Molecular Microbiology (1992) 6(23), 3501 -3510

Characterization of the minimal origin required for replication of the streptococcai plasmid plP501 in Bacillus subtilis S. Brantl^ and D. Behnke Institute for Moiecuiar Biology, BeutenbergstraBe 11, 0-6900 Jena, Germany. Summary By using deletional analysis the origin of replication, oriR, of the streptococcai plasmid plP501 in Bacillus subtilis has been mapped at a position immediately downstream of the repR gene. Determination of both the right and left border of oriR allowed the definition of a sequence of a maximum of 52 nucleotides which theoretically constitutes the minimal origin of replication. Recently, the start point of leading-strand synthesis of the closely related plasmid pAM^I has been mapped at a position which is located exactly in the middle of this sequence (Bruand et al., 1991). The function of ohR did not depend on its location downstream of the repR gene. Translocation of oriRcontaining fragments to other regions of the plasmid proved to be possible. The smallest translocated fragment that still reconstituted autonomous replication was 72bp in size. This fragment was also active in directing the replication of an Escherichia coli piasmid in B. subtilis when the RepR protein was supplied in trans from a repR gene integrated into the host chromosome. The transformation efficiency of piasmids carrying translocated oriR fragments showed a certain dependence on the fragment length and orientation. The DNA sequence of oriR inciuded an inverted repeat, both branches of which appeared to be essential for oriR function. The repeats of oriR shared sequence similarity with a repeat located upstream of promoter pil, which has been suggested to be involved in autoregulation of repR expression.

Introduction The molecular anaiysis of replication functions of broadhost-range plasmids appears to be particularly interesting, since such plasmids must have evolved structural and regulatory properties that are compatible with a Received 20 March, 1992; revised and accepted 24 July. 1992. 'For correspondence. Tel. (78) 853727; Fax (78) 852252.

variety of specific replication machineries in different hosts. Plasmid plP501 (Horodniceanuefa/., 1976; Behnke etai, 1981) belongs to such a group of broad-host-range plasmids, which also includes two other streptococcai replicons, namely pAMp1 (Clewell ef at., 1974) and pSM19035 (Behnke et ai, 1979; Behnke and Ferretti, 1980). These plasmids can be propagated in a wide range of Gram-positive bacterial hosts (Gibson et ai. 1979; Engel et ai, 1980; Schaberg et at., 1982; Gonzalez and Kunka, 1983; Buu-Hoi et at., 1984; Oultram and Young, 1985; de Vos, 1987) including B. subtilis and exhibit a pronounced segregational and structural stability (Janniere et ai, 1990, our unpublished data). Certain derivatives of these plasmids have been used to stably clone large fragments of foreign DNA (Behnke ef ai, 1981; Harlander and MoKay, 1984; Rabinovich ef at., 1985). All three plasmids share a high degree of sequence homology between their replication regions (Brantlef a/., 1989; 1990; Sorokin and Khazak, 1990; Swinfieid etai, 1990). They have recently been shown to replicate via a theta-type mechanism (Bruand ef ai, 1991) rather than by the rolling-circle type mechanism known from the small staphylococcal plasmids frequently used as cloning vectors in Bacillus sp. (Alonso, 1989; Gruss and Ehrlich, 1989; Bron, 1990). Transcriptional and mutationai analysis of the plP501 replication region has recently revealed at least two different levels of copy-number control (BrantI and Behnke, 1992a; BrantI et ai, 1992). These levels involve a non-essential small protein, CopR, an antisense RNA of 136 nucleotides (RNAIM) and an inverted repeat structure located upstream of promoter pil, which directs transcription of the essential repR gene. The target of all regulatory mechanisms appears to be the repR gene, the product of which is required for initiation of plasmid replication. Recently the start points for the leading strand synthesis and the termination point of the lagging strand synthesis have been located downstream of the rep gene on the related plasmid pAMpi (Bruand ef a/., 1991). In this communication we present evidence that the origin of replication {oriR) and the RepR protein of plP501 are the only two plasmid components essential for replication of plP501. The oriR sequence consisted of a maxmimum of 52 nucleotides immediately following the stop codon of the repR gene. The function of oriR was, however, not linked to this position, as or/fl-containing

3502

S. BrantI and D. Behnke Pv

HSP

X 8 X

X Pv

Pv

pPS9 phleo

Pspac

repR

oriR

bla

Rg. 1. Linear map of plasmid pPS9. The basic E. co//replicon is pUC18. phleo, phleomycin-resistartce gene from pUBIIO; Pspac: SPAC promoter (Yansura and Henner. 1984); repR, coding sequence of the plP501 repR gene including its natural SD sequence and downstream region; oriR, origin of replication of plP501; bla, ampfcillin-resistance gene. Restriction sites: Pv, Pvull; H, HindW; S, Sph\\ P. Psfl; X, Xbal, B, BamHl; E, Eco Rf.

fragments translocated to other regions of the plasmid were aotive as well. Furthernnore, oriR was found to be active when RepR was supplied in trans.

number was determined to be approximately 50 molecules per cell. The functional replacement of this region by PSPAC proved that neither oriR nor essential sequences other than promoter pil (BrantI et at., 1992) was/were present upstream of repR. Consequently, only two possible locations were left for the origin of replication of plP501, either within the coding sequence of repR or downstream of this gene within a segment of approximately 150bp. Thus by using polymerase chain reaction (PCR) techniques a deletion was constructed that removed all DNA sequences immediately downstream of the repR stop codon leaving, however, the repR gene functionally intact. The resulting plasmid pPR2111 (Fig. 2) failed to transform 6. subtitis. We have previously noted the presence of a weak inverted repeat immediately downstream of repR (BrantI ef ai, 1990). In order to rule out the possibility that the deletion impaired a possible role of this structure in terminating or stabilizing the repR transcript, the bacteriophage fd transcriptionai terminator was inserted into the unique EcoRI site of pPR21l1 located immediately downstream of repR(pPR2111T; Fig. 2). The presence of this terminator, however, did not

Results The origin of replication ofplP501 is located downstream of repR In a previous communication v^e proposed that the origin of replication (oriR) of plasmid plP501 may be located upstream of the repR gene (BrantI ef ai, 1990). This conclusion was, however, jeopardized by subsequent transcriptional analysis (BrantI and Behnke, 1992a; BrantI et al., 1992). In order to conclusively rule out a location of oriR upstream of repR, the entire upstream region up to the repR Shine-Dalgarno (SD) sequence was replaced by a DNA fragment carrying the PSPAC promoter (Yansura and Henner, 1984). The resulting plasmid, pPS9 (Fig. 1), transformed 6. subtilis efficiently and was capable of autonomous replication. Plasmid pPS9 reisolated from B. subtilis did not show structural alterations and its copy

EPv

PvHSP

XB K

Pv

H

pPR t

bla

phleo

pil

repR

oriR

pPR22'l 3 pPR2210 pPR2195 pPR2179 pPR2167 pPR2164 PPR2153 pPH211t pPR2111T fdterm

Fig. 2. Deletional analysis to map the right border of the origin of replication of plasmid plP501. + and - indicate the ability of the deletion derivative to replicate in B. subtilis: plasmid numbers correlate with nucleotide positions of right deletion endpoints; nucleotide numbering is identical to that of the repH sequence originally pubiished (BrantI et al.. 1990) except for the insertion of an additional C residue beyond nucleotide 233 resulting in a one-nucleotide shift for all downstream positions; pll/repR, repRgene of plP501 under control of its natural promoter. Restriction sites: K, Kpn\\ fd term, transcriptional terminator of £. coli bacteriophage fd; for other abbreviations see the legend to Fig. 1.

Origin of reptication of piasmid ptP501 3503 Pv H S P

X ax

X Pv

HE

Pv

I

pPS 10 phleo

Pspac

repH

bla Length olthe oriR fragments inseried into the BamHl site of pPS10

pPS2025

245bp

*

pPS2O73

197bp

+

pPS2064

iS6bp

*

PPS2096

174bp

+

pPS2108

162 bp

+

pPS2113

157bp

+

pPS2120

150bp

pPS136

136 bp

pPSI03

Fig. 3. Deletional analysis to map the left border of the origin of replication of plasmid plP501 (A), to define the minimal size of onR that functions after translocation to a new position (B), and to detemiine the orientation dependence of oriR in its natural location (C). + and - at the right margin refer to the ability of plasmids to replicate in 8. subtilis. Plasmid numbers in (A) correlate with nudeotide positions of left deietion endpoints (BrantI etai. 1990). repR, SD and coding sequence of the plP501 repR gene, the downstream sequences of which were replaced by an EcoR\ site; for other abbreviations see the legends of Figs 1 and 2.

103 t)p

pPS72

72 bp

B pPS2179-1

pPS2179-2

Insertion otthe 72 bp oriR EcoRI tragment ol pUC72 in bo\h onenlaKons " ' ° '^^ EcoRI sue ol pPSIO

restore the ability of pPR2111 to transform B. subtilis. Reoonstitution of the sequenoes downstream of repR again allowed the plasmid to replicate autonomously in B. subtitis. This effect, however, was not observed when the downstream sequences were present in an inverted orientation (plasmids pPS2179-1 and pPS2179-2; Fig. 3o). From these data, therefore, we ooncluded that on plP501 oriR is located either downstream of, or overlapping with, the 3' end of the repR gene.

Determination of the right and teft border ofonH In order to narrow down the location of oriR the region downstream of repR was systematioally shortened by using PCR as described in the Experimentat procedures. In this way plasmids pPR2153. -2164, -2167, -2179, -2195, -2210, and pPR2243 (Fig. 2) were constructed which carried progressive deletions downstream of repR.

Numbering of these plasmids was derived from the deletion endpoints and based on the nucleotide positions of the originally published DNA sequence of the plP5G1 replication region (BrantI etat., 1990). Except for plasmid pPR2153 all other derivatives were able to transform B. subtilis and to replioate autonomously in this host (Fig. 2). These data suggested that the right border of orlR was located somewhere between nucleotides 2153 and 2164, i.e. 42-53 nucleotides 3' of the repR stop codon. The close proximity of oriR and the 3' end of repR made mapping of the left border of or/ff complicated, as oriR had to be displaced from its original position in order to leave the coding sequenoes of the essential repR gene intact. For this purpose plasmid pPS10 (Fig. 1) was constructed which now lacked all sequences downstream of repR. In addition, on pPSI 0 the sequences upstream of repR were replaced by the PSPAC promoter. As expected, this plasmid failed to replicate in S. subtilis. Consequently, DNA

3504

S. BrantI and D. Behnke

fragments, that restored the ability of pPSI 0 to replicate in B. subtiiis can be expected to carry functionally intact oriR sequences. In order to prove that sequences downstream of repR have this capacity, and to map the left border of oriR, several subcloning steps and/or PCRs (for details see the Experimental procedure^ were used to generate a number of fragments, which covered part of the 3' sequenoes of the repR gene and/or its downstream region. All of these fragments had a fixed 5' end (right border, nucleotide position 2269), while the 3' ends were progressively shortened. Insertion of these fragments into the unique SamHI site of pPSIO, which was located upstream of repR, gave rise to plasmids pPS2025, -2073, -2084, -2096, -2108, -2113, and pPS2120 (Fig. 3a, plasmid numbers again reflect nucleotide positions of deletion endpoints as mentioned above). Except for pPS2120 all plasmids regained the ability to replicate autonomously in B. subtitis (Fig. 3a), thus proving that the cloned sequences possessed oriR activity. Both insertion orientations were found for all fragments. Although the ability to replicate was restored in all instances the efficiency of transformation was to some degree dependent on the orientation of the inserted fragments. The failure of pPS2120 to replicate in B. subtitis allowed us to allocate the left border of oriR to a position between nucleotides 2113 and 2120, i.e. between the stop codon of repfl and 12 nucleotides (nt) downstream of it. In conclusion, these data confirm that the origin of replication of plP501 {oriR) is located immediately downstream of the essential repR gene. The function of oriR, however, is not dependent on this location, as we were able to translocate oriR to an artificial location upstream of repR, where it still maintained its full ability to direct plasmid replication. The left and right borders of oriR mapped to nucleotide positions 2113-2120 and 2153-2164, respectively. Thus the minimal oriR of plP501 theoretically spans a region of maximally 52 nucleotides. Examination of the DNA sequence within this oriR region reveals that the left and right borders of oriR enclose the weak inverted repeat structure detected previously. Complete removal of either branch of the inverted repeat inactivated oriR, thus indicating that these sequences may serve an essential function. This may apply, however, only to certain parts of the inverted repeat, as plasmids pPR2164 and pPS2113, both of which lacked the outer three nucleotides of either the right or left branch of the inverted repeat, were still able to replicate in S. subtiiis.

Minimai tength of an in vivo active oriR The mapping data presented above suggested that a fragment spanning the DNA sequence between the right and left border of oriR may be sufficient to drive autonomous replication of plasmid DNA in B. subtiiis In

the presence of RepR. To test this assumption three small SamHI fragments covering oriR sequences were generated by PCR and inserted into pPSIO upstream of repR(plasmids pPSI 36, -103, and-72; Fig. 3b). The three fragments had a constant left side (nt 2108) and encompassed 136, 103, and 72 bp of the repR downstream region including the inverted repeat. All three fragments restored the ability of pPS10 to replicate in 6. subtitis. A transformation efficiency reduced 10-100-fold was noted for all three plasmids when compared with plP501 derivatives carrying oriR in its natural arrangement or carrying larger translocated oriR fragments. This reduction in transformation efficiency was most pronounced with plasmid pPS72. Nevertheless, these results confirmed that a sequence as small as 72 nt was sufficient to drive autonomous plasmid replication in S. subtitis. In fact, as will be presented below, this sequence was sufficient to facilitate replication of an Escherichia coli plasmid in S. subtitis as long as the RepR protein was provided in trans. In all instances in which or/R fragments were inserted into the SamHI site of pPSIO, plasmids were recovered that carried the oriR fragment in either one of the two insertion orientations. Those piasmids carrying oriR opposite to its natural orientation exhibited a lower efficiency of transformation.

oriR is active when the RepR protein is supplied in trans On all plasmid derivatives studied so far the repRgene and the oriR sequence were arranged in a cis configuration. This was also true for the pPSIO derivatives, where oriR was displaced from its natural position. In order to find out whether oriR also functions with RepR supplied in trans, three derivatives of £ co//plasmid pBT48 (pORI9, -10, -11; Fig. 5) were constructed. These E. coii plasmids (based on pUC18) carried only oryR fragments of various length and a marker gene that allowed selection in B. subtilis. The smallest oriR fragment spanned only 72 bp and completely included the inverted repeat sequenoes (Figs 4 and 5). To supply RepR in trans the repR gene was inserted into the chromosome of 6. subtitis DB104 by using the integration vector pDH32 (Shimotsu and Henner, 1986). The repR gene devoid of any downstream sequence and controlled by the PSPAC promoter was recovered as a SamHI-EcoRI fragment from pPSIO and inserted into pDH32 to yield pDH32/8 (Fig. 5). Successful integration of pDH32/8 by a double crossing over was confirmed by screening the chloramphenicol-resistant transformants for the absence of a-amylase production. Transformation of S. subtitis DB104::pDH32/8 was successful with all three plasmids (pORI9, -10, -11). However, while pORI9 and -10 transformed S. subtitis with the same efficiency as plP501 derivatives with or/Rand repRin c/s configuration, drastically reduced numbers of transformants were

Origin of reptication of ptasmid piP501 Asp Thr Gly TER 5 ' - GAT ACA GGC TGA AAATAAAACC CGCACTATGC CATTACATTT 2100 I 2120 2140 AtATCTaTGA TACGTGTTTG TTTTTCTTTG CTGT - 3 "^^ ?160 • 2175

t

Fig. 4. DNA sequence downstream of Ihe repR gene of plasmid plP501 containing the origin of replication. The left and right borders of oriR ate indicated by arrows. The inverted repeat sequences constituling putative RepR-binding sites are underlined. Nucleolides showing homology lo the E. coli IHF-binding motif are shown in italics. The amino acids represent the C-terminus of RepR.

obtained with pORIII. This observation was consistent with the behaviour of piasmid pPS72 carrying the same 72 bp oriR sequence in a cis position upstream of repR. The copy numbers of plasmids pORI9 and pORHO were found to be approximately fivefold lower than that of plasmid pC0P4 (BrantI and Behnke, 1992a), which carried all regulatory components involved in copy control of ptP501 in a natural arrangement. Both plasmids could be reisolafed from S. subtitis DB104::pDH32/8 and their structural integrity was confirmed by restriction analysis. Integration of the plasmids into the chromosome can be excluded, as no overlapping sequences were present at least on pORIIO or pORIII and the inserted plasmid pDH32/8. An additional plasmid, pORI4, was constructed and included in the experiments as a negative control. This plasmid carried a 432 bp fragment covering sequences located upstream of repR (for details see the Experimental

3505

procedures). As expected, pORl4 failed to transform 6. subtitis DB104::pDH32/8, thus confirming that replication of pORI9-11 in this host was not an artefact. In summary, the ability of pORI9-11 to replicate in S. subtiiis DB104::pDH32/8 proved that RepR can function in trans and that under such conditions a sequence as small as 72 bp is sufficient to drive autonomous replication of plasmid DNA in the Gram-positive host, ln addition these data confirm our previous assumption that the only plasmid components essential for plP501 replication are RepR and oriR.

Discussion The replication functions of plasmid plP501 include two proteins, CopR and RepR, a small antisense RNA of 136 nucleotides, RNAIII, and the origin of replication, oriR (BrantI et ai, 1990; BrantI and Behnke, 1992a; BrantI et ai, 1992). From the data presented in this communication it is now clear that the RepR protein and oriR are the only two components essential for autonomous replication of plasmids derived from plP501. CopR and RNAlll are merely regulatory components controlling the amount of RepR produced. The availability of RepR is, in fact, the limiting factor determining the plasmid copy number, as we have recently been able to show that artificial regulation of repR expression via the PSPAC promoter in the presence of Lac repressor and varying IPTG concentrations led to plasmid copy numbers that positively correlated with the amount of inducer added (BrantI and Behnke, 1992b).

X B

c

PDH32/8 amy back

cat

repR

PSPAC

tacZ

amy front

I size of the oriR fragment pORI9

319 bp

1951 pORIIO 162 bp phleo

2269

oriR

phleo

2269 oriR 2179

2108

pORI 11 bla

Fig. 5. Integration of Ihe psPAc-'-epR gene into the B. subtilis chromosome as part ot the vector pDH32/8 (upper part) and the oriR-containing £ col\ plasmids used to demonstrate interaction of oriR and RepR in trans (lower part). The repR gene on pDH32/8 was identical to that on pPSIO (see Fig. 3). amy front and amy back, 5'- and 3'-sequences of the S. subtilis a-amylase gene, respectively; cat. chloramphenicol-resistance gene; IacZ, E. coli ^-galactosidase gene; for the other abbreviations see the legends to Figs 1 and 2.

3506

S. BrantI and D. Behnke

IR pll 225 IR' pll 259 IR oriR 2120 IR'oriR 2157 consensuB

5 5 555-

GCTTT OCTTA GTTTT GTTTG GYTT-

TTTCT TTTTT ATTTT TTTTT -TTYT

TC TT CA CT Y-

- 3' - 3' - 3' - 3' - 3'

214 269 2108 2168

Fig. 6. Compilation of putative binding sites for the RepR protein of plP501. IR pll, inverted repeat located upstream of promoter pll directing fBpfl expression; IR oriR, inverted repeat located within the plP501 origin of replication.

In contrast to our previous hypothesis (BrantI et at., 1990) the location of oriR can now be assigned conclusively to a position downstream of the repR gene. Determination of the left and right border of oriR allowed us to narrow down the essential region to a sequence of maximally 52 nuoleotides immediately following the stop codon of the repR gene. The actual sequence necessary for efficient in vivo functioning of oriR, however, may be slightly larger, ranging somewhere between 52 and 72 bp since the transformation efficiency of plasmids became progressively lower as the minimal size of oriR was experimentally approached. Thus the size of oriR of plP501 was surprisingly small being comparable to that of the leading strand origins of the rolling-circle type plasmids pT181 {43bp, Gennaro et ai, 1989), pC194 (55bp, Gros et ai, 1987) or pUB110 (approximately 30 bp, Alonso etat., 1988). Plasmid plP501 replicates, however, via a theta-type mode like most of the well-studied £ ooli plasmids. The minimal origins of these plasmids are, however, considerably larger, ranging from 188bp (RI, Masai and Arai, 1988), and 250 bp (pSC101, Vooke and Bastia, 1983a; Yamaguchi and Yamaguchi, 1984a), to 430bp(RSF1010, Kim etai, 1987) or even 617bp (RK2, Stalker ef a/., 1981). The location of or/7? on plP501 coincided well with the recent mapping of the replication start points of the closely related plasmid pAMf31 (Bruand etai, 1991). The replication regions of both plasmids share a high degree of sequence homology. The two Rep proteins as well as the DNA sequences downstream of the rep genes are almost completely identical (BrantI et ai, 1990; Swinfieid et ai, 1990). Leading strand synthesis of pAMpi was found to start at a single position 27 nt downstream of the repf? stop codon (Bruand etai., 1991). From this point replication proceeded unidirectionally by a theta-type mechanism. Lagging strand synthesis was found to terminate 13-16 nucleotides further downstream, leaving a short region of single-stranded DNA at the replication origin (Bruand ef ai, 1991). By extrapolating from these data replication of plP501 would initiate exactly in the middle of the DNA sequence defined here as the minimal in vivo aotive origin. This is clearly different from the situation found in plasmids

RI or RI 00. Here, leading strand synthesis has been found to initiate approximately 380 bp downstream of oriR. However this region was not essential for R1/R100 replication as oriR was found to function with random downstream sequenoes (Miyazaki ef ai, 1988; Masai and Arai, 1989). The coincidence of oriR and leading strand initiation site on plP501 is likely to reflect a different mechanism of replication initiation. In contrast to R1 the oriR of plP501 does not contain any DnaA-binding sites, and thus the RepR protein must somehow compensate for the DnaA function in priming replication. Analysis of the DNA sequence within the oriR region reveals two interesting properties. First, the sequence includes an inverted repeat which apparently was essential as on/?activity was lost when either branch was deleted. Second, a DNA sequence with striking similarity to the consensus binding sequence of the E. coti integration host factor (IHF) is present within this region (Fig. 4). The two branches of the inverted repeat are 30 bp apart from each other. Since the repeats are not perfect and very AT-rich it seems unlikely from an energetical point of view that they form a secondary structure. Rather, they may serve as recognition sequenoes for binding of the RepR protein. It is interesting to point out that a similar inverted repeat is located upstream of, and partially overlapping with, promoter pll, which directs repRtranscription. This repeat not only shares considerable homology with the oriR (Fig. 6) repeat, but the two branches of this repeat are also located apart from each other at a similar distance. These sequence similarities, in fact, may not be purely accidental, since we have previously reported that the inverted repeat upstream of promoter pll is involved in control of repR expression, most likely via an autoregulatory mechanism. Since plasmid pORI4, which carried the repeat upstream of pll but not oriR, failed to replicate in B. subtitis, it can be excluded that there are two origins present within the plP501 replication region. All four repeats together with a possible consensus sequence (5'-GYTT-TTYTY-3') are summarized in Fig. 6. Mutational analysis as well as DNA-footprinting studies will be necessary to test this hypothesis. A dual function, like the one proposed for RepR of plP501, has been demonstrated for the replication initiator protein RepA of plasmid pSCIOI. However, on plasmid pSCIOI the promoter of the repA gene and the ori region overlap (Vocke and Bastia, 1983b; Yamaguohi and Yamaguchi, 1984a,b; Vocke and Bastia, 1985). It also remains speculative at this point whether there Is any significance attached to the presence of the IHF-binding motif, since no equivalent protein has yet been reported to exist in B. subtilis. IHF-binding sites have been observed, however, within the replication origins of several E. co//plasmids (Som and Tomizawa, 1983; Abeles ef a/., 1984; Gamasefa/., 1986; Masai and Arai, 1987) and at least for plasmids pSCIOI

Origin of replication of ptasmid ptP501 3507 and R6K., it has been shown that IHF is an essential host protein for plasmid replication (Gamas etai, 1986; Stenzel etai, 1987; Filutowiczand Appelt, 1988). In contrast to the origins of theta-type £ coii plasmids oriR was not only much smaller but also lacked an iteron structure typical for all of these plasmids (Kiies and Stahl, 1989). The ability of RepR somehow to substitute for the function of DnaA during priming of replication together with the simple topology of oriR may explain why plP501 and its related plasmids pAMpi or pSM19035 are able to replioate in such a broad range of bacterial hosts (Gibson etai., 1979; Engel ef at., 1980; Schaberg ef ai, 1982; Gonzalez and Kunka, 1983; Buu-Hoi ef a/., 1984; Oultram and Young, 1985). The ability of oriR to drive autonomous plasmid replication was not linked to its location downstream of repfland, moreover, RepR and oriR were shown to function efficiently in trans. Apparently, oriR oould be translocated to other regions of the plasmid without losing its activity, although a certain dependence on the orientation of oriR was noted. This orientation dependence was most pronounced when oriR was inverted at its natural location downstream of repR. It is tempting to speculate that this effect may be linked to transcriptional activities running into the origin region. It has recently been shown for oriC plasmids of £ coli that the activity of flanking promoters may severely affect the efficiency of replication initiation. On these plasmids the orientations of promoters and thus the direction of transcription towards oriC was crucial for the effects observed (Asai etai, 1992). Inversion of or/Rat its natural position downstream of repR resulted in convergent directions of replication and repfl transcription. In addition to causing steric problems the positive supercoiling induced in front of the migrating RNA polymerase (Wu etai, 1988) may lead to destabilization and abortion ofthe replication initiation complex at oriR. In its natural orientation transcription of repR may have a dual effect on oriR. As long as the RNA polymerase is travelling upstream of or/ff the presence of positive supercoils may reduce the initiation frequency. However, once it has reached or passed oriR either opening of the region by RNA polymerase or the generation of negative superooils behind it may then facilitate replication which proceeds in the same direction as repfl transcription. On the pPSIO derivatives carrying the translocated onR fragments divergent transcription from the spac and phieo promoters also induced negative supercoiling, which would facilitate replication initiation. However, since replication proceeds unidirectionally from oriR, its orientation towards the flanking promoters may influence its activity differently; this is similar to the effect seen with the oriC plasmids of £ coii (Asai etai, 1992). Further experiments will be required to elucidate whether the transcription of repR is in fact crucial to the efficiency of replication initiation at oriR.

Experimental procedures Bacterial strains, plasmids and growth conditions The plasmids used in this study to construct new derivatives of plP501 are listed in Table 1. Plasmids were propagated in B. subtilis DB104 (Kawamura and Doi, 1984), and E. co//'strain TG2 (Sambrook etat., 1989) was used for subcloning and mutagenesis experiments. All strains were routinely grown on TY medium (Sambrook et at., 1989). TY agar plates were supplemented with 0.15% amylopectin azur (Calbiochem) to discriminate between a-amylase-positive and -negative B. subtilis transformants.

Table 1. Plasmids used in this study. Plasmid

Description

Reference

pSPAC

B. subtilis, E. coli shuttle source of PSPAC promoter Vector for integration into the amy locus of S. subtilis E. CO//cloning vectors, Ap", MCS

Yansura and Henner (1984) Shimotsu and Henner (1986) Sambrook e( al. (1989) BrantI et al. (1990)

PDH32 PUC18/19 pUCIIB-F

pBT48

pC0P4 pMET24

pUC118, carrying the plP501 replication region as a 2.3 kb KpnI-fcoRI fragment pUCIB, carrying the phleomycinresistance gene of pUB110 in the Sa/GI sile pBT48, carrying the complete replication region of plP501, Pm'' pUC18 carrying the bacteriaphage fd transcriptional terminator as EcoRI fragment

Bra nil ef a/. (1990) BrantI ef ai (1992) T. Ellinger, unpublished

Ap'^, ampicillin resistant; MCS, multi-cioning site; Pm", phleomycin resistant.

DNA preparation and maniputation The isolation of plasmid DNA from B. subtitis has been reported (BrantI ef at., 1990). The copy number of pIP501 derivative plasmids in B. subtilis strain DB104 was determined as described before (BrantI and Behnke, 1992a). DNA manipulations, like restriction enzyme cleavage, ligation and filling-in reactions with Klenow fragment of DNA polymerase I, were carried out at the conditions specified by the manufacturer or according to standard protocols (Sambrook ef al., 1989). A GenAmp PCR Kit from Perkin Elmer/Cetus was used for PCRs. DNA sequencing ot all mutations or fragments generated by PCR was performed according to the dideoxy chain termination method (Sanger etai. 1977) with a Sequenase kit from US Biochemicals.

Construction of plasmids expressing repR under control of PSPAC

The construction of plasmid pPS9 (for a map see Fig. 1), expressing repR constitutively under the control of the PSPAC promoter, has been described in detail elsewhere (Branti and Behnke, 1992b). An intermediate plasmid, pPS8, which carried the repR gene under the control of PSPAC but was devoid of all sequences downstream of the repR stop codon, was generated by joint cloning ofthe eamHI-H/ndlll fragment of pPS9 and the 641 bp EcoRI-H/ndlll fragment of pPR2111 into pUCI 9 digested

3508

S. Brantt and D. Behnke

with SamHI and EcoRI. Subcloning of this 1.62 kb SamHI-EcoRI fragment of plasmid pPS8 into the pBT48 vector gave rise to plasmid pPSI 0. Cloning of the same fragment into the integration vector pDH32 yielded plasmid pDH32/8, which was subsequently used for inserting pspAc-repR into the chromosome of B. subtilis DB104.

Construction of plasmids with deietions downstream of repR Derivatives of plP501 deleted downstream of the repR gene were all constructed by PCR. Initially the desired fragment was amplified by using pUC118-F (BrantI etai, 1990) as template, the universal sequencing primer, and one thtj following mutagenic oligonucieotides: 5'-TCTAGAGAATTCTCAGCCTGTATCGTA-3' (pPR2111); 5'-TCTAGAGAATTCTATCATAGATATAA3' (pPR2153); 5'-GGATCCGAATTCAAAGAAACACGTATC-3' (PPR2164}; 5'-GGATCCGAATTCGAAAAACAAACACG-3' (pPR2167); 5'-TCTAGAGAATTCTAAACAGCAAAGAA-3' (pPR2179); 5'-GGATCCGAATTCTGCTAATCATTC-3' (pPR2195); 5'-GGATCCGAATTCTTACTCTGTATAT-3' (pPR2210); 5'-GGATCCGAATTCTCTCTCCTTCTCC-3' (pPR2243). The amplified fragments were cleaved with SamHI and 5coRI and cloned into pBT48 linearized with the same pair of enzymes. Piasmid pPR2111T was constructed by inserting a 400bp EcoRI fragment of plasmid pMET424 (T. Ellinger, unpublished), comprising the bacteriophage fd transcriptional terminator, into the unique EcoRI site of pPR2111.

Construction ofpPSIO derivatives carrying oriR fragments upstream of repR For the insertion of oriR fragments into the unique SamHI site upstream of PspAc-repR on pPSIO a number of intermediate plasmids was generated. The intermediate plasmid pCRI6 was constructed by cloning an Rsal-EcoRI fragment, covering nucleotides 2025-2269 of the plP501 replication region (BrantI et at., 1990) initially into pUCl 8 cleaved with Smal and EcoRI. It was subsequently transferred as an Xbal-EcoRI (filled-in) fragment into the Smal site of pUC19 to yield plasmid pOB6, which now contained the oriR fragment flanked by two SamHI sites. Eventually this fragment was inserted into pPSIO to give rise to plasmid pPS2025. For the other pPS plasmids initially the intermediate pOB5 was oonstructed by subcloning an Xba\EcoRI (filled-in) fragment from pORi5 (covering nucleotides 2073-2269 of the plP501 replication region, see below) into the SmaI site of pUC 18. From pOB5 the or/Rfragment was recovered as a SamHI fragment and inserted into pPSIO to yield pPS2073. In a second step a faql-EcoRI (filled-in) fragment from pOB5 covering nucleotides 2084-2269 ofthe plP501 replication region was again subcloned into the Smal site of pUC18 to yield pUC2084. Similarly, plasmid pUC2096 was constructed by using an Alu\-Eco^\ (filled-in) fragment from pOB5 covering nucleotides 2096-2269 of the plP501 replication region. Transfer of the OriR fragments, now flanked by SamHI sites, from pUC2084 and pUC2096 to pPS10 gave rise to pPS2084 and pPS2096. The intermediate plasmids pUC2108, pUC2113, and pUC2120 were obtained by cloning SamHI-cleaved PCR fragments into the SamHI site of pUC18. These PCR fragments were generated by using plasmid pOB5 as a template, the universal sequencing

primer, and one of the following mutagenic oligonucieotides: 5'-GTCTAGAGGATCCTGAAAATAAAACCC'3' (pUC2108); 5'TCTAGAGGATCCAATAAAACCCGCACT-3' (pUC2113); 5'GTCTAGAGGATCCCGCACTATGCCA-3' (pUC2120). The oriR fragments were subsequently recovered from pUC2108, pUC2113, and pUC2120 as SamHI fragments and inserted into pPSlO to yield plasmids pPS2108, pPS2113, and pPS2120. Plasmids pPS136, pPS103, and pPS72 were also constructed initially by using PCR to amplify the respective fragments, which were then cleaved with EcoRI and subcloned into the EcoRI site of pUC18. The fragments were amplified by using pUC2108 as a template, the universal sequencing primer, and the oligonucieotides used to generate plasmids pPR2179, pPR2210, or pPR2243. Recloning of the amplified fragments, now flanked by SamHI sites, from pUC72, pUC103, and pUC136 into the SamHI site of pPSIO finally gave rise to plasmids pPS72, pPS103, and pPS136. For all pPS 10 derivatives with orifl fragments upstream of repR both insert orientations were obtained. The insert orientation as well as the identity of the desired mutations were confirmed by DNA sequencing in all cases. For control purposes, the 72 bp oriR fragment of pUC72 was recovered as an EcoRI fragment and placed at its natural position downstream of repR by cloning it into the unique EcoRI site of pPSIO. Both insert orientations were Isolated and the plasmids were designated pPS2179-1 and pPS2179-2.

Construction ofpORI plasmids The intermediate plasmid pORI3 was obtained by cloning of an AvaW (filled-in)-fcoRI fragment of plasmid pUC118-F, covering nucleotides 2073-2270 of the plP501 replication region, into pUC19 cleaved with Smal-EcoRI. The oriR segment, which could now be recovered as a SamHI-EcoRI fragment, was subsequently inserted into pBT48 to generate plasmid pORI5. Similarly, plasmid pORI7 was constructed by inserting a Cla\EcoRI (filled-in) fragment of pUC118-F, covering nucleotides 1951-2269 of the plP501 replication region, into the Smal site of pUCI 8. Plasmid pORI9 was then obtained by subcloning this oriR region as a SamHI-EcoRI fragment from plasmid pORt7 into pBT48. Plasmids pORIIO and pORMI encompassing or/Rfragments of 162 and 72 bp (nucleotides 2108-2269 and 2108-2179, respectively) were generated by cloning a 1 kb Xbal-Psfl fragment, containing the phleomycin-resistance gene of pBT48, into plasmids pUC2108 and pUC72 (see above) digested with the same pair of enzymes. Plasmid pORI4 was constructed as a negative controi for the frans-complementation experiments. A 432bp SamHI-Pi'ull fragment of pPR3 (BrantI and Behnke, 1992a) covering nucleotides 182-613 of the plP501 replication region (located upstream of repR) was initially cloned into the EcoR! (filled-in)-SamHI site cf pUC19 to yield pORI2, and subsequently inserted as a SamHI-EcoRI fragment into pBT48, giving rise to pORI4.

Acknowledgements We would like to thank R. Breitling for helpful discussions, E. Birch-Hirschfeld for synthesizing numerous oligonucieotides, and Ina Poitz (deceased) for excellent technical assistance. This work was supported by grants from the Max-Planck-Society and the Fonds der Chemischen Industrie (to D.B.).

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Characterization of the minimal origin required for replication of the streptococcal plasmid pIP501 in Bacillus subtilis.

By using deletional analysis the origin of replication, oriR, of the streptococcal plasmid pIP501 in Bacillus subtilis has been mapped at a position i...
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