Molec. gen. Oenet. 168, 1-25 (1979) © by Springer-Verlag 1979
Plasmid Replication Functions II. Cloning Analysis of the
RepA Replication Region of Antibiotic Resistance Plasmid R6-5 *
Isabel AndrOs 1 **, Patrick M. Slocombe l, Felipe Cabello 1 ***, Joan K. Timmis 1, Rudolf Lurz ~, Hans J. Burkardt 2, and Kenneth N. Timmis 1 Max-Planck-lnstitutfiir Molekulare Genetik, Ihnestral3e 63-73, D-1000 Berlin 33 2 Lehrstuhl fiir Mikrobiologieder Universit~itErlangen-NSrnberg, Erlangen, BRD
Summary. R6-5 is a low copy number, conjugative, FII incompatibility group plasmid that has a molecular length of 102 kb and that specifies resistance against several antibiotics (chloramphenicol, fusidic acid, kanamycin, streptomycin and sulphonamide) and mercury salts. By means of in vitro cloning procedures, mini plasmids have been generated that contain a D N A segment from the essential region of R6-5 that is only 2.6 kb in length. This DNA segment, which consists of two P s t I fragments that are adjacent in the parent plasmid, carries all genes and sequences required for the regulated replication and incompatibility properties of R6-5, including its origin of replication, O r i V , an essential function that has been designated R e p A , and the copy control function, Cop. Three different polypeptides, having monomer molecular weights of 23,000, 10,000 and 9,500 daltons, are synthesized in detectable quantities by minicells carrying pBR322 hybrid plasmids that contain DNA segments from the R6-5 essential region. A spontaneous deletion derivative of a pBR322 hybrid plasmid that carries the R6-5 origin of replication was isolated. Heteroduplex analysis of this derivative plasmid indicates that the deleted DNA segment carries the R6-5 replication origin and that its termini consist of short inverted repeat sequences.
Introduction Intensive investigation of the mechanism of DNA replication over the past few decades has provided *
Paper I in this series is Timmis et al., 1978b
** Current address: Department of Biochemistry, University of
Santander, Santander, Spain *** Current address ."Departmentof Microbiology,New York Medical College,Valhalla, New York, USA For offprints contact: Dr. K.N. Timmis
a detailed outline of this fundamental biological process (Kornberg, 1974). Nevertheless, the control of DNA replication is still poorly understood despite the elaboration of a number of models which suggest plausible systems that could effect such control (Jacob et al., 1963; Pritchard et al., 1969; Pritchard, 1978). The main problem in analyzing the control of DNA replication is the identification and isolation of the individual components of the control system in order to study their interaction. One component is probably a D N A sequence present on the chromosome with which the controlling element interacts. Because of their large size, intact chromosomes are difficult both to isolate and to use in biochemical studies. Furthermore, controlling elements are likely to be soluble proteins present to the extent of a small number of molecules per cell or to be membrane components whose functioning may depend upon membrane integrity. Plasmids would appear to be useful model systems for studying the control of D N A replication: they are relatively small and consequently simple to isolate as intact structures and their physical properties and molecular structure have been extensively characterized (Falkow, 1975). Furthermore, D N A cloning techniques should allow the isolation and amplification of genes and their products which are involved in the control of plasmid replication (Timmis et al., 1978e). The control of replication of some plasmids, notably the large low copy number plasmids, appears to be strictly maintained and is tightly integrated into the cellular division cycle (NordstrSm et al., 1972; Pritchard, 1978). Thus far, the following plasmidcoded functions have been identified as being essential for controlled plasmid D N A replication: (a) an origin of replication, O r i V , (b) an essential replication function, the R e p A function (Yoshikawa, 1974), and (c) a copy number control function, Cop.
0026-8925/79/0168/0001/$05.00
2 The p r o p e r t y of p l a s m i d i n c o m p a t i b i l i t y , Inc, which prevents the stable coexistence a n d coinheritance of two related plasmids in the same host cell is also u s u a l l y associated with p l a s m i d replication f u n c t i o n s ( T i m m i s et al., 1975; Lovett a n d Helinski, 1976). One d i s a d v a n t a g e of those large plasmids which are a p p r o p r i a t e models for the study of D N A replication c o n t r o l is that they are genetically p o o r l y characterized. The usefulness of such plasmids w o u l d therefore be e n h a n c e d , a n d their genetic analysis simplified, if they could be s u b s t a n t i a l l y reduced in size. Recently, we reported the g e n e r a t i o n in vitro of m i n i p l a s m i d derivatives of the fertility p l a s m i d F'lac a n d the transmissible a n t i b i o t i c resistance plasm i d R6-5 ( T i m m i s et al., 1975). The c o n s t r u c t i o n of these m i n i - p l a s m i d s , designated respectively p S C 1 3 8 a n d pSC135 (see Fig. 1), d e m o n s t r a t e d that all essential f u n c t i o n s for c o n t r o l l e d a u t o n o m o u s replication a n d p l a s m i d i n c o m p a t i b i l i t y are clustered o n single E c o R I - g e n e r a t e d D N A fragments c o m p r i s i n g only 6%, in the case of F'lac, a n d 13%, in the case of R6-5, of the total p l a s m i d D N A . I n the case of R6-5, the E c o R I f r a g m e n t carrying essential replication functions, the R e p A E c o R I fragment, is 13 kb in length, could code for a p p r o x i m a t e l y 17 proteins, a n d is therefore a m e n a b l e to genetic a n d f u n c t i o n a l analysis. This c o m m u n i c a t i o n reports the c l o n i n g a n d f u n c t i o n a l analysis of s u b s e g m e n t s of the R e p A E c o R I f r a g m e n t of R6-5 a n d the g e n e r a t i o n of a regulated a u t o n o m o u s m i n i R6-5 replicon that c o n t a i n s only 2.6 k b (or 2.6%) of R e p A E c o R I D N A sequences. These experiments a n d others r e p o r t e d in an acc o m p a n y i n g c o m m u n i c a t i o n (Synenki etal., 1979) have allowed us to m a p precisely the origin of replication, the i n c o m p a t i b i l i t y d e t e r m i n a n t a n d the R e p A f u n c t i o n of the R6-5 plasmid.
I. Andr6s et al. : Replication Functions of R6-5. II
Table 1. Bacterial strains used in this work Strain
Characteristics
Reference
E. coli K-12 C600
thr-1, leu-6, thi-1, Bachmann, 1972. supE44, lacY1, tonA21, ,~-
E. coli K-12 W1485
met 2 , 2r
Bachmann, 1972.
E. coli K-12 JC411 mal +, thy-, polAt~214, ColE1 -
heat-cured ColE1derivative of T S 2 1 4 : thy, his, argA, metB, leu, xyl, lacY,
Kingsbury and Helinski, 1970; Timmis et al., 1974.
E. coli DS410
sup + lac + derivative of P678-54; minA, minB, rpsL
Dougan and Sherratt, 1977.
E. coIi M2124
lac-, pro- deletion of DS410
Thompson and Achtman, 1978.
strA, polAts214
Media and Buffers L-Broth (LB) and 56/2 were prepared as described by Miller (1972), and Achtman (1975) respectively. 56/2 was supplemented with glucose (0.5%), thiamine (0.2 gg/ml) and proline (70 gg/ml). AM3 consists of Difco Antibiotic Medium No. 3 solidified with 1.5% Difco Agar, and enriched M9 medium is M9 glucose medium (Miller, 1972) supplemented with 0.5% Difco Casamino Acids and 10 gg/ml thiamine. Where appropriate, antibiotics were added to liquid or solid media at the following concentrations: ampicillin (Ap) 20 gg/ml, kanamycin (Km) 50 gg/ml, tetracycline (Tc) 5 ~tg/ ml, and sulfonamide (sulfathiazole: Su) 200 gg/ml. The latter antibiotic was always used in conjunction with Difco Mueller-Hinton (M-H) agar containing 5% lysed horse blood, BSG medium consists of 0.85% NaC1, 0.03% KH2PO4, 0.06% Na2HPO4 and 0.01% gelatin. TE buffer is 10 mM tris-HC1, pH 8.0 1 mM EDTA; TM is 10 mM tris-HC1, pH 7.4 - 10 mM MgC12; and TM-Sal is 8 mM tris-HC1, pH 7.6 6 mM MgC12 - 0.2 mM EDTA - 150 mM NaCI 50 gg/ml bovine serum albumin (Hamer and Thomas, 1976).
Isolation of Plasmid DATA
Materials and Methods Bacteria and Plasmids Bacterial strains and plasmids used in this study, and their relevant properties, are listed in Table 1. EeoRI and HindIII restriction endonucleases were prepared according to Greene et al. (1974) and Roberts (personal communication), respectively, and PstI was kindly provided by H. Mayer. BglI, BglII, HpaI, KpnI, SalI, XhoI, HincII, AvaI, Sinai, HaeII, and PvuII, were purchased from New England Biolabs, SstI was obtained from Bethesda Research Labs, and T4 DNA ligase was obtained from Miles. Kanamycin sulfate, chloramphenicol, tetracycline hydrochloride, sulfathiazole and Brij 58 were purchased from Sigma, Ampicillin (Binotal) was a gift from Bayer Leverkusen. Agarose was obtained from BioRad and Seakem.
Plasmids that exhibit relaxed replication (Timmis et al., 1974) in the presence of chloramphenicol were prepared from small volume cultures of bacteria grown in LB and treated with chloramphenicol (Cm) as described previously (Timmis et al., 1978d), except that the lysis solution was Brij 58-deoxycholate (Clewell and Helinski, 1969). Plasmids that do not amplify during Cm treatment were isolated from large volume cultures of bacteria grown in enriched M9 medium by a similar procedure except that plasmid DNA in the cleared lysate was concentrated by polyethyleneglycol (Humphreys et al., 1975) prior to equilibrium centrifugation in caesium chloride density gradients containing ethidium bromide.
DNA Cleavage with Restriction Endonucleases Enzyme reactions involving EcoRI or SalI were performed in TMSal buffer whereas reactions involving other restriction endonucleases were performed in TM buffer. DNA concentrations were
I. Andr6s et al. : Replication Functions of R6-5. II usually between 5 and 30 gg/ml. Reactions were carried out at 37 ° for 30 60 min and were terminated either by heating to 70 ° for 5 min, if the D N A fragments were subsequently to be ligated, or by the addition of 0.2 volumes of a 60% solution of sucrose containing 20 m M E D T A and 0.025% bromophenol blue, if the fragments were to be analyzed by electrophoresis through an agarose gel.
Ligase Reactions The solutions of restriction endonuclease-generated D N A fragments (10 30 pg/ml) were placed on ice for 60 rain after the 70 ° heat pulse used to inactivate the restriction endonuclease. Solutions containing D N A fragments generated by EcoRI or SalI were diluted with an equal volume of 20 m M tris-HC1, pH 7.4 - 10 m M MgC12 prior to addition of ligase reagents. A T P (100 gM), dithiothreitol (10 mM), and T4 D N A ligase (5 units/ml) were then added and after a further 60 min on ice, the solutions were incubated at 14° C overnight.
3 incubated at 3 7 ° C with shaking until its optical density A59 o reached 0.6 whereupon it was chilled in ice/water for 30 rain. Bacteria were sedimented, washed with 50 ml of ice-cold 10 m M NaCI, and resuspended in 50 ml of ice-cold 30 m M CaCI2. The suspension was kept on ice for 20 min, after which the bacteria were collected by sedimentation and resuspended in 4 ml of ice-cold 30 m M CaCI~. 200 gl quantities of competent cells prepared in this manner were then added to chilled tubes containing 100 gI a m o u n t s of 30 m M CaCI2 and up to 50 gl of D N A solutions in TE buffer. Control tubes either lacked D N A (cell control) or contained D N A but did not receive cells ( D N A control). Tubes were kept on ice for 3 0 m i n and were then subjected to a 3rain heat pulse at 42°C. 3 ml quantities of LB supplemented with 0.2% glucose and 10 ],tg/ ml of thiamine were then added to all tubes. These were incubated at 3 7 ° C for 90 min after which the bacterial suspensions were diluted and plated on selective media. A standard plasmid D N A , usually pBR322 (Bolivar et al., 1977), was included in every transformation experiment to determine the level of competence of the prepared bacteria.
Plasmid Requirement of DNA Polymerase I Agarose Gel Electrophoresis Analytical agarose gel electrophoresis was performed in an apparatus similar to that described by Studier (1973) containing slabs of agarose at concentrations which are indicated in the figure legends. The gel and running buffers were tris-borate (TB : 90 m M tris-2.5 m M E D T A - 90 m M boric acid, pH 8.3) or tris-phosphate (TP: 36 m M Tris - 30 m M NaHzPO4 10 m M EDTA, pH 7.5) containing 0.5 pg/ml of ethidium bromide. Electrophoresis was carried out either at 50 mA/80 V for 150 min (TB) or at 50 m A / 20 V for 14 h (TP). D N A bands which were visualized by short wave ultraviolet light were photographed on Polaroid PN55 film using a Polaroid MP4 camera, fitted with a Wratten No. 21 orange filter (Kodak). Molecular weights of D N A fragments were calculated by comparison of their mobilities with the mobilities of ,~ D N A fragments generated by digestion with HindIII and EcoRI endonucleases. The molecular weights of 2 EcoRI/HindIII fragments were taken from M u r r a y and Murray (1975). Preparative agarose gel electrophoresis was carried out in gels containing tris-acetate buffer (TA: 4 0 r a M Tris - 2 0 m M C H 3 C O O N a - 2 m M E D T A - 18 m M NaC1, p H 8.0) containing 0.5 pg/ml ethidium bromide at 50 mA/25 V for 5 h. D N A bands were visualized by long wave ultraviolet light from a Transilluminatot (UV Products, San Gabriel) and segments of the gels containing required bands were cut out. Large D N A fragments (greater than 5 kb) which form sharp bands that can be easily visualized in potassium iodide density gradients containing ethidium bromide were isolated from gel segments by the method of Blin et al. (1975) whereas small fragments that were difficult to visualize by this method were extracted by the method of McDonnell et al. (1977). In this case, gels composed of 0.8% Seakem H G T (P) agarose were used. D N A isolated from such gels by electrophoresis was dialysed against TE buffer prior to phenol extraction and concentration by precipitation with ethanol. R6-5 RepA EcoRI fragment E-2 was purified by the method of Blin et al. (1975) following preparative electrophoresis of EcoRIcleaved pKT097 plasmid D N A (Table 2).
Transformation A modification of the Cohen et al. procedure (1972) was used. 0.5 ml of a fresh overnight culture of bacteria in LB was added to a flask containing 50 ml of fresh medium. The culture was
Plasmids were introduced by transformation into E. coli polAtszl 4 and the strains were grown at a temperature (32 ° C) where the cellular level of functional D N A polymerase I is sufficient for stable inheritance of those plasmids that require high cellular levels of D N A polymerase I (Kingsbury and Helinski, 1970, 1973). Fresh colonies of purified clones of plasmid-containing bacteria were suspended in 1 ml of LB supplemented with 10 ~tg/ml of thymidine and the suspensions diluted i0 - 4 in fresh medium. Each culture was divided into two samples one of which was placed at 32 ° C and the other of which was placed at 42 ° C. Both cultures were incubated overnight, i.e. for 13 to I4 bacterial generations. They were then diluted and plated on AM3 agar plates at 32 ° to give isolated colonies. A b o u t 100 colonies from each culture were subsequently transferred by toothpick to AM3 agar plates at 32 ° containing an antibiotic to detect the continued presence of the plasmids originally carried. In general, R6-5 derivative plasmids that do not require high cellular levels of D N A polymerase I are present in greater than 90% of the clones derived from bacteria grown at 42 ° C whereas those plasmids that do require high cellular levels of D N A polymerase I are present in less than 1% of clones obtained from such bacteria.
Detection of Plasmid Incompatibility Test plasmids were introduced by transformation (Cohen et al., 1972) into E. coli K-12 C600 bacteria containing one of the two standard plasmids indicated and the bacteria were plated on media selective for the transforming plasmid. After purification by single colony isolation, 20 transformant clones were tested for the presence of the original standard plasmid. Negative signs indicate that > 9 0 % of the clones examined had lost the standard plasmid.
Analysis of Plasmid-coded Proteins Minicells from 150 ml stationary phase LB cultures of strain DS410 or M2104 containing the indicated plasmids were harvested by centrifugation at 13,000 rpm for 10 min, washed with BSG, resuspended in l ml of BSG and purified by velocity sedimentation through two successive sucrose gradients. Minicells were washed twice with 56/2 and resuspended in this medium to a final 0D60 o of 0.5-0.6. 1.2 ml of the minicell suspension was preincubated for
I. Andr+s et al. : Replication Functions of R6-5. II
4 Table 2. Plasmids used in this work
Plasmid
Structure and relevant properties
Reference
R6-5 R100-1 pSC102 pSC135 pKT015 pML21 pKT002 pKT017 pBR322 pKT098 pKT093 pKT097 pKT081 pKT086 pKT029 pKT056 pKT032 pKT031 pKT059 pKT060 pKT036 pKT037 pKT039 pKT061 pKT040 pKT057 pKT042 pKT043 pKT044 pKT045 pKT047 pKT049 pKT052 pKT053 pKT054 pKT055 pKT071
tra + , FinOP + , IncFII + , Cop + , RepA+, C m r, Km r, Tcs, Sm r, Su r, Hg ~ tra + , FinO-, IncFII + , Cop + , RepA + , Cm r, Tc~, Smr, Sur, Hg r R6-5 EcoRI fragments E-2, E-6 and E-8*; Km ~, Sur, IncFII +, Cop + , RepA + , FinO+ R6-5 EcoRI fragment E-2+E-2 from pi258; Ap r, IncFII ÷, Cop +, RepA ÷ R6-5 HindIII fragments H-l, H-7 and H-8; Smr, Sur, IncFII +, Cop +, RepA + mini ColE1 + R6-5 EcoRI fragment E-6; Km r pML21 +R6-5 HindIII fragment H-2; Cm ~, Km r pML21 ÷R6-5 HindIII fragment H-2 reverse; Cm ~, Km ~ Tcr Ap r pBR322 + pSC102 HindIII fragment H-l; Ap r, IncFII + PBR322+pSC102 HindIII fragment t-I-2; Ap ~, Sff pBR322+pSC102 HindIII fragments H-1 and H-2; Ap r, Sur, IncFII + pBR322 +pSC102 SalI fragment S-2; Ap r, IncFII + pBR322 ÷ pSC 102 SalI fragment S-3; Ap ~, Km r pBR322 + pSC102 PstI fragment P - l ; Tc r, Km ~ pBR322 + pSC102 PstI fragment P-1 reverse; Td, Km r pBR322 + pSC102 PstI fragment P-2; Tc r, FinO + pBR322 + pSC 102 PstI fragment P-2 reverse; Tc r, FinO ÷ FinO- mutant of pKT030 FinO- mutant of pKT030 pBR322 + pSC 102 PstI fragment P-3 ; Tcr pBR322+pSC102 PstI fragment P-3 reverse; TcF pBR322 + pSC 102 PstI fragment P-4; Tcr pBR322+pSC102 PstI fragment P-4 reverse; Tcr pBR322+pSC102 PstI fragment P-5 ; Tcr pBR322+pSC102 PstI fragment P-5 reverse; Td pBR322+pSC102 PstI fragment P-6; Tc~, IncFII + pBR322+pSC102 PstI fragment P-6 reverse; Td, IncFII + pBR322 + pSC 102 PstI fragment P-7 ; Tc~ pBR322+pSCI02 PstI fragment P-7 reverse; Tc~ pBR322+pSC102 PstI fragment P-8 ; Tcr pBR322+pSC102 PstI fragment P-8 reverse; Tc~ pBR322+pSC102 PstI fragment P-9; Tc~ pBR322+pSCI02 PstI fragment P-9 reverse; Tc~ pBR322+pSC102 PstI fragment 10; Tc ~ pBR322+pSC102 PstI fragment 11; Tcr pSC102 PstI fragments P-1 +P-4+P-6; Km r, IncFII +, Cop + , RepA +
Silver and Cohen, 1972. Nishimura et al., 1967. Cohen et al., 1973. Timmis et al., 1975. Timmis et al., 1978d. Lovett and Helinski, 1976. Timmis et al., 1978d. Timmis and Cabello, unpubl. Bolivar et al., 1977. This paper.
Timmis et al., 1978b.
This paper.
tra: transmissible; fin: fertility inhibition; Inc: Incompatibility; Cop : copy control; Rep : replication. 60 min at 37° C before adding 25 ~tCi of 14C protein hydrolysate (Amersham; 55 gCi/milliatom carbon) or 25 gCi of 35S_methionine (Amersham; about 700 Ci/mmol). Plasmid-coded proteins synthesized by the minicells were analyzed by electrophoresis through 15-25% polyacrylamide gels containing SDS followed by autoradiography or fluorography (Kennedy et al., 1977). Results
Localization o f Restriction Endonuclease Cleavage Sites in the p S C I 0 2 Plasmid T w o m i n i p l a s m i d s t h a t c o n t a i n the R e p A E c o R I - g e n e r a t e d f r a g m e n t o f R 6 - 5 h a v e b e e n c o n s t r u c t e d in v i t r o ( C o h e n et al., 1973; T i m m i s et al., 1975; see Fig. 1). T h e p S C 1 0 2 p l a s m i d c o n t a i n s t h r e e E c o R I generated fragments of R6-5, namely EcoRI fragm e n t s E-2, E-6 a n d E - 8 * w h i c h specify t h e f u n c t i o n s f o r a u t o n o m o u s r e p l i c a t i o n , r e s i s t a n c e to k a n a m y c i n
a n d r e s i s t a n c e to s u l f o n a m i d e , r e s p e c t i v e l y . F r a g m e n t E - 8 * is n o t a bona fide E c o R I f r a g m e n t 8 o f R 6 - 5 b u t is d e l e t e d for p a r t o f this f r a g m e n t s u c h t h a t it n o l o n g e r e x p r e s s e s t h e s t r e p t o m y c i n r e s i s t a n c e enc o d e d b y f r a g m e n t 8 ( T i m m i s et al., 1978d). T h e p S C 1 3 5 p l a s m i d ( T i m m i s et al., 1975) c o n t a i n s o n l y o n e R 6 - 5 f r a g m e n t , t h e R e p A E c o R I f r a g m e n t E-2 ( T i m m i s et al., 1977) b u t c o n t a i n s in a d d i t i o n E c o R I f r a g m e n t 2 o f t h e Staphylococcus aureus p l a s m i d p i 2 5 8 w h i c h specifies r e s i s t a n c e to a m p i c i l l i n ( C h a n g a n d C o h e n , 1974).
HindIII, BglIi, a n d SalI D i g e s t i o n o f p S C 1 0 2 w i t h E c o R I g e n e r a t e s its t h r e e component R6-5 fragments, namely E-2 ( M W 8.5 m D ) , E-6 ( M W 4.5 m D ) , a n d E - 8 * ( M W 3.1 m D , F i g . 2, t r a c k A ) w h e r e a s d i g e s t i o n o f p S C 1 0 2
I. Andr6s et al. : Replication Functions of R6-5. II E c o R I AND Hind TIT RESTRICTION MAP OFR6-5
Om
RepB
Km cb
Fa
,?
IS1a
Sm Su
(% Hg oa Is~b
43.7 142.4 48.9 48.9 ITcl
w,
533
I+5
100.6 - 1
49.8
52.0
ori V Rep A, tnc
RepC m cb
/
/
/
"5"
,e
E-5
frQ
with HindIII endonuclease generates two D N A fragments (Fig. 2, track D). Double digestion with EcoRI and HindIII showed that one HindIII cut is approximately in the middle of E-6 whereas the other is close to one end of E-8* (track B). Comparison of these fragments of p s c 1 0 2 with those generated by double digestion of a hybrid plasmid, designatedpKT093 (see below and Fig. 4), that is composed of pBR322 plus the smallest HindIII fragment (H-2) of pSC102 (track C) allowed us to position the HindIII cleavage sites of pSC102 relative to its EcoRI sites. The pSC102 plasmid contains a unique cleavage site for the BglII endonuclease (Fig. 2, track G) and a double digestion with BglII and EcoRI (track I) or BgIII digestion of purified fragment E-2 (track F), demonstrated that the cleavage site is located within E-2, 2.5 mD from one end. Because double digestion of pSC 102 with BgIII and HindIII generates, in addition to the HindIII-HindIII fragment, a HindIII-BglII D N A fragment slightly larger than 2.5 mD and another about 8.5 mD (track H), then the BglII site must be located 2.5 mD from the E2/E8* junction.
HI0 = 0.36 kb
Fig. 1. The R6-5 plasmid. Localization of the R6-5 functions an EcoRI (wavy lines) and HindIII (straight lines) restriction endonuclease cleavage sites on the plasmid is describedin Timmiset al. (1978d)
Digestion of pSC102 D N A with the SalI endonuclease generates three fragments having sizes of 6.8 mD (S-l), 6.2 mD (S-2), and 3.0 mD (S-3), (Fig. 3, track C). Treatment of purified E-2 D N A generates three fragments having sizes of 6.2 mD (S-2), 2.0 mD and 0.2 mD (track D). Thus, two of the three SalI cleavage sites of pSC102 are present in E-2. Double digestion of purified fragment E-2 with SalI and BglII results in a shortening of the 2.5 mD BglII-EcoRI fragment by 0.5 mD and the appearance of a corresponding 0.5 mD fragment (track E). Thus, one SalI site is located 0.5 mD anticlockwise from the unique BglII site ofpSC102 and the other is therefore located 0.2 mD from one of the two EcoRI termini of the E-2 fragment. Because SalI digestion of pSC102 does not generate a 1.8 mD D N A fragment, the second SalI site in E-2 must be located 0.2 mD from the E-2/E-6 junction. Double digestion of pSC102 with SalI and EcoRI demonstrated that the third SalI cleavage site is located in E-6: E-8* remains intact after treatment with SalI (track B). The S-3 fragment thus runs clockwise from the SalI site close to the E-2/E-6 junction to a position clockwise of the Hin-
I. Andr6s et al. : Replication Functions of R6-5. II
A
A BCD
E FG
B
C
D
E
F
G
H
H I
Fig. 2. Location of HindIII and BglII cleavage sites in pSC102. Purified D N A was digested with restriction endonucleases as described in Materials and Methods, and subjected to electrophoresis through a 0.6% TB agarose slab gel. A: pSCIO2/EcoRI; B: pSClO2/HindIII + EcoRI; C: pKTO93(pBR322 + H-2)/HindIII + EeoRI ; D : pSC 102/HindIII ; E : 2/EcoRI + HindIII ; F : purified E-2 fragment/BglII ; G: pSC 102/BglII ; H: pSC i02/BglII + HindIII ; I : pSC 102 :BglII + EcoRI
Fig. 3. Location of SalI cleavage sites in pSC102. Digested DNAs were subjected to electrophoresis through a 0.6% TB agarose slab gel. A: pSCIO2/EcoRI; B: pSClO2/EcoRI+SalI; C: pSClO2/SalI; D: purified E-2/SalI; E: E-2/SalI+BglII; F: E-2/BglII; G: pBR322/HindlII + SalI; H : pKT086 (pBR322 + S-3)/HindIII + SalI
dIII site present in E-6. Digestion of hybrid plasmid pKT086 (pBR322 + pSC102 fragment S-3) with SalI and HindIII endonucleases generated, in addition to the two fragments from the pBR322 vector (track G), two fragments having molecular weights of 2.6 mD and 1.1. mD (track H). The third SalI cleavage site pSC102 therefore lies 1.1 mD clockwise from the HindIII cleavage site of E-6. Figure 4 shows the locations of the SalI and HindIII cleavage sites of pSC102 and indicates the fragments and designations of some of the pBR322 hybrid plasmids containing cloned pSC102 segments that have been used in this work.
Localization of Restriction Endonuclease Cleavage Sites in the R6-5 RepA EcoRI Fragment (E-2)
SstI, XhoI, KpnI, HpaI The endonuclease SstI cleaves the pSC102 plasmid at a single location whereas XhoI, KpnI and HpaI, all cleave pSC102 at two locations. None of these enzymes cleaves the pBR322 vector plasmid (Slocombe and Timmis, 1978). The locations of the cleavage sites of these enzymes were determined by methods described above. A pSC102 plasmid map showing the EcoRI, HindlII, SalI, SstI, BglII, XhoI, KpnI, and HpaI cleavage sites is shown in Fig. 5.
HincII Digestion of purified E-2 DNA with HincII generates 6 DNA fragments having sizes of 3.5, 2.1, 1.5, 0.9, 0.23 and 0.05 mD (Fig. 7, track I). Because the SalI and HpaI recognition sequences are also HincII recognition sequences, the previously determined locations for the SalI and HpaI cleavage sites accounts for 4 of the 5 HincII cleavage sites and therefore 4 of the 6 HincII fragments. The position of the fifth HincII cleavage site was determined by digestion of purified E-2 fragment with HincII+BglII (Fig. 7, track H). As can be seen, the third largest HincII fragment is cut by BglII into two fragments having sizes of 0.5 and 0.9 mD. This result positions the remaining HincII cleavage site 0.9 mD to the left of the BglII cleavage site.
PstI Digestion of the pSC102 plasmid with the PstI endonuclease generates 11 DNA fragments having sizes
I. AndrOs et al. : Replication Functions of R6-5. II
EcoRILA
- -
7
So~I
% Fig. 4. pSC102 fragments present in some hybrid plasmids used for endonuclease cleavage mapping. Each of the fragments indicated with an arrow was cloned into pBR322 as described in Materials and Methods
% Km
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Fig. 5. Restriction endonuclease cleavage m a p of pSC102
I. Andr6s et al. : ReplicationFunctions of R6-5. II A
B
C
D
E
F
G
H
I
CDEFG
A B --1
--2 --3 --4 --5 --6
--7 --8,9 --10 --11 Fig. 6. Location of PstI cleavage sites in pSC102. Digested DNA samples were subjected to electrophoresis through a 0.8% TBagarose slab gel. A: E-2 fragment/PstI; B: pSClO2/PstI; C: E-2/ PstI + HpaI ; D : E-2/PstI + SalI; E : E-2/PstI + BglII ; F: E-2/PstI ; G : 2/EcoRI + HindIII
Fig. 7. Location of PstI and HincII cleavage sites in the RepA EcoRI fragment.DigestedDNA sampleswere subjectedto electrophoresis through a 0.8% TB agarose slab gel. A: pBR322/PstI+ EcoRI; B: pKT035 (pBR3224-P-3)/PstI+EcoRI; C: pKT029 (pBR322+ P-1)/PstI+ EcoRI ; D : pBR322/PstI + SalI; E : pKT081 (pBR322 + S-2)/PstI + SalI ; F: E-2/PstI; G: E-2/PstI + HincII ; H: E-2/HincII + BglII; I : E-2/HincII
of 7.0 (P-l), 2.95 (P-2), 2.0 (P-3), 1.07 (P-4), 0.9 (P-5), 0.69 (P-6), 0.46 (P-7), 0.26 (2 fragments, P-8 and P-9), 0.17 (P-10), and 0.11 (P-11) mD (Fig. 6, track B). The P s t I cleavage sites in the pSC102 molecule were located using several approaches including double digestion with enzymes whose cleavage sites had been previously mappea, digestion of purified plasmid fragments, and single and double digestions of hybrid plasmids containing specific fragments of pSC102. Comparison of the D N A fragments generated by PstI digestion of pSC102 and purified E-2 fragment shows that the E-2 fragment contains 8 of the 11 PstI-generated fragments of pSC102 and hence 9 of the 11 cleavage sites (Fig. 6, tracks A and B). pSC102 fragments P-l, P-3, and P-11 are not generated by PstI digestion of the E-2 fragment, but instead two new fragments having molecular weights of 0.15 mD and 0.9 mD are generated. These two fragments are the terminal P s t / E c o R I fragments of E-3. P s t I fragment P-6 contains the B g l I I and one of the SalI cleavage sites of the E-2 fragment: in double digestion of purified E-2 fragment, the P-6 fragment was reduced in size by 0.15 mD by digestion with P s t I and B g l I I (Fig. 6, track E), and 0.05 mD by digestion with P s t I and SalI (track D). Fragment P-7 contains the second SaII site of the E-2 fragment and also one of the H p a I sites. In double digestions of purified E-2 fragment DNA, fragment
P-7 is reduced by 0.02 mD by digestion with PstI and SalI (Fig. 6, track D) and by 0.04 mD by digestion with P s t I and H p a I (track C). Thus, one of the termini of P-7 is located 0.15 mD from the E-2/E-6 E c o R I junction and the other 0.46 mD towards the BglII cleavage site. In the H p a I - P s t I double digest of the E-2 fragment (Fig. 6, track C), one of the 0.26 mD fragments (P-8) also disappears and two new bands of almost equal size, 0.12 mD and 0.13 roD, appear. This localizes the termini of the P-8 fragment. The position of fragment P-2 was deduced from a H i n c I I - P s t I double digestion: fragments P-2, P-6, P-7, and P-8 are cleaved with H i n c I I (Fig. 7, track G). Because P-6, P-7, and P-8 contain either SalI or H p a I cleavage sites, P-2 must contain the fifth H i n c I I cleavage site. This fragment is cleaved into 2 fragments 2.2 mD and 0.7 mD in size, which indicates that fragments P-2 and P-6 are adjacent fragments in the pSC102 molecule and provides the position of the BglII-distal P s t I terminus of fragment P-2. Digestion of hybrid plasmid pKT081 (pBR322+ S-2 ; Fig. 4) with P s t I and SalI showed that P s t I fragments P-2, P-5, P-8, and P-9 fragments are contained within SalI fragment S-2, (Fig. 7, track E). P s t I fragment P-4 is therefore located outside of S-2 and must be situated between P-6 and the P-3 fragment which contains the E-2/E-8* junction. P-3 is a 2.0 mD fragment, 0.9 mD of which is present on
I. Andr6s et al. : Replication Functions of R6-5. II E-2 (Fig. 7, track B). Thus, 1.1 mD of P-3 must be contained on E-8*. Digestion of hybrid pKT029, which consists of pBR322 and P-1 (Fig. 13), with PstI and EcoRI generated 5 D N A fragments, three of which are derived from P-1 and two of which come from the vector molecule (Fig. 7, tracks A and C). Hence P-1 contains the complete E-6 fragment, the major part of E-8* and the 0.15 mD terminal PstI/EcoRI fragment of E-2. Because fragment P-11 is not located on E-2 or E-6, it must be located on E-8* between P-1 and P-3. Digestion of pKT093 (pBR322 + H-2) with PstI endonuclease generated a D N A fragment 0.11 m D in size, which confirms that fragment P-11 is located between P-1 and P-3 (not shown). At this stage of the mapping it seemed very likely that PstI fragment P-10 is located between P-7 and P-8. Direct confirmation of this was obtained by PstI cleavage of purified small (0.9 roD) HpaI fragment D N A which yielded the expected partial fragments of P-7 and P-8 and the complete P-10 fragment (Fig. 8, track C). In addition, a fourth very small fragment can be seen in the gel indicating the existence of a twelfth PstI cleavage site in the pSC102 plasmid that is located very close to one of the two termini of fragment P-10. Finally, it remained to determine the order of the two fragments P-5 and P-9 which, from the mapping of the other PstI fragments of pSC102, must be adjacent fragments situated between P-8 and P-2. For this purpose, BglI fragment B-2 (see below and Fig. 9) D N A was purified and digested with PstI. Figure 8 (track A) shows that three D N A fragments with molecular weights of 0.5 mD (P-2 partial), 0.26 mD (P-9) and 0.2 mD (P-5 partial) were obtained, which demonstrates that the order of PstI fragments in this region is therefore, from left to right, P-5, P-9, and P-2. The complete map of PstI cleavage sites in the pSC102 plasmid is shown in Fig. 5.
9
A
B C
D
Im
Fig. 8. Location of ambiguous PstI cleavage sites. Digested DNA samples were subjected to electrophoresisthrough a 0.8% TB agarose slab gel. A: BglI fragment B-2 (Fig. 9) DNA was obtained by preparative electrophoresisthrough a 0.8% TA agarose slab gel of Bg/I-digested RepA EcoRI fragment, followed by electrophoretic elution from the gel slice and ethanol precipitation, and was digested with PstI (the DNA is not completely digested by PstI although the final digestion products are visible and are indicated by the arrows); B: E-2/PstI; C: the small HpaI fragment of E-2 was obtained by sedimentation of 3H-thymidine-labelled HpaI-cleaved pKT081 (pBR322+ S-2) DNA through a 5-20% neutral sucrosegradient, followedby dialysisand ethanol precipitation, and was digested with PstI; D: E-2/PstI+HpaI
0.1, and 0.07 mD. Complete cleavage maps of the RepA fragment for Bgll, PvuII, AvaI, and Sinai, and a partial cleavage map for HaeII, that were derived using methods similar to those described above, are presented in Fig. 9.
Determination of the Orientation of Fragment E-2 in the Parent R6-5 Plasmid Molecule BglI, PvulI, Ar.aI, Sinai and HaeII Digestion of purified E-2 fragment D N A with the BglI endonuclease generates six fragments with sizes of 2;1, 1.95, 1.90, 1.25, 0.75, and 0 . 4 m D ; digestion with PvuII generates 11 fragments with sizes of 1.85, 1.45, 1.3, 0.9, 0.7, 0.65, 0.45, 0.4, 0.28, 0.15, and 0.1 roD; digestion with AvaI generates 13 fragments with sizes of 1.8, 1.5, 0.9, 0.7, 0.65, 0.6, 0.5, 0.45, 0.4, 0.35, 0.15, 0.12, and 0.1 mD; digestion with Sinai generates 11 fragments with sizes of 2.35, 1.8, 1.1, 0.7, 0.65, 0.5, 0.4, 0.35, 0.15, 0.12, and 0.1 mD; and digestion with HaeII generates 11 fragments with sizes of 2.1, 1.55, 1.45, 1.0, 0.7, 0.7, 0.25, 0.21, 0.2,
In order to relate the map positions of endonuclease cleavage sites and plasmid functions that have been identified on pSC102 or on the E-2 fragment (see below) to corresponding locations on the parent R6-5 plasmid molecule, it was necessary to determine the orientation of E-2 within R6-5 or, expressed in another way, to determine the ISlb-proximal EcoRI terminus of E-2. We therefore asked whether the asymmetric BglII cleavage site in E-2 is proximal or distal to IS lb. For this purpose we used a hybrid plasmid, designated pKT015 (Timmis et al., 1978d), that contains three HindIII fragments of R6-5, namely H-1 (replication), H-7 (Sm-resistance), and H-8 (Su
10
I. Andr6s et al. : Replication Functions of R6-5. II i--i rr
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resistance (Fig. 10). R6-5 H-1 fragment contains several EcoRI fragments but, important for our purpose, the ISlb-proximal EcoRI fragment is E-2. Furthermore, the HindIII and EcoRI cleavage sites delineating the ISlb-proximal termini of R6-5 H-1 and E-2 are located very close to one another, i.e. within a 0.25 mD sequence. Thus, digestion of pKT015 with ItindIII and BglII endonucleases should yield a D N A fragment having a molecular weight of either 3.0 mD or 6.5 mD, depending on the orientation of E-2 within R6-5. Figure 10 (track C) shows that this type of digestion generates a 3.1 mD fragment. Thus, the BglII cleavage site in E-2 is located close to the ISlbproximal EcoRI terminus of this fragment and the orientation we have chosen for representing E-2 corresponds to the orientation of E-2 within R6-5 as it is drawn in Figs. 1, 9 and 10.
Structure of the Sm-Su Regions of R6-5, pKTOI5 and pSCI02 Digestions of pKT015 D N A with BglII, BglII+ EcoRI, EcoRI, and BglII + HindIII (Fig. 10, tracks B, E, F, C) allow mapping of the locations of cleavage sites of these endonucleases, and a comparison of the Sm-Su regions of R6-5 and pKT015 is shown in Fig. 10. The pSC102 plasmid contains an R6-5
Su-resistance EcoRI fragment, although this does not appear to be the bona fide R6-5 E-8 fragment because it no longer possesses the central single HindIII and BglII cleavage sites and no longer expresses resistance to Sm. This fragment, called E-8 * (Timmis et al., 1978d) does nevertheless contain a HindIII cleavage site close to the Sff end of the fragment (Fig. 5). The Su'EcoRI fragment of pSC102 therefore appears to have evolved by two (or more) polynucleotide sequence changes that involved (a) deletion of a central segment that carries resistance to Sm and single HindilI and BglII cleavage sites and (b) insertion of a new HindIII cleavage site close to one of its ends. It should be noted that in pSC135, but not in pSC102, there is a HindIII cleavage site within the RepA fragment and close to its ISlb-proximal EcoRI terminus. F r o m our earlier observations on sequence inversion in this region of R6-5 (Timmis et al., 1978c), we would conclude that the new HindIII cleavage site in fragment E-8* of pSC102 was probably generated by transfer of the E-2 HindlII site to E-8 * by inversion of a D N A sequence at the E-2/E-8* junction. Also noteworthy is a small stalk and loop structure between the E-6/E-8* and P-l/P-11 junctions which shows the presence of inverted repeat sequences in the neighbourhood of the E-8* deletion (Fig. 16). Whether this transposon-like structure promoted the Sm" deletion event is currently unknown.
I. Andr6s et al. : Replication Functions of R6-5. II
A
B
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'~-7
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Bg~Tr Fig. 10. Orientation of the RepA EcoRI fragment in R6-5 and structure of the Sm-Su region in R6-5 and its mini-plasmids, Digested DNA samples were electrophoresed through a 0.9% TB agarose slab gel. pKT015 is a mini-plasmid generated from R6-5 and contains HindIII generated fragments H,I+H-7+H-8 (Timmis et al., 1978d) A: pFC009 (ColEI+R6-5 E-6)/EcoRI; B: pKTO15/BglII; C : pKT015/BglII + HindIII ; D : pKT015/HindIII ; E : pKT015/EcoRI + BglII ; F : pKT015/EcoRI ; G : E-2/BglII ; H : pS C 102/Pstl Solid lines: HindIII cleavage sites; dotted lines: EcoRI cleavage sites
Cloning of Sub-fragments of the Mini R6-5 Plasmid p S C l 0 2 In o r d e r to facilitate m a p p i n g o f r e s t r i c t i o n e n d o n u clease cleavage sites (see above) and r e p l i c a t i o n functions o f the R6-5 R e p A EcoRI f r a g m e n t , a n d to o b tain large q u a n t i t i e s o f R e p A D N A sequences, we c l o n e d a variety o f r e s t r i c t i o n e n d o n u c l e a s e - g e n e r a t e d
f r a g m e n t s of pSC102 on the high c o p y n u m b e r v e c t o r p l a s m i d p B R 3 2 2 (Bolivar et al., 1977). The HindIII a n d SalI r e s t r i c t i o n e n d o n u c l e a s e s cleave the p S C 1 0 2 p l a s m i d into two and three D N A f r a g m e n t s , respectively, w h e r e a s the PstI e n z y m e cleaves this p l a s m i d into 11 f r a g m e n t s (see a b o v e ) ; these enzymes were t h e r e f o r e a p p r o p r i a t e for g e n e r a t ing D N A f r a g m e n t s suitable for cloning. The SalI,
12 A
I. Andr6s et al. : Replication Functions of R6-5. lI B
C
D
E
F
G
H
Fig. 11. Cloning of HindIII and SalI fragments of pSC102, pSC102 and pBR322 DNAs were cleaved with HindlII or SalI endonuclease, mixed, ligated and transformed into E. coli K-12 C600. Plasmid DNAs were prepared from ampicillin-resistant tetracycline-sensitive transformant clones, cleaved with the particular enzyme used for construction, and analyzed by electrophoresis through 0.7% TB agarose gels. A: pBR322/HindIII; B: pKT093/ HindIII; C: pKTO97/HindIII; D: pSClO2/HindlII; E: pKT086/ SalI; F: pSClO2/SalI; G: pKTO81/SalI; H: pBR322/SaII
A
B C
D
E
F G
H
I
.I K
L
M
Fig. 12. Cloning of PstI fragments of pSC102, pSC102 and pBR322 DNAs were cleaved with PstI endonuclease, mixed (except for pKT071, track L), ligated and transformed into E. coli K-12 C600. Plasmid DNAs were prepared from tetracycline-resistant ampicillin-sensitive transformant clones (except for pKT071, track L), cleaved with Pstl, and analyzed by electrophoresis through a 1% TB agarose slab gel. In addition to cloning individual fragments ofpSCl02 into the pBR322 vector plasmid, mini pSC102 plasmids (e.g. pKT071, track L) were generated that contain only pSC102 DNA fragments, i.e. no pBR322 vector molecules were present during the ligation. Transformants carrying mini pSC102 plasmids were selected on kanamycin medium. A: pSC102; B: pKT054; C: pKT052; D: pKT047; E: pKT044; F: pKT043; G: pKT040; H: pKT039; I: pKT036, J: pKT032; K: pKT029; L: pKT071; M: pBR322
identified by means of insertional inactivation (Timmis et al., 1974; Timmis et al., 1978e) of the tetracycline resistance (cloning of SalI and HindIII fragments) or the ampicillin resistance (cloning of PstI fragments) determinants of the vector molecule. Figures 11 and 12 show that both HindIII fragments, all PstI fragments, and two of the three SalI fragments have been cloned in the pBR322 plasmid. Although a number of the hybrid plasmids originally constructed contain more than one pSC102 D N A fragment, those shown in Figs. 11 and 12, and used throughout this work, contain single D N A fragments of the pSC102 plasmid. Detection of a pSC102 function on a hybrid plasmid therefore identifies the particular pSC102 fragment that carries the function of interest. One exception to this generalization is the pKT097 plasmid which consists of pBR322 plus both HindIIIgenerated fragments ofpSC102 (Fig. 11). Because this hybrid was the only one isolated which contains the whole of the R e p A EcoRI sequence, and which can be amplified by chloramphenicol treatment of host cells, it was employed to obtain large amounts of R e p A EcoRI fragment D N A (Materials and Methods). It is noteworthy that a number of the cloned pSC102 fragments contain unique cleavage sites for some of the newer restriction enzymes. Several of the hybrid plasmids are therefore useful vector molecules for D N A fragments generated by these enzymes (Slocombe and Timmis, 1978). Cloning m a y separate some plasmid structural genes from their natural promoters. Such genes may therefore be expressed in recombinant plasmids as a result of the activity of an unnatural promoter, for example the fi-lactamase promoter of the pBR322 vector plasmid, and m a y be expressed only when inserted in one particular orientation into the vector molecule. To detect such orientation effects on the functions and proteins encoded by the pSC102 replicon, we generated pSC102/pBR322 recombinant plasraids containing pSC102 PstI fragments in each of the two possible orientations within the vector plasmid. Figure 13 is a diagramatic representation of the constructed recombinant plasmids and how the two possible orientations of the cloned fragments were distinguished.
Generation of Mini Plasmids in vitro HindIII, and PstI fragments of pSC102 were introduced into the unique SalI, HindIII, and PstI cleavage sites of the pBR322 vector plasmid by standard recombinant D N A procedures (for review see Timmis et al., 1978 e). After transformation of E. coli K-12 C600 bacteria with recombinant D N A mixtures, bacterial clones carrying hybrid plasmids were
In order to estimate the size and identify the location of the essential D N A sequence involved in the autonomous replication of R6-5 and to have smaller, more easily analyzed plasmids, we attempted to generate mini pSC102 plasmids. Preliminary experiments demonstrated that the PstI enzyme does not cleave
I. Andr6s et al. : ReplicationFunctionsof R6-5. II within the kanamycin structural gene of pSC102. Mini plasmids were therefore constructed by cleavage of pSC102 DNA with PstI followed by random joining of the fragments by treatment with DNA ligase, and subsequent transformation of E. coli K-12 strain C600. Plasmid DNA was prepared from kanamycin resistant transformants and analyzed by cleavage with PstI, followed by agarose gel electrophoresis. Figure 12 shows that the mini pSC102 plasmid pKT071 contains PstI fragments P-1 +P-4+P-6. All of 12 independently isolated mini plasmids of this type contain the same pSC102 DNA fragments. From the endonuclease cleavage map of pSC102 (Fig. 5) it can be seen that the P-1 fragment carries the kanamycin resistance determinant but very little (less than 0.1 mD) of the RepA EcoRI fragment. It therefore presumably does not contribute any functions for autonomous replication. On the other hand, fragments P-4 and P-6 are adjacent fragments derived exclusively from the RepA EcoRI fragment of pSC102. These findings indicate that both P-4 and P-6 are essential for the autonomous replication of the mini plasmid, and hence R6-5 itself.
Functions Encoded by the R6-5 RepA EcoRI Fragment Table 3 and Fig. 14 show the map positions of, and the endonuclease fragments that carry the identified functions of the pSC 102 plasmid and the RepA EcoRI fragment of R6-5. In an accompanying paper we describe experiments which demonstrate that OriV is located approximately in the middle of the P-4 fragment (Synenki etal., 1979), and in an earlier communication we showed that one function involved in the FII incompatibility expressed by R6-5 is encoded by the P-6 and S-2 fragments of plasmid pSC102 (Timmis et al., 1978b). We have examined hybrid plasmids containing all cloned fragments of pSC102 in both possible orientations and find that only those which contain the DNA sequence common to P-6 and S-2 (Fig. 5) express detectable incompatibility (Table 4). Examination of two hybrid plasmids composed of pKT043 (pBR322 + P-6) plus BglII-generated fragments derived from the F plasmid (Thompson and Achtman, 1978) revealed that insertion of DNA into the BglII cleavage site of pKT043 does not inactivate its IncFII property (data not shown). IncFII is therefore located between the BglII cleavage site at R6-5 coordinate 97.0 kb and the SalI cleavage site at R6-5 coordinate 97.7 kb. It is highly probable that one contribution to the incompatibility expressed by this fragment is made by the R6-5 copy control function, Cop.
13
RepA It has been shown by several groups that expression of plasmid-coded functions is not obligatory for replication of ColEl-type plasmids (Tomizawa et al., 1975; Donoghue and Sharp, 1978; and Kahn and Helinski, 1978), and it has been concluded that this plasmid requires only origin DNA sequences for its self-duplication. This does not, however, seem to be the case for the large, strictly regulated plasmids of the R6-5 type because essential plasmid-encoded replication functions have been identified (Yoshikawa, 1974; K. Timmis and F. Cabello, unpublished experiments). The evidence presented above that fragment P-6, in addition to the origin-carrying fragment P-4, is essential for autonomous replication of the R6-5 plasmid is consistent with a requirement for the expression of at least one function in addition to the presence of the origin itself. This requirement was demonstrated directly by examining the DNA polymerase I-requirement of a pBR322 hybrid plasmid containing the R6-5 origin PstI fragment P-4. R6-5 and its miniplasmids pSC102 and pKT071 replicate in and are stably inherited by mutant bacteria deficient in DNA polymerase I, whereas the ColEl-like pBR322 plasmid cannot replicate in, and is rapidly lost from, such bacteria (Table 5). If all functions obligatory for the autonomous replication of R6-5 were present on PstI fragment P-4 of this plasmid, then the pBR322 hybrid plasmid should replicate stably in DNA polymerase I-deficient bacteria. In fact, this hybrid plasmid was shown to be incapable of replication in such bacteria, indicating that it is not able to replicate using the R6-5 origin of replication and therefore that functions not contained on the P-4 fragment are obligatory for R6-5 replication. It should be noted that no hybrid plasmid composed of pBR322 and individual PstI or SalI DNA fragments from pSC102 was capable of replication in bacteria deficient in DNA polymerase I (Table 5). Furthermore no hybrid plasmid was capable of replication in such bacteria that concurrently carried the parent pSC102 or the pSC135 plasmids. We conclude that low copy number mini R6-5 plasmids either fail to express levels of a trans-acting essential replication protein that are sufficient for R6-5 origin usage by P-4-carrying hybrid plasmids, or that a cis-acting function not encoded by P-4 is required for R6-5 origin usage. Because pSC102 fragments P-4 and P-6 are both necessary for replication, it was important to determine whether sequence continuity at their junction in the R6-5 and pSC102 plasmids was preserved in the miniplasmids or whether a different orientation of one fragment relative to the other was possible. The relative order and orientation of the PstI frag-
14
I. AndrOs et al. : Replication F u n c t i o n s of R6-5. II l
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1.9 MD 1.45MD 0.14 NID 0.11 MD
1.45 MD 1.06MD 0.95 MD 0.14 MD
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Fig. 13. Orientation of cloned pSC102 fragments in the pBR322 vector molecule. Each PstI fragment of pSC102 has been inserted into the pBR322 plasmid in both possible orientations with respect to the vector molecule. For each type of hybrid plasmid the essential restriction endonuclease cleavage sites used for distinguishing orientations of the cloned fragments are shown
I. AndrOset al. : Replication Functions of R6-5. II J/ P-6
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P-8
-.•
P-8 ~
,
/.
.,,'"
• ",
Hindrlf/HpaI
fragment sizes
pK___T0~7
pK~o4g 2 . 3 7 MD
2 . 3 6 MD
0.58 M D
0.59 MD
EcoRI
restriction site
Hi ndllI restriction site ___> Hp~ I restriction site
• ,~, P-9
P-9
@ "
""
. ,~ / ~ _ . . ~ .
,~, HindlII/SmaI
fragment sizes
pKTO53
pKTOS2
2,55MD O.35ND
2.3MD O.6f4D
EcoRI
restriction
site
Hi ndTrf restriction
site
___> S m ~ I
ments in four miniplasmids were therefore determined by a series of double digestions with pairs of nucleases. Figure 15 shows the four possible orientations of the P-4 and P-6 fragments and the expected subfragment sizes obtained by digestion with EcoRI+ BgllI and HindIII + BgllI. Such digestions of one miniplasmid, designated pKT071, demonstrated that orientation B in Fig. 15 is correct, i.e. that fragment P-6 has the same orientation in pKT071 as it has in pSC102, and that fragments P-4 and P-6 have the
restriction site
same order in pKT071 as they have in pSC102. The orientation of fragment P-4 was determined by comparing the P~uII+BglII double digest products of pKT071 and pSC102: all D N A fragments generated from pKT071 by this combination of endonucleases comigrated with fragments generated from pSC102. This indicates that the orientation of P-4 relative to P-6 is the same in the pKT071 miniplasmid as it is in pSC102 and hence R6-5. This was also found to be the case for three other miniplasmids. It seems
I. A n d r 6 s et al. : Replication Functions of R 6 - 5 . I1
16
Table 3. Functions coded by restriction fragments of p S C 1 0 2 Fragment
PstI-1 PstI-2 PstI-3 PstI-4 PstI-5 PstI-6 PstI-7 PstI-8 PstI-9 PstI-10 Pstl- 11
Function a
Fragment
Function
Test plasmid
Standard plasmid
Stability of standard
Km r
EcoRI-1 EcoRI-2 EcoRI-3
RepA, OriV, IncFlI
1/2 K m ~, RepA, OriV, IncFII
pSC135 Apr " pSC102 Km r " pSC135 Apr
-
HindIII-1 HindIII-2
1/2 K m ~, Su r
(Sa/I-1)
(Su)
SalI-2 SalI- 3
IncFII, FinO
pSC102 pKT071 pBR322 pKT081 pKT029 pKT056 pKT032 pKT031 pKT036 pKT037 pKT039 pKT061 pKT040 pKT037 pKT042 pKT043 pKT044 pKT045 pKT047 pKT049 pKT052 pKT053 pKT034 pKT055
FinO b
Km r Su r
OriV ~ IncFII
Km ~
A b b r e v i a t i o n s : K m ~, S u r, resistance to kanamycin and sulfonamide, respectively; FinO, fertility inhibition; OriV, origin of vegetative replication; IncFII, incompatibility towards FII-type plasmids
"
b
Table 4. Location of b~cFII on cloned fragments
see T i m m i s et al., 1 9 7 8 a see Synenki e t a l . , i 9 7 9
highly probable therefore that sequence continuity at the P-4/P-6 junction is essential for the autonomous replication of the R6-5 plasmid suggesting that an essential function, most likely RepA, is located at this junction.
Km r (P-1 + P - 4 + P - 6 ) K m r Tc r (pBR322+S-2) Ap r (pBR322+P-1) Km r . . . . (pBR322+P-2) Tc r . . . . ( p B R 3 2 2 + P-3) Tea . . . . (pBR322+P-4) Tc r . . . . (pBR322+P-5) Tc' . . . . (pBR322+P-6) Tc r . . . . (pBR322+P-7) Tc r . . . . ( p B R 3 2 2 + P-8) T c r . . . . (pBR322+P-9) Td . . . . (pBR322+P-10) Tc r (pBR322+P-11) Tc r
pSC102 Km r " " "
+ + + + + + + + + + +
" + + + + + + + +
" " " " "
See Materials and Methods' for experimental details
Plasmid Instability Sequence, Ins During the cloning of EcoRI and HindIII fragments of R6-5 into the high copy number vector plasmids ColE1 and pML21, we experienced considerable difficulty in obtaining recombinant molecules containing RepA-containing D N A fragments: only one such recombinant was obtained and this proved to be unstable (K. Timmis and F. Cabello, unpublished experiments). Similar difficulties occurred during the cloning of HindIII and SalI-generated fragments of the RepA-containing pSC102 plasmid: of 29 pBR322 recombinant plasmids containing HindIII fragments of pSC102, 26 contained fragment H-2 and only three contained H-l, and of 15 hybrids containing SaII fragments of pSC102, 4 contained fragment S-2, 11 contained S-3, but none contained S-1. These results suggest that a sequence in the RepA region of R6-5, when cloned into a high copy number vector plasmid, causes the hybrid plasmid to be unstable. This plasmid instability sequence (designated Ins) appears to be located between the S-1/S-2 junction and the ISlb
Table 5. Ability of hybrid plasmids to replicate in bacteria deficient in D N A p o l y m e r a s e I Plasmid
pSC102 pKT071 pBR322 pKT081 pKT038 pKT036 pKT039 pKT040 pKT043 pKT044 pKT050 pKT053 pKT054
Replication at 42 ° in
(P-1 + P - 4 + P - 6 ) ( p B R 3 2 2 + S-2) ( p B R 3 2 2 + P-2) ( p B R 3 2 2 + P-3) ( p B R 3 2 2 + P-4) (pBR322 +P-5) ( p B R 3 2 2 + P-6) ( p B R 3 2 2 q- P-7) ( p B R 3 2 2 + P-8) ( p B R 3 2 2 + P-9) (pBR322 + P-10
SC294
SC294 (pSC102)
+ + -
ND ND ND ND ND ND ND ND ND ND ND
See Materials and Methods for experimental details
replication
L v
~P-- ~ra . . . . . . .
finO I n c RepA ori
117 ola I 5 191 87.5 k b
Fig. 14. Functional map of the R 6 - 5 RepA EcoRI fragment
I 6 t
~
ISlb
I
3
....
100.8 kb
R-det
I. Andr+s et al. : Replication Functions of R6-5. II
17
ABCDE
FRAGMENT SIZES Hind rn" - Bgl g 4.75 ND 4.0
EcoRI -BgIII
Hind rrt - Bglrr
5.0 N D 2.15 1.65
6.3 MD 2.5
EcoRI - B9111 5.0 3.6 0.3
restriction sites
--EcoRI
,'A
FRAGMENT Hind I ] I - Bg[ II 5.1 MD 3.65
EcoRI- Bgl II 5.0 MD 2.5 1.3
wvw H i n d m
,,
,,
- -
Pst I
,,
,,
---~,
Bgirr
,,
,,
SIZES Hind rrm- Bgl rr 5 3 ~ID 3.0
Eco RI- Bgl II 5.0 3.2 0.7
Fig. IS. Structure of the mini pSC102 plasmid pKT071. Digested D N A s were subjected to electrophoresis through a 1% TB agarose slab gel. A : pKT071/BglII + HindIII ; B : pKT071/BglII + EcoRI ; C: pKT071/BglII + PvuII ; D : pSC102/BglII + PvuII ; E: 2/HindIlI + EcoRI. This gel shows that structure B is correct.
proximal end of the RepA EcoRI fragment. Because no plasmid instability was observed with recombinant plasmids containing PstI-generated fragments of pSC102, it is likely that Ins contains a PstI cleavage site such that no single PstI-generated fragment of pSC102 contains the complete instability sequence. Since there are only two PstI cleavage sites within the segment between the S-1/S-2 and E-2/E-8* junctions, we conclude that Ins must be located either at the P-6/P-4 junction or at the P-4/P-3 junction. We investigated the effects of Ins on plasmid replication by examining the properties of pBR322 hybrid plasmids containing the H-1 fragment of pSC102. As indicated above, three such recombinant plasmids were obtained. One of these rapidly underwent internal sequence rearrangements during propagation of the host bacteria on selective media such that the original restriction endonuclease cleavage pattern of the plasmid was greatly altered; this plasmid was not studied further. Another hybrid, plasmid pKT097, contained not only fragment H-1 but also fragment
H-2: this plasmid was quite stable, was maintained at the high cellular level typical of pBR322, could amplify in the presence of chloramphenicol, and could replicate in DNA polymerase I-deficient bacteria. The third plasmid, pKT098, contained only fragment H-l, was extremely unstable, could replicate to a limited Table 6. Stability of pBR322-pSC102 hybrid plasmids and their ability to replicate in chloramphenicol Plasmid
Cloned fragment
Stability
Relative increase in chloramphenicol
pBR322 pKT097 pKT098
H-1 + H-2 H-1
100 100 4
10 10 1.6
Stabilities of hybrid plasmids were determined as described in Materials and Methods for testing plasmid requirement for D N A polymerase I, except that bacterial cultures were incubated at 37 ° throughout. The relative increase in plasmid D N A in chloramphenicol-treated cells was measured as described by Timmis et al. (1974)
18
I. Andr6s et al. : Replication Functions of R6-5. I1
A
extent in D N A polymerase I-deficient bacteria, but could not amplify in the presence of chloramphenicol, i.e. the ability of the pBR322 component replicon to replicate relaxedly in the presence of chloramphenicol was inhibited (Table 6). We have been unable to demonstrate any structural differences in the H-1 fragments of pKT097 and pKT098 and therefore are not at this time able to provide a definitive explanation for the differences in replication behaviour of these two hybrid plasmids.
One additional function that has been identified on the RepA EcoRI fragment of R6-5 is the finO cistron, f/nO is one component of the FinOP system of fertility inhibition that reduces the expression of tra genes involved in plasmid fertility, finP has been shown to be located on R6-5 on the promoter proximal side of the tra operon and close to the presumed site of action of the FinOP repression system (Timmis et al., 1978a; see Fig. 1). In contrast, the cistron for the other component of this system, namely finO,
19
I. Andr6s et al. : Replication Functions of R6-5. II 0.g
was f o u n d to be l o c a t e d on the RepA EcoRl f r a g m e n t s o m e 3 5 k b f r o m FinP ( T i m m i s etal., 1978a). finO has been m a p p e d a c c u r a t e l y to the IncFII-proximal end of the PstI f r a g m e n t a d j a c e n t to P-6, i.e. P-2. It t h e r e f o r e lies within 1-2 k b of IncFII a n d the replic a t i o n f u n c t i o n s o f R6-5.
IR2 II 1~0
Orientation of E-8* within pSCI02 and Location of Su-resistance Determinant
B
P r e l i m i n a r y e x p e r i m e n t s s h o w e d t h a t the HindIII Suresistance f r a g m e n t o f R6-5, H-8, c o n t a i n s two Pstl cleavage sites, w h e r e a s the S m - r e s i s t a n c e f r a g m e n t , H-7, c o n t a i n s n o n e (not shown). T h e Su region o f E-8* is t h e r e f o r e p r o x i m a l to the E-8*/E-2 j u n c t i o n a n d the Sm region is p r o x i m a l to the E-8 */E-6 j u n c tion. F i g u r e 5 shows t h a t there are two PstI cleavage sites within the E-8 * s u l f o n a m i d e resistance f r a g m e n t o f R6-5. Because neither P-1 n o r P-3 was f o u n d to express s u l f o n a m i d e resistance, we c o n c l u d e t h a t p a r t o f the d e t e r m i n a n t for this resistance m u s t be l o c a t e d on P-11. This finding locates the Su-resistance gene precisely on the pSC102 m a p .
Fig. 16A and B. Heteroduplex analysis of the R6-5 origin deletion-
Spontaneous Deletion of the R6-5 Origin of Replication in a pBR322/P-4 Hybrid Plasmid
derivative hybrid plasmid pKT062. A Purified DNAs of plasmid pKT071 (pSC102 PstI fragments P-I+P-4+P-6) and pKT062 (pBR322+deleted pSCI02 PstI fragment P-4) were denatured, mixed, allowed to anneal, and spread for microscopy using the cytochrome C method (Davis et al., 1971 ; panels 2 and 3) or the protein-free method of Vollenweider et al. (1975; panels 1, 4, 5, 6 and 7). With the latter method, DNA molecules were spread on a water hypophase and positively stained with uranylacetate in acetone prior to shadowing. Double-stranded regions of homology are indicated by DS (panels 2-7) and the previously identified inverted repeat structure that is associated with Km-Nm resistance is indicated by IR2. Internal molecular standards used were the stem (for double-stranded DNA: l kb) and loop (for singlestranded DNA: 0.9 kb) of IR2 (Skurray et al., 1976a). A newly identified inverted repeat structure is designated IR4 in the singlestranded pKT071 molecule in panel 1. IR4 is located in the Sm~ region of R6-5. The DNA segment deleted from the P-4 fragment in pKT062 is shown by a single-stranded deletion loop in the middle of the region of homology between pKT071 and pKT062. Initial heteroduplexes obtained with nicked circular molecules always showed a highly wound single-stranded deletion loop (e.g. panel 3, arrow). Linearization of the molecules by treatment with the BgllI (pKT071) and BamHI (pKT062) endonucleases prior to denaturation permitted visualization of an uncoiled singlestranded deletion loop and revealed the presence of a short stem at its base. A diagrammatic representation of a heteroduplex is shown in part B. The size of the deleted origin segment is 0.54 kb and has termini located 0.63 kb from the BglII-proximal terminus and 0.36 kb from the BglII-distal terminus of P-4. The termini of the deletion therefore have R6-5 coordinates of 98.5 kb and 99.0 kb. The R6-5 origin of replication has been mapped at 98.6 kb (Synenki etal., 1979), and therefore is contained on the deleted segment
In o u r initial cloning e x p e r i m e n t s we o b t a i n e d only one p B R 3 2 2 h y b r i d p l a s m i d , p K T 0 3 9 , t h a t c o n t a i n e d the R6-5 o r i g i n - c a r r y i n g PstI f r a g m e n t , P-4. In o r d e r to generate a h y b r i d p l a s m i d with P-4 in the reverse o r i e n t a t i o n in the v e c t o r molecule, we d i g e s t e d p K T 0 3 9 D N A with PstI, re-ligated the f r a g m e n t s a n d c l o n e d new h y b r i d s b y t r a n s f o r m a t i o n . O n e o f these h y b r i d p l a s m i d s , d e s i g n a t e d p K T 0 6 1 , was o f the required type. A n o t h e r h y b r i d , however, d i d n o t contain P-4 b u t r a t h e r a s m a l l e r D N A f r a g m e n t . Restriction e n d o n u c l e a s e cleavage analysis of the l a t t e r plasmid, d e s i g n a t e d p K T 0 6 2 , d e m o n s t r a t e d t h a t the c l o n e d f r a g m e n t is P-4 t h a t is deleted for a p p r o x i m a t e l y 1/a o f the central sequences (Fig. 16), one o f the two HaeII sites, a n d the R6-5 origin o f r e p l i c a t i o n . Heteroduplex formation between pKT062 and p K T 0 7 1 D N A s c o n f i r m e d the l o c a t i o n of the d e l e t e d s e g m e n t and, f u r t h e r m o r e , s h o w e d t h a t the t e r m i n i o f this s e g m e n t are p r o b a b l y i n v e r t e d r e p e t i t i o n s : a s h o r t d u p l e x stem is o b s e r v e d at the base o f the deletion l o o p in this h e t e r o d u p l e x (Fig. 16). C o n s i s t e n t with this is the f i n d i n g t h a t the P-4 d e l e t i o n f r a g m e n t has lost entire HpaII a n d HaeIII f r a g m e n t s o f P-4, w i t h o u t r e t a i n i n g a n y d e t e c t a b l e p a r t i a l HpaII o r HaeIII f r a g m e n t s , which suggests t h a t the d e l e t i o n t e r m i n i are G - C rich r e p e a t e d sequences t h a t c o n t a i n
a53
20
I. Andr6s et al. : Replication Functions of R6-5. II
1
2
1
2
3
4.
4
5
5
6
7
8
9
10 11 12 13
14. 15 16 17
A
3
6
7
8
9
10
11 12
13
14
15
16
17
18
19
20
B Fig. 17A and B. Proteins of minicells carrying pSC102 hybrid plasmids Part A. Total minicell proteins: Coomassie blue-stained gels. 1:pKT054 (P-10); 2: pBR322; 3:pKT060 (P-2 FinO-); 4:pKT059 (P-2 FinO ); 5:pKT031 (P-2); 6:pKT032 (P-2 reverse); 7: pSC102; 8:pKT017 (R6-5 H-2 reverse); 9:pKT002 (R6-5 H-2); 10: pML21; 11:pKT056 (P-1 reverse); 12:pKT029 (P-l); 13: pKT071; 14:pKT085 (S-3); 15: pSCI02; 16:pKT054 (P-10); 17: pBR322. The arrows indicate the positions of protein bands that are new or are of greater intensity than corresponding proteins from the plasmid-negative minicells. Part B. Radioactive plasmid proteins: autoradiograms 1:pKT017 (R6-5 H-2 reverse); 2:pKT002 (R6-5 H-2); 3:pML21 (R6-5 E-6); 4:pKT056 (P-1 reverse); 5:pKT029 (P-l); 6:pKT071 (P-I+P-4+P-6); 7:pKT085 (S-3); 8:pSC102 (R6-5 E-2+E-6+E-8*); 9:pKT040 (P-5); I0:pKT036 (P-3); 11: pKT032 (P-2 reverse); 12:pKT031 (P-2); 13:pKT054 (P-10); 14: pBR322; 15:pKT043 (P-6); 16:pKT039 (P-4); 17:pKT052 (P-9); 18:pKT049 (P-8 reverse); 19:pKT047 (P-8); 20:pKT044 (P-7)
HpaII and HaeIII cleavage sites. Similar origin deletion m u t a n t s of R1 hybrid plasmids have been obtained by Kollek et al. (1978).
pSClO2-specified Plasmid Proteins A variety of plasmids derived f r o m R6-5 (Table 2) were introduced into the minicell-producing strain DS410 ( D o u g a n and Sherratt, 1977) or its lac- dele-
tion derivative M2124 ( T h o m p s o n and A c h t m a n , 1978) for the purpose of identifying mini R6-5 plasmid proteins. Minicells were p r e p a r e d f r o m purified t r a n s f o r m a n t s and labeled with 14C a m i n o acids or 35S-methionine. The proteins extracted f r o m these minicell preparations were subjected to electrophoresis through 15-25% polyacrylamide gradient gels which were subsequently stained with C o o m a s s i e blue (to show total proteins) and a u t o r a d i o g r a p h e d (to show plasmid-specified proteins).
I. AndrOs et al. : Replication Functions of R6-5. II
The majority of minicell preparations exhibit similar total protein profiles that are identical to that of minicells prepared from plasmid negative bacteria. On the other hand, minicells that carry kanamycin resistance plasmids, such as pSC102, pKT071, pML21, etc. (Fig. 17A, tracks 9-15), produce large amounts of a new protein band that migrates with an apparent monomer molecular weight of 27,500 daltons and that is identical to a plasmid-specific radioactive protein that is probably the kanamycin-neomycin inactivating enzyme. This protein is not seen in minicells carrying pKT017 (track 8) which is identical to pKT002 (track 9), except that it contains an inactivated kanamycin resistance determinant. pKT002 and pKT017 both encode resistance to chloramphenicol and minicells carrying these plasmids show a strong band representing a new protein having a monomer molecular weight of 22,600 daltons (tracks 8 and 9) which is the monomer molecular weight of chloramphenicol acetyltransferase (Fitton et al., 1978). A strong protein of the same molecular weight is also seen in the autoradiograph, indicating that the 22,600 dalton protein is plasmid encoded. It can also be seen that pBR322-carrying minicells exhibit an increased intensity of a visible protein band having a molecular weight of 27,000 daltons (tracks 2, 17) and this is assumed to result from the presence of a pBR322-encoded/Mactamase polypeptide at this position. In addition to these presumptive antibiotic inactivating enzymes, some minicells that carry mini R6-5 hybrid plasmids also' exhibit several other changes in the standard protein profile. Minicells carrying plasmids pKT031, pKT059 and pKT060 (pBR322 + P-2) exhibit a strong protein band having a molecular weight of 27,100 daltons (Fig. 17A, tracks 5, 4 and 3). This protein is not thefinO protein which is known to be encoded on P-2 (Timmis et al., 1978a), because (a) the finO protein has a molecular weight of only 22,500 daltons, (b) the protein is made by the finO- mutant plasmids pKT059 and pKT060 that do not produce a finO-sized protein, and (c) the protein is not exhibited by pKT032 (track 6), a finO + plasmid that contains the P-2 fragment in the orientation opposite to that of pKT031 with respect to the pBR322 cloning vector, and that directs the production of amounts of finO protein that are equivalent to that of pKT031. At present it is not known whether the protein present in increased amounts in minicells carrying pKT031, pKT059 and pKT060 is plasmid-or-host specific (see also Iyer, 1977). Figure 17B shows autoradiograms of polyacylamide gels containing the radioactive proteins synthesized by purified minicells carrying pSC102 hybrid plasmids and Table 7 lists the apparent monomer too-
21 Table 7. Plasmid-specified proteins detected in minicells pSC102 protein
Molecular enweightlo2 coded by
Designation
Molecular Identified weight~yb function
1 2 3
37.500 29.500 27.500
P-1 P-3 P-I
P1-A P3-A P1-B
27.500
4 5 6 7 8 9 10 11 12
26.400 25.200 24.700 23.200 22.500 21.000 17.900 15.400 14.400
P-2 P-1 P-1 P-5 P-4 P-2
P2-A P1-C P1-D P5-A P4-A P2-B
13
13.500
14
10.000
15
9.000
16
6.200
P-3 P-1 P-1 P-3 P-1 P-2 P-1 P-4 P-6 P-7 P-8 P-9 P-1
P3-B PI-E P1-F P3-C P1-6 P2-C P1-H P4-B P6-A P7-A P8-A P9-A Pl-I
U m r (Km-Nm phosphotransferase)
27.800
fin 0 15.600 14.500 I3.400 13.000 10.100 10.000 9.400 8.200 7.800 7.000 5.800
Molecular weight102 is the molecular weight of a protein synthesized by minicells containing the pSC102 plasmid; Molecular weightilyb is the molecular weight of a protein synthesized in minicells containing a pSC102/pBR322 hybrid plasmid or the mini pSC102 pKT071
lecular weights of these proteins. The three main protein bands (M.W. 29,500, 27,000 and 24,000 daltons) synthesized by minicells containing pBR322 are not expressed by any pBR322 hybrid plasmid with DNA inserted at the PstI cleavage site and which fails to express ampicillin resistance. One or more of these three bands is therefore concluded to be the /Mactamase polypeptide. All of the minicells with hybrid plasmids containing pSC102 fragment P-1 (e.g. pKT071, pKT029 and pKT056) exhibit protein bands that comigrate with pSC102 bands 2 (27,500), 3 (26,400) and 4 (25,200). These bands are also exhibited by other plasmids that contain the R6-5 Km-Nm resistance determinant (Tn601) such as pML21 and pKT002 (pML21 + R6-5 H-2), although none of these bands is expressed by the Kin-sensitive plasmid pKT017, which is identical to pKT002, except that the orientation of H-2 in pML21 is reversed. It is concluded that pSC102 band 2 is the kanamycin-neomycin phosphotransferase which has a published molecular weight of 27,500 daltons (Mitsuhashi et al., 1976). Bands 3 and 4 may be either degradation prod-
22 ucts of this protein or may be distinct proteins that are under the control of the Km resistance promoter. Plasmids carrying fragment P-2 direct the synthesis of three proteins, P-2A, P-2B and P-2C, having molecular weights of 27,800, 22,500 and 13,000 daltons. From earlier experiments (Timmis et al., 1978 a) it seems likely that P-2B is the finO protein. Differences in the orientation of cloned fragments had in general little effect on the proteins expressed by hybrid plasmids. The exceptions to this were (a) in one particular orientation, P-2 directs the synthesis of a 24,000 dalton protein that does not correspond to any obvious pSC102 protein band; and (b) in one particular orientation, P-8 directs the synthesis of a 7,800 dalton protein that could correspond to a pSC102-determined band. As can be seen in Fig. 17 and Table 7, the incompatibility fragment P-6 directs the synthesis of only one protein in detectable amounts. Insertion of D N A fragments into the BglII cleavage site of pKT043 does not cause loss of either the Inc function or of the 9450 dalton protein. This protein may be the copy control/incompatibility protein of R6-5 but definitive identification will require the isolation of Inc- and Cop mutants. The DNA fragment that carries the R6-5 origin of replication, P-4, expresses two detectable proteins having molecular weights of 23,200 and 10,000 daltons. Sequence continuity at the junction of P-4 and P-6 appears to be essential for R6-5 plasmid viability and hence a replication function may be located at this junction. In this case the mini pSC102 plasmid pKT071 may express a protein not expressed by hybrid plasmids carrying individual P-I, P-4 and P-6 fragments. No such protein was detected in this series of experiments and we conclude that the D N A sequence containing the P-4/P-6 junction either does not encode a protein or that the protein is not made in detectable quantities. This latter possibility is consistent with the inability of R6-5 miniplasmids to permit R6-5 origin usage in P-4-containing hybrid plasmids (see above). Finally, it should be noted that the pSC102-specific-proteins that are expressed in detectable quantities by pSCI02 hybrid plasmids account for 50% of the coding capacity of pSC102.
Discussion
Extensive genetic analysis of plasmids has until recently been severely inhibited because of plasmid incompatibility functions which prevent the creation of stable plasmid diploids and consequently the use of complementation analysis. Advances that have been made were possible due to the use of special
I. Andr6s et al. : Replication Functions of R6-5. II techniques (construction of transient diploids: Achtman et al., 1972; integrative suppression : Yoshikawa, 1974) that do not have general applicability. However, the development of recombinant DNA techniques now allows the cloning of small DNA fragments that are readily analyzed onto vector molecules that are compatible with the plasmid under investigation, and has facilitated a range of genetic analyses of plasmid functions (e.g. Skurray et al., 1976; So et al., 1976; Timmis et al., 1978d). Although different plasmids code for a wide varie t y of functions, such as antibiotic resistance, fertility, toxins, catabolic pathways, etc., all have the basic property that they replicate autonomously in, and are inherited stably by, host bacteria. Furthermore, plasmid replication is carefully regulated such that each plasmid species is maintained at a constant cellular concentration in dividing bacteria. It is now well established that plasmids make extensive use of host replication functions (e.g. Collins et al., 1975) although they do encode some plasmid-specific functions, including the copy control function and an origin of replication. The identification of a plasmidspecified essential replication function of R100 (RepA; Yoshikawa, 1974) and the isolation of temperature-sensitive replication mutants of R6-5 (K. Timmis and F. Cabello, unpublished experiments) demonstrate that some, though not all, plasmids specify additional replication functions that are obligatory for plasmid viability. It is our objective to understand in molecular terms the overall process of regulated autonomous plasmid replication and inheritance. This communication presents some initial steps towards this goal namely, construction of a detailed restriction enzyme cleavage map of the RepA region of R6-5, identification of the specific plasmid segment essential for replication by the in vitro generation of miniplasraids that contain a minimal amount of DNA from the RepA region, mapping of all of the functions currently known to be involved in the replication process (OriV, RepA, Cop), and cloning on high copy amplifiable vector plasmids of small DNA segments that separately carry OriV and Inc. Hybrid plasmids of this latter type are of considerable utility and can be used to obtain large quantities of both DNA fragments and gene products for the structural and functional analysis of plasmid vegetative replication. The first point to be made is that there is a very high degree of clustering of essential replication functions in R6-5:pSC102 PstI fragments P-4 and P-6 are together sufficient for replication. The maximum length of DNA required for autonomous replication is therefore 2.6 kb (although the minimum length might be somewhat less, perhaps 1.0 kb if sequences downstream of the replication origin are not essential)
I. Andr+s et al. : Replication Functions of R6-5. II
and within this region are the origin of replication and the RepA, Cop, and Inc functions. Recent experiments (Slocombe and Timmis, 1979) have shown the presence of three RNA polymerase binding sites within the essential region, one in P-6, one in P-4 close to the P-4/P-6 junction, and one at a site in the middle of P-4 that is indistinguishable from the origin of replication. It is possible that this latter site is the promoter for origin primer R N A synthesis, that the site in P-6 is the promoter for the incompatibility/copy control gene, and that the site close to the P-4/P-6 junction is the promoter of a replication gene(s), possibly RepA. It should be noted that the size of the essential region of R6-5 is considerably larger than that of ColEl-like plasmids (Backman et al., 1978) and presumably this reflects the finding that ColEl-like plasmids do not appear to require any plasmid-encoded functions for their replication (Tomizawa et al., 1975; Donaghue and Sharp, 1978 and Kahn and Helinski, 1978). Figurski et al. (1978) and Kollek et al. (1978) have also found tight clustering of replication functions in the F, R6K and R1 plasmids. Secondly, despite this clustering and although the origin of replication is located in the middle of a 1.6 kb PstI fragment of pSC102, P-4 (see accompanying paper by Synenki etal., 1979), this fragment is not itself sufficient for autonomous replication but requires in addition the presence of the adjacent PstI fragment, P-6, which encodes the incompatibility/copy control function. This is evidenced by the fact that all the mini pSC102 plasmids that were isolated consist of both P-4 and P-6, and by the fact that pBR322 hybrid plasmids carrying P-4 alone, unlike the parent pSC102 plasmid, cannot replicate in DNA polymerase I-deficient bacteria. Although it is not clear whether Cop and Inc are essential replication functions, it is likely that an additional obligatory replication function is partly encoded by P-4 and partly by P-6 because sequence continuity of P-4/P-6 junction is always preserved in pSC102 miniplasmids. This additional function is probably RepA. Although only one PstI fragment of the pSC102 plasmid, P-6, has been shown to express FII-incompatibility, the incompatibility behavior exhibited by P-6-containing hybrid plasmids can be interpreted as resulting from two distinct functions namely, copy control (Cop) and plasmid partition structure recognition (Timmis et al., 1978c; Timmis et al., 1979), although confirmation of this will require the analysis of plasmid Inc and Cop mutants. In two other F-incompatibility group conjugative plasmids, two distinct plasmid segments have been shown to encode Inc functions (Manis and Kline, 1978 and W. Goebel, personal communication). As has been observed pre-
23
viously (Cabello et al., 1976; Eichenlaub et al., 1977), the origin of replication is not itself an incompatibility determinant although it probably is involved in copy control-mediated incompatibility reactions. Although we have been able to identify a number of pSC102 encoded proteins that collectively account for about 50% of the coding capacity of this plasmid, it is not possible at this time to assign with certainty particular proteins encoded by the essential region to identified replication or incompatibility functions, although only one 9,400 dalton protein was expressed in detectable quantities by the incompatibility fragment P-6, and it is tempting to speculate that this protein may be the copy control/incompatibility protein. We have recently isolated a number of incompatibility and other replication mutants of R6-5 derivative plasmids (H. Danbara and K. Timmis, unpublished experiments) and are currently analyzing the proteins expressed by these mutant plasmids in order to relate definitively replication genes with their gene products. During the cloning of the P-4 origin-carrying fragment into pBR322, a hybrid plasmid containing a deleted P-4 fragment was isolated. This fragment lacks 0.36 mD of the central DNA sequences of P-4 including the origin of replication and an RNA polymerase binding site that has been located close to the origin of replication and that may be the promoter for the origin RNA primer sequence (Slocombe and Timmis, 1979). Minicells containing the origin deletion fragment/pBR322 hybrid plasmid failed to synthesize the two identified P-4 encoded proteins but instead synthesized two slightly smaller polypeptides. Heteroduplex formation between BglII-cleaved mini pSC102 plasmid pKT071 DNA and BamHl-cleaved origin deletion hybrid plasmid pKT062 DNA showed the presence of a short duplex stalk at the base of the deletion loop, indicating the presence of short inverted repeat sequences at the termini of the segment that has been deleted from P-4. Similar findings were recently obtained with the R1 plasmid (Kollek et al., 1978). The origins of replication of the two FII incompatibility group plasmids R6-5 and R1 are therefore located on transposon-like structures: whether these structures are predisposed to deletion formation is an interesting question that is currently under investigation. Acknowledgements. We thank W. Goebel and R. Kollek for communicating their results prior to publication, D. Vogt for valued technical assistance, M. Achtman for helpful advice on minicell methodology, H. Mayer for gifts of HindIII and PstI restriction endonucleases and Bayer-Leverkusen for a gift of ampicillin (Binotal). I.A. and P.M.S. were supported by fellowships from the Spanish Ministry of Education and the European Molecular Biology Organisation, respectively.
24
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Communicated by T. Yura Received July 26, 1978