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Applied and Environmental Microbiology, January 2001, p. 231-238, Vol. 67, No. 1
INRA Laboratoire de Recherche de Technologie
Laitière1 and INRA Laboratoire
de Génétique Animale,2 35042 Rennes Cedex, France
Received 9 June 2000/Accepted 13 October 2000
Dairy propionic acid bacteria, particularly the species
Propionibacterium freudenreichii, play a major role in the
ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated
from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric
bacteriophage type analysis. The genomic DNA of 76 strains were
hybridized with the DNA of nine bacteriophages isolated from Swiss type
cheeses, and the DNA of 25 strains exhibited strong hybridization.
Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of P. freudenreichii was
readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in P. freudenreichii.
Based on habitat, the genus
Propionibacterium can be divided into two groups: cutaneous
and dairy (or classic) propionic acid bacteria (dairy PAB). Dairy PAB
are gram-positive, non-spore-forming, catalase-positive, nonmotile,
facultatively anaerobic organisms (18). They are used in
the manufacture of Swiss type cheeses to ensure eye formation and the
development of a typical flavor (19).
Like members of many other bacterial genera, dairy PAB are infected by
viruses (14). Two types of bacteriophages which can infect
dairy PAB have been isolated in Swiss type cheeses. One type belongs to
group B1 of Bradley's classification, and the other is, to our
knowledge, the first infective filamentous virus found in gram-positive
bacteria (16). The existence of bacteriophage DNA able to
replicate and express itself in the genus Propionibacterium enabled us to determine the efficiency of electrotransfection for
developing a cloning vector (13).
The isometric type of bacteriophages that infect dairy PAB have
frequently been shown to be present in Swiss type cheeses; the
levels of contamination range from 14 to 7 × 105
PFU/g of cheese, depending on the cheese and the indicator strain used
for detection. These bacteriophages are produced during multiplication of dairy PAB during cheese ripening and may multiply on either endogenous or starter strains (15). In order to understand
and control the multiplication of bacteriophages during cheese
production, it is important to determine their source in the
manufacturing plant. We have shown that raw milk can be a source of
free bacteriophages (15), although lysogenic strains may
also produce them.
Lysogeny is widespread in nature, but in the context of the genus
Propionibacterium, only lysogeny of Propionibacterium
acnes has been studied previously (33, 35).
The purpose of this work was to demonstrate lysogeny of dairy PAB.
Studies of putative lysogeny are hindered by problems with finding the
lysogen and sensitive strains. It is also difficult to determine the
optimum amount of mutagenic agent and the optimum bacterial growth
stage for massive prophage induction. There is some homology between
the virulent and temperate bacteriophages which infect
Lactococcus and Lactobacillus species (24,
26). The strategy which we chose was to hybridize the genomes of
76 dairy PAB strains with DNA from nine bacteriophages isolated from Swiss type cheeses. Some putative lysogenic strains of
Propionibacterium freudenreichii subsp. shermanii
harboring genomic sequences exhibiting homology with bacteriophages
were chosen to determine the optimal induction parameters and further
confirm lysogeny in this species.
(This research was conducted by C. Hervé in partial fulfillment
of the requirements for a Ph.D.)
Bacterial strains and culture conditions.
The strains used
for screening (Table 1)
came from the American Type Culture
Collection (ATCC), Rockville, Md.); from the Centre National de
Recherches Zootechniques (CNRZ), Institut National de la Recherche
Agronomique, Jouy en Josas, France; from the Collection du Laboratoire
de Recherche de Technologie Laitière (TL), Institut National de
la Recherche Agronomique, Rennes, France; from the Collection
bactérienne de l'Institut Pasteur (CIP), Paris, France; from the
National Collection of Food Bacteria (NCFB [=NCDO]), Aberdeen, United
Kingdom; from the Deutsche Sammlung von Mikroorganismen (DSM),
Braunschweig, Germany; from the National Collection of Industrial
Bacteria (NCIB), Aberdeen, United Kingdom; from the Anaerobe Culture
Collection of Virginia Polytechnic Institute (VPI), Blacksburg; from
different university collections (T. Britz in South Africa and B. A. Glatz, in the United States); and from a commercial starter
(industrial starter). Cells were stored at
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.1.231-238.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
First Evidence of Lysogeny in
Propionibacterium freudenreichii subsp.
shermanii
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
80°C in YEL broth
containing 15% glycerol. Prior to use, they were subcultured twice
(2% inoculum) in YEL broth for 48 h at 30°C. Colonies were
grown anaerobically (anaerocult A; Merck-Eurolab, Nogent sur Marne,
France) on YEL agar (YELA) (12 g of agar [Merck-Eurolab] per liter)
for 1 week at 30°C (17).
TABLE 1.
Dairy PAB strains used for lysogeny screening
Bacterial genome preparation: dot blot experiments. Bacterial DNA were extracted from 500-ml cultures (optical density at 650 nm [OD650], 1 to 2). Cells were harvested, resuspended in 50 ml of buffer (10 mM Tris base, 2 mM EDTA, 100 mM NaCl, 5% Triton X100; (pH 8.0) containing 120 mg of lysozyme (CHR-Hansen, Arpajon, France), and then incubated for 30 min at 43°C. A longer incubation period (2 h) was sometimes necessary to achieve optimum lysis. Ten milligrams of proteinase K (Amresco, Solon, Ohio) and 2.5 ml of sarcosyl (5% [wt/vol]; Sigma Chemical Co., St. Louis, Mo.) were added to the cell material, and then each preparation was incubated at 40°C for 1 to 2 h. Proteins were eliminated by solvent extraction (28), and DNA was precipitated from the aqueous phase by adding 0.1 volume of 3 M sodium acetate and 0.7 volume of propan-2-ol. Nucleic acids were then dissolved in 5 ml of TE (10 mM Tris base, 1 mM EDTA; pH 8.0).
Pulsed-field gel electrophoresis (PFGE). Dairy PAB cells were embedded in agarose to prepare a DNA insert (plug), as described by McClelland et al. (25). Ten milliliters of a culture was harvested at an OD650 of 0.3 and treated as previously described (15). The electrophoretic conditions employed are described in the figure legends.
Dot blot hybridization experiments. A positively charged Hybond membrane (Appligen-Oncor, Illkirch, France) was humidified with 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7) and placed in a vacuum. The bacterial DNA was denatured for 10 min at 100°C and placed in ice for 10 min. One volume of 20× SSC was added. Bacterial DNA samples were placed in each well and subjected to a vacuum. The DNA was then fixed to the membrane for 5 min under UVc light (312 nm), and the membrane was dried at room temperature and stored at 4°C.
Southern blot hybridization experiments. Southern blotting was performed after PFGE. All DNA were transferred to a positively charged Hybond membrane (Appligen-Oncor) by capillary blotting (29). Each DNA was fixed to the membrane by treating it for 5 min with UVc light (312 nm).
For preparation of the bacteriophage genome probe, 25 ng of DNA was incubated at room temperature for 3 h with [
-32P]dCTP (3,000 Ci/mmol) in the presence of DNA
polymerase (Amersham, Piscataway, N.J.). Labeling was halted by adding
150 µl of TE (10 mM Tris base, 1.0 mM EDTA; pH 8) and 25 µl of
carrier DNA in a final volume of 200 µl; unbound labeled nucleotides
were separated from labeled DNA by exclusion chromatography with a Sephadex G-50 column (Amersham). Immediately before use, the labeled DNA was denatured by boiling it for 5 min and then rapidly cooled in
ice in the presence of 25 µl of herring sperm DNA.
The positive nylon membrane (Appligene-Oncor) was prehybridized in
Denhardt solution for 4 h at 42°C in the presence of 250 µg of
denatured herring sperm DNA. Hybridization was carried out for 18 h at 42°C. The membrane was then washed once for 30 min in 3× SSPE
(1× SSPE is 0.15 M NaCl, 10 mM NaH2PO4
· H2O, and 1 mM EDTA [disodium salt]) containing 0.1%
sodium dodecyl sulfate (SDS), twice for 30 min in 1× SSPE-0.1% SDS,
and, if any noise remained, once for 30 min in 0.5× SSPE-0.1% SDS
and twice for 30 min in 0.1× SSPE-0.1% SDS. The membrane was exposed
overnight and read with a phosphoimager (Hewlett-Packard, Avondale,
Pa.).
Prophage induction: spontaneous liberation. In order to demonstrate spontaneous bacteriophage liberation, each bacterial culture obtained at the end of growth (48 to 72 h) was centrifuged at 10,000 × g for 20 min. The supernatants were treated with DNase I (final concentration, 1 µg/ml; Roche Diagnostics, Meylan, France) and RNase I (final concentration, 10 µg/ml; Roche Diagnostics) at 37°C for 1 h and then filtered though a 0.45-µm-pore-size filter (type HA; Millipore, Molsheim, France). The soft agar layer method described by Adams (1) was used to detect bacteriophages; 0.1 ml of each filtrate or 0.1 ml of a decimal dilution was added to 0.2 ml of a mid-log-stage sensitive (or potentially sensitive) bacterial culture. After incubation for 15 min at 30°C, 3 ml of YELA (8 g of Bacto Agar [Difco Laboratories, Detroit, Mich.] per liter) was added. The resulting mixture was spread on a YELA plate. After 2 to 3 days of anaerobic incubation at 30°C, the plaques were enumerated. In order to differentiate spontaneous liberation from the carrier stage, the lysogenic culture was reisolated three times in succession, and if bacteriophages were always present in the culture supernatant, spontaneous liberation was accepted.
UVc irradiation. A mid-log-stage bacterial culture was centrifuged, washed in sterile water, and then resuspended in saline solution. Irradiation was performed with constant stirring by using a 254-nm UV lamp (Fisher-Bioblock Scientific, Illkirch, France). UV fluences were measured with a VLX-254 radiometer (Vilber-Lourmat, Marne la Vallée, France). Irradiated samples were then mixed in double-strength YEL broth. After incubation at 30°C for 24 h, the cells were pelleted, and the supernatant was treated with DNase I and RNase I at 37°C for 1 h. The supernatant was then filtered through a 0.45-µm-pore-size filter. A nonirradiated culture served as the control. The presence of bacteriophages in the supernatant was then determined as described above.
MC. Different concentrations of mitomycin C (MC)(Roche Diagnostics) were added to a mid-log-stage bacterial culture. Turbidity (OD650) was monitored by spectrophotometry. After incubation at 30°C for 24 h, the cells were centrifuged and the supernatant was treated as described above for induction by UVc irradiation. MC-free cultures were used as controls. The presence of bacteriophages was determined as described above.
Purification and concentration of bacteriophages. A lysis plaque was removed from the soft agar layer and incubated with 3 ml of an early-log-stage sensitive culture at 30°C for 24 h. After centrifugation, the supernatant was filtered with a 0.45-µm-pore-size filter and tested again with the sensitive strain. Following incubation, a plaque was removed and used to infect a culture of the same sensitive strain (designated the propagating strain). After titration, a new plaque was removed and propagated again on the propagating strain. The filtrate obtained was titrated and served as a bacteriophage stock suspension.
High-titer lysates (1010 to 1011 PFU/ml) were required for bacteriophage DNA extraction or electron microscopy observation. These high-titer lysates were prepared by infecting an early-log-stage sensitive culture (100 ml) with phage at multiplicity of infection of 0.1. The infected culture was incubated at 30°C for 24 h and centrifuged, and then the supernatant was treated as described above for spontaneous liberation. In order to obtain the desired concentration of bacteriophages with this method or after treatment of bacterial cultures (100 ml) with mutagenic agents (MC, UVc), each filtrate was centrifuged at 30,000 × g for 4 h. The pellet was dissolved in 500 µl of TM buffer (50 mM Tris base, 10 mM MgSO4; pH 7.5) overnight.Electron microscopy. Bacteriophage particles purified on continuous CsCl gradients were stained with uranyl acetate 5 UA (2% [wt/vol], pH 4.5). Stained grids were observed with a Zeiss EM.10 electron microscope operating at 80 kV.
Extraction of bacteriophage DNA. One hundred microliters of STEP buffer (50 mM Tris base, 0.4 M EDTA, 0.5% SDS, 1 mg of proteinase K per ml; pH 7.5) (22) was added to 500 µl of a high-titer bacteriophage suspension. The suspension was incubated at 55°C for 1 h and extracted twice with Tris-saturated phenol-chloroform-isoamyl alcohol (25:24:1). DNA was precipitated from the aqueous phase by adding 0.1 volume of 3 M sodium acetate (pH 7.0) and 0.7 volume of propan-2-ol. The precipitated DNA was recovered by centrifugation (20 min, 15,000 × g), washed in 70% (vol/vol) ethanol, dried, and resuspended in 30 µl of TE.
Restriction of bacteriophage DNA. Digestion of bacteriophage DNA with restriction endonucleases was performed as recommended by the manufacturer (Roche Diagnostics). DNA fragments were separated on a 0.8% agarose gel (Life Technologies, Paisley, Scotland) in 1 × TBE buffer (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA; pH 8.3).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Screening of putative lysogenic strains by dot blot
hybridization.
Nine virulent bacteriophages infecting P. freudenreichii and representing the bacteriophages isolated from
Swiss type cheeses were chosen for this study. Their genomes were used
as probes. These bacteriophages (
110E1t, 110E1c, 19E4,
110D6, 110B3, 19E1, 19B3, 52, and 142) belong to
group B1 of Bradley's classification (4). They are
similar to many of the bacteriophages which infect lactic acid
bacteria, and the genome of each of them consists of a linear
double-stranded DNA molecule that is 36 to 40 kb long. Cohesive ends
were detected for one of the bacteriophages (B22) (14).
The host spectra of these bacteriophages differed and could be
distinguished by using DNA restriction patterns (15). However, the bacteriophages exhibited a high degree of DNA homology (16).
110B3, 110E1c, 110Elt, and 110D6
hybridized with the genomes of numerous strains, whereas probes from
bacteriophages 19E1 and 19E4 did not hybridize with bacterial DNA. Other bacteriophage probes hybridized with only one or a small
number of bacterial DNA.
In terms of putative lysogeny with dairy PAB, the results are somewhat
dubious because although they indicate that there is homology between
some bacteriophages and bacterial genes, they do not formally prove
that prophages are present on the bacterial chromosome. Indeed, the
bacterial cultures employed during this study may have been
contaminated with virulent or carrier stage bacteriophages, so that the
bacterial DNA extracted could have been contaminated with DNA of
bacteriophage which were not at the prophage stage.
In order to circumvent these problems with the dot blot procedure,
Southern blot DNA hybridization was used to confirm our results.
Southern blot DNA hybridization. Positive hybridization results obtained in the dot blot analysis were studied thoroughly by performing a Southern analysis. The 29 bacterial DNA exhibiting homology with one or more bacteriophage DNA were digested with endonuclease XbaI, which cut the dairy PAB chromosome into several fragments. These fragments were separated by PFGE. The patterns obtained were made up of 10 to 16 bands ranging in size from approximately 10 to 1,000 kb, which were easier to analyze (12).
The four bacteriophage genomes (110B3, 110E1t, 110E1c, and
110D6) that exhibited homology with numerous bacterial DNA in dot
blot experiments were used as probes.
Southern blot analysis confirmed the results obtained by dot blot
hybridization. A total of 25 of the 29 strains studied (86%) exhibited
homology on their chromosomes with the DNA of bacteriophages (Table 1).
Only four bacterial DNA (DNA from P. jensenii TL1 and
P. thoenii TL39, TL45, and TL115) which produced positive signals in the dot blot hybridization analysis did not hybridize with
the corresponding bacteriophage DNA probes. When the strain patterns
obtained by PFGE were similar, the strains were closely related and
their hybridization patterns were identical (TL22 and TL24, TL166 and
TL164, TL154 and TL170) (data not shown).
Spontaneous prophage induction.
Three strains (TL4, TL146, and
TL170) were chosen to study spontaneous prophage induction. They were
selected because they had produced strong hybridization signals during
the Southern blot analyses. Their hybridization profiles were made up
of between three and six different bands. A hybridization band between
30 and 40 kb was observed in all cases, and this band could have corresponded to free bacteriophages in the culture. We therefore tried
to demonstrate spontaneous bacteriophage liberation by using the
sensitive strains in our collection (TL18, TL19, TL21, TL29, TL105,
TL110, TL301, and TL302). A bacteriophage capable of infecting strain
TL110 was detected in a culture of strain TL146. After three successive
isolations of this strain on YELA, the bacteriophage (designated
146) was still present. This result suggests that spontaneous
liberation probably occurred. The concentration of temperate
bacteriophages liberated at the beginning of the stationary stage was
106 PFU/ml of culture.
Optimization of prophage induction.
In order to optimize the
conditions for prophage induction of mutagenic agents, UVc irradiation
and MC were chosen because they are the mutagenic agents that are most
commonly used in this type of study and because MC has been used
successfully for P. acnes prophage induction
(33). Strain TL146 was probably lysogenized with a
bacteriophage which could be detected by sensitive strain TL110, so we
decided to optimize the conditions under which UVc irradiation and MC
were used with this strain. A culture of strain TL146 at three
different growth stages was treated with several MC concentrations and
different intensities of UVc irradiation (Fig.
1). Whatever level of mutagenic agent was
used, a culture of strain TL146 at an OD650 of 1 (end of
the log stage) exhibited the greatest bacteriophage liberation. Maximum
liberation was obtained with an MC concentration of 0.1 µg/ml and a
UVc intensity of 60 J/m2.
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146
liberation was the same (data not shown). Thus, for strain TL146, it
appears that the efficacy of induction depended mainly on the number of
cells capable of division after treatment.
As a rule, massive prophage induction and liberation in a culture are
accompanied by cell lysis. The culture of strain TL146 was treated
under optimum conditions (UVc irradiation and MC) at the end of the log
stage (OD650, 1.0) (Fig. 2a).
In parallel, the temperate bacteriophages liberated were enumerated
(Fig. 2b). During the first 12 h after treatment, no difference in
OD650 was noted between controls and treated cultures.
Thereafter, the OD650 values fell more markedly in cultures
treated with mutagenic agents than in control cultures. A possible
explanation for this difference is induction by MC of another
undetected prophage.
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146 liberation occurred 7 h after induction
treatment. The majority of viruses had been liberated after 24 h
(Fig. 2b).
The same induction parameters were applied successfully to two other
strains (TL4 and TL170), which released bacteriophages 4 and 170.
Characterization of temperate bacteriophages 4, 146, and
170. (i) Morphology.
Electron microscopy observations showed
that three temperate bacteriophages (146, 4, and 170) had
morphological characteristics similar to those of virulent
bacteriophage B22 isolated from an industrial cheese and described
in a previous study (14; data not shown). These
bacteriophages could be considered members of group B1 in Bradley's classification.
(ii) Genome.
The genomes of temperate bacteriophages were
digested with restriction endonuclease PstI, which was
selected during a previous study (15) (Fig.
3). The lengths of the 4, 146, and
170 genomes were 30, 32, and 34 kb, respectively, as calculated by
adding the lengths of restriction fragments. The genome restriction
profiles of temperate bacteriophage 146 and virulent bacteriophage
B22 were found to be very closely related.
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Prophage-cured derivatives and relysogenization. In a lysogenic bacterial culture, a fraction of the population was naturally prophage cured. There were few such cured derivatives, so it was necessary to reduce the number of lysogenic cells in order to isolate them. Treatment of a culture with one or more mutagenic agents was required to enable prophage induction. As the cured derivatives lost their immunity to infection, they became sensitive to a temperate bacteriophage and had to be isolated on solid agar medium. Finally, to complete the demonstration that lysogeny occurred, it was necessary to relysogenize the cured derivatives by using the same bacteriophage (21).
Temperate bacteriophages4 and 170 could not be detected by a
sensitive strain in our collection, so they may have been defective. In
this case, identification of a cured derivative would have been
impossible. We therefore decided to demonstrate lysogeny in dairy PAB
by using strain TL146. Initially, strain TL146 cells in the log stage
and a decimal dilution were plated directly onto YELA and then treated
with a prophage-inducing dose of UVc irradiation. A mortality rate of
99% was obtained. One hundred clones that survived UV exposure were
tested for sensitivity to the induction filtrate of strain TL146. Only
one clone was found to be sensitive. When this variant was evaluated
for spontaneous bacteriophage liberation or after MC treatment, no
viruses were detected in the culture.
Relysogenization of the cured derivative.
In order to complete
our formal demonstration of lysogeny, the prophage-cured derivative was
relysogenized by using the same bacteriophage. The cured derivative was
infected with temperate bacteriophage 146, and bacteria from the
center of a lysis plaque were plated onto YELA. Several colonies were
screened for bacteriophage sensitivity. Seventeen clones were purified,
and 15 were found to exhibit restored infectious immunity and
spontaneous liberation.
Demonstration of prophage insertion.
In order to obtain
physical evidence that the clone isolated was a prophage-cured
derivative of strain TL146, genomes of several isogenic strains were
examined for the presence of 146 DNA sequences. Genomic DNA from
strain TL146, from a cured variant, and from one relysogenized clone
were digested with the low- restriction enzyme
XbaI, and the fragments were separated by PFGE. This
analysis revealed that the two XbaI restriction fragments at
54 and 164 kb, which were present in the lysogenic strain, had
disappeared in the cured variant and a new band at 186 kb had appeared
(Fig. 4a). Bacteriophage 146 DNA had
one XbaI site, and its absence from the cured variant
removed one XbaI site from the bacterial chromosome. The
combined size of the two smaller fragments (54 and 164 kb) minus the
size of the bacteriophage genome (32 kb) corresponded to the size
of the largest fragment (186 kb). This result demonstrated that the
integration site of prophage 146 was on the bacterial
chromosome. Restriction analysis of several relysogenized variants
produced identical results, suggesting that there was a specific
integration site for the genome of prophage 146 on the chromosome of
strain TL146. With this in mind, the DNA of 146 was labeled with
[32P]dCTP and used as a hybridization probe to confirm
that the insertion site really was on the bacterial chromosome (Fig.
4b). Hybridization signals were obtained for strain TL146 and the
relysogenized clone. However, chromosomal XbaI fragments of
the cured derivative did not exhibit homology with the DNA of
bacteriophage 146.
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146 was integrated in
strain TL146 and relysogenized clonal chromosomes and was absent from
the cured derivative. Thus, our data provide the first formal
demonstration of lysogeny in the genus Propionibacterium.
The presence of a prophage in the bacterial genome endows the genome
with certain properties. For instance, immunity to infection by other
related bacteriophages is more widespread. In order to study this
phenomenon, we infected strain TL146 and the cured derivative with four
bacteriophages (110E1t, B22, 110D6, and 110B3). All of the
bacteriophages were able to propagate on the cured derivative, and none
was able to propagate on strain TL146. This result suggested that there
was homoimmunity among these bacteriophages.
Another property found in lysogenic bacteria is conversion by the
integrated prophage (20, 23, 31). In order to study whether conversion was present in strain TL146 because of bacteriophage 146 integration, several metabolic activities (API 50 CH strips) and
growth kinetics on YEL medium were compared in the lysogenic strain and
the cured derivative. No differences were observed between the two cell types.
To complete this work, we attempted to lysogenize sensitive strain
TL110 with bacteriophage 146. Twelve clones were isolated from a
lysis plaque. All of these clones were resistant to 146 and
liberated it spontaneously. Genomic DNA from strain TL110 and one clone
lysogenized with 146 and digested with the low-restriction enzyme
XbaI produced PFGE patterns similar to those produced by the
cured derivative and strain TL146, respectively. One restriction fragment present in strain TL110, the XbaI fragment at 186 kb, was not present in the lysogenized derivative, while two bands appeared at 54 and 164 kb (data not shown). The gel was Southern blotted, and the 146 genome was used as a hybridization probe. Identical hybridization patterns were obtained for the lysogenized clone of TL110 and strain TL146, indicating that similar integration sites were present in strains TL110 and TL146 (Fig. 4b and c).
Conclusion and perspectives.
Obtaining a cured derivative and
relysogenization of this derivative with the same bacteriophage enabled
us to demonstrate lysogeny for the first time in P. freudenreichii subsp. shermanii. Spontaneous induction
could be detected with prophage 146. We identified a temperate
bacteriophage-sensitive strain system which allowed optimization of the
conditions for prophage induction. The MC concentrations used were
similar to or lower than those used for P. acnes,
Lactococcus spp., Leuconostoc oenos,
Lactobacillus spp., and Streptococcus
thermophilus (2, 3, 5, 6, 8, 9, 27, 33). When UVc
irradiation was used, the optimum intensities used for dairy PAB were
higher than those employed for lactococcal and Lactobacillus
strains (7, 8, 11). This result agreed with the
antimutagenic and reactive activities against UVc irradiation described
for dairy PAB (32).
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ACKNOWLEDGMENTS |
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This work was supported by funds provided to C. Hervé by the Brittany Region.
We thank Françoise Michel for assistance with the electron microscopy observations and Jane Hall for correcting the English in the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: INRA Laboratoire de Recherche de Technologie Laitière, 65, rue de Saint Brieuc, 35042 Rennes Cedex, France. Phone: 00-33-2-23-48-55-82. Fax: 00-33-2-23-48-75-78. E-mail: michel.gautier{at}agrorennes.educagri.fr.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Applied and Environmental Microbiology, January 2001, p. 231-238, Vol. 67, No. 1
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.1.231-238.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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