The EMBO Journal vol.14 no.11 pp.2642-2650, 1995 assembly site in Bacillus subtilis

Claude Bruand1, S.Dusko Ehrlich and involving the Rep protein, was proposed (Bruand et al., Laurent Janniere 1993). In contrast, little is known about the requirements for lagging strand synthesis of pAMf1-related plasmids. Laboratoire de Genetique Microbienne, Institut de Biotechnologie, It was reported that it may depend on the dnaD gene in INRA - Domaine de Vilvert, 78352 Jouy en Josas Cedex, France Bacillus subtilis (Ceglowski et al., 1993), but the function 'Corresponding author of this gene is unknown. Initiation of lagging strand synthesis is well documented A single-strand initiation site was detected on the in rolling circle replicating genomes such as single stranded Enterococcus faecalis plasmid pAMP1 by its ability to (ss) DNA phages and ss DNA plasmids. In these replicons, prevent accumulation of single stranded DNA of a lagging strand synthesis initiates at an origin called sso rolling circle plasmid, both in Bacillus subtilis and (for single-strand origin; Baas and Jansz 1988; Gruss and Staphylococcus aureus. This site, designated ssiA, is Ehrlich 1989). The sso belong to the family of single- located on the lagging strand template, -150 bp down- strand initiation sites (ssi), defined as sequences which stream from the replication origin. ssiA priming activity direct primer synthesis on ss DNA templates, either by requires the DnaE , the DnaC replication fork RNA polymerase or by a DNA primase. ssi were also , as well as the of the dnaD products dnaB, and detected in certain theta replicating plasmids. In some dnal genes of B.subtilis, but not the RNA polymerase. these sites were shown to be involved in The primase and the instances, the replication fork helicase require- priming of either leading or lagging strand DNA synthesis ments indicate that ssiA is a primosome assembly site. (Minden and Marians, 1985; Masai and Arai, 1989b; Interestingly, the pAM01 lagging strand synthesis is Masai etal., 1990; Honda etal., 1992; Kubotaetal., 1993). inefficient when any of the proteins involved in ssiA In all ssi activity is but occurs in Gram-positive bacteria, found so far originate mutated, efficiently the absence from rolling circle plasmids, and the majority of them of ssiA. This suggests that normal plasmid replication RNA requires primosome assembly and that the primosome require polymerase. Here we describe a new type can assemble at of ssi, present in the theta replicating plasmid pAMP1. not only ssiA but also elsewhere on This is the plasmid. This work for the first time describes a site, designated ssiA, located -150 bp downstream primosome in a Gram-positive bacterium. Involvement of the replication origin, on the lagging strand template. of the B.subtilis proteins DnaB, DnaD and DnaI, which ssiA activity is dependent on DnaE primase, DnaC helicase do not have any known analogue in Escherichia coli, and the products of the dnaB, dnaD and dnaI genes of raises the possibility that primosome assembly and/or B.subtilis, but not on RNA polymerase. Primase and function in B.subtilis differs from that in E.coli. helicase requirements indicate that ssiA is a primosome Key words: Bacillus subtilislDNA replication/helicase/ assembly site. Requirement for proteins without known primase/primosome assembly site homologue in E.coli (the B.subtilis DnaB, DnaD and DnaI) suggests that primosome assembly and/or function in B.subtilis differ from those in E.coli or that the E.coli analogues exist but have not yet been identified. Proteins Introduction involved in ssiA activity are required for efficient lagging strand synthesis of plasmids derived from pAMP1, which pAMP1 is a 26.5 kb plasmid isolated from Enterococcus suggests that assembly of a primosome is necessary for faecalis (Clewell et al., 1974). It belongs to a large family normal pAM,B1 replication. of closely related, broad host range plasmids, which includes pIP501 from Streptococcus agalactiae and pSM 19035 from Streptococcus pyogenes (see Janniere Results et al., 1993 for a review). Replication of these plasmids is dependent on a plasmid encoded protein (Rep) and Detection and mapping of ssiA proceeds by a unidirectional theta mechanism, initiated In order to detect a ssi on pAM31 we made use of plasmid downstream of the rep gene, within an origin of <50 bp pHV1610 (Boe et al., 1989). This plasmid is a hybrid (Figure lA; Swinfield et al., 1990; Bruand et al., 1990, between pC194 and pUC19 (Figure IC) and replicates in 1991, 1993; Brantl and Behnke 1992a,b; Ceglowski et al., B.subtilis by a rolling circle mechanism, using the pC194 1993; Le Chatelier et al., 1993; E.Le Chatelier, personal (Figure IB; Gros et al., 1987). The sso of pC 194 communication). Leading strand synthesis, which has been is inactive in this host and the plasmid accumulates ss studied in some detail, is initiated by DNA polymerase I DNA (Gruss et al., 1987). Insertion of an active ssi into (Pol I), a property in common with Escherichia coli pHV1610 reduces this accumulation, in an orientation- plasmids of the ColEl and ColE2 families. A model of specific manner (Figure IB; Boe et al., 1989). The plasmid initiation, based on that of ColE l-type plasmids but carries a convenient multiple cloning site (MCS), and

2642 K Oxford University Press ssiA of plasmid pAM,B1

A D repE ssiA

0.2 kb

B C

pHV1 61 0-1 5, pHVl 61 0-2

Ap

44 'dso pC194 x

I ori pUC repA

Fig. 1. (A) Schematic representation of a 2.8 kb HpaI-EcoRI segment of pAMPl, present in plasmid pIL253, which contains the minimal replicon. Open reading frames are represented by arrows and the replication origin (ori) and direction of replication by an open triangle. ssiA is represented as a black box. (B) Description of rolling circle replication. A strand-specific nick is introduced by the plasmid replication protein in the double-strand origin (dso, indicated by a bent arrow). The 3' end at the nick is used as a primer for leading strand elongation, while the lagging strand template is displaced. After one round of replication, the replication protein introduces a second nick at the same site in ori, and ligates the DNA ends, producing a ds and a ss DNA molecule. The ss DNA is then converted to a ds form. This step is stimulated if the ss DNA molecule carries a ssi, represented by a black box. (C) Schematic representation of plasmid pHV1610 and its derivatives pHVI610-1 and 2. Symbols are as in (A) and (B). replicates in E.coli, which facilitates construction of 1610-1 1610-2 1610 different derivatives. A segment of pAM,1 encompassing 57 bp of the 3' W,,* XF9Mw ...... end of the repE gene, the replication origin and -450 bp of the downstream region (Figure IA), was inserted in both orientations into the MCS of pHV1610. The resulting HMW plasmids, pHVI610-1 and pHVI610-2 (Figure IC), were introduced into the B.subtilis strain 1012 and tested for ss DNA accumulation. The amount of ss DNA of pHV1610-2 was similar to that of the parental plasmid, whereas that of pHV 1610-1 was much lower (Figure 2, two independent transformants were tested for each construct). These results -m %P. -o -m suggest that pAM,1 carries a ssi, designated ssiA, on the ccc lagging strand template. To map ssiA more precisely, the effect of different short pAM, segments on ss DNA accumulation was tested as above. The results, summarized in Figure 3A, show that SS --tom s ssiA is active in plasmids Al and AD5.18 but not in plasmids AG3.8 and AD5.24. The sequence of the relevant pAM,B1 region and the end-points of the segments used are shown in Figure 3B. ssiA is located in a 145 bp Fig. 2. Detection of ssiA. Bacillus subtilis 1012 cells harbouring segment (coordinates 4712-4856 of to plasmids pHV1610, pHV1610-1 or pHV1610-2 were grown to mid-log pAMPI according phase and their total DNA was extracted and analysed by Southern Swinfield et al., 1990), which can form a hairpin structure. hybridization, using 32P-labelled pHV1610 DNA as probe. For pHVI610-1 and pHV1610-2, two independent constructs were tested. ssiA activity does not require RNA polymerase CCC, covalently closed circular monomer; ss, single stranded DNA; HMW, high molecular weight plasmid which accumulate in Most ssi described so far in Gram-positive bacteria require multimers, B.subtilis when pUC sequences are joined to rolling circle plasmids the host RNA polymerase. To test whether ssiA activity (Gruss and Ehrlich, 1988). also requires this enzyme, we determined the effect of rifampicin, a specific RNA polymerase inhibitor, on the accumulation of pHV1610-1 ss DNA. For this purpose on RNA polymerase (pHV1611, Boe et al., 1989), was B.subtilis 1012 cells harbouring the plasmid were grown used as control. pHV1610-1 did not accumulate ss DNA to mid-log phase, and rifampicin was added. The amount upon addition of rifampicin, even after prolonged incuba- of ss DNA was determined in samples withdrawn at tion, whereas pHV1611 did, as early as 10 min after different times after drug addition. A pHV1610 derivative addition of the drug (Figure 4). These results indicate that carrying the ssoU of plasmid pUB 110, which is dependent ssiA activity does not depend on RNA polymerase.

2643 C.Bruand, S.D.Ehrlich and L.Janniere

p,HVI611 pHV1610-1 A ssi Nhel AseI EcoRI activity 0 10 20 30 60 0 10 20 30 60 p6or ssiA pHVl1610-1 D~~ 1+

Al + AG3.8

AD5.18 + AL Aik AIL AD5.24 ccc Vow 0.1 kb

B G AA A G A.T G G T. A C.G A G Fig. 4. Effect of rifampicin on ssiA activity. Bacillus subtilis 1012 G G cells 1611 or were to G A harbouring plasmids pHV pHV 1610-1 grown T. A mid-log phase, and then incubated with erythromycin and rifampicin T. A C.G (100 gg/ml each) for 1 h. Samples were taken at time intervals G G GAGCGAAGCGAA pAMP1 (ssiA) indicated above the wells (in minutes; 0 was taken before rifampicin T T GAGCaAcGCGAA R100(ssiB) Tr.A addition) and total DNA was analysed as described in the legend to G.C GAGCaAAGCGAg F (ssiC) Figure 2. ATT aAGCGAAGCGcg *X174 T. A T. A T. A (Figure SA bottom) indicated that the amount of C C.G pHVI610-1 ss DNA was -50% of that of pHVl610 in A T A C A G the mutant cells at 42°C, whereas it was -15% at 30°C. A A This amount was not significantly different in wild-type AG3.8 ( G)LC.GG Al (+) CG AD5.24 (-) AD5.18 (+) cells at the two temperatures (-25%). In contrast, the G.C amount of ss DNA of plasmid pHV1611, which carries TAATTATTAGGGGGAGAAGGAGAGAGTAr AGTGACGACAACTTGATTMAATMTCTATCTTT-TATAGGm 4712 4M66 the RNA polymerase-dependent ssoU, was essentially pIP501: C G G A pSM19035: C G G constant in all conditions (Figure 5B). These results indicate that ssiA, but not ssoU, depends on the DnaE Fig. 3. Localization and sequence of ssiA. (A) DNA segments tested for ssi activity. The pAM,Bl NheI-EcoRI segment and subsegments primase. carried by the various pHV1610 derivatives used in this study are We attempted to confirm these observations by carrying shown. Direction of pC194 replication in these derivatives is from left out temperature shift-up experiments, from 30 to 46°C, to right (see Figure 1 and its legend). Relevant restriction sites are the restrictive temperature for growth of mutant cells. indicated. + and - indicate ssi activity or inactivity, respectively. However, this was not (B) ssiA sequence and possible secondary structure. Coordinates are as approach successful since ssiA is defined by Swinfield et al. (1990). Nucleotide divergences of plasmids slightly thermosensitive at 46°C, and the conversion of ss pIP501 (Pujol et al., 1994) and pSM19035 (Sorokin and Khazak., DNA lacking ssiA to a double stranded (ds) form is 1987) are shown below the pAMfl sequence. End-points of segments stimulated by the temperature shift-up (unpublished present in pHV1610 derivatives (see A) are indicated by angled lines, results). The conjunction of these two phenomena resulted + and - indicating ssi activity or inactivity, respectively. Nucleotides conserved in the n'-pas of 4X174, F (ssiC) and RIOO (ssiB) are boxed in a difference between the amounts of ss DNA with and and a sequence comparison is shown in the inset (Imber et al., 1983; without ssiA which was too small to allow reliable Greenbaum and Marians, 1984; Nomura et al., 1991). Small letters measurement of ssiA activity. correspond to residues not present in all sequences. The first G of the OX174 subsequence was found by methylation protection assay to be in contact with the n' protein (Greenbaum and Marians, 1984). ssiA is dependent on the DnaC helicase and the products of the dnaB, dnaD and dnal genes of B.subtilis ssiA is dependent on the B.subtilis DnaE primase Two types of primase-dependent ssi were previously RNA polymerase-independent ssi sites require DNA described, and are denoted G sites and primosome primase. The B.subtilis DNA primase is encoded by the assembly sites (pas). They have different requirements for dnaE gene and thermosensitive mutants are available host functions, the former depending on primase only and (Karamata and Gross 1970; Wang et al., 1985). In order the latter on several additional proteins, including the to determine whether ssiA requires the primase, one replication fork helicase. In order to assign ssiA to one of such mutant, dnaE20, was used. Mutant cells harbouring the two types, its requirement for proteins other than pHV1610-1 or pHVl610, which do or do not carry primase was tested. Thermosensitive mutants affected ssiA, respectively, were grown in steady state cultures at in proteins involved in different steps of chromosomal permissive (30°C) or semi-permissive (42°C) temperatures replication were used (Karamata and Gross, 1970; Moriya (the restrictive temperature is 46°C), and the accumulation et al., 1990): dnaA (the E.coli dnaA analogue), dnaB and of plasmid ssDNA was measured. Significantly higher dnaD (without known homologues in Ecoli), which are amounts of pHV1610-1 ssDNA were detected at 42°C required during initiation of DNA replication, dnaC (the than at 30°C in dnaE20 cells but not in wild-type cells probable counterpart of E.coli helicase DnaB), involved (Figure 5A top). Phosphorlmager-mediated quantification in elongation, as well as dnaI, acting at an unknown step. 2644 ssiA of plasmid pAMPI

A wt dnaE20 dnaA1 dnaB 19 dnaC14 dnaD23 dr7al2 30 42 30 42 30 42 30 42 30 42 30 30 C pHV1610-1 *, ' , _a pHV1610 at * - _ *b _b-d ccc ;I* *to~~~~~~~~~~~~~~~~~_ _.

Ss

0 - ssiA

O Cd 100 }0crC> m fn > 0>o

0: > Vw dnaE20 dnaA1 dnaB 19 dnaC 14 dnaD23 dnal2

-? 30oC 42oC B

o o, 100 ssoU

Z T U n C- 50

X00 > C) _ cics >u0 wt dnaE20 dnaA f dnaB19 Q dr7aCl4 dnaD23 dnaf2

Fig. 5. Activity of ssiA in B.subtilis replication mutants. Various B.subtilis strains harbouring plasmids pHV1610 or pHV1610-1 were grown to mid- log phase at 30 or 42'C, then total DNA was prepared and analysed as described in the legend to Figure 2. (A) Top: autoradiograms of Southern blots. Only the bottom of the autoradiogram is presented, and the position of pHV1610 and pHVI610-1 CCC plasmid monomers is indicated. The position of migration of ss DNA varies because the results of different electrophoreses are presented. Growth temperatures and names of strains are indicated above the wells. Bottom: amount of ss DNA accumulated by pHV1610-1, expressed in percentage of the amount of ss DNA accumulated by pHV1610 in the same strain at the same temperature (ss DNA amount was normalized relative to the amount of corresponding ds DNA). Data were obtained by direct quantification of the blots using a Molecular Dynamics Phosphorlmager, and each value presented is the average of results of at least two independent experiments. Bars indicate standard deviation. (B) Amount of ss DNA accumulated by pHV 1611, which carries the pUB I 10 ssoU (Boe et al., 1987). Data were obtained as for pHV1610-1.

None of these proteins is involved in replication of pC 194, require any of the proteins tested. We conclude that ssiA from which derive the plasmids used to test ssiA activity belongs to the pas type of ssi. (Forough et al., 1987). The activity was tested by propagat- ing plasmid-harbouring mutants at permissive or semi- Role of ssiA in pAM3 1 replication permissive temperatures, as described below. ssiA is located close to the replication origin, on the Plasmid pHV1610-1, which carries ssiA, accumulated lagging strand template of pAMP1, and could therefore as much ss DNA as plasmid pHV1610, which lacks ssiA, be involved in priming of pAMf I lagging strand synthesis. in the dnaD and dnaI mutants already at 30°C, and in the Its inactivation should therefore affect plasmid replication. dnaB and dnaC mutants at 42°C (Figure 5A). In contrast, Interestingly, we observed that pIL253, a pAMi1 deriva- the dnaA mutation had no effect on pHV1610-1 ss DNA tive carrying ssiA, accumulates ss DNA in the dnaB, accumulation at any temperature. Since the temperature dnaC, dnaD, dnaE and ! mutants, but not in a wild- restrictive for the dnaA mutant is higher than that for the type strain, at 30 or 37°C (Figure 6A; ss DNA was clearly other mutants (49 instead of 46°C), ssiA activity was also visible in the dnaE strain upon longer exposure, not tested in the dnaA and wild-type cells at 46°C. ssiA shown). This ss DNA corresponds to the plasmid lagging activity was similar in both strains at this temperature (not strand template, as revealed by hybridization with strand- shown). In control experiments, the amount of ssDNA specific oligonucleotide probes (Figure 6B). These accumulated by pHV 161 1, which carries ssoU of pUB 10, observations indicate that ssiA might be involved in was always <20% of that accumulated by pHV1610 under pAM 1 lagging strand synthesis. However, a pIL253 the same conditions (Figure 5B). These results indicate derivative lacking ssiA (pIL253AAS) did not accumulate that ssiA requires the replication fork helicase DnaC and any ssDNA in wild-type B.subtilis cells, and accumulated the products of the dnaB, dnaD and dnaI genes, in addition no more ssDNA than pIL253 in the dnaB, dnaC, dnaD, to the DnaE primase, whereas the pUB 1O ssoU does not dnaE and dna! mutants (not shown; the dnaA mutant

2645 C.Bruand, S.D.Ehrlich and L.Janniere

A wt dnaE20 dnaB19 dnaC14 dnaD23 dnal'2 0 0~~ (0 30 37 30 37 30 37 30 37 30 37 30 37 C CD 0~~ CD CD 0 C)0- ia < Q 0 30 0 30

CCe M

Ss ccc ONmS - CCW1- 1- 1- - CSa ss 4 g B ss.

GCC Fig. 7. ssiA activity in S.aureus. Staphylococcus aureus cells RN4220 harbouring plasmids pCl94, pADG6406 or pADG6406-1 were grown to mid-log phase. For pADG6406-1 and pCI94, rifampicin was then added (100 ,ug/ml each) and total DNA was analysed before (0) or SS after 30 min (30) of incubation with the drug.

Host range of ssiA Fig. 6. Accumulation of ss DNA by plasmids derived from pAMP3l. pAM 1 has a large host range among Gram-positive Bacillus subtilis cells harbouring pIL253 were grown to mid-log phase bacteria. We therefore tested the activity of ssiA in a host at 30 or 37°C and their total DNA content was analysed as described For this ssiA was in the legend to Figure 2, except that the probe used was an other than B.subtilis, S.aureus. purpose oligonucleotide complementary to the leading strand template (A) or inserted into plasmid pADG6406, a pC 194 derivative the lagging strand template (B, only the bottom of the autoradiogram lacking its own sso (Gruss et al., 1987), giving the plasmid is shown). Growth temperatures and names of strains are indicated pADG6406- 1. The ratio of ss to ds DNA of pADG6406- 1 above the wells. The material migrating slightly more slowly than the was lower than that of pADG6406 in S.aureus cells CCC monomeric forms corresponds to replication intermediates (Bruand et al., 1993; L.Janniere, unpublished data). ss DNA (Figure 7), which indicates that ssiA is active in this host. accumulated in the dnaE20 strain was clearly visible on overexposed This activity is independent of RNA polymerase, since autoradiograms. Oligonucleotides used as probes were: 5'-GCTACT- pADG6406- 1 did not accumulate ss DNA upon incubation CTCTCCTTCTCC (in A) and 5'-GGAGAAGGAGAGAGTAGC with rifampicin (Figure 7). As expected, ss DNA of the (in B), (position 4724-4741 on the pAMPI sequence). control plasmid pC194 did accumulate in the presence of rifampicin, since the pC194 sso is sensitive to this drug (Boe et al., 1989). Since ssiA is active in at least two Gram-positive could not be correctly tested because pAM 1 replication bacteria, we tested its activity in the Gram-negative is thermosensitive at the temperature restrictive for the organism E.coli. ssiA was inserted in plasmid pHV33AHae, mutant; Bruand et al., 1993). Furthermore, the copy a pC194-pBR322 hybrid lacking the ssiB site of pBR322, number of pIL253AAS was similar to that of pIL253 in which accumulates ss DNA in E.coli (te Riele et al., four different hosts, B.subtilis, Efaecalis, Lactococcus 1986b). The presence of ssiA did not affect ss DNA lactis and Staphylococcus aureus (unpublished results). accumulation (not shown), which suggests that it does not These results indicate that ssiA either does not affect function in Gram-negative bacteria. pAMPl replication or it does, but in a more subtle way than can be detected by the tests used. Examination of the sequence of pIL253AAS did not reveal the presence Discussion of sequences similar to ssiA. We therefore assume that in Plasmid pAM,B 1 carries a single-strand initiation site, the absence of ssiA, lagging strand synthesis is initiated designated ssiA, on the lagging strand template, -150 bp by one or several altemative mechanisms. Since this downstream of the leading strand initiation site. The site synthesis is inefficient in the dnaB, dnaC, dnaD, dnaE contains a potential secondary structure and is relatively and dnal mutants, these altemative mechanism(s) are GC rich (42 against 32% in the replication region; Figure dependent on the corresponding proteins, as is the ssiA 3B). It is conserved in two plasmids related to pAMPI, function. pIP501 and pSM19035, as judged by sequence comparison

2646 ssiA of plasmid pAM,1

(Figure 3B) and functional assay (pIP501 ssi only was former occurs at the so-called n'-pas, found in OX174 and tested, C.Bruand, unpublished results). It is probably active in a number of plasmids, including pBR322, and requires in many Gram-positive hosts, since it functions in B.subtilis seven proteins: the DnaB and PriA (termed also and S.aureus, and is present on broad host range plasmids n' or factor Y), the DnaG primase and the products of isolated from E.faecalis (pAMPl), S.agalactiae (pIP501) genes dnaT, dnaC, priB and priC (see Komberg and Baker and S.pyogenes (pSM19035) (see Janniere et al., 1993 for 1992 for a review; Arai and Kornberg, 1981; Zipursky review). However, it is not functional in a Gram-negative and Marians, 1981). The ABC primosome assembles on host, E.coli. ssiA of plasmid R6K, a site carrying a hairpin structure ssiA is the first ssi detected on a theta replicating with a DnaA box at the stem, and requires DnaA, DnaB, plasmid in Gram-positive bacteria. It differs from the sso DnaC and DnaG proteins (Masai et al., 1990). The previously identified in rolling circle plasmids from these primosome assembled on ssiA of pAM,B1 is probably organisms by the following criteria: (i) it shares no more related to that of OX174 than to the ABC primosome, sequence homology with these sites, which are grouped for the following reasons: (i) it does not require the DnaA in four classes according to their primary structure (Gruss protein, (ii) ssiA does not contain a DnaA box, (iii) ssiA and Ehrlich 1989; Janniere et al., 1993), (ii) it does not contains a 12 bp motif present in the n'-pas of OX174, depend on RNA polymerase, (iii) it depends on the product R100 (ssiB) and F (ssiC), that has not been noticed of the dnaE gene, the putative B.subtilis primase (Wang previously (Figure 3B). This motif is located at similar et al., 1985). Nevertheless, it should be noted that two positions in all ssi, 5-8 bp downstream from a stem-loop sso were previously reported to be independent of RNA structure, and contains a G residue which is protected polymerase. However, they were different from ssiA since against methylation when the primosomal protein PriA is their activity was either replicon specific (Boe et al., 1991) bound to the pas in OX174 (Imber et al., 1983; Greenbaum or host specific (Leenhouts et al., 1991). Taken together, and Marians, 1984; Nomura et al., 1991). In spite of this these data indicate that ssiA acts via a mechanism different resemblance to n'-pas, ssiA is inactive in E.coli, suggesting from that of previously described ssi of Gram-positive that it is not recognized by PriA. bacteria. Another indication that ssiA is a pas is its location on Two types of primase-dependent ssi are known in E.coli, pAM31, -150 bp downstream of the origin. pAM 1 G sites and primosome assembly sites (pas). Both are leading strand synthesis is initiated by Pol I (Bruand et al., active on templates coated with the single-strand binding 1993), which generates displacement loops (L.Janniere, protein, but G sites direct the synthesis of a single primer unpublished results). This mode of initiation implies a by the primase alone, whereas the pas direct the loading specific mechanism of entry for the replication fork of several proteins into a complex, the primosome. This helicase and the primase required for the coupled synthesis complex contains, among other proteins, the replicative of the two DNA strands by Pol III. In plasmids related to DnaB helicase which renders the complex mobile and ColEl, from Gram-negative bacteria, replication is also able to capture the primase. The primosome can thus initiated by Pol l, and loading of the helicase and primase move along the DNA and synthesize primers at different is mediated by the assembly of a primosome, taking place locations on the template. Three lines of evidence indicate at a n'-pas located -150 bp downstream from the origin, that ssiA of pAM1 is a primosome assembly site. First, on the lagging strand template (Zipursky and Marians, ssiA does not show any sequence homology with the well 1981; Minden and Marians, 1985; Masai and Arai 1988). conserved motifs of G sites (Masai et al., 1990; Tanaka We propose that ssiA and pas are functionally equivalent et al., 1994). Second, and more important, ssiA requires and that the early stages of pAM,B1 replication take place the B.subtilis dnaC gene product. This gene encodes a as represented in Figure 8. protein homologous to the Ecoli DnaB helicase (42% Like the n'-pas of pBR322 (Van der Ende et al., 1983), identity), and is thus probably the replicative helicase of ssiA of pAMP1 is not essential for plasmid replication: it is B.subtilis (Ogasawara et al., 1994; N.Ogasawara personal located outside the minimal replication region of pAMP1, communication). Finally, ssiA requires at least three other pIP501 and pSM19035 in B.subtilis (Brantl and Behnke, proteins, the B.subtilis dnaB, dnaD and dnaI gene products. 1992a; Bruand et al., 1993; Ceglowski et al., 1993) and Homologues of these proteins are not known in Ecoli is not required for maintenance of a pAM,B1-derived (Ogasawara et al., 1986; Hoshino et al., 1987; Bruand plasmid in three different hosts. Furthermore, deletion of and Ehrlich, 1995; Bruand et al., 1995) and their function ssiA in pAMf 1 derivatives does not lead to ss DNA remains to be elucidated. However, the purified DnaB accumulation in B.subtilis. In E.coli, several alternative, protein was reported to be a very strong non-specific ss n'-pas-independent, mechanisms of primosome loading DNA binding protein, present both in the cytosol and in have been described (Masukata et al., 1987; Seufert and the membrane in association with other proteins (Laffan Messer, 1987; Ma and Campbell, 1988; Masai and Arai, and Sueoka, 1993; Sueoka et al., 1993). We propose that 1989a). We have therefore to assume that in B.subtilis, as these proteins may be components of the primosome in E.coli, alternative mechanisms of primosome loading assembled at ssiA. An alternative possibility is that these also exist. proteins are subunits of the DNA polymerase III (Pol III) Although ssiA is not required for pAMP1 replication, involved in the conversion of ss to ds DNA. However, the primosome appears to be, for two reasons. First, in this hypothesis is less likely, since none of the proteins is the absence of proteins involved in ssiA activity, plasmid required when this conversion is primed by the RNA ss DNA accumulates (Figure 6). A similar accumulation polymerase at ssoU of pUB 110. was observed in dnaD, dnaI and dnaC mutant strains for Two types of primosome are known in E.coli, the pSM19035, a plasmid highly related to pAMP1 (Ceglowski oX174 type and the ABC primosome. Assembly of the et al., 1993, J.Alonso, personal communication). This 2647 C.Bruand, S.D.Ehrlich and L.Janniere

iritiation site functions involved in ssiA activity were originally characterized as deficient in chromosomal DNA replication -1 (Karamata and Gross, 1970). The subsequent sequence RNA pol analysis clarified the role of dnaC and dnaE gene products, RepE given their homology with the DnaB helicase and DnaG primase of E.coli, respectively (Wang et al., 1985; Ogasawara et al., 1994). However, no clear function has 2 been assigned to B.subtilis DnaB, DnaD and DnaI proteins, in spite of sequence determination (Ogasawara et al., 1986; Hoshino et al., 1987; Bruand and Ehrlich, 1995; Bruand et al., 1995). At least two of these, DnaB and 3 DnaD, are involved in initiation of replication (Gross et al., 1968). In the light of this work, we propose that the function of these proteins could possibly be to direct the replication fork helicase DnaC towards oriC via Po!o primosome assembly. It is interesting to note in this 4 context that DnaB and the termination protein Rtp have a short amino acid motif in common (Kralicek et al., poloe 1991). Since Rtp interacts with the replicative helicase (at least in E.coli; Kaul et al., 1994), it is possible that DnaB does so as well. 5 Escherichia coli homologues of DnaB, DnaD and DnaI proteins are not known, indicating either that they have not yet been identified in this organism, in spite of the primosome 9\<. -- >Ql;l numerous studies of DNA replication, or that they are not present. Their absence could account for the lack of ssiA activity in E.coli. DNA hybridization experiments revealed sequences homologous to B.subtilis dnaB in many organ- 6 isms, including Archaebacteria and eukaryotic cells, but not in Ecoli (Sueoka et al., 1993). If the presence of this gene is indicative of a common mechanism of initiation of DNA replication, the one used by B.subtilis may be much more widely distributed than that of E.coli. 7 Materials and methods Bacterial strains and growth conditions Fig. 8. Model for the initiation of pAMPl replication. (1) The host The strains used were (i) B.subtilis 1012 (leuA8 metB5 r m+; Saito RNA polymerase approaches the site of initiation of DNA synthesis. et al., 1979), L1430 (ilvA metC /vs-21), L1432 (dnaBI9 mnetC ilvA), (2) A DNA-RNA heteroduplex is formed in the region of the L1433 (dnaCI4 mnetC /vs-21), L1434 (dnaD23 metC lys-21), L1435 initiation site. (3) The transcript is converted into a primer by site- (dnaE20 metC ilvA) and L1439 (dnal2 metC ilvA) (Mauel and Karamata, specific RNA cleavage. RepE could be involved in the formation of 1984); the dnaA I mutation described in Moriya et al. (1990) was moved the heteroduplex and/or in the site-specific cleavage. (4) Pol I to the L1430 background by congression with the lys+ allele; (ii) E.coli recognizes the 3' OH end of the primer. (5) Pol I carries out initial strain MT102 (strA araDl39 leu lacX74 galU galK hsdR; Casadaban leading strand DNA synthesis. (6) ssiA is activated on the displaced and Cohen, 1980); (iii) S.aureus RN4220 (Kreiswirth et al., 1983); (iv) strand. (7) A primosome is assembled, allowing initiation of lagging L.lactis subsp. lactis IL1403 (Simon and Chopin, 1988) and (v) E.faecalis strand synthesis and duplex unwinding in front of the fork, thus JH2-2 (Jacob and Hobbs, 1974). Luria broth medium was used for enabling Pol III to replace Pol I. growing B.subtilis or Ecoli cells, M17 glc (glucose 5 mg/ml) or brain heart infusion were used for growing the other bacteria. Media were supplemented, when necessary, with agar (15 mg/ml), chloramphenicol accumulation presumably results from inefficient lagging (5 gg/ml), erythromycin (0.3-5 ,ug/ml) or ampicillin (100 ,ug/ml). strand synthesis, allowing displacement loops of the For detection of ss DNA, overnight cultures at 37°C (or 30°C for thermosensitive mutants) were diluted 100-fold in the same medium and plasmid size to form. Lagging strand synthesis could be grown to OD650 -0.6-0.8 at 370C (30, 37 or 42°C for the mutants). inefficient because of deficient primer synthesis, due to primase inactivation (in the dnaE mutant), or inability of Plasmids the primosome to attract the primase (in the dnaC mutant). Plasmid pIL253 (Simon and Chopin, 1988) essentially contains the In other mutants, the primosome may be unable to replication region of pAMPI shown in Figure IA plus the erythromycin assemble. If the appropriate helicase is not loaded, dis- resistance gene of pAM,I. Plasmids pHV 1610, pHVI611, pC194, pADG6406 and pHV33AHae were described previously (Horinouchi placement loops could be due either to the strand displace- and Weisblum, 1982; te Riele et al., 1986b; Gruss et al., 1987; Boe ment activity of Pol I or to the unwinding activity of et al., 1989). pHV1610-1 was constructed by inserting the Nhel-HpaII another, unidentified, DNA helicase. Second, it was (blunted) segment of pIL253 into the XbaI-SalI (blunted) cut pHV1610. reported that proteins involved in ssiA activity may be pHV1610-2 was constructed by inserting the NheI-EcoRI segment of pIL253 into the XbaIlEcoRI cut pHV1610. Plasmid Al was constructed required for replication of pSM19035 (Ceglowski et al., by inserting the AseI (blunted)-Pstl segment of pIL253 into the Small 1993). PstI cut pHV 1610. Plasmids AG3.8, AD5.24 and ADS. 18 were generated The B.subtilis thermosensitive mutants used to analyse by introducing unidirectional nested deletions from the left or right end

2648 ssiA of plasmid pAMP1 of the insert of plasmid pHV1610-1 by ExolII/S I treatment (Sambrook coli replication factor Y with complementary strand origins of DNA et al., 1989), using the KpnI-BamHI or PstI-SalI sites, respectively. replication. J. Biol. Chem., 259, 2594-2601. Plasmid pIL253AAS derives from pIL253 by a deletion between the Gros,M.-E, te Riele,H. and Ehrlich,S.D. (1987) Rolling circle replication Asel and Smal sites. Plasmid pADG6406- 1 was constructed by inserting of single stranded DNA plasmid pC194. EMBO J., 6, 3863-3869. the Smnal-HincII segment of pHV 1610-1 into the PvuII site of Gross,J.D., Karamata,D. and Hempstead,P.G. (1968) Temperature- pADG6406. ssiA was inserted in the EcoRI site of plasmid pHV33AHae sensitive mutants of B.subtilis defective in DNA synthesis. Cold as an EcoRI segment from pHV1610-1. Spring Harbor Symp. Quant. Biol., 33, 307-312. Gruss,A., Ross,H. and Novick,R. (1987) Functional analysis of a DNA analysis palindromic sequence required for normal replication of several All standard DNA manipulations were carried out essentially as described staphylococcal plasmids. Proc. Natl Acad. Sci. USA, 84, 2165-2169. in Sambrook et al. (1989). Single stranded plasmid DNA was detected Gruss,A. and Ehrlich,S.D. (1988) Insertion of foreign DNA into plasmids as described by te Riele et al. (I 986a,b). DNA sequencing was done from Gram-positive bacteria induces formation of high-molecular- using the Sequenase kit from USB. Quantification of ss and ds DNA weight plasmid multimers. J. Bacteriol., 170, 1183-1190. amounts on Southern blots was performed using a Phosphorlmager SI Gruss,A. and Ehrlich,S.D (1989) The family of highly interrelated single apparatus from Molecular Dynamics. stranded deoxyribonucleic acid plasmids. Microbiol. Rev., 53, 231-241. Honda,Y., Nakamura,T., Tanaka,K., Higashi,A., Sakai,H., Komano,T. and Bagdasarian,M. (1992) DnaG-dependent priming signals can Acknowledgements substitute for the two essential DNA initiation signals in oriV of the broad host-range plasmid RSFIOIO. Nucleic Acids Res., 20, We thank N.Ogasawara for providing the dnaA strain, J.C.Alonso for 1733-1737. kindly communicating unpublished results, N.Goupil and P.Tortosa for Horinouchi,S. and Weisblum,B. (1982) Nucleotide sequence and help in isolating exonuclease III-mediated deletions, and C.Anagnosto- functional map of pCI94, a plasmid that specifies chloramphenicol poulos, E.Le Chatelier, B.Michel, M.-F.Noirot-Gros and M.-A.Petit for resistance. J. Bacteriol., 150, 815-825. stimulating discussions and critical reading of different versions of this Hoshino,T., McKenzie,T., Schmidt,S., Tanaka,T. and Sueoka,N. (1987) manuscript. This work was supported, in part, by EEC grant BIOT Nucleotide sequence of Bacillus subtilis dnaB: a gene essential for CT910268. DNA replication initiation and membrane attachment. Proc. Natl Acad. 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Received on January 27, 1995; revised on March 27, 1995

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