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Previous Article | Next Article 
Applied and Environmental Microbiology, May 2001, p. 2235-2239, Vol. 67, No. 5
Department of Food Science and Human
Nutrition, Iowa State University, Ames, Iowa 50011
Received 27 July 2000/Accepted 29 January 2001
Polyclonal antibodies against the bacteriocin propionicin PLG-1
were produced in rabbits at high titer (256,000 to 512,000, as
determined by indirect enzyme-linked immunosorbent assay [ELISA]). Anti-PLG-1 antiserum neutralized the antimicrobial activity of PLG-1 preparations in a well diffusion assay. Cross-reacting
protein was detected using an indirect ELISA of the culture supernatant from a fed-batch fermentation of the producer strain
Propionibacterium thoenii P127 within the first 24 h
of incubation, but bacteriocin activity was not detected in the same
culture until 217 h of incubation. Culture supernatants from 156 strains of classical dairy propionibacteria were tested by indirect
ELISA at 5 and 12 days of incubation for production of cross-reacting
protein and by well diffusion assay for bacteriocin activity.
Cross-reacting protein was detected in 52 strains: all of the
tested strains of P. thoenii, most of the strains of
Propionibacterium jensenii, and a minority of
the Propionibacterium acidipropionici and
Propionibacterium freudenreichii strains. Of these 52 strains, only 4 strains of P. thoenii showed bacteriocin activity in a well diffusion assay. Eight
bacteriocin-negative mutants of strain P127 were negative in both
ELISA and well diffusion assays. Western blot analysis showed that
three protein bands bound anti-PLG-1 antibodies in culture
supernatants: a 9.1-kDa band that is assumed to be the
PLG-1 monomer and 16.2- and 27.5-kDa bands that may be precursors,
multimers, or complexes of PLG-1.
The best characterized bacteriocin
from the dairy propionibacteria is propionicin PLG-1, which is produced
by Propionibacterium thoenii P127 (14). This
bacteriocin is moderately heat-stable, sensitive to proteolytic
enzymes, and stable at pH 3 to 9 (15). It contains 99 amino acid residues, has a molecular mass of 9,328 Da, and seems
unrelated to other bacteriocins from lactic acid bacteria based on a
comparison of its N-terminal amino acid sequence to others in the
SWISS-PROT data bank (21). Propionicin PLG-1 is rapidly
bactericidal against other dairy propionibacteria and lactic acid
bacteria (14, 16). Methods for PLG-1 production and
purification have been optimized (10, 21).
The use of immunological methods in bacteriocin research has been
limited. Recent attempts to produce bacteriocin-specific antibodies
have had varied results (1, 3, 4, 12, 17, 18). The
relatively low molecular mass (<5,000 Da) of many bacteriocins makes
them poorly or nonimmunogenic (22); conjugation of small bacteriocins to carrier proteins can improve their immunogenicity. Immunological techniques based on immunoblotting and enzyme-linked immunosorbent assays (ELISA) can be useful in investigating details of
bacteriocin production, structure, and function (17, 18). Production of bacteriocin-specific antibodies has enabled development of sensitive immunoassays for nisin (7, 22) and pediocins (1).
The objective of this work was to produce polyclonal antibodies against
propionicin PLG-1 to enable immunoassay development. These assays were
used for detection of PLG-1 and other cross-reacting proteins, which
might be novel bacteriocins or other forms of PLG-1.
Microorganisms and media.
All cultures were from the
Iowa State University Department of Food Science and Human Nutrition
culture collection. P. thoenii P127 was the PLG-1 producer
strain. Bacteriocin-negative mutants of P127 obtained by
nitrosoguanidine mutagenesis have been described previously
(15). All propionibacteria were propagated in sodium lactate broth (NLB) at 32°C (14). Indicator strain
Lactobacillus delbrueckii ATCC 4797 and other lactic acid
bacteria were propagated in lactobacilli MRS broth (Difco Laboratories,
Detroit, Mich.) as previously described (10). Other
bacterial strains were propagated in tryptic soy broth (Difco)
statically at 37°C. Working cultures were stored on the appropriate
agar medium with 1.5% Bacto agar (Difco) added. Long-term storage was
at
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2235-2239.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of the Bacteriocin Propionicin PLG-1
with Polyvalent Anti-PLG-1 Antiserum
and
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 the appropriate medium with 20% glycerol added
(10). Viable counts of propionibacteria were obtained on
sodium lactate agar (NLA) after 5-day anaerobic incubation at 32°C
(14).
20°C until
required. The numbers of strains of each organism tested for
cross-reactivity were as follows: P. thoenii, 8;
Propionibacterium jensenii, 26; Propionibacterium
acidipropionici, 19; Propionibacterium freudenreichii
subsp. shermanii, 79; P. freudenreichii subsp. freudenreichii, 14; unclassified
Propionibacterium strains, 10; bacteriocin-negative
(bac
) mutants of P. thoenii P127, 8;
Lactobacillus bulgaricus, 3; L. delbrueckii, 1;
Lactobacillus casei, 1; Streptococcus cremoris, 1; Streptococcus diacetylactis, 1; Streptococcus
lactis, 1; Streptococcus thermophilus, 1;
Bacillus cereus, 1; Escherichia coli, 1;
Pseudomonas aeruginosa, 1; and Staphylococcus
aureus, 1.
Bacteriocin production and purification.
Strain P127 was
grown in 14-day fed-batch fermentations in the Iowa State University
Fermentation Facility. The cells were removed by centrifugation
(8,000 × g; 30 min), and propionicin PLG-1 was
harvested from the cell-free supernatant and purified using ammonium
sulfate precipitation, ion exchange chromatography, and reverse-phase
high-performance liquid chromatography as described previously
(21). Purification was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of aliquots of
the sample, followed by silver staining (21). The gel was overlaid with the indicator organism L. delbrueckii ATCC
4797 to confirm that the stained bands had biological activity, as described by Bhunia and Johnson (2). Purified PLG-1
batches were combined and stored at 80°C until required for immunizations.
Bacteriocin assay. Bacteriocin activity was determined in an agar well diffusion assay (10, 21) using L. delbrueckii ATCC 4797 as an indicator organism. To test the ability of the polyclonal antiserum to neutralize bacteriocin activity, serial dilutions of bacteriocin were mixed with an equal volume of undiluted antiserum in each well prior to adding the overlay. Preimmune serum and sterile deionized water were mixed with bacteriocin in the control wells. All tests were run in duplicate.
Immunization and serum harvest.
All procedures were
conducted at the Iowa State University Cell and Hybridoma Facility.
Three New Zealand white rabbits were immunized both subcutaneously and
intramuscularly with 1 ml of antigen solution containing approximately
200 µg of PLG-1, using either Freund's complete adjuvant for the
initial immunization or Freund's incomplete adjuvant for subsequent
booster immunizations at 4-week intervals. After titers had reached
acceptable levels, one rabbit was chosen for antibody production and
maintained for production bleeds (a 30-day cycle of immunization
followed by three 50-ml bleeds at 10-day intervals). All blood samples
were refrigerated for 24 h and centrifuged (450 × g; 10 min) at room temperature. The serum was then stored at
20°C.
Indirect ELISA. An indirect ELISA based on the methods of Engvall and Perlmann (6) and similar to that described by Fan and Chu (8) was used for antigen detection. Antigen-containing samples (100 µl) were dispensed into duplicate wells of COSTAR polystyrene microtiter plates (Corning, Inc., Corning, N.Y.), which were then sealed in plastic bags and incubated at 4°C overnight. Wells were washed twice with a solution consisting of phosphate buffered saline (PBS), 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 154 mM NaCl (pH 7.4) and 0.05% (vol/vol) Tween 20 (Sigma, St. Louis, Mo.) (PBST), blocked with blocking buffer (PBST + 2% [wt/vol] casein) for 30 min at 37°C, and washed twice with PBST.
Primary antibody (100 µl of rabbit serum diluted 1:20,000 in PBS) was then added to the wells and incubated for 120 min at 37°C. Wells were washed three times with PBST before the secondary antibody (100 µl of goat anti-rabbit immunoglobulin G [IgG]-horseradish peroxidase conjugate [Sigma]; 1:5,000 dilution in PBS) was added. After 90 min of incubation at 37°C, wells were washed three times with PBST, and then o-phenylenediamine (Sigma) substrate solution at a concentration of 0.66 mg/ml plus 0.012% (vol/vol) H2O2 was added, and plates were incubated at room temperature for 10 min in the dark. The reaction was stopped by addition of 0.5 M sulfuric acid (Fisher) and the absorbance (492 nm) of each well was read with a Bio Kinetics EL 340 microtiter plate reader (Bio Tek Instruments, Inc., Winooski, Vt.). Average absorbance due to nonspecific binding of secondary antibody was subtracted from the absorbance values of all samples. Purified PLG-1 solutions at concentrations of 12.5 and 6.25 µg/ml in PBS were used as standards. To allow comparison of values across different plates, all values were normalized to a standard value of 0.500 for the 6.25 µg of PLG-1 per ml control. Concentrations of PLG-1 greater than 0.08 µg/ml were detected. The titer of the rabbit antiserum was determined by testing serum diluted in PBS against purified PLG-1 (2 µg/ml in PBS) using indirect ELISA. Titer was defined as the reciprocal of the highest dilution with an absorbance at least twice the average absorbance for all dilutions of preimmune serum. Rabbit IgG at concentrations greater than 0.23 ng/ml was detected.SDS-PAGE and Western blot. Gel electrophoresis was performed using the buffer system described by Laemmli (13) in 10 to 20% gradient polyacrylamide gels (mini-PROTEAN II ready gels; Bio-Rad Laboratories, Hercules, Calif.). Proteins were separated at 180 V for 45 min by using the Bio-Rad mini-PROTEAN II electrophoresis apparatus. Gels were fixed in 30% ethanol-10% glacial acetic acid and silver-stained according to the manufacturer's instructions (Bio-Rad).
For Western blotting (5), the separated proteins were transferred to a polyvinyl difluoride membrane (Immobilon-P; Millipore Corp., Bedford, Mass.) by using a Bio-Rad Trans-Blot electrotransfer apparatus according to the manufacturer's instructions. After blocking with Tris-buffered saline (TBS) (20 mM Tris base, 0.5 M NaCl [pH 7.5]) plus 5% (wt/vol) nonfat dry milk at room temperature for 1 h, the membrane was incubated, first with primary antibody (rabbit antiserum diluted 1:2,000 in TBS plus 1% nonfat dry milk plus 0.3% Tween 20) and then with secondary antibody (goat anti-rabbit IgG horseradish peroxidase conjugate diluted 1:2,000 in the same buffer). Each incubation was at room temperature for 1 h with gentle agitation. Bound antibodies were visualized with diaminobenzidine (Sigma).Colony blot. The protocol for the colony blot assay was modified from a procedure described by Noel et al. (20). Organisms were grown on NLA for 5 days at 32°C. A moist nitrocellulose membrane disk cut to fit the petri dish was pressed gently onto the colonies, and the dish was incubated for 45 min at room temperature. The disk was then removed and reacted first with primary antibody (rabbit antiserum diluted 1:2,000 in TTBS [Tris-buffered saline plus 0.05% Tween 20] plus 1.0% gelatin) for 2 h at room temperature and then with secondary antibody (goat anti-rabbit IgG horseradish peroxidase conjugate diluted 1:2,000 in TTBS plus 1.0% gelatin) for 90 min at room temperature. Antibodies bound to colonies were visualized with diaminobenzidine.
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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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Antibody production. The titers of polyclonal antibodies against propionicin PLG-1 were at 256,000 to 512,000 in all three rabbits after the first booster immunization (34 days), and they remained at this level throughout the antiserum production cycle (152 days). Propionicin was large enough (9.3 kDa) to be immunogenic itself; conjugation with a carrier protein was not needed. Antiserum from a single rabbit was used for all of the experiments reported here.
Neutralization of bacteriocin activity.
Undiluted antiserum
mixed with an equal volume of a PLG-1 preparation reduced bacteriocin
activity by 98 to 100% in the well diffusion assay; preimmune serum
had no effect on activity (Table 1).
Partially purified bacteriocin samples contained much more total
protein than did the purified propionicin; this may have interfered
with antibody binding to PLG-1. These results suggested that specific
antibodies against PLG-1 were present in the antiserum and that these
antibodies interacted with the active bacteriocin molecules.
|
Propionicin PLG-1 detection in fermentation supernatant.
A
fed-batch fermentation of P. thoenii P127 in NLB with a
feeding of 0.6% sodium lactate every 12 h was monitored for
viable counts, PLG-1 content (indirect ELISA), and bacteriocin activity (well diffusion assay). Viable counts and bacteriocin activity levels
were similar to those previously reported by Paik and Glatz (21) (Fig. 1A). Cell numbers
plateaued at approximately 60 h of incubation, but bacteriocin
activity was not detected in the supernatant until 217 h, a
pattern typical of secondary metabolite synthesis (15).
However, supernatant samples from as early as 5 h of incubation
gave a response in the indirect ELISA (Fig. 1B). The response was high
and relatively constant for the duration of the fermentation. All
components of NLB as well as 0.1 and 1.0% (wt/vol) solutions of
propionic and acetic acid (typical concentrations produced by
propionibacteria in fermentation) produced no response in the ELISA.
|
Immunoblotting of PLG-1 and fermentation supernatant.
To
determine what components in the fermentation supernatant reacted with
anti-PLG-1 antibodies, the proteins in supernatants from two different
fermentations as well as purified and partially purified preparations
of PLG-1 were separated by SDS-PAGE and probed in a Western blot
procedure using anti-PLG-1 antiserum (Fig.
2).
|
Detection of cross-reactive proteins from other bacteria. A total of 156 strains of propionibacteria and representative cultures of other gram-positive and gram-negative bacteria were tested for bacteriocin activity in the well diffusion assay and for production of proteins that reacted with anti-PLG-1 antibodies in the indirect ELISA. Culture supernatants harvested after 5 and 12 days of incubation were used.
Only four strains (all strains of P. thoenii), P15, P20, P126, and P127, produced zones of inhibition in the well diffusion assay. Both the 5- and 12-day supernatants of strain P126 were active; this strain produces the bacteriocin jenseniin G (9). Only the 12-day supernatants of the other strains were active. Bacteriocin activity is typically seen at 12 days for P127, the PLG-1 producer strain (15). This was the first time that strains P15 and P20 were seen to produce bacteriocin-like activity. Supernatants of bac mutants of P127 and of all other bacteria tested did
not produce zones of inhibition.
A supernatant was considered to be reactive with anti-PLG-1 antiserum
in the indirect ELISA if its absorbance was at least 10-fold the
average nonspecific absorbance value of 0.010. Using this criterion,
supernatants from 52 strains of propionibacteria were considered to be
reactive at one or both of the testing times (Table
2).
|
mutants of
P. thoenii P127 and for the other bacterial strains tested.
The only exception was the 5-day supernatant from P. aeruginosa, whose absorbance value (0.13) was considered a
marginally positive response.
These results suggest that protein(s) immunologically similar to
propionicin PLG-1 are unique to the propionibacteria and perhaps to
certain species of propionibacteria. It may be possible to use
anti-PLG-1 antiserum in a simple screening test to tentatively identify
P. thoenii and P. jensenii strains.
Intensity of response in the ELISA did not correlate with bacteriocin
activity. The absorbance values of several strains with no detectable
bacteriocin activity were much higher than those of the four strains
that were biologically active. It is likely that the bacteriocin
activity of strain P126 was due to the presence of jenseniin G, a
different bacteriocin, but the ELISA response of this strain was very
similar to that of strain P127. The nature of the cross-reacting
material in supernatants that gave a positive response in the ELISA was
studied further by Western blot analysis.
Western blot analysis of reactive supernatants.
The same
5- or 12-day supernatants from strains that had moderate to
strong ELISA reactions also were subjected to SDS-PAGE and probed with
anti-PLG-1 antibodies using the Western blot procedure. Some
supernatants from ELISA-negative strains, including the
bac mutants of strain P127, also were tested, as was the
marginally reactive supernatant from P. aeruginosa. Results
are summarized in Table 3.
|
mutants of P127. The other two bac mutants contained very
faint 27.5-kDa bands.
These results suggest that many strains of propionibacteria produce
proteins similar to the proteins produced by strain P127 that bind
anti-PLG-1 antibodies. If these are inactive precursors or
multimers of the bacteriocin, strains without bacteriocin activity must
lack the mechanism required to release the active form of the
bacteriocin. The failure to find a reactive 9.1-kDa band, hypothesized
to be the active form of PLG-1, in biologically active supernatants,
particularly in strain P127, is puzzling. The protein might be present
at low concentration, below the detection limit of the Western blot
assay, or it may readily form complexes with other proteins that remain
intact in SDS-PAGE. The absence or low intensity of reactive protein
bands in bac mutants of P127 supports the hypothesis that
these bands represent precursor or multimer forms of PLG-1.
Colony blot analysis.
To test whether anti-PLG-1 antiserum
could be used in a colony blot procedure as a relatively rapid, simple
test for production of bacteriocin or cross-reacting protein, several
colonies each of strain P127, two bac mutants of
P127, two ELISA-negative strains of P. freudenreichii and
P. acidipropionici, and a strain of B. cereus were examined using the colony blot procedure after 5 days
of growth on NLA.
mutant gave dark reactions, whereas the colonies
of the other bac mutant gave very faint reactions.
Perhaps the bac mutants produced levels of cross-reactive
proteins that were undetectable in culture supernatants by the ELISA or
Western blot procedures but were concentrated at the colony surface on
solid medium and thus were visible in the colony blot. Alternatively, growth on solid medium may favor production of these proteins.
After colony growth and transfer of proteins to nitrocellulose
membranes for probing with antiserum, the L. delbrueckii
indicator organism was overlaid onto the plates to detect bacteriocin
activity in the colonies. No inhibition of the indicator organism was
seen for any colony. This was expected for all colonies except those of
strain P127. It is possible that a sufficient quantity of active PLG-1
had not yet accumulated in the P127 colonies to give a zone of
inhibition (the assay was done after 5 days of incubation, when
biological activity typically only starts to appear) or that the
blotting procedure removed much of the active form from the plate.
The colony blot assay was able to detect cross-reactive proteins; like
the ELISA, it did not distinguish which or how many proteins were
reactive. Several colonies could be grown on each plate and probed
simultaneously on the same membrane. No specialized equipment was
necessary to complete the assay. This procedure may be useful as a
qualitative test to screen propionibacteria for bacteriocin analog production.
In conclusion, propionicin PLG-1 is antigenic in rabbits. Polyvalent
anti-PLG-1 antiserum neutralizes biological activity of PLG-1 in the
well diffusion assay and reacts not only with the bacteriocin itself
but also with precursors, multimers, and/or complexes of the
bacteriocin in indirect ELISA, Western blot, and colony blot
procedures. Many strains of propionibacteria, especially P. thoenii and P. jensenii strains, produce cross-reacting proteins that do not seem to have antimicrobial activity. The nature of
these proteins and their function(s) in the propionibacteria must still
be elucidated.
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ACKNOWLEDGMENTS |
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This research was supported by research grant US-2614-95C from BARD, the United States-Israel Binational Agricultural Research and Development Fund. Project 3475 is supported by Hatch Act and State of Iowa funds.
We are deeply indebted to Joan Cunnick, Department of Microbiology, Iowa State University, for her advice and for the use of her laboratory facilities to carry out much of this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: 2312 Food Sciences Bldg., Iowa State University, Ames, IA 50011. Phone: (515) 294-3970. Fax: (515) 294-8181. E-mail: bglatz{at}iastate.edu.
Journal paper No. J-18987 of the Iowa Agriculture and Home
Economics Experiment Station, Ames, Iowa, Project 3475.
Present address: Medarex, Inc., Annandale, NJ 08801.
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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, viable counts
(log CFU/ml); 
, bacteriocin activity in well diffusion assay
(activity units/ml). (B) 
, absorbance at 492 nm of supernatant
samples that reacted with anti-PLG-1 antiserum in the indirect ELISA.
