Characterization of a single-strand origin, ssoU,required for broad host range replication ofrolling-circle plasmids

M. Gabriela Kramer,1 Manuel Espinosa,2 Tapan K.

Misra3 and Saleem A. Khan1*1Department of Molecular Genetics and Biochemistry,

University of Pittsburgh School of Medicine, Pittsburgh,

PA 15261, USA.2Centro de Investigaciones Biologicas, CSIC, Velazquez,

144, E-28006 Madrid, Spain.3Department of Microbiology and Immunology, University

of Illinois College of Medicine, Chicago, IL 60612, USA.

Summary

Single-stranded DNA (ssDNA) promoters are the key

components of the single-strand origins (ssos) of

replication of rolling-circle (RC) replicating plasmids.

The recognition of this origin by the host RNA poly-

merase and the synthesis of a short primer RNA are

critical for initiation of lagging-strand synthesis. This

step is thought to be a limiting factor for the establish-

ment of RC plasmids in a broad range of bacteria,

because most of the ssos described are fully active

only in their natural hosts. A special type of sso, the

ssoU, is unique in the sense that it can be ef®ciently

recognized in a number of different Gram-positive

hosts. We have experimentally deduced the folded

structure and characterized the ssDNA promoter pre-

sent within the ssoU using P1 nuclease digestion and

DNase I protection assays with the Bacillus subtilis and

Staphylococcus aureus RNA polymerases. We have

also identi®ed the RNA products synthesized from

this ssDNA promoter and mapped the initiation points

of lagging-strand synthesis in vivo from ssoU-contain-

ing plasmids. Through gel mobility shift experiments,

we have found that ssDNA containing the ssoU sequ-

ence can ef®ciently interact with the RNA polymerase

from two different Gram-positive bacteria, S. aureus

and B. subtilis. We have also realigned the narrow

and broad host range sso sequences of RC plasmids,

and found that they contain signi®cant homology. Our

data support the notion that the strength of the RNA

polymerase±ssoU interaction may be the critical factor

that confers the ability on the ssoU to be fully func-

tional in a broad range of bacteria.

Introduction

Single-stranded DNA (ssDNA) intermediates are generated

during the rolling-circle (RC) mode of DNA replication of

most small, drug resistance plasmids in Gram-positive bac-

teria and of many in Gram-negative bacteria (for reviews,

see Gruss and Ehrlich, 1989; Novick, 1989; Khan, 1997).

Similar to the ®lamentous coliphages, the conversion of

the ssDNA ! double-stranded DNA (dsDNA) involves the

synthesis of an RNA primer by the host RNA polymerase

in most cases (Dempsey et al., 1995; Kramer et al., 1997,

1998a). A group of ssDNA promoters are the key compo-

nents of the single strand origins of replication (ssos) that

are essential for ef®cient initiation of lagging-strand synth-

esis in these plasmids. Four types of ssos, ssoA, ssoT,

ssoU and ssoW have been described in bacterial RC plas-

mids (Gruss et al., 1987; Boe et al., 1989; Devine et al.,

1989; Leenhouts et al., 1991). Defects in the sso activity

lead to accumulation of ssDNA in vivo (del Solar et al.

1987; Meijer et al., 1995; Kramer et al., 1995; 1998b). We

have previously analysed the function of the ssoA-type

origins present in plasmids pE194 and pLS1 (Kramer et

al., 1997; 1998a). The ssoA and ssoW type origins are

fully active only in their native hosts (te Riele et al., 1986;

Gruss et al., 1987; del Solar et al., 1987). On the other

hand, lagging-strand replication from the ssoU-type origins

is very ef®cient in various Gram-positive bacteria such

as Bacillus subtilis, Staphylococcus aureus, Streptococcus

pneumoniae and Lactococcus lactis (Boe et al., 1989; Van

der Lelie et al., 1989; Kramer et al., 1995; Meijer et al.,

1995; Seegers et al., 1995).

Much is known about the mechanism of initiation of RNA

transcription involving dsDNA promoters and the bacterial

RNA polymerase (for reviews, see Helman and Chamber-

lin, 1988; Lonetto et al., 1992). It is well established that

initiation of RNA synthesis requires a speci®c interaction

between the RNA polymerase and its cognate promoter

sequence, and that the sigma subunit of the holoenzyme

is responsible for this speci®city (Helmann, 1994). The con-

sensus ÿ10 (58-TATAAT-38) and ÿ35 (58-TTGACA-38)

promoter regions that are critical for gene expression

have been described for Escherichia coli, B. subtilis and

Molecular Microbiology (1999) 33(3), 466±475

Q 1999 Blackwell Science Ltd

Received 5 February, 1999; revised 13 April, 1999; accepted 19 April,1999. *For correspondence. E-mail Khan@pitt.edu; Tel. (�1) 412 6489025; Fax (�1) 412 624 1401.

S. aureus RNA polymerases (McClure, 1985; Lisser and

Margalit, 1993; Helmann, 1995). These holoenzymes are

associated with the s70(sD), sA and sSA factors, respec-

tively, which are the most commonly used sigma subunits

in these organisms (Collado-Vides et al., 1991; Rao et al.,

1995; Deora and Misra, 1995; 1996). In a few cases, RNA

polymerases are also known to recognize promoters pre-

sent within ssDNA. Examples of ssDNA promoters for

gene expression were described in the coliphage N4

(Haynes and Rothman-Denes, 1985). These promoters

are recognized by the virion RNA polymerase, and share

sequence homology in a region that contains essential

inverted repeats for promoter activity (Glucksmann et al.,

1992). A different kind of ssDNA promoter, Frpo, found in

the F plasmid of E. coli is used for gene expression and

appears to play a role during plasmid conjugation (Masai

and Arai, 1997).

Another class of ssDNA promoters is involved in the

conversion of the circular ssDNA to the dsDNA (replicative

form) of ®lamentous phages of E. coli (Geider and Korn-

berg, 1974; Geider et al., 1978; Kaguni and Kornberg,

1982). In this case, the host RNA polymerase recognizes

a promoter sequence on the minus strand replication ori-

gin and synthesizes a 20-nt-long primer RNA that is utilized

by DNA polymerase III for lagging-strand synthesis (Higa-

shitani et al., 1993; 1997). Interestingly, no evident ÿ35

and ÿ10 consensus sequences were found and mutations

that changed the putative ÿ10 region into a perfect cano-

nical sequence did not affect the ef®ciency of transcription

from this promoter (Higashitani et al., 1997).

In this work, we have analysed the ssoU-type origins

of lagging-strand replication and the molecular basis of

their broad host range function. Previous in vivo studies

localized the ssoU to within a 270 nt region that has the

potential to form an extensive secondary structure (Boe

et al., 1989). This origin is present in the staphylococcal

pUB110, streptococcal pMV158 and Bacillus pTB913 RC

plasmids (Viret and Alonso, 1988; Boe et al., 1989; van

der Lelie et al., 1989). In this paper, we provide experi-

mental evidence for the secondary structure of the ssoU

using nuclease P1 and DNase I cleavage analysis. We

have identi®ed the RNA polymerase-binding region within

the ssoU promoter by footprinting analysis. We have also

identi®ed the primer RNA synthesized by the S. aureus

and B. subtilis RNA polymerases in vitro and localized

the initiation points of lagging-strand synthesis in vivo. The

ssoU promoter was active when present within a ssDNA,

but was inactive when contained in the dsDNA. We discuss

the existence of a common RNA polymerase interaction

region in all the sso types described to date, based on a

realignment of their sequences and footprinting data.

Our results also suggest that a strong interaction between

the ssoU and RNA polymerases from different bacterial

hosts is an important factor in determining the broad

host range replication of ssoU-containing RC plasmids

and this may contribute to the horizontal spread of drug

resistance plasmids in nature.

Results

In vitro RNA synthesis from the ssoU and mapping of

the transcription initiation points

The DNA sequence of the pMV158 and pUB110 plasmids

has been published and their accession numbers are

M29725 and M19465 respectively (Lacks et al., 1986;

McKenzie et al., 1986; Priebe and Lacks, 1989). Both

plasmids contain the ssoU sequence, and an alignment

of these regions showed differences in the sequences

between nucleotides 3355 and 3374 of pMV158 and

1372±1395 of pUB110 (Priebe and Lacks, 1989; van der

Lelie et al., 1989). However, during our initial studies, we

resequenced the pMV158 ssoU region and found several

errors in its published sequence. All the errors were con-

®ned to the above variable region, and the corrected sequ-

ence showed that the ssoU sequences were identical in

the pUB110 and pMV158 plasmids (see Fig. 5). The cor-

rected sequence added four nucleotides to the pMV158

genome that were missing in the published sequence.

Throughout this study, the same pUB110/pMV158 ssoU

was used in our experiments.

It is known that ssoU-type origins can support ef®cient

lagging-strand replication in S. aureus and B. subtilis. To

study whether RNA polymerases from these bacterial

hosts were able to recognize ssoU as a template for primer

RNA synthesis, ssDNA fragments containing the ssoU

were tested in in vitro transcription experiments using

both the B. subtilis and the S. aureus RNA polymerases.

As the ssos are strand speci®c, we also used a ssDNA frag-

ment containing the sequence complementary to ssoU

(ssoU ÿ) as a negative control. Three major RNAs of

44±46 nt were synthesized by both RNA polymerases

when the ssDNA containing the active ssoU was used

as the template (Fig. 1A). No transcripts were generated

when the ssoU ÿ sequence was used, con®rming the

strand speci®city of this origin of replication. When dsDNA

containing the ssoU sequence was used in the above

experiment, no RNA was synthesized (Fig. 1A). These

results showed that the promoter present within the ssoU

sequence could be recognized by two different RNA poly-

merases for RNA synthesis and these enzymes recognize

the promoter region on the ssoU only when it is present in

a ss form.

To localize the 58-end of the RNA synthesized from the

ssoU, primer extension analysis was performed. The tem-

plate used for reverse transcription was ssoU-directed,

unlabelled de novo synthesized RNA obtained from the in

vitro transcription reactions (see Experimental procedures ).

Q 1999 Blackwell Science Ltd, Molecular Microbiology, 33, 466±475

ss origin of rolling-circle plasmids 467

Primer extension analysis using an oligonucleotide (nt

1320±1344 on the pUB110 map) corresponding to the

region downstream of the predicted in vivo initiation site

of lagging-strand DNA synthesis generated a single band

of 27 nt (Fig. 1B). These data showed that RNA synthesis

from the ssoU initiates with a G residue at position 1318

of pUB110 (nt 3432 of pMV158) and terminates at posi-

tions 1361±1363 (nt 3387±3389 for pMV158) located at

the end of hairpin III (see Fig. 4B).

In vivo mapping of the initiation points of DNA

synthesis from the ssoU

To identify the initiation points of lagging-strand DNA synth-

esis in vivo from the ssoU in different Gram-positive bac-

teria, repeat primer extension analysis was carried out.

Plasmid pMV158 and pUB110 DNA was isolated from S.

aureus and/or S. pneumoniae. In addition to fully repli-

cated supercoiled DNA, these preparations are expected

to contain trace levels of replicative intermediates in which

the lagging-strand synthesis has not been completed. In

such cases, this DNA strand would contain a ®xed 58 end

corresponding to the initiation site of synthesis and random

38 ends. A 58-end-labelled oligonucleotide primer (corre-

sponding to position 1372±1392 of pUB110) was used

for polymerase chain reactions (PCRs). This oligonucleo-

tide was chosen so as to anneal several nucleotides down-

stream of the predicted start point of lagging-strand

synthesis within the ssoU. The PCR would allow extension

synthesis from the primer oligonucleotide until it reaches

the 58 end of the lagging strand. We have previously

shown that the RNA primers required for initiation are

removed from the DNA prior to the completion of lagging-

strand synthesis (Dempsey et al., 1995; Kramer et al.,

1998b). Thus, the repeat primer extension assay should

yield band(s) that correspond to the distance between

the primer hybridization site and transition point(s) from

RNA to DNA synthesis. On the other hand, primer exten-

sion from fully replicated plasmid molecules would yield

a smear of large DNA products depending upon the ef®-

ciency and processivity of the Taq polymerase. Analysis

of the reaction products on a DNA sequencing gel showed

major bands of 60, 89, 91 and 96 nt along with the larger

products (LPs) (Fig. 2). These bands were absent when

plasmid DNA from pLS1, a pMV158 derivative that lacks

the ssoU, was used in the above experiment (Fig. 2). The

smallest, 60 nt, band positioned the initiation site of lag-

ging-strand DNA synthesis at the T residue of the template

(nt 1333 for pUB110 and 3417 for pMV158; see Fig. 4B).

The 89, 91 and 96 nt bands may correspond to additional

points of initiation of lagging-strand synthesis. In vitro tran-

scription experiments did not generate the additional tran-

scripts that would suggest initiation at these sites, indicating

that the above bands may represent `minor' initiation sites

that are observed only upon their ampli®cation by primer

extension, which is a qualitative assay. Based on the map-

ping of the transcription initiation site in vitro (Fig. 1A and

Q 1999 Blackwell Science Ltd, Molecular Microbiology, 33, 466±475

Fig. 1. RNA polymerase-directed synthesis of RNAs from the ssoUand determination of their 58-end.A. ssDNAs containing the pUB110/pMV158 ssoU in the functional(�) or non-functional (ÿ) orientation were used as templates.Reactions were carried out using the S. aureus (S.au) or B. subtilis(B.su) RNA polymerase (RNAP). ds, double-stranded DNAcontaining the ssoU sequence; ACGT, sequencing laddergenerated by the Sanger method. Numbers indicate sizes innucleotides.B. In vitro synthesized, ssoU-directed RNA was used as a templatefor primer extension analysis using an oligonucleotide primer. Theposition of the 27 nt product (P) is indicated. C and T, sequencingladders generated by the Sanger method.

Fig. 2. Determination of the in vivo initiation points of lagging-strand synthesis from the ssoU sequence in S. aureus and S.pneumoniae. The products of repeat primer extension assays usingthe indicated plasmids puri®ed from S. aureus or S. pneumoniae(S. pn) were separated on a denaturing polyacrylamide±urea geland their sizes are indicated. LP, large products (see Results );ACGT, sequencing ladder.

468 M. G. Kramer et al.

B), it is most likely that the 60 nt band represents the

primary RNA to DNA transition point. The above results

show the existence of speci®c initiation positions of lag-

ging-strand DNA synthesis from the ssoU origin of both

the pMV158 and pUB110 plasmids in S. aureus and S.

pneumoniae. Moreover, the major initiation points for

both of these RC plasmids were identical in the two bac-

terial hosts tested.

B. subtilis and S. aureus RNA polymerases interact

with the ssoU

The ssoU-type origins can function ef®ciently in vivo in a

number of Gram-positive bacteria, and we have previously

reported that ssoA±RNA polymerase interactions play

an important role in the host speci®city of RC plasmids

(Kramer et al., 1998a). Therefore, we wished to determine

whether ssoU could interact ef®ciently with the RNA poly-

merase from different hosts. To address this issue, we per-

formed electrophoretic mobility shift assays (EMSAs)

using a 58-end-labelled ssoU DNA and the two available

RNA polymerases from B. subtilis and S. aureus. These

enzymes bound equally well to the ssoU in the functional

orientation, while their interaction with the complementary

ssoUÿ sequence was much weaker (Fig. 3). These results,

together with our previous data, suggest that the ability of

the host RNA polymerases to bind ef®ciently to the promo-

ter sequence present within the plasmid ssoU is a major

determinant of the high in vivo activity of ssoU in B. sub-

tilis, S. aureus and other Gram-positive organisms.

Determination of the secondary structure of ssoU

and identi®cation of the RNA polymerase binding site

As folding of the ssDNA containing the ssos is likely to

be important for their function (Gruss et al., 1987; Kramer

et al., 1997), an enzymatic analysis was carried out to

determine the ssoU structure. For this, we used nuclease

P1, which preferentially digests single-stranded regions in

the DNA. Treatment of the 58-end-labelled ssoU DNA with

the P1 nuclease generated several discrete bands (Fig.

4A). In the presence of low levels of P1 (0.03 units), two

major cleavage sites were present in regions 5 and 6

located within loops of hairpin II (see Fig. 4C). At higher

levels of P1, major cleavage sites were present in regions

6 and 10 of hairpin II as well as in additional ss regions of

the ssoU. We also performed nuclease P1 mapping experi-

ments using a 38-end-labelled ssoU DNA (Fig. 4B). This

allowed structural mapping of the ssDNA regions located

near the 38 end of ssoU within hairpins III and IV (Fig.

4C). Treatment of the ssoU DNA with DNase I, which pre-

ferentially cleaves dsDNA, generated a large number of

bands (Fig. 4B), suggesting that ssoU contains extensive

double-stranded regions. The predicted folded structure

of the ssoU (Fig. 4C) was generally consistent with the

results of the nuclease P1 and DNase I cleavage analysis.

The structure of hairpin V is based on computer analysis

and this region was not affected in the above experiments

(data not shown).

The ssoU sequence does not appear to contain any

region with signi®cant homology to the canonical promoter

sequences. To identify the RNA polymerase-binding region

within the ssoU, we performed footprinting experiments

using both 58-and 38-end-labelled ssoU DNA substrates

for DNase I and P1 nuclease digestion (Fig. 4B). The

nucleotides protected from DNase I digestion by the B. sub-

tilis RNA polymerase are indicated in Fig. 4C. Two major

protected regions were identi®ed within hairpin II when 58-

end-labelled ssoU DNA was used (Fig. 4B and C). Using

38-end-labelled ssoU DNA, we identi®ed the RNA polymer-

ase-binding sequences within hairpin III. No protection was

observed when the P1 nuclease was used (Fig. 4B), indi-

cating that RNA polymerase does not appear to interact

with the ssDNA regions of the ssoU. Similar results were

obtained when the S. aureus RNA polymerase was used

in these experiments (data not shown). Overall, the ssoU

structure predicted by computational methods was found

to be consistent with the experimental results, and we

mapped the host RNA polymerase binding sites within

the ssoU, which is likely to act as the promoter for primer

RNA synthesis.

Discussion

The ssoU origin is unique in the sense that it functions

ef®ciently as a signal for ss ! dsDNA conversion during

Q 1999 Blackwell Science Ltd, Molecular Microbiology, 33, 466±475

Fig. 3. EMSA of S. aureus and B. subtilis RNA polymerases boundto labelled ssDNA fragments. The top panel shows EMSA usingB. subtilis RNA polymerase and the ssoU in the functional (ssoU)or non-functional (ssoUÿ) orientation while the bottom panel showsEMSA using the S. aureus RNA polymerase. The ssDNAs wereincubated with the indicated amounts of RNA polymerase (innanograms). Positions of the bound (B) and free (F) ssDNAs areindicated.

ss origin of rolling-circle plasmids 469

Q 1999 Blackwell Science Ltd, Molecular Microbiology, 33, 466±475

470 M. G. Kramer et al.

plasmid RC replication in a broad range of Gram-positive

bacteria. Therefore, the presence of the ssoU sequence

is an important factor in determining, at least in part, the

promiscuity of RC plasmids. In the present study, we

have de®ned the RNA polymerase binding site within the

ssoU sequence and localized the RNAs synthesized from

the ssoU. These RNAs (44±46 nt) are likely to be the pri-

mers used by the host DNA polymerases for lagging-strand

synthesis, as shown previously for the ssoA-type origin of

pLS1 (Kramer et al., 1997). We have also mapped the tran-

sition points from RNA to DNA synthesis in vivo in S. aureus

and S. pneumoniae in plasmids containing the ssoU origin.

Our in vitro transcription experiments revealed that the

B. subtilis and S. aureus RNA polymerases recognize a

ssDNA promoter within the ssoU for RNA synthesis (Fig.

1A). These enzymes were unable to initiate transcription

when the ssoU was present within dsDNA (Fig. 1A). In a

previous work, we have shown that the single-stranded-

ness of the conserved RSB sequence in the ssoA origins

was necessary for RNA polymerase activity (Kramer et

al., 1997; also see below). Other examples of ssDNA

promoters that are inactive when present in dsDNA

include those present in the N4 coliphage and in the F

plasmid (Frpo promoter), which function only when the

DNA template is supercoiled or denatured (Haynes and

Rothman-Denes, 1985; Masai and Arai, 1997). In general,

supercoiled templates are transcribed more ef®ciently than

linear DNA, and it is known that the interaction of the s70

subunit of E. coli holoenzyme with dsDNA promoters

involves a speci®c ssDNA-binding domain (McClure,

1985; Callaci and Heyduk, 1998). Also, it has been

reported that the B. subtilis sA as well as the E. coli s70

holoenzymes bind with high selectivity to ssDNA contain-

ing the non-consensus ÿ10 sequence (Roberts and

Roberts, 1996; Huang et al., 1997). Therefore, ssDNA

promoters may have a functional advantage because of

their ability to bind more strongly to the RNA polymerase.

This possibility is consistent with the observation that the

af®nity of the E. coli RNA polymerase for the minus-strand

origin of ®lamentous bacteriophages is about 16-fold

higher than for the duplex lacUV5 promoter (Higashitani

et al., 1997).

Previously, four different groups of plasmid ssos, namely

ssoA, ssoU, ssoT and ssoW, were described. No nucleo-

tide sequence homology was noted between the four sso

groups, but sequence and structure similarities between

ssos belonging to the same group were reported (Gruss

et al., 1987; Novick, 1989; Dempsey et al., 1995). As repli-

cation from these ssos is inhibited by rifampicin, a role for

RNA polymerase in lagging-strand synthesis has been

postulated in all the cases (Boe et al., 1989; Birch and

Khan, 1992; Dempsey et al., 1995; Kramer et al., 1995;

Seegers et al., 1995). We wished to investigate the mole-

cular basis for the ability of a RNA polymerase to recog-

nize many different promoter sequences present within

apparently unrelated ssos. We performed a realignment

of the ssoA, ssoU, ssoT and ssoW sequences (Fig. 5).

In contrast to previous reports, a considerable degree of

homology between the four sso types was observed,

especially in the conserved RSB region, which was origin-

ally thought to be present only in the ssoA-type origins

(Novick, 1989). In all the ssos aligned, the RSB or RSB-

like regions were located in the stem of a hairpin structure.

This conserved sequence is known to be important for the

binding of RNA polymerase to the ssoA (Kramer et al.,

1997). Our structural analysis of the ssoU by P1 nuclease

and DNase I demonstrates that ssoU has an extensive

secondary structure that is very similar to that predicted

by using computer programs. DNase I footprinting analy-

sis showed that RNA polymerase binds to the ssoU in

the ds regions of hairpins II and III (Fig. 4B and C). One

of the protected regions mapped within the region that

shares homology with the RSB sequence (Fig. 5). Based

on earlier studies and the results presented in this paper,

we propose that the regions conserved among the four

types of ssos, especially the RSB or RSB-like sequence,

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Fig. 4. Nuclease P1 and DNase I mapping and identi®cation of the RNA polymerase binding sites within the ssoU, and its deduced secondarystructure.A. Nuclease P1 cleavage analysis of the ssoU using indicated amounts of the enzyme. An ssDNA fragment containing the ssoU (nt 1204±1551 of pUB110) was labelled at its 58 end with 32P and used in this experiment. The digestion products were analysed on a denaturingpolyacrylamide±urea gel. 5 and 6 refer to P1 nuclease-sensitive subregions in the folded structure of ssoU (C).B. Nuclease P1 and DNase I footprinting of ssoU. The ssoU was labelled at its 58 or 38 end with 32P and treated with either the P1 nuclease(0.3 units) or DNase I (15 units) or both in the absence (0) or presence (0.5 and 2 mmg) of the B. subtilis RNA polymerase. The digestionproducts were analysed on a denaturing polyacrylamide±urea gel. I, II, III, IV and V refer to the ®ve hairpin regions of ssoU shown in C.a and b, two major regions of ssoU protected from DNase I cleavage in the presence of the RNA polymerase; E1 and E2, sites of enhancedP1 nuclease cleavage in the presence of RNA polymerase; 1±16, P1 nuclease-sensitive subregions in the folded structure of ssoU (C); ACGT,DNA sequencing ladder.C. Deduced secondary structure of ssoU. Information obtained from the nuclease P1 and DNase I cleavage patterns was used in conjunctionwith the PC FOLD sequence analysis program to deduce the ssoU folded structure. The nucleotide coordinates correspond to those of thepUB110 plasmid. I, II, III, IV and V refer to the ®ve hairpin regions of the ssoU fragment used in this study. The initiation sites of in vitro primerRNA synthesis and in vivo DNA synthesis are indicated. Subregions 1±16 of ssoU are indicated in green along with the sites of enhanced P1nuclease cleavage shown in B. Nucleotides protected by RNA polymerase from DNase I cleavage are shown in red and sequencesresembling the CS-6 and RSB of ssoA origins are indicated.

ss origin of rolling-circle plasmids 471

may play a crucial role in RNA polymerase binding to all

the origins. Although the RSB-like sequence is located

<161±174 nt downstream of the transcription initiation

site (Fig. 4C), these regions may be in close proximity in

the folded, three-dimensional structure of ssoU. Thus,

the folding of ssDNA containing the promoter may allow

proper positioning of the RNA polymerase with respect

to the transcription initiation site.

Another conserved sequence, termed CS-6 (58-

TAGCGT-38), present within the ssoA of some RC plasmids

was previously shown to be involved in the termination of

primer RNA synthesis (Kramer et al., 1997). This sequence

is localized in the loop of the ssoA and ssoW hairpins (del

Solar et al., 1987; Leenhouts et al., 1991). Partially homo-

logous CS-6 regions were found in the loop of the ssoU

and ssoT structures (Figs 4C and 5). This region could

act as a transcriptional terminator for RNA synthesis.

In vitro transcription analysis showed that primer synth-

esis initiates at position 1318 and terminates at positions

1361±1363 of pUB110, which is consistent with termi-

nation within the loop of hairpin III (Fig. 4C). The major

transition point from RNA to DNA synthesis in vivo was

localized to position 1333 in the stem of hairpin III (Fig.

4C). This places the initiation of DNA synthesis 16 nt

downstream of the primer RNA initiation site. It is possible

that the 44±46 nt primer RNAs may be processed in vivo

by an enzyme such as RNase H to an approximate length

of 15 nt, which is then recognized by the DNA polymerase

for lagging-strand synthesis. We found another sequence,

partially homologous to the CS-6 region (58-AAGGGT-38;

boxed nucleotides in Fig. 5), present at the transition point

of RNA to DNA synthesis that could act as a transcriptional

terminator. However, it is located in the non-template

strand for RNA synthesis, and represents the left arm of

the stem of hairpin III. Further experiments are necessary

to identify the events involved in the termination/process-

ing of the primer RNA.

Previously, it was thought that speci®c host factors were

required for the activity of individual ssoAs in their native

hosts, and the absence of such factors was responsible

for their poor functionality in heterologous hosts (del Solar

et al., 1987; Gruss and Ehrlich, 1989). Our recent studies

with ssoA-type origins suggest that, rather than speci®c

host factors, a better interaction between a particular ssoA

and its homologous RNA polymerase determines the host-

speci®c function of these origins (Kramer et al., 1998a).

We have also noted that non-conserved nucleotides in

ssoAs that function in different Gram-positive bacteria

may be critical for their host-speci®c recognition. In the

case of ssoU, at least two possible models can be pro-

posed for its ef®cient function in many different bacterial

hosts: (1) the lack of requirement for a host-speci®c factor

needed for replication from such origins or (2) the ability

of ssoU to interact ef®ciently with a variety of RNA poly-

merases. Taking into account our data with the ssoA-type

origins, and the lack of a puri®ed in vitro system for lag-

ging-strand replication of RC plasmids, we investigated

the ssoU±RNA polymerase interactions in vitro. The S.

aureus and B. subtilis RNA polymerases interacted equally

well with the ssoU (Fig. 3). Twenty nanograms of RNA poly-

merase was suf®cient to bind 3 ng of the ssoU probe used

in each reaction. The same amount of S. aureus RNA poly-

merase was required to bind 3 ng of the staphylococcal

pE194-ssoA DNA (Kramer et al., 1998a). As the ssoU

Q 1999 Blackwell Science Ltd, Molecular Microbiology, 33, 466±475

Fig. 5 Comparison of the ssoA , ssoU, ssoT and ssoW sequences found in various rolling-circle plasmids. The sequences were aligned byusing the CLUSTALW Multiple Sequence Alignment software. The sequences conserved in either four or all the ®ve ssos shown are shaded.The underlined nucleotides correspond to those in ssoU that are bound by the RNAP. The conserved RSB sequence and the proposed ÿ10and ÿ35 sequences of ssoA (Kramer et al., 1997) are indicated. Boxed regions represent the conserved CS-6 sequences found in ssoA ortheir homologues in the other sso types.

472 M. G. Kramer et al.

origins function ef®ciently in both hosts, we propose that

this functionality is determined, at least to a signi®cant

extent, by their ability to bind strongly to different RNA

polymerases. The possibility that other host factors are

also important in the broad host range function of ssoU

cannot be ruled out at present, and the availability of puri-

®ed replication proteins for lagging-strand replication in the

future should allow further investigation of this issue.

It is interesting to note that the ssoT of plasmid pBAA1,

which is fully active in B. subtilis and S. aureus (Seery and

Devine, 1993), has 69% homology to the ssoU sequence

(Fig. 5). On the other hand, the extent of homology between

ssoU and the ssoAs of plasmids pE194 and pLS1 and the

ssoW of pWV01, which function ef®ciently only in their

native hosts, is 52%, 50% and 59% respectively. Therefore,

it is likely that regions that are conserved between ssoU

and ssoT but absent in the ssoAs and ssoW are critical

for broad host range replication and plasmid promiscuity.

Such sequences may be important for interaction with var-

ious RNA polymerases, and possibly other host proteins.

As the function of ssoAs is host dependent whereas that

of ssoU is host independent, it is possible that ssoU and

ssoT may have evolved from the ssoAs by acquiring

sequences that allowed their stable maintenance in the

new bacterial hosts. Alternatively, ssoAs may have evolved

from ssoU as an `speciation' phenomenon.

Experimental procedures

Enzymes, plasmids and DNA preparations

The B. subtilis and S. aureus RNA polymerases were puri®edas described previously (Deora and Misra, 1995; 1996; Menciaet al., 1996). Plasmid DNAs were isolated from S. aureusRN4220 and S. pneumoniae 708 as described previously(Kramer et al., 1998a). The ssDNA fragments used in thiswork were prepared by asymmetric PCR essentially as pre-viously described (Kramer et al., 1997). The primers used forDNA ampli®cation were the same as those for DNA sequencing(see below). The ssDNA products (348 nt long) containing thedesired sequence (ssoU or ssoU ÿ) were isolated from a 2.5%agarose gel as described previously (Kramer et al., 1997).When required, labelling of the 58- or 38-end of the ssDNAwith 32P was performed using either T4 polynucleotide kinaseor terminal deoxynucleotidyl transferase as described by Sam-brook et al. (1989).

Sequencing of the pMV158 and pUB110 ssoUs

Both DNA strands of the ssoU region of the plasmids weresequenced by the dideoxy method of Sanger et al. (1977)either manually using the Sequenase Version 2.0 DNA Sequ-encing Kit (US Biochemicals) or by an automated ABI Sequen-cer. The following oligonucleotide primers surrounding thessoU region were used: 58-GCGCGATTGCTGAATAAAAG-38 (pUB110 positions 1551±1532) and 58-AGTGCCGACCA-ACCACATAA-38 (positions 1204±1223).

In vitro transcription and identi®cation of the 5 8-end

of primer RNA

In vitro transcription from ssDNA templates was carried outusing RNA polymerases from B. subtilis and S. aureus asdescribed previously (Kramer et al., 1997; 1998a). Reactionmixtures (50 ml) containing 0.1 pmol of ssDNA or dsDNA,200 mM NTPs, 5 mg heparin, 5 units of the RNase inhibitorRNasin (Promega) and 1 pmol of RNA polymerase (preincu-bated with saturating amounts of the puri®ed s factor) wereincubated at 378C for 15 min in 1 ´ binding buffer (50 mM potas-sium glutamate, 25 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1 mMCaCl2, 650 mM NaCl, 1 mM DTT, 0.05% Nonidet P-40, 40%sucrose and 90 mM ammonium sulphate; Kramer et al., 1997;1998a). When labelled RNA was to be synthesized, 80 mM[a-32P]-UTP (3000 Ci mmolÿ1) was included in place of coldUTP. The desired RNA products were puri®ed by electrophor-esis on 10% sequencing gels and the bands were visualized byautoradiography (Sambrook et al., 1989). To determine thetranscriptional initiation points of RNAs generated from thessoU templates, unlabelled newly synthesized products wereused for primer extension analysis. The DNA±RNA hybridswere denatured at 908C for 30 min followed by digestion ofthe ssDNA template with 10 units of RNase-free DNase I(Life Technologies) for 20 min at 378C. RNA was then recov-ered by phenol±chloroform extraction and alcohol precipita-tion, and used as template for reverse transcription by AMVreverse transcriptase. Initially, nine different oligonucleotideswere used in a pilot experiment to identify the appropriate pri-mer for primer extension analysis. The reaction conditionswere as previously described (Kramer et al., 1997) and the[a-32P]-dNTP used in the reaction was chosen so as to corre-spond to the ®rst nucleotide expected to be incorporated inaccordance with the ssoU sequence.

Electrophoretic mobility shift assays

To study the RNA polymerase±ssoU interactions in vitro,EMSAs were performed. Reaction mixtures (10 ml) containing3 ng of 58-end-labelled ssDNA (25 000 cpm), 1 mg of polydA,0.5 mg of polydI±dC, 1 mg of bovine serum albumin (BSA) in1 ´ binding buffer were incubated on ice for 5 min After theaddition of 2 ml of 30% glycerol, the samples were electro-phoresed on a 3.8% polyacrylamide minigel for 1 h at 100 V.The bands were visualized by autoradiography of dried gels.

Determination of the initiation sites of lagging-strand

synthesis in vivo

To map the initiation points of DNA synthesis from the ssoU,plasmid DNA preparations containing both supercoiled andpartially replicated products were subjected to repeat primerextension analysis (Bernander et al., 1992; Grohmann et al.,1997) essentially as described (Kramer et al., 1998a). Twohundred to six hundred nanograms of plasmid DNA prepara-tions was used as template for PCR using only a single 58-end-labelled primer oligonucleotide (50 pmol). Each reactionwas performed using the Taq polymerase for 30 cycles con-sisting of 908C/1 min, 488C/1 min and 728C/1 min. The primercorresponded to positions 1372±1392 of pUB110 predicted tobe downstream of the replication initiation site, having the

Q 1999 Blackwell Science Ltd, Molecular Microbiology, 33, 466±475

ss origin of rolling-circle plasmids 473

sequence 58-GCAAGGGTTTAAAGGTGGAGA-38. Severalprimers were initially tried in pilot experiments to identify theappropriate oligonucleotide to be used in these experiments.The DNA products were puri®ed by phenol±chloroformextraction and alcohol precipitation, and analysed on 8%sequencing gels (Sambrook et al., 1989). The bands gener-ated by elongation of the radiolabelled primer were visualizedby autoradiography.

DNase I and P1 nuclease protection analysis

The 58- and 38-end-labelled ssDNA fragments were used assubstrates for DNase I and P1 (obtained from BoehringerMannheim) nuclease digestion. The speci®c activity of P1used was 300 units mgÿ1. The reaction mixtures contained5 ng of DNA, 1 mg of polydA, 0.5 mg of polydI±dC and 1 mgof BSA in 1 ´ binding buffer. For footprinting experiments,RNA polymerase was ®rst added to the reaction mixturesand incubations were carried out on ice for 5 min for the RNApolymerase±ssoU complexes to form. DNase I cleavage reac-tions were carried out by the addition of 15 units of the enzymefollowed by incubation at room temperature for 5 min. For P1cleavage reactions, either the indicated amounts (Fig. 4A) or0.3 units (1 mg) of this enzyme were added and the digestioncarried out at 378C for 3 min. Initially, various amounts ofDNase I and P1 nuclease and different incubation times weretested in pilot DNA cleavage reactions. When 58-end-labelledDNA was used, a DNA sequencing ladder obtained with thessoU DNA was run on the same gel to identify the protectedregions and P1 nuclease-sensitive sites. When 38-end-labelledfragments were assayed, a different DNA sequence was usedas a size marker for the mapping of the above-mentionedregions within the ssoU sequence.

Acknowledgements

We thank members of our laboratories and G. Wagner forhelpful discussions. Special thanks are due to R. Deora forproviding the S. aureus RNA polymerase, M. Salas, M. Menciaand J. M. Lazaro for their gift of B. subtilis RNA polymeraseand S. Lacks for providing streptococcal strains. This workwas supported by Grant GM31685 from the National Institutesof Health (to S.A.K.), ComisioÂn Interministerial de Ciencia yTecnologõÂa Grant BIO97±0347 (to M.E.) and by a BenedictResearch Foundation grant, University of Illinois at Chicago(to T.K.M.).

References

Bernander, R., Krabbe, M., and Nordstrom, K. (1992) Map-ping of the in vivo start site for leading strand DNA synth-esis in plasmid R1. EMBO J 12: 4481±4487.

Birch, P., and Khan, S.A. (1992) Replication of single-strandedplasmid pT181 DNA in vitro. Proc Natl Acad Sci USA 89:290±294.

Boe, L., Gros, te Riele, H., Ehrlich, S.D., and Gruss, A.(1989) Replication origins of single-stranded-DNA plasmidpUB110. J Bacteriol 171: 3366±3372.

Callaci, S., and Heyduk, T. (1998) Conformation and DNAbinding properties of a single-stranded DNA binding region

of s70 subunit from Escherichia coli RNA polymerase aremodulated by an interaction with the core enzyme. Bio-chemistry 37: 3312±3320.

Collado-Vides, J., Magasanik, B., and Gralla, J.D. (1991) Con-trol site location and transcriptional regulation in Escherichiacoli. Microbiol Rev 55: 371±394.

Dempsey, L.A., Zhao, A.C., and Khan, S.A. (1995) Localiza-tion of the start sites of lagging-strand replication of rolling-circle plasmids from Gram-positive bacteria. Mol Microbiol15: 679±687.

Deora, R., and Misra, T.K. (1995) Puri®cation and character-ization of DNA dependent RNA polymerase from Staphylo-coccus aureus. Biochem Biophys Res Commun 208:610±616.

Deora, R., and Misra, T.K. (1996) Characterization of theprimary s factor of Staphylococcus aureus. J Biol Chem271: 21828±21834.

Devine, K., Hogan, S., Higgins, D., and McConnell, D. (1989)Replication and segregational stability of Bacillus plasmidpBAA1. J Bacteriol 171: 1166±1172.

Geider, K., Beck, E., and Schaller, H. (1978) RNA transcribedfrom DNA at the origin of phage fd single strand to replica-tive form conversion. Proc Natl Acad Sci USA 75: 645±649.

Geider, K., and Kornberg, A. (1974) Conversion of the M13viral single-strand to the double-stranded replicative formsby puri®ed proteins. J Biol Chem 249: 3999±4005.

Glucksmann-Kuis, M.A., Makiewicz, P., Malone, C., andRothman-Denes, L.B. (1992) Speci®c sequences and ahairpin structure in the template strand are required for N4virion RNA polymerase promoter recognition. Cell 70:491±500.

Grohmann, E., Zechner, E.L., and Espinosa, M. (1997) Deter-mination of speci®c DNA strand discontinuities with nucleo-tide resolution in exponentially growing bacteria harboringrolling circle-replicating plasmids. FEMS Microbiol Lett 152:363±369.

Gruss, A., and Ehrlich, S.D. (1989) The family of highly inter-related single-stranded deoxyribonucleic acid plasmids.Microbiol Rev 53: 231±241.

Gruss, A., Ross, H.F., and Novick, R.P. (1987) Functionalanalysis of a palindromic sequence required for normalreplication of several staphylococcal plasmids. Proc NatlAcad Sci USA 84: 2165±2169.

Haynes, L.L., and Rothman-Denes, L.B. (1985) N4 virionRNA polymerase sites of transcription initiation. Cell 41:597±605.

Helmann, J.D. (1994) Bacterial sigma factors. In Transcrip-tion: Mechanisms and Regulation. Conway, R.C., andConway, J.W. (eds). New York: Raven Press, pp. 1±17.

Helmann, J.D. (1995) Compilation and analysis of Bacillussubtilis sA-dependent promoter sequences: evidence forextended contact between RNA polymerase and upstreampromoter DNA. Nucleic Acids Res 23: 2351±2360.

Helmann, J.D., and Chamberlin, M.J. (1988) Structure andfunction of bacterial s factors. Annu Rev Biochem 57:839±872.

Higashitani, N., Higashitani, A., and Horiuchi, K. (1993)Nucleotide sequence of the primer RNA for DNA replica-tion of ®lamentous bacteriophages. J Virol 67: 2175±2181.

Higashitani, A., Higashitani, N., and Horiuchi, K. (1997) Minus-strand origin of ®lamentous phages versus transcriptional

Q 1999 Blackwell Science Ltd, Molecular Microbiology, 33, 466±475

474 M. G. Kramer et al.

promoters in recognition of RNA polymerase. Proc NatlAcad Sci USA 94: 2909±2914.

Huang, X., Lopez de Saro, F.J., and Helmann, J.D. (1997) sfactor mutations affecting the sequence±selective inter-action of RNA polymerase with ÿ10 region single-strandedDNA. Nucleic Acids Res 25: 2603±2609.

Kaguni, J.M., and Kornberg, A. (1982) The s subunit of RNApolymerase holoenzyme confers speci®city in priming M13viral DNA replication. J Biol Chem 257: 5437±5443.

Khan, S.A. (1997) Rolling-circle replication of bacterial plas-mids. Microbiol Mol Biol Rev 61: 442±455.

Kramer, M.G., del Solar, G., and Espinosa, M. (1995) Lag-ging-strand origins of the promiscuous plasmid pMV158:physical and functional characterization. Microbiology 141:655±662.

Kramer, M.G., Khan, S.A., and Espinosa, M. (1997) Plasmidrolling circle replication: Identi®cation of the RNA polymer-ase-directed primer RNA and requirement of DNA polymer-ase I for lagging-strand synthesis. EMBO J 16: 5784±5795.

Kramer, M.G., Espinosa, M., Misra, T.K., and Khan, S.A.(1998a) Lagging strand replication of rolling-circle plasmids:Speci®c recognition of the ssoA-type origins in differentgram-positive bacteria. Proc Natl Acad Sci USA 95:10505±10510.

Kramer, M.G., Khan, S.A., and Espinosa, M. (1998b) Lag-ging-strand replication from the ssoA origin of plasmidpMV158 in Streptococcus pneumoniae : In vivo and in vitroin¯uences of mutations in two conserved regions. J Bacteriol180: 83±89.

Lacks, S.A., Lopez, P., Greenberg, B., and Espinosa, M.(1986) Identi®cation and analysis of genes for tetracyclineresistance and replication functions in the broad-host-range plasmid pLS1. J Mol Biol 192: 753±765.

Leenhouts, K.J., Tolner, B., Bron, S., Kok, J., Venema, G.,and Seegers, J.F.M.L. (1991) Nucleotide sequence andcharacterization of the broad-host-range lactococcal plas-mid pWVO1. Plasmid 26: 55±66.

Lisser, S., and Margalit, H. (1993) Compilation of Escherichiacoli mRNA promoter sequences. Nucleic Acids Res 21:1507±1516.

Lonetto, M., Gribskov, M., and Gross, C.A. (1992) The s70

family: sequence conservation and evolutionary relation-ships. J Bacteriol 174: 3843±3849.

McClure, W.R. (1985) Mechanism and control of transcriptioninitiation in prokaryotes. Annu Rev Biochem 54: 171±204.

McKenzie, T., Hoshino, T., Tanaka, T., and Sueoka, N. (1986)The nucleotide sequence of pUB110: some salient featuresin relation to replication and its regulation. Plasmid 15: 93±103.

Masai, H., and Arai, K.-I. (1997) Frpo: a novel single-stranded

DNA promoter for transcription and for primer RNAsynthesis of DNA replication. Cell 89: 897±907.

Meijer, W.J., van der Lelie, D., Venema, J.G., and Bron, S.(1995) Effects of the generation of single-stranded DNAon the maintenance of plasmid pMV158 and derivativesin Lactococcus lactis. Plasmid 33: 91±99.

Mencia, M., Monsalve, M., Rojo, F., and Salas, M. (1996)Transcription activation by phage f29 protein P is mediatedby interaction with the a subunit of Bacillus subtilis RNApolymerase. Proc Natl Acad Sci USA 93: 6616±6620.

Novick, R.P. (1989) Staphylococcal plasmids and their repli-cation. Annu Rev Microbiol 43: 537±565.

Priebe, S.D., and Lacks, S.A. (1989) Region of the strepto-coccal plasmid pMV158 required for conjugative mobiliz-ation. J Bacteriol 171: 4778±4784.

Rao, L., Karlis, R.K., and Betley, M.J. (1995) In vitro tran-scription of pathogenesis-related genes by puri®ed RNApolymerase from Staphylococcus aureus. J Bacteriol 177:2609±2614.

Roberts, C.W., and Roberts, J.W. (1996) Base-speci®crecognition of the nontemplate strand of promoter DNAby E. coli RNA polymerase. Cell 86: 495±501.

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) MolecularCloning: A Laboratory Manual, 2nd edn. Cold Spring Har-bour, NY: Cold Spring Harbor Laboratory Press.

Sanger, F., Nicklen, S., and Coulson, A.R. (1977) DNA sequ-encing with chain-terminating inhibitors. Proc Natl Acad SciUSA 74: 5463±5467.

Seegers, J.F.M.L., Zhao, A.C., Khan, S.A., Pearce, L.E.,Meijer, W.J.J., Venema, G., and Bron, S. (1995) Structuraland functional analysis of the single-strand origin of replica-tion from the lactococcal plasmid pWVO1. Mol Gen Genet249: 43±50.

Seery, L., and Devine, K.M. (1993) Analysis of features con-tributing to activity of the single-strand origin of Bacillusplasmid pBAA1. J Bacteriol 175: 1988±94.

te Riele, H., Michel, B., and Ehrlich, S. D. (1986) Single-stranded plasmid DNA in Bacillus subtilis and Staphylo-coccus aureus. Proc Natl Acad Sci USA 83: 2541±2545.

del Solar, G., Puyet, A., and Espinosa, M. (1987) Initiationsignals for the conversion of single stranded to doublestranded DNA forms in the streptococcal plasmid pLS1.Nucleic Acids Res 15: 5561±5580.

Van der Lelie, D., Bron, S., Venema, G., and Oskam, L.(1989) Similarity of minus origins of replication and ¯ank-ing open reading frames of plasmids pUB110, pTB913and pMV158. Nucleic Acids Res 17: 7283±7294.

Viret, J.F., and Alonso, J.C. (1988) A DNA sequence outsidethe pUB110 minimal replicon is required for normal replica-tion in Bacillus subtilis. Nucleic Acids Res 16: 4389±4406.

Q 1999 Blackwell Science Ltd, Molecular Microbiology, 33, 466±475

ss origin of rolling-circle plasmids 475