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Applied and Environmental Microbiology, March 1999, p. 1236-1240, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Organization of the Gene Cluster for Biosynthesis
of Penicillin in Penicillium nalgiovense and Antibiotic
Production in Cured Dry Sausages
Federico
Laich,1
Francisco
Fierro,1,2
Rosa
Elena
Cardoza,1 and
Juan F.
Martin1,2,*
Institute of Biotechnology (INBIOTEC), 24006 León,1 and Area of Microbiology,
Department of Ecology, Genetics and Microbiology, Faculty of
Biology, University of León, 24071 León,2 Spain
Received 14 July 1998/Accepted 3 December 1998
 |
ABSTRACT |
Several fungal isolates obtained from two cured meat products from
Spain were identified as Penicillium nalgiovense by their morphological features and by DNA fingerprinting. All P. nalgiovense isolates showed antibiotic activity in agar diffusion
assays, and their penicillin production in liquid complex medium ranged from 6 to 38 µg · ml
1. We constructed a
restriction map of the penicillin gene cluster of P. nalgiovense and found that the organization of the penicillin biosynthetic genes (pcbAB, pcbC, and
penDE) is the same as in Penicillium
chrysogenum and Aspergillus nidulans. The
pcbAB gene is located in an orientation opposite that of
the pcbC and penDE genes in all three species.
Significant amounts of penicillin were found in situ in the casing and
the outer layer of salami meat during early stages of the curing
process, coinciding with fungal colonization, but no penicillin was
detected in the cured salami. The antibiotic produced in situ was
sensitive to penicillinase.
| |
INTRODUCTION |
Penicillium nalgiovense
Laxa is one of two species in the Penicillium subgenus with
white or pale green colonies (21, 23). P. nalgiovense was originally isolated from cheese (23)
and cured meat products (10). In Spain, Italy, France,
Switzerland, Hungary, Rumania, Germany, and some other European
countries, dry sausages are usually ripened with molds. The development
of molds on the surface of these sausages is required before they are
considered to be cured and ready for consumption (26).
P. nalgiovense gives a typical white appearance and
characteristic odor and flavor to cured sausages and is available
commercially as a starter culture for dry sausages (12).
Fungal isolates from meat products have appeared to be very frequent
producers of penicillin (2, 7). However, in situ penicillin
production by fungi growing on meat products has not been demonstrated.
Genes involved in a particular biosynthetic pathway for secondary
metabolites are often clustered in bacteria and fungi (11, 16). This phenomenon is especially common in the so-called
dispensable pathways, i.e., pathways that are not required for normal
growth or are required only under certain conditions. The genes
involved in the biosynthesis of 
-lactam antibiotics are located in
clusters in filamentous fungi as well as in actinomycetes (1, 13, 15). In Penicillium chrysogenum, the penicillin
biosynthetic genes pcbAB, pcbC, and
penDE are clustered together in a 17-kb DNA fragment
(6, 24). The long pcbAB gene (11.2 kb) is located in an orientation opposite that of pcbC and
penDE.
Our objectives in this study were (i) to identify penicillin-producing
isolates from cured sausages and (ii) to determine if those isolates
have a penicillin gene cluster similar to that in P. chrysogenum and Aspergillus nidulans. We use these
results to determine the role of penicillin in the control of
undesirable bacteria during the curing process.
| |
MATERIALS AND METHODS |
Cured sausage types.
Filamentous fungi were isolated from
the surface of salami (two different types, fuet artisan and fuet
petit) and chorizo. Both contain minced pork meat, salt, and spices,
and chorizo also contains paprika. Both products are fermented by
lactic acid bacteria and then cured.
Isolation and identification of fungi.
The strains that we
used are listed in Table 1. Isolates from
salami and chorizo were obtained from the surface of the sausages with
a sterile cotton swab impregnated with saline solution (0.9% NaCl)
(19). Each sample was submerged in 1 ml of saline solution and homogenized by agitation. For colony isolation, 0.1 ml of the
sample was plated on malt extract agar (MEA) (21) and
incubated at 25°C for 7 days. Isolates obtained were identified by
use of a taxonomic key based on morphological and pigmentation
characteristics (21) and by DNA fingerprinting
(18).
DNA fingerprinting analysis.
Total DNA from all fungal
strains was extracted by a small-scale procedure (8). Total
DNA was digested with EcoRI and electrophoresed in a 0.7%
agarose gel. Subsequent treatments for fixing the DNA to the gel were
carried out as described previously (4). The probe used in
the fingerprinting analysis was the oligonucleotide (GTG)5.
Labeling of (GTG)5 with [
-32P]ATP and
hybridization were done essentially as described by Meyer et al.
(18).
Determination of -lactam antibiotic production in pure
cultures.
For each strain, spores obtained from three petri dishes
of potato dextrose agar medium (Difco, Detroit, Mich.) were inoculated into Erlenmeyer flasks with 100 ml of defined growth medium
supplemented with yeast extract (containing, in grams per liter, the
following: glucose, 40; NaNO3, 3; yeast extract, 2; KCl,
0.5; MgSO4 · 7H2O, 0.5; and
FeSO4 · 7H2O, 0.01; pH adjusted to 6.0)
(3) and incubated at 25°C with agitation (250 rpm) for
48 h. Ten milliliters of each culture was transferred to 100 ml of
complex penicillin production medium (CPM) (containing, in grams per
liter, the following: corn steep liquor, 20; lactose, 55;
MgSO4 · 7H2O, 3; CaCO3, 10;
KH2PO4, 7; and 64% potassium phenylacetate,
6.25 ml; pH adjusted to 6.8) (25) or liquid minimal medium
(MM) (7). These cultures were incubated for 120 h at
25°C in an orbital shaker at 250 rpm. One-milliliter samples were
taken at 24-h intervals from each culture. Each sample was centrifuged
(10,000 × g for 15 min), and the supernatant was used
for the quantitative determination of -lactam antibiotic activity.
Micrococcus luteus ATCC 9341 was used as a bacterial
indicator in agar diffusion assays with tryptic soy agar (TSA) medium (Difco) containing 1% agar. Solutions with increasing penicillin G
(Antibióticos S.A., León, Spain) concentrations were used as controls. Simultaneously, control plates with penicillinase (2 µg
from a Bacillus cereus UL1 penicillinase preparation [about 1 µg of protein] per ml of TSA medium) were inoculated with M. luteus to test if the antibiotic produced was degraded by penicillinase.
Penicillin production in situ in cured salami at different stages
of ripening.
The production of penicillin in situ by P. nalgiovense was tested with three salami products obtained from
the same manufacturer and inoculated with P. nalgiovense
at different stages of ripening: (i) soft, unpalatable salami (not
cured) with initial fungal colonization in which isolated P. nalgiovense colonies were observed; (ii) semidry (not fully cured)
salami covered with a nonhomogeneous fungal mat; and (iii) cured, dried
salami showing good organoleptic properties and a complete mat of
fungal growth.
The production of penicillin was tested directly in the casing with
9-mm-diameter disks of salami casing, salami meat outer
surface (0 to 4 mm deep), inner surface (4 to 8 mm deep), and
inner core (>10 mm
deep). The disks were assayed directly on a
penicillin-sensitive
M. luteus surface culture on 1% TSA medium.
The antibiotic
was allowed to diffuse in the plates in the cold
(3 h at 5°C), and
the cultures were incubated at 30°C for 24
h.
Alternatively, the penicillin was extracted with organic solvent as
follows. Samples taken as described above from the casing,
outer
surface layer (0 to 4 mm deep), inner surface layer (4 to
8 mm deep),
and inner core (>10 mm deep) of each type of salami
were frozen in
liquid nitrogen and ground in a mortar. The triturated
material was
suspended in 20 ml of saline solution (0.9% NaCl)
(pH 7.0) and stirred
at 5°C for 15 min. The solid material was
removed by centrifugation
at 3,000 ×
g and 5°C for 30 min. The
aqueous
solution was taken to pH 3.0 (with 1.0 N HCl) and extracted
with an
equal volume of ethyl acetate. The organic phase (free
of the lipid
interface) was collected, dried in a vacuum evaporator,
and redissolved
in 0.2 ml of distilled water at pH 8.0. Bioassays
with 50-µl aliquots
of the extracts were performed with
M. luteus as indicated
above. Similarly, bioassays were also run with 60-µl
aliquots of the
aqueous phase or the lipid
interface.
Southern blotting and hybridization.
Total DNA was extracted
by the same small-scale procedure as that used for the DNA for
fingerprinting (8). DNA fragments were separated by
electrophoresis and blotted by standard techniques (22). The
probe (a 7.2-kb NaeI fragment containing the
penDE and pcbC genes and the 5' end of
pcbAB) was labeled by nick translation with
[
-32P]dCTP as recommended by the manufacturer (Promega
Corp., Madison, Wis.). This probe allowed us to check for the presence
of the three penicillin genes and to map the restriction sites by
comparing the band patterns and sizes. Prehybridization and
hybridization were done with 30% formamide standard buffer
(22) at 4°C. After hybridization, the membrane was washed
once at room temperature for 20 min with 2× SSC (1× SSC is 0.15 M
NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate and
twice at 42°C for 15 min each time with the same solution.
| |
RESULTS |
Identification of the fungal isolates from cured meat
products.
Four different Penicillium strains were
isolated from cured meat products (Table 1) and identified by the
criteria of Pitt and Hocking (21). The strains isolated from
fuet artisan and fuet petit salami were morphologically similar to the
P. nalgiovense commercial starter culture INBCC 100. Colonies on MEA were flat (15 to 17 mm in diameter) and lacked exudate
or soluble pigment. The reverse of the colony was strongly colored
orange-brown. Colonies on Czapek yeast extract agar (CYA) (30 to 35 mm
in diameter) were radially sulcate and had a clear exudate. The reverse
of the colony was pale yellow. Colonies on MEA, CYA, and 25%
glycerol-nitrate agar (21) were always white, even in old cultures.
Isolates from chorizo on MEA (25 to 30 mm in diameter) were white to
yellow in the center and green at the borders. The reverse
of the
colony was yellow-brown but lighter than for
P. nalgiovense NRRL 911. Colonies on CYA (35 to 40 mm in diameter) were white
but had
a pale brown center and were radially sulcate but less
so than the
colonies of the isolates from fuet. The reverse of
the colony was pale
yellow and very different from the dark brown
for
P. nalgiovense NRRL 911. Colonies of the isolates from chorizo
were
larger than those of the isolates from fuet and NRRL 911
on all culture
media.
All isolates had a neutral to weakly acid reaction on
creatine-sucrose neutral agar (
20), except that strains
isolated from
fuet artisan salami showed a strongly acid
reaction.
DNA fingerprinting.
By fingerprinting of
EcoRI-digested total DNAs of different isolates with the
(GTG)5 probe, we were able to distinguish different species
and in some cases to find minor differences between different isolates
of the same species (Fig. 1). P. chrysogenum and Penicillium notatum (4), two
species that have been classified as P. chrysogenum by Pitt
and Hocking (21), had similar hybridization patterns. The
P. nalgiovense isolates in Fig. 1, lanes 3, 5, 6, 7, and 8, had almost identical patterns. No differences were found between isolates from fuet petit, fuet artisan, and chorizo. Only a minor difference in the intensity of one band was observed between the commercial starter culture and the salami isolates. Strain NRRL 911 showed three differences in band patterns compared to the rest of
P. nalgiovense isolates; these different bands (either absent or at a much lower intensity in NRRL 911) are indicated in Fig.
1. Differences in the intensities of DNA fingerprinting bands with the
(GTG)5 probe have also been reported for some P. chrysogenum strains (4).
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FIG. 1.
DNA fingerprinting of several isolates from cured
sausages and control species of the genus Penicillium. Total
DNAs were digested with EcoRI and probed with labeled
oligonucleotide (GTG)5. Lanes: 1, P. notatum
ATCC 9478; 2, P. chrysogenum AS-P-78; 3, P. nalgiovense INBCC 102 isolated from fuet petit; 4, P. viridicatum NRRL 963; 5, P. nalgiovense INBCC 101 isolated from fuet artisan; 6, P. nalgiovense NRRL 911; 7, P. nalgiovense INBCC 100 commercial starter culture; 8, P. nalgiovense INBCC 200 isolated from chorizo; 9, P. expansum NRRL 976; 10, P. hirsutum NRRL 2032; 11, P. commune NRRL 845. Bands that are different between
P. nalgiovense strains and other strains are indicated by
arrows (see the text for details).
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|
Other
Penicillium species used in this study had different
hybridization patterns and were clearly distinguished from
P. nalgiovense.
Antibiotic production.
Isolates from cured meat products, the
commercial starter culture, and strain NRRL 911 were tested for their
ability to suppress the growth of M. luteus.
All the isolates examined produced antibiotics in CPM (Fig.
2A). The commercial starter culture
produced significant levels
of antibiotics in MM (Fig.
2B), while no
inhibitory effect could
be detected when
P. nalgiovense NRRL
911 was grown in the same
medium. The penicillin production levels of
most
P. nalgiovense isolates fell within the range for
P. chrysogenum NRRL 1951 (wild
type) under the same
fermentation conditions (
9), while the
penicillin production
level of
P. nalgiovense NRRL 911 was similar
to the average
level observed for isolates of
A. nidulans (
17).
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FIG. 2.
Penicillin production in flask cultures of different
P. nalgiovense isolates in CPM (A) and MM (B). The isolates
were obtained from fuet artisan (INBCC 101) ( ), fuet petit (INBCC
102) ( ), chorizo (INBCC 200) ( ), commercial starter culture INBCC
100 ( ), and NRRL 911 ( ). In MM, only the commercial starter
culture and NRRL 911 were tested.
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|
The antibiotic activity obtained after fermentation in CPM and MM was
completely inactivated by the addition of penicillinase
(see below) in
agar diffusion assays with
M. luteus, indicating
that it
corresponds to a
penicillin.
Presence of the penicillin gene cluster in P. nalgiovense isolates.
Hybridization signals with the 7.2-kb
pcbC-penDE probe, corresponding to the penicillin genes,
appeared in all P. nalgiovense isolates (all of them shown
previously to be penicillin producers) but were absent in a
Penicillium subgenus Penicillium isolate which
did not produce the antibiotic (Fig. 3).
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FIG. 3.
Southern hybridization of total DNAs from different
P. nalgiovense isolates and P. chrysogenum
AS-P-78 with a probe internal to the penicillin biosynthetic gene
cluster. The probe used was a 7.2-kb NaeI fragment
containing the pcbC and penDE genes and the 5'
region of the pcbAB gene (see Fig. 4). The enzymes used for
the DNA digestions are indicated above each panel. DNA size markers (in
kilobases) are indicated at the left. (A) Lanes 1, 5, 8, and 11, P. chrysogenum AS-P-78; lanes 2, 6, 9, and 12, P. nalgiovense NRRL 911; lanes 3, 7, 10, and 13, P. nalgiovense INBCC 100 (commercial starter culture); lane 4, Penicillium sp. strain INBCC 300 isolated from cecina
(nonproducer of penicillin; note that there is no hybridization with
the penicillin probe). (B) Lanes 1, 5, 9, and 13, P. chrysogenum AS-P-78; lanes 2, 6, 10, and 14, P. nalgiovense INBCC 200 isolated from chorizo; lanes 3, 7, 11, and
15, P. nalgiovense INBCC 102 isolated from fuet petit; lanes
4, 8, 12, and 16, P. nalgiovense INBCC 101 isolated from
fuet artisan.
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|
Organization of the penicillin biosynthesis gene cluster in
P. nalgiovense isolates.
Hybridization signals common
to P. nalgiovense and P. chrysogenum in
BamHI and EcoRI fragments indicated that the
three penicillin biosynthetic genes are clustered in P. nalgiovense and that many restriction sites are conserved within
the cluster in P. nalgiovense compared to P. chrysogenum. Taking into account the hybridization patterns of
both species, we constructed a restriction map of the P. nalgiovense cluster (Fig. 4). Most
BamHI and EcoRI sites were conserved in the
mapped region with respect to P. chrysogenum, while a new
EcoRI site appeared in the P. nalgiovense penDE
gene.
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FIG. 4.
Restriction maps of the P. chrysogenum and
P. nalgiovense gene clusters for penicillin biosynthesis.
The orientation of the pcbAB, pcbC, and
penDE genes is conserved in both species. The P. chrysogenum NaeI probe used in the Southern hybridizations (Fig.
3) is indicated on the P. chrysogenum map.
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Relevance of in situ penicillin production by P. nalgiovense in meat products.
Results of bioassays of
solvent-extracted antibiotic production by P. nalgiovense
growing in situ on the cured and semicured sausages are shown in Fig.
5. As shown in panel 1 of Fig. 5, there was moderate penicillin production in solvent extracts of the casing
and in the lipidic interface from semicured salami. No detectable
levels of penicillin were found either in the casing or in the meat of
cured salami. Similarly, using direct bioassays of penicillin
production in the casing or the salami meat, we found significant
levels of penicillin in the soft salami, namely, 1.25 µg/cm2 of casing, 0.66 µg/g of meat in the outer
surface layer, and 0.08 µg/g of meat in the inner surface layer. The
antibiotic produced was completely degraded by penicillinase and was
extracted with ethyl acetate, indicating that the side chain in the
penicillin molecules was not polar.
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FIG. 5.
Bioassays of antibiotic produced in situ by P. nalgiovense growing in salami. (Panel 1) Solvent-extracted
antibiotic from cured or semicured salami. A, Casing from dry cured
salami; B, casing from semicured salami (0.013 µg/cm2 of
casing); C, lipidic interface from the solvent extraction of cured
salami; D, aqueous phase from the extraction of semicured salami; E,
aqueous phase from the extraction of cured salami; center, lipidic
interface from semicured salami. (Panel 2) Direct assays of soft salami
(not cured). A, Salami casing; B, inner core of salami; C, casing
(region different from that in A); D, outer salami layer; center, inner
salami layer.
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| |
DISCUSSION |
Molds grow on a variety of food products. In many cases, they are
deleterious, producing undesirable alterations of the food or feed
products, e.g., aflatoxin production in corn or peanuts. However, in
some cases (e.g., cheese ripening), the proteolytic and lipolytic
activities of fungi are clearly beneficial, contributing decisively to
the organoleptic characteristics of traditional foods. Molds may also
produce antibacterial agents that help to preserve foods.
Dry sausages are ripened with molds in many countries, but the exact
role of fungi in the salami casing is unknown. Our results clearly
indicate that there is a significant level of penicillin production by
P. nalgiovense isolates in cured meat products. The
conserved arrangement of the penicillin biosynthetic genes among
different species indicates that the penicillin gene cluster is derived
from a common ancestor of P. nalgiovense, P. chrysogenum, and A. nidulans. Clustering also may be
important in the regulation of expression of the three genes
(14).
We found that penicillin is produced during fungal colonization of the
salami casing in the early stages of the curing process, in agreement
with previous reports (2, 7). This is one of the first
examples of the production of an antibiotic in a natural substrate and
provides support for the role of antibiotics as antagonists in nature
(5). The penicillin in the salami casing diffuses to the
outer layer of the meat and becomes undetectable in the salami core.
Cured salami (as available in the market) had no detectable levels of
penicillin. The reason for the decay of penicillin during the curing
process is unclear, although it may be due to inactivation by exposure
of the antibiotic to an acid pH for prolonged periods of time.
Colonization by P. nalgiovense of salami during the early
stages of the curing process may be beneficial by preventing the growth
of undesirable bacteria on the surface of the soft sausage. Cured
salami does not contain detectable levels of penicillin, excluding the
possible induction of undesirable cross-resistance to -lactam
antibiotics from penicillin present in the meat. Disruption of the
penicillin gene might be useful for obtaining starter strains unable to
produce penicillin, although the ability to colonize the salami also
must be maintained in the disarmed strains.
| |
ACKNOWLEDGMENTS |
This research was supported by a grant from the Diputación
de León (León, Spain). F. Laich was supported by a MUTIS
fellowship from the Agencia Española de Cooperación
Internacional (AECI).
We thank B. Martín and J. Merino for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Area of
Microbiology, Department of Ecology, Genetics and 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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Applied and Environmental Microbiology, March 1999, p. 1236-1240, Vol. 65, No. 3
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Abstract
Introduction
