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Journal of Bacteriology, February 1999, p. 1181-1188, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Gene Targeting in Penicillium
chrysogenum: Disruption of the lys2 Gene Leads to
Penicillin Overproduction
Javier
Casqueiro,1
Santiago
Gutiérrez,1,2
Oscar
Bañuelos,2
Maria Jose
Hijarrubia,2 and
Juan
Francisco
Martín1,2,*
Area of Microbiology, Faculty of Biology,
University of León, 24071 León,2
and
Institute of Biotechnology (INBIOTEC), 24006 León,1 Spain
Received 14 October 1998/Accepted 30 November 1998
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ABSTRACT |
Two strategies have been used for targeted integration at the
lys2 locus of Penicillium chrysogenum. In the
first strategy the disruption of lys2 was obtained by a
single crossing over between the endogenous lys2 and
a fragment of the same gene located in an integrative plasmid.
lys2-disrupted mutants were obtained with 1.6%
efficiency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly,
lys2-disrupted mutants were obtained by a double
crossing over (gene replacement) with an efficiency of 0.14% by using
two lys2 homologous regions of 4.3 and 3.0 kb flanking the
pyrG marker. No homologous recombination was observed when
the selectable marker was flanked by short lys2 homologous
DNA fragments. The disruption of lys2 was confirmed by
Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. The
lys2-disrupted mutants lacked
-aminoadipate reductase activity (encoded by lys2) and
showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The -aminoadipic acid precursor is
channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the -aminoadipate reductase level.
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INTRODUCTION |
In Penicillium
chrysogenum the pathways for the biosynthesis of lysine and
penicillin have several steps in common (Fig.
1). -Aminoadipic acid is the branching
intermediate where both routes diverge; in the lysine pathway
-aminoadipic acid is converted into
-aminoadipate-
-semialdehyde by the -aminoadipate reductase (24, 25), whereas in the penicillin pathway -aminoadipic acid is condensed with L-valine and L-cysteine
to form the tripeptide -L-(-aminoadipyl)-L-cysteinyl-D-valine
(ACV) by the ACV synthetase. -Aminoadipic acid has a key function in
penicillin biosynthesis, since the addition of exogenous
-aminoadipate (11) or other conditions that increase the
internal -aminoadipic acid pool (15) increase the rate of
ACV and penicillin biosynthesis. High-level penicillin-producing
strains of P. chrysogenum exhibit higher intracellular
-aminoadipic acid pool levels (17) and a reduced conversion rate of -aminoadipic acid to lysine (15).
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FIG. 1.
Biosynthetic pathways of lysine and penicillin in
P. chrysogenum. -Aminoadipate is the branching point
intermediate. The conversion of -aminoadipate into -aminoadipic
semialdehyde is catalyzed by -aminoadipate reductase encoded by
lys2. Note that the disruption of the lys2 gene
(indicated by the bold X on one pathway) directs the -aminoadipate
pool toward penicillin biosynthesis (thick arrows). Acetyl-CoA, acetyl
coenzyme A.
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It should be possible to increase the pool of -aminoadipic acid
available for penicillin biosynthesis by the disruption of the
lys2 gene (Fig. 1). Transformation in P. chrysogenum occurs, in most cases, by the ectopic integration of
donor DNA into chromosomal loci (4). In most fungi, the
relative frequencies of integration via homologous and nonhomologous
recombination vary according to the extent of genetic homology between
donor and recipient DNAs, the conformations of the DNA molecules, and
the intrinsic genetic properties of the organism being transformed
(23, 27).
The targeted disruption of genes has not been reported for
P. chrysogenum. In the present work we describe the
targeted disruption of lys2 of P. chrysogenum, by using two different techniques, and the
effect of this mutation on penicillin production.
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MATERIALS AND METHODS |
Microorganisms.
P. chrysogenum Wis 54-1255, a
low-level penicillin-producing strain, and a P. chrysogenum pyrG1
mutant, a uridine auxotroph obtained from Wis 54-1255 by
mutation with nitrosoguanidine and selection for 5'-fluoroorotic acid
resistance (8), were used as recipient strains in
transformation experiments. P. chrysogenum L2, a lysine
auxotroph blocked in the first part of the -aminoadipate pathway
(9), was used as the control strain in nutritional experiments. Escherichia coli DH5 was used for
high-frequency plasmid transformation (107 to
108 transformants/µg of DNA). Micrococcus
luteus ATCC 9341 was used for the penicillin quantification.
Plasmids.
pBluescript I KS(+) phagemid (Stratagene) was used
for routine subcloning experiments. pAC43 and pJL43 (12),
containing the ble (phleomycin resistance) gene, were used
for transformation of Penicillium protoplasts. pB*G
(13) was used as the control in transformation experiments
with Penicillium by complementation of the uridine
auxotrophy and as a source of the pyrG gene. pBL2a and
pBL2CX (6) were used to subclone fragments of the
lys2 gene of P. chrysogenum.
Media and culture conditions.
Spores of P. chrysogenum were collected from plates of Power medium
(10) after having grown for 5 days at 28°C. For penicillin production studies, spores from one plate were inoculated in defined inoculation (DI) medium (solution A: 10 g of citric acid, 2.5 g of acetic acid, 3 g of ethylamine, 5 g of
(NH4)2SO4, 1 g of KH2PO4, 0.5 g of MgSO4
· 7H2O, 0.05 g of FeSO4 · 7H2O, 0.01 g of ZnSO4 · 7H2O, 0.01 g of CuSO4 · 5H2O, 0.01 g of MnSO4 · 4H2O, 0.005 g of CoSO4, 0.001 g of NaCl in 800 ml of distilled water, pH 5.5; solution B: 20% glucose in 200 ml of
distilled water; both solutions were sterilized separately and mixed
before use [80 ml of solution A with 20 ml of solution B in a 500-ml
flask]). After 48 h of incubation at 25°C and at 250 rpm, 10 ml
of the culture in DI medium was added to a 500-ml flask containing 100 ml of defined production (DP) medium (9). The cultures were incubated for 168 h at 25°C and at 250 rpm; 1-ml samples were taken every 24 h to measure penicillin production.
Stability of the lysine auxotrophy.
Spores of the
transformants grown in Power medium with lysine (0.87 mM) were
collected; serial dilutions were plated in Power medium with lysine and
incubated at 28°C for 7 days to establish the concentration of viable
spores. To study the stability of lysine auxotrophs, between
108 and 109 spores were plated in minimal
Czapek medium (9a) with uridine (100 mg/liter) without lysine.
Nucleic acid manipulations.
Total DNA of P. chrysogenum was extracted as described previously (10).
All other nucleic acid manipulations were performed by standard methods
(26).
Transformation of P. chrysogenum.
The transformation
of P. chrysogenum protoplasts was performed as described
previously (5, 10). Transformants were selected in Czapek
medium with 0.7 M KCl (for the pyrG marker) or in Czapek medium (9a) with 1 M sorbitol supplemented with 30 µg of
phleomycin per ml (for the ble marker).
Preparation of cell extracts and determination of
-aminoadipate reductase activity.
Cultures of P. chrysogenum Wis 54-1255 and the disrupted mutants TD10-195 and
TD7-115 were grown in MPPY medium (containing 40 g of glucose,
3 g of NaNO3, 2 g of yeast extract, 0.5 g of KCl, 0.5 g of MgSO4 · 7H2O,
0.01 g of FeSO4 · 7H2O in 1 liter of distilled water, pH 6.0) with or without lysine (4 mM) at 25°C in
an orbital shaker at 220 rpm for 22 h.
Crude enzyme preparations were obtained by grinding the cells with a
mortar in liquid nitrogen. The supernatant extract was dialyzed against
0.01 M Tris-HCl buffer (pH 8.0) for 12 h at 4°C before use
(31).
The
-aminoadipate reductase activity was assayed by the procedure of
Sagisaka and Shimura (
25), as described by Suvarna
et al.
(
33). Reaction mixtures lacking
-aminoadipic acid were
used as controls. The reaction mixtures were incubated at 30°C
for
1 h and terminated by the addition of 1 ml of 2%
p-dimethylaminobenzaldehyde
in 2-methoxyethanol. One unit
was defined as the enzymatic activity
that produced an increment of 0.1 in the absorbance at 460 nm
per
min.
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RESULTS |
Strategies for disruption of lys2 by a single crossing
over.
Disruption by single integration was obtained by
recombination between the endogenous target gene and a fragment of the
same gene located in a plasmid (Fig. 2A).
The fragment of the target gene inserted in the plasmid lacked
both the 5' and 3' ends of the gene; after the recombination
between the fragment of the target gene and the endogenous gene, two
inactive copies of the target gene were generated, one of them lacking
the 5' end and the other the 3' region of the gene.
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FIG. 2.
Strategies for gene disruption by a single crossing over
(A) or gene replacement (B) with the pyrG gene as a marker
in P. chrysogenum.
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Two plasmids, pDL1 and pDL7, were designed for this technique (Fig.
3) which differed in (i) the selectable
marker and (ii)
the size of the DNA region homologous to the
target included in
the plasmids, which allowed the determination
of the relationship
between the size of the homologous region and the
frequency of
homologous recombination. Plasmid pDL1 contains an
internal 2.1-kb
SacI-
XhoI fragment of the
lys2 gene from pBL2a (
6). pDL1 was
linearized
with
BstEII to improve the efficiency of recombination
between the digested homologous fragment of the plasmid and the
endogenous
lys2 gene (Fig.
3).
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FIG. 3.
Plasmids pDL1, pDL2, pDL7, and pDL10 constructed for
targeted disruption of the lys2 gene (the P. chrysogenum genome is shown at the top, and lys2 is
shown as an arrow inside). The DNA fragments homologous to the
lys2 region are indicated below it. ble,
phleomycin resistance gene of Streptoalloteicus hindustanus
expressed from the A. chrysogenum pcbC promoter.
pyrG, pyrG gene of P. chrysogenum. S, SalI; BXI, BstXI; Sc,
SacI; B, BamHI; EV, EcoRV; P,
PstI; Xh, XhoI; Xb, XbaI; E,
EcoRI; BS, BstEII. B* indicates a frameshift
mutation at the BamHI site. The 1.3-kb EcoRV
fragment used as a probe is shown at the very top of the figure.
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pDL7 contains a 4.9-kb insert (from pBL2a) that lacks the 3' end of the
lys2 gene but contains the whole 5' end. To get the
disruption (i.e., two inactive copies of the target gene), a mutation
in a
BamHI site located in the 5' end of the
lys2
gene was introduced
by digestion with
BamHI and filling in
with the Klenow fragment
of the
E. coli DNA polymerase I,
resulting in a frameshift mutation.
pDL7 was linearized with
BstEII (a restriction site located between
the mutated
BamHI site and the 3'-truncated end of the
lys2
gene)
to enhance the recombination at this
point.
pDL7 contains the
pyrG gene of
P. chrysogenum as
a selectable marker, whereas pDL1 includes the
ble
(phleomycin resistance)
gene.
Lysine auxotrophs obtained by transformation of P. chrysogenum with pDL1 and pDL7.
Of 495 transformants tested
2 clones were lysine auxotrophs (Table
1), suggesting that the integration
occurred mostly by random recombination. Both lysine auxotrophs, TD7-88
(for transformant disrupted with construction 7) and TD7-115, were
obtained with the pDL7 plasmid, but none was obtained with pDL1, which
contained the short 2.1-kb insert homologous to lys2. Both
TD7-88 and TD7-115 were unable to grow in Czapek medium
supplemented with -aminoadipic acid, while P. chrysogenum L2 (a lysine auxotroph blocked in the first part of
the -aminoadipate pathway and used as the control strain) grew when
-aminoadipic acid or lysine was added to the medium. These results
suggest that TD7-88 and TD7-115 are disrupted in the lys2
gene.
Molecular analysis of the integration in transformants TD7-88 and
TD7-115.
The recombination between pDL7 and the genomic
lys2 gene should give rise to a modification of the
restriction endonuclease pattern, due to the generation of two copies
of the lys2 gene in the P. chrysogenum genome.
One of these copies has been inactivated by the frameshift
mutation at the BamHI site, and the other is also
inactive due to the lack of a 1-kb fragment of the 3' end of the
lys2 gene (Fig. 4A).
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FIG. 4.
Disruption of lys2 by a single crossing over
and molecular analysis of the transformants. (A) Disruption by
integration of pDL7. B* indicates a frameshift mutation at the
BamHI site. The 8.0-kb BamHI fragment in the
genome and 2.9- and 13.0-kb BamHI fragments obtained after
single crossing over are indicated by solid bars. S, SalI;
B, BamHI; Xh, XhoI. (B) Southern blot
hybridizations of BamHI-digested total DNA of several
transformants with a 1.3-kb EcoRV probe internal to
lys2. Lane 1, HindIII-digested lambda DNA;
lane 2, DNA from a nonauxotrophic transformant; lane 3, TD7-88; lane 4, TD7-115; lane 5, P. chrysogenum Wis 54-1255. The
sizes (in kilobases) of the hybridizing bands are indicated by arrows
on the right.
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The lack of the
BamHI site in one of the copies was
visualized by Southern hybridization of total DNA extracted from the
transformants
and digested with
BamHI, with a 1.3-kb
EcoRV fragment internal
to the
lys2 gene as a
probe (Fig.
3).
As shown in Fig.
4B, Southern hybridization with the genomic
DNA of the transformants showed the expected pattern for single-copy
integrants. The hybridization of
BamHI-digested DNA of
untransformed
P. chrysogenum Wis 54-1255 (Fig.
4B, lane 5)
with the
lys2 probe
gave rise, as expected, to one single
band of 8.0 kb. Transformants
TD7-88 and TD7-115 lacked the 8.0-kb
hybridization band (Fig.
4B, lanes 3 and 4). The lack of this 8.0-kb
band indicates that
recombination events have occurred at this
point.
In transformant TD7-88, the integration has occurred by a single
crossing over and in one copy (the 8.0-kb
BamHI band has
been changed, giving two bands of 13 and 2.9 kb, as expected).
In
transformant TD7-115 the hybridization pattern is more complex;
it
lacks the 8.0-kb band, and in addition to the 13.0-kb band
it contains
other large-sized bands, indicating that besides disruption
of the
lys2 gene other recombination processes have occurred.
The
hybridization pattern for one nonauxotrophic transformant
(negative
control) with integration at heterologous loci obtained
with pDL7
showed that, in addition to the 8.0-kb band, two other
bands were
obtained (Fig.
4B, lane
2).
Disruption of lys2 by double recombination.
In
this strategy an endogenous target gene is replaced by an in
vitro-manipulated gene. The inactivation of the target gene is obtained
by the insertion of one marker within the gene (Fig. 2B).
Two plasmids, pDL2 and pDL10, were constructed for double crossing over
experiments (Fig.
3). Plasmid pDL2 contained the same
2.1-kb DNA
fragment internal to
lys2 used in pDL1, with a 1.3-kb
EcoRV internal fragment of the
lys2 gene replaced
by a 1.5-kb
XhoI-
EcoRI fragment containing the
phleomycin resistance gene
(
ble) under the control of the
Acremonium chrysogenum pcbC promoter.
A linear 2.3-kb
XhoI-
BamHI fragment from pDL2, in which two
regions
(0.36 and 0.43 kb) homologous to the 5' and 3' regions of
lys2 flanked the
ble gene, was used for the
transformation.
In plasmid pDL10 the
pyrG gene was inserted to inactivate
the
lys2 gene, and in addition an internal
PstI-
EcoRV fragment of
200 bp was removed to
avoid the reversion of the
lys2 mutation
by further
recombination processes. A linear 8.8-kb
NotI-
KpnI
fragment from pDL10, in which two
regions (4.3 and 3 kb) homologous
to the
lys2 region are
located at both sides of the
pyrG gene,
was used for the
transformation.
Transformation of P. chrysogenum with pDL2 and pDL10
results in lysine auxotrophs.
Nine hundred sixty-four
transformants were tested for lysine auxotrophy. As observed in Table
1, one lysine auxotroph, named TD10-195, was obtained. This
transformant was unable to grow in Czapek medium with
-aminoadipic acid, while the control strain, P. chrysogenum L2, was able to grow.
The replacement of the endogenous
lys2 gene by the fragment
that contains the mutated
lys2 gene with the
pyrG
insertion produced
a change in the restriction pattern (Fig.
5). As expected, in
the disrupted
transformant TD10-195, the 8.0-kb hybridization
band of the parental
strain was converted to a band of 2.1 kb
(Fig.
5B, lane 2); the
genomic DNA of the nondisrupted prototrophic
transformant TD10-C (lane 3) showed, in addition to the intact
8.0-kb
band, other bands that indicate random integrations.
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FIG. 5.
Disruption of lys2 by a double crossing over
and molecular analysis of the transformants. (A) Disruption by gene
replacement with pDL10. The BamHI fragments modified by the
recombination events are indicated by solid bars. S, SalI;
B, BamHI; Xh, XhoI; Xb, XbaI; P,
PstI. (B) Southern blot hybridizations of
BamHI-digested total DNA from several transformants with
the same labelled probe internal to lys2 shown in Fig. 3.
Lane 1, P. chrysogenum Wis 54-1255; lane 2, TD10-195; lane 3, a nonauxotrophic transformant; lane 4, HindIII-digested lambda DNA. The sizes (in kilobases) of
the hybridizing bands are indicated by arrows on the right.
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Stability of the lys2-disrupted mutants.
Transformants TD10-195 and TD7-115 were very stable, showing
no detectable reversion rate (less than 1 in 109 and 1 in 108, respectively), in contrast with TD7-88, which has a
very low level of stability (reversion frequency of 1.2 in
104 transformants).
The lys2-disrupted mutants lack -aminoadipate
reductase activity.
To confirm that the lys2-disrupted
mutants were really altered in the -aminoadipate reductase, the
activity of this enzyme was determined. Results (Table
2) showed that the disrupted stable mutants TD10-195 and TD7-115 lacked detectable levels of
-aminoadipate reductase, whereas the parental strain, Wis 54-1255, showed considerable -aminoadipate reductase activity.
Supplementation of the culture medium with 4 mM lysine did not affect
the -aminoadipate reductase activity of Wis 54-1255.
Penicillin production by the disrupted mutants TD10-195 and
TD7-115.
The stable transformants TD7-115 and TD10-195 were used
to study the effect of the lys2 disruption on penicillin
production. The growth of the disrupted transformants in the defined
production medium (containing 4.0 mM lysine) was slower than the growth
of the parental strain, reaching a cell density close to 10 mg/ml after
72 h of culturing, while in the parental strain, growth reached
the same level at about 24 h (Fig.
6). The growth of the disrupted
transformants was better in cultures supplemented with lysine
concentrations above 10 mM, but at these concentrations lysine feedback
inhibited penicillin biosynthesis (19, 20).
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FIG. 6.
Growth kinetics (A) and penicillin production (B) of
lys2-disrupted mutants in DP medium are shown: parental
nondisrupted strain, P. chrysogenum Wis 54-1255, supplemented with 4 mM lysine ( ) or alone (); transformant
TD7-115 ( ); transformant TD10-195 ( ).
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The penicillin level for the Wis 54-1255 strain was low at 24 h of
culture and increased at 48 h in cultures without lysine,
while in
the cultures supplemented with lysine the penicillin
levels were low at
all times, possibly due to the feedback regulation
exerted by the
lysine on the homocitrate synthase (
19,
20),
which is the
first enzyme involved in the lysine
biosynthesis.
The disrupted mutants showed penicillin levels that were double those
observed in the parental strain at 96, 120, and 144
h and
approximately threefold higher at 168 h (Fig.
6B).
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DISCUSSION |
Targeted gene disruption is dependent upon the degree of
homologous recombination. While in yeast cells, homologous integration is virtually the rule, in mammalian cells, site-specific integrations are rare (21, 22, 32). As shown in this work, P. chrysogenum seems to behave similarly to mammalian cells, since
only a very low proportion of recombination events occur at the
homologous site (1.6% with pDL7 and 0.14% with pDL10).
It has been reported that gene disruption in yeast is affected by
several factors, including the size of the homologous region and the
DNA topology. One of the most important parameters determining the
efficiency of gene disruption in several organisms is the length of the
homologous fragment. About 23 to 27 bp of homology and 70 bp of
homology are sufficient for homologous recombination in E. coli and Bacillus subtilis, respectively (18,
29), whereas larger fragments of 472 bp are required in murine
embryonic stem cells (14). In Saccharomyces
cerevisiae as few as 4 bp were shown to direct homologous
recombination (28).
In filamentous fungi there are no detailed studies on the minimal
length of DNA fragment required for gene disruption. In this work we
found 1.6% of disruption events with 4.9 kb of homologous DNA for the
single-integration technique and a lower efficiency (0.14%) with the
double crossing over (one-step) gene disruption technique. To our
knowledge this work represents the first deliberate gene disruption in
this economically important organism. About 82% of disruption events
have been reported in Alternaria alternata with a homologous
region of 3.1 kb (30), 4% in Aspergillus
nidulans with a homologous length of 1 kb (34), and
15% in Glomerella cingulata with 500 bp (3). Our
results showed that the integration of exogenous DNA in
P. chrysogenum occurs mainly by nonhomologous recombination. Increasing the length of the homologous fragment leads
to homologous recombination, although with a low frequency. We used
linearized plasmids, since double strand breaks have been shown to have
a positive effect on homologous integration in S. cerevisiae
(21) and A. alternata (30) and no
effect in Neurospora crassa (1, 7) or A. chrysogenum (16, 35). A new factor affecting gene
disruption efficiency, the target locus, has been reported by Bird and
Bradshaw (2); targeting to the niaD locus is at
least fivefold more efficient than targeting to the amdS locus. It is possible that the low frequency observed for
lys2 disruption in this work is due to the targeted locus.
New targets are being disrupted in P. chrysogenum
for the purpose of studying this parameter. An additional parameter
affecting the efficiency of integration may be the P. chrysogenum strain employed in the disruption experiments.
In this paper, we also describe a successful new strategy for
increasing penicillin production. This strategy was based on the
observation that high-level penicillin-producing strains have a larger
pool of -aminoadipic acid than the lower-level producers (17). In addition, Hönlinger and Kubicek
(15) observed that in the higher-level-producing strains,
the rate of conversion of -aminoadipic acid to lysine is lower than
in the lower-level producers. The increased levels of penicillin
production are related to a higher availability of -aminoadipic acid
(17).
Our work shows that the disruption of the lys2 gene favors
penicillin production. When the lysine pathway is interrupted at a
point after -aminoadipic acid, all the synthesized -aminoadipic acid in the disrupted strain is able to be used for penicillin biosynthesis.
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ACKNOWLEDGMENTS |
This work was supported by grants from the CICYT, Madrid, Spain
(BIO97-0289-CO2-01) and Antibióticos, S.p.A. (Milan, Italy). O. Bañuelos and M.-J. Hijarrubia received fellowships from the Basque Government (Vitoria, Spain).
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FOOTNOTES |
*
Corresponding author. Mailing address: Area of
Microbiology, Faculty of Biology, University of León, 24071 León, Spain. Phone: (34 987) 291505. Fax: (34 987) 291506. E-mail: degjmm{at}unileon.es.
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Journal of Bacteriology, February 1999, p. 1181-1188, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
