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International Journal of Food Microbiology 90 (2004) 237–248
High efficiency transformation of Penicillium nalgiovense with
integrative and autonomously replicating plasmids
Francisco Fierroa,b, Federico Laicha, Ramon O. Garcıa-Ricoa, Juan F. Martına,b,*
a Instituto de Biotecnologıa de Leon (INBIOTEC), Parque Cientıfico de Leon, Avda. del Real, no. 1, 24006 Leon, SpainbArea de Microbiologıa, Facultad de Ciencias Biologicas y Ambientales, Universidad de Leon, 24071 Leon, Spain
Received 2 January 2003; received in revised form 19 May 2003; accepted 30 May 2003
Abstract
Penicillium nalgiovense is a filamentous fungus that is acquiring increasing biotechnological importance in the food industry
due to its widespread use as starter culture for cured and fermented meat products. Strains of P. nalgiovense can be improved by
genetic modification to remove the production of penicillin and other potentially hazardous secondary metabolites, to improve
its capacity to control the growth of undesirable fungi and bacteria on the meat product, and other factors that contribute to the
ripening of the product in order to get safer and better quality foods. Genetic manipulation of P. nalgiovense has been limited by
the lack of molecular genetics tools that were available for this fungus, particularly for ‘‘self-cloning’’ avoiding the use of
exogenous DNAs. In this article we describe a series of vectors, selectable markers and transformation methods that can be used
for efficient transformation of P. nalgiovense, gene cloning and expression. A uridine auxotrophic P. nalgiovense mutant with
an inactive pyrG gene has been isolated. The P. nalgiovense wild-type pyrG gene was cloned and sequenced, and vectors
carrying the gene were shown to complement the pyrG mutant. Autonomously replicating plasmids carrying the AMA1 region
from Aspergillus nidulans transformed P. nalgiovense very efficiently; these plasmids were shown to be maintained as stable
extrachromosomal elements in P. nalgiovense and could be rescued in Escherichia coli. The mitotic stability of self-replicative
AMA1 plasmids in P. nalgiovense was higher than that reported for Penicillium chrysogenum.
D 2003 Elsevier B.V. All rights reserved.
Keywords: Food starters; Penicillium nalgiovense; Transformation; Autonomous replication
1. Introduction the product (Grazia et al., 1986; Lucke, 1986). How-
In the elaboration of dry sausages (salami) and dry
curedmeat products, a layer of fungi develops naturally
on the surface of the product during the curing/ripening
process, which is considered to be beneficial due to
their positive effects on the flavour and appearance of
0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0168-1605(03)00306-4
* Corresponding author. Instituto de Biotecnologıa de Leon
(INBIOTEC), Parque Cientıfico de Leon, Avda. del Real, no. 1,
24006 Leon, Spain. Fax: +34-987-210388.
E-mail address: [email protected] (J.F. Martın).
ever, many of these fungi are mycotoxigenic (Sutic et
al., 1972; Leistner and Pitt, 1977; Leistner, 1984;
Pestka, 1995) and their presence should be avoided to
get a product suitable for human consumption. The use
of the filamentous fungus Penicillium nalgiovense as
starter for cured and fermented meat products is be-
coming a routine in the food industry as a way to
prevent growth of undesirable microbiota (Fink-Grem-
mels et al., 1988; Leistner, 1990; Berwall and Dincho,
1994). Some of the features that make P. nalgiovense
suitable as a starter are (i) it contains enzymatic
F. Fierro et al. / International Journal of Food Microbiology 90 (2004) 237–248238
activities like proteases (Geisen, 1993a), which con-
tribute to the ripening of the product, and (ii) it has not
been found to produce known mycotoxins. Geisen
(1993b) described the requirements that a given strain
should fulfill to be used as starter, among them the lack
of production of antibiotics and mycotoxins and the
ability to antagonize the growth of other undesirable
microorganisms on the product. Despite its widespread
use, P. nalgiovense does not totally fulfill Geisen’s
requirements, as it produces the antibiotic penicillin
(Andersen and Frisvad, 1994; Farber andGeisen, 1994;
Laich et al., 1999), and other secondary metabolites
like isocoumarins (Larsen and Breinholt, 1999, and
references therein), whose potential risk on humans has
not been tested yet. In addition, P. nalgiovense is not
able to prevent completely the development of other
fungi and bacteria on some cured products like Spanish
Cecina (Laich et al., unpublished results), though it
causes a reduction in their number.
P. nalgiovense strains can be improved in many
ways to be used as starter. Strains should be obtained
that do not produce penicillin and, if proven toxic, other
secondary metabolites (Laich et al., 2003). Proteases,
lipases and other activities that contribute to the ripen-
ing and development of the organoleptic features
typical of the product can be enhanced or introduced
by transformation with plasmids containing adequate
genes. This method can also be used to introduce genes
that help to inhibit the development of other undesir-
able microorganisms.
In this article, we describe the development of
several genetic tools for P. nalgiovense, namely aux-
otrophy and antibiotic resistance markers, pyrG host
strains and integrative as well as self-replicative
vectors. These tools will greatly facilitate the research
at the molecular level on this fungus in order to
characterize secondary metabolite biosynthetic path-
ways and will also be useful to obtain improved
strains that can function as better and safer starters
than the currently used ones.
2. Materials and methods
2.1. Fungal strains
P. nalgiovense 16a, a biotype 6 strain isolated from
Cecina (Laich et al., unpublished results), was used as a
source of DNA for the genomic library, as recipient for
transformation, and in mutagenesis experiments to
obtain the pyrG mutant.
2.2. Mutagenesis of P. nalgiovense 16a with UV light
Thirty milliliters of a suspension of 1.5� 107 co-
nidia/ml were submitted to UV light radiation of 30 W
and 253.7 nm wavelength from a UV source placed at
30 cm above the conidia on a petri dish (without cover),
inside a laminar flow hood. Samples of 1 ml were taken
at initial time (t = 0) and every 5 s for 2.5min. Irradiated
conidia were kept in the dark. From each sample, 100
Al were taken, and serial dilutions up to 10� 6 were
made, which were plated on petri dishes with MEA
medium (containing in g/l: malt extract, 20; peptone, 1;
glucose, 20; agar, 20; pH 5.6) and incubated at 25 jCfor 5 days in the dark. The number of colonies in each
plate was counted and the percentage of survival with
respect to t = 0 was calculated for each irradiation time.
Samples showing an 80% ofmortality were selected for
screening of pyrG mutants.
2.3. Screening of the irradiated conidia for pyrG
mutants
An auxotroph enrichment method was used, mod-
ified from Bos and Stadler (1996). A suspension
containing 1�107 viable conidia was inoculated in
100 ml Czapeck (Cz) minimal medium in flasks and
incubated in the dark at 25 jC, 250 rpm, for 48 h. The
culture was then filtrated through nylon membranes
(20-Am pore size), which allowed retention of germi-
nated conidia. Nongerminated conidia were collected
by centrifugation at 8000� g for 15 min, resuspended
in 0.8% NaCl at a concentration of 1�103 viable
conidia/ml, and 100 Al of this suspension were plated
on petri dishes with Cz solid medium supplemented
with uridine (140 Ag/ml final) and incubated in the dark
at 25 jC for 7 days. The colonies obtained were picked
individually with a toothpick on Cz and Cz + uridine
solid media, respectively, and the plates were incubated
at 25 jC for 5–7 days. Uridine auxotrophs were chosen
and streaked on Cz + uridine + 5-fluoroorotic acid (5-
FOA, 1 mg/ml final concentration) to select those
clones resistant to 5-FOA, which would lack the
orotidine-5V-phosphate decarboxylase, encoded by
the pyrG gene.
of Food Microbiology 90 (2004) 237–248 239
2.4. Screening of the genomic library
Lysis plaques obtained after infection of Escher-
ichia coli LE392 with a P. nalgiovense 16a genomic
library (constructed in the vector Lambda GEMR-12,Promega, Madison, WI, USA) were transferred onto
nitrocellulose membranes (Protan BA 85, Schleicher
and Schuell, Dassel, Germany). The membranes were
hybridized by standard procedures (Sambrook et al.,
1989) with a 1.5-kb HindIII DNA probe containing
the pyrG gene of Penicillium chrysogenum (Fierro et
al., 1996). The probe was labeled by the nick trans-
lation system (BioRad, Hercules, CA, USA) with
[32P]-dCTP. Positive phage plaques were taken out
from the plate as plugs for a second and third
screening, after which, pure clones with DNA frag-
ments containing the pyrG gene were isolated.
2.5. Extraction of DNA from recombinant phages
Recombinant phages isolated after the screening of
the genomic library were used to infect E. coli LE392
at a proportion of 1�108 pfu per 5� 109 E. coli cells,
and incubated at 37 jC for 20–30 min. The infection
mixture was transferred to a flask containing 22 ml of
TY (2� ) medium (containing in g/l: bacto-triptone,
20; yeast extract, 10; pH 7.2) and incubated at 37 jC,250 rpm, until the bacterial culture was totally lysed
by the phage (approximately 5 h). Then, 5 Ag/ml
RNAse, 10 Ag/ml DNAse and 1 Ag/ml lysozyme were
added to the lysate, which was incubated at 37 jC and
100 rpm for 30 min and centrifuged at 11,000� g at 4
jC for 10 min. The supernatant was transferred to
CorexR tubes (Corning, Big Flats, NY, USA) and the
phages were precipitated by adding 1.4 g NaCl and
6.25 ml of 50% PEG 6000 (w/v), incubated for at least
60 min in ice and centrifuged 7500� g, 4 jC for 30
min. The precipitated phages were then resuspended
in 3 ml of TE buffer and extracted three times with
chloroform–isoamyl alcohol (CIA, 24:1). Three milli-
liters of 4% SDS (w/v) were added and the mixture
was incubated for 20 min at 70 jC; afterwards, 3 ml
of 2.5 M potassium acetate, pH 4.8, was added, mixed
thoroughly and incubated in ice for 10 min. The
mixture was centrifuged at 26,500� g, 4 jC, for 10min and the supernatant filtered through a nylon
membrane (30 Am in diameter) to eliminate the debris.
The released DNA was precipitated with 1 volume of
F. Fierro et al. / International Journal
isopropanol (30 min at room temperature) and centri-
fuged at 7500� g for 30 min. The DNA pellet was
washed with 70% ethanol, resuspended in 500 Al TEbuffer and treated with RNAse (100 Ag/ml final) at 37
jC for 60 min. Finally, the RNAse-treated DNA
solution was extracted successively with 1 volume
phenol–CIA (1:1) and 1 volume CIA, precipitated
with ethanol at � 20 jC, washed with 70% ethanol
and resuspended in 50 Al TE buffer.
2.6. Plasmid constructions
Plasmids for P. nalgiovense transformation were
constructed following the standard methods. Compe-
tent cells of E. coli DH5a were used for high
efficiency transformation and isolation of plasmid
DNA.
2.7. Sequencing of DNA
Sequencing clones were generated by unidirection-
al deletions using the Erase-a-baseR system (Prom-
ega) following the manufacturer’s instructions.
Sequencing reactions and automatic sequencing were
performed with the AutoReadk system (Pharmacia,
Uppsala, Sweden).
2.8. Fungal transformation
Protoplasts of P. nalgiovense 16a were obtained as
described by Wang et al. (1999). Plasmid DNA (1 Ag)was mixed with 100 Al STC buffer (1 M sorbitol, 50
mM CaCl2, 2.5 mM Tris–HCl, pH 7.5) containing
108 protoplasts/ml and 10 Al of 66% polyethylene
glycol (PEG) 3350 solution in STC buffer; this
mixture was incubated for 20 min in ice. Then, 500
Al of 66% PEG 3350 in STC buffer were added, and
the transformation mixture was incubated at room
temperature for 20 min and mixed with 600 Al of
STC buffer afterwards. Serial dilutions (up to 10� 4)
of the transformation mixture were made with KTC
buffer (0.6 M KCl, 50 mM CaCl2, 2.5 mM Tris–HCl
pH 7.5), and 1 ml of each dilution was mixed with 10
ml of molten Cz–KCl (Cz medium supplemented
with 0.6 M KCl) and plated onto petri dishes con-
taining 10 ml Cz–KCl as base medium.
When resistance to antibiotics (phleomycin) was
used as selective marker, the transformation mixture
F. Fierro et al. / International Journal of Food Microbiology 90 (2004) 237–248240
was incubated with 5 ml of CM medium (in g/l: yeast
extract, 5; malt extract, 5; glucose, 5) containing 1 M
sorbitol at 25 jC for 8 h with gentle shaking. Aliquots
of 1 ml were then mixed with 9 ml of molten CM agar
(1.5% w/v) containing 1 M sorbitol and 15 Ag/ml
phleomycin (Cayla, Toulouse, France) and plated onto
petri dishes containing 10 ml of the same medium.
2.9. Plasmid recovery in E. coli
One microgram of total DNA from P. nalgiovense
transformants was used to transform competent E. coli
DH5a cells (Hanahan, 1986). Extraction and analysis
of DNA from the E. coli transformants was performed
by standard procedures.
2.10. Plasmid stability studies
Plasmid stability analysis of P. nalgiovense trans-
formants was performed essentially as described pre-
viously (Fierro et al., 1996), but using MEA (see
composition above) instead of POWER as sporulation
medium. MEA does not support growth of P. nalgio-
vense uridine auxotrophs. Transformants were grown
on MEA under selective pressure conditions (without
supplement of uridine) or without selective pressure
(MEA supplemented with 140 Ag/ml uridine). Conid-
ia were then collected, diluted to a suitable concen-
tration and plated onto Cz minimal medium with or
without uridine, and the colonies growing on each
condition were counted.
2.11. Southern blotting and hybridization
Total DNA from the transformants was extracted
and Southern blotting to Hybond N membranes
(Amersham, Little Chalfont, Buckinghamshire, Eng-
land) were performed as described by Fierro et al.
(1996). The probe was labeled with the DIG DNA
labeling Mix (Boehringer Mannheim, Mannheim,
Germany) according to the manufacturer’s protocol.
Prehybridization and hybridization were done with
40% formamide standard buffer (Sambrook et al.,
1989) at 42 jC. After hybridization, the membrane
was washed for 15 min at room temperature with 2�SSC, 0.1% sodium dodecyl sulfate (SDS), 15 min at
42 jC with 0.1� SSC, 0.1% SDS, and 3 min at 65
jC with 0.1� SSC, 0.1% SDS. The signals were
visualized with a chemiluminescent substrate for al-
kaline phosphatase, according to the manufacturer’s
protocol (CDP-Star, Roche, Mannheim, Germany).
3. Results
3.1. Isolation of a P. nalgiovense pyrG mutant
Transformation of auxotrophic mutants using the
wild-type P. nalgiovense pyrG gene offers clear
advantages over transformation with vectors based
on antibiotic resistance markers for use in the food
industry (see Discussion). Therefore, our first aim was
to get a P. nalgiovense pyrG mutant to establish a
transformation system for this fungus based on aux-
otrophy complementation.
The possibility that more than one copy of the
pyrG gene was present in the genome of P. nalgio-
vense 16a was tested by Southern analysis, but the
results indicated that there was one single copy of
the gene (data not shown). If there is only one copy
of the pyrG gene, it should be feasible to get uridine
auxotrophs mutated in this gene. Using a method
based on an enrichment of auxotrophs after UV light
treatment (see Material and Methods), six uridine
auxotrophs were isolated from 1800 colonies
screened. One of these auxotrophs was resistant to
5-FOA (Fig. 1), suggesting that it was mutated in the
pyrG gene. The pyrG auxotrophy was finally con-
firmed by transformation with plasmid pAMPF9L
(Fierro et al., 1996), containing the pyrG gene of P.
chrysogenum, which was able to complement the P.
nalgiovense mutant. This mutant, named P. nalgio-
vense 16a pyrG-1, had the same morphology as the
parental P. nalgiovense 16a strain and did not show
any phenotypic difference with respect to the paren-
tal strain other than the auxotrophy of uridine.
3.2. Cloning of the P. nalgiovense pyrG gene
The pyrG gene of P. nalgiovense 16a was cloned
by screening of a genomic library with a probe
containing the P. chrysogenum pyrG gene. Positive
clones were isolated and purified, and their DNAs
analyzed by Southern to locate the gene among the
different restriction fragments obtained (Fig. 2). Fi-
nally, an EcoRI 3.1 kb and an XhoI 2.3 kb fragments
Fig. 2. Southern blot hybridization with a pyrG probe of the DNA of
recombinant phages Y6 and Y10, isolated after screening of the
genomic library, digested with different restriction enzymes. A 1.5 -
kb HindIII fragment containing the P. chrysogenum pyrG gene was
used as probe. The enzyme NotI was used to release the
recombinant DNA from the arms of the phage vector. DNAs
digested with enzymes SalI, XbaI, SacI and BamHI gave two
hybridizing bands, whereas digestion with enzymes EcoRI and XhoI
gave only one, suggesting that the hybridizing EcoRI or XhoI DNA
fragments contained the whole P. nalgiovense pyrG gene.
Fig. 1. Growth on Czapeck medium containing 140 Ag/ml uridine
and 1 mg/ml 5-FOA of six P. nalgiovense mutants auxotrophs of
uridine. After 10 days of incubation at 28 jC, only one of the
mutants, named P. nalgiovense 16a pyrG-1 (strain 1 in the
photograph), was able to grow in presence of 5-FOA, therefore
indicating that it is defective in orotidine 5V-phosphate decarboxy
lase activity, encoded by the pyrG gene.
F. Fierro et al. / International Journal of Food Microbiology 90 (2004) 237–248 241
apparently containing the whole P. nalgiovense pyrG
gene were subcloned in the vector pBluescript-SK+
(Stratagene), generating, respectively, the plasmids
pBPnYE12 and pBPnYX19.
The nucleotide sequence of the XhoI 2.3 kb DNA
fragment was determined. This fragment contained an
ORF of 892 bp that showed 94.3% identity at the
nucleotide level with the P. chrysogenum pyrG gene
and 69.5% with the Aspergillus niger pyrG gene. The
ORF contained one intron in the same position as in
the P. chrysogenum gene and encoded a polypeptide
of 276 amino acids with a deduced molecular mass of
29,924 Da. Comparisons of the amino acid sequence
with proteins in databases showed that the protein
encoded by the putative P. nalgiovense pyrG gene is
unequivocally an orotidine 5V-phosphate decarboxyl-
ase. The functionality of the cloned P. nalgiovense
pyrG gene was confirmed by transformation of the
strain of P. chrysogenum Wis54-1255 pyrG1 (Dıez et
al., 1987), with the plasmids pBPnYE12 and
pBPnYX19. Both plasmids were able to complement
the mutation of the P. chrysogenum pyrG strain and
reverted its auxotrophy, thus confirming the function-
ality of the cloned P. nalgiovense pyrG gene.
The sequence of the P. nalgiovense pyrG gene has
been deposited in GenBank, accession number
AF510725.
3.3. Transformation of P. nalgiovense with integrative
and autoreplicative plasmids by uridine auxotrophy
complementation
The uridine auxotroph P. nalgiovense 16a pyrG-1
was used as recipient strain for transformation with
two different constructions containing the P. nalgio-
vense pyrG gene: plasmid pBPnYSX15 (integrative),
which was a pBluescript-SK+ derivative carrying a
XhoI–EcoRI 2.2 kb fragment with the cloned pyrG
Fig. 3. Integrative and autoreplicative plasmid constructions to transform strain P. nalgiovense 16a pyrG-1. The P. nalgiovense pyrG gene is
shown with an arrow, the AMA1 fragment (see text) is indicated with a black box, and the grey boxes correspond, respectively, to pBluescript-
SK+ sequences (in pBPnYSX15) and pBC-KS+ (Stratagene) sequences (in pAMPF2-H and pAMPn2). Plasmid pAMPF2-H is a derivative of
pAMPF2 (Fierro et al., 1996) obtained by HindIII digestion and auto-religation of pAMPF2. The following restriction sites are shown: ApaI (A),
BamHI (B), BglII (Bg), EcoRI (E), EcoRV (EV), HindIII (H), KpnI (K), SacI (Sc), SalI (S) and XhoI (X). Steps to construct plasmid pAMPn2
were as indicated in the figure. The designation AM is used in constructions or transformants containing the autonomous replication AMA-1
sequence.
F. Fierro et al. / International Journal of Food Microbiology 90 (2004) 237–248242
Table 1
Transformation efficiencies of P. nalgiovense with integrative and
autoreplicative plasmids
Strain Selection
marker
Plasmid Transformants/
Ag DNA per
107 protoplasts
P. nalgiovense pyrG gene pBPnYSX15 749 (F 216)
16a pyrG-1 pAMPn2 45,000 (F 7000)
P. nalgiovense ble gene pULJ43 104 (F 16)
16A pAMPF21 768 (F 134)
The figures are the average of three independent experiments; the
standard deviation is shown in parentheses.
F. Fierro et al. / International Journal of Food Microbiology 90 (2004) 237–248 243
ORF and its promoter region, and plasmid pAMPn2,
which contained the same pyrG fragment and the
AMA1 region of Aspergillus nidulans (Fig. 3). The
AMA1 region (Gems et al., 1991) has been shown to
confer autonomous replication capacity to plasmids in
several filamentous fungi (Fierro et al., 1996; Alek-
senko and Clutterbuck, 1997).
Transformation frequencies were very different for
each of these plasmids (Table 1). With pBPnYSX15, a
transformation efficiency typical of an integrative
Fig. 4. Plasmid pAMPn2 is self-replicative and plasmid pBPnYSX15 is in
strains and different transformants electrophoresed in an agarose gel. L
nalgiovense 16a pyrG-1 (parental untransformed strain); 3, transformant A
marker (E-phage DNA digested with AccI); 11, plasmid pAMPn2 extracted
marker (E-phage DNA digested with HindIII). (B) Hybridization of the gel
kb fragment consisting of plasmid pAMPF2-H linearized by digestion with
on the hybridized membrane, the sizes of the different bands are indicated
plasmid (Cantoral et al., 1987; Dıez et al., 1987)
was obtained, but when pAMPn2 was used, the
efficiency was about 60-fold higher, in the range of
the efficiencies described for AMA1-based autono-
mously replicating plasmids in different fungi (Gems
et al., 1991; Fierro et al., 1996). The percentage of
protoplast regeneration was in all experiments be-
tween 26% and 38%, as measured by the number of
colonies growing on nonselective minimal medium
with respect to the initial number of protoplasts in the
transformation reaction.
3.4. Transformation of P. nalgiovense with integrative
and autoreplicative plasmids by resistance to
phleomycin
The use of the antibiotic phleomycin and the ble
gene conferring resistance to this antibiotic is an
alternative transformation method for several fila-
mentous fungi (Kolar et al., 1988; Austin et al.,
1990). It was of interest to compare the transfor-
mation efficiencies of P. nalgiovense by phleomycin
tegrative in P. nalgiovense. (A) Total, undigested DNA from control
anes: 1, size marker (E-phage DNA digested with HindIII); 2, P.
M1; 4, AM2; 5, AM3; 6, AM4; 7, AM5; 8, AM6; 9, AM7; 10, size
from E. coli; 12, transformant YSX1; 13, YSX2; 14, YSX3; 15, size
in panel A transferred to a nylon membrane. The probe was an 8.6-
NotI. The size marker in lane 15 was pre-stained and thus it is visible
in kilobases at the right of the panel. See text for details.
Fig. 5. Hybridization of a nylon-blotted agarose gel containing SalI-
digested DNAs from control strains and different transformants with
plasmids pAMPn2 and pBPnYSX15. The probe was the same as in
Fig. 4. Lanes: 1, P. nalgiovense 16a (parental untransformed strain);
2, transformant AM1; 3, AM2; 4, AM3; 5, AM4; 6, AM5; 7, AM6;
8, AM7; 9, plasmid pAMPn2 extracted from E. coli and digested
with SalI; 10, transformant YSX1; 11, YSX2; 12, YSX3; 13, size
marker (E-phage DNA digested with HindIII). The sizes of the size
marker bands are indicated in kilobases at the right of the panel. See
text for details.
F. Fierro et al. / International Journal of Food Microbiology 90 (2004) 237–248244
resistance, with those obtained using the pyrG
system. Two different plasmids, pULJL43 and
pAMPF21 (Fierro et al., 1996), were used for
transformation of strain P. nalgiovense 16a. Both
plasmids contain the ble gene expressed under the
promoter of the pcbC gene of P. chrysogenum
(Barredo et al., 1989); plasmid pAMPF21 contains
in addition the AMA1 fragment. The transformation
efficiency obtained with plasmid pAMPF21 (700–
800 transformants/Ag of DNA) was about eightfold
higher than that obtained with the integrative vector
pULJL43 (Table 1), which clearly suggests that
plasmid pAMPF21 functions as an autonomously
replicating plasmid in P. nalgiovense. In general,
the plasmids with the ble gene as selection marker
showed a lower transformation efficiency than those
with the pyrG, especially in the case of the AMA1-
carrying plasmids.
3.5. Southern analysis of P. nalgiovense transform-
ants with the autoreplicative plasmids
To confirm that AMA1-carrying plasmids are
maintained extrachromosomally and replicate auton-
omously in P. nalgiovense, total DNA was extracted
from seven transformants obtained with plasmid
pAMPn2 and three transformants obtained with plas-
mid pBPnYSX15, and Southern analysis was per-
formed both with total undigested DNA (Fig. 4) and
with SalI-digested DNA (Fig. 5). The results of the
hybridizations showed that plasmid pAMPn2 is
maintained as an extrachromosomal circular DNA
in all transformants (AM1 to AM7); no rearrange-
ments were found in at least five of the seven
transformants analyzed (transformants AM3 to
AM7), as shown by the same SalI restriction pattern
in the transformants with respect to the original
plasmid pAMPn2 (Fig. 5). In transformants AM1
and AM2, an additional weaker hybridization signal
of a different size in each case appeared in the
hybridization of the SalI-digested DNAs, which
suggests that a rearrangement of the plasmid DNA
has taken place in at least some of the nuclei in the
mycelia of those transformants; this was confirmed
by the results of the hybridization of the undigested
DNAs (Fig. 4), where transformants AM1 and to a
lesser extent AM2 showed a different band pattern
from that of the rest of transformants and from that
of the pure plasmid. In all the transformants with
plasmid pAMPn2, with the exception of transformant
AM1, the pattern of bands in the hybridization of the
undigested DNAs is very similar to that of pure
pAMPn2 (extracted from E. coli by alkaline lysis).
This result indicates that in the fungus, plasmid
pAMPn2 adopts topological conformations similar
to those in E. coli, although there are also differ-
ences, like the absence in the fungus of a plasmid
form present in E. coli, and vice versa. Another
difference is the higher intensity of the high molec-
ular weight form (upper band) in the fungus with
respect to E. coli; this upper band appears to be a
multimeric form of the plasmid; the presence of
multimers of pAMPn2 seems to be more frequent
in the fungi than in E. coli.
F. Fierro et al. / International Journal of Food Microbiology 90 (2004) 237–248 245
In the area where total undigested DNA is
located in the gel, a very faint signal appears in
some of the transformants with plasmid pAMPn2.
This can be interpreted as a very low rate of
integration of the plasmid in the genome of the
fungus, though the possibility that some multimeric
form co-migrates with the total DNA and causes
that signal cannot be excluded. In contrast, trans-
formants obtained with plasmid pBPnYSX15
(YSX1, YSX2 and YSX3) showed a strong hybrid-
ization signal in the area of the total undigested
DNA (Fig. 4, lanes 12–14), indicating that plasmid
pBPnYSX15 integrates into the genome. However,
faint hybridization signals of much smaller size are
also visible in the three transformants; the position
of these bands does not agree with the expected
size of the different topological forms of plasmid
pBPnYSX15.
Fig. 6. Nature of the plasmids rescued in E. coli from P. nalgiovense tr
extracted from 28 E. coli clones was double-digested with the enzymes Xho
membrane was then hybridized with a 2.9-kb pBluescript probe (panel A) o
digestion with XhoI and EcoRI) (Panel B). Lane C (control) corresponds to
indicate that a few rescued plasmids (clones 11, 13 and 24) seem to contain
similar to that plasmid. The rest of the rescued plasmids show sizes betw
sequences and one or two fragments that hybridize with the pyrG probe, an
pBPnYSX15. The rest of the clones contain pBluescript sequences plus u
two transforming pBPnYSX15 plasmids (not involving pyrG sequences) o
In the filter with the SalI-digested DNAs (Fig. 5),
the three transformants with plasmid pBPnYSX15
show different hybridization patterns that are indica-
tive of different locations and number of copies
integrated in each case.
3.6. Recovery of the plasmid in E. coli
The definitive confirmation that plasmid pAMPn2
is maintained extrachromosomally in P. nalgiovense
was provided by transformation of E. coli with total
DNA extracted from transformants AM4 and AM6.
About 550–650 transformant colonies per microgram
of DNA were obtained. The analysis of plasmids
present in the E. coli transformants showed that the
restriction pattern in all clones tested was the same as
that of the original plasmid (not shown), indicating
that no rearrangements had occurred in any of them.
ansformants with plasmid pBPnYSX15 (see text). Plasmidic DNA
I +EcoRI, electrophoresed and blotted onto a nylon membrane. The
r with a 2.2-kb pyrG probe (obtained from plasmid pBPnYSX15 by
plasmid pBPnYSX15 digested with the same enzymes. The results
exclusively pBluescript sequences and have a size identical or very
een 3 and 4.5 kb. Clones 2, 12, 18, 21 and 22 contain pBluescript
d therefore they most likely represent truncated forms of the original
nidentified DNA, and might have arisen by recombination between
r between plasmid pBPnYSX15 and the chromosome of the fungus.
Table 2
Mitotic stability of plasmid pAMPn2 in P. nalgiovense
AM5 (%) AM7 (%) AM9 (%) YSX2 (%)
With selective
pressure
93.6
(F 5.9)
81.8
(F 8.2)
88
(F 7.6)
95.4
(F 4.2)
Without selective
pressure
84.3
(F 6.9)
70.6
(F 3.5)
67.9
(F 7.9)
96.9
(F 3.1)
Transformant YSX2 was obtained with the integrative plasmid
pBPnYSX15 (see text). Nonselective or selective conditions during
the development of the cultures for one generation were established
by adding or not adding uridine to the MEA sporulation medium.
The percentages represent the number of colonies growing on Cz
minimal medium with respect to the total number growing on Cz
supplemented with uridine.
The figures are the average of five independent experiments;
standard deviations are shown in brackets.
F. Fierro et al. / International Journal of Food Microbiology 90 (2004) 237–248246
The same experiment performed with total DNA
from the P. nalgiovense transformants with plasmid
pBPnYSX15 (YSX1, YSX2 and YSX3) yielded 25–
35 E. coli transformants per microgram of DNA.
When plasmid DNA extraction was attempted from
these E. coli transformants, no plasmid molecules
could be observed in some of them, while in other
transformants, plasmids around 3–4.5 kb in size were
obtained, a size which is smaller than the 5.2 kb of
plasmid pBPnYSX15. This range of sizes is in accor-
dance with the size of the bands present in the
hybridized undigested DNAs (Fig. 4). These results
suggested that the small bands most likely represent
truncated forms of plasmid pBPnYSX15, containing
essentially pBluescript sequences, which are able to
replicate autonomously in P. nalgiovense and con-
serve sequences that allow their recovery in E. coli. To
test this hypothesis, 28 rescued plasmids extracted
from the E. coli clones were analyzed by Southern,
with plasmid pBluescript or with a 2.2-kb pyrG-
containing fragment as probes (Fig. 6). The results
indicated that a few of the rescued plasmids contained
exclusively pBluescript sequences, while others
contained also fragments 0.3–1 kb in size, comprising
pyrG sequences. Therefore, all these plasmids repre-
sent truncated/rearranged forms of pBPnYSX15. In
other plasmids, unidentified sequences were present
linked to the 3.0-kb pBluescript fragment, and they
might have arisen from recombination processes be-
tween two pBPnYSX15 molecules or between
pBPnYSX15 and the chromosome of the fungus.
3.7. Mitotic stability of autoreplicative plasmids in P.
nalgiovense
The mitotic stability of the autoreplicating plasmid
pAMPn2 in P. nalgiovense was studied, as indicated
in Material and Methods, using conidia from trans-
formants AM5, AM7 and AM9, obtained after one
generation of growth in MEA medium with or without
selective pressure. The results (Table 2) showed that
plasmid pAMPn2 is highly stable in P. nalgiovense:
between 82% and 94% of conidia kept the plasmid
after one generation under selective pressure. This
percentage is roughly indicative of the number of
mitosis in which the plasmid is distributed between
the phialide and the generated conidia. After one
generation without selective pressure, between 68%
and 84% of conidia conserved the plasmid. The
integrative plasmid pBPnYSX15 showed a higher
stability, 96.9% after one generation without selective
pressure, which correlated well with the fact that it
integrates into the genome.
4. Discussion
Genetic manipulation of P. nalgiovense has been
limited, so far, to the introduction of the lysostaphin
gene from Staphylococcus staphylolyticus (Geisen et
al., 1990) and the glucose oxidase gene from A. niger
(Geisen, 1995, 1999) with the aim of increasing the
antibacterial properties of the fungus. The amdS gene
of A. nidulans was used as selection marker in all
cases (Geisen and Leistner, 1989).
The purpose of this work was to develop various
molecular tools useful for efficient transformation and
gene cloning in P. nalgiovense, in order to facilitate
the molecular studies about secondary metabolite
biosynthesis and other metabolic aspects related to
the use of this fungus as starter in the food industry.
The availability of a pyrG mutant strain and a
transformation system based on uridine auxotrophy
complementation with the homologous pyrG gene
described in this article offers several advantages over
transformation based on antibiotic resistance or on
acetamidase utilization using the amdS gene (Geisen
and Leistner, 1989; Geisen et al., 1990; Geisen, 1995):
(1) The transformation efficiency is much higher
when using the pyrG gene than with either of the
F. Fierro et al. / International Journal of Food Microbiology 90 (2004) 237–248 247
other systems; (2) the use of the homologous pyrG
gene as selection marker allows counter-selection
using 5-FOA, which is very useful to perform suc-
cessive transformations on the same strain, for in-
stance, to disrupt several genes, eliminating the
previously used pyrG gene by recombination and 5-
FOA selection (d’Enfert, 1996); (3) any construction
can be introduced in a single copy at the pyrG locus
by gene targeting using a mutant pyrG gene (contain-
ing a point mutation) in the transforming vector
(Gouka et al., 1995; Kosalkova et al., 2000); (4) there
is much concern about the use of heterologous genes
to get new improved strains of microorganisms used
in the food industry, and in many countries, strict
regulations on this issue have been implemented; the
use of a homologous gene as selection marker, such as
the pyrG, overcomes this problem.
The use of autonomously replicating vectors in P.
nalgiovense is also of great interest. The transforma-
tion efficiency achieved with autonomous-replicating
vectors and the possibility of recovering these plasmids
in E. coli make them excellent tools for constructing P.
nalgiovense genomic libraries to clone genes by com-
plementation, which will help the molecular studies on
secondary metabolite pathways and genes of interest in
this fungus. The behaviour of AMA1-based plasmids in
P. nalgiovense is similar to what has been previously
reported for P. chrysogenum (Fierro et al., 1996).
However, there are differences in the mitotic stability
of the plasmids and in the multimeric forms that occur
in both Penicillium species. Mitotic stability is higher
in P. nalgiovense, and the multimeric forms are clearly
predominant in P. chrysogenum, whereas in P. nalgio-
vense, monomeric and multimeric forms appear at a
similar frequency (Fig. 4). In both fungi, AMA1-based
plasmids show a low degree of reorganization and can
be easily rescued in E. coli.
An interesting result is the presence of small
plasmids, apparently derived from the integrative
plasmid pBPnYSX15, which are maintained auton-
omously in P. nalgiovense. Plasmid pBPnYSX15
integrates in the genome of the fungus, as shown
by the results of the Southern analysis (Figs. 4 and
5) and by its mitotic stability (Table 2), but small-
size DNA molecules also appear when undigested
DNA is hybridized (Fig. 4). These small plasmids
correspond to truncated forms of pBPnYSX15 that
conserve E. coli sequences enabling them to be
rescued in E. coli at low rate (Fig. 6). There are
reports on plasmids lacking known replication ori-
gins that replicate autonomously in other fungi, for
instance, plasmid pUT737 in Botrytis cinerea
(Santos et al., 1996). Autonomous plasmid replica-
tion occurs much more frequently in mucoraceous
fungi than in other groups of filamentous fungi
(Wostemeyer et al., 1987).
In summary, novel plasmids containing homolo-
gous DNA sequences and P. nalgiovense host strains
have been developed and are now available for
modification of this filamentous fungus widely used
in the food industry.
Acknowledgements
This work was supported by grants of the
Diputacion de Leon (DLE 01/97) and Proyecto
Generico of the ADE (Junta de Castilla y Leon,
Valladolid, Spain) (10-2/98/LE/0003). F. Laich re-
ceived a fellowship of the Agencia Espanola de
Cooperacion con Iberoamerica (AECI) of the Ministry
of Foreign Affairs (Madrid, Spain). We thank J.
Merino, B. Martın and M. Corrales for excellent
technical assistance.
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