Functional analysis of the Streptomyces ambofaciens element pSAM2

PLASMID

25,40-52 (199 I)

Functional Analysis of the Streptomyces

ambofaciens

Element pSAM2

TAMARASMOKVINA,*~'FR~D~RICFWCXARD,*JEAN-LUCPERNODET, ANNICKFRIEDMANN,ANDMICHEL GU~RINEAU Laboratoire de Biologic et GCnlique Moltkdaire, URA CNRS Dl354, B&t. 400, UniversitC Paris-&d, F-91405 Orsay Cedex, France

Received June 8, 1990; revised December 4, 1990 pSAM2 is an 1I-kb element integrated in the Streptomyces ambofaciens ATCC23877 genome and found additionally as a free replicon present at several copies per chromosome in strain J13212,the derivative ofATCC23877 isolated after uv irradiation. In spite of its small size, this element specifies numerous functions including maintenance, site-specific integration, selftransmissibility, pock formation, and mobilization of chromosomal markers. After transfer of the free form of pSAM2 to Streptomyces lividans, the free and the integrated forms coexist. A functional map of pSAM2 was deduced from phenotypes exhibited in S. lividans by numerous deletion or insertion derivatives. In addition to the previously characterized regions sufficient for site-specific integration we have shown that separate regions are involved in either plasmid maintenance as a free molecule, plasmid transfer, and pock formation. Transfer of pSAM2 could depend on its ability to be maintained in a free form, since plasmids deficient in this function are transferred at very low frequency. Deletions of some regions of the plasmid are lethal for the plasmid or the host, but if some other regions are deleted simultaneously, transformants can be obtained. 0 1991 Academic Press, IX

Many naturally occurring plasmids have been isolated from actinomycetes (for review, see Hopwood et al., 1986). The majority of them specify common functions, such as those necessary for replication, transfer, and pock formation. Contrary to the situation encountered with plasmids of gramnegative bacteria, only a few kilobases located on the plasmid are involved in the conjugation event. Other genetic elements, mainly found among actinomycetes, have the properties of plasmids but exist as integrated sequences in the chromosome (for review, see Hopwood et al., 1986; Htitter and Eckhardt, 1988; Omer and Cohen, 1989; Kieser and Hopwood, 1990). These elements are capable of transfer by conjugation and site-specific integration in the chromosome of the recipient strain.

Particular interest has been focused on this class of elements because of their academically interesting features and the possibility of constructing integrative vectors which are stably inherited through generations at one copy per chromosome. pSAM2 is an 1 1-kb integrated element from Streptomyces ambofaciens ATCC23877 (strain Bl) and ATCC15154 (strain B2, an uv derivative of strain Bl) and is found additionally as ccc (covalently closed circular) DNA in the strain J132 12 (strain B3, another uv derivative of strain B 1). This element is self-transmissible, able to elicit the lethal zygosis reaction (Pernodet et ul., 1984) and to enhance the recombination of chromosomal markers (Smokvina et al., 1988). Both the free and the integrated forms can integrate site-specifically after transfer in the genome of S. lividans and of S. ambofaciens DSM40697 (strain A, naturally devoid of pSAM2) (Boccard et al., 1988). To characterize in more details the different functions of pSAM2 and to study their relationship, it was first useful to determine a

r Present address: John Innes Institute, Colney Lane, Norwich NR4 7UH, UK. ’ Present address: The Sainsbury Laboratory, John lnnes Center for Plant Science Research, Colney Lane, Norwich NR4 7UH, UK. 0147-619X/91 $3.00 Copyright 0 1991 by Academac Press, Inc. All rights of reproduction in any form reserved.

40

FUNCTIONAL

ANALYSIS

functional map of this element. The site-specific integration event of pSAM2 has been partially characterized. A 2.3-kb fragment sufficient to mediate the integration event (Boccard, 1988; Kuhstoss et al., 1989; Smokvina et al., 1990) contains the integrase (int) and putative excisionase @is) genesas well as the plasmid attachment site, attP (Boccard et al., 1989a). The recombination event occurs through a 58-bp sequencepresent in pSAM2 (attP) and in the chromosome (attB) (Boccard et al., 1989b; Kuhstoss et al., 1989). The attB site overlaps a putative tRNA gene conserved among actinomycetes (Mazodier et al., 1990). Here we report a functional map of the free form of pSAM2 originating from the S. ambofuciens strain B3. Deletion and insertion derivatives were analyzed in S. lividans, pSAM2 regions involved in maintenance of the free form, integration, transfer, and pock formation were determined. MATERIALS

AND METHODS

Strains and Culture Conditions pSAM2 was extracted from the S. ambofaciens strain J13212 (strain B3) (Pert&et et al., 1984). S. lividuns TK64 (pro-2, str-6) (Hopwood et al., 1983) and JT46 (pro-2, str-

OF pSAM2

41

tion, protoplasts were regenerated on R2YE medium at 30°C. Transformants were selected using 50 pg/ml nosiheptide (nos) or 400 pg/ml spectinomycin (spc). The concentration of spectinomycin in HT medium was 100 pg/ml. Nosiheptide was a kind gift of RhBne Poulenc-Sante and spectinomycin a kind gift of Upjohn Co.

Vectorsand Plasmid Constructions pIJ39 (pBR322 with a 1.8-kb BamHI fiagment insert containing the thiostrepton resistance gene (tsr) from S. azureus: Thompson et al., 1980) was used for the construction of the shuttle plasmid pTS39, tsr also confers resistance to nosiheptide. pBR329 (Covarrubias and Bolivar, 1982) was used for the construction of pOSl1. The Q interposon containing the str (streptomycin)/spc resistance gene was obtained from plasmid pHP45Q (Prentki and Krisch, 1984). Constructions of pSAM2 derivatives were performed by various deletions, Q interposon insertions and Klenow filling in (Table 1). One type of deletion was carried out by cleavage of pSAM2 at the unique BstEII site (after filling in the BstEII site of the tsr-containing fragment) and using ExoJII and ExoVII enzymes as described by Maniatis et al. (1982). For DNA ligation T4 DNA ligase was used. Another type of deletion was obtained by removing fragments between two restriction sites, blunting the ends by DNA polymerase I Klenow fragment and recircularizing. The Q interposon was extracted from pHP45Q using EcoRI, BumHI, SmaI, or Hind111 digestions, purified by agarosegel electrophoresis and inserted at different restriction sites of pTS39. Mutations were also obtained by digestion of plasmids at different restriction sites and filling in with DNA polymerase I Klenow fragment.

6, deficient in intraplasmid recombination) (Tsai and Chen, 1987) were used asrecipients in transformation experiments. S. lividans TK64, TK54 (his-2,leu-2, spc-1) (Hopwood et al., 1983) and TK22 (t-if-l) (T. Kieser, unpublished results) were used in mating experiments. Mating experiments were performed on plates of R2YE medium (Thompson et al., 1982) in the presence of an excessof the recipient strains. Escherichia coli strains for cloning were HBlOl (Boyer and RoullandDussoix, 1969) and DHl (Hanahan, 1983). S. lividans strains were grown at 30°C on R2YE medium and Hickey-Tresner (HT) DNA Manipulations medium (Hickey and Tresner, 1952). Before Plasmid DNA from E. coli was isolated by protoplasting, S. lividans strains were grown at 28°C in YEME liquid medium containing the cleared lysate method (Maniatis et al., 34% (w/v) sucrose, 5 mM MgC&, and 0.5% of 1982) and from Streptomycesby alkaline lyglycine (Bibb et al., 1977). After transforma- sis (Kieser, 1984). Transformation of Strepto-

42

SMOKVINA

ET AL.

TABLE 1 pSAM2 DERIVATIVES AND THEIR cHARAcrtIRIsncs Name poS7 pos11 pTS33 pTS39 pD5 PD~A PD~B PD8 pDl0 pD19 pTS4 1 pTS42 pTS60 pTS64 pTS65 pTS66 pTS67 pTS68 pTS69 pTS7 1 pTS74 pTS79 pTS83 pTS84 pTS86 pTS88 pTS89 pTS90 pTS94 pTS96 pTS105 pTS106 pTS.107

Construction’

Stat$

Form’

Pocks

Transfeti

de]. BamHI( 15)-BumHI(25) of pSAM2, insert. of tsr gene (BarnHI fragment) pBR329 insert in EcoRI of pOS7 EcoRI(ZO)-EcoRI(31) of pSAM2 in EcoRI of pIJ39 pIJ39 insert. in EcoRI(20) of pSAM2 0.5-kb de]. from BstEII(5) of pTS39 0. I-kb de]. from BstEII(5) of pTS39 2.3-kb de]. from BstEII(5) of pTS39 5.0-kb del. from BstEII(5) of pTS39 1.O-kb de]. from BstEII(5) of pTS39 2.5-kb del. from BstEII(5) of pTS39 Q interposon in EgfII(26) of pTS39 s2interposon in BglII( 14) of pTS39 BglII( 14) fill in of pTS39 Q interposon in KpnI( 16) of pTS39 del. KpnI( 16)-EcoRI(20) of pTS39 (G) Q interposon in KpnI(9) of pTS39 EcoRI( 3 1)-XbaI( 18) of pSAM2 in EcoRI of pIJ39 Q interposon in BstEII(5) of PTS39 0 interposon instead of SucI( 12)-SacI( 13) of pTS39 del. NotI(24)-NotI(29) of pD19 BclI(28) fill in of pTS60 del. BstEII(S)-EcoRI(20) of pTS60 del. &WI(~)-KpnI( 16) of pD19 6% C, D, E, F) Kpn I(9) fill in of pTS65 de]. &x1( 12~&zcI( 13) of pTS39 (D) de]. &zcI( 12)-SacI( 13) and KpnI( 16)EcoRI(20) of pTS39 (D, G) del. KpnI(9)-KpnI( 16) of pTS39 (C, 4 E, F) del. SacI( 12)-S&( 13) and KpnI( 16)EcoRI(20) of pD19 (A, D, G) XbaI( 18) fill in of pTS39 del. BglII( 14)-EcoRI(20) of pTS39 (F, G) de]. SucI( 12)-&#I( 14) of pTS4 1 (D, E) de]. SucI( 12~%I( 13) of pD 19 (A, D) de]. KpnI(9)-SucI( 13) and KpnI( 16)EcoRI(20) of pD19 (A, C, D, G)

V.

free, int.

+

ND

V.

free, int. int.

+ -

ND ND

free, int. free, int. free, int. int. int. free, int. free, int. free free, int. free, int. free, int. free, int.

+ defective defective defective + + defective +

1 3 x 10-3 8 x IO-* 5 x lo+
free, int. ND

-

4 x 10-4 ND

ND int. int.

-

ND 7 x 10-6 ND

int. ND ND

+ +

4 x 10-6 ND ND

v.

int.

-

4 x 10-6

V.

free, int. free, int.

+ +

1 1

ND int.

-

ND
V.

V. V. V. V. V. V. V. V. V. V. V. V.

n.t. n.t. V.

a.t. n.t. V. V.

n.t. V.

a.t. a.t. n.t.

V.

n.t. a.t. V.

’ de]., deletion; (number), position of restriction site (see Fig. 1); boldface letter, deleted region (see Fig. 2). b v., viable; a.t., “abortive” transformants; n.t., no transformants. ’ int., integrated; ND, not determined. d Transfer is calculated from the ratio of recipient colonies containing the plasmid and those without plasmid. ND, not determined.

FUNCTIONAL Pvull(1)

ANALYSIS OF pSAM2

43

O/ 16.95

FIG. 1. Restriction map of pTS39. pSAM2 is represented by a thick line and pIJ39 by a thin one. The restriction sites of pSAM2 are numbered clockwise from the unique AwII site.

myces was carried out as described by Hop

previous pSAM2 derivative, pOS7, in which

wood et al. (1985). Streptomycestotal DNA the two adjacent BamHI fragments of 0.9 was prepared as described by Hintermann et and 1.0 kb (containing the EcoRI(20) site) al. (198 1). For Southern blot analyses, di- were replaced by tsr gene, shows the same gestedDNA was blotted on Hybond-N filters features as pSAM2 (Simonet et al., 1987) in(Amersham) as recommended by the manufacturer. The 7.5-kb PstI fragment of the S. lividans chromosome containing the pSAM2 attB site (Boccard et al., 1988), the pOS210 plasmid (7.6kb BamHI fragment of pSAM2 cloned in pBR329, Smokvina et al., 1990) and pTS39 were used as probes alter being labeled by nick-translation (Rigby et al., 1977) using [&‘P] dCTP and enzymes from Amersham. Filters were washed as described previously (Boccard et al., 1989b).

dicating that the EcoRI(20) site and its surrounding regions are not involved in features we intended to study. The constructed shuttle plasmid, called pTS39, was tested for its ability to confer nosiheptide resistance (nosR) in S. lividans, to exist as a free plasmid, to integrate in the host chromosome, to form pocks, to transfer, and to produce small circular molecules in S. lividans.Afier transformation of S. lividans with pTS39, nosR clones were isolated and tested for their plasmid content. They all contained RESULTS pTS39 as cccDNA without any detectable modification. Hybridization analysis of total Construction of the Shuttle Plasmid pTS39 DNA from S. lividans transformants (Fig. 2) In order to study pSAM2 we first con- showed that pTS39 was, in addition to the structed a shuttle plasmid able to replicate in free form, present integrated at the specific E. coli and to be maintained in Streptomyces. attB site in the chromosome, like pSAM2 The pIJ39 plasmid, containing the tsr gene from strain B3 used for its construction. on a 1.8-kb BamHI fragment, was cloned When a mutation in the coding sequence of into the EcoRI(20) site of pSAM2 (Fig. 1). the int genewas introduced, using the Q interThis insertion site was chosen because a poson or filling in DNA at the BglII(26) re-

44

SMOKVINA

ET AL.

BamHl

A

----J BarnHI,,*;' 1,'

,’

"\\ '\

,

circular pSAM2 derivative

FIG. 2. Status of the pSAM2 derivatives in S. lividans. The status of the pSAM2 derivatives was determined by Southern hybridization of the “P-labeled BumHI fragment of pSAM2, which contains the attP site, with BumHI-cleaved total DNA from S. lividans transformed by the pSAM2 derivatives. (A) Schematic representation of the different types of fragment revealed by the probe. The region of pSAM2 derivatives variable in the different derivatives is represented by a dotted line, the region unchanged in all derivatives is represented by a continuous line. When a pSAM2 derivative is integrated the two junction fragments (fragment A constant in length and fragment B variable in length depending on the derivative) are revealed by the probe. When a pSAM2 derivative is also free a third fragment (fragment C) is revealed by the probe. (B) Southern blot analysis. The “P-labeled BarnHI fragment of pSAM2, which contains the attP site, was hybridized with BarnHI-cleaved total DNA from S. lividam transformed by: (I) pTS39; (2) pDS; (3) pD6B; (4) pD8; (5) pDlO; (6) pD19; (7) pTS42; (8) pTS74; (9) pTS84; (10) pTS90; (1 I) pTS96. Arrows indicate fragments corresponding to the circular free form of the pSAM2 derivatives.

striction site, pTS41 and pTS40 were obtained, respectively. These two plasmids could not integrate into the S. lividans chromosome but existed in the free form (Boccard et al., 1989a). The same result was obtained when the attP site of pTS39 was deleted (Smokvina et al., unpublished results). pTS39 affected in its integration functions is present in the free form and maintained without selective pressure in the liquid medium. Thus, pTS39 is probably able to replicate autonomously with or without the integrated copy. The morphology of pocks formed by pTS39 on a lawn of an S. lividans strain was unchanged compared to pocks of pSAM2. To test the frequency of transfer of pTS39 during mating with a plasmid-free strain, S. lividans TK64 (stP) containing pTS39 was mated with S. lividuns TK54 (spcR). The TK54 exconjugants were selected on plates containing spectinomycin and the efficiency of transfer was calculated from the ratio of

SpcR+ NosR colonies among SpcRcolonies. The transfer of pTS39 was as efficient as that of pSAM2 (100%: Table 1). pTS39 also produces small circular molecules (scm) in S. lividuns as described for pSAM2 (Simonet et al., 1987). In order to study functions carried by pSAM2 and their locations on the plasmid, we constructed various deletion or insertion derivatives of pTS39. Another type of mutant was obtained by insertions of the Q interposon in different sites or by filling in restriction sites (seeMaterials and Methods). These approaches led to location of different pSAM2 functions in arbitrarily delimited regions which were designated A to J (Table 1 and Fig. 3).

pSAM2 Determinants for the Free and Integrated Forms pTS39 integrates site-specifically into the chromosome of different Streptomycesspe-

FUNCTIONAL K EcoRI(31)

ANALYSIS OF pSAM2

45

and F regions and, second, sequenceslocated near the K region and the beginning of J (Fig. 3). To confirm that these two regions are involved in pSAM2 maintenance as a free plasmid, further derivatives were constructed. When the right EcoRI( 3 1)-XbuI( 18) fragment was cloned into pIJ39 (pTS67), no nosR clones were obtained after transformation of S. lividuns JT46, reinforcing the hypothesis that some functions involved in maintenance ” . ., (, ,, _, ,_. .’ .’ of a free copy are situated on the left of the F
46

SMOKVINA

regions (seebelow), involvement of the E region in maintenance of a free copy was not tested. The derivative pOS7, which has two adjacent BumHI fragments (between BamHI( 15) and BamHI(25) sites) replaced by the tsr gene, contains a unique EcoRI(31) site (Simonet et al., 1987). To construct a shuttle Streptomyces-E. coli plasmid, pBR329 was inserted into the EcoRI site, forming pOS 11. After transformation of S. lividans TK64 with pOSl1, plasmid DNA was extracted from several nosRclones. Gel electrophoresis showed that the plasmid was smaller than pOSl1. This plasmid, named pOSl1 A, lacked pBR329 but also about 600 bp of pOS7 DNA on both sides of the EcoRI site where pBR329 was inserted. The amount of extracted plasmid DNA was always higher by a factor of 10. It must be noticed that the plasmid yield is not always correlated to the real number of plasmid copies because the yield can be affected by changes in membrane composition or growth rate. For pOS 11A the increase in plasmid yield that we observed, being important, could reflect an increase of copy number. In this case,we can imagine that the deleted region is directly involved in the plasmid copy number control. However, the change of plasmid copy number can be also due to changes in plasmid organization which do not directly alter elements controlling plasmid copy number. The sequence analysis of the region surrounding this EcoRI site of pSAM2 revealed the presence of a 30-bp sequencedirectly repeated at 200 bp to the right and 400 bp to the left of the EcoRI site. Restriction analysis of this region in pOS 11A suggestedthat the deletion of pBR329 with a fragment of pOS7 occurred by recombination between the two 30-bp direct repeats. Although the I region is sufficient for integration, very few transformants were obtained. The transformation efficiency with plasmids containing the 3.5-kb BamHI(25)EcoRI(3 1) fragment (I, J, K regions) was higher than those obtained with the minimal integrative fragment (I region) by a factor of

ET AL.

10. The BamHI(25)-EcoRI(3 1) fragment was used for the construction of the integrative vector pTS55 (Smokvina et al., 1990). The addition of the EcoRI(20)-BamHI(25) fragment (H region) enhanced by a factor of 20 the transformation efficiency, and transformants contained only integrated plasmid DNA (pTS33 in Table 1). The differences in transformation efficiency obtained with different deletion derivatives existing only in integrative form suggestthat the C region also affects the number of transformants. The efficiency of transformation with pTS 107 (lacking A, C, D, G) was IO-fold lower than with pTS90 which has a deletion of the A, D, and G but not the C region. This difference in transformation efficiency was also observed between pD6B and pD8. pD6B, which contains the C region, gave rise to IO-fold more transformants than pD8. Thus, regions (H, J, K) on both sides of the minimal integrative fragment (I), as well as region C, increase the number of transformants obtained with integrative plasmids.

pSAM2 Determinantsfor Plasmid Existence When some fragments of pTS39 were deleted, the resulting constructions could not give rise to transformants although they contained the entire region necessary and sufficient for integration. This suggested that these derivatives lacked regions essential for their existence in both integrated and free form. Deletion of the D and E regions (pTS 105), of C, D, E, and F (pTS89), or of A, C, D, E, and F (pTS83) did not allow recovery of transformants, but deletions of the A region alone (pD19), of G (pTS65) or F and G (pTS96) had no effect on the recovery of transformants. The presence of the B region, when D and E are deleted, could be responsible for the absence of transformants because plasmids lacking A, B, C, D, and E (pD8) or A, B, C, D, E, F, and G (pTS79) could transform S. lividans (Fig. 4A). Another construction obtained by the insertion of the Q interposon in the KpnI(9) site

FUNCTIONAL

A

.. .. ‘.

B

EcoRI(31) ,

NorI(29).

,,

_. .’ . . ,. ,. _. ,.

. ” ”

47

ANALYSIS OF pSAM2 EcoRI(3I)

..- \

EcoRI(20) EcoRf(20)

FIG.4. Deletion and insertion derivatives of pTS39 giving (A) viable transformants or not able to give transformants; (B) viable transformants or those called “abortive” (see text). Arcs represent deleted fragments. Lines indicate filling in or insertion of a in restriction sites. Pattern of arcs or lines indicate the observed phenotype. Boldface letters show regions of pSAM2 deleted in different derivatives.

(pTS66), situated between the B and C regions, did not allow recovery of transformants, whereas filling in this site (pTS84) gave transformants containing integrated plasmid. This suggeststhat a region included in a putative transcriptional unit overlapping the KpnI(9) site could be also involved in the recovery of pSAM2 transformants. Other derivatives, which we called “abortive,” yielded transformants which appeared 2 of 3 days later than others and their morphology was different from that of normal S. lividans transformants: they were darkcolored, never grew very much, and did not sporulate. When these transformants were picked or replica plated on selective media they failed to grow. Deletion of the D region (pTS86) or its replacement by the Q interposon (pTS69) gave rise to such “abortive” transformants (Fig. 4B). The deletion of D either with A (pTS106) or with G (pTS88), respectively, also yielded “abortive” colonies after transformation. However, the combined deletion of D with A and G (pTS90) or with A, C, and G (pTS107) did not affect plasmid existence because transformants were recovered at identical efficiency. This suggeststhat the presence of the D region is

necessary to control functions encoded by the A and G regions which could be toxic for the host. The derivative pD8, which lacks the A, B, C, D, and E regions but contains F and G, is able to give transformants, suggesting that the effect of G could require another region(s) of pSAM2 which are deleted in pD8.

pSAM2 Determinants for the Transfer and Pock Formation To test the pSAM2 derivatives for the ability to yield pocks, protoplasts of S. lividans TK64 after transformation with each deleted plasmid were plated on R2YE without adding the antibiotic layer and the morphologies of pocks were compared to that of pSAM2 pocks. pD6B, pD8 and pD19 did not yield any pocks but pD5, pD6A, and pD10, containing short deletions (between 100 and 1000 bp) all located in the A region (Fig. 5) showed variations in their pock-forming ability. pD6A yielded almost normal pocks like those of pTS39, but was affected in transfer ability (seebelow); pD5 formed small pocks, and pocks generated by pDl0 were visible only after replica plating of pD 1O-containing patches. The extent of the deletions in pD6A,

48

SMOKVINA

P “Drmd pocki ..dlfferenlpar . . . . . . . . . . . “Opock,

FIG. 5. Deletion and insertion derivatives of pTS39 able to form normal or different (“small”) pocks and those not able to give pocks. Arcs with patterns corresponding to different phenotypes indicate deleted fragments. Phenotypes of Q insertions and Klenow filling in derivatives are also marked with patterned lines showing the corresponding phenotype.

ET AL.

The pTS74 derivative which exists only as integrated plasmid and pTS84 which is affected in the free form maintenance could not form pocks, and transfer during mating was 10b6. The phenotypes of pTS74 and pTS84 showed that two distant regions involved in the pSAM2 maintenance as a free plasmid were also required for its transfer. A plasmid deleted in the D region gave rise to “abortive” nosR transformants but could also be detected by pock formation. This suggeststhat the D region does not contain plasmid functions essential for the formation of pocks. pTS65 and pTS96, lacking the G and F and G regions, respectively, could form normal pocks and transfer at 100% as well as pTS94 (Klenow filling in at the XbaI( 18) site located in the G region). The results showed that two separated regions are involved in pSAM2 conjugal transfer and pock-forming: first, a part of the A region extending to the B region and, second, the E and F regions.

pD5, and pDl0 correlates with the ability to form normal pocks. The transfer efficiency of DISCUSSION each deletion derivative plasmid was deterFunctional analysis of the free form of mined, as described for pTS39 (Table 1). Very low plasmid transfer ( 10e5- 1Oe6)was pSAM2, originating from the mutant strain observed with the derivatives pD6B, pD8, B3 of S. ambofuciens, was carried out in S. and pD19, containing large deletions (2.3-5 kb). The transfer efficiency of small deletion derivatives (pD5, pD6A, and pDl0) was rather low, between 8 x lo-’ and 5 X 10m3. When the D interposon was inserted into the BstEII(5) restriction site (pTS68), no pocks were observed and the transfer of this plasmid was 4 X 10w4.These results indicated that the BstEII site is situated in a region carrying pock-forming functions and involved in the conjugative transfer of pSAM2. The insertion of the 0 interposon into the BglII( 14) restriction site (pTS42) also abolished pock formation, and transfer of the plasmid decreasedto 10e3.However, bluntending DNA with Klenow fragment at the BglII( 14) site (pTS60) slightly affected pocks FIG. 6. pSAM2 functional map deduced from the phenotypes of different insertion and deletion derivatives. and transfer was rather efficient (75%).

FUNCTIONAL

ANALYSIS

lividans strains using the shuttle derivative pTS39. Regions involved in plasmid maintenance as a free copy, site-specific integration, transfer, and pock formation were identified (Fig. 6). pSAM2 probably also carries functions which can be lethal for the plasmid or the host. The D and E regions are absolutely required for the recovery of transformants but when they are deleted together with the A, B, and C regions, transformants were obtained. The A, B, and G regions seem to be responsible for the nonexistence of D and E deletion derivatives. We cannot explain this phenomenon, which requires further analysis. However, several hypotheses can be proposed. A kil-kor system, originally found on RK2 and some others plasmids of gramnegative bacteria and also described in the well-characterized Streptomyces plasmid pIJ 101 (Kendall and Cohen, 1987), could be responsible for the nonexistence of some derivatives. In pIJ 101, genetic loci necessaryfor plasmid transfer contain loci lethal to the plasmid and/or host. Stein et al. (1989) suggested that this serves as a genetic switch to control the transfer process.In the same way, plasmids of gram-negative bacteria have been shown to contain potentially lethal genesnecessaryfor replication or conjugal transfer (reviewed by Thomas and Smith, 1987; Kues and Stahl, 1989). The A region is involved in pock formation and in transfer of pSAM2. The recovery of “abortive” transformants with plasmids containing the A region and lacking the D region is reminiscent of pIJ 101 derivatives defective in kil-override (kor) functions. The D region of pSAM2, as well as the region surrounding the KpnI(9) site, is involved in the maintenance of a free copy. The G region does not seem to be involved in pock formation, transfer, integration, or maintenance of a free form of pSAM2. However, its effect is also overridden by the D region, suggesting that a similar phenomenon could be responsible for the nonexistence (“abortive” transformants) of mutants containing G and lacking D. Another hypothesis, involving deregulation of plasmid mainte-

OF pSAM2

49

nance as a free copy, could also be proposed. Integrated plasmids which replicate could interfere with the replication of the chromosome and induce lethality of such transformants. In the case of SLPl it has been proposed that the imp (inhibition of maintenance of the plasmid) function inhibits the ability of SLPl to replicate and/or to be maintained as an extrachromosomal element (Grant et al., 1989). pSAM2 from the B2 mutant strain of S. ambofacienshas never been detected in the free form, whereas pSAM2 in strain B3 exists simultaneously in both free and integrated forms. The mutation is situated on the plasmid becausein S. lividans the same situation is present. We do not know the nature or the location of the mutation but it is probable that the integrated copy is not replicated. Changes in this regulation could be lethal for the host. A previously described region involved in site-specific integration of pSAM2 contains the int and the putative xis genes. We determined that regions upstream of the xis gene and downstream of attP increase integration efficiency and we speculated (Smokvina, 1990) that transcriptional signals located in theseregions could be responsible for this phenomenon (Smokvina et al., to be published). The C region also increased the transformation efficiency of integrated plasmids. This region is involved in pSAM2 maintenance in a free form. We still do not know if the increasedtransformation efficiency of such derivatives could be due to an abortive replication. We identified two regions necessaryfor the maintenance of pSAM2 as a free plasmid. One, of 1.5 kb, is situated upstream of the int and putative xis genes. The other is situated within a 2.7-kb fragment included in the B, C, D, and E regions. These two regions are separatedby a region involved in transfer and pock formation. We can suppose that the maintenance of the free copy of pSAM2 is due to its ability to replicate, but we do not have basis for distinguishing between replication and maintenance in the free form. In

50

SMOKVINA

many other Streptomyces plasmids replication functions are clustered together, allowing isolation of minimal replicons of between 2 and 5 kb (Hopwood et al., 1986). In the S. coelicofor SLPl.2 plasmid (Bibb et al., 198I), two regions of 1.4and 2.7 kbinvolved in replication of the plasmid are also separated one from the other (Kieser cited in Omer et al., 1988). It was suggestedthat in SLPl.2, which replicates only in the absenceof an integrated copy, one of the regions necessaryfor replication (or survival) may represent a kor locus required to override lethal loci on the plasmid (Hopwood et al., 1986). In pSAM2 we were able to distinguish the functions needed for maintenance of a free plasmid from those required for existence because mutants which do not exist in the free form can still integrate into the chromosome. It remains to investigate whether the two regions necessary for the maintenance of the free plasmid are components of the replication or some other system of pSAM2. It is also possible that one of the two regions could encode a replication regulator. One can imagine that this putative regulator, of which presence is necessary for replication, is a positive one but it must be noticed that in most prokaryotic systems already described, replication is negatively regulated (Scott, 1984; Novick, 1987). Moreover if the increase of plasmid yield after spontaneous deletion in pOSl1 A corresponds to an increase of the copy number, the hypothesis that the deleted region is involved in copy number control implicates that the replication is also under negative control. Transfer and pock-forming regions of pSAM2 deduced from the properties of deletion and insertion derivatives were mapped in the BstEII(S)-BglII( 14) fragment of 4.5 kb. Two regions involved in this phenomenon are located on both sides of the region necessary for the maintenance of a free plasmid. The region located near the BstEII site contains several loci responsible for transfer and pock morphology. It has been shown that the mutants which give rise to small defective pocks are transferred at low frequency (5 x 10m3-8 X lo-‘). In pIJ101, defective

ET AL.

pocks could be induced by mutations in different loci (kilB, SpdA, and SpdB; Kieser et al., 1982; KendalI and Cohen, 1987). Spread (Spd) mutants are still transferred at 100% whereas transfer of kilB mutants is reduced. pSAM2 mutants in the A region are similar to kilB mutants of pIJ 101 becausethis region contains functions which can be toxic for the host and/or the plasmid and mutations in that region decreasethe efficiency of plasmid transfer. Two derivatives (pTS74 and pTS84) affected in a maintenance as a free plasmid are also transfer and pock mutants. The two mutations, in the KpnI(9) and &/I(3 1) sites, respectively, are probably in loci involved in a maintenance of the free copy. The inability of these mutants to transfer and to form pocks may be a consequence of their inability to maintain as free plasmids. We propose that pSAM2 needs to be maintained in a free form (probably replicative) for its transfer and that during mating the integrated plasmid is excised and replicated. The presence of mutants that cannot exist as free plasmids and that are always pock mutants too and the presence of pock mutants that can exist as free plasmids, as well as the absence of mutants that can forms pocks and transfer normally but are not maintained as free plasmids, contribute to this hypothesis. We cannot say if the low transfer frequency ( 1Op6)of pTS74 represents plasmid transfer or the frequency of basic chromosomal recombination as a consequence of mating. ACKNOWLEDGMENTS We thank T. Kieser for the kind gift of strains and A. Sabatier for interest in this work. T.S. and this work were supported by the CHVP (Couple H&e-Vecteur Performant) Program.

REFERENCES BIBB, M. J., FREEMAN,R. F., AND HOPWOOD,D. A. ( 1977).Physical and genetical characterization ofa second sex factor, SCP2 for Streptomyces coelicolor A3(2). Mol. Gen. Genet. 154, 155-166. BIBB,M. J., WARD,J. M., KIESER,T., COHEN,S. N., AND HOPW~~D, D. A. (1981). Excision of chromosomal

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ANALYSIS OF pSAM2

DNA sequencesfrom Streptomyces coelicolor forms a novel family of plasmids detectable in Streptomyces lividans. Mol. Gen. Genet. 18,230-240. BOCCARD,F. (1988). These de Doctorat, University of Paris VII, Paris, France. BOCCARD,F., PERNODET,J.-L., FRIEDMANN,A., AND GUBRINEAU, M. (1988). Site-specific integration of plasmid pSAM2 in Streptomyces Iividans and Streptomyces ambofaciens. Mol. Gen. Genet. 212,432-439. BOCCARD,F., SMOKVINA,T., PERNODET,J.-L., FRIEDMANN, A., AND GUBRINEAU,M. (1989a). The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages. EMBO J. 8,973-980. B~CCARD,F., SMOKVINA,T., PERNODET,J.-L., FRIEDMANN, A., AND GUI?RINEAU,M. (1989b). Structural analysis of loci involved in pSAM2 site-specific integration in Streptomyces. Plasmid 21, 59-70. BOYER,H. W., AND ROULLAND-Du%?.oIx,D. (1969). A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 170, 2287-2295.

COVARRUBIAS,L., AND BOLIVAR, F. (1982). Construction and characterization of new cloning vehicles VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication. Gene 17, 7989.

GRANT, S. R., LEE, S. C., KENDALL, K., AND COHEN, S. N. (1989). Identification and characterization of locus inhibiting extrachromosomal maintenance of the Streptomyces plasmid SLPI. Mol. Gen. Genet. 217, 324-331. HANAHAN,D. ( 1983). Studies on transformation ofEscherichia coli with plasmids. J. Mol. Biol. 166,557-580. HICKEY, R. J., AND TRESNER,H. D. (1952). A cobalt containing medium for sporulation of Streptomyces species.J. Bacterial. 64, 89 I-892. HINTERMANN, G., CRAMERI, R., KIESER, T., AND HOTTER, R. (198 1). Restriction analysis of the Streptomycesglaucescensgenome by agarosegel electrophoresis. Arch. Microbial. 130,2 18-222. HOPWOOD,D. A., KIESER, T., WRIGHT, H. M., AND BIBB,M. J. (1983). Plasmids, recombination and chromosome mapping in Streptomyces lividans 66. J. Gen. Microbial. 129,2257-2269. HOPWOOD,D. A., BIBB, M. J., CHATER,K. F., KIESER, T., BRUTON, C. J., KIEXR, H. M., LYDIATE, D. J., SMITH, C. P., WARD, J. M., AND SCHREMPF,H. (1985). “Genetic manipulation of Streptomyces. A Laboratory Manual.” John Innes Foundation, Norwich, UK. HOPWOOD,D. A., KIESER,T., LYDIATE,D. J., AND BIBB, M. J. (1986). Streptomyces plasmids: Their biology and use as a cloning vector. In “Antibiotic-Producing Streptomyces: The Bacteria” (S. W. Queener and E. Day, Eds.), Vol. 9, pp. 159-229. Academic Press,New York. H~~TTER,R., AND ECKHARDT,T. (1988). Genetic manip

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ulation. In “Actinomycetes in Biotechnology” (M. Goodfellow, S. T. Williams, and M. Modarski, Eds.), pp. 89- 184. Academic Press,London. KENDALL, K. J., AND COHEN, S. N. (1987). Plasmid transfer in Streptomyces lividans: Identification of a kil-kor system associated with the transfer region of pIJlO1. J. Bacterial. 169,4177-4183. KIESER, T., HOPWOOD,D. A., WRIGHT, H. M., AND THOMPSON,C. J. (1982). pIJlO1, a multicopy broad host-range Streptomyces plasmid: Functional analysis and development of DNA cloning vectors. Mol. Gen. Genet. 185,223-238. K~ESER,T. (1984). Factors affecting the isolation of ccc DNA from Streptomyces lividans and Escherichia coli. Plasmid 12, 19-36. KIESER,T., AND HOPWOOD,D. A. (1991). Genetic manipulation of Streptomyces: New integrating vectors and methods for gene replacement. In “Methods in Enzymology.” In press. KUES, U., AND STAHL, U. (1989). Replication of plasmids in gram-negative bacteria. Microbial. Rev. 53, 491-516. KUHSTO~& S., RICHARDSON,M. A., AND RAO, R. N. (1989). Site-specific integration in Streptomyces ambofaciens: Localization of integration functions in S. ambofaciens piasmid pSAM2. J. Bacterial. 171, 1623.

MANIATIS, T., FRITSCH, E. F., AND SAMBROOK,J. ( 1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, New York. MAZODIER, P., THOMPSON,C. J., AND BOCCARD,F. ( 1990).The chromosomal integration site of the Streptomyces element pSAM2 overlaps a putative tRNA gene conserved among actinomycetes. Mol. Gen. Genet. 222,43 l-434. NOVICK, R. P. (1987). Plasmid incompatibility. Microbiol. Rev. 51,38 l-395. OMER,C. A., STEIN,D., AND COHEN,S. N. (1988). Sitespecific insertion of biologically functional adventitious genes into the Streptomyces lividans chromosome. J. Bacterial. 170, 2 174-2 184. OMER,C., AND COHEN,S. N. (1989). SLPI: A paradigm for plasmids that site-specifically integrate in the Actinomycetes. In “Mobile DNA” (D. E. Berg and M. M. Howe, Eds.), pp. 289-296. Am. Microbial., Washington, DC. PERNODET,J.-L.,SIMONET,J.-M.,ANDGUBRINEAU,M. (1984). Plasmids in different strains of Streptomyces ambofaciens: Free and integrated forms of plasmid pSAM2. Mol. Gen. Genet. 198, 35-41. PRENTKI,P., AND KRISCH,I. L. M. (1984). In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29,303-3 13. RIGBY, P. W. J., DIECKMANN, M., RHODES,C., AND BERG, P. (1977). Labelling deoxyribonucleic acid to high specific activity in vitro by nick-translation with DNA polymerase I. J. Mol. Biol. 113,237-25 1.

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%orr, J. R. (1984). Regulation of plasmid replication. Microbial. Rev. 48, l-23. SIMONET,J.-M., BOCCARD,F., PERNODET,J.-L., GAGNAT, J., AND GU~RINEAU,M. (1987). Excision and integration of a self transmissible replicon of Streptomyces ambofaciens. Gene 59, 137- 144. SMOKVINA,T. (1990). These de Doctorat, University of Paris XI, Orsay France. SMOKVINA,T., FRANCOU,F., AND LUZZATI, M. ( 1988). Genetic analysis in Streptomyces ambojiiiens. J. Gen. Microbial. 134, 395-402. SMOKVINA,T., MAZODIER, P., E~OCCARD, F., THOMPSON,C. J., AND GUBRINEAU,M. (1990). Construction of a series of pSAM2-based integrative vectors for use in actinomycetes. Gene 94, 53-59. STEIN,D. S., KENDALL,K. J., AND COHEN,S. N. ( 1989). Identification and analysis of transcriptional regula-

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tory signals for the kil and kor loci of Streptomyces plasmid pIJ101. J. Bacterial. 171, 5768-5775, THOMAS,C. M., AND SMITH,A. C. (1987). Incompatibility group P plasmids: Genetics, evolution and use in genetic manipulation. Annu. Rev. Microbial. 41, 71101. THOMPSON,C. J., WARD, M. J., AND HOPWOOD,D. A. (1980). DNA cloning in Streptomyces: Resistance genes from antibiotic-producing species. Nature 286, 52-527. THOMPSON,C. J., WARD, J. M., AND HOPWOOD,D. A. ( 1982).Cloning ofantibiotic resistanceand nutritional genes in Streptomyces. .I. Bacterial. E&668-677. TSAI, J. F.-Y., AND CHEN, C. W. (1987). Isolation and characterization ofStreptomyces lividans mutants deficient in intraplasmid recombination. Mol. Gen. Genet. 208,211-218. Communicated by David A. Hopwood