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Copy number of the broad host-range plasmid R1162 is determined by the amounts of essential plasmid-encoded proteins
.J. Mol. Biol.
(1985) 185, 755-767
Copy Number of the Broad Host-range Plasmid R1162 is Determined by the Amounts of Essential Plasmid-encoded Proteins Kyunghoon Kim and Richard J. Meyer University of Texas Department of Microbiology Austin, Tex. 78712-1095, U.S.A. (Received 5 November 1984, and in revised form
5 April
1985)
DNA of the broad host-range plasmid Rl162 contains a 1700 base-pair segment essential for plasmid maintenance. This region, RepI, consists of two cotranscribed genes encoding polypeptides with molecular weights of 29,000 and 31,000. Fusion of Rep1 to the strong tat promoter results in greatly increased amounts of at least one of these polypeptides. In tram, this construction has two other properties: it can raise the copy number of Rl162, and it can protect this plasmid from loss due to incompatibility. Both effects require intact Rep1 genes. These properties of the Rep1 region, along with those of an origin-linked region described earlier, are discussed with respect to current models for control of plasmid copy number.
1. Introduction
from right to left as drawn in Figure 1. The promoter-proximal gene, which occupies a position within 1.7 to 2.63 kb, encodes an essential polypeptide (M, 31,000) (Meyer et al., 1985). We have identified a second essential polypeptide (M, 29,000) encoded by the remaining DNA of RepI. In addition to Rep1 and RepII, the oriV-Inc region (Fig. 1) is required for plasmid replication. This region contains the origin of DNA replication, and the determinant for plasmid incompatibility (Lin & Meyer, 1984). A 370 base-pair fragment of DNA specifying this incompatibility is involved in control of plasmid copy number (Lin & Meyer, 1984). In this paper, we show that the amounts of Rep1 proteins determine the amount of RI 162 plasmid DNA in the cell: when the Rep1 genes are fused to a strong promoter, more Rep1 polypeptides are made, and the plasmid DNA content per cell rises to greater than normal values. In addition, the plasmid becomes more resistant to elimination from the cell through incompatibility. We discuss these results in terms of models for copy-number control.
Rll62, is a high copy-number plasmid, originally isolated from Pseudomonas aeruginosa, but with a broad host-range among Gram-negative bacteria (Barth & Grin&, 1974; Bryan et al., 1972). It is very similar or identical to several independently isolated plasmids (Barth & Grinter, 1974; Grinter & Barth, 1976); the most thoroughly studied of these is RSFlOlO from Escherichia coli (Guerry et al., 1974). The plasmids in this group are small, consisting of about 9000 base-pairs of DNA. They to resistance streptomycin and encode sulfonamides, and are not self-transmissible, although they can be mobilized readily during conjugative transfer of certain other plasmids (Barth & Grinter, 1974). A genetic map of Rl162 is shown in Figure 1. Two regions, designated Rep1 and RepII, encode products required for plasmid maintenance (Meyer et al., 1985). Proteins encoded by the corresponding regions in RSFlOlO have been isolated by Scherzinger et al. (1984), who showed that they are necessary for plasmid DNA replication in vitro. The genetic organization of the RepI region has been determined partially (Meyer et al., 1985). The essential DNA lies within the co-ordinates 0.91 to 2.63 kbt (Fig. 1). The entire region is transcribed
2. Materials and Methods (a) Bacterial
strains
and plasmids
The strains of E. coZi K12 used in this study are: MVlO
(thr Zeu thi ZacY supE44 tonA AtrpE5) (Hershfield et al., 1974); GM1 (aruA(Zac pro) thi/F”(Zac pro) lucIq L8) (Miller et al., 1977); and P678-54 (thr leu thi lacY minA m&B gal
t Abbreviations used: kb, IO3 base-pairs; kd, M, x 10-3; IPTG, isopropyl-B-n-thiogalactoside. 0022-2836/85/200755-13
$03.00/O
755
0 1985 Academic Press Inc. (London) Ltd.
756
K. Kim and R. J. Meyer
RP RI162 0.00
I
P
Et
BC
Bc
Bc
I I
I I
I I
I I
I
30
7.4
83
08
I I
,
,-’
p
R
87kb
mRNA
Polypeptldes
31,000
Figure 1. Genetic map of R1162. Shown are the approximate locations of genes for resistance to sulfonamides (Su’) and streptomycin (Smr). Rep1 and Rep11 are regions of the plasmid encoding products necessary for plasmid maintenance; o&V-Inc indicates the position of the origin of plasmid DNA replication and the associated determinant for incompatibility. The locations of a few restriction enzyme cleavage sites (in kb) from the single EcoRI site, are shown also. Abbreviations: Bc, B&I; Bt, B&EII; P, P&I; and R, EcoRI. Below the map of R1162, the Rep1 region has been expanded to show the locations of the genes encoding 29,000 M, and 31,000 JZ, polypeptides and the direction of transcription through these genes.
rpsL) (Adler et al., 1967). In the last case, a thymineand their requiring variant was used. Plasmids derivations are listed in Table 1. (b) General procedures Bacteria were grown at 37°C in TYE broth (1% (w/v) Bacto-tryptone, 05% (w/v) Bacto yeast extract, 0.5% (w/v) NaCl) or in M63 minimal medium (Miller, 1972) supplemented with 0.4% (w/v) glucose, 1 mM-MgSO,.and 0901~0 (w/v) thiamine* HCl. When necessary, the following antibiotics were added to the indicated final concentrations: carbenicillin (200 pg/ml), streptomycin sulfate (25 pg/ml), trimethoprim (l&l pg/ml) and tetracycline hydrochloride (10 pg/ml).
Plasmid DNA was generally prepared by the rapidboiling method of Holmes & Quigley (1981). Transformation of cells with plasmid DNA was done by the procedure of Cohen et al. (1972). Restriction enzymes, BuZ31 exonuclease and phage T4 polynucleotide ligase were obtained from a commercial source (New England Biolabs), and used according to the instructions provided by the supplier. For limited digestion of linearized plasmid DNA with BaZ31, approximately 10 pg of DNA in 56 ~1 of buffer was incubated at 30°C with 2 units of enzyme, and samples taken every 10 min. Procedures for running 0.8% (w/v) agaroae gels and visualizing the DNA bands are standard and have been described (Meyer et al., 1975, 1982).
Table 1 Plasmids used in this study Plasmids Cloning
Relevant
properties
Source or derivation
vectors:
pACYC184 pRR322 pMC1403 psc101 ptacl2
Rep(p15) Cm’ Tc’ Rep(ColE 1) Cb’ Tc’ Rep(ColE1) Cb’ lucz’ lacy TC’ Rep(ColE1) tac( ) Cb’
R1162 and derivatives: IncQ Su’Sm’ R1162 pMS178 Rep(p15) RepI+ Km’ &Z(a)
Chang & Cohen (1978) Rolivar el al. (1977) Casadaban et al. (1980) Cohen et al. (1973) Amann et al. (1983) Rarth & Grinter (1974) (Fig. 1) Insertion of DNA fragment containing 0+3.0 kb R1162 DNA into BarnHI site of pMC489 (Casadaban & Cohen, 1980) (Fig. 3)
Copy Number
of Plasmid
R1162
Table 1 (continued) Plasmids
Relevant
properties
Source or derivation
pMR228 pMR245 pMS319
Rep(R1162) IncQ (dupl) Tp’ Rep(R1162) IncQ Tp’ Rep(Co1El) IncQ Cb’
pi%342
Rep(ColE1) RepI+ Cb’ lac%’ lacy
pMS34X
Kep(ColE 1) Cb’ lacZ’ ZaeY
pVT182
Rep(ColE1) tac(
pf.TT 184 and pVT18.5
Rep(ColEl
Rep(ColE1) Cb’ 1acZ’ 1ocY
pIyT191
Rep(ColEl
plTT236 and pl’T23X
Rep(ColE1) RepI+ tac(O.O-2.83) Cb’
prT”37 and pI’T”39 pVTP47 and plT248
) Cb’
RepI+ Cb’ 1acZ’ lacy
tac(
Rep(ColE1)
) Cb’
RepI+ tac(2.83-0.0) Cb’
Rep(ColE1) tac(OG2.2)
Cb’
Rep(ColE1) tae(2.2-0.0) Cb’ Kep(p15) tac(2.2-0.0) Tc’
Rep(pl5)
RepI+ tac(2.83~0.0) Tc’
pl’T25l
Rep(pSC101) RepI+ tac(2.83-0.0)
plTT2.52
Kep(pSC1O1) tac(2.240)
pl.T278
Rep(ColE1) tae(2.83-0.0. A1.1) (‘b’
pI’T282
Rep(p15) tae(2.83-0.0. A1.1) Tc’
pITT3 16
Rep(p15) RepI+ Tc’
Tc’
Tc’
Lin & Meyer (1984) Lin & Meyer (1984) Derivative of pBR322 containing 370 basepair DNA fragment derived from R1162 and specifying incompatibility (unpublished result) Insertion of DNA fragment containing 0.0-2.83 kb R1162 DNA, generat,ed by sequential digestion of pMS178 DNA with SalI, Ba/31 (partially) and EcoRI. into pMC1403, by replacement of EcoRI-SmaI DNA linker (Fig. 3) As for pMS342, except that inserted DSA contains only 0.0-2.2 kb R1162 DS.1 (Fig. 3) ” Insertion of 11 base-pair Seal-Hincll linker DXB. containine a BansHI cleavage site, from -M13mp8(RFI) (Messing & Vieira. 1982) into PwcII site of ptacl2 (Fig. 2) Insertion of 42 base-pair EcoRI-generated DNA fragment. containing mult,iplr restriction enzyme cleavage sites. from M13mp7(RFI) (Messing et al., 1981) into EcoRI sites of pMS342 and pMS348. respectively (Fig. 3) Replacement of small EcoRI + Bar/~H1generated DNA fragment of pBR322 with DNA fragment from pUT182 and cow taining tat promoter (Fig. 2) Insertion of DNA fragment containing 0.0-2.83 kb Rl162 DNA, generated by BamHI digestion of pUT184 DNA. into BarnHI site of PUT191 in each of 2 orientations (Fig. 3) As for pLlT236. pCT238: Barr&HI-generated DNA fragment containing 0.0-2.2 kb from pVT185 R1162 DNA derived (Fig. 3) Insertion of DNA fragment containing Inc (2.2-0.0) R1162 DNA fusion. generated by EcoRI digestion of pUT239 DNA. into EcoRI site of pACYC184 (Fig. 4) As for pUT247. pUT248. except EcoRlgenerated DNA fragment, containing tat (2.X3-0.0) R1162 DNA fusion. derived from pITT238 (Fig. 4) As for pVT249. plT250. except DNA fragment inserted into EroRI site of pSClO1 (1 orientation only) (Fig. 4) As for pLlT247. pUT248. except DXA fragment inserted into EcoRI site of pSClO1 (1 orientation only) (Fig. 4) Deletion in R1162 DNA across 1.1 kb b> limited digestion of BstEIl-cleaved pTT238 DXA with Ra/31 rxonucleasr (Fig. 5) Insertion of DNA fragment containing R1162 DSA, generated by EcoRI digestion of pUT278, into EcoRI site of pAC\-(‘184 (Fig. 5) Insertion of DNA fragment containing 0.0-2.83 kb R1162 DNA. generat,ed by EcoRI +BamHI digestion of pMS342. into EcoRI site of pACYC184 (Fig. 5)
Abbreviations: Rep ( ), replication system derived from naturally occurring plasmid in parentheses; tac( ), contains tax promoter sequence adjacent to R1162 DNA with co-ordinates and orientation indicated in parentheses; IncQ, member of Q incompatibility group; (dupl), IncQ locus duplicated; RepI+, contains entire Rep1 region of R1162: Cb’, Cm’. Sm’, Su’. Tc’, Tp’. encodes resistance to carbenicillin, chloramphenicol, streptomycin, sulfonamides. tetracycline, trimethoprim. respectively. Other abbreviations are standard or defined in the text.
758
K. Kim (c) Estimation
and R. J. Meyer
of plasmid content in cells
Plasmid-containing derivatives of GM1 were grown overnight in TYE brot,h. then diluted 1 : 50 (v/v) into M63 medium. Growth in M63 medium requires the presence of the F prime facbor in the cell; other plasmid species were maintained by supplementation of the medium with antibiotics. The cultures were incubated at 37°C to mid-log phase (about 3 x lo* to 5 x lO’/ml), then divided and one portion induced by the addition of isopropyl-j-n-thiogalactoside to a final concentration of 1 mM. Incubation of the cells was continued to stationary phase (about 2 x 109/ml). Plasmid DNA was then isolated, digested with a restriction endonuclease to facilitate subsequent identification, and displayed by electrophoresis through a O+Jo/0agarose gel. The relative amounts of each plasmid were estimated from the intensities of the plasmid DNA bands appearing in each lane. Each sample well contained DNA extracted from approximately 2 x lo9 cells. (d) IdentiJication
of plasmid-encoded polypeptides
Minicells from plasmid-containing strains of P678-54. grown to stationary phase in 250 ml of TYE b&h containing appropriate antibiotics for selection of plasmids, were isolated by 2 successive centrifugations through sucrose gradients, according to the method of Frazer & Curtiss (1973). Preparations contained less than I whole cell per 10’ minicells. Proteins being synthesized in the minicells were radioactively labeled by incubation of approximat’ely 109 minicells in I ml of labeling medium (Rothstein & Reznikoff. 1981) containing 30 to 50 PCi of r,-[35S]methioninc (> 1000 mCi/mmol). After I I1 incubation at 37°C. the minicells were pelleted by centrifugation, resuspended in TYE broth, and incubated for 10 min at 37°C. Finally, the labeled minicells were pelleted, resuspended in 20 ~1 of 9.59/, (v/v) glycerol containing 0.0625 M-Tris HCl (pH 6+3), 5% (v/v) P-mercaptoethanol, 2.3% (w/v) sodium dodecyl sulfate and 0.002% (w/v) bromphenol blue. Samples were boiled for 2 to 3 min, then stored at -20°C before electrophoresis. Samples were applied to a polyacrylamide gel (11% (w/v) acrylamide. 0.276 (w/v) bis-acrylamide, 0.1% sodium dodecyl sulfate) prepared according to the method of Lugtenberg et al. (1975). Electrophoresis was carried out at room temperature and at constant current (30 mA). Gels were stained with Coomassie blue. destained, and then dried. Proteins were visualized by autoradiography using Kodak X-Omat XRP-1 film with an intensifying screen, and with an exposure time of 3 days at room temperature.
by the parental vector (Fig. 6(b), lane b). The 29,000 J!& and 27,000 M, polypeptides probably represent unprocessed and processed forms of fl-lactamase (Ambler Br Scott, 1978; Sutcliffe. 1978). We have identified the 31,000 LWrpolypeptide as t,he product of an essential gene in Repl, lying within 1.7 to 2.63 kb (Meyer et al.. 1985). Cloned Rl 162 DNA containing the Rep1 genes was fused at position 2.83 kb to DNA encoding the strong tat promoter (Amann et al.. 1983). The resulting plasmid, pUT23X (Fig. 3), specifies substantially greater relat)ivr amount,s of thv 31,000 ~21~protein (Fig. 6(a), lanes h and v). This confirms the direction of transcription of t hr gcncl for this protein (Fig. 1). A4dditional essential DNA is cotransvrihetl but promotjer-distal to this gene (Meyer et (~1.. 1985). The polypeptides encoded in minicells by Rl 162 11?1;A4(04 to 2.2 kl,) cloned int’o pMC’1403 (pMS318, Fig. 3) were examined on polyacv-glamidr gels (Fig. 6(a). lane g), As expected. the 31 .OOO N, species was absent. since onl)- a portion of the gent’ had been cloned. We observed no other Keplencoded proteins. which was cxpccted also, since, the Rep1 promoter (Fig. 1 ) was missing from the SmBHc
cbr$y~
6
PV
Sm+Hc / R Cb’
Cb’
R+Bh
R+B Cb’ f+y (
3. Results (a) B,UOO and 37,000 Mr polypeptides by the Rep1 region
are encoded
The plasmid pMS342 (Fig. 3) consists of 0.0 to 2.83 kb R1162 DNA cloned into the vector pMC1403. The polypeptides encoded by this plasmid are shown in Figure 6(a). lane d. Two polypeptides with molecular weights of approximately 29,000 and 27,000 are made, as well as several smaller and one larger (Mr 31,000) species. All except the 31 .OOO Jfr polypeptidr are encoded
B PUT191
Figure 2. Construction of recombinant plasmids used in this study. The different molecules are not drawn to scale. The locations of genes encoding resistance to carbenicillin (Cb’) and tet,racycline (Tr’) are indicated. as well as the positions of relevant restriction enzyme caleavage sites. A section elevated with broken lines from the plasmid map represents a small linker DNA containing multiple restriction enzyme cleavage sites. Portions of the circular maps drawn with heavier lines indicate tax promoter DNA, with the direction of transcription shown by the arrow. The abbreviations for restriction enzyme cleavage sites are: B, BarnHI; He, HincII; Pv, PvuII; R, EcoRI; S, SalT; and Sm, SmaI.
759
Copy Number of Plasmid R1162 R
Sm
B
R (0
-,--.-.,.
\I
R(G0)
0) NO 0)
CbrA
Y
pMS348
pMS342
1) RBSPSBR
ABSPSBR
RBSPSBR
R Cd B :2 2)
Figure 3. Construction of recombinant plasmids (continued). The double-lined portion of the plasmid sketches indicate the presence of R1162 DNA between the kb co-ordinates (Fig. 1) given in parentheses. Y and Z’ indicat,e the locations of the ZacY and la& genes; Km’, the gene encoding resistance to kanamycin. Other abbreviations are defined in Figs I and 2.
cloned DNA. In an attempt to visualize the other essential gene products, we fused the R1162 DNA at 2.2 kb to the tat promoter (pUT239, Fig. 3). Complementation in L&JO by pUT239 of a replication-defective deletion derivative of R1162 demonstrated that under these circumstances the additional Rep1 DNA is expressed, whereas pMS348 is unable to complement (Meyer et al., 1985). However, no new polypeptides are observed for pUT239 (Fig. 6(a), lanes e and f). We thought that an additional Rep1 protein might be obscured by the /3-lactamase polypeptides. 26
inserted the DNA fragments We therefore containing the tat-RI1 162 DNA fusions at 2.83 kb and 2.2 kb into a different cloning vector, pACYC184 (Fig. 4). DNA was introduced at the EcoRI site of pACYC184, which lies in the gene for chloramphenicol acetyltransferase (Alton & Vapnek, 1979). In order to identify possible hybrid polypeptides arising as a consequence of intragenic insertion, molecules containing the cloned DNA in each orientation were obtained. The polypeptides encoded by these plasmids were t’hen examined, and the results are shown in Figure 7. The vector
760
K. Kim and R. J. Meyer Cm’ R
pACYCl84
0
TC’
B(2 83) TC’
Figure 4. Construction of recombinant plasmids (continued). Cm’ indicates the region of the plasmid encoding resistance to chloramphenicol. The notation used in the Figure and other abbreviations are described in the legends to Figs 2 and 3.
pACYC184 encodes a 25,000 M, polypeptide corresponding to chloramphenicol acetyltransferase (Fig. 7, lane b) (Shaw et al., 1979); this band is missing when the gene is interrupted by cloning. Derivatives containing the tat-2.83 fusion DNA intense 31,000 M, band, and an show an incompletely resolved band at 29,000 M, (Fig. 7, lanes e and f). The latter, 29,000 M, band, is clearly observed for the tat-2.2 fusion, in which case the gene encoding the 31,000 M, polypeptide is absent (Fig. 7, lanes c and d). We conclude that the promoter-distal essential gene in Rep1 encodes a 29,000 M, polypeptide. A protein band with a molecular weight of approximately 27,000 is observed for pUT247 (Fig. 7, lane c). We cannot account simply for this
second product. It is probably not a fusion polypeptide containing part of chloramphenicol acetyltransferase at the amino-terminal end. The tat sequence contains nonsense codons in all three reading frames (Amann et al., 1983). Since the of the chloramphenicol acetylinterruption transferase gene is in the codon for the seventythird amino acid (Alton & Vapnek, 1979), such a possible fusion polypeptide is calculated to have a molecular weight much less than 27,000. The size of the Rep1 region, estimated from the analysis of deletions (Meyer et al., 1985), is between 1.50 kb and 1.72 kb. This is in good agreement with the amount of non-overlapping DNA required to encode proteins with molecular weights of 29,000 and 31,000.
761
Copy Number of Plasmid RI162
R (0.0)
B(2.83)
2’
B(2.83) n B B-R
R linker
Figure 5. Construction of recombinant plasmids (continued). The notation and abbreviations used are given in the legends to Figs 1 to 4. The broken double line indicates the location of the BaZOl-generated deletion in R1162 DNA. The BumHI-EcoRI linker DNA is derived from M13mp7RFI (Messing et al., 1981).
(b) Increased transcription
in the RepI region elevates the copy number of R1162
A comparison of the amounts of 31,000 M, polypeptide synthesized in minicells containing pMS342 (Fig. 6(a), lane d) and pUT238 (Fig. 6(a), lanes b and c) shows that the tat fusion at 2.83 kb results in substantially greater amounts of this essential polypeptide. We do not have direct evidence for increased amounts of the 29,000 M, polypeptide, although it would seem likely that the amount of this product, made from a cotranscribed
gene, would increase also. Indirect evidence for this is presented later (section c, below). We asked whether increased transcription through Rep1 would affect the copy number of R1162. To test this, we used two plasmids, pMS228 and pMS245 (Lin & Meyer, 1984), in which additional DNA has been inserted into R1162. The first of these, pMS228, contains a DNA fragment both encoding resistance to trimethoprim, and also including a 370 base-pair segment derived from the region of the replicative origin of R1162. Thus, pMS228 is diploid for a fragment of DNA near the
762
K. Kim and R. J. Meyer a
b
c
d
e
f
a
(a)
b
(b)
Figure 6. Polyacrylamide gel electrophoresis of 35S-labeled polypept,ides synthesized in purified minicells. Plasmids in the minicell-producing strains were: (a) none (lane a); pUT238 (lanes b and c); pMS342 (lane d); pUT239 (lanes e and f); pMS348 (lane g). (b) None (lane a); pMC1403 (lane b). Polypeptide
(iM, 14,000), soybean trypsin inhibitor serum albumin
markers (not shown) were from Bio-Rad:
lysozyme
(M, 21,500), carbonic anhydrase (M, 31,000), ovalbumen (M, 45,000), bovine
(A& 66,200). and phosphorylase
B (M, 92,500). These were unlabeled and visualized
origin; the effect of this duplication is to lower the copy number of the plasmid (Lin & Meyer, 1984). The plasmid pMS245 differs from pMS228 only in that it lacks the cloned second copy of the 370 basepair sequence, and therefore retains the same copy number as the parental DNA. Strains were constructed containing either pMS228 or pMS245, and pUT238 (Fig. 3), which has the tat promoter adjacent to the Rep1 region at 2.83 kb, or PUT191 (Fig. 2), the parental tat vector lacking any cloned R1162 DNA. We then estimated the relative amounts of the different plasmid DNA species in these strains by extracting the plasmid DNA, cleaving it with a restriction endonuclease, and subjecting it to electrophoresis through 0.8% agarose. The results are shown in Figure 8. In the presence of the control plasmid pUTl91, the amount of coextracted pMS228 is substantially less than for pMS245 (Fig. 8, lanes a and e). In the presence of pUT238, cellular amounts of both pMS228 and pMS245 increase (Fig. 8, lanes c and g). From the examination of different amounts of DNA from several independent experiments, we estimate a four- to sixfold increase in pMS228 plasmid DNA, and a two- to threefold increase in pMS245. This result contrasts with the earlier observation that if Rep1 DNA (including its own promoter) is cloned into a plasmid with a copy number similar to that of Rl162 and then
by staining.
introduced into cells containing pMS228 or pMS245, no increase in the copy number of these plasmids is observed (Lin & Meyer, 1984). Evidently, the increased transcription originating from the tuc promoter is required for the higher copy number. Expression of the tat promoter is sensitive to the regulatory system of the lac operon (Amann et al., 1983). If the higher copy number of pMS228 and pMS245 is in fact due to transcription from the tat promoter, then the copy number should vary depending on the level of expression of this promoter. Initially. we looked at plasmid copy numbers for cultures grown in the presence of IPTG, a gratuitous inducer of the lac operon (Fig. 8, lanes b, d, f and h). Under these conditions, the tat promoter should be maximally active. However, no further increase in copy numbers of pMS228 or pMS245 was observed. It is possible that, because pUT238 and PUT191 are themselves high copy-number plasmids, the lac repressor is always substantially titrated, IPTG then having little effect. Therefore, we recloned the DNA containing the tat promoter-Rep1 fusion into pSC101, which has a much lower copy number than PUT191 (Cohen et al., 1973). We then tested the effect of this plasmid, pUT251 (Fig. 4), on the copy numbers of pMS228 and pMS245, both in the presence and absence of IPTG (Fig. 9). The results show that higher copy numbers are achieved by
Copy Number
0
27,000
-
b
c
d
e
of Plasmid a
f
763
RI 162 b
C
d
e
f
a
h
C C
cat -
gel electrophoresis of Figure 7. Polyacrylamide 35R-labeled polypeptides svnthesized in purified minicells. Plasmids in the minicell:producing strains were: none (lane a); pACYC184 (lane b); pUT247 (lane c); pUT248 (lane d); pUT249 (lane e); pUT250 (lane f). Polypeptide markers (not shown) are given in Fig. 6. The label cat refers to rhloramphenicol acetyltransferase encoded by pA(‘Y(‘184.
bot’h pMS228 and pMS245 when IPTG is added t,o the culture medium (Fig. 9, compare lanes c and h with d and i). A similar effect is not observed for these plasmids in the presence of the control plasmid, cloning vector pSClO1 (Fig. 9, compare lanes a and f with b and g). We conclude that transcription from t,he tar promoter into Rep1 is responsible for the higher copy numbers of the R1162 derivatives pMS228 and pMS245. (c) The elevated copy number of the R1162 derivative pMS228 depends upon increased transcription through both genes in Repl The fusion of Rep1 DNA and tat promoter DNA at R1162 co-ordinate 2.83 should result in increased transcription through both the promoter-proximal gene for the 31,000 M, polypeptide, and the distal gene for the 29,000 LV~ polypeptide. Which of these genes is responsible for the increased copy number of pME228? We examined the copy number of pMS228 in the presence of plasmids in which the tat promoter was linked to the entire Rep1 region (31+29’), or to only one of the two genes (31’29or 31-29+). The results (Fig. 10) show that neither the 31+29plasmid pUT282 (Fig. 5), nor the 31-29+ plasmid pUT247 (Fig. 4), increases the copy
Figure 8. Agarose gel electrophoresis of EcoRI-cleaved plasmid DNA isolated from E. coli GM1 strains. Cells were grown in the presence (lanes b. d, f and h) or absence (lanes a, c, e and g) of IPTG. Marker fragments (lane i) are HindIII-cleaved lambda bacteriophage DNA (23.7. 9.5; 6.7, 4.3, 2.3. 2.0 kb; Allet & Bukhari. 1975). Gel lane
Plasmid
Properties
DNA fragments
a. b
pMS228 PUT191
IncQ(dup1) 31-29-
1. 4 2
c. d
pMS228 pUT238
IncQ(dup1) 31+29+
I, 4 2, 3
e. f
pMS245 PUT191
IncQ 31-29
I, 5 2
g. h
pMS245 pUT238
IncQ 31+29+
1, 5 2, 3
Plasmid DNAs were isolated from cells grown in the presence (lanes b, d, f and h) or absence (lanes a. c, e and g) of IPTG.
number of pMS228 beyond that observed in the presence of control plasmid, the cloning vector pACYC184 (Fig. 10, compare lanes a, b and c). In contrast, the 31+29+ plasmid pUT249 (Fig. 4) clearly results in a higher copy number for pMS228 (Fig. 10, lane d). The higher copy number does not require cvtranscription of both genes from the same DNA. When the 31-29+ plasmid PUT239 (Fig. 3) is introduced into a cell containing pMS228 and pUT282 (31+29-), or when the 31+29plasmid pUT278 (Fig. 5) is introduced into a cell containing pMS228 and pUT247 (31-29+), the copy number of pMS228 is increased (Fig. 10, lanes f and h). This is not due to an effect of the cloning vector PUT191
764
K. Kim a
b
c
and R. J. Meyer d
e
f
h
2 3
4
Figure 9. Agarose gel electrophoresis of BarnHI-cleaved plasmid DNA isolated from E. coli GM1 strains. Cells were grown in the presence (lanes b, d, g and i) or absence (lanes a, c, f and h) of IPTG. Markers (lane e) are HindIII-cleaved
lambda DNA (Fig. 8). Gel lane
Plasmid
Properties
a. b
pMS228 psc101
Inc&(dupl) 31-29-
1 3
c, d
pMS228 pUT25 1
IncQ(dup1) 31+29+
1 3, 4
f, g
pMS245 psc101
IncQ 31-29-
2 3
h. i
pMS245 pUT25 1
IncQ 31+29+
2 3, 4
DNA fragments
Plasmid DNAs were isolated from cells grown in the presence (lanes b, d, g and i) or absence (lanes a. c, f and h) of IPTG.
used in the construction of pUT239 or pUT278, since this molecule is present in the strains used for lanes a to d. Moreover, the copy number of pMS228 is not elevated when pUT239 or pUT278 are present along with only the parental cloning vector for pUT247 and pUT282, pACYC184 (Fig. 10, lanes g and i). We conclude that increased transcription through both the Rep1 genes is required for an increase in the copy number of pMS228. The simplest interpretation is that greater amounts of both gene products are necessary for the increase.
determinant for incompatibility (Lin & Meyer, 1984). When this fragment is cloned into pBR322 and introduced into a cell containing R1162, the resident plasmid is rapidly lost from the culture during subsequent growth. We tested whether the tat promoter-Rep1 fusion that increases the copy number of R1162 also confers protection against the cloned incompatibility determinant. To do this, we transformed strains containing R1162 and a tat promoter plasmid with pMS319, a pBR322
derivative
fragment. (d) Increased confers
transcription through the Repl region protection against expression of a cloned determinant for incompatibility
Incompatibility is the failure of two plasmids to be coinherited stably. The phenomenon is thought to reflect, in many cases at least, a common mechanism of copy-number control (Pritchard, 1978). The 370 base-pair fragment of R1162 DNA that affects copy number also contains a
incoming
containing
Transformed plasmid,
the
370 base-pair
cells were selected for the
and colonies
tested
for retention
of R1162 by screening for resistance to streptomycin. The results are shown in Table 2. The plasmid pUT249 (Fig. 4), which contains the tat promoter fused to the complete Rep1 region, protects R1162 from pMS3 19-mediated incompatibility. The complete Rep1 region with the normal promoter (pUT316) is ineffective, as are fusions
in which
the gene for either
31,000 M, polypeptides
the 29,000
is absent. Thus, sensitivity
or
765
Copy Number of Plasmid R1162 b
a
a
d
e
f
a
h
I
Figure 10. Agarose gel electrophoresis of EcoRI-cleaved plasmid DNA isolated from E. co& GM1 strains. A11 cultures were grown in the presence of IPTG. Markers (lane e) are HindIII-cleaved lambda DNA (Fig. 8).
Plasmid
Properties
pMS228 PUT191 pACYC184
IncQ(dup1) 31-2931 -29-
1, 6
b
pMS228 PUT191 pUT282
IncQ(dup1) 31-2931 +29-
1, 6 2 2, 4
c
pMS228 PUT191 pUT247
IncQ(dup1) 31-2931 -29+
1, 6 2 2, 5
d
pMS228 PUT191 pUT249
IncQ(dup1) 31-2931 +29+
1, 6 2 2, 3
f
pMS228 pUT239 pUT282
IncQ(dup1) 31-29+ 31+29-
1, 6 2, 5 2. 4
g
pMS228 pUT239 pACYC184
IncQ(dup1) 31-29+ 31-29-
1, 6 2, 5 2
h
pMS228 pUT278 pUT247
IncQ(dup1) 31+2931 -29+
1, 6 2, 4 2, 5
i
pMS228 pUT278 pACYC184
IncQ(dupl) 31+2931-29-
1, 6 2, 4 2
Gel lane a
to the cloned incompatibility determinant parallels the copy-number effect.
exactly
4. Discussion Enhanced transcription through the Rep1 region elevates the copy number of R1162. We assume
DNA fragments
2 2
that this is due to an increase in the amounts of the Rep1 gene products. If this is so, the products of both the Rep1 genes must be increased in order to raise the amount of plasmid DNA (Fig. 10). Analogous polypeptides encoded by plasmid RSFlOlO are required for plasmid DNA replication in vitro (Scherzinger et al., 1984). We envision that
766
K. Kim and R. J. Meyer
Transformation
Table 2 with pMS319 DNA
Number of streptomycin-resistant Resident plasmids R1162, RI 162, R1162, Rl162, Rl162,
pACYCl84 pUT249 pIJT247 pCT282 pUT316
A 1 20 8 0 4
13 0 20 6 2 5
No two plates contained sibling tested from each of 5 plates.
colonies
C
D
E
0 20 2 3 2
0 20 3 0 2
0 20 7 0 4
transformants:
20 colonies
the Rep1 polypeptides, in the form of their native proteins, participate in the initiation of DNA synthesis at the replicative origin (oriV) of R1162. These proteins could act co-operatively at a single step in the initiation of replication, or could independently affect different steps in this process. In addition, we have shown that a 370 base-pair fragment of DNA from the oriV region both specifies plasmid incompatibility and affects plasmid copy number (Lin & Meyer, 1984; Meyer et a,Z., 1985). If this DNA fragment is duplicated at a novel position in RIl62, the copy number of the resulting plasmid is substantially lowered. Increasing the copy number of the Rep1 genes does not noticeably reverse this effect (Lin & Meyer, 1984). An examination of the base sequence of the 370 base-pair fragment, reveals no large open reading frames, but does show the presence of a cluster of directly repeated sequences similar in size to t’hose observed for several other plasmids (Murotsu et al., 1981; Stalker et aE., 1979, 1981; Churchward et al., 1983; Kamio & Terawaki, 1983; Abeles et al., 1984). One possibility that might account for these observations is that the 370 base-pair fragment encodes an inhibitor that regulates expression of the Rep1 genes. In view of the base sequence of t,his fragment, such an inhibitor would probably not be a polypept)ide, but could be a small RNA. The role of small RNAs in the regulation of plasmid DNA replication is well-established. In the case of ColEl, such a molecule affects production of an RNA primer for DNA replication (Tomizawa et al., 1981); for the plasmid Rl, translation of the transcript’ of the essent’ial repA gene is modulated (Light, & Molin. 1983). Several observations argue against the existence of a small RNA inhibitor. The increased amount of this hypothetical inhibit,or that would be specified by plasmids containing a, duplicated 370 base-pair fragment is apparently not titrated by a commensurate increase in Rep1 gene dosage (Lin & Meyer, 1984). In addition, both RepI genes have been fused at numerous positions to ZacZ DXA lacking its normal transcriptional and translational start) signals (Meyer et aZ., 1985; Kim & Meyer. unpublished results). In no case is expression of the
la& gene, measured in terms of units of J?-galactosidase activity, affected by the presence in the cell of multiple copies of the 370 base-pair fragment. Finally, the amount of the 31,000 M, polypeptide synthesized in minicells containing pMS342 is not decreased when the minicellproducing strain also contains multiple copies of a plasmid with the 370 base-pair DNA (data not shown). We favor the idea that the Rep1 genes are under some form of autogenous regulation. If the concentrations of the Rep1 products are thus kept’ constant during growth, one way that their activity could be regulated is by binding to a region of the 370 base-pair DNA. Because the number of these regions would increase with plasmid copy-number, a large number of plasmid copies would result in a small amount of unbound Rep1 protein for replication, and so a regulatory circuit would be established. This model has been proposed for the F factor by Tsutsui et al. (1983). Evidence in support of this titration model has come from studies on replication of the F factor (Tsutsui et al., 1983) and the phage Pl (Chattoraj et al., 1984). The presumptive titrating sites in both cases are 19 base-pair, directly repeated sequences. For R1162, titration of either of the two Rep1 proteins would be sufficient to affect control. If this model is correct, it could provide an explanation for t,he directly repeated, 20 base-pair sequences within the 370 base-pair fragment. The direct repeats of several other plasmids have been shown to be binding sites for essential, plasmid-encoded proteins (Germino &, Bastia, 1983; Vocke & Bastia. 1983; C’hattoraj et al., 1984). Increased transcription through Rep1 protects. in trans, R1162 from expression of the strong incompatibility determinant encoded by a cloned 370 base-pair fragment. The simplest interpretation is that both incompatibility and copy-number control are different manifestations of the same functional activity. For R1162, we think that incompatibility occurs because a Rep1 gene product is bound to a target site on the 370 base-pair fragment. Thus, free Rep1 protein is unavailable for replication of R1162, and the plasmid is lost from the cell. Increased transcription through Rep1 spares RI 162, because the autoregulation of the Rep1 region is overcome, and more protein is available for plasmid replication. In summary, two regions of R1162, one encoding essential plasmid proteins and the other linked to 0riV and also exerting incompatibility, can functionally interact to affect copy number. The exact regulatory relationship between these regions in the control of plasmid copy number is not understood, however, and further experiments will be required for a detailed model.
This work was supported, in part, by National Science Foundation grant PCM-8219916 and, in part, by grant F-964 from the Robert A. Welch Foundation.
Copy Number
of Plasmid
References Abeles, A. L.. Snyder, K. M. & Chattoraj, D. K. (1984). J. Mol. BioZ. 173, 307-324. Adler, H. I.. Fisher, W. D., Cohen, A. & Hardigree, A. A. (1967). Proc. Nat. Acad. Sci., U.S.A. 57, 321-326. Allet, B. & Bukhari. A. I. (1975). J. Mol. Biol. 92, 529540. Alton, pu’.K. & Vapnek, D. (1979). Nature (London), 282, 864-869. Amann? E.. Brosius, J. & Pt’ashne, M. (1983). Gene, 25, 167-178. Ambler, R. P. & Scott, G. K. (1978). Proc. Nat. Acad. 8ci., 1r.S.A. 75, 3732-3736. Bart)h. P. T. & Grinter. 1\;.J. (1974). J. BacterioZ. 120. 618-630.
Bolivar. F.. Rodriguez, R. L., Greene, P. J., Betlach. $1. C., Heyneker, H. L., Boyer, H. W., Crosa, J. H. & Falkow, S. (1977). Gene, 2, 95-113. Bryan, L. E.. van den Elzen, A. M. & Tseng, J. T. (1972). Antimicrob. Ag. Chemother. I, 22-29. Casadaban. M. J. & Cohen, S. N. (1980). J. Mol. BioZ. 138, 179-207.
Casadaban, M. J.. Chou? J. & Cohen, S. K. (1980). J. Bacterial. 143, 971-980. (‘hang, A. (‘. Y. & Co hen, S. plj. (1978). J. Bacterial. 134, 1141-I 156. (‘hat,toraj. I).. Cordes, K. & Abeles, A. (1984). Proc. Nut. dcad. tici., U.S.A. 81, 6456-6460. Churchward, G.. Linder, P. & Caro, L. (1983). NucZ. Acids Res. 11. 5645-5659. Cohen, S. N.> Chang, A. C. Y. & Hsu, L. (1972). Proc. Nut. Acad. tici., U.S.A. 69, 2110-2114. Cohen, S. K‘.. Chang, A. C. Y., Boyer, H. W. & Helling, K. B. (1973). Proc. AVat. Acad. Sci., U.S.A. 70, 32403244. Fra.zrr. 4. C. & Curtiss, R’. III (1973). J. Bacterial. 115. 615-622.
Germino, J. & Bastia, D. (1983). Cell, 32, 131-140. Grinter, pu’. J. & Barth. P. T. (1976). J. BacterioZ.
128,
394-400.
Guerry, P.. van Embden, J. & Falkow, S. (1974). ,/. BacterioZ. 117, 619-630. Hershtield. V.. Bayer. H. W.. Yanofsky, C., Lovett, M. A. & Helinski, D. R. (1974). Proc. Nat. Acad. Sci., i:.S.A.
71, 3455-3459.
Holmes, D. 8. & Quigley, M. (1981). Anal. B&hem. 193~-197.
114.
R1162
Kamio, Y. & Terawaki, Y. (1983). J. Bacterial. 155, 11851191. Light’, J. & Molin, S. (1983). EMBO J. 2. 93-98. Lin, L.-S. & Meyer, R. J. (1984). Mol. Gen. Genet. 194, 423-431. Lugtenberg, B., Meijers, J., Peters, R:, van der Hoek, P. & van Alphen, L. (1975). FEBS Letters, 58, 254-258. Messing? J. & Vieira, J. (1982). Gene, 19, 269-276. Messing, J., Crea. R. & Seeburg, P. H. (1981). ‘VucZ.Acids Res. 9. 309-321.
Meyer, R’. J., Figurski, D. & Helinski, D. R. (1975). Science, 190, 1226-1228. Meyer, R. J.! Hinds, M. & Brasch. M. A. (1982). J. Bacterial. 150, 552-562. Meyer. R. cJ., Lin. L.-S., Kim, K. & Brasch, M. A. (1985). In Plasmids in Bacteria (Helinski. I). R.. Cohen. S. N.. Clewell. D. B.. Jackson, D. A. 8r Hollaender. A.. eds). pp. 173-188, Plenum Press. Pu’ewYork. Miller. J. H. ( 1972). Experiments in MoZecuZar Genetics, p. 431, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Miller, J. H.. Ganem. D., Lu, P. & Sehmitz. A. (1977). J. Mol. BioZ. 109, 275-301. Murotsu. T.. Matsubara, K., Sugisaki, H. & Takanami, M. (1981). Gene, 15, 257-271. Prit’chard, R. H. (1978). In DNA Synthesis Present and Future (Molineux, I. & Kohiyama. M.. eds). pp. l-26, Plenum Press, ru’ew York. Rothstein. S. J. & Reznikoff. W. 6. (1981). Cell, 23, 191199. Scherzinger, E., Bagdasarian, M. M., Scholz, P., Lurz, R., Ruckert, B. & Bagdasarian, M. (1984). Proc. Nat. Acad. Sci., Cr.&‘. A. 81, 654-658. Shaw, W. V.? Packman, L. C., Burleigh, B. D., Dell, A., Morris. H. R. & Hartley, B. S. (1979). Nature (London),
282, 870-872.
Stalker, D. M., Kolter, R. & Helinski, D. R. (1979). Proc. ,Vat. Acad. Sk., I!S.A. 76, 1150-1154. Stalker. 11. M., Thomas, C. M. & Helinski. D. R’. ( 1981). Mol. Gen. Genet. 181, 8-12. Sutcliffe, J. G. (1978). Proc. Nat. ilcad. Sci., I:.S.A. 75, 3737-3741. Tomizawa, J.-T., Itoh. T., Selzer, G. & Som, T. (1981). Proc. Nat. Acad. Sci., U.S.A. 78, 1421-1425. Tsutsui, H., Fujiyama, A., Murotsu, T. & Matsubara, K. (1983). J. RacterioZ. 155, 337-344. Vocke. C. & Bastia. D. (1983). Cell, 35, 495-502.
Edited by M Gottesman
(1985) 185, 755-767
Copy Number of the Broad Host-range Plasmid R1162 is Determined by the Amounts of Essential Plasmid-encoded Proteins Kyunghoon Kim and Richard J. Meyer University of Texas Department of Microbiology Austin, Tex. 78712-1095, U.S.A. (Received 5 November 1984, and in revised form
5 April
1985)
DNA of the broad host-range plasmid Rl162 contains a 1700 base-pair segment essential for plasmid maintenance. This region, RepI, consists of two cotranscribed genes encoding polypeptides with molecular weights of 29,000 and 31,000. Fusion of Rep1 to the strong tat promoter results in greatly increased amounts of at least one of these polypeptides. In tram, this construction has two other properties: it can raise the copy number of Rl162, and it can protect this plasmid from loss due to incompatibility. Both effects require intact Rep1 genes. These properties of the Rep1 region, along with those of an origin-linked region described earlier, are discussed with respect to current models for control of plasmid copy number.
1. Introduction
from right to left as drawn in Figure 1. The promoter-proximal gene, which occupies a position within 1.7 to 2.63 kb, encodes an essential polypeptide (M, 31,000) (Meyer et al., 1985). We have identified a second essential polypeptide (M, 29,000) encoded by the remaining DNA of RepI. In addition to Rep1 and RepII, the oriV-Inc region (Fig. 1) is required for plasmid replication. This region contains the origin of DNA replication, and the determinant for plasmid incompatibility (Lin & Meyer, 1984). A 370 base-pair fragment of DNA specifying this incompatibility is involved in control of plasmid copy number (Lin & Meyer, 1984). In this paper, we show that the amounts of Rep1 proteins determine the amount of RI 162 plasmid DNA in the cell: when the Rep1 genes are fused to a strong promoter, more Rep1 polypeptides are made, and the plasmid DNA content per cell rises to greater than normal values. In addition, the plasmid becomes more resistant to elimination from the cell through incompatibility. We discuss these results in terms of models for copy-number control.
Rll62, is a high copy-number plasmid, originally isolated from Pseudomonas aeruginosa, but with a broad host-range among Gram-negative bacteria (Barth & Grin&, 1974; Bryan et al., 1972). It is very similar or identical to several independently isolated plasmids (Barth & Grinter, 1974; Grinter & Barth, 1976); the most thoroughly studied of these is RSFlOlO from Escherichia coli (Guerry et al., 1974). The plasmids in this group are small, consisting of about 9000 base-pairs of DNA. They to resistance streptomycin and encode sulfonamides, and are not self-transmissible, although they can be mobilized readily during conjugative transfer of certain other plasmids (Barth & Grinter, 1974). A genetic map of Rl162 is shown in Figure 1. Two regions, designated Rep1 and RepII, encode products required for plasmid maintenance (Meyer et al., 1985). Proteins encoded by the corresponding regions in RSFlOlO have been isolated by Scherzinger et al. (1984), who showed that they are necessary for plasmid DNA replication in vitro. The genetic organization of the RepI region has been determined partially (Meyer et al., 1985). The essential DNA lies within the co-ordinates 0.91 to 2.63 kbt (Fig. 1). The entire region is transcribed
2. Materials and Methods (a) Bacterial
strains
and plasmids
The strains of E. coZi K12 used in this study are: MVlO
(thr Zeu thi ZacY supE44 tonA AtrpE5) (Hershfield et al., 1974); GM1 (aruA(Zac pro) thi/F”(Zac pro) lucIq L8) (Miller et al., 1977); and P678-54 (thr leu thi lacY minA m&B gal
t Abbreviations used: kb, IO3 base-pairs; kd, M, x 10-3; IPTG, isopropyl-B-n-thiogalactoside. 0022-2836/85/200755-13
$03.00/O
755
0 1985 Academic Press Inc. (London) Ltd.
756
K. Kim and R. J. Meyer
RP RI162 0.00
I
P
Et
BC
Bc
Bc
I I
I I
I I
I I
I
30
7.4
83
08
I I
,
,-’
p
R
87kb
mRNA
Polypeptldes
31,000
Figure 1. Genetic map of R1162. Shown are the approximate locations of genes for resistance to sulfonamides (Su’) and streptomycin (Smr). Rep1 and Rep11 are regions of the plasmid encoding products necessary for plasmid maintenance; o&V-Inc indicates the position of the origin of plasmid DNA replication and the associated determinant for incompatibility. The locations of a few restriction enzyme cleavage sites (in kb) from the single EcoRI site, are shown also. Abbreviations: Bc, B&I; Bt, B&EII; P, P&I; and R, EcoRI. Below the map of R1162, the Rep1 region has been expanded to show the locations of the genes encoding 29,000 M, and 31,000 JZ, polypeptides and the direction of transcription through these genes.
rpsL) (Adler et al., 1967). In the last case, a thymineand their requiring variant was used. Plasmids derivations are listed in Table 1. (b) General procedures Bacteria were grown at 37°C in TYE broth (1% (w/v) Bacto-tryptone, 05% (w/v) Bacto yeast extract, 0.5% (w/v) NaCl) or in M63 minimal medium (Miller, 1972) supplemented with 0.4% (w/v) glucose, 1 mM-MgSO,.and 0901~0 (w/v) thiamine* HCl. When necessary, the following antibiotics were added to the indicated final concentrations: carbenicillin (200 pg/ml), streptomycin sulfate (25 pg/ml), trimethoprim (l&l pg/ml) and tetracycline hydrochloride (10 pg/ml).
Plasmid DNA was generally prepared by the rapidboiling method of Holmes & Quigley (1981). Transformation of cells with plasmid DNA was done by the procedure of Cohen et al. (1972). Restriction enzymes, BuZ31 exonuclease and phage T4 polynucleotide ligase were obtained from a commercial source (New England Biolabs), and used according to the instructions provided by the supplier. For limited digestion of linearized plasmid DNA with BaZ31, approximately 10 pg of DNA in 56 ~1 of buffer was incubated at 30°C with 2 units of enzyme, and samples taken every 10 min. Procedures for running 0.8% (w/v) agaroae gels and visualizing the DNA bands are standard and have been described (Meyer et al., 1975, 1982).
Table 1 Plasmids used in this study Plasmids Cloning
Relevant
properties
Source or derivation
vectors:
pACYC184 pRR322 pMC1403 psc101 ptacl2
Rep(p15) Cm’ Tc’ Rep(ColE 1) Cb’ Tc’ Rep(ColE1) Cb’ lucz’ lacy TC’ Rep(ColE1) tac( ) Cb’
R1162 and derivatives: IncQ Su’Sm’ R1162 pMS178 Rep(p15) RepI+ Km’ &Z(a)
Chang & Cohen (1978) Rolivar el al. (1977) Casadaban et al. (1980) Cohen et al. (1973) Amann et al. (1983) Rarth & Grinter (1974) (Fig. 1) Insertion of DNA fragment containing 0+3.0 kb R1162 DNA into BarnHI site of pMC489 (Casadaban & Cohen, 1980) (Fig. 3)
Copy Number
of Plasmid
R1162
Table 1 (continued) Plasmids
Relevant
properties
Source or derivation
pMR228 pMR245 pMS319
Rep(R1162) IncQ (dupl) Tp’ Rep(R1162) IncQ Tp’ Rep(Co1El) IncQ Cb’
pi%342
Rep(ColE1) RepI+ Cb’ lac%’ lacy
pMS34X
Kep(ColE 1) Cb’ lacZ’ ZaeY
pVT182
Rep(ColE1) tac(
pf.TT 184 and pVT18.5
Rep(ColEl
Rep(ColE1) Cb’ 1acZ’ 1ocY
pIyT191
Rep(ColEl
plTT236 and pl’T23X
Rep(ColE1) RepI+ tac(O.O-2.83) Cb’
prT”37 and pI’T”39 pVTP47 and plT248
) Cb’
RepI+ Cb’ 1acZ’ lacy
tac(
Rep(ColE1)
) Cb’
RepI+ tac(2.83-0.0) Cb’
Rep(ColE1) tac(OG2.2)
Cb’
Rep(ColE1) tae(2.2-0.0) Cb’ Kep(p15) tac(2.2-0.0) Tc’
Rep(pl5)
RepI+ tac(2.83~0.0) Tc’
pl’T25l
Rep(pSC101) RepI+ tac(2.83-0.0)
plTT2.52
Kep(pSC1O1) tac(2.240)
pl.T278
Rep(ColE1) tae(2.83-0.0. A1.1) (‘b’
pI’T282
Rep(p15) tae(2.83-0.0. A1.1) Tc’
pITT3 16
Rep(p15) RepI+ Tc’
Tc’
Tc’
Lin & Meyer (1984) Lin & Meyer (1984) Derivative of pBR322 containing 370 basepair DNA fragment derived from R1162 and specifying incompatibility (unpublished result) Insertion of DNA fragment containing 0.0-2.83 kb R1162 DNA, generat,ed by sequential digestion of pMS178 DNA with SalI, Ba/31 (partially) and EcoRI. into pMC1403, by replacement of EcoRI-SmaI DNA linker (Fig. 3) As for pMS342, except that inserted DSA contains only 0.0-2.2 kb R1162 DS.1 (Fig. 3) ” Insertion of 11 base-pair Seal-Hincll linker DXB. containine a BansHI cleavage site, from -M13mp8(RFI) (Messing & Vieira. 1982) into PwcII site of ptacl2 (Fig. 2) Insertion of 42 base-pair EcoRI-generated DNA fragment. containing mult,iplr restriction enzyme cleavage sites. from M13mp7(RFI) (Messing et al., 1981) into EcoRI sites of pMS342 and pMS348. respectively (Fig. 3) Replacement of small EcoRI + Bar/~H1generated DNA fragment of pBR322 with DNA fragment from pUT182 and cow taining tat promoter (Fig. 2) Insertion of DNA fragment containing 0.0-2.83 kb Rl162 DNA, generated by BamHI digestion of pUT184 DNA. into BarnHI site of PUT191 in each of 2 orientations (Fig. 3) As for pLlT236. pCT238: Barr&HI-generated DNA fragment containing 0.0-2.2 kb from pVT185 R1162 DNA derived (Fig. 3) Insertion of DNA fragment containing Inc (2.2-0.0) R1162 DNA fusion. generated by EcoRI digestion of pUT239 DNA. into EcoRI site of pACYC184 (Fig. 4) As for pUT247. pUT248. except EcoRlgenerated DNA fragment, containing tat (2.X3-0.0) R1162 DNA fusion. derived from pITT238 (Fig. 4) As for pVT249. plT250. except DNA fragment inserted into EroRI site of pSClO1 (1 orientation only) (Fig. 4) As for pLlT247. pUT248. except DXA fragment inserted into EcoRI site of pSClO1 (1 orientation only) (Fig. 4) Deletion in R1162 DNA across 1.1 kb b> limited digestion of BstEIl-cleaved pTT238 DXA with Ra/31 rxonucleasr (Fig. 5) Insertion of DNA fragment containing R1162 DSA, generated by EcoRI digestion of pUT278, into EcoRI site of pAC\-(‘184 (Fig. 5) Insertion of DNA fragment containing 0.0-2.83 kb R1162 DNA. generat,ed by EcoRI +BamHI digestion of pMS342. into EcoRI site of pACYC184 (Fig. 5)
Abbreviations: Rep ( ), replication system derived from naturally occurring plasmid in parentheses; tac( ), contains tax promoter sequence adjacent to R1162 DNA with co-ordinates and orientation indicated in parentheses; IncQ, member of Q incompatibility group; (dupl), IncQ locus duplicated; RepI+, contains entire Rep1 region of R1162: Cb’, Cm’. Sm’, Su’. Tc’, Tp’. encodes resistance to carbenicillin, chloramphenicol, streptomycin, sulfonamides. tetracycline, trimethoprim. respectively. Other abbreviations are standard or defined in the text.
758
K. Kim (c) Estimation
and R. J. Meyer
of plasmid content in cells
Plasmid-containing derivatives of GM1 were grown overnight in TYE brot,h. then diluted 1 : 50 (v/v) into M63 medium. Growth in M63 medium requires the presence of the F prime facbor in the cell; other plasmid species were maintained by supplementation of the medium with antibiotics. The cultures were incubated at 37°C to mid-log phase (about 3 x lo* to 5 x lO’/ml), then divided and one portion induced by the addition of isopropyl-j-n-thiogalactoside to a final concentration of 1 mM. Incubation of the cells was continued to stationary phase (about 2 x 109/ml). Plasmid DNA was then isolated, digested with a restriction endonuclease to facilitate subsequent identification, and displayed by electrophoresis through a O+Jo/0agarose gel. The relative amounts of each plasmid were estimated from the intensities of the plasmid DNA bands appearing in each lane. Each sample well contained DNA extracted from approximately 2 x lo9 cells. (d) IdentiJication
of plasmid-encoded polypeptides
Minicells from plasmid-containing strains of P678-54. grown to stationary phase in 250 ml of TYE b&h containing appropriate antibiotics for selection of plasmids, were isolated by 2 successive centrifugations through sucrose gradients, according to the method of Frazer & Curtiss (1973). Preparations contained less than I whole cell per 10’ minicells. Proteins being synthesized in the minicells were radioactively labeled by incubation of approximat’ely 109 minicells in I ml of labeling medium (Rothstein & Reznikoff. 1981) containing 30 to 50 PCi of r,-[35S]methioninc (> 1000 mCi/mmol). After I I1 incubation at 37°C. the minicells were pelleted by centrifugation, resuspended in TYE broth, and incubated for 10 min at 37°C. Finally, the labeled minicells were pelleted, resuspended in 20 ~1 of 9.59/, (v/v) glycerol containing 0.0625 M-Tris HCl (pH 6+3), 5% (v/v) P-mercaptoethanol, 2.3% (w/v) sodium dodecyl sulfate and 0.002% (w/v) bromphenol blue. Samples were boiled for 2 to 3 min, then stored at -20°C before electrophoresis. Samples were applied to a polyacrylamide gel (11% (w/v) acrylamide. 0.276 (w/v) bis-acrylamide, 0.1% sodium dodecyl sulfate) prepared according to the method of Lugtenberg et al. (1975). Electrophoresis was carried out at room temperature and at constant current (30 mA). Gels were stained with Coomassie blue. destained, and then dried. Proteins were visualized by autoradiography using Kodak X-Omat XRP-1 film with an intensifying screen, and with an exposure time of 3 days at room temperature.
by the parental vector (Fig. 6(b), lane b). The 29,000 J!& and 27,000 M, polypeptides probably represent unprocessed and processed forms of fl-lactamase (Ambler Br Scott, 1978; Sutcliffe. 1978). We have identified the 31,000 LWrpolypeptide as t,he product of an essential gene in Repl, lying within 1.7 to 2.63 kb (Meyer et al.. 1985). Cloned Rl 162 DNA containing the Rep1 genes was fused at position 2.83 kb to DNA encoding the strong tat promoter (Amann et al.. 1983). The resulting plasmid, pUT23X (Fig. 3), specifies substantially greater relat)ivr amount,s of thv 31,000 ~21~protein (Fig. 6(a), lanes h and v). This confirms the direction of transcription of t hr gcncl for this protein (Fig. 1). A4dditional essential DNA is cotransvrihetl but promotjer-distal to this gene (Meyer et (~1.. 1985). The polypeptides encoded in minicells by Rl 162 11?1;A4(04 to 2.2 kl,) cloned int’o pMC’1403 (pMS318, Fig. 3) were examined on polyacv-glamidr gels (Fig. 6(a). lane g), As expected. the 31 .OOO N, species was absent. since onl)- a portion of the gent’ had been cloned. We observed no other Keplencoded proteins. which was cxpccted also, since, the Rep1 promoter (Fig. 1 ) was missing from the SmBHc
cbr$y~
6
PV
Sm+Hc / R Cb’
Cb’
R+Bh
R+B Cb’ f+y (
3. Results (a) B,UOO and 37,000 Mr polypeptides by the Rep1 region
are encoded
The plasmid pMS342 (Fig. 3) consists of 0.0 to 2.83 kb R1162 DNA cloned into the vector pMC1403. The polypeptides encoded by this plasmid are shown in Figure 6(a). lane d. Two polypeptides with molecular weights of approximately 29,000 and 27,000 are made, as well as several smaller and one larger (Mr 31,000) species. All except the 31 .OOO Jfr polypeptidr are encoded
B PUT191
Figure 2. Construction of recombinant plasmids used in this study. The different molecules are not drawn to scale. The locations of genes encoding resistance to carbenicillin (Cb’) and tet,racycline (Tr’) are indicated. as well as the positions of relevant restriction enzyme caleavage sites. A section elevated with broken lines from the plasmid map represents a small linker DNA containing multiple restriction enzyme cleavage sites. Portions of the circular maps drawn with heavier lines indicate tax promoter DNA, with the direction of transcription shown by the arrow. The abbreviations for restriction enzyme cleavage sites are: B, BarnHI; He, HincII; Pv, PvuII; R, EcoRI; S, SalT; and Sm, SmaI.
759
Copy Number of Plasmid R1162 R
Sm
B
R (0
-,--.-.,.
\I
R(G0)
0) NO 0)
CbrA
Y
pMS348
pMS342
1) RBSPSBR
ABSPSBR
RBSPSBR
R Cd B :2 2)
Figure 3. Construction of recombinant plasmids (continued). The double-lined portion of the plasmid sketches indicate the presence of R1162 DNA between the kb co-ordinates (Fig. 1) given in parentheses. Y and Z’ indicat,e the locations of the ZacY and la& genes; Km’, the gene encoding resistance to kanamycin. Other abbreviations are defined in Figs I and 2.
cloned DNA. In an attempt to visualize the other essential gene products, we fused the R1162 DNA at 2.2 kb to the tat promoter (pUT239, Fig. 3). Complementation in L&JO by pUT239 of a replication-defective deletion derivative of R1162 demonstrated that under these circumstances the additional Rep1 DNA is expressed, whereas pMS348 is unable to complement (Meyer et al., 1985). However, no new polypeptides are observed for pUT239 (Fig. 6(a), lanes e and f). We thought that an additional Rep1 protein might be obscured by the /3-lactamase polypeptides. 26
inserted the DNA fragments We therefore containing the tat-RI1 162 DNA fusions at 2.83 kb and 2.2 kb into a different cloning vector, pACYC184 (Fig. 4). DNA was introduced at the EcoRI site of pACYC184, which lies in the gene for chloramphenicol acetyltransferase (Alton & Vapnek, 1979). In order to identify possible hybrid polypeptides arising as a consequence of intragenic insertion, molecules containing the cloned DNA in each orientation were obtained. The polypeptides encoded by these plasmids were t’hen examined, and the results are shown in Figure 7. The vector
760
K. Kim and R. J. Meyer Cm’ R
pACYCl84
0
TC’
B(2 83) TC’
Figure 4. Construction of recombinant plasmids (continued). Cm’ indicates the region of the plasmid encoding resistance to chloramphenicol. The notation used in the Figure and other abbreviations are described in the legends to Figs 2 and 3.
pACYC184 encodes a 25,000 M, polypeptide corresponding to chloramphenicol acetyltransferase (Fig. 7, lane b) (Shaw et al., 1979); this band is missing when the gene is interrupted by cloning. Derivatives containing the tat-2.83 fusion DNA intense 31,000 M, band, and an show an incompletely resolved band at 29,000 M, (Fig. 7, lanes e and f). The latter, 29,000 M, band, is clearly observed for the tat-2.2 fusion, in which case the gene encoding the 31,000 M, polypeptide is absent (Fig. 7, lanes c and d). We conclude that the promoter-distal essential gene in Rep1 encodes a 29,000 M, polypeptide. A protein band with a molecular weight of approximately 27,000 is observed for pUT247 (Fig. 7, lane c). We cannot account simply for this
second product. It is probably not a fusion polypeptide containing part of chloramphenicol acetyltransferase at the amino-terminal end. The tat sequence contains nonsense codons in all three reading frames (Amann et al., 1983). Since the of the chloramphenicol acetylinterruption transferase gene is in the codon for the seventythird amino acid (Alton & Vapnek, 1979), such a possible fusion polypeptide is calculated to have a molecular weight much less than 27,000. The size of the Rep1 region, estimated from the analysis of deletions (Meyer et al., 1985), is between 1.50 kb and 1.72 kb. This is in good agreement with the amount of non-overlapping DNA required to encode proteins with molecular weights of 29,000 and 31,000.
761
Copy Number of Plasmid RI162
R (0.0)
B(2.83)
2’
B(2.83) n B B-R
R linker
Figure 5. Construction of recombinant plasmids (continued). The notation and abbreviations used are given in the legends to Figs 1 to 4. The broken double line indicates the location of the BaZOl-generated deletion in R1162 DNA. The BumHI-EcoRI linker DNA is derived from M13mp7RFI (Messing et al., 1981).
(b) Increased transcription
in the RepI region elevates the copy number of R1162
A comparison of the amounts of 31,000 M, polypeptide synthesized in minicells containing pMS342 (Fig. 6(a), lane d) and pUT238 (Fig. 6(a), lanes b and c) shows that the tat fusion at 2.83 kb results in substantially greater amounts of this essential polypeptide. We do not have direct evidence for increased amounts of the 29,000 M, polypeptide, although it would seem likely that the amount of this product, made from a cotranscribed
gene, would increase also. Indirect evidence for this is presented later (section c, below). We asked whether increased transcription through Rep1 would affect the copy number of R1162. To test this, we used two plasmids, pMS228 and pMS245 (Lin & Meyer, 1984), in which additional DNA has been inserted into R1162. The first of these, pMS228, contains a DNA fragment both encoding resistance to trimethoprim, and also including a 370 base-pair segment derived from the region of the replicative origin of R1162. Thus, pMS228 is diploid for a fragment of DNA near the
762
K. Kim and R. J. Meyer a
b
c
d
e
f
a
(a)
b
(b)
Figure 6. Polyacrylamide gel electrophoresis of 35S-labeled polypept,ides synthesized in purified minicells. Plasmids in the minicell-producing strains were: (a) none (lane a); pUT238 (lanes b and c); pMS342 (lane d); pUT239 (lanes e and f); pMS348 (lane g). (b) None (lane a); pMC1403 (lane b). Polypeptide
(iM, 14,000), soybean trypsin inhibitor serum albumin
markers (not shown) were from Bio-Rad:
lysozyme
(M, 21,500), carbonic anhydrase (M, 31,000), ovalbumen (M, 45,000), bovine
(A& 66,200). and phosphorylase
B (M, 92,500). These were unlabeled and visualized
origin; the effect of this duplication is to lower the copy number of the plasmid (Lin & Meyer, 1984). The plasmid pMS245 differs from pMS228 only in that it lacks the cloned second copy of the 370 basepair sequence, and therefore retains the same copy number as the parental DNA. Strains were constructed containing either pMS228 or pMS245, and pUT238 (Fig. 3), which has the tat promoter adjacent to the Rep1 region at 2.83 kb, or PUT191 (Fig. 2), the parental tat vector lacking any cloned R1162 DNA. We then estimated the relative amounts of the different plasmid DNA species in these strains by extracting the plasmid DNA, cleaving it with a restriction endonuclease, and subjecting it to electrophoresis through 0.8% agarose. The results are shown in Figure 8. In the presence of the control plasmid pUTl91, the amount of coextracted pMS228 is substantially less than for pMS245 (Fig. 8, lanes a and e). In the presence of pUT238, cellular amounts of both pMS228 and pMS245 increase (Fig. 8, lanes c and g). From the examination of different amounts of DNA from several independent experiments, we estimate a four- to sixfold increase in pMS228 plasmid DNA, and a two- to threefold increase in pMS245. This result contrasts with the earlier observation that if Rep1 DNA (including its own promoter) is cloned into a plasmid with a copy number similar to that of Rl162 and then
by staining.
introduced into cells containing pMS228 or pMS245, no increase in the copy number of these plasmids is observed (Lin & Meyer, 1984). Evidently, the increased transcription originating from the tuc promoter is required for the higher copy number. Expression of the tat promoter is sensitive to the regulatory system of the lac operon (Amann et al., 1983). If the higher copy number of pMS228 and pMS245 is in fact due to transcription from the tat promoter, then the copy number should vary depending on the level of expression of this promoter. Initially. we looked at plasmid copy numbers for cultures grown in the presence of IPTG, a gratuitous inducer of the lac operon (Fig. 8, lanes b, d, f and h). Under these conditions, the tat promoter should be maximally active. However, no further increase in copy numbers of pMS228 or pMS245 was observed. It is possible that, because pUT238 and PUT191 are themselves high copy-number plasmids, the lac repressor is always substantially titrated, IPTG then having little effect. Therefore, we recloned the DNA containing the tat promoter-Rep1 fusion into pSC101, which has a much lower copy number than PUT191 (Cohen et al., 1973). We then tested the effect of this plasmid, pUT251 (Fig. 4), on the copy numbers of pMS228 and pMS245, both in the presence and absence of IPTG (Fig. 9). The results show that higher copy numbers are achieved by
Copy Number
0
27,000
-
b
c
d
e
of Plasmid a
f
763
RI 162 b
C
d
e
f
a
h
C C
cat -
gel electrophoresis of Figure 7. Polyacrylamide 35R-labeled polypeptides svnthesized in purified minicells. Plasmids in the minicell:producing strains were: none (lane a); pACYC184 (lane b); pUT247 (lane c); pUT248 (lane d); pUT249 (lane e); pUT250 (lane f). Polypeptide markers (not shown) are given in Fig. 6. The label cat refers to rhloramphenicol acetyltransferase encoded by pA(‘Y(‘184.
bot’h pMS228 and pMS245 when IPTG is added t,o the culture medium (Fig. 9, compare lanes c and h with d and i). A similar effect is not observed for these plasmids in the presence of the control plasmid, cloning vector pSClO1 (Fig. 9, compare lanes a and f with b and g). We conclude that transcription from t,he tar promoter into Rep1 is responsible for the higher copy numbers of the R1162 derivatives pMS228 and pMS245. (c) The elevated copy number of the R1162 derivative pMS228 depends upon increased transcription through both genes in Repl The fusion of Rep1 DNA and tat promoter DNA at R1162 co-ordinate 2.83 should result in increased transcription through both the promoter-proximal gene for the 31,000 M, polypeptide, and the distal gene for the 29,000 LV~ polypeptide. Which of these genes is responsible for the increased copy number of pME228? We examined the copy number of pMS228 in the presence of plasmids in which the tat promoter was linked to the entire Rep1 region (31+29’), or to only one of the two genes (31’29or 31-29+). The results (Fig. 10) show that neither the 31+29plasmid pUT282 (Fig. 5), nor the 31-29+ plasmid pUT247 (Fig. 4), increases the copy
Figure 8. Agarose gel electrophoresis of EcoRI-cleaved plasmid DNA isolated from E. coli GM1 strains. Cells were grown in the presence (lanes b. d, f and h) or absence (lanes a, c, e and g) of IPTG. Marker fragments (lane i) are HindIII-cleaved lambda bacteriophage DNA (23.7. 9.5; 6.7, 4.3, 2.3. 2.0 kb; Allet & Bukhari. 1975). Gel lane
Plasmid
Properties
DNA fragments
a. b
pMS228 PUT191
IncQ(dup1) 31-29-
1. 4 2
c. d
pMS228 pUT238
IncQ(dup1) 31+29+
I, 4 2, 3
e. f
pMS245 PUT191
IncQ 31-29
I, 5 2
g. h
pMS245 pUT238
IncQ 31+29+
1, 5 2, 3
Plasmid DNAs were isolated from cells grown in the presence (lanes b, d, f and h) or absence (lanes a. c, e and g) of IPTG.
number of pMS228 beyond that observed in the presence of control plasmid, the cloning vector pACYC184 (Fig. 10, compare lanes a, b and c). In contrast, the 31+29+ plasmid pUT249 (Fig. 4) clearly results in a higher copy number for pMS228 (Fig. 10, lane d). The higher copy number does not require cvtranscription of both genes from the same DNA. When the 31-29+ plasmid PUT239 (Fig. 3) is introduced into a cell containing pMS228 and pUT282 (31+29-), or when the 31+29plasmid pUT278 (Fig. 5) is introduced into a cell containing pMS228 and pUT247 (31-29+), the copy number of pMS228 is increased (Fig. 10, lanes f and h). This is not due to an effect of the cloning vector PUT191
764
K. Kim a
b
c
and R. J. Meyer d
e
f
h
2 3
4
Figure 9. Agarose gel electrophoresis of BarnHI-cleaved plasmid DNA isolated from E. coli GM1 strains. Cells were grown in the presence (lanes b, d, g and i) or absence (lanes a, c, f and h) of IPTG. Markers (lane e) are HindIII-cleaved
lambda DNA (Fig. 8). Gel lane
Plasmid
Properties
a. b
pMS228 psc101
Inc&(dupl) 31-29-
1 3
c, d
pMS228 pUT25 1
IncQ(dup1) 31+29+
1 3, 4
f, g
pMS245 psc101
IncQ 31-29-
2 3
h. i
pMS245 pUT25 1
IncQ 31+29+
2 3, 4
DNA fragments
Plasmid DNAs were isolated from cells grown in the presence (lanes b, d, g and i) or absence (lanes a. c, f and h) of IPTG.
used in the construction of pUT239 or pUT278, since this molecule is present in the strains used for lanes a to d. Moreover, the copy number of pMS228 is not elevated when pUT239 or pUT278 are present along with only the parental cloning vector for pUT247 and pUT282, pACYC184 (Fig. 10, lanes g and i). We conclude that increased transcription through both the Rep1 genes is required for an increase in the copy number of pMS228. The simplest interpretation is that greater amounts of both gene products are necessary for the increase.
determinant for incompatibility (Lin & Meyer, 1984). When this fragment is cloned into pBR322 and introduced into a cell containing R1162, the resident plasmid is rapidly lost from the culture during subsequent growth. We tested whether the tat promoter-Rep1 fusion that increases the copy number of R1162 also confers protection against the cloned incompatibility determinant. To do this, we transformed strains containing R1162 and a tat promoter plasmid with pMS319, a pBR322
derivative
fragment. (d) Increased confers
transcription through the Repl region protection against expression of a cloned determinant for incompatibility
Incompatibility is the failure of two plasmids to be coinherited stably. The phenomenon is thought to reflect, in many cases at least, a common mechanism of copy-number control (Pritchard, 1978). The 370 base-pair fragment of R1162 DNA that affects copy number also contains a
incoming
containing
Transformed plasmid,
the
370 base-pair
cells were selected for the
and colonies
tested
for retention
of R1162 by screening for resistance to streptomycin. The results are shown in Table 2. The plasmid pUT249 (Fig. 4), which contains the tat promoter fused to the complete Rep1 region, protects R1162 from pMS3 19-mediated incompatibility. The complete Rep1 region with the normal promoter (pUT316) is ineffective, as are fusions
in which
the gene for either
31,000 M, polypeptides
the 29,000
is absent. Thus, sensitivity
or
765
Copy Number of Plasmid R1162 b
a
a
d
e
f
a
h
I
Figure 10. Agarose gel electrophoresis of EcoRI-cleaved plasmid DNA isolated from E. co& GM1 strains. A11 cultures were grown in the presence of IPTG. Markers (lane e) are HindIII-cleaved lambda DNA (Fig. 8).
Plasmid
Properties
pMS228 PUT191 pACYC184
IncQ(dup1) 31-2931 -29-
1, 6
b
pMS228 PUT191 pUT282
IncQ(dup1) 31-2931 +29-
1, 6 2 2, 4
c
pMS228 PUT191 pUT247
IncQ(dup1) 31-2931 -29+
1, 6 2 2, 5
d
pMS228 PUT191 pUT249
IncQ(dup1) 31-2931 +29+
1, 6 2 2, 3
f
pMS228 pUT239 pUT282
IncQ(dup1) 31-29+ 31+29-
1, 6 2, 5 2. 4
g
pMS228 pUT239 pACYC184
IncQ(dup1) 31-29+ 31-29-
1, 6 2, 5 2
h
pMS228 pUT278 pUT247
IncQ(dup1) 31+2931 -29+
1, 6 2, 4 2, 5
i
pMS228 pUT278 pACYC184
IncQ(dupl) 31+2931-29-
1, 6 2, 4 2
Gel lane a
to the cloned incompatibility determinant parallels the copy-number effect.
exactly
4. Discussion Enhanced transcription through the Rep1 region elevates the copy number of R1162. We assume
DNA fragments
2 2
that this is due to an increase in the amounts of the Rep1 gene products. If this is so, the products of both the Rep1 genes must be increased in order to raise the amount of plasmid DNA (Fig. 10). Analogous polypeptides encoded by plasmid RSFlOlO are required for plasmid DNA replication in vitro (Scherzinger et al., 1984). We envision that
766
K. Kim and R. J. Meyer
Transformation
Table 2 with pMS319 DNA
Number of streptomycin-resistant Resident plasmids R1162, RI 162, R1162, Rl162, Rl162,
pACYCl84 pUT249 pIJT247 pCT282 pUT316
A 1 20 8 0 4
13 0 20 6 2 5
No two plates contained sibling tested from each of 5 plates.
colonies
C
D
E
0 20 2 3 2
0 20 3 0 2
0 20 7 0 4
transformants:
20 colonies
the Rep1 polypeptides, in the form of their native proteins, participate in the initiation of DNA synthesis at the replicative origin (oriV) of R1162. These proteins could act co-operatively at a single step in the initiation of replication, or could independently affect different steps in this process. In addition, we have shown that a 370 base-pair fragment of DNA from the oriV region both specifies plasmid incompatibility and affects plasmid copy number (Lin & Meyer, 1984; Meyer et a,Z., 1985). If this DNA fragment is duplicated at a novel position in RIl62, the copy number of the resulting plasmid is substantially lowered. Increasing the copy number of the Rep1 genes does not noticeably reverse this effect (Lin & Meyer, 1984). An examination of the base sequence of the 370 base-pair fragment, reveals no large open reading frames, but does show the presence of a cluster of directly repeated sequences similar in size to t’hose observed for several other plasmids (Murotsu et al., 1981; Stalker et aE., 1979, 1981; Churchward et al., 1983; Kamio & Terawaki, 1983; Abeles et al., 1984). One possibility that might account for these observations is that the 370 base-pair fragment encodes an inhibitor that regulates expression of the Rep1 genes. In view of the base sequence of t,his fragment, such an inhibitor would probably not be a polypept)ide, but could be a small RNA. The role of small RNAs in the regulation of plasmid DNA replication is well-established. In the case of ColEl, such a molecule affects production of an RNA primer for DNA replication (Tomizawa et al., 1981); for the plasmid Rl, translation of the transcript’ of the essent’ial repA gene is modulated (Light, & Molin. 1983). Several observations argue against the existence of a small RNA inhibitor. The increased amount of this hypothetical inhibit,or that would be specified by plasmids containing a, duplicated 370 base-pair fragment is apparently not titrated by a commensurate increase in Rep1 gene dosage (Lin & Meyer, 1984). In addition, both RepI genes have been fused at numerous positions to ZacZ DXA lacking its normal transcriptional and translational start) signals (Meyer et aZ., 1985; Kim & Meyer. unpublished results). In no case is expression of the
la& gene, measured in terms of units of J?-galactosidase activity, affected by the presence in the cell of multiple copies of the 370 base-pair fragment. Finally, the amount of the 31,000 M, polypeptide synthesized in minicells containing pMS342 is not decreased when the minicellproducing strain also contains multiple copies of a plasmid with the 370 base-pair DNA (data not shown). We favor the idea that the Rep1 genes are under some form of autogenous regulation. If the concentrations of the Rep1 products are thus kept’ constant during growth, one way that their activity could be regulated is by binding to a region of the 370 base-pair DNA. Because the number of these regions would increase with plasmid copy-number, a large number of plasmid copies would result in a small amount of unbound Rep1 protein for replication, and so a regulatory circuit would be established. This model has been proposed for the F factor by Tsutsui et al. (1983). Evidence in support of this titration model has come from studies on replication of the F factor (Tsutsui et al., 1983) and the phage Pl (Chattoraj et al., 1984). The presumptive titrating sites in both cases are 19 base-pair, directly repeated sequences. For R1162, titration of either of the two Rep1 proteins would be sufficient to affect control. If this model is correct, it could provide an explanation for t,he directly repeated, 20 base-pair sequences within the 370 base-pair fragment. The direct repeats of several other plasmids have been shown to be binding sites for essential, plasmid-encoded proteins (Germino &, Bastia, 1983; Vocke & Bastia. 1983; C’hattoraj et al., 1984). Increased transcription through Rep1 protects. in trans, R1162 from expression of the strong incompatibility determinant encoded by a cloned 370 base-pair fragment. The simplest interpretation is that both incompatibility and copy-number control are different manifestations of the same functional activity. For R1162, we think that incompatibility occurs because a Rep1 gene product is bound to a target site on the 370 base-pair fragment. Thus, free Rep1 protein is unavailable for replication of R1162, and the plasmid is lost from the cell. Increased transcription through Rep1 spares RI 162, because the autoregulation of the Rep1 region is overcome, and more protein is available for plasmid replication. In summary, two regions of R1162, one encoding essential plasmid proteins and the other linked to 0riV and also exerting incompatibility, can functionally interact to affect copy number. The exact regulatory relationship between these regions in the control of plasmid copy number is not understood, however, and further experiments will be required for a detailed model.
This work was supported, in part, by National Science Foundation grant PCM-8219916 and, in part, by grant F-964 from the Robert A. Welch Foundation.
Copy Number
of Plasmid
References Abeles, A. L.. Snyder, K. M. & Chattoraj, D. K. (1984). J. Mol. BioZ. 173, 307-324. Adler, H. I.. Fisher, W. D., Cohen, A. & Hardigree, A. A. (1967). Proc. Nat. Acad. Sci., U.S.A. 57, 321-326. Allet, B. & Bukhari. A. I. (1975). J. Mol. Biol. 92, 529540. Alton, pu’.K. & Vapnek, D. (1979). Nature (London), 282, 864-869. Amann? E.. Brosius, J. & Pt’ashne, M. (1983). Gene, 25, 167-178. Ambler, R. P. & Scott, G. K. (1978). Proc. Nat. Acad. 8ci., 1r.S.A. 75, 3732-3736. Bart)h. P. T. & Grinter. 1\;.J. (1974). J. BacterioZ. 120. 618-630.
Bolivar. F.. Rodriguez, R. L., Greene, P. J., Betlach. $1. C., Heyneker, H. L., Boyer, H. W., Crosa, J. H. & Falkow, S. (1977). Gene, 2, 95-113. Bryan, L. E.. van den Elzen, A. M. & Tseng, J. T. (1972). Antimicrob. Ag. Chemother. I, 22-29. Casadaban. M. J. & Cohen, S. N. (1980). J. Mol. BioZ. 138, 179-207.
Casadaban, M. J.. Chou? J. & Cohen, S. K. (1980). J. Bacterial. 143, 971-980. (‘hang, A. (‘. Y. & Co hen, S. plj. (1978). J. Bacterial. 134, 1141-I 156. (‘hat,toraj. I).. Cordes, K. & Abeles, A. (1984). Proc. Nut. dcad. tici., U.S.A. 81, 6456-6460. Churchward, G.. Linder, P. & Caro, L. (1983). NucZ. Acids Res. 11. 5645-5659. Cohen, S. N.> Chang, A. C. Y. & Hsu, L. (1972). Proc. Nut. Acad. tici., U.S.A. 69, 2110-2114. Cohen, S. K‘.. Chang, A. C. Y., Boyer, H. W. & Helling, K. B. (1973). Proc. AVat. Acad. Sci., U.S.A. 70, 32403244. Fra.zrr. 4. C. & Curtiss, R’. III (1973). J. Bacterial. 115. 615-622.
Germino, J. & Bastia, D. (1983). Cell, 32, 131-140. Grinter, pu’. J. & Barth. P. T. (1976). J. BacterioZ.
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Guerry, P.. van Embden, J. & Falkow, S. (1974). ,/. BacterioZ. 117, 619-630. Hershtield. V.. Bayer. H. W.. Yanofsky, C., Lovett, M. A. & Helinski, D. R. (1974). Proc. Nat. Acad. Sci., i:.S.A.
71, 3455-3459.
Holmes, D. 8. & Quigley, M. (1981). Anal. B&hem. 193~-197.
114.
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