Copyright© 1989, American Society for Microbiology
Stability of Bacillus Plasmid pBAA1
KEVIN M. DEVINE,* STEPHANE T. HOGAN, DESMOND G. HIGGINS,AND DAVIDJ. McCONNELL DepartmentofGenetics, Trinity College,Dublin 2, Ireland
Received31May 1988/Accepted 13 October 1988
Acrypticplasmid, pBAA1,wasidentified inanindustrial Bacillus strain. The plasmid is 6.8 kilobasesinsize and ispresentincellsatacopynumberof approximately 5perchromosomeequivalent. The
p!asmidhas been maintained under industrialfermentation conditions withoutapparentselectivepressureandsoisassumedto bepartitionproficient. The minimal repliconwaslocalizedtoa1.4-kilobasefragment which also contains the functions required for copynumber control. The verylow level ofsegregational instability of the minimal replicon suggests that it also contains functions involved in plasmid maintenance. Comparison with other plasmids indicates that pBAA1 belongs to the group of small
gram-positiveplasmids which replicate by a
cycle-typemechanism. Asequence was identified which isrequired for the efficient conversion of the
single plus strandtothedouble-stranded form during plasmidreplication. Deletionof thissequenceresulted in alowlevelof segregationalplasmid instability.
The development ofcloning vectors for use in Bacillus
subtilis has reliedonplasmidsisolated fromStaphylococcus
aureus(6, 7, 14, 15). Both structural (10, 15, 19, 20, 27)and
segregational (1, 2, 10, 19, 28) instabilities were frequently
observed whenrecombinantvectors weretransformed into
B. subtilis. These instabilities ledtointense investigation of the modeof replication and ofthestability functions ofthese
plasmids. It has emerged that many of these plasmids
replicate byarolling cycle-type mechanism (11, 12, 31).The
essential features of this modeofreplicationare(i) anorigin of plus-strand synthesis, (ii) a replication protein which interacts with the plusorigintogenerate anickwhich allows displacement synthesis of the plus strandtooccur, and(iii)a
signal for efficient conversion of the single strand to the
double-stranded form. These features have been identified
fora variety of plasmids,
includingpT181 (12, 16, 25) and pC194 (11, 12).
Our aim is to analyze the
plasmids inB. subtilis. Itwas
segre-gational instability of manyS. aureusplasmidsin B.subtilis is due,atleast inpart, to asuboptimalhost-plasmid
relation-ship.Thus, wechosetoanalyze thestability functions ofa
plasmid, pBAA1, which wasresident inan industrial strain ofB. subtilis. pBAA1 has beenmaintainedunderindustrial
fermentation conditions without apparent selection
pres-sure. Preliminary experiments demonstrated that the copy
numberwaslow (approximately 5 perchromosome
equiva-lent), soitwas assumed that pBAA1mustencode an active
partitioning function. Inthis paper, we report that allof the
functions required for replication,copy number control, and
fragment.The DNA sequence and other evidence show that pBAA1
repli-catesbyarolling-circlemechanism and that somefeaturesof itsreplicationfunctions are related both to
gram-positive plasmids pC194,
Bacterial strains and plasmids. The bacterial strains and
plasmidsusedinthis study are described in Table 1. Bacteria
were grown in LB (22). Medium was supplemented with antibiotics to thefollowingfinal concentrations: ampicillin, 50pug/ml; chloramphenicol, 5
Manipulation of DNA. All manipulations of DNA were
performedasdescribed by Maniatisetal.(21). All enzymes
werepurchasedfrom commercial suppliers.Sequencingwas
carried out by the
Sanger et al. (29). A sequencing kit was used (Amersham
Corp., U.K.); reactions were carried out according to the
instructions of the manufacturer. In some cases, 17-mer
Manchester Institute of Science andTechnology)wereused
Analysis ofsingle-stranded DNA production.Lysateswere
prepared by the method ofteRieleetal. (31). Lysateswere
0.5 ,ugof ethidium bromidepermlfor
4 V/cm. Todetect single-stranded plasmid DNA only, gels
weretransferredtoBiodyne (PallUltrafine FiltrationCorp., EastHills, N.Y.) withoutprior denaturation bythemethod ofte Riele et al. (31). All other gels were denatured with
diffusion withlox SSC(lx SSC is0.15 MNaCI plus 0.015
citrate) accordingto the instructions of the
manufacturer.Prehybridization, hybridization, andwashing
Autoradiograms were made with RX medical X-ray film
Film,Japan) with intensifying screensat room temperature.
Copynumberdeterminations.Plasmidcopy numbers were
determinedasfollows. TotalDNAwasprepared separately
fromcellscontainingtheplasmids of interestand was double
digested with BamHI-HindIII, which cuts the plasmids to
giveapBAA1 fragment of approximately2.2kb. Thisdouble
digestioncuts chromosomal DNA, releasinga fragment of
approximately 4 kb which contains the ,-glucanase gene.
ThedigestedDNA wasseparatedon a
wastransferred to Biodyne. This filterwas thenhybridized
with a nick-translated pJG14 plasmid (which contains both 1.5kbof
P-glucanaseDNAand 1.4 kbofpBAA1 DNA).The
filter was then washed and exposed to X-ray film. The
developed film was scanned densitometrically with a
BACILLUS PLASMID pBAA1 1167
TABLE 1. Strains and plasmids used
Strainorplasmid Description Source or reference
E.colitgl K-12A(lac-pro) supE thi hsdRF' traD36 proABlacIlacZAM15 Amersham B. subtilis
S0113 trpC2 amy-3 26
8G-5 trpC2 tyr ade his nic ura rib met highly transformablederivative ofB. subtilis168 3
pBAA1 6.8-kb cryptic plasmid isolated from B. subtilis BAA1 BioconLtd., Ireland;
this work pDE68 pBAA1 with theCmrgene frompBD64 cloned into anEcoRIsite(7.8 kb) Thiswork pDE37 The 3.7-kbEcoRl fragment of pBAA1 plus the Cmrgene of pBD64 (4.7 kb) Thiswork pDE22 The 2.2-kbHindl.l fragment ofpBAA1 plus theCmrgeneof pBD64 (3.2 kb) This work pDE14 The 1.4-kb pBAA1 HindIII-BamHI fragment inpRP14 plus the Cmrgene of Thiswork
pBD64.This plasmid underwenta100-bpdeletion attheBamHIsite which does notdetectablyaffectthereplicationproficiency copy number orchloramphenicol resistance oftheplasmid(2.3kb)
pBD64 Plasmid derived frompUB110encoding Kmrand Cmr 13
pRP1 Replicon probe plasmid derived by insertion of theHpaIIfragment of pBD64 Thiswork containing theCmrgeneinto theEcoRIsite of pUC18
pRP2 Replicon probe plasmid derived by insertion of theHpaIIfragment of pBD64 containing theCmrgeneinto theAccIsite of pUC18
pRP3 Replicon probe plasmid derived by cutting pRP2 withSmaI andinserting aHindIII Thiswork linker
pJG14 pRP14containing a 1.5-kb fragment on which is located the P-glucanasegene Thiswork
M13mpl9 M13-derived vectors used in the dideoxy sequencing method
matogram densitometer CD50 (DESAGA, Heidelberg, Fed-eral Republic of Germany) with an integrator to estimate peak area to determine the intensities of signals from the
3-glucanaseandpBAA1 fragments. Three quantities of total DNA (0.1, 1.0, and 4 ,ug) were loaded onto the gel, and the
filterwasexposed for various lengths of time to ensure that the signals were within the linear range of response of the
film. The similar sizes of the
P-glucanaseandpBAA1 frag-ments in the gel ensure that the efficiencies of transfer of
both fragments to Biodyne should be very similar, if not
identical. Likewise, the similar sizes of the ,B-glucanase and pBAA1 fragments ontheprobe plasmid ensure comparable
efficiencies of hybridization. Since the probe contains both the
P-glucanaseandpBAA1homologous DNAs, thespecific
activity of the probe is identical for both species of DNA. Thus the ratio of signals of pBAA1 to 3-glucanase is an
estimate ofthe number of copies ofpBAA1 per chromosome
Analysis of the segregational stability of plasmids. Cells
containing plasmids were grown in LB in the presence of
antibiotic. These cells wereused toinoculate antibiotic-free medium. Ten successive transfers (approximately 100
gen-erations) were performed in antibiotic-free medium over a
period of80 h. Approximately 105 cellswere transferredat
eachstage. Thecellswerethen platedonantibiotic-free LB agar, and individual colonies were tested for resistance to
antibiotic. At every second transfer, 100 colonies were
tested for eachplasmid. Plasmids whose stabilities were to
Delimitation of the minimal replicon of pBAA1. Plasmid
pBAA1 was isolated from an
Bacillus strain. It is 6.8 kb in size,andits restriction map is shown in Fig. la. This plasmid has no known selectable
marker but has been maintained stably under industrial fermentation conditions without apparent selection pres-sure.When marked with achloramphenicol resistance gene
(derived from pC194) and transformed intoB. subtilis, the
resultant plasmid pDE68 was found to be segregationally stable. The copy number ofpBAA1 is approximately 5 per chromosomeequivalent. It is thus assumed that theplasmid isstably maintained byan active partitioning system.
To delimit the minimal replicon, fragments of pBAA1
weresubcloned ontothe replicon probe plasmidpRP1 (Fig.
lb)andweretestedfortheirabilitytosupportreplication by transformation into B. subtilis and selection for
antibiotic-resistanttransformants. It wasfound that plasmids pRP37,
pRP22, and pRP17 containing 3.7, 2.2, and 1.7 kb,
respec-tively, of pBAA1 replicatedin B. subtilis. To further delin-eatetheminimal replicon, progressive deletion of each end of the 1.7-kb HaeIIIfragmentofpBAA1 wascarriedoutby
using Bal 31 (Fig. 1c). The precise endpoints of the Bal
31-deleted derivatives were determined by sequencing.
Re-sults showed that deletion of 350 base pairs (bp) from one end of thefragment allowedreplication,whereasdeletion of afurther 230bp(regionA) abolished theabilitytoreplicate. Similarly,deletionof356
bp (regionB)from theother endof
the fragment abolished the
whetherpUC18 containedonthe pRPplasmids contributed
to their replication proficiency,
pUC18does notaffect thereplication
plasmids.It was concluded that the minimal replicon of
contained on a 1.4-kb
regionsA and B
contain functions essential for
pBAA1in B. subtilis.
Sequence of the 2.2-kb HindIIIfragmentofpBAA1 which contains theminimalreplicon. The 2.2-kb
(a) L r
H v RIH
H H RI
1kb ,PIHP EI EH ,,
A AA E/
M_ ,, /
(c) E A A A ORF A
>j pRP17 rep+
E pRP14 rep+
FIG. 1. (a)Linearizedrestriction mapofplasmid pBAA1. (b)The 3.7-kbEcoRI,2.2-kbHindlIl,and 1.7-kb HaeIIIfragmentsofpBAA1 wereinserted into compatible restriction sites in the multiple cloning site ofthe replicon probeplasmid pRP1 andwere tested for their replication proficiency inB. subtilis. (c) pRP17 wasseparately digestedwith HindIII andBamHI anddigestedwith Bal31todelimit the minimal replicon. After endfilling and ligation, the plasmids weretransformed into Escherichia coli and the sizes of the deletions were
characterized.Aseries ofplasmids withincreasingamountsofpBAA1 deletedweretested for theirabilitytoreplicateinB.subtilis. Deletion ofregionA or Babolished theabilitytoreplicatein B. subtilis. Thesequenceof theregionsessential forreplicationisgiveninFig.3.rep'
andrep-, Replication proficient anddeficient,respectively. A,RsaI;B,BamHI;E, HaeIII;H,HindIll;P, PstI; RI,EcoRI; V,PvuII.
methodofSanger et al. (29). Shotgun-cloned AluI andRsaI
fragments and Bal 31-generated deletion derivatives were
sequenced. Syntheticoligonucleotides wereusedasprimers to sequence regions of nonoverlap. Both strands were
se-quenced, and each base pair was sequenced at least four
times. The sequence is shown in Fig. 2. Analysis of the
sequence reveals an open reading frame (ORF A) of307
amino acids. ORFAispreceded byaputative&43promoter
(P1). Two lines of evidence indicate that this protein is
involved in replication ofpBAA1.First, deletion of region B,
which extends 334 bp from the 3' end ofthe open reading
frame(Fig.2,regionB),abolishesthereplicationproficiency ofthe plasmid. The second line of evidence derives from a
comparison of this protein with the replication proteins of
pUB110, pC194, and pFTB14 (23) (Fig. 3). The
92%homologous attheamino acidlevel
with the replication protein of pFTB14 (homology not
shown) and homologous to alesser though very significant extent with the replication proteins of pUB110 and pC194
(Fig. 3). Thus, theprotein coded by the open reading frame ofpBAA1 is designated RepA.
A 230-bp fragment (Fig. 1c and 2, region A) was also
shown tobeessential for replication of pBAA1. Analysis of the sequence in this region shows four sets of inverted repeats ranging in size from 4 to 14 bp. To discover the sequencesessential for replication, a homology search was
performedto determinewhether there are sequences within
this region conserved among other Bacillus and S. aureus
plasmids known to replicate in B. subtilis. Results (Fig. 4) showed that a 15-bp sequence was conserved in pBAA1,
pUB110,pC194, and pFTB14. No significant homology was
found with pT181, pC221, or pE194. This conserved se-quence is found in the55-bp region of pC194 shown by Gros etal.(11) to have originactivity. Within this 15-bp conserved sequence are two additional features. (i) The sequence CTTGATA is the sequence at which nicking of the plus
strandoccursin the initiation ofreplicationof the coliphage XX174. (ii) An 18-bp sequence (containingthese conserved 15bp) has been shownby Grosetal. (11)tocontainasignal sufficient to terminate replication ofplus-strand synthesis. Thesedatastrongly suggestthat this15-bp conservedregion is part of the origin of pBAA1 and is one of the features within region A which is important for replication. In addi-tion, by analogy with 4X174 and pC194, it suggests that pBAA1 replicates byarolling circle-typemechanism.
Copynumberdetermination ofparentaland deletion deriv-atives of pBAA1. The copy number of pBAA1 and the deletion derivatives were determinedas describedin Mate-rials and Methods.Densitometricscanning of the autoradio-gram indicates that the copy number of pBAA1 in the industrial strain BAA1 is 2 to 3 chromosome equivalents. The copy numbers of pDE68, pDE37, pDE22, and pDE14 in
the laboratory strain S0113 are the same as that of the
parental plasmid in the industrial strain BAA1. Under these
conditions,the copynumberofpC194 is11perchromosome
equivalent,approximatelyhalf the reported value. If normal-izedtothereportedvalue of pC194, then the copy number of pBAA1 and its derivatives is approximately 4 to 6 per chromosomeequivalent. Itcanbe concluded that the
mini-mal replicon of pBAA1 contains thefunctions required for
copy number control.
Some deletion derivatives of pBAA1 produce
BACILLUS PLASMID pBAA1 1169
ATAC Al~~lllpJlCKX]OOCSTATAATATl=rAT~A rI-l-AC-l-l-AAA IZH
Aax~lA~r~r.A~rGAC.~ri~r~ru7A~r~sA.AcT. TrCAACACi&'t~jWlMIyiM.A 284A _ X A l A l < M l . 3b44
AygAGI#AATAGOAGATl".AAXCAAITAYACA1A(TIATITXTIGACI.IrT1XCTArAAT;AAxt(~TrAG0A-AI---.AJJ-AJOT,-MA.iIIGA-1-1-1m GAT GAT'AAATx r-1-TAlTAVJIA 52444448
AAATrAlrmrAAZAArA6ATiVl-t'n rttFAT01CrITAcAmrI s-*AArmo-XIrrcAAAEroA(,ITAAA 542
A>(rlTI7'Asfi ~ i---lli-mT~ r ~ YXI.Al;-IMI Ail*(Ci;]T(TATAT-ViAA J4
ATrA~ll7'rrAAAAIY'A(.l-lAA1.nrT.r71TIAA(XXTrAr.Gy.rr~.%Al5YrlAAAAi n'RAlATT 92H
Al 'AIlAATAhATAATrVk- l 1A A-OMTAAr',rrrTr.AM~rAlrAA OOMH
A(1GATTfATAGCAT¶TJ.AATAAAAM'AArXTAAAGT2.A1OOAA IrATaAAJYC I(M1m
AIMGX'GGAG CATTMA GM(AMA TTAGAGAM MAGATTOG(GOCA(ITTKTAT OCAM AMG 1144
MetAla Glu HisTyr Gl AlaIue Giti HSer Lys Ile Gly Ala Pro Tyr Tyr Gly Lys lys 211
OCTGM AA CTAATTACTTT(rAGAGTAT TI= TTTMAGAGA(G aM GAGMO;GOC 12044 AlaCluG ysly u lieSeriyeAlaGluTyrlpu Ser PheLyeArtAspProGlu ThrGly 40
AAG T!A MAAi TATCM 1rrCAT mTOT MA GTGAGO TTA1MC(LAM1m7TOrr,10 1268 Lys1-ulyeleu Tyr GinAla HisPheCys LysVat ArgLouCysProMpt Cys Ala Trp 60
(tIC AGO TL TTA MA ATT OCT TATCMrMTMGTTG ATCCTAGAGGM OCC MT(TC CAG 1324 6rgArg Ser Leou lys I e AlaTyrHis AsnLyslpu I eVal Clu Glu AlaAsn ArtGin 80
TAC00C 10C OGAT1GAT!TTiTCAM MAM1M,CTGMT GTAMCGOA GM(TI CLI01 1384 Tyr Gly Cys GlyTrp lie ThLeuLe rlouThrVal Art Asn Val Lys Gly Glu Arg lou 100
AG CACMATT TIT GAGAMGAMGGM00 Tm AML AM11 Tit CAITA AAAMACIA 1448 LysProGin leSerGluMetMet Glu GlyTheAr1 Lys iJu The GinTyr Lys Lys Val 120
AAMT1MT MT `TT 0GGATTT07 AGOTTTAGAGATT AM MT CATGM GM GAT 1I50M Lys Thr Ser Val Ieu Gly PhePheArgAla LUGClulieThrLysAn His Glu Glu Ap 140 ArATAC CATCOT CAT mTT CAT 1G7 TTG ATACrA OTA AOG A MT TATTTT00MAAMC 1568
ThrTyrHis ProHis Te His VaIt u lePro Vat Arg LysAsnTyr PheGly Lysmn 160 TAT ATT MG CAG0OM2 GAG TOG ACGOCCTTTMG AA MG MAG AAATTG GATTMr AT 1628 Tyr IleLye GinAlaGluTrpT'r Ser leuTrpLyelysAiaMet Lys Leu AspTyrTIr 180 .= ATT CT! GATATT17CGA G1TGMA MAOATAMMGATT GAC(X1rGM C1G ATTlAA 1688
Pro TIe ValAsp Ile ArgArgVat Lys GlyLyseAlaLysIle AmpAlaGluLeuIleGlu 200
Mr CATG17; (00 (A WA AM AlT WA0CMMAA OCTTO1TT (T GAAAT!OGAA TAT 1748
AMn AspValArg GluAlaMet Met GluGin1y Aia Val Leu Glu lie|erLys 220
G'"AAGGATUMGAT]GTT 1r. oCAoC MAT AAG G1 mT GMGMCMAAT CMAMrA01a 1808
Asl1.3mApilir AspVal ValArgGlyAsn lys Val Thr Glu Asp Asn Leu mn ThrVal 240
CTT TM TT CATGAT xr.T2 OrA OCT(rr2 AOGTTA ATTOGA TAC017 aOt ATTTT AAGI164
IouTyr IojAsp Asp AlaLeuAlaAlaArgArgtuI le Gly Tyr Gly Gly Ile Leu1 260 GAGATA CATMAAGAG IOMTCT 017 GAT HOMGAGGfGOC GAT(C 1710 MGATT AG 1928
CliIlie HisleGblu lpuAnn luC.ly AspAlaGlu AspGlyAspltuValLyslIeGlu 280
AAA;A4A1; ATOMO; MA(i17 CAAAMr G-Ml CATTTG AGOTTA151 ("l ATT4 (rAlT 1lGCAT GliiGhIsAspUp Clii ValAia AnnGlyAlaCe GluVal Met AlaTyr TrpHlisProGly A1TAAAAAT TAr ATA ATr AAATM44.AAWAACAGa(1TMiJ C A.AMAAl-CCArTC 7AT
I lI.)AsnTyr IIe l1e yeStop 8aie8
FIG. 2. Sequence of the 2.2-kb HindIII fragment ofpBAA1.Regions A and B, which areessential for replication,arebracketed.Inverted repeats aremarked witharrowsdirectlyover thesequence, while direct repeats are boxed. Theputative active site oftheprotein isboxed, and the conserved tyrosine residue is underlined.P1isa consensus(,41promoter; aputativeribosome-binding site is underlined. Thesequence conservedinplasmidspC194,pUB110,andpFTB14(Conserved15-mer)isshown; the arrowwithin thissequenceindicates the siteatwhich nicking oftheplusstrand occursinphage fX174.
strandedplasmid DNA. Intherolling-circle model of plasmid replication, the plus strand is synthesized as a covalently closed circular single strand of DNA. Synthesis of the second strand then initiates at a minus-strand origin se-quence, generating a double-stranded plasmid DNA mole-cule (for a review, see reference 18). Such minus origins,
which are characterized by their potential for secondary
structure formation, havebeen identified forthecoliphages
M13 (9), G4 (8) and (X174 (30). In addition, minus-origin
sequences have been identified for a variety of plasmids,
e.g., pT181,pC221, andpC194 (12). Deletionof the
plasmid DNA. IfpBAA1 replicates by a rolling cycle-type
mechanism, asissuggested by thehomologyto 4X174and
pC194,then(i)itshouldhaveaminusoriginand(ii)deletion of this minus origin should lead to the accumulation of
single-strandedplasmidDNA. Todetermine the location of
the minus origin ofpBAA1, cells containing the pRP and
pDE series of plasmids were tested for the presence of
single-stranded plasmid DNA, asdescribedin Materials and
withoutprior denaturation of the DNA with NaOH(31). In
the absence of denaturation, only single-stranded nucleic
acid binds to the filter. Figure 5 shows the results for the pDE series ofplasmids. Plasmids pBAA1, pDE68, pDE37,
pDE22, and pDE14 show two major
probably corresponding to CCC monomeric and dimeric
plasmid species.These bands didnotbindto
Biodynein the absence of denaturation. Plasmid pDE14, in addition to thesebands,hasaband(arrow)which
double-stranded DNAspeciesand which boundto
in the absence of denaturation. This fast-migrating band is
preferentially degraded by S1 nuclease under conditions
which cause no detectable degradation of the double-stranded forms (data not shown). These data indicate that this band is single-stranded plasmid DNA. For the pRP series of plasmids, the single-stranded plasmid DNA was observed in cellscontaining pRP17 butnot in cells contain-ing pRP22 or pRP37 (data not shown). Thus, sequences presenton the 2.2-kbHindIII fragmentbut absent from the 1.7-kb HaeIIIfragmentarerequired for normal replication of theplasmid.
Two regions of DNAaredeletedfrom the 2.2-kbHindIII
fragment to give the 1.7-kb HaeIII fragment: a 355-bp
fragment from the left-hand (5') terminus and a 197-bp
fragment from the right-hand (3') terminus (Fig. 2). To
determine the location of the minus origin on the 2.2-kb
HindIII fragment, the sequences of these regions were
analyzed.The355-bpfragmentdeleted in the 1.7-kb HaeIII
subfragmenthas thepotentialfor complexsecondary
struc-tureformation. Sincenounusual featurewasevident in the
197-bp right-hand sequence, we decided to concentrate on
the355-bp sequence. Three
(200 bp), EagI (353 bp), and NaeI (770 bp) were used to
generatemutations in the left-hand end of the 2.2-kb HindIII
fragment. Plasmid pRP22 was restricted with
pRP22F, which had a 5-bp insertion into the BstEII site. Plasmid pRP22 was also
BstEII-EagIand withEagI-NaeI,the endsweremade blunt,
and the vector was
religated.This yielded plasmids
pRP22BE and pRP22EN, with 152- and
lysatesweremade of cells
1170 DEVINE ET AL.
PW"IISSKWUPIMCE I. T
BilHpSK--- Is i
P11 S6ZU LLHIL --' ! l
r% SL WL6tKQ XE-FI HEE
HL C6~ 1 tFLTLMR1KRLa
Em!LKDYTPIt .UZ.I+LItE" I OTl
I---- --lT aTol~
t--S- --- YLS-t
ULH @+ EK1
ODE R RL .-I T e
FIG. 3. Comparison of the amino acidsequencesof the
replica-tion proteins of pBAA1, pC194, and pUB110. The amino acidsin
boxesareconserved between thepBAA1 replication proteinandthe replication proteins of pC194 andpUB110. The amino acidsequence
of thereplication protein of pFTB14 is 92% homologous tothat of
pBAA1 (homology notshown). Dashes representgaps inserted to optimize the protein alignment.
single-stranded plasmid DNA, whereas plasmids pRP17, pRP22F, and pRP22BE produce detectable quantities of single-stranded plasmid DNA (data not shown). Similarly, deletion of theBstEII-EagI fragment from plasmid pDE22 resulted in the production of single-stranded plasmid DNA (data not shown). Interestingly, the amount of single-strandedplasmid DNA produced by pRP22F is less than that produced by pRP22BE, suggesting that the 5-bp insertion attenuatesbut doesnottotally abolish this function. Itcanbe concluded thatsequences which when deleted result in the
production of plasmid single-stranded DNA, overlap the BstEII site (atbp 200; Fig. 3) but donotextendasfarasthe
EagI site (at bp 352; Fig. 2). Thus, asignal required for the
efficient conversion of the single-stranded plasmid DNAto thedouble-stranded form during replication is locatedtothis region.
MF3TI 6TCTTTT CTTATCTT ACTATATAGCAACT
IIII I Il I I
FIG. 4. Homology at the origin for plus-strand synthesis be-tween plasmids pBAA1, pFTB14,pUB110, and pC194. The 15-bp sequenceconserved in all four plasmids is in boldface. Thesequence
CTTGATA, which is the site atwhich nicking of the plus strand
occursin4.X174, is underlined. Nickingoccursbetween the G and
A residues of this sequence. Vertical lines indicate bases not
homologous with pBAA1.
FIG. 5. Detection ofsingle-stranded plasmidDNAin cells
con-tainingpBAA1 andits deletion derivatives. Whole-celllysates ofB. subtiliscontainingnoplasmid (lanesAandA'),pBAA1 (lanes B and B'), pDE68 (lanes C andC'), pDE37 (lanesDandD'),pDE22(lanes EandE'),orpDE14(lanesFandF')wereseparated byagarosegel electrophoresis, denatured with NaOH (panel 1) or notdenatured (panel 2), and transferredtoBiodyneasdescribed in Materials and Methods. The filterwasthenhybridizedto32P-labeled pRP14. The bandofsingle-stranded plasmid DNA(4)isshown.
Segregational stability of pBAA1 and the deletion deriva-tives. Since pBAA1 wasstably maintained under industrial
fermentation conditionswithout apparentselective pressure, it is assumed to be segregationallystable. Thesegregational stabilities of the deletion derivatives were analyzed as de-scribed in Materials and Methods to detectplasmid-encoded
functions involved in this process. Results(Table2) demon-strate that pRP22 and pDE22 aretotally stable under these conditions. Plasmids pRP17 and pDE14, both of which
produce single-stranded plasmid DNA, displayeda lowbut
reproducible level of instability, with 5 to 20% of the cells
losing plasmid duringthegrowth period. The deletion ofthe
BstEII-EagI fragment from pRP22, which resulted in the
productionofsingle-stranded plasmidDNA, also resulted in
instability.Noinstabilitywasobservedforthe5-bp insertion into the BstEII site (pRP22F) despite the production of
single-stranded plasmid DNA. It can be concluded that
removal ofthe entire 355bpHindIII-HaeIIIfragment results in a low level of instability. This instability does not,
however, correlate with the production of single-stranded
TABLE 2. Segregational stabilityof pBAA1 derivatives
Plasmid Production ofssDNA' Stability(%)
pDE14 + 79
pRP17 + 95
pRP22F + 100
pRP22BE + 88
assDNA,Single-strandedDNA.Symbols:+,production of ssDNA;-, no
B L PLA1MID
A newplasmid, pBAA1, has been identified in an
indus-trial Bacillus strain. Results presented in this study demon-strate that pBAA1 belongs to the class of small, naturally occurring plasmids of gram-positive bacteria. Although pBAA1 is different in size and restriction map from pLS11
(4) and pFTB14 (23), a high degree of homology exists
between these plasmids in regions involved in plasmid replication. Homology also exists between the plus-strand origin and Rep protein regions of pBAA1 and those regions ofpUB110and pC194, the greaterlevelof homology being found withpUB110. No homologywasdetected between the
replication regionof pBAA1 and other gram-positive
plas-mids pT181, pC221, and pE194.
The minimal replicon of pBAA1 was found to reside withina 1.4-kb fragment of DNA. The copy number ofthis
plasmid is similarto that of the parental plasmidat
approx-imately 5 per chromosome equivalent. In addition, this
plasmid is very stable, with over 80% of cells retaining
plasmid after 100 generations in the absence of selection. It
canbeconcluded that all functions required forcopynumber
determinationarecontained within the minimal replicon. In
addition, the lowcopy numberper chromosome equivalent
suggeststhat an active partitioning system may also reside onthisfragment.
Abody of evidence supports thehypothesis that pBAA1 replicates by a rolling circle-type mechanism. Key features
of thismodeof replication include (i)anorigin of plus-strand
synthesis containingasiteatwhich the DNA isnicked;(ii)a
replication protein which nicks the plus strand, forms a
covalent linkage with the DNA, and renicks and ligates the newly synthesized plus strand afteroneroundofreplication;
and (iii) an origin sequence for initiation of minus-strand
synthesis. The replication functions of pBAA1 have several features homologous to the replication functions of coli-phage 4X174 and S. aureus plasmid pC194, both of which
replicate by a rolling cycle-type mechanism (11, 18). The
similarities between(X174, pC194, and pBAA1 include all thefeatures essentialtothis mode of replication. For
exam-ple, thereareconservation ofsequenceattheplus origin and conservation of amino acidsat the active site of the replica-tion protein in all threecases. Inaddition, all three containa
genetic function which although notsimilaratthe sequence
levelis involved in efficientconversionof the single strandto thedouble-stranded form. In summary, these data strongly suggestthatpBAA1 replicates byarolling cycle-type mech-anism.
Itwas observed that deletion or mutation ofa sequence
overlapping the BstEII site results in the production of large quantities of single-stranded plasmid DNA. In view of the strong likelihood that pBAA1 replicates bya rolling
circle-typemechanism, it is probable thatthissequenceisinvolved in the efficient conversion ofthe single plus strand to the double-stranded plasmid form. Palindromic plasmid
se-quenceswith thisactivity have previously beenreportedfor gram-positive plasmids, e.g., pT181, pC221, pUB110, and pC194 (12), and forpLS1 (5). This region ofpBAA1has no
homology with the previously reportedminusorigins. Thus, thissequenceconstitutesanovelminusoriginfor thisgroup
The HindIII-EagI 350-bp fragment ofpBAA1 is almost identicaltothesequenceofthefragmentofpLS11, reported to contain a partition function(4). There are differences in thesequencesatonlyfourpositions:therearesix C residues between bases 306 and 311 in pBAA1 but only four C
pLS11;there is a T residue at
plasmidconstructions suggest that the par function (of
minus-origin activity(of pBAA1) may be the samefunction.
predictedtofunction in oneorientation only (12); Changet al. (4) report that their par sequence functions only in one orientation. (ii) The minus
overlapthe BstEII site but do not extend as far as the
(iii)Deletion of neitherparnorthe minus
plasmidcopy num-ber. A crucial difference betweenthese
is the effect of their removal on
stability.Removal ofpar resultsin adramatic reduction in the
pLS11 constructions,whereas inactivationor deletion of the minus
onlyalow level of
instability.These differences may be caused
plasmidconstruction differences. Itcan be
concluded, however,that increases in the
plasmids.As shown in this
produces single-stranded plasmidDNA but is not
plasmidDNA in B.
pE194isvery unstable in this
detectablyunstable after 100
generationsin the absence of selection
This work was supported by European Economic Community
wassupported bycontract BAP-0137-IRL.
We are grateful to Dusko Ehrlich and co-workers, especially Sandy Gruss, HeinteRiele, andMarie-FrancoiseGros,for
provi-sion ofdata prior topublication andfor manystimulating discus-sions. WearealsogratefultoBiocon Ltd. forprovisionofpBAA1.
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