© INSTITUrPAStEI:r/EtSEVtEr

Reg. Microbiol. 1991, 142, 869-873

Paris 1991

Plasmid replication and structural stability in Bacillus subtilis S.D. Ehrlich, C. Bruand, $. Sozhamannan, P. Dabert, M.-F. Gros, L. Janni&e and A. Grass Laboreloire de G#n6tfqae Mierobienne, lnstftltt de Biotechnologte, INRA-Domaine de Vilvert, 78352 3ouy en Josas Cede.,: (France)

Introduction Plasmids isolated from different Gram-positive bacteria fall into two categories, small and large. For example, 19 plasmids originating from Staphylococcus aureus were small, ranging in size from 1.3 to 4.6 kb, with an average of 3.3 kb, and 5 were large, ranging between 19.7 and 52 kb, with an average of 33 kb (Noviek, 1989}. More generally, the cut-of f size between the two classes is about 10 kb. Many pinsraids from the first class and several from the second were studied in some detail, in their natural hosts or in laboratory strains of l~a~cillus subtilis, which are normally devoid of plasmids. Results of these studies indicate that plasmids of the two categories differ not only in size hut also in mode of replication. Smal! and large plasmids appear to use rol!ing circle and theta replication, respectively, and have low and high stntetural stability, respectively. We suggest that the mode o f replication determines plasmid structural stability.

Small plasmids A number of small plasmids have been observed to accumulate circular single-stranded (ss) DNA during replication (re Riele et al., 1986a). Many more small piusmids were examined since the original report, and, in all cases, ssDNA was detected (reviewed by Gruss and Ehrlich, 1989). Several of these plasmids were studied in some detail, and shown to replicate by a rolling circle mechanism (Koepsel et aL, 1985 ; te Riele eta[., 1986b ; Grog el aL, 1987, 1989; Boa et aL, 1989; Sozhamannan et aL, 1990), and less studied plasmids are believed to use the same replication mechanism (see below). The rolling circle mechanism has been previously well characterized in Eseheriehia coli ssDNA bac-

teriophages (reviewed by Bans and Jansz, 1988; Model and Russell, 1988). It requires two replication origins, one called "plus", used for the synlhesis of the ss replication intermediate, the other called "minus", used for conversion of the intermediate into a mature double-stranded (ds) molecule. Initiation of replication at the two origins is not simultaneous, and results in the accumulation of the replication intermediate. Replication of ssDNA plasmids is initiated by a plasmid-encoded replication protein, which in. troduces a nick at the specific site in the !31us replication origin. The T-hydroxyl end thus generated is used as a primer for DNA synthesis ; while one DNA ~irand is copied, the other is simultaneously displaced. At the end of a round of replication, the origin is nicked again by the same protein and the displaced single strand is thereby detached from the fully dsDNA molecule. The protein seals the ends of the displaced strand, which generates a circular ss replication intermediate. Replication is completed by the conversion of ss intermediates into dsDNA form. The conversion synthesis appears to be initiated by RNA polymerase, which uses the minus origin as a template to synthesize a primer (Boe et aL, 1999)~ Elongation of this primer by the hosl DNA replication machinery generates the ds plasmid form. Sequence comparisons of the plus replication origins and the cognate replication proteins have revealed four families of replication region~ (Gruss and Ehrlich, 1989); a compilation o¢ 32 plasmids is shown in table 1. The smallest of these families has 5 members, which suggests that not many new families remain to be identified. There is extensive homology between replicatiolt regions of the same family. For example, there is an 18-bp sequence within the 55-bp replication origin of plasmid pC194, which is also found in the Jrigin of plasmid pUB110. Likewise,

S.D. E H R L I C H E T A L .

870

respectively, In addition, 6 out of 9 amino acids in the active site of the ~X174 replication protein are homologous to a regina of the putative active site of the pC194 replication protein (Gros er ~rl., 1987 and unpublished results). These observations suggest an evolutionary relationship between plasmids from Gram-positive bacteria and phages from Gramnegative bacteria. Many plus origins can function in a wide variety of hosts, transcending the Gram barrier (Ehrlich, 1977; Goze and Ehrlich, 19801 Kok et el., 1984). It is remarkable tb.at such a broad host range is achieved by interplay of only two pIasmld-encoded elements, a protein and its target sequence, which must therefore be capable of interacting with the DNA.synthesizlng machinery of many different organisms. Three families of minus origins, with 2, 3 and 8 known members, have been identified uo )a .na'~ (Gross et el., 1987; Boe el ,',!., !999; Devine et el., 1989). These origins are longer than the plus origins (close to 200 bp), and are characterized by numerous imperfect inverted repeats. Surprisingly, their host range seems to be generally rather narrow, For example, the origins of the p a l A family function in S. aureus but not in B. subtilis (Grusset eL, 1987). However, at least one of the minus origins, that of pUB110, can function in severat hosts (Boe et aL, 1989). Aspecific initiation, probably mediated by RNA polymerase (at least in B. subtilis; Boe et eL, 1989), can be used for the lagging strand synthesis, and thus assure plasmid maintenance in hosts where the minus origin is not active. Such initiation is efficient enough to maintain the plasmid copy number at its usual level in B. subtilis but not in 5:. aureus or E, coil (Gruss et eL, 1987 ; te Riele, personal communication).

Table I. Small plasmids from Gram-positive bacteria. Plasmid Size (kb)

Original host

pTlgl pT127 pC22i pC223 pSt94 pUBII2 peW7 pTZI2 MI3 I'l

4.4 4,4 4.6 4.6 4.4 4.1 4.2 2.5 6.4

5:. aureus 5:. attretls 5:. aureus S. attreu3 S. attreus S. aureus S. aureus Corynebaprerhtm xerosis E. coil

pC 194 pUBII0 pOX6 pLSI I pTAI060 pBAA I pFTB14 pBCl6 pCBI01 pLP I plJ 101 pC30il • Xi~4 I')

2,9 4.5 3.2 8,4 8.4 ft.8 8.2 4.6 6.0 2. t 8.8 2. i

S. attrezts 5:. attreus S. attreus B. subtilis B. subtilis B, S~tbtilis Bacillus amyloliqnefaeiens Bacillus cereus Clostridinm butyrieum Laclobacillus planturtt~ Streptamy~es fividans L. plantarum

5.3

E. CO//

pE,~94 pMV 15;8 pWV0I pSH71 pLB4

.3.7 5.5 3.3 2.1 3.5

S, aureus Streptococcus agalactiae Lactococcus laetis subsp, cremoris Lactococcns lactis L. plantarum

1.3

5:. aureus S. aureus 5:. aureus S. aureus S. aureus Staphylococcus epidermidis B. subtilis

pSN2 pE12 pE5 pT48 pTCS1 pNE 13 ! [zlM.13

~.2 2.1

2.1 1.3

2.1 2.1

Small plasmlds have a modular organization. This is deduced from observations that, in different plasmids, plus origins of a given family are associated with minus origins of different families and the same combination of plus and minus origins is associated with different antibiotic-resistance markers (Gross and Ehdich, 1989). This modular organization suggests that small plasmids have a propensity to exchange genetic information by recombination. Plasmid recombination can be mediated by sitespecific systems (Noviek et el., 1984; Gennaro et eL, 1987) and by non-specific host functions.

Plasmids are grouped in familiesa,.a;mdingta their plus oriOn and/of the Repproteit~.The familiesare plier,namedaccording to lhe first member (e.~. pCI94 family). (*} E. coil phages.

the replication proteins of the two plasmids are about 30 % homologous (Gros et el., 1987). Interestingly, two ssDNA )~b,a.ges from E. coli, OX174 and MI3, may he distoe:t? related to two of these families 12/13 and 9/I 1 nueleutides flanking the nick site in these two phages correspond to nucleotides flanking the nick site in plasmid.s pC:94 and pTI8l,

Several lines of evidence indicate that DNA sequences present on small plasmids recombine with a high frequency. First, directly repeated segments of 4 kb reeombined about 100 times more efficient-

-

ds

- double-~trzndcd.

[

ss

- single-stranded.

MODE OF REPLICA T/ON A N D S T A B I L I T Y Ob B. SUBTI LIS PLASMIDS

ly when carried on plasmids than when on the B. subtills chromosome (Niaudet et al., 19841. Next, transposon Tn5 underwent precise excision 150 to 1,500 times more frequently from plasmids than f ' o m the chromosome (Janniere and Ehrlich, 19871, The excision was due to recombination of 9-bp repeats which flank the transposon. Finally, foreign DNA segments inserted into ssDNA plasmids often undergo deletions, a phenomenon termed p!a';mid structural instability {Ehrlich et al., 1982, 19861. Frequent recombination of plasmid-carried sequeflces, which might lead to modular plasmid organization and which underlies plasmid structural instability, seems to result from the rolling circle mode of plasmid replication. Recombination of chromosomally-carried direct repeats, which is relatively infrequent, was stimulated up to 450 times whoa a Oiusmiu was it:s~.,:?cl in the vicinity of the repeats and allowed to replicat," (No[rot et el., 1987). It was suggested that ssDNA generated during plasmid replication stimulates recombination. Furthermore, errors of the replication protein, which nicks the replication origin, were shown :o generate deletions (Michei and Ehrlich, 19861. Also, plasmid forms of high molecular weight frequently arise upon insertion of foreign DNA segments in:o rolling circle plasmids (Gruss and Ehrlich, 19881. Such forms may be inefficiently maintained in the hOSt cell and also provide recombination substrates. Structural instability, in conjunction with selection for efficient maintenance, might account for the small size of ssDNA plasmids from Gram-positive bacteria (Gruss and Ehrlich, 1989").

Large plasmids Indirect evidence suggests that several large plusraids from Gram-positive bacteria {table II) do not

Table II. Large plasmids from Gram-positive bacteria, known or supposed to use the 0 mode of replication. Plasmid pAM[~I pSMIg035 plPS01 piP404 pHT1030 pi524 p43 p44 p60 pTBI9

Size (kb)

Original host

26.5 27,5 30.2 10.2 15 31.8 65 69 95 26.5

Enterococcusfaecalis Streptococcus pyogenes Streptococcus agalacrine Clostridium perfricrgens Bacillus thuringiensls S. aureus B. thuriagiensis B. rhuringiensis B. thuriagiensis Bacillus stearothert~tophilus

87t

replicate as rolling circles. First, their replication regions show no homology with any of the small pEasmid families (Gamier and Cole, 1988a,b; Brant[ et al., 1990; Swinfield er aL, 1990;/anni6re et al., t990 ; Bantu, Lereclus, personal communications). Next, small plasmids derived from piP404, pAM[31, pHT1030 and the repA region of pTB19 do not accumulate ssDNA during replication (Garnier and Cole, 19ggb; Janniere et at., 1990 and unpublished data; Lereclus, personal communication). Finally, radioactivity-labelling experiments suggest that pl524 replicates bidirectionally (Novick, persona! communication). To determine the mode of replication of such plasmids we studied a broad host raege peasmid, pAM~I. The minimal replication region of pAMpa| comprises one open reading frame, which encodes the replieatio:l protein (RepEL and a contiguous downstream region of about 300 bp (Swinfield et ol, 1990; Le Chatelier, personal communication). Two lines nf evidence show that pAMI31 uses a unidirectional theta replication mode (Bruaad et aL, 1991}, First, 0-form molecules accumulated when the terminus region of the B. subtilis chromosome (Tel) was inserted into a small plasmid derived from pAM~I. Both forks were at constant positions in these molecules, one at ~he terC site where chromosomal replication is normally arrested, the other at the pinsmid origin, as determined by electron microscopy. This shows that replication ocurs by a tOmechanism. Second, ~wo-dimensional gel elcctrophoresis of another small derivative plasmid has revealed replication intermediates having a 9 form. In the intermediates, one of the forks was always at the same position, close ro the downstream end of the repE gene, and the other further downstream, at variable positions, This indicates that replication initiates at the constant sltc and progresses unidirectionally in the direction of repE transcription. We mapped the site of initiation of pAM[31 leading-strand synthesis tO a nueleotlde, using the 0-form molecules accumulated by Ter insertion. Only two inverted and two direct repeats flank this site, in contrast to the more complex structures found in replication origins of Gram-negative bacteria, or in the B. subtitis chromosomal origin (rrzn|tiple direct repeats, AT-rich regions and DnaA boxes ; Bramhill and Kornberg, 1988; Ogasawara er eL, 19901. The sites of arrest of the lagging-strand synthesis were also mapped. Three sites were detected, Focalized 13-15 bp downstream of the initiation site of leading-strand synthesis, in a region displaying no homology with the known replication pause sites (Lewis and Wake, this volume). These observations suggest that pAM}t replication mechanism differs from the previously studicd 0-type mechanisms.

872

S.D. EHRLICH

Two mher large plasmids, piPS01 and pSMI9035 (table II), have replication regions highly homologous to that of pAM,31 (Sorokin and Kai~ak, 1987; Btm;tl et at., 1990), which indicates that they also use the 0 mode of teplicalion. If the smatl size of ssDNA ptasmids is due to their mode of replication, which slffrtulates recombination and therefore causes structural instability (see above) the mode of replication of large plasmids should stimulate recombination much less. Indeed, excision of Tn5 was 400- to 900-fold less frequem from pAM[Xl-derived plasmids than from a repllcon which uses a rolling circle replication, pC194 (Jannicre et aL, 19901. A similar result was obtained with plasmlds using the r e p A region of oTBIg, another large piasmid. Also, short direct repeats carried on pAM~,I recombined 10- to 5e-fold less efficiently than the same repeats carried on a small rolling circle pinsntJd (Bron et a t , 19991. Furthermore, large DNA segments (up to 40 kb) could be efficiently clol~cd and propagated, on pTBI9 and pAM[~l-derived plasmids in g . sttbfitis (Jannic~re et al., 19901. These results suggest that the replication ef large plasmids causes much less instability than that of small plasmids and lead us to speculate that the evolutionary appearance of large gcnomcs, plasmids and oh! omosomes, may have been contingent on the evolution of the 0-mode of replication. From the practical point of view, the resubs also show that long DNA segments can be efficiently cloned in plasmid vectors which do not replicate as rolling circles. We suggest that such cloning vectors should he nsed in any organism in which the stable maintenance o f large segments is sought. The approaches described above and also elsewhere {Braand e t a L , 19901 may help to ~dentify and characterize the necessary 0-replicating plasmids. K e y - w o r d s : Replication, Bacillus sublilis, Plasmid; Structural stability, Mode of replication.

References Boas, P. & Jansz, H. (1988l, Single-stranded DNA phage origin. Curt. Top. Microbial lmmunol., 136, 31-70, Bee, L , Gros, M.-F., te Ride, H., Ehrllch. S.D. & Gruss, A. (1989), Replicationorigins of single-stranded DNA p~asmid pUBIIQ. J. Boer., 171, 3366-3372. Bramhill, D, & Kornberg, A. (1988), Duplex opening by dnaA protein at r~ovelsequences in initiation of replicamionat the origin of the E. colt chromosome. Ceil, 52, 743-755. Brantl, S., Belmke, D. & Alonso~3.C. (19901, Molecular analysis of the replication region of the eonjugative Streptococcu~ ugaluctiue plasm~dpiPS01 in Bacillus sub[ills. Comparison with plasmids pAM[31 and pgMIg035. Nat.L Acids. Bvs.. 18. 4783-4790,

ET AL.

Bron, S., Pcijnenburg, A., Peelers, B., Haimn, P. & Venema, G. (1989), Cloning and plasmid tin)stability in Bacillus subrilis, in "Genetlc transformation and expression" (L.O. Butler, C. Harwood & B.E.B. Moseley) (pp. 205-2191. Intercept, Andover. Bruand, C., Ehrllch, S.D. & Janni~re, L. (lqg0}, A method for detecting unidirectional[hera replication in Bay{lIns sublilis plasmids, in Genetics and bin[ethnology of bacilli (Ganesan. A.T., Hach, J.A. & Zukowski, M.M.) (pp. 123-1291. Academic Press, New York. Bru,'uld, C., Ehriieh, S.D. & Janni~re, L. (19911, Unidirectional theta replication of the structurally stable Eaterococcus faecalis plasmid pAMBI. Etnbo J,, 10, 2171-217"/. Devine, K., Hogan, S., bliggins, D. & McConneg, D. (19891, Replication and segtegational stability of the Bacillus plasmid pBAA I. J. Bact., 171, 1166-1172. Ehrlich, S.D. (19771, Replication and expression of pinsraids from Staphylococcus aureus in Bacillus subtiIts. Prec. nat. Acad. Set. (Wash.), 74, 1680-1682. Ehrlich, S.D., Niaudet, B. & Michel, B. (1982), Use of pinsraids from Slaphytoeoce~ts aureus for cloningof DNA in Bacillus aubtilis. Curt. TOp. MtcrobioL ImmunoL, 96, 19-30. Ehrlich, S.D., No{rot, Ph., Petit, M.A., Jarmi~re, L,, Michel, B. &te Ricie, H. (1986), Structural instability of ,?aeitlussubtilis plasmids, in "Genetic engineering, VOI. 8" {J.K. So[low & A. Hollaender) {pp. 7t-83). Plenum Press, New York. Gamier, T. & Cole, S.T. (l 9g8a), Complete nucleotide sequence and genetic organization of the batter{pc{nogenie plasmid, piP404, from Closrridlum aerfringeas. Pierre{d, 19, 134-150. Gamier, T. & Cole, S.D. (1988b), Identification and molecular genetic analysis o[ replication functions of the bacteriocinogenicpiasmid piP404 front Clostridiuta perfringens, P&smid, 19, 151-160. Gennaro, M.L., Kornblnrn, J. & Novick, R,P. (1987), A site-specific recombination [unction in Slapkylueoecn$ anreas plasmids. J. Bacr., 169, 2601-2610. Go~:, A. & Ehrlich, S.D. (19801, Replication of plasmids from Staphylococcus aureus in Eschar{chin coil Prec. nut, Aead, Sei. {Wash,), 77, 7333-733% Gros, M.-F., te Ride, H. & Ehrlieh, SD. (1987l, Rolling circle replication of single-stranded DNA plasmid pC194. E M B O £, 6, 3863-3869. Gros, M.-F., te Ride, H. & Ehrllch, S.D. (19891, Replication origin of single-stranded DNA plasmid pC194. E M B O J., 8, 2711-2716. Gruss, A. & Ehrlich, S.D. (19881, Insertion of foreign DNA into plasmids from Gram-positive bacteria induce~ formation of high molecular weight plasmid multimers. J. Bact., 170, 1183-1190. Gruss, A. & Ekrlich, S.D. riga9), The family of highly interrelated single-strandeddeoxyrlbonucleleacid plasmids. MicrobioL Bey., 53, 231-241. Gruss. A,D., Ross, H.F. & Novick, R.P, (19871,Functional analysis of a palindromic sequence required for normal replication o1 ~everal staphylococcal plasmids. Proc. ;*at. Aead. ScL (Wash.). 84, 2165-2169. Jannibre, L. & Ehrlich, S.D. (1997), Recombination between short repeated sequences is more frequent in plasmids than in the chrnmnsome of Bacillus subtilig. MoL sen. Generics, 210, 116-121.

MODE OF REPLICATION

AND STABILITY

Janni~re, L,, Bruand, C. & EhrLich, S.D, (1990L Strut turally stable Bacillus .~ubtilis ck:ning vectors. Gene. 87, 53-61. Keepsel, R., Murray, R., Rosenblum, W. & Khan, S. (19:85), The reolication initiator protein of p!a.zmid pTl$1 has sequence-specific endonuclease and topoisometase-!ike anti'dries. Pro['. nat, At'ad. Sci. (Wash.), 82, 6845-6849, Kok, J., van der Vossen, J.M.B.M. & Venema, G. (1984), Construction of plasmid ctonirtg vectors for lactic streptococci in Bucillus subtilis and Escherichie cob. AppL environm. MicrobioL. 48, 77.6-731. Michel, B. & Ehrlich, S.D. (1986), Illegitimate recombination occurs between the replication origin of the plasmid pCI94 and a progressing replication fork, E M B O J., 5, 3691-3696. Model, P. & Russel, M. (1988), Filamentous bacteriophage, ia "The bacteriophages" (R. Calender) (pp. 375-456L Plenum Press, New York. Ninudet, B., Janni~re, L. & Ehrlich, S.D. (1984), Recombination between repeated DNA .sequences occurs more often in plasmids than in the chromosome of Ban:flus subtilis. MoL gen. Genetics. 197.46-54. Noirot, Ph., Petit, M.-A, & Ehrlich, S.D. 11987L Plasmid replication stimulates DNA recomblnadon in Bacilhis subtilis. .L tool BioL, 196, 39-48. Noviek, R.P. (1989), Slaphylococcal plasmids and their replication. Ann. Rev. Microbiol., 43, 537-565. Noviek, R.P., Adler, G.K., Projan, S.J., Carlelon, S.. Highlander, S.K., Grass, A., Khan. S,A. & Iordanes

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ca, S. (1984), Control of pTI81 replication, - - 1. The prig1 copy control function acts by iahibilillg !ht: ~;ynthesis of a replication protei.;, f3t1.1t10 1., 3. 2399-2405. Ogasav, ara, N,, Fujita. M.Q.. Motiya, S., Fukuoka, T . Hirano, M. & Yoshika~a, H. (1990). Comparative anatomy of o n C or enbaeteria, ill "The bacterial chromosome" (Drlica, K. & Riley, M.) (pp. 287-295). American Society for Microbiology. Washington D.C. Sorokin. A.V. & Khaz..ak, V.E. (1987). Structure of pBMlg035 replication region and MLS-resislance gene, in "Genetic transformation and expression" (Buller, k.O., Nartwood, C. & Moseley, B.E.B.) (pp, 269-2gi), Intercept, Andover, Sozhamannan, S.. Dabert, P., Moretto, V.. Ehrlich. S.D. & Grass, A. (1990), Plus-origin mapping of singlestranded DNA plasmid pE194 and nick-site homolo gins with other plasmlds..L [:l~ct.. 172, 4543-4548. Swinfield, T.J,, Oullram, J.D., Thompson, D.E., Brehm, J, K. & Mi~lon, N.P. (1990). Physical characterization of the replication region of the Streptococcus faecalis plasmid pAMI31. Gene, 87, 79-90. te Ride, H., Michel B. & Ehrlich, S,D. (1986a}, Sir~gle~tranded p~,asmid DNA in Bacillus subtilis and Stvphyloeoecus aureu~. Pron. net. At'ad. Sci. {Wash.), 83, 2541-2545. te Riele, H.. Michel, B. & Ehrllch, S.D. (1986b), Are singlestranded circles intermediates in plasmid DNA repli cation? EMBO J., 5, 631-637.

Plasmid replication and structural stability in Bacillus subtilis.

© INSTITUrPAStEI:r/EtSEVtEr Reg. Microbiol. 1991, 142, 869-873 Paris 1991 Plasmid replication and structural stability in Bacillus subtilis S.D. Eh...
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