~-) INSTITUTPAStEUR/[~.t.S[:VU~R Paris 1991

Res. Mierobiol. 1991, 142, 875-883

Plasmid instability and molecular cloning in Bacillus subtilis S. Bron t*~, W. Metier, S, Holsappel and P. Haima Department o f Genetics, Center o f Biological Sciences, Kerklaan 30, 9751 N N Haren eThe Netherlands)

Introduction Both from fundamental and applied points of view, bacilli are importhm organisms. Their major iuterest in application is their ability to secrete large amounts of industrially important enzymes into the medium. Model studies are usually conducted with Bacfflus subtilis. This organism has also become the paradigm for fundamemal research on other bacilli and Gram ~ bacteria in generai, e.g. for studies of gene expression, genetic stability, competence and transformation. For the further development of B. subtilis as a model organism for research and application, stable and efficient molecular cloning systems are needed. However, the development of suitable plasmid vector systems for B. subtHis and other bacilli has met with considerable difficulty. [t has become clear that the commonly used plasmid vectors, which have mainly been derived from Staphylococcus aureus, are i'ar from optimal for B. sublilis. With these plasmids, it is frequently difficult to clone foreign DNA, in particular large DNA fragments. In addition, high levels of plasmid instability has frequently been observed (Alonso et al., 1987 ; Bron, 1990; Bron and Luxcn, 19851Bron etal,, 1988, 1989, 1991; Dubnau, 1983; fihrlich, 1989; Ehrlich et al., 1986). Two kinds of instability occur frequently: segregational instability (the loss of the entire plasmid population from a cell) and structural instability (rearrangements [n the pinsmid. most frequently deletions). Considerable progress has recently been made in the understanding of plasmid instability, and the difficulties encountered in molecular cloning in B. subtilis and other Gram + bacteria. A key observation was that small plasmids from Gram* bacteria replicate via a rolling-circle (RC) mechanism, generating single-stranded DNA (ssDNA) intermediates

(~1 Correspondingaulhor.

(re Riele et aL, 1986; Gruss and Ehrlich, 1989). In the following, we will denote these plasmids as ssDNA plasmids. Evidence has accumulated thal the RC mode of replication, in particular the generation of plasmid ssDNA, is an important factor in both segregational and structural instability. The main purpose of this paper is 1o describe the role of RC replication in plasmid instability in B. subtills. We a!so describe efficient and stable plasmidcloning systems, based on the endogenous B. $ublilis plasmid pTAI060, in which most of the problems observed with standard ssDNA plasmid vectors have been solved (Haima et at., 1990a,b,c). Plasmids used in B. sublilis and their mode of replication B. subtili~ 168M, the eommordy used transformable strain, is naturalIy devoid of plasmids. Although plasmids are present in several other bacilli, these are usually cryptic. This is one of the reasons that plasmid-doning vectors for B. sabtilis were originally taken from other Gram + bacteria, such a.~ S. aureus. Severr.l of these, like pCI94 pE194 and pUB110, appeared to replicate in B. yubtilis and to express their antibiotic resistance markers (Dubnau, 1983; Ehrlich, 1977). It is now clear that most small plasmids (< 10 kb) from Gram ~ bacteria generate ssDNA intermediates, most likely by RC replication (Gruss and Ehrlich, 1989). In the conversion of ssDNA to double-stranded plasmid DNA (dsDNA) minus origins (MO) of replication play an important role. They function as initiation sites for complementary strand synthesis. Although their primary role is in replication, MO have ¢onsiderable effects on plasmid stability (see the following 2 chapters).


S. B R O N E T A L.

M e arc non-coding, highly palindromic sequences, usually about 200-300 bp, which fnnction in one orientation. In their absence, plasmid ssDNA accumulates. M e arc usually nou-essent]al, since in their absence, plasmid replication can continue. This suggests that alternative structures may function as (inefficient) initiation sites for complementary strand symhesis. MO are normally functional in a limited number of bacterial species. Of relevance to the topic of this paper is that most M e from S, aureus plasnlid~ are non-functional in B. subtilis (Gruss and Ehrlich, 1989). This may be an important factor in explaining the high levels of instability observed with these plasmids in B. subtilis. It should also be said that during many vec,or constructions, M e have accidentally been deleted. Three families of M e are now well-known. The first, denoted palM, is present in several S. altreas plasmids. This family of Me, the members of which have non-identical sequences, has only limited activity in B. subrilis (Gruss and Ehrlich, 1989). Tire second group of Me, denoted as palU (formerly denmed as BA]-type Me), is different from palm MO and is present in, lk)r instance, the S. aureus plasmid pUB110 (Boeet al., 1989; Bronet ai., 19g8 ', Vircl and Alonso, 1987), the streptococcal plasmid pMVI58 (van der Lelie el al., 1989), and the Bacillus plasmid pTB913 (van der Lelie etaL, 1989). The sequences of this family of M e are nearly identical. Contrary topalA, palU MO are functional in B. subtills (Bee et aL,1989; Bran el aL, 1988, 1991). The third ciass of M e , denoted pttlT, is present on the Bacillus plasmids pTA 1060 (Bran, 1990; Bran eral., I989, 1991) and pBAA1 (Devine etaL, 1989). The palT MO have no sequence similarity with paIA or palU, Like other Me, the sequence ofpalTis highly palindromic and comprizes about 250 bp. The growing realization that plasmids from other non-homologous G,ram + bacteria are not optimal for B. subtilis, has prompted us to study endogenous Bacillus plasmids. We reasoned that with homologous systems, a better matching between host and plasmid Oanetions ought to e~ist, which should contribute to better plasmid stability. For this purpose, we chose the cryptic Bacillus plasmid pTAI060 (Bran, 1990; BronetaL, 1987, 1989, 1991). Although pTAI060 appeared to be an ssDNA plasmid, it was far more stable than S. aureus-derived plasmids and enabled the coust ruction of efficient cloning vectors (see the 3rd following chapter).

DR direct repeal ds = double~tranded. HMW - highr~olecularweight. IR = ]nvcrt~repeal.

Although in this paper we will concentrate on ssDNA plasmids, in particular pTAt060, it should be mentioned that plasmids not replicating via ssDNA intermediates are receiving increased attention at present, The best studied is the streptococcal plasmid pAM~31, which replicates via a unidirectional lheta mechanism (Bruand eraL, 1991). Derivatives of pAM[~t are highly efficient for cloning in B. subtilis (Janni~re etal., 1990) and recombinant ptasmids arc slructurally stable. For scgrcgational stability, it is important to have present a gone which presumably encodes a site-specific resolvase (Swinfield et al., 19~9).

Segregatiunal plasmid instability From the onset of cloning attempts, it was observed that recombinant S. aureus pIasmlds were often poorly maintained in B. subtilis (Dubnau, 1983). Similar observations have since been made by many researchers working with B. subtilis, Also, it has been diffieuh to clone large DNA inserts, After it was realized that these plasmids were of tile ssDNA type, the een'iral question wa~ whether the RC mode of replication caused the high levels of instability and cloning problems, In addition, a log!col question was whether the absence of a functional M e , and therefore the generation of increased amounts of ssDNA, increased the levels of instability. We have addressed these questions using 3 p]asmid model systems: S. anreus plasmid p U B l l 0 ; Streptococcus agalactiae plasmld pMV 158. and B. subtilis plasmid pTA1060. DNA fragments of various size extracted from Escheriehia coil were inserted at several non-essentlal positions in pUBl l0 (Bran and Luxen, 1985; Bran etal., 1988). The effects of the inserts on plasmld maintenance in B. subtilis were measured during approximately 100 generations of growth under nonselective conditions. Typical results are summarized in figure IA. It was concluded that, whereas the 5.7-kbp derivative pEBI was stably maintained, derivatives of increased size were not. The levels of instability were dearly related to the size of the plasraids. These results showed that even with the entire pUB110 OoalU present), the larger derivatives were segregatJonally highly unstable in a size-dependent way. In general, plasmids with DNA inserts larger than about 3 kbp were poorly maintained; which may

MO RC as

: : =

m i n u s origin. r o l l i n s ¢ir¢1¢, sin#c-stranded.


pEB1 , (5 7kbp) ~

1C (6.gkbp)

tC (6.9kbp)

1O 0


t 2g',,

,, \ < I-to


(B. 1kbp),,o \ ~'\



w IT

3C (9.9kbp) i




(B.Bkbp) ~. \ i ~1


\ \, '\


~. c~




\ 1C z~alU % (6.4kbp]


\ •









Fig. I. Effect of DNA inserts on the maintenance of pUBI I0, with IA) and without (B) the MO region. Fragments of various size were cloned at several positions in pUB110, pEBI (5.7 kbp) is pUBI tO carrying an additinnal Ent~ gone. Inserts and plasrnid sizes are indicated in the figure. B. s~btilis cells carrying Ihc plasmids were cultured for about 100 generations in the absence of selectiveantibiotic pressore. Segregational stabilities, expressed at, the fraction of anlibintic-resistant cells, were mcastned by plating nn selective agars.

be an important factor in causing the low efficiencies of cloning. To study whether plasmid maintenance is Further decreased by the accumulation of ssDNA, similar experiments were carried out with pUB110 derivatives lacking MO. The resuhs (fig. 1B) sho~ved that the absence ofpalU resulted in a considerable reduction of plasmid maintenance. The kinetics or the appearance of plasmld-free cells showed a biphasic pattern ; the most drastic effects being observed during the later stages of the experiment. The broad-host-range streptococcal plasmid pMV]58, which replicates in B. sublilis (copy number 5 per chromosome), was of interest since it contains two MO, one of the pttlA type (del Solar et uL, 1987) and the other of the palU type (van dot Lelie et aL, 1989). Both are active in B, subtilis, although palA isto a lower extent than.oulU(Meijer and Bran, in preparation), pMVIS8 offered an attractive model system, since MO can be deleted either separately or together, and the effects on plasmid maimenance can be studied.

Experiments of this sort showed thai the absence or MO was generally correlated with instability (Meijer and Bran, in preparation). High levels of instability were obtained when both MO were abscm (large amounts o f accumulated ssDNA), whereas in the absence of one MO, the levels nf instability were moderate (low amounts of accumulated ssDNA). Taking into consideration that ssDNA plasmids from other Gram" bacteria are not optimal for B. subtilis, we reasoned that plasmids naturally present in B. snbtilis might be better adapted to this host. Therefore, we analysed the cryptic B. subtili.~ plasmid pTA 1060 (8.6 kbp; copy number 5 per chromosome), pTAI060 was labelled with antibioticresistance markers /fig. 2A), resulting in plasmid pBB2 (11.3 kbp; Bran, 1990; Bran et al., 1987, 1991). Several small derivatives, like pHPI3 and pHPSg, ~,ere also constructed (Haima et al_, 1987; 1990a,b,c). Like other small plasmid-', from Gram +bacter in, pTA 1050 appeared to be of the ssDNA type. The palT MO was contained in a 250.bp fragment, Milch


S. B R O N E T A L .

pBB2 .~ ( 1 1.3 kbp)"] ~?~' // ~ J S /"t_


~ lOO

T/a/i: C/a~ k~,,'nd/~






i J N;...-

-'O- 0 pBB3(ApalT) (9.8 kbp)

Iq) Eco~/




(15.5 kbp)


f-7'tnd/tt Ncol


Fig. 2. (A)Pla~midspBB2 and pBB3, and (BI maintenance of their derivative~in ceilsgrowing under non-selective conditions. A) pBl$2consists ol Ihe entire plasmid pTABI060 (circular part or lhe figure), into which Cm R and A'ttt~:~¢ne~ ~ere inlrodaee4. Primary replication functions (Rap) and palTare indicated. Inserts ~ere placed tr~ the t3gtlI site (Kinr~genO. pBB3 is oBB2 from v,hich the pa/Tregion has been deleted. B) Inserts and pJasmid sizes are ind,iealed ta the figure. For details, see legends to figure 1.

was not linked to theessential replication functions, and had. a highly palindromic sequence (Bron, 1990; Bron and Holsappel, in preparation). In variants (tike pHPI3 and pl-lPS9) lackingpalT, about 30% of the plasmid, molecules are present in the ssDNA form. pBB3 was obtained from pBB2 by deleting the MO region. Typical results of segregarional stability assays of pTA 1060 derivatives are shown in figure 2B. Two conclusions were drawn. First, the DNA inserts which rendered pUB110 highly unstable, hardly aff~zted the stability o f pBB2 (the effect of the 4,2-kbp E. ~,//DNA fragment 3C is shown as an example). This means that, despite their larger size, the stabilily of pTAI060 derivatives is far superior to those of oUB110 derivatives. Second, removal of the MO (plasmid pBB3) reduced the level of maintenance (about 50070 of the cells carried the plasmid after about ll30 generations). This showed that, as for pUB110 and pMV 158, Ihe MO contributes to segregational stability. It was also obvious, however, that the effect of the removal of p e l t from pTAIO60 was far less draslie than that of the removal of patU from pUB[10. Altogether, it is evident that the generation of ssDNA by RC replication, in particalar in the absence

of a functional MO, is an important factor in segregational plasmid instability in B. sztOtilis. It is also clear that the endogenous B. subtilis plasmid pTAI060 is superior to several other plasmids of helerologous origin. At least two questions remain to be answered, however, First, what is the mechanism causing segregational instability of ssDNA plasmlds; and second, why is pTAI060 superior to several other ssDNA plasmids? One obvious explanation for the effects of ssDNA accumulation would be a reduction in ds plasmid forms as a consequence of inefficient conversion. This is not likely to be the case, however, since the d e l e t i o r t o f p a l U a n d p a l T h a s only a slight effect on plasmid copy numbers (Bran el aL, 1987; 1988), A more likely explanation is that the accumulation of ssDNA interferes with cell physiology. It can be con.. calved that ssDNA titrates out (as yet unidentified) cellular components essential for functions such as chromosomal replioation. This would result in a growth disadvantage and the cells would be rapidly outnumbered by plasmid- free cells. The effect of MO on plasmid maimenance would thus be indirect. The biphasic nature of the segregation kinetics curves



(fig. 1) supports this view. Such curves arc predicted when a growth disadvantage occurs in a subpopulotion of cells (Boe et al., 1987). In addition to ssDNA, the accumulation of large amounts of other plasmid replication intermediates, composed of high-molecular-weight linear head-totail multimerg (HMW DNA), may also contribute to the observed instability. Such DNA has been observed with several recombinant ssDNA plasmids (Grass and Ehtlich, 1988). In addition, the formation of linear plasmid muhlmcric forms is markedly affected by several host mutations (Viret and Ainuso, 1987). Grass and Ebrlich (198g) showed that the same inserts which caused high levels of plasmid instability in our studies (Bron et aL, 1985, 1988), also caused high levels of HMW DNA accumulation. How HMW DNA might interfere with plasmid stability is not dear. One possible explanation is that, as postulated for ssDNA, HMW DNA titrates out essential cellular components, resulting in a growth disadvantage. The reason why pTA 1060 derivatives are segregationally superior to pUB110 derivatives is not fully understood. One important difference is the plasmld copy number (50 and 5 per chromosome for pUB110 and pTAI060, respectively). Lower copy numbers will result in less HMW and ssDNA and therefore cause less physiological stress. In agreement with this idea is the observation that a low-copy variant of pUB110 permits the cloning of genes which cannot be cloned in the high.copy variant (Leonhardt, 1990). Whether the relatively low copy number can fully account for the stability of pTAI060, is doubtful, however. We are presently investigating whether additional stability determinants are present in the plasmid.

Structural plnsmid instability Structural rearrangements, most frequently deletions, also constitute a considerable problem in B. subtilis. Since the target sequences for deletion formation do not contain extended regions of DNA ho-



mo[ogy, this process seems to result from illegilimate recombination. Nevertheless, many deletions (up to about 5007o in some cases) seem to occur by recombination c,f short directly repeated sequences, usually from 3-20 bp long. Two classes of mechanism have been proposed to explain deletions in B. subrili$ (Ehrlich, 1989; Ehrlich et al., 1986). The first involves copy choice replication errors, resulting from fork slippage between short direct repeats (DR). The consequence of this template switch is that one of the repeats and the sequences between the repeats are not copied and become deleted in part of the progeny molecules. The presence of DR and the generation of ssDNA is essential in these models. As suggested by Ehrlich et al. (1985), ssDNA intermediates generated during RC replication, are likely to promote these replication errors. This would, at least in hart, explain why ssDNA plasmids are structurally unstable. Although copy choice replication errors may explain one class of deletion, other mechanisms must also exist, for instance to account for deletions between non-repeated sequences. These are frequently considered to be of the breakage-reunion type (Ehrlich, 1989), A key question in the context of th!s paper is whether RC replication and the generation of ssDNA affect the structural stability of plasmids in B. subtilts. Again, the question was addressed as to whether MO play a role in this process. To this end, we used two model systems: one involvingshort DR, and the other, random sequences as deletion targets. A frequently used model system for deletion formation between DR is precise excision of transposons (Ehrlich, 1989; Ehrlich et aL, 1986; Janni&e and Ehrligh, 1987). In a variant of this system, a set of deletion units consisting of DR (9, 18, or 27 bp) which flanked inverted repeats (IR) and a selectable marker, was inserted into a Cm n gene encoded by the pTAI060 derivative pHP700 (Bron et al., 1991 ; Petters ef aL, 1988); this inactivated the marker. The in vivo removal of the deletion unit by recombination at DR, restored the Cm a phenotype, which was positively scored. If this process was in fact stimuluted by the generation of ssDNA, it was predicted

Table I. Effects of ssDNA on deletion formation between DR. pTAI060 (pHP700) pal* 0

Replicon % ssDNA

Apal 30


1.3 x 10 -7 2.g x 10 4

3.3 x 10 ~ 8.8 × l0 6


pAM~I 0

1.2 x 10-: 4.4 × l0 4

1.2 x 10 -~ 2.5 × 10 6

Ratio Apal/pAM~ 1 .~.pal/pal + 11 It0

4 32

Deletion frequencies(fractionof Cm~ cells)are ~ivenper cell/plasmidcopy. ANal(Lenotc~the pTAICfi0dcri~ati~.¢pHPT00(Bron el M., 1991)lackingparT; pal" and pal denotepPH700 carryingputt ill the functionalalld Bon functionaloriginationwhh respocl to ssDNAconversion,respectively. Resultsare given fur tile deletion millscarrying 18-bp DR and I8-bp DR ÷ 300-bp IR.


S. B R O N E T A L.

that the frequencies of deletion formation would be related Io Ihc amounts of ssDNA formed. To test this prediction, the amount of plasmid ssDNA was varied, (I)by using replicons generating different amounts of ssDNA, and (2) by using pTAI060 derivatives in wifich IhepalTMO was either present or absent, in both systems, the deletion frequencies (rraction of Cm k cells} were determined. Results of representative experiments arc summarized in table 1. In the absence of the M e , deletion frequencies with pHP700 were 11- and 110-fold higher for the DR and DR + IR series, respectively, than for those with the thorn-type plasmid pAMI~I which does not generate ssDNA. About 300/0 of the pFIP700 (Apal) plasmid molecules were in the ssDNA form. Analogous results were obtained with other ssDN/, plasmids (Bran et at, 1991 ; Jannidre and Ehrlich, 1987). It ~.vas concluded that the generation of ssDNA by RC replication stimulates deletion formation between short DR, in particular if flanking IR are present. The results in table 1 also show that the deletion frequencies with pHP700 were considerably reduced when palT was inserted in only one of the orientations. In the other orientation, paIT did not affect the frequency of deletion formation. In the correct orientation ofpalT(no ssDNA detectable), pHP700 was only slightly more unstable than pAM[31. The effect of palTwas most pronounced when ]R flanked the DR, This is to be expected if template switching errors during complementary strand synthesis underlie this deletion event: the formation of stem-loop structures by complementary base pairing would bring the DR into close proximity and facilitate slippage of the replication machinery. In conclusion, the results described in this section indicate that the generation of ssDNA by RC replication is an important factor in tee deletion formation between short DR and that these deletions most probably result from copy choice replication errors. At least one other class of deletions has been attributed to the RC mode of replication. These dole. irons result from errors made by Rep, the replication initiation protein (Ehrlich, 1989; Gruss and Ehrlich, 1989). Replication is initiated by nicking the origin sequence and, after one round o f plus-strand synthesis, termination occurs at this sequence by the closing activity of the Rep protein. Aberrant initiation or termination at sites other than the origin nick site underlies a significant number of deletions in ssDNA plasmids {Ballester et at, 1989; E hrlich, 1989; Michel and Ehrlich, 1986). Clearly, this class of deletion event~ belongs to the breakage-reunion type. Deletions between seq~vences other than DR or origin nick sites, cannot be explained by the nmehanisms described above and are generally considered to

be the result of breakage-reunion by topoisomerases (Ehrlich) 1989). A system whleh wa~ useful for the analysis of random deletions (Peijnenburg etal., 1988; 1989} was based on a lecZ-gene fusion, in which deletions were recognized by the appearance of white colonies on X-gal-eontaining plates. Analysis of deletion endpoints suggested that topoisomerase I might be involved, To test whether the RC mode of replication affected this deletion event, pTAI060 and pAMI~I derivatives were compared. No differences in deletion frequencies were observed. Also, paITdid not affect deletion flequencies in pTAI060 derivatives (Bran and Relano, in preparation), This indicated that RC replication does not affect this deletion event.

Cloning vectors based on pTAI060

In the foregoing chapters, we described plasmids derived from pTAI060 which were segregationally more stable than other ssDNA plasmids. Even io the absence of their M e , pTAI060 plasmids showed only moderate levels of instability (fig. 2B). These results prompted us to construct a series of cloning vectors based on pTAI060 (Harms et at, 1987, 1990a,b,c). pHPI3 (Haima el al., 1987) is a B. $ubtilts/E. ¢oli shuttle plasmid which carries the replication functions of pTA1060 and pBR322. Although palT is not present in pHP13, very high efficiencies of shotgun cloning of E, coil DNA were obtained in restrictiondeficient B. subtilis host cells. The fraction of recombinant clones may amount to over 30%. In contrast to other ssDNA pIasmids, large inserts were relatively abundant (26°'/0 of the clones contained inserts ranging from 6-15 kbp). A more versatile variant of pHPl 3 is pHPS9. This plasmid enabled ~-galactosidase-lacZa complementation in B. subtilis (Haima etal., 1990b; 1990c). To achieve this in p}IPSg, the tacZ~ gone was fused to Gram +.expresslon signals (promoter P59 from Lactecoccu~ ?actis and the N-terminal part of the Bacillux cat86 gone). The other component of the complementation system, the lacZZkA,ll5 gone, was also placed under the control of Gram*-expression signals (promoter P23 from L. lactis and translation initiation signals from the spoOF gerte of B. subti/is). To stabilize the/aeZZXM15 gone, it was integrated as a single copy into the chromosome by double cross-over replacement recombination. The resulting strain was denoted 6GMIS. The complementation system is schematically shown in figure 3. This system has several advantages over other cloning systems used so far in B. subtilis. (l) The blue/white assay on X-gal plates enables the direct selection of recombinant clones. (2) Several cloning sites are available in the lacZ= gene. (3) The efficiency








SphI c0t88::lucZe(e~x~'d'wll .

vsu, le c r o s s - o v e r





Fig. 3. ~-Galactosidase tacZa-comptemcnration system in B. subtilis. A) Plasmid pI.4PS9, containing the pTA 1060 and pDC repiieatinn functions. The pelT MO is absent. The lacZ= gene is fused (in frame} to cat86 sequences. B) Integrated copy o f the lacZZXMI5 gene in the g. subtilis chromosome. The expression of both the lacZ~ and the lacZAMI5 gene is controlled by Gram ~ transcription/translation signals.

of cloning in restriction-deficient competent B. subtilts cells is high (under optimal conditions up to 30-60% o f the transformants are recombinant). (4) The average insert length is high and inserts up to about 30 kbp have already been successfully cloned (larger inserts have not been tested). (5) The structural stability of the recombinant clones is high; no deleted plasmid forms were detected after I00 generations of growth under selective pressure. A general factor limiting cloning effieieneies in B. subtilia is the requirement for plasmid multimers in competent cell transformation. We made one further variant o f the pHPS9-1aeZa eomplementation system to avoid this complication. It was based on homo[ogy~faci[itated plasmid marker rescue transfor~ mating. The donor plasmid was again pHPS9. The recipient cell was 6GMI5 carrying a resident plasmid, pHPS9R, which, except for a deletion removing the lneZo~ gene and part of the flanking seleetable Cm R

marker, was identical to pHPSg. Homologous recombination between donor and resident plasmid resulted in Cm•/lueZa transformants (Haima el al., 1990a). [n addition to the advantages mentioned above, the marker rescue system enabled forced cloning experiments to be carried nut and competent cell transformation with plasmid monomers to occur. These cloning systems can he obtained through the Bacillus Genetic Stock Center, Columbus, Ohio (strain 6GMI5: IA718; ptasmid pHPS9 in E. colt: ECE51 ; strain 6GM 15 (pHPS9R) : 1E52). One further improvement may be beneficial: the introduction o f p a l T in the cloning vectors. Although the segregational stabilities of large recombinant plasmids obtained with pHPS9 have not yet been tested, it is anticipated that a fmlctional MO will improve their maintenance. Such vectors are now being construtted.


S. B R O N E T A L .

Conclusions Many piasmid-cloning vectors used for B. s,.btih s are derived front othc~ G r a m ÷ bncterla. These are frequently highly unstable, both segregationally and structurally. These plasmlds gcnetate coitsiderable amounts o1" ssDNA iniermedig'~es by RC replication. Plasmid s s D N A is a major factor causing segregational instability and at least one form o f structural instability (deletions between short direct repeats). The absence o f functional M e o f replication in many cloning vectors increases the levels o f instability considerably. RC replication also underlies a class of deletions caused by aberrant initiat i o n / t e r m i n a t i o n reactions o f the replication initiation protein. The B. subtiliy plasmid pTA 1060, although generating ssDNA, is fat more stable, in particular when its MO is present. A set o f versatile, efficient and stable cloning vectors had been constructed from pTAt060. K e y - w o r d s : Bacillus subtilis, Plasmid ; Vectors, ssDNA, Instability, Molecular cloning.

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Plasmid instability and molecular cloning in Bacillus subtilis.

~-) INSTITUTPAStEUR/[~.t.S[:VU~R Paris 1991 Res. Mierobiol. 1991, 142, 875-883 Plasmid instability and molecular cloning in Bacillus subtilis S. Bro...
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