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Applied and Environmental Microbiology, September 2001, p. 4361-4364, Vol. 67, No. 9
Department of
Microbiology1 and Department of Food
Science,2 Southeast Dairy Foods Research
Center, North Carolina State University, Raleigh, North Carolina
27695
Received 22 February 2001/Accepted 30 May 2001
An efficient method is described for the generation of
site-specific chromosomal integrations in Lactobacillus
acidophilus and Lactobacillus gasseri. The
strategy is an adaptation of the lactococcal pORI system (K. Leenhouts,
G. Venema, and J. Kok, Methods Cell Sci. 20:35-50, 1998) and
relies on the simultaneous use of two plasmids. The functionality of
the integration strategy was demonstated by the insertional
inactivation of the Lactobacillus acidophilus NCFM
lacL gene encoding Lactobacillus acidophilus
and related lactobacilli represent an important group of lactic acid
bacteria which are commonly found in association with the human
gastrointestinal tract and are delivered as health-promoting bacteria
in foods and probiotics. Recent research efforts have focused on
expanding the use of probiotic lactobacilli to include biotechnological
applications such as enzyme and vaccine delivery systems (reviewed in
reference 12). The results to date are promising, but
successful implementation of this technology depends on the ability
to identify and genetically engineer proper candidate strains.
Unfortunately, efficient integration systems are not available for many
important lactobacilli. In order to study gene function and
chromosomally stabilize expression cassettes, an integration strategy
is needed for thermophilic lactobacilli that is independent of
transformation frequency, based on a broad-host-range replicon,
and stably maintains integrants at the desired temperature (37°C).
Conditionally replicating plasmids based on the broad-host-range
lactococcal pWV01 replicon have been used to make a variety of
integrations into lactococcal chromosomes. Law et al. (4) described an integration strategy for Lactococcus that
utilizes pWV01-derived vectors from which the repA gene has
been removed. These "Ori+" integration
vectors replicate only in strains providing repA in
trans. One method to establish these vectors in a desired
host strain is to supply repA from a second
temperature-sensitive helper plasmid. When a DNA fragment with homology
to the host chromosome has been cloned on the
Ori+ vector, a subsequent temperature shift
selects for loss of the helper plasmid and integration of
Ori+-plasmid DNA. Despite the potential
usefulness of this system, its application to thermophilic lactobacilli
was still limited by the low functional temperature range of the
available helper plasmid, pVE6007.
Creation and temperature sensitivity of plasmid pTRK669.
In
order to increase the host range of this system to include thermophilic
lactobacilli, we sought to combine the useful features of the
Ori+ vectors with a more suitable pWV01-based
helper plasmid. Previous experiments in our laboratory have
indicated that the wild-type pWV01 replicon, while not completely
temperature sensitive, is moderately unstable at temperatures of
>42°C in thermophilic lactobacilli, similar to what has been
reported for the related plasmid, pE194, in Bacillus
subtilis (14). While not suitable for performing traditional temperature-sensitive integration experiments, this feature
made it possible to adapt the two-plasmid lactococcal integration
strategy for use in Lactobacillus. A helper plasmid, based
on pGK12, was created to provide repA in trans
for the replication of pORI28-based plasmids (7). This
helper plasmid, pTRK669, retains the pWV01 replicon including the
origin of replication and repAC genes as well as the
cat gene (Table 1). The
erythromycin resistance (Emr) marker was removed
in order to make the new plasmid compatible with pORI28, which harbors
its own Emr gene. Plasmids pGK12 and pTRK669 were
transformed into L. acidophilus, where their stability was
monitored during growth at 37 and 43°C in the absence of antibiotic
selection (Fig. 1). The results indicate that both plasmids are stable at 37°C but not at 43°C. An almost 4-log reduction of plasmid-containing cells was observed at 43°C over
the course of 30 generations. Similar results were obtained using
L. gasseri cells (data not shown). The ability of pTRK669 to
supply RepA for the replication of pORI28 was tested by transferring pORI28 into L. acidophilus and L. gasseri cells
with or without pTRK669. Emr clones could only be
recovered from cells which also carried pTRK669, indicating that
replication of pORI28 was supported by pTRK669.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4361-4364.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Efficient System for Directed Integration into the
Lactobacillus acidophilus and Lactobacillus
gasseri Chromosomes via Homologous Recombination
ABSTRACT
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References
-galactosidase and of the Lactobacillus gasseri ADH gusA gene
encoding -glucuronidase.
TEXT
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TABLE 1.
Bacterial strains and plasmids
View larger version (12K):
[in a new window]
FIG. 1.
Stability of pGK12 ( 
) and pTRK669 (
) in L.
acidophilus NCFM at 37°C (closed symbols) and 43°C (open
symbols). The percentage of Cmr cells in each culture was
determined by plating on MRS versus MRS plus chloramphenicol.
Inactivation of the L. acidophilus lacL gene.
In order to verify the functionality of the integration system, plasmid
pTRK670 was designed to disrupt the L. acidophilus lacL
gene, encoding -galactosidase. The primers bgalF
(5'-GACTGGATCCTGCCGAACGAGCCATGTATG-3') and bgalR
(5'-GACTGAATTCCCGGCATAAGATTCGTTTCC-3'), based on
the previously reported lacL gene of L. acidophilus JCM 1229 (GenBank no. AB004867), were used to
amplify a 945-bp internal region of lacL from L. acidophilus NCFM. This fragment was then cloned via the
BamHI/EcoRI sites (underlined) into pORI28, an
Ori+ RepA
integration
plasmid (4). DNA sequencing confirmed that the sequence of
the NCFM fragment was 100% identical to the JCM1229 lacL
sequence. Plasmid pTRK670 was then introduced by electroporation into
L. acidophilus(pTRK669). One Emr,
chloramphenicol-resistant (Cmr) transformant,
carrying both plasmids, was propagated overnight once at 37°C in MRS
broth plus 5 µg of erythromycin and 5 µg of chloramphenicol per ml.
The culture was then transferred three times at 43°C (1% inoculum)
in MRS broth plus 5 µg of erythromycin/ml (ca. 30 generations). After
this enrichment, the culture was plated with a Whitley Automatic Spiral
Plater (Don Whitley Scientific Limited, West Yorkshire, England) on
MRS-X-Gal
(5-bromo-4-chloro-3-indolyl--D- galactopyranoside)-galactose
plates with either erythromycin, chloramphenicol, or no antibiotic, and
incubated for 48 h at 37°C. The results of the plating indicated
that the final culture contained ~107
Emr cells per ml and
<107 Cmr cells per ml.
All of the recovered colonies were white, indicating the loss of
-galactosidase activity. Conversely, a control culture, which had
been enriched at 37°C instead of at 43°C, still contained a large
percentage of LacL+, Cmr cells. One
Emr Cms clone, designated
NCK1398 was chosen for further confirmation. To confirm the loss of
-galactosidase activity, ONPG
(o-nitrophenyl--D-galactopyranoside) assays (11) were performed on log-phase cultures of
L. acidophilus NCFM and NCK1398. In order to induce
-galactosidase production, cultures were grown with galactose as the
sole carbon source. Upon analysis, no measurable -galactosidase
activity could be detected from NCK1398 compared to 2,652 ± 167 U
from NCFM. In order to demonstrate that the disruption of
-galactosidase was the result of integration of pTRK670 in the
lacL gene, Southern hybridizations were performed using the
945-bp lacL fragment as a probe (Fig.
2). The lacL probe hybridized
to an EcoRI fragment of ca. 11.5 kb in L. acidophilus NCFM. In NCK1398, this band disappeared and was
replaced by junction fragments of ca. 5.3 and 8.8 kb due to the
presence of a single EcoRI site present in the pTRK670 plasmid sequence. In addition to the two junction fragments, another band was observed that corresponded to the amplified copies of the
2.6-kb pTRK670 sequence. Further confirmation was obtained by
hybridization to HindIII digestions because no
HindIII sites were present within the pTRK670 sequence.
The lacL probe hybridized to an ca. 3.2-kb fragment in
L. acidophilus NCFM. In NCK1398, this band disappeared and
was replaced with bands of ca. 5.8, 8.4, and 11.0 kb, corresponding to
insertions of one, two, and three plasmid copies, respectively. These
results were identical in each of the clones tested, indicating that
each individual clone gave rise to a mixed population of cells
harboring different numbers of plasmid copies. This variation in
plasmid copy number has been observed in other lactic acid bacteria
(9, 10). Leenhouts et al. (8) reported
L. lactis clones that had generated one, two, or three
chromosomal plasmid copies after the insertion of a pORI-type plasmid.
This amplification of plasmid sequences has generally been attributed
to recombinatory activity between the flanking DNA regions of homology
that result from campbell-like integration and is influenced by
a number of factors, including the nature and location of the insertion
event.
|
-HindIII marker. DNA sizes are indicated in
kilobases. (B) Schematic representation of the relevant regions of the
NCFM and NCK1398 chromosomes. Chromosomal DNA is represented by a heavy
line, plasmid DNA is represented by a thin line, the
lacL gene is represented by an arrow, and the internal
lacL fragment is represented by a solid box. The
EcoRI (E) and HindIII
(H) sites are indicated. The repeating unit represents
the plasmid DNA present in various copies ("n"). The diagram is not
drawn to scale.
Inactivation of the L. gasseri gusA gene.
The
complete sequence of the L. gasseri ADH gusA
gene, encoding -glucuronidase, has been reported recently
(13). A 777-bp internal region of the gusA gene
was amplified by PCR using the primers GUS1F
(5'-GACTTCTAGAACAGTTGACGAATACACAGAT-3') and
GUS1R (5'-GTGAATTCAGGCGATGAGAAGAAGATAATG-3').
This fragment was then cloned into pORI28 as an
XbaI/EcoRI fragment using the sites added to the
5' ends of the primers (underlined). The resulting plasmid, pTRK685,
was then transferred by electroporation into L. gasseri ADH(pTRK669), and the integration strategy was carried out as described
above. The results were similar to those obtained for lacL;
only Emr Cms colonies were
obtained, and disruption of the gusA gene was evidenced by
the formation of only white colonies. One clone, designated NCK1423,
was subjected to further testing. Confirmation of the GusA phenotype was obtained by performing
-glucuronidase assays (13) on stationary-phase L. gasseri ADH and NCK1423 cells. No measurable -glucuronidase
activity could be detected with NCK1423 compared to 97.2 ± 11 U
in ADH. Integration of plasmid DNA into the gusA gene was
confirmed by Southern hybridization (Fig.
3). The results of the Southern
hybridization revealed that integration had occurred within the
gusA gene. However, unlike the previous integration into
L. acidophilus lacL, the number of plasmid DNA sequences integrated into the gusA gene appeared to be fixed at two
copies.
|
Stability of integrations.
To determine the stability of the
integrated plasmids, NCK1398 and NCK1423 were propagated in MRS broth
in the absence of antibiotics for ca. 50 generations and plated on
media with the appropriate chromogenic substrate, i.e., either X-Gal or
X-Gluc (5-bromo-4-chloro-3-indolyl--D-glucuronic
acid). Loss of the integrated plasmid was assessed by restoration of
the LacL+ or GusA+
phenotypes, as indicated by the formation of blue colonies. After 50 generations, the percentages of revertants were 5.36% for
lacL and 0.72% for gusA, corresponding to
frequencies of 0.11% and 0.01% per generation, respectively. These
results indicated that, while plasmid DNA is stably integrated in both
strains, the integrated DNA is ca. 10-fold more stable in the
gusA gene of L. gasseri NCK1423.
Advantages of the system. The advantages of the method reported here over other integration strategies for lactobacilli are that (i) it is not dependent on transformation efficiency, (ii) it allows growth of mutant strains at preferred growth temperatures, (iii) it allows efficient recovery of stable integrants, and (iv) it is likely to function across a broad range of species. While this study has focused on the use of a single integration event to insertionally inactivate functional genes, a number of other applications are envisioned. For example, this strategy can be easily modified to construct and integrate expression cassettes into preferred sites in the chromosome. However, the primary disadvantage of the current design is that plasmid DNA and antibiotic markers remain integrated in the chromosome, leading to potential complications in food-grade applications. Recently, the pORI system has been used to create unlabeled gene replacements in L. lactis and B. subtilis (7) and to create a food-grade multiple-copy integration system (6) that has been used to overexpress proteins in Lactococcus (5). It is expected that the protocol described here could be easily modified to facilitate similar strategies in lactobacilli.
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ACKNOWLEDGMENTS |
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This research was supported by The Southeast Dairy Foods Research Center; Dairy Management, Inc.; Rhodia, Inc.; and the North Carolina Dairy Foundation. W. Michael Russell was supported in part by a U.S. Department of Education GAANN Biotechnology Fellowship.
We thank Jan Kok, University of Groningen, for kindly providing pORI28 and E. coli EC1000. We also thank Evelyn Durmaz, Eric Altermann, Olivia McAuliffe, and Michael Callanan for their help and for critical review of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: North Carolina State University, Department of Food Science, Box 7624, Raleigh, NC 27695. Phone: (919) 515-2972. Fax: (919) 515-7124. E-mail: klaenhammer{at}ncsu.edu.
Paper no. FSR01-7 of the Journal Series of the Department of Food
Science, North Carolina State University, Raleigh, NC 27695-7624.
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Applied and Environmental Microbiology, September 2001, p. 4361-4364, Vol. 67, No. 9
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.9.4361-4364.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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