404 is described in Materials and Methods. We have analyzed the sites of Tn7 insertion into the R-prime, pRPS404. by Southern hybridization using Tn7 probes. These experiments require a brief description of the Tn7 restriction map and the subcloned Tn7 probes. Lichtenstein & Brenner (1981,1982) have determined the sequence of the ends of Tn7, mapped restriction sites, and characterized transposition properties. Tn7 has asymmetric direct repeats and is 13 kb in length. EcoRI cleaves the transposon once, generating an 8.9 kb left fragment and a 4.3 kb right fragment bearing all of the antibiotic TAHH 1 Bacterial strains and plasmids strain

MYI2 SEC0100 SEC( ) I 25 SK1592 SK1593 SK2267 UF(‘100 I 1 : : Tni ’ H EC‘404 IX404 V.4# l.AE#

Relevant

properties

pro - . lru-. thr-, lnrY-. hsdJ-. hsdK-. rrc.4 - Sm’ thi-. thr-, Iv.-. trp-. wt.-l-. /WYMVl2. RI’ MV12. Sl’ gal-. thi-, hsdMlil+, hsdtl(:alactos~,-insensitive derivative of SK 1592 SK1592, rec.4 NIWO100, Tni insertion at minute 82 HB101 (pRP8404) SEC”0100 : : Tn7 (pRPS404) Library of SK2267 (pRPS404: :Tnl) Library of NIX’0125 (pRPS404 : : Tn7)

Khodops~/“l~~t~/~~~~~~s cnpsulnttr SK1093 Rf’ Y142 R(‘-. LHII, Sm’ l.AR # Library of SB1003 (pRPS404 : : Tn7) r-AR#l Cousins? resulting from I’AR # Plasmids pLM2 RPl derivative. Km’. ema’, Team. Amam pBLM2 pLM2 derivative, Km’. cma’. Team, Am”” pRPS404 pBLM2 derivative. Km’. Team, Amam bears PSA genes. crt1X233 C’olEl : : Tn7 Sm’. Sp’, Tm’ pW’T14 pWTI5 Ir:coRI deletion derivative of pVCT14 Sm’. Sp’. Tm’ Rl’l ,aAm* Km’. To’. Am’. rep,, RI’1 ,,Am” bearing Tni RPl,, : : Tni

Reference/Source

S. Cohen S. Panapoulos This work This work S. Kushner S. Panopoulos S. Kushner This work Marrs (1981) This work This work This work Yen CGMarrs (1976) Drews et (I/. (1976) This work This work Mindch et al. (1976) Marrs (1981) Marrs (1981) Lichtmstein Lichtmstein

& Brenner (1981 & Brenner (1981

Sakanyan et nl. (1978) Sate et al. (1981)

t UAR # 1 strains are the repurified transconjugants from the corresponding UAR # merozygotes resulting from crosses between a particular UAE# donor and SBl003 recipients. The lower-case letter (I) after the strain number (#) designates a particular, independently repurified transconjugant. A set, of independently repurified transconjugants arising from the same donor and bearing the same strain number are termed cousins. Abbreviations: ampicillin (Am), kanamgcin (Km), naladixic acid (NI). rifampicin (Rf). spectinomycin (Sp). streptomycin (Sm). trimet,hoprim (Tm). chromosomal mobilizing abilit,y (c-ma). and photosynthetic apparatus (WA).

22

I). (‘. YOIT\‘AS

ET

.-I/,

resistance genes. There are two internal BarnHI sites generating a 0.9 kb internal fragment. In mapping Tn7 insertions with these two enzymes. we expect the loss ot the R-prime target fragment, which is now part of two new bands. WC have obtained TnT probes in ColEl derivatives: pVCT1-t bears all of Tn7 and pV(‘T1.5 is an EcoRT delet,ion derivative containing only the “right)” EcoRI fragment. Thertl is no homology between VCTl5 and the left’ and internal HamHI fragments. In fGoR1 digests of Tn7 mutagenized R-primes, we expecat t,he \‘C’T11 and YC’TI 5 probes to hybridize to two bands and one band. respect,ively. The same is true for Harm HI digests, with the addition of the small internal fragment self-hybridizing w&h the VCT14 probe. In 23 out, of 24 TnT mutagenized R-primes (pRYS404 : : Tn7). the dat,a are consistent with the insertion of a single transposon in the R-prime and t,he lack of any detectable rearrangements. R-primes bearing multiple insertions have not been observed. With one exception (UAE91). we observe only one missing R-prime BcoRT or HamHI band in each digest. New bands are generated by the ‘l’n7 a,nd lZprime junctions that hybridize to the \-(‘T14 probe as expec%ed : t,\vo fragmrnt,s hybridize in the EcoRT digests and two fragments plus the internal fragment hybridize in the RnmHI digests (Fig. 1). In ea,ch case. only one of the pair of These dat,a may be used to fragments hybridizes with the right-end probe, V(“‘I’I5. position and orient the Tn7 insert.ions. Since the rrstric*tion fragmer1t.s involved arts large and the maps are at low resolution, positioning is not, pre&e. Marfa acLcuratt> data will be available after subclones are rnade and the R-prime and Tni restriction maps are refined. We present here tht> transposon insertion data in tabular form. specifying only the target RamHI and EcoRI fragments (Table 2). The insertion in UAE70 is of particular interest, since it is within t,he genetically mapped repiou (Taylor of (II., 1982). (b) Chromosomnl

Tn7 irjsrrtion

.sitps

As shown in the previous section, hybridization of a ‘l’n7 probe to blots of’ mutagenized R-primes reveals two major bands in both RcrrrrHI and EcoRT digests. since both of the enzymes cleave the transposon into two fragments (neglecting t,he small internal BarnHI fragment). When total USA. rather tJhan episomal DNA. is extracted from strains bearing Tn7 mutagenized R-primes. twice the expected number of hybridizing bands are observed (Fig. 2). (‘ontrols indicate that neither Tn7 nor the ColEl vector has homology with the XE~‘O125 background bearing the mutagenized R-primes (UAE strains). This phenomenon has been explained b> Lichtenstein $ Brenner. They have observed a chromosomal “hot spot” for Tni insertion in the El:. co& chromosome at, minute 82. Tn7 occupation of this preferred site in a unique orientation was shown to generate two Er:oKI genomic fragments hybridizing with a Tn7 probe. Six out) of seven of the genornica EcoKl digest)s displayed in Figure 2 possess two bands of approximately 17 kb and 105 kb. in close agreement witah the previously observed bands in E’. coli. When t,hesf mutagenized R-primes are crossed into R. capszcttlto and t,he genomie blots a.r’r repeated, again we observe two extra bands. These bands have a constant size in seven out of seven strains analyzed in Figure 2. 105 kb and A.7 kb. Presumably. this is an example of a preferred Tn7 integrat’ion site in another chromosome.

Tn7 insertiom

iu pRPM04

The YAE strains are NM”0125 backgrounds containing Tni mutagenized pRPS404 R-primes. The position of the Tn7 insert is designated by the RrrmHI and EcoRl fragments of the R-prime that bear the inwrt.. The R-prime restriction fragments are designated by alphabetic* upper case let.ters starting with the largest fragment. which is designated A. SD. no det,ectable perturbation of the pRPS404 restriction pattern (due to lack of resolution in the screening gel for large and small fragments or comigration of ,junvtion fragments and an K-prime fragment): ST. no test.

Whether the R. capsulata site has homology with the E. coli preferred site has not) been determined. Apart from the insertion of the Tn7 into the preferred chromosomal site, the data in Figure 2 are consistent with the interpretation t’hat Tn7 is inserting semirandomly into the R-prime. (c) Conjugation

of Tn7 mutagrnized

R-primes

into R. capsulata

The next step in the site-directed mutagenesis scheme is to conjugate the Tn7 mutagenized R-primes into wild-type R. capsulata. where recombination may introduce the transposon ink) the PM genes. We term t,his process “clonal” sitedirected mutagenesis as opposed to “shotgun” site-directed mutagenesis, since the mutagenized R-primes have been purified and stored in E. coli intermediates (UA E library). Shotgun mut,agenesis implies a direct cross between CS404 and R. capsdata. In the clonal scheme, individual UAE donors are crossed with a strain of R. capsuhta bearing a wild-type PSA (SR1003). The resultant R. capsulatn transconjugants are designated the VAR strains, with a strain number identical to t,he lME donor.

2-3 2-O

24

kb

M

R

b

BUrnHI

d

e

VCT I4 probe

b C

d

EcoRI e

M

M

R a b

BOt77HI d

e

a

VCT 15 probe

b d

ECORI

e

R

o

b

c

defghijklmnopqrs

VCT 14 probe

R. coPsuloio E. coli MobcdefghijkImnopgrS

VCT 15 probe R. cop~ul~t~

FOG.2. Insertion of Tn7 into a preferred site in the chromosomes of E. coli and R. cupsdata. Total DNA was extracted from 9 strains of E. coli and R. cupsulat.a (UAE and UAR strains, respectively) bearing Tn7 mutagen&d pRPS404. DNA was digested with EeoRI, electrophoresed on a 0.7% agarose gel, transferred bidirectionally to nitrocellulose, and hybridized with nick-translated, Tn7-specific probes. Left: VcT14 probe bearing the intact transposon. Right: VCTI5 probe bearing the antibiotic marked end of Tn7 borne on the terminal EcoRI fragment. Lane M, Hind111 lambda DNA markers: 24, $8,6.9.2.3.2.0 kb. Lanes a to i. total DNA from E. coli strainsdigested with EcoRI: lane a, NECOlOO: : Tn7; b, UAE18; c, UAE19; d, UAE22; e, UAE39; f, UAEBT; g, ITAE70: h. UAE78; i, LlAE115. Lanes j to s, total DNA from R. cupsdata strains digested with EcoRI: lane j, SB1003; k, UARlSf; 1, UARl9e; m. L’AR22h: n, UAR39f; o, UAR67h; p, UAR7Oe; q, UAR7&; r, UARllBh; s, U23. E, fragments generated by preferential Tn7 insertion at minute 82 in thr E. eoli chromosome (17 and 10.5 kb). R, fragments generated by preferential Tn7 insertion at a unique site in the R. capsdata chromosome (10.5 and 6.7 kb).

M

E. coli

M

b C

d

e h

k m

n 0

P

M

TRASSPOSOS

MUTAGENESIS

OF THE

PHOTOSYNTHETIC

APPARATI’S

27

Twenty-four UAE strains were mated with SB1003 recipients, and selection was made for the transfer of the mutagenized R-prime into the SB1003 background by selection for streptomycin on RCV plates, counterselecting the donors by auxot,rophy. Four transconjugants from each cross were repurified several times on RCV medium containing streptomycin. These independently repurified transronjugants arising from a mating with a clonal donor are termed cousins. Different cousins are designated by a lower-case letter following the strain number. Matings are sufficiently efficient (lo-’ per recipient) that the probability that any two cousins are siblings is far less than loo/&. This estimate is made based on the worst case, where all transconjugants were generated at the beginning of the mating and multiplied exponentially until the mating was disrupted. Cousins may or may not, have identical phenotypes. This depends on the particular recombination events that occur in the Ret+ merozygote. We have investigated the fate of the Tn7 mutagenized DNA in R. capsulata by comparing genomic blots of the UAE strains with the corresponding UAR strains probed with Tn7. Figure 3 clearly reveals that the position of the Tn7 relative to the adjacent prime restriction sites is unchanged. The integrity of the Tni” Recombinational event,s mutagenesis site is maintained in the Reef background. that may have incorporated the mutagenized prime into the chromosome have preserved the adjacent prime restriction sites. We can estimate the plasmid copy number by comparing the intensities of the preferred insertion bands in the chromosome with the bands corresponding to insertion in the PSA DNA. In most csases. it appears that the PSA insertion signal is two to four times greater than the chromosomal preferred site signal, hence we must be observing one to three copies of the mutagenized R-prime sequences in addition to the copy that has recombined int,o the chromosome. That this is indeed the case will be shown in later sections by genomic blots using the R-prime as a probe. (d) Phenotypes of thr UAR strains (“ousins, independently repurified transconjugants arising from clonal donors, have either identical phenotypes or no detectable change in the PSA phenotype. No selectZion was made in the picking and repurification of mutants: i.e. typical colonies were picked. Identical phenotypes among cousins is very strong evidence t’hat our mutagenesis scheme is indeed site-directed and that the mutant, phenotype is the result of the Tn7 insertion. Mutants may be either green or red, depending on whether or not the R-prime cdl lesion was successfully recombined into the chromosome. An exception to this rule occurs when the Tn7 lesion reduces the overall expression of carotenoids. Of the 24 different mutagenized R-primes crossed into SB1003, we have found PSG- cousins in six of the UAR series. These mutations and the positions of the transposons relative to the R-prime EcoRI and RnlnHI bands are listed in Table 3. Four out of four of the independently repurified transconjugants from t’he UAE70 x SB1003 cross have identical PSA mutations. All of these cousins lack the RC and LH polypeptides and accumulate a bacteriochlorophyll precursor that absorbs at 66.5 nm, characteristic of a lesion in the bchA gene. Results from absorption spectroscopy and SDS/polyacrylamide gel electrophoresis of

1). (‘

\iOl‘VAS TAHLE

Phenotypes Strain [JAR 18f 22h 39f 39h 7OP

70f 7% 70h 11.5e IlSf IlSg 115h 219r

Irwxtion RamHI EcoRI F1 E I) I) (‘ (’ (’

(’ E E E F1 E

b E F F H H H H E: Ix E E E

6’f 3

of R. capsulata Polypeptidrs

(blor K (: (: t: t: (; t: t; R. (: (: (: (:

28

24

21

,112.

PM:

rmtants

(M, x 10m3) 14

12

r\bsorptiorr

10

x

+++-+++

-

5 + + +

-

+ + + +

-

+ + + +

-

-

-

+ + + +

-

-

+ + + +

+ + + +

(m)

590

x00

X60

t ~ ~ ~ ~ .+ + + +

-. --. I. 1, IA I‘ -

+ ~ ~ ~ ~~ + + + + -

I’lns

liti3 665

663 665 8X0 X80 xx0 XX0

Twenty-four I’AE strains with mapped Tn7 insertions were crossed into SRIOF)3 and 4 cousins were selected from each cross. Of the 96 cousins assayed. 13 were PS(F and further analyzed by absorption spectroscopy and SDfi/polyacrylamide gel electrophoresis. The I’SA lesions are list.ed in t,erms of missing polypeptides and altered infrared absorption. The FAR70 series is of particular interest. since the Tn7 insertion in the Barn C -/Cco H fragment maps near or within the hrhd and hchP genes. and the mutant phenotype (a bacteriochlorophyll precursor absorbing at 665 nm) is characteristic of R h&d lesion. CAR18f is lacking only the 14.OOOM, LH I1 polypeptide. suggesting that this LH I I gene may br in thti Barn E-&o E fragment.

chromatophores from the UAR strains are summarized in Table 3. The CAE70 Tn7 insertion maps to the Barn C-Eco H fragment of pRPS404. In the physical-genetic map recently determined (Taylor et al., 1982), this lesion maps in the bchC gene. which is immediately adjacent to t’he hch,.-l gene. B. L. Marrs (personal communication) has observed lesions that, map 11ia GTA to the bch(’ gene and cause a bchA phenotype. Presumably, in both cases, the lesion in the bch(’ gene has a polar effect on the expression of t,he bchA gene. In the case of the transposon mutant UAR70, this is expected if the insertion is between the promoter and the bch.4 gene. Four out. of four of the cousins arising from the CAEl16 donor have identical lesions in the LH II antenna. Three are green and one is red. These mutants have abnormally low absorption at 800 nm and higher absorption at 880 nm. The visible spectra are virtually superimposable. wit’h the except’ion of the difference in the carotenoids associated with the cdl lesion. All of t)hese cousins lack t,he 14.000 M, LH II polypeptide, which may be involved in the assembly of the LH II antenna by facilitating the binding of bacteriochlorophyll to the 8000 and 10,000 N, polypeptides, which generates t,he 800 nm absorption band. The UAEll5 Tn7 insertion maps to the Barn E-Eco E fragment. which is outside of t,he known genetic map. One of four of the UAR18 cousins. UARl8f. is PSG- and has an insertion in the Ram E-Eeo E fragment. This insertion maps approximately 2 kb away from the site in the UARI 15 mutants. CARlXf has almost no absorption at, 800 nm, and lacks only the 14,000 M, LH II polypeptide. It has wild-type

TRANSPOSON

MUTAGENESIS

OF THE

YHOTOSYSTHETI~

APPARATITS

29

carotenoid coloration and no other detectable PSA lesions. These data suggest that the Barn E-Eco E fragment contains genes affecting the LH II antenna. We believe that the UAE18 insertion may be near the structural gene for the 14,000 M, polypeptide. Five other mutants listed in Table 3 lack bacteriochlorophyll and all PSA polypeptides. These Tn7 lesions map outside of the genetic map. Some may be in other bacteriochlorophyll biosynthetic genes, the inactivation of which turns off the expression of the PSA. Testing four cousins from each cross and finding no altered PSA phenotypes does not rule out the possibility that the position of the transposon in the DNA is mutagenic. As will be shown later, prime DNA bearing the Tn7 insertion may be deleted before recombination with the chromosome. We have found that infrared photography of colonies can be used to screen for mutants lacking reaction centers. These mutants are highly fluorescent in the near infrared (Youvan et al., 1982). This technique will facilitate the screening of large numbers of cousins, so that less frequent recombination events leading to the loss of reaction centers can be observed. (e) RecA’dependent

deletion of prime DNA mediated by IS21 elements

In the previous section, we demonstrated that both the transposon lesion and the c&D lesion may be expressed in R. cupsulatu transconjugants. Apparently, the wild-type alleles are not complementing these lesions in trans as expected from a reciprocal homologous recombination. One explanation for the loss of the wild-type phenotype after recombination with the mutagenized R-prime is that the chromosomal wild-type alleles, transiently on the R-prime, have been deleted. This is not due to the loss of the entire R-prime, since the kanamycin marker is stable for at least 200 generations without selection. Mobilization of DNA using the R-factor pBLM2 results in direct repeats of IS21 flanking the prime DNA (Clark et al., 1982). This suggests a mechanism for the deletion of prime DNA and loss of wild-type alleles. Intramolecular recombination between the IS21 elements should regenerate the parent R-factor pLM2 bearing one IS21 element and a circular piece of DNA bearing the prime DNA and the other IS21 element. The circular prime DNA is subsequently lost, since it is not a replicon. The structure of pLM2 and pBLM2 regarding the tandem duplication of IS21 elements is analogous to the structural changes that have been well-characterized for R68 and R68.45, which also result in enhanced chromosomal mobilizing ability (Rieb et al., 1980). To test for prime deletion, we conjugated pRPS404 from a recA- background (HBlOl) into two isogenic strains: SK2267 (recA-) and SK1593 (recA+). Transconjugants were selected by kanamycin and the donor was counterselected by auxotrophy on M9 medium. These strains were repurified twice on M9 medium containing kanamycin. In the E. coli background, intermolecular recombination between the R-prime and the chromosome is eliminated, since there is no DNA homology as assayed by Southern hybridizations. Plasmids were prepared from these strains by the alkaline/SDS procedure described in Materials and Methods. Plasmids were subjected to restriction digestion and agarose gel electrophoresis : 18 independently repurified strains of SK2267 (pRRS404) are stable, as assayed by

IV

d

e

.

h k

n

v

F)t:. 4. Deletion of the prime sequence from pRPS404 in ReeA+ E. cdi. The R-primepRPS404 was conjugated from HBlOl (recA-) into SK1593 (rrc.4 + ) and .I593 (pRPS404) transconjugants were repurified by streaking for isolated colonies twice. Plasmids were extracted from 15 of these strains and analyzl ed by vR1 digestion and electrophoresis on a OG’*!nagarose gel (stained with ethidium bromide). Higher than stoichiometric amounts of the R-factor sneeri& IXWXlS * seen in all of the samples. Lanes a. b. c, d. g.?. 1 and m demonstrate that some of these strayns are deleted almost entirely to the parental K-fact,ors. (:)thcr, lan t’s: (a. f. h. j. k. m and o reveal various mixtures of’R-factor and R-primes. Lane M. Hind111 lambda markers: 24, 9%. 69. 29 and 2.9 kb. Lane R. R-1nrinir PR,l’SWI with bands labeled to the right (A to I,. with R-factor specific band8 underlined). d refers tu the deleted product of the Ecn A and Era R ha .nds

b

B

L

0

b

c

d e

t 9

h

i i

k

I

ml 0

0

Prc:. 5. Deletion of the R-prime pRYS404 to the parental R-factor pLM2. These plasmid samples are identical to those in Fig. 4, except in this case the DN.4 has been digested with Hind111 and SalI. Digests of the parental R-factors pBLM2 (two IS21 elements) and pLM2 (one IS21 element) are shown in lanes B and I,. Bands labeled L and B refer to the HindIIT-SaZ1 fragment (4640 kb) spanning the IS21 element in pLM2 and the HindIII-SaZI fragment (6760 kb) spanning the tandem IS21 elements in pBLM2. Lanes a to o reveal that plasmids from all of the SK1593 (pRPS404) strains arc deleting to pLM2. Lane M. Hind111 i~~mbtia rnarkws.

M

3”

I). (‘. \‘() Ci\‘A N ET &A/,

EcoRI restriction (data not shown) : 15 out of 15 strains of SK1593 (pRPS404) are in various stages of prime deletion (Fig. 4). This DNA has been replicated approximately 50 generations between conjugation and plasmid extraction. We interpret the non-stoichiometric ratios among bands to represent mixed populations of the deleted R-factor and undeleted R-prime. The most intense bands map to the R-factor (Eco C, G and K). The ratio of the intensities of the Eco G band to the Eco F or Eco H bands is a measure of the ratio of total plasmids to undeleted plasmids. The same DNA samples from SK1593 (pRPS404) were subjected to a HindIII-Sal1 double digest. Analogous to R68 and R68.45, a 4640 bp fragment is characteristic of one IS21 element (pLM2) and a 6760 bp fragment is characteristic of two IS21 elements (pBLM2). All plasmids analyzed were deleting to pLM2, as indicated by a 4640 bp HindIII-Sal1 fragment (Fig. 5). Deletion of pRPS404 to parental R-factors has also been observed in the homologous organism. Similar to the E. coli deletion experiments described above. transconjugants pRPS404 was conjugated from HBlOl into SB1003. R. capdata were selected on RCV medium containing kanamycin and the E. coli donor was counterselected by auxotrophy. After two stages of repurification, DNA was extracted by the modified alkali/SDS procedure and only R-factor bands were observed with EcoRI digestion (data not shown). These digests are obscured by endogenous plasmid bands. However, the fusion of the R-factor EGOA and Eco B bands into a higher molecular weight band characteristic of pLM2 was observed. The following experiment was designed to avoid the problems generated by the endogenous plasmid bands. Kanamycin-resistant plasmids were rescued from the UAR strains by conjugation into a RecA- background, NEC0125. NEC0125 strains bearing plasmids were selected by exposure to naladixic acid and kanamycin. It is critical to note that, up to this point, the UAR strains have been maintained under constant streptomycin selection. For matings, the UAR strains were subcultured without antibiotic selection. We have observed that kanamycin resistance is stable in the UAR strains for at least 200 generations without selection but that the level of resistance to streptomycin is significantly reduced after subculturing without selection for streptomycin. Strains originally resistant to 15 pg of streptomycin per ml on plates form only pin-point colonies on these plates after the colonies have been subcultured in the absence of streptomycin. This is genetic evidence for the deletion of the multi-copy mutagenized R-prime Tn7 marker. Figure 6 demonstrates that this is due to IS21-mediated deletion of the prime DNA. Of the 12 plasmids independently isolated from PSG- UAR strains. t,he characteristic HindIII-Sal1 fragment indicates that seven plasmids havr deleted to pLM2 and five plasmids have deleted to pBLM2. Two lanes displa) plasmids that, upon further analysis, were found to have some undeleted prime sequences. An alternative explanation is that 12 plasmids deleted to pLM2 and tive of them underwent a second transposition event generating another copy of IS21. In a later section we will show that deletion of the chromosomal copy of the PSA genes occurs immediately after recombination but that other copies of the mutagenized R-prime do not delete until the streptomycin selection is discontinued. This is due to a gene-dosage effect giving a selective advantage to bacteria containing undeleted R-primes and multiple copies of Tn7.

M

l3

L

0

b

c

d

e

t

h

i

I

m

n 0

8

L

FIG. 6. Deleted R-primes rescued from the UAR strains. Thirteen PSG- UAR strains were conjugated with NEC0125 and kanamycin-resistant transconjugants were selected. Plasmids were extracted from 1 transconjugant in each of the 13 crosses. Each DNA sample was digested with EcoRI and electrophoreaed on a 67% agarose gel (ethidium bromide stain). Both pLM2 and pBLM2 deletion products were observed. Lanes El and L and bands marked B and 1, are identical to those described in the legend to Fig. 5. Lanes a and b contain samples identical to those displayed in Fig. 5. Lanes c to 0 contain digests of plasmids rescued from the IJAR strains: c, UARl8f; d, IJAR22h; e, UAR39f; f. IJAR39h: g, UAR7Oe; h, UAR76f; i, UAR70g: j, UAR70h; k, UARllAr: I. IrARl15f: m. UARl Ifig; n, UARl1.5h: o. UAR219e. In 2 cases, plasmids bear prime sequences: UAR22h and UAR70h.

2.3

6.9

24

kb

(f) Analysis

qf 7’n7

insrrtiovs

into

the

Ii. vapsulata chromosnmr

Southern hybridizations to total DNA from the UAR strains blotted bidirectionally and probed with the pRPS404 and Tn7 are shown in the left and right panels of Figure 7. With the R-prime probe, we expect that the target PSA restriction fragment will be missing in each lane due to the insertion of the t,ransposon, which has internal BamHI and EcoRI sites. This is indeed the case, and is evidence for recombination, incorporation of the Tn7 lesion into the chromosomal PSA genes and loss of the wild-type alleles. This Figure can be compared to the insertions listed in Table 2 to verify that the target fragment is missing from the blot probed with the R-prime. Of particular interest are strains UAR18f, CAR70e and UAR115h, all of which possess mutant PSA phenotypes. Both UAR18f and the entire UARllS series have LH II lesions with reduced 800 nm absorption and loss of the 14,OOOM, polypeptide. Tn7 was mapped to t)hr Ram E-Eco E fragment in the corresponding donors: UAE18 and UAE115. and t,hese fragments are missing in the genomic blot (lanes c and i, respectively). UAR7Oe accumulates a bacteriochlorophyll precursor characteristic of a bchA lesion. This is consistent with the insertion of Tn7 into the Barn C-EGO H fragment in the genetically mapped region. The R-prime probed blot (lane g) reveals tha,t both of these fragments are missing. Eco A, Eco B, Barn A and Barn B fragment,s characteristic of the undeleted R-prime are present in all of the UAR strains in Figure 7 except UAR19e (lane d). All of the strains have been under continuous streptomycin selection, which selects against deletion of prime sequences bearing Tn7 insertions. The Tn7 insertion in UARl9e is in the Barn A-EGO G fragment which is part, of the R-factor, hence deletion of the prime sequence does not affeqat the level of streptomycin resistance. Most of the signal in this blot is due to undeleted R-primes, but we know that there is sufficient sensitivity to observe an> chromosomal copies of the fragments since the prime-chromosome junction fragments can be observed in the genomic digest of SB1003 cut with EcoRI and probed with pRPS404. In the right half of Figure 7 the same samples have been probed with Tn7 (VCT14). EcoRI and BamHI digests of US404 (sample a) reveal hybridizing bands characteristic in size of the Tn7 insertion into the E. co/i preferred site at minute 82. Sample b is total DNA4 from SB1003, which shows no cross-hybridizat,ion with the Tn7 probe or ColEl vector. All of the UAR strains have three common BamHI bands: the upper two common bands are characteristic of the chromosomal preferred insertion site and the lower common band is the internal RamHl fragment from Tn7. The two common bands in the EcoRI digests are also due t,o the chromosomal preferred insertion site. Other bands in each digest are charac%eristic of the junction fragments between the ends of Tn7 and the target fragment’. (g) Ikletion

of prime

sequences in the absence of dreptomyein

selection

Assuming that, deleted and undeleted plasmids transfer with equal frequencies, the analysis of deleted plasmids in the UAR strains by conjugal transfer into a RecA- E. coli background suggests that approximately 80~~~of the mutagenized R-primes have deleted. However. the experiments described in the preceding

ghiMobcdefghi

probe

&OR1

J ,K,L M

F G H

P

2.0

2.3

6-9

24

kb MabcdefghiMobcdefghi

BOrnHI

Tn7

probe EcoRI

FIG:. 7. Analysis of Tn7 insertions in the PSA genes of R. capsulnta.Total DNA prepared from R. capsulate strains that received different Tn7 mutagenized Rprimes 1)~ conjugation (UAR strains) were subjected to digestion with EcoRl or HnmHI restriction endonucleases, electrophoresed on a 070/, agarose gel, and blotted bldirectionally to nitrocellulose. One filter was hybridized with an R-prime specific probe prepared by nick-translation of pRPS404 DNA ; the other with Tn7 specific probe similarly prepared from pVCT14 DNA. Left: R-prime specific probe. I&ters A to K to the left of the blot and letters B to M on the right refer to the RnntHl and EeoRI restriction fragments from pRPS404. Lanes marked $1 refer to HiwdIIT lambda markers with the fragment sizes (in kb) listed on the left of the right panel. As each UAR strain is listed, the strain number will be followed by 2 upper-case letters, which designate the R-prime BnmHI and E:roRI restriction fragment. respectively. into which the transposon has inserted (also. SW Table 2). Strain I’AR87e has no detectable PSA lesion and the corwsponding chromosomal fragment is intact. Sample a. US404: b. SKlOO3: c. I’.4R18f (E. I?): d. Y.ARlCk (A. G): V. VARSSf (D, F); f, rAR6ih (F. F): g. IYAR70e (C. H): h. 1~11187~~(F. F): i. Y.AKll.jh (E. E)

J K

FG,H

D E

C

Mobcdef

BornHI

R-prime

EcoRI

j

k

abcdefghi

probe BO/VHI

AB

A3

obcdefghi

EcoRI

R-factor

jkobcdefg

probe BornHI

hi

PIG. 8. Deletion of prime sequences in TAR strains suhcultured without streptomycin selection. Total DNA from E. coli donors (UAE strains) and the corresponding R. r*cpsuluta mutants (UAR strains) was digested with EcoRI or BnnzHI. electrophoresed on a 0,70/o agarose gel, blotted bidirectionally tc nitrocellulose and probed. The left panel was probed with the R-prime pRPS404 and the right panel was probed with the R-factor pBI,MZ. Samples alternate in the order: IJAE donor, UAR mutant (cultured continuously under streptomycin selection). and CAR mutant (further subcultured without streptomycin selection). Deletion of the prime sequence tin IS21 -mediated homologous recombination is indicated by the loss of the 2 largest EcoRT and BumHI bands (A and 1%.in hoth digests) and the generation of a larger band (designated A). This is due to the loss of A’coRI restriction sites in the prime DNA. Samples on the left side of each panel were digested with EcoRI and the samples on the right side of each panel were digested with RnmH I Symbols following the CAR strain numIwr designate lrhrther st,wptomyvin was present in the subculture from which the total I)54 was extracted: a. I’AE18: 11. 1’BItlgf (+ ): c. I.AR18f (- )’ cl. l~AE:l 1.5: (x. I-SRI I.?h (+): f. IThRI I.‘,h (-- ): g. IT.AE70: h. l~AK7Oe ( t ): i. l’.%KiOg (-): j. IW404: k. SBIOO3.

A.B

obcdefghi

R-prime

TRANSPOSOS

MUTAGENE:SIS

OF THE

YHOTOXYNTHETIC

AYYARATCJS

37

section utilizing R-prime probed total UAR DNA suggest that a much smaller proportion of the R-primes has been deleted. This apparent inconsistency is resolved by comparing blots of UAR strains that have been under continuous streptomycin selection to strains subcultured without streptomycin selection. Figure 8 reveals that the strains subcultured without streptomycin delete to t,he parental R-factor, as indicated by the loss of the Eco A and Eco B bands, and appearance of a higher molecular weight band predicted from prime deletion and fusion of the EGOA and Eco B arms. Of the three strains tested in t,his experiment. two bear only deleted plasmids (UARlSf and UAR115h), and one strain contains a mixture of deleted and undeleted plasmids (UAR7Og). Presumably, R-primes in UAR70g are still in the process of deleting. ISBl-mediated deletion of the unselected prime sequence explains the deleted plasmids observed in the conjugal rescue experiment. In order to mate with a streptomycin-sensitive recipient, the UAR donors were subcultured without streptomycin and apparently deleted at that time.

4. Discussion Our scheme for site-directed mutagenesis in R. capsulata using an R-prime bearing the genes for anoxygenic photosynthesis is shown in Figures 9 and 10. The genetic data and physical Southern hybridization data supporting this model have been presented above. Transposon Tn7 is inserted into pRPS404 in a RecA- E. coli background and then the R-prime : : Tn7 is conjugated into the homologous may transfer the mutagenized DNA into the organism, where recombination chromosome. IS21-mediated intramolecular recombination on the recombinant Rprime subsequently deletes the wild-type alleles. The following is a brief summary of the clonal site-directed transposon mutagenesis scheme. US404 contains a chromosomal copy of Tn7 in the preferred insertion site at minute 82 and the R-prime, pRPS404, bearing the PSA genes. All of the E. coli strains used in this scheme are RecA-. The prime sequence of pRPS404 specifies most of the genes necessary for the differentiation of the PSA and is marked by a crtD lesion, which results in the accumulation of a green colored carotenoid. Tn7 is semirandomly inserted into pRPS404 and conjugated into a recipient, SK2267, by selection for streptomycin, and the donor is counterselected. The overall efficiency is 10e6 per SK2267 recipient. Twenty-four mutagenized plasmids in the UA library have mapped Tn7 insertions. Insertions are semirandom but there is some preference for the Barn E fragment. SK2267 was selected because it is a highly efficient recipient, but it cannot be counterselected in crosses with SB1003. The UA library was transferred into a more auxotrophic strain, NEC0125, by selection for streptomycin and naladixic acid. Next, individual UAE strains (NEC0125, pRPS404 : : Tn7) are crossed with wild-type R. capsulata strain SB1003 and merozygotes are selected by streptomycin. These merozygotic colonies develop green sectors as the c&D lesion is recombined into the chromosome and the wildtype allele is deleted. The frequencies of these events are sufficiently high that the perimeter of the colonies becomes entirely green. Upon repurification, stable red and green colonies result. Independently repurified transconjugants stemming from

3X

NECOlOO::Tn7 pRPS404

I

Select Sm Km Counterselect donor

SK2267 pRPS404::Tn

x SK2267 I

/I

UA# (iibrory)

7

Chromosome

11 R-factor Chromosomal PSA yews

II

Select

Sm Km Nl

I

x NECOl25 I sma

NECOl25 pRPS404::

II

UAE# ( 1Ibrary)

Tn7

Select Sm Counterselect

Ip

Transposon b

IS21

-D

crt

0

Tn7

Tn7

D lesion hot

spot

x SB1003

donor

SBlOO3 t1RPS404:: Tn 7 II

R-prime PSA qenes

I

rd’

iiL?

UAR #

Repurify

Cousinsa, b,c,d

UAR#a

UAR#b

UAR#c

UAR#d XBL

823-8829

VI<:. 9. A model for the mrvhanism of clonal site-directed transpoaon mutagtwrsis in H. ccc~~~lr~trr. The R-prime pRI’R404 is mutagrnizrd in a Rec.\- background of 15’.co/i and then t,ransferwd to wildtype R. urpsulcltn by conjugation, whereby homologous recombination may exchange the mutagenized PSA sequences for the rhromosomal copy. IWl-mediated intramolecular homologous recombination deletes the wild-type alleles from the R-prime. Four possible outcomes are shown for indrptmdentl~ repurified tmnsronjugants arising from a single donor. These cousins possess identical Tni induced lesions in the PSA if t,he transposon has rrcombinrd into thr chromosomr. Thr indrpendentl? segregating rrtl1 lesion results in a green phrvmtype in some of thr cwsirls. ‘I’hr fate of only onca mutagenized R-prime is shown in this Figure. SW the Discussion and Fig. IO for mow drtails.

I.

Recombination

Select

Sm

II.

Wild- type alleles delete

Select

Sm Kill

Ilf.

All primes delete

I

No selection

NC:. 10. A model for recombinat,ion and deletion of transposou Tni mutagrnized PSA genes and c*hromosomal alleles. This model details t,he recombination and deletion processes occurring in step IV of Fig. 9. which leads to the successful recombination of both the transposon and the crtl) lesion into the K. cnpsulota chromosome. Consistent with data from Southenl hybridizations and genetic data on phrnotypit, expression of mutagenizrd alleles. I of 3 mutagenized K-primes recombines with the chromosome and only the recombinant R-prime bearing the wild-type WA alleles deletes while under streptou~yrin selection. At this point. both the transposcln-intiucrd lesion and the crtl) lesion are t~xpresscd. while other mutagenized K-primes are undrlrted. Since all R-primes are mutagenizeti. the> do not complement the chromosomal mutations. These rnutagenizrd R-primes will also delete to the parent,al K-factor when the streptomycin selevtiorr is removed and the strains are subcultured. l’msumabl~. maintenance of undrlrtrd R-primes under streptomyck selection is due to a gene-dosage phrnomrnon H hrrehy bacteria with more copies of Trr7 yrcw f:dst,er in st.reptom,vrin medium.

40

I). (‘. YOlTVAN

K7’ dl!

one donor are termed cousins. Since an E. coli library was used in this scheme, the mutagenesis is termed clonal. Cousins bear mutagenized R-primes with identical Tn7 insertions but their phenotypes may differ depending on whether or not, the crtD lesion and the Tn7 lesion are integrated into the chromosome. Four cousins arising from a single cross are shown in Figure 9. Let us assume that the Tn7 insertion is in a gene essential for photosynthetic growth. Different lower-case letters designate cousins from this cross : a, PSG - green ; In, PKG+ red : c, PSG red : d, PSG+ green. The Tn7 preferred insertion site is occupied in all of the strains in Figure 9 because transposition to this site occurs very soon after any Tn7-bearing plasmid is introduced into the background. There is no detectable phenotypic effect due to this insertion in either E. coli or El. capsdata. Figure 9 is an oversimplification of the recombination and deletion processes, in that only one copy of the mutagenized R-prime is shown ; only the R-prime that will recombine is depicted. After recombination, the wild-type alleles are deleted from this particular recombinant R-prime and the c&D and Tn7 mutations in the chromosomes are phenotypioally expressed. Other copies of mutagenized R-primes do not, delete until the streptomycin selection is terminated and the bacteria are subcult,ured (Fig. 10). Wh i 1e under selection, multiple copies of Tn7 yield higher levels of antibiotic resistance, hence undeleted plasmids are selected. Ruvkun & Ausubel (1981) proposed a general scheme for site-directed mutagenesis using homologous recombination with transposon mutagenized plasmids. Recombined plasmids bearing wild-type alleles must, be excluded t,o reveal the mutagenized chromosomal gene. This requires a second cross in which a second, incompatible plasmid is selected. This was our original plan until WV observed the spontaneous deletion of wild-type alleles. We have shown that the deletion is due to intramolecular homologous recombination between IS21 elements flanking the prime sequence. This process is dependent on the RecA protein in E. coli and, presumably, a similar protein in R. capsulata. Selection for streptomycin selects against deleted mutagenized R-primes because of a genedosage effect increasing the level of resistance with increasing numbers of transposons. This enhances recombination with the chromosome. since multiple copies of t,he mutagenized alleles are maintained. After recombination with the chromosome, the wild-type alleles. transiently carried by an R-prime, are deletjed &a intramolecular recombination between the flanking IS21 elements. This deletion is immediate, since the prime bears no resistance marker. An alternative explanation is that this plasmid is lost by competition with the other R-primes that contribute to the antibiotic resistance. Remaining prime sequences delete after t.hr antibiotic is removed and the mutants are subcultured. Under kanamycin selection alone, merozygotic colonies of SB1003 (pRPS404) develop only infrequent and narrow green sectors, since there is no selection against strains bearing deleted R-primes.

This work was supported by Environmental Research Division contract no. W-7405FINC48.

the Director of Energy Research. of the United States Department

Hiomrdiual of Energy

and under

THANSPOSON MUTAGENESIS

OFTHE

PHOTOSYNTHETICAPPARATVS

41

REFERENCES Barth, P. T. & Grinter, N. J. (1977). In DNA Insertion Elem,mts, Plasmids, and Episomes (Bukhari, A. I., Shapiro, J. A. & Adhya, S. L., eds), pp. 675-677, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Barth, P. T., Dalta, N., Hedges, R. W. & Grinter, N. J. (1976).
(i.

(1978). Curr.

Topics

Bioenerg.

8, 161-207.

G.. Dierstein. R. & Schumacher, A. (1976). FEBS Letters. 68. 132-136. Lichtenstrin, (1. & Hrenner, S. (1981). Mol. Gen. G’enet. 183, 380-387. Lichtenstrin, C. & Brenner, S. (1982). Nature (London) I 297, 601-603. Maniatis. T., Jeffrey, A. & Kleid, D. G. (1975). Proc. iVat. Acad. Sci., US.-4 72: Marmur, J. (1961). J. Mol. Biol. 3. 208-218. Marrs, B. L. (1978u). Curr. Topics Bioenerg. 8, 261-294. Bacteria (Clayton, R. K. & Sistron, Marrs, B. L. (19786). In The Photosynthetic pp. 873-883, Plenum Press, New York and London. Marrs, B. L. (1981). .J. Bacterial. 146, 1003~1012. Marrs, B. I,.. Kaplan, S. & Shepard, W. (1980). Methods Enzymol. 69, 29-37. Mind&, I,., Cohen. J. & Weisburd, M. (1976). J. Bactrriol. 126, 177-182. Rieb. (:., Holloway, B. W. &, Puhler, A. (1980). &net. Res. 36. 99-109. Ruvkun. U. B. & Ausubel, F. M. (1981). LVature (London), 289. 85-88. Sakanyan. V. A., Yakubov, I,. Z.. Alikhanian, S. I. & Stepanov, A. I. (1978). Mol. 165, 331-341. Sato. 31.. Staskawicz. B. ,J., Panopoulos, N. J., Pet.ers, S. & Honma, M. (1981).

1184--l 188.

W. R., eds)

Gen. Genet. Plasmid,

6,

Schumacher, A. & Drews, G. (1978). Biochim. Biophys. Acta, 501, 183-194. Scolnik. 1’. A., Walker, M. A. & Marrs. B. L. (1980a). J. Biol. ChewA. 255, 2427-2482. Scolnik. 1’. A.. Zannoni, D. & Marrs. B. L. (198Ob). Biochim. Biophys. Acta, 563, 230-24.0. Shiozawa. ?J.A.. Cuendet, P. ,4.. Dress. 6. & Zuber. H. (1980). Eur. J. Biochem. 111. 4X-460. Smith. S. E. & Summers. M. D. (1980). ilnal. Biochem. 109, 123-129. Solioz. M., Yen. H. C. & Marrs, B. L. (1975). J. Bactrriol. 123, 651-657. Taylor. I). P., Cohen, 8. N., Clark. W. G. & Marrs. B. L. (1982). .J. Bucteriol. In the press. Yen. H. (‘. & Marrs, B. L. (1976). J. Bacterial. 126, 619429. Yen. H.-c’. & Marrs, B. (1977). Arch. Biochem. Biophys. 181, 411-418. Yen, H.-C., Hu, S. T. & Marra, B. I,. (1979). J. Mol. Biol. 131, 157-168. Youvan. D. (y., Zsebo, K.. Marrs, B. L. & Hearst. ,J. E. (1982). J. Bacleriol. In the press. Note added in proof: At the time this manuscript was written, no photosynthetic: apparatus mutations had been observed in strain UAR87. The data in Figure 7 reveal that the transposon mutagenized Barn F-Eco F fragment had not recombined into the chromosome. Subsequent subculturing of the UAR87 merozygotes and observation with infrared fluorescence photography have led to the isolation of an enhanced fluorescence mutant lacking all three reaction center polypeptides. The UAR87 transposon insert in the Barn F-&o F restriction fragment defines the reaction center rscB locus. This locus has been further characterized by marker-rescue experiment,s: see Youvan et al. (1982). Edited

by S. Brenner