K. Leenhouts .G. Buist. A. Bolhuis. A. ten Berge J. Kiel. I

Springer- Verlag]
MoJ Gen Genet (1996) 253:217-224
K. Leenhouts .G. Buist. A. Bolhuis.
J. Kiel. I. Mierau .M. Dabrowska
G. \'enema. J. Kok
A general
in
bacterial
system
tor
996
A. ten Berge
generating
uniabelled
gene
replacements
chromosomes
Received- 2 AprjJ 1996/ Accepted- 15 JuJy 1996
Abstract A genera] system is described that facilitates
gene rep]acements such that the recombinant strains
are not labelled with antibiotic re sistance gelles. The
method is based on the conditiona] replication of derivatives of the lactococcal plasmid pW\lOl, which lacks
the repA gene encoding the replication
initiation
protein. Rep]acement vectors can be constructed in and
iso]ated from gram-positive and gram-negative helper
strains that provide RepA in rrans. Cointegrate formation of the integration vectors with the chromosome of
the tare:et strain is selected bv antibiotic resistance.
Reso]ution of the cointegrate structure is identified in
the second step of the procedure by the ]oss of the lacZ
reporter gene present in the de]ivery vector. The second
recombination event results either in gene replacement
or in restoration of the original copy of the gene. As no
antibiotic resistance marker is present in the genome of
the mutant the system can be used to introduce mu]tiple mutations in one strain. A feasibility study was
performed using Lacrococcus lacris and Bacillus subrilis
as model organisms. The resu]ts indicate that the
method shou]d be app]icable to any non-essential gene
in numerous bacterial species.
Key words Conditional
replication
M utagenesis .lacZ reporter
Communicated
.Gene
replacement
by w. Goebei
K. Leenhouts 1 .G. Buist,
A. Bolhuis.
A. ten Berge
J. Kiel .1. ~ierau .M. Dabrowska .G. Venerna. J. Kok (8)
Department of Genetics. Biomolecular Sciences and Biotechnology
Institute. Universily of Groningen. Kerklaan 30.
9751 NN Haren. The Netherlands
Presen/ address:
1 International
Centre for Genetic Engineering
Department of Molecular
1-34012 Trieste, Italy
Pathology,
Padriciano
and BiotechnoJogy.
99.
.
Introduction
The study of chromosoma] genes in mo]ecu]ar bio]ogical research or the modification of bacterial strains
with applications in the (medical) industry of ten requires the rep]acement, by homo]ogous recombination,
of a chromosomal gene by a mutated version, The
simplest and most efficient method of achieving this is
to exchange the target gene by a copy that is inactivated by the insertion of an antibiotic resistance gene.
However. the introduction of an antibiotic resistance
marker cou]d be undesirab)e for various reasons..
A common strategy that avoids the use of an antibiotic
resistance marker in the rep]aced gene is a two-step
procedure described by Hamilton et al. (1989). In general, in the first step homo]ogous recombination between the delivery vector and the chromosome results
in the formation of a cointegrate that is se]ected by
positive selection. The second step is usuaJly based on
negative se]ection and, depending on the nature of the
de]ivery vector, is achieved af ter a temperature shift or
a period of non-selective growth. Reso)ution of the
cointegrate results either in reversion to the parental
chromosomal structure or in gene replacement. Bioassays or ana]yses of the chromosome are required to
distinguish between the two possibilities. The use of
temperature-sensitive(Ts) rep]icons is high]y efficient in
this..strategy (Hami]ton et a]. 1989: Biswas et al. 1993).
but Ts plasmids cou]d have restricted host ranges or
may not be thermosensitive in certain hostso Therefore.
the deve]opment of an efficient non-replicative delivery
system with a wide spectrum of potential target organisms is justified.
We have previously described a conditionaJly rep]icating vector derived from the broad-host-range roJIing circle-type lactococca] p]asmid pWVOl (Leenhouts
et a]o 1991a)o From this vector (pORI)the gene encoding the replication initiation protein RepA VI'as removed. Therefore, pORI is unable to replicate in any
18
Table 1 Bacterial
strains and plasmids
Relevant properties
Strain
or
Saurce
ar reference
plasmid
Strain
E.~('heri('hia
('{)li
NM522
supE,
MCIOOO
ECJOOO
F-, araDl39 (aru ABC-Ieu)7679, fJullo, fJaIK.lac).'74. r.\pLo rhi
RepA+ MCJOOOo Km'. carrying a single copy of the pWVO1 repA gene
in the gIfJB gene
Bacillu.'i
rhi, LI(lar-proAB),
Llhsd5(r-.m~J
[F',
pruAB.I(/rlqZLlMJ.'J
Stratagene
(La .JoIJa. Calir.)
Wcinstock
This
et al. (1983)
work
.~uhtili.~
RepA+ OBI04 (Kawamura and Ooi 19841. Cm'. carrying
the pWVOI r(!pA gene in the (Jdd gene region
BSJIO
multiple
copies of
rrpC2. p/Je. spoOH.1Hind
rrpC2, degU32(H)') : : ap/JA3
QB4371
Lactococcus
unpublished
GTyczan and Dubnau
(]982)
WeiT et al. (1984)
Msadek et al. (1991)
rrpC2, 1)'5, rhyA. rh.\'B
]5233
Leenhouts.
lams
MGI363
MGI363acmAJl
LLIOS
LL302
Plasmid-free NCDO712
Derivative of MGI363 containing a 703 hp Sacl-Spel dele1ion in acmA gene
RepA. MG1363. Cm'. carrying multiple copies of the pWV01 repA gene
in the random chromosomal fragment A ( leenhouts el al. 19901
RepA+ MG1363, carrying a single cop)' of 1he pWV01 repA gene in
the pepX p gene
Gasson
Apr. carrying the pWVOI repA gene under control of promoter P13
Kmr, Tc', pKVB2 (Kiel et al. 1987) carrying the rep.4 gene ofpWVOI
Apr, Emr, pUCI9 (Yanisch-Perron
et al. 19851 carrying the Emr of pEI94
(Horinouchi
and Weisblum 1982) in the Smal restriction site
Apr, Tc', pMTL25 (Chambers et al. J988) carrying the Tcr of pLSI
(Lacks el al. J986) in the Smal restriction site
Kmr. lacZa
Kmr, Tcr, pUK2J carrying the Tcr gene of pTC1 in the );ïlOI restriction site
Em', pMG36e carrying the E. co/i lacZ gene
Cryptic plasmid of L. lactis Wg2
Tcr. ori+ of pWVO1. replicates onJy in strains providing rep.4 in trans
Emr, ori+ of pWVOI, repJicates only in strains providing repA in trans
Tcr. LacZ~, ori+ of pWVOI. replicates only in strains providing rep.4 in trans
Emr. LacZ+, ori~ of pWVOJ. replicates only in strains providing repA in trans
Ernr, derivative of pORl280 specific for integrations in the L. lactis pepX p gene
Emr, derivative of pORl280 specific for integrations in the L. lacti.~ nrdD gene
Tcr, derivative of pORI240 specific for integrations in the B .~ubtiHs spo OH gene
Emr. derivative of pORl280 specific for integrations in the B. subtili.~ degL' gene
Em'. derivative of pORl280 specific for integrations in the L. lactis acmA gene
leenhouts e.t al. (1991a)
law et al. (1995)
laboratory
col1ection
Buist
(1983)
et al. (1995)
Leenhouts,
unpublished
Leenhouts.
unpublished
Plasmid
pUC23rep3
pECI
pUCI9E
pUK21
pUK24
pMG60
pWVOI
pORI24
pORI28
pORI240
pORI280
pORI280-pepXP
pORI280-nrdD
pORI240-spoOH
pORI280-degU32(hy)
pINT AA
bacterial strain unless RepA is provided in trans. The
construction
of inlegration
vectors from
pORI
plasmids is possible by the availability of Escherichia
coli, Bacillus subrilis and Lacrococcus lacris helper
strains that produce the replication
prolein from
a chromosomaIly incorporated copy of repA (Leenhouts el al. 1991 a; Law el al. 1995; Leenhouts, unpublished data). To simplify cloning procedures and to
extend the application ofthe pORI vectors, we describe
in the present paper the modular construction of pORI
\'ectors that can be conveniently used for gene replacement strategies. The vectors carry an erythrom)'cin
(Em) or a tetracycline (Tc) resistance gene, a multiple
cloning site (m cs) and the E. coli lacZ reporter gene.
The feasibility of the system is demonstrated by the
Laboratory
col]ectjon
Vieira and Messing (199J )
This work
van de Guchte et al. (1991)
Leenhouts et al. (199Jc)
This work
This work
This work
This work
This work
This work
Thjs work
Thjs work
Buist et aJ. (J995)
replacement of several genes in L. lacris and B. subrili.'\.
In principle an)' non-essential chromosomal gene il1
a vast repertoire of transformabIe gram-positive and
gram-negative bacteria can be replaced by a mutated
copy using the system described here.
Materials and methods
BacteriaJ strains and gro\\'1h conditions
The bacterial strains used in this study are listed in Table I. E. coli
and B. subrili.~ were grown in TY medium (Rottlander and Trautner
1970). and L Jaclis was growp in M 17 medium (Terzaghi and
Sandine 1975) containing 0.5% glucose (GM 17). L. Jacri.~was plated
219
onto G M 17 agar containing 0.3 M sucrose arter electrotransrormation. Arter transformatjon
B. suhlilis competent cells were plated
onto minimal medium (Smith et al. 1983) containing lysine. tryptophan and thymidine
Kanamycin. Em and Tc were used at final
concentrations or 50. JOOand 1:?J.!g!ml. respectjvely. for E. (.mi. Tc
was used at a final concentration of 8 J.!g!ml for B. suhlilis. Chloramphenicol and Em were used at final concentrations of 5 J.!g!ml for
B. .~uhlili.~ and L. I(lclis. 5-bromo-4-chJoro-3-indolyl-galactoPYranosidc (X-gal) was used at a final concentration
of 80 J.!g'ml for B
.~uhlili.~ and L. I(lcris.
containing the mutation produce hal
whereas wild-type colonies do nol
DNA
isolation
and
Chromosomal
h()uts
method
1990).
was
isolated
Plasmid
of Birnboim
and Doly
purification
as pre\.iously
DNA
was
extracted
( 19791 \\'ith
Seegers et aJ. ( ] 994). Large-scale
gradient
typc
of agar
pJatc.
anaiysis
DNA
et al.
on this
the m()dification
pJasmid
iso)atjon
et
1982).
(Maniatis
described
aJ.
(Leen-
according
was done
DNA
t() the
made
by
by CsCI
transfer
to
Qiabrane
NyJon
Plus membranes
(Qiagen.
Diisseldorf.
Germanyl
was according
to the protocoJ
of Chomczynski
and Qasba ( 19841.
Transformation
E. l"oli and L lal"ri.~ were transformed by electrotransformation
as
described by Zabarovsky and Winberg (1990) and Holo and Nes
(1989). respectively. Competent celIs of B. subrilis were transformed
by the method of Bron and Venerna (1972)
Probes
and
of
were labeJJed using
probe
the
detection
ECL
Amersham.
poJymerase
BiotechnoJogy,
were
system
UK).
The
the ECL
carried
Label)ing
out
manufacturer
PCR
according
to
Cambridge.
UK)
and hybridization
to the instructions
(Amersham
was carried
the
Kit
according
out
with
instructions
of
on chromosomal
]nternationaJ.
super
the
Taq
suppJier
DNA
(HT
DNA.
Plasmids and oligonucleotides
The plasmjds used il1 this study are listed jn Table ]. Po]ymerase
chail1 reaction (PCR) primers 5'-GGA CTT GTC TGT TGA AG
al1d 5'-A TT A TT TGA TTG GAG TT were used to ana]yse the
MG]363::lIrdD
integrants. MG]363I1rdD..acmA
integrants were
checked lisil1g the primers 5'-CAA GGT TAA GTC CAC G al1d
5'-A TT A TT TGA TTG GAG TT. The presence of the 507 bp
deletjol1 in IIrdD or 1he 70] bp de]etjon il1 acmA in the different
integrants was analysed using the PCR primers 5'-GAC TAT TTA
ACG AAC TG al1d 5'-GGC AAG AGC A TC A TG TG. The
sequel1ces of the primers are based on sequences present in the
EMBL Database under accessjon number X56954 (pWVO] I and the
GenBank accession numbers U] 7696 (acmA) and U73336 (m,ID ).
The PCR primers used for the cioning of the mutant spoOH gene
of B. SUblilis IS233 were 5'-GGG AGA TCT GAG AGA GGT AGA
AAC G and 5'-TTA TCT AGA TTC TCA TGC CAT TAC ACC.
Primers 5'-AGA AAA GAT CTA TAT ACA ACC GAG G and
5'-GCA TCT AGA GCA CTT CAC A TT CCC GC \\'ere used 10
clone the degl' 32(h.ï) mulation of B. Sublilis QB4371. The sequences
of the primers are based on the sequences present in the Genbank
under accession numbers M29693 (spoOH) and M23558 (defJU ). In
both PCRs a fragment of] kb was generated with the mutation
approximate]y in the middle of the fragment.
Selection for gene replacemenl
Transformants
of L. lactis and B. subtilis in which p)asmids had
integrated via a single crossover were grown overnight in GM ] 7 or
minjmal medium. respectjvely. with the appropriate antibiolics. Cultures were then diluled in medjum wilhoUI antibiolics 10 a densily of
]-]0
cel]s/m] of medium, fo]]owed by growlh for approximately
30-40 generations. Dilutions of the cultures were spread onto agar
p)ates containing X-gal. Afler ]2-48 h white colonies were selected
for further analysis.
Bioassays
PepXP-deficjent mutants were identified usjng a PepXP plate assay
as descrjbed before (Leenhouts et al. 1991 b). AcmA actj\,jty was
analysed as described by Bujst et al. (1995). Spo0H-deficjent
mutants were jdentjfied as prevjously descrjbed (Schaeffer et al
1965).
The degU32(h.\") mutation was identified by transferring
njes to TY agar plates cOntajning 0.8% skimmeo milk.
the CoJoC010nies
SDS-paJyacrylamide
geJ electrapharesis
V..'estern bJattjng and immunadetectian
(SDS-PAGE\.
Ce]] extracts of overnjghl cu]lures were prepared as descrjbed by van
de Guchle el al. ( 1990) Prolejn samp]es \Vere subjecled to SDSPAGE accordjng 10 the protoCO] descrjbed by Laemmli (1970) wjlh
a Prolean II minigel syslem (Biorad Laboralories.
Rjchmond.
Ca]if.). Proteins separated by SDS-PAGE were transfered 10 BA85
njlrocellu]oSe membrane (Sch]ejfer & Scheull, Dassel. Germany) by
the method described by Towbin el al. (1979). Endopeplidase anligen \Vas delecled \\.ith pO]yC]onaJ endopepljdase antjbodies (T.an
el al. 1991) di]uted I: 8000 and aJkaline phosphalase-conjugaled
goal anli-rabbil
antibodies (Promega Corporalion.
Madison. Wjs.)
according 10 the manufacturer.s jnslrucljons.
Results and discussion
Construction
of pORI240
and pORI280
Derivatives of the lactococcal plasmid pWV01. the
so-calIed pORI vectors, lack the gene encoding the
replication initiation
protein RepA and are, thus,
unable to replicate unless RepA is provided il1 tral1s
(Leenhouts et al. 1991a). The vectors pORI24 and
pOR128, which were used as the basis for the integration vectors pORI240 and pORI280, were constructed
as follows. The Tc resistance gene was isolated from
plasmid pTC2 as a 1.6 kb Bal/1HI fragment and plasmid pUK21 was digested with XhoI. Prior to ligatjon
of the two fragments. blunt ends were generated by
Klenow enzyme treatment. The ligation resulted in
pUK24. in which the Tcr gene is flanked by two Xho]
restriction sites. The 1.7 kb Spe] fragment of pUK24
carrying the Tcr gene was treated with Klenow enzyme
to create blunt ends. A 601 bp T aq] fragment of
pWV01. which carries the plus origin of replication
(Ori + ) but lacks the gene encoding the replication initiation protein (repA ). was also treated with Klenow
enzyme and both fragments were ligated. resulting in
pORI24. The XhoI fragment of pORI24 carrying the
220
/
W
t
---~--~
T
ORJ+ ~
pORI240/280
P'."C1
R
R
Em
"'
/
/
Hpall
S~I
Ncol
SphI
BglII
CRI+
TCR ar EmR
*
A~B
X
~
A
,
==:>
~LacZB
Fig. ] Plasmid map or the OTiRepApWVOJ deTi\,ati\,es
pORI240 (5.9 kb) and pORl280 (5.3 kb). The TestTiction sites indicated aTe unique. [Tc'/Em'
eTythTqmycin OT tetTacycline Tesistance gene. lacZ {J-galactosidase gene or E. (.0/i expTessed undeT the
contToI or lactococca] pTomoteT P31 (P31 ), T terminatoT or the
lactococcal pToteinase gene pnP. opel1 square (ORJ+) oTjgin or
Teplication or lactococcal plasmid pWVO1. Prepc pTomoteT rTom the
repC gene or p]asmid pWVOl (Leenhouts et al. 1991c)J
Tcr gene was replaced by the Emr gene from plasmid
pUCI9E, located on a I kb Sa1l fragment, which resulted in plasmid pORI28. Vectors like pORI24 and
pORI28 have a low copy number in the E. coli RepA+
strain ECI000, in contrast to RepA+ pWVOl derivatives, which have a high copy number in E. coli (Kok et
al. 1984}.
To enable the detection of resolution of integrated
plasmids in genes for which no simple detectable
phenot)'pic difference exists between the wild-type and
mutant strains, the E. coli lacZ reporter gene was
cloned in pORI24 and pORI28. The lacZ expression
unit from pMG60 (van de Guchte et al. 1991) was
isolated a& a 3.5 kb Sa/1 fragment and, af ter treatment
of Lhe blunt ends with Klenow enzyme, the fragment
was ligated into the StuI sites of pORI24 and pORI28.
resulting in pORI240 and pORI280 (Fig. 1). The lacZ
gene in these two integration plasmids is under the
control ofthe lactococcal promoter P32 which is recognized in gram-positive and gram-negative bacteria (van
der \1ossen et al. 1987}. Expression of lacZ from
pORI240 and pORI280 resulted in blue colonies on
X-gal plates ofthe RepA+ strains LUO8, LL302, BSII0
and ECI000.
in L. lactis
We previously conducted gene replacement studies in
the pepXP gene of L. lactis using a pVC derivative.
Frequencies appeared to be relatively low with vafues
as low as 10-6 per generation (Leenhouts et af. 1991b).
Therefore, the pepX P region was chosen to test the
pOR] delivery system. Two 1.5 kb fragments of pepX P
we re clohed in pOR]280. Fragment A carried the pro-
T cS ar Ems
t )ntegratian
*
,~
CRI+
~~~
lacZ
Emr
LacZ+
A
~
Gene replacernent
40 ar
CRI280
XbaI
No11
XmallI
B gIl
BamHI
Ndel
Acc]
MluI
Pst]
HindIll
Smal
~
~
B
TcRorEmR
Fig. 2 Schematic representation of a two-step procedure to obtain
gene-replacement recombination. The lollipopsaslerisks
indicate the
presence of a mutation or a deJetion or. alternativeJy. of a gene to be
jnserted in 1he chromosome. A and B two consecutive fragments on
the plasmids and on the chromosome thTough which recombination
could take place. In step I depicted here. only recombination
via A is
visualized. The end resuJt in II would be the same ir an integration
had taken place through B. Open har gene (fragmentlof
interest.
\\,ar.ï line chromosome. See Fig. 1 for a detailed description of the
pORI vectors
moter and the 5' end of the gene and fragment B the
3' end. In the fina] construct, pORl280-pepX P, pepX P
]acks an interna] 716 bp fragment. pORI280-pepXP
was used to transform L. lactis M G 1363 by the tV.'Ostep strategy outlined in Fig. 2. Co]onies were screened
for LacZ activity but not for PepXP activity during this
procedure to mimic a situation in which a bioassay is
]acking. We first determined through which fragment
pORl280-pepX P had integrated. Southern b]ots were
used to ana]yse 28 transformants: 19 had integrated
through fragment A (MG 1363: : pep.)(P-A) and 9 through
fragment B (MG1363 : : pepX P-B). One transformant of
each type was taken and grov.'n for 35 generations
under non-se]ective conditions to a!lov.' reso]ution Qf
the cointegrate structure. Approximate]y 20000 co]ony
forming units (4000 cfu per plate of 15 cm diameter) of
each cu]ture were p]ated onto agar medium containing
X-gal. Five LacZ- co]onies were detected among.the
MG1363::pepXP-A
co]onies and nine among those
derived from MG1363::pepXP-B.
A!I LacZ- colonies
were Em' and b)' Southern blot analysis (data not
shown) it was shown that the numbers of gene replacements were one and three for the M G 1363: : pepX P-A
and MG1363::pepXP-B
cu]tures, respecti\'e]y. The
mutant nature of the co]onies was confirmed b)' the
PepXP plate assay. Taken together, the frequencies by
which gene replacements were generated in the
221
Table
2 Numbers
frequencies
forming
and
of LacZ-
colony
uni1s (:li/ ) and
rcplacements
r..;umber
Strain
lacZ-
gene.
of
Frequenc~
cru
~umber
of
LacZ-
ar
Frequencyof
mutants
mutants
cru
MGl363
5
9
.:P('J1>.'P-A
::J1ep>.'P-B
ï.J
~5
)9
: .nrdD-A
::/1/ïID-B
x
].4 x 1(1-"
4.2 x] 0- "
10-1.
1.3xI0-o
3
::J
x
10-..
4.6
x
10-
~.5
x 10
< 2.1 x 10-~
9.1 x 10- ;
()"
..
.,.,
BD393
6
: : sJ1('OH
3.ti
x
10
BD393.!;J1{10H
:.deIIL'31(h,ï)
Ten LacZ-
~
.Ox 10-01
1.5 x ]0
cru were lesled
MGJ363::pepXP-A
al1d MGJ363::pepXP-B
cultures
ral1ged betweel1 1.4 x 10-6 and 4.2 x 10-6 per generation (Table 2). These frequenciesare in agreement with
our pre\'ious results {Leenhouts et al. 1991b) and demonstrate that the pORI system can be used to generate
gel1e replacements in regions with low recombination
freq uencies.
To demonstrate ul1ambiguously that with the present system gel1es can be replaced for \\!hich a phenotypic distil1ction is lacking. we chose the anaerobic
ribonucleotide reductase ge;ne nrdD as a target. The
nrdD gel1e (2.24 kb) of L. lactis had not yet been described. but was idel1tified upstream of the major peptidoglycan hydrolase gene (acInA) {Buist et al. 1995).
Plasmid pORI280-nrdD carries the promoter and 5'
end of the gene in fragment A (0.8 kb) and a 3' internal
part of the gene in fragment B (1.2 kb). In this vector
nrdD lacks an interl1al EcoRV fragment of 507 bp. After
introduction ofpORI280-nrdD
into strainMG1363.
20
LacZ+ Emr cfu were analysed by PCR. Fifteen transformal1ts (MG1363 : : nrdD-A) had integrated \'ia fragment A, while five had il1tegrated through fragment
B (MG1363::nrdD-B).
One transformant ofeach type
was taken and grown non-selectively (28 generations).
after which LacZ- Em" cfu were identified among
4500 cfu of each culture (Table 2). Ten colony forming
units of each type were analysed by PCR and two
replacement mutants were identified origil1ating from
the MG1363::nrdD-B
culture (9.1 x 10-5 per generation). This result was col1firmed by Southelï1 hybridization (data not showl1). The fact that nrdD could be
replaced by a mutant cop)' indicates thatthe gene is not
essel1tial under the experimental conditions applied. i.e.
aerobic growth. A detailed characterization of the nrdD
locus of L': la('tis. wiIJ be published elsewhere (G. Buist
et al., mal1uscript in preparation).
Interestingly, frequel1cies of recombination between
the fragments carryil1g the promoter and 5' end of both
pepX P and nrdD were two to three times higher in both
steps of the procedure. This is in agreemel1t \\,ith earlier
observations tliat homologous
fragmel1J.s carrying
1
M
3
2
4
3238
2731
2537
2030
Fig.
3 AgaTose
tion
(PCRI
(1ane
MG
of
( 1.2%)
pToducts
geJ electTophoTesis
foTmed
1). MG1363nrdD
1363nrdDacmA
bacteTiophage
maTkeT.
maTgins.
(1ane
2).
SPP]
DNA
DNAs
MG1363acmAL11
(1ane 4). Sizes of DNA
]ane M) and of PCR
of polymeTase
on chTOmOsomal
cut
pToducts
with
(1ane
fTagments
EcoR]
aTe shown
chain
Teac-
of MG1363
3). and
(in base paiTs)
(moleculaT
weight
in the ]eft and Tight
Tespectively
a promoter
sho\v higher rates of recombination
than
similar.sized
fragments
without
a promoter
(Biswas
et al. 1993; Buist et al, 1995).
To show that the system can be used to introduce
multiple
mutations
in one strain. pINT AA (Buist et al.
1995) was used to introduce a deletion in the QcmA gene
of MG(nrdD).
In this case integration
and excision
events were not closely examined. The presence of the
deletions in the genomes of the three different mutants
MGI363(nrdD).
MGI363(G('mAL11).
and the double
mutant
MG1363(nrdD
GcmA) was checked by P CR.
The lengths of the P CR fragments
clearly show the
presence of the different
delelions
in the mutants
(Fig, 3), These results were confirmed
by Southern hybridization
and. for the Qcm.4 mutation.
by an activity
assay (results not shown).
Frequently.
genes are organized
in operons and insertions in one gene can cause polar effects on downstream genes, This could preclude the construction
of
a mutation
in any gene if it is folIowed by an essential
gene under the same transcriptional
control,
In the
pORI
plasmids
the repC promoter
(PrepC)
from
pWVO1 (Leenhouts
et al. 1991c) is present between
PrepC (Fig.4A.
lil1e 5). A Weslern
blolOf
cel]-free
extracls of the \'arious slrail1s is presented il1 Fig. 4B.
The anti-PepO
antibodies used also recognize a second
homologous
endopeptidase
(PepO.:.), which is somewhat largerthan
PepO and specified else\\'here on the
chromosome
(Mierau
el al. ] 993 ). As is clear from
Fig. 4B. lanes 4 and 5. PrepC is active on lhe chromosome of L. Jaclis. The smal] amount of PepO. in lane
5 is probably
caused by degradalion
of the truncaled
protein.
Proteolytic
breakdown
combined
with the
antisense orientation
of PrepC relative
lO PA could
explain the absence of the PepO' antigen in Fig. 4B.
lane 2. In conclusion.
polar effects due to the insertjon
of the pORI vectors can be avoided by employing
the
transcriptional
activity of PrepC.
A
~t:iJPA
Prepc
2
4
PA
5
B kD
80
~
Prepc
~
Prepc
4
Gene replacement
.I
\"
PepO2
PepO
PepO'
Fig. 4A Schematic representation
of the chromosoma]
regions
around the pepO gene of the five L. /actis MG1363 deri\.atives
examined on the Western b)ot in B. The numbers cQrrespond to the
lane numbers in B. PA and PrepC represent the oppA and repC
promoter. respectively.For
reasons ofclarity.
the oppA gene js not
drawn between PA and pepO. Thej(Jgged ends represent deletjon end
pojnts. The restrjctjon sites used to make these deletions are shown
in )ine 3: E EcoRI. S ScaI. B Western b)ot analysis of cell-free
extracts of MG1363 and four sing)e crossover integrants in pepO (for
details. see text). Equa] amounts of protein in each )ane were subjected to SDS po)yacry)amide (12.5%) gel electrophoresis. The size
of a molecu]ar weight marker js indicated on the Jeft. PepO. PepO2
and PepO' are indicated jn the right margin
Ori... and the mcs (Fig. 1). The lactococcal endopeptidase gene pepO was chosen as a chromosomal target
to examine whether this promoter would be strong
enough to express a gene downstream of the insertion
point. pepO is normally transcribed from promoter PA
upstream of oppA, the gene immediately preceding
pepO (Mierau et al. 1993: Tynkkynen et al. 1993; see
Fig. 4A, line 3). A DNA fragment carrying L1pepO',with
a truncation of the last 50 codons of pepO and a large
deletion removing codons 37 to 285 internal to pepO.
was cloned in two orientations
behind PrepC in
pORI280. Integration
of the two pORI-pepO constructs via sequences preceding the internal deletion
results in L1pepO' being transcribed from PA (Fig. 4A,
lines 1, 4). An intact copy of pepO is either uncoupled
from p A (Fig. 4A, line 1) or located immediately downstream of PrepC (Fig. 4A, line 4). In the case of integration of the two pORI-pepO constructs via sequences
behind the deletion, pepO' is under the control of PA
while the internally deleted form (L1pepO) is either devoid of-a promoter (Fig. 4A, line 2) or transcribed from
in B. subti/is
Ta demonstrate that the pORl system also operates in
another gram-positive organism even under moderate
transformation
frequency conditions, B. subrilis was
chosen for the consecutive introduction of the spoOR
and degU 32(/1.\') mutations into the same strain, The
mutated spoOR gene was cloned as a 1 kb fragment by
P CR from strain 15233 into pOR1240, with the mutation approximately in the middle of the P CR fragment,
The resulting plasmid pOR1240spoOR was used ta
transfarm B. subtilis BD393, The route of integration
was nat investigated in this case. A Tc' LacZ- transformant was taken and grawn non-selectively for
40 generations befare plating, Amang 2000 cfu. 6
LacZ- colanies appeared. 3 of which were found ta be
5po- in a sparulation assay (3.8 x 10- 5 per generation)
(Table 2).
The degU gene from strain QB371. carrying the
degU32(/1y) mutation, was claned as a 1 kb fragment
by P CR in pOR1280. 5train BD393 : : spoOR was transformed with the resulting plasmid pORI280de?JL' 32.
An Ems LacZ + colany was taken and grawn nanselectively for 40 generations. Eight LacZ- colanies
were identified amang 2000 cfu generated from this
culture {Table 2). Two of the LacZcolanies had
retained the degU32{/1y) mutation {2.5 x 10-5 per generation) as judged by a halo assay using agar plat~s
containing skimmed milk.
Camments
an the methad
The vectors used in this stud)' lack their cognate rep
gene. Thus, this type of integration vector does not rely
on replication in the target host and does not depend
on temperature sensitivity of a replication protein in
that host. Apart from the functionality
of the pORI
vectors in L. lactis and B. .\"ubtilis, these vectors have
been used in E. co/i, albeit for single crossover integrations (1. MulIer, unpublished data). It is conceivable
that the vectors can be used in practically any bacterial
host. An exception may be some lactococcal strains
harbouring pWV01-like plasmids, which could provide
RepA in trans. A plasmid-curing step prior to the use of
the pORI vectors will be necessary. Adjustment of the
vectors for use in some bacterial species may be required, such as insertion of another selectable marker
and replacement (of the promoter) of the reporter gene.
This can easily be achieved by virtue of the modular
design ofvectors pORI24 and pORI28.
The pORI plasmids have the advantage over other
non-replicalive vectors that they allow cloning of the
target gene {fragment) in gram-positive or gram-negalive backgrounds. This may minimise cloning problems. If such problems should persist, construction of
a homologous RepA + background may be considered.
The availability of a repA expression cassette is convenient for this purpose.
A drawback of the melhod is that transformation
frequencies are required that are sufficiently high to
obtain at least a few integrants. Therefore, it may be
necessary to optimise transformation
prolocols for
poorly transformabIe strains.
In the present system the presence or absence of an
expressed lacZ gene enabled a simple blue/white
screening of colonies to visualize recombination events
in the second step of the .procedure. However, this
s~'stem is still based on negative selection, which may
result in extensive screeninQ of colonies if recombination frequencies in the target gene are very Iow { < 10- 6
per generation). As already indicated above, the reporter gene may be replaced by a gene that allows positive
selection, if available. Nevertheless, the pORI vectors
described here \\'ere found to be highly efficient. In
addition to the mulations described, the pORI system
has been used to mulale the dtpT, pepO, pepT and
acmA genes of L. lactis {Mierau el al. 1993, 1994;
Hagling el al. 1994; Buist el al. 1995).
The fact that no antibiotic resistance marker is lef 1
on the chromosome of a mulated strain makes the
strain not on]y more desirable for applied purposes bul
also Ieaves the possibility of introducing more desired
mutations (or genes).
Acknowledgements This work was supported by Unilever Research,
Vlaardingen
and by the CEC V ALUE
Programme.
We thank
Henk M ulder for preparing the figures. Jan Kok is the recipient of a
fellowship of the Royal Netherlands Academy of Arts and Sciences
(KNAW).
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