Applied and Environmental Microbiology, June 1999, p. 2281-2286, Vol. 65, No. 6
Departments of Animal
Science1 and Molecular
Biology,2 University of Wyoming, Laramie,
Wyoming 82071
Received 25 August 1998/Accepted 9 March 1999
The properties of the pediocin AcH precursor, prepediocin AcH, have
been studied to gain insight into how producer cells may protect
themselves from the activity of intracellular prebacteriocins. The
native 62-amino-acid precursor and the 44-amino-acid mature species
were expressed in Escherichia coli host strains that lack the leader peptide processing enzyme, PapD. Both forms inhibited the
growth of the test bacterium Listeria innocua Lin11,
indicating that the native precursor is biologically active. The two
species also were synthesized in the context of maltose-binding protein chimeric proteins to facilitate the measurement of their relative specific activities. The chimeric form of the precursor was ~80% as
active as the chimeric mature species. Of relevance to cell protection
and pediocin AcH production, it was determined that the precursor is
strongly susceptible to inactivation by reducing agents and to
degradation by chymotrypsin and endogenous E. coli proteases. Taken together, the results indicate that the activity of
prepediocin AcH may have to be controlled prior to secretion to prevent
toxicity to the host. Perhaps producer cells avoid membrane damage by
maintaining the precursor in a reduced inactive state or by degrading
molecules whose secretion is delayed.
The bacteriocins produced by
gram-positive bacteria are gene-encoded antimicrobial peptides that
kill target cells by forming pores in the cytoplasmic membrane (1,
3, 4, 14). Nearly all bacteriocins contain N-terminal leader
peptides that direct precursors to dedicated, ABC-export system
proteins that translocate the active C-terminal prodomain across the
cytoplasmic membrane and remove the leader peptide (2, 6, 29,
31). Currently, it is unclear why specialized export systems are
used for secretion, and it has been shown that pediocin AcH (which is
the same as pediocin PA-1 [17]) can be secreted via
the Escherichia coli sec machinery when targeted to it via
linkage to a standard secretory protein (18, 19). The only
bacteriocin that normally is secreted by the general sec
machinery of the cell is divergicin A (32).
Leader peptides are structurally and functionally distinct from signal
sequences of sec-dependent secretory proteins
(14). They contain 23 to 30 amino acids for nisin-like
prebacteriocins and 18 to 24 amino acids for pediocin-like
prebacteriocins. Although leader peptide sequences differ considerably
between these two families, the members of each group show a great
degree of sequence identity, suggesting that conserved residues have
functional significance (6, 14). For example, amino acids in
the Leader peptides may perform other functions besides directing the
interaction of prebacteriocins with ABC-export systems. These could
include guiding and maintaining conformation during posttranslational
modification of lantibiotics such as nisin, stabilizing prebacteriocin
molecules to degradation prior to translocation, and/or keeping
molecules inactive against the cytoplasmic membranes of producer cells
prior to secretion (6, 14, 15, 30). With regard to the
latter point, it has been shown that the fully modified nisin precursor
(29) and a pediocin-like prebacteriocin, preleucocin A
(10), are inactive, and that another pediocin-like prebacteriocin, precarnobacteriocin B2, is 125-fold less active than
its mature form (24). In contrast, the prepediocin PA-1 precursor appears to display activity (31), but its activity relative to that of mature pediocin PA-1 has not been measured. In
cases where precursors are inactive, leader peptides may alter the
conformations of propeptide domains or interfere with their interaction
with the cytoplasmic membrane of target as well as producer cells
(6, 10, 12, 24).
In this study, we have examined possible auxiliary roles played by the
pediocin AcH leader peptide during secretion. It has been determined
that the precursor is nearly as active as the mature species and is
highly susceptible to protease degradation and disulfide bond
reduction. The implications of the results for cell protection and
pediocin AcH production are discussed.
Bacterial strains, plasmids, and growth conditions.
The
E. coli host strain BL21(DE3) was used for expression of the
mature and precursor forms of pediocin AcH (Table
1). This strain contains the T7
bacteriophage RNA polymerase gene cloned on the chromosome under the
control of the isopropyl-
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Pediocin AcH Precursor Is Biologically
Active
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2 and 1 positions relative to the processing site are highly
conserved, probably because they comprise the recognition site for
processing enzymes (6, 12, 14, 28). Leader peptides
generally are relatively hydrophilic and may assume an amphipathic
-helical structure in lipophilic environments (6, 9, 14, 26,
27). Currently, the features of the leader peptide that are
recognized by ABC-export system proteins are unknown. At least some of
these features may be in common because a given prebacteriocin
sometimes can be secreted by an unrelated ABC-export system
(28). After their removal, leader peptides may be secreted
or degraded, as occurs for signal sequences of sec-dependent
secretory proteins (21).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-D-thiogalactopyranoside (IPTG)-inducible lac promoter. The papA and
prepapA genes, which encode the respective mature and
precursor species, were cloned into a T7 RNA polymerase promoter
plasmid, pET3c, and the resulting plasmids were introduced into
BL21(DE3) for expression. Transformed BL21(DE3) strains were grown at
37°C in Luria-Bertani (LB) broth containing 100 µg of ampicillin
per ml.
TABLE 1.
Bacterial strains and plasmids
Construction of expression plasmids.
The method used for the
construction of plasmid pPR6823 was similar to that used for the
construction of pPR6821 (18). The prepapA coding
region of plasmid pMBR1.0 was amplified by PCR by using a 5' PCR primer
(5'-AAAAAAATTGAAAAATTAACTGAAAAAGAAATG-3') that begins with
the Lys
17 codon of the prepapA DNA sequence
and a 3' PCR primer (5'-GGGTCGACCTAGCATTTATGATTACCT-3') that
contains a SalI restriction endonuclease site located
downstream of the prepapA termination codon. The conditions
used for DNA synthesis and the methods used for DNA fragment
manipulation and cloning have been described in detail previously
(18). After synthesis of the DNA fragment, it was treated
with SalI restriction endonuclease and ligated between the
StuI (blunt end) and SalI restriction
endonuclease sites of pPR682 to construct pPR6823. As a result of the
cloning steps, the Lys17 codon of the leader peptide
coding sequence was fused in frame to a 14-amino-acid linker
peptide-coding sequence located between the malE and
prepapA genes. The sequence of the prepapA region was confirmed by double-stranded DNA sequencing by using Sequenase DNA
polymerase (U.S. Biochemicals).
18 of the leader peptide region and
also contains an NdeI restriction endonuclease site for
ligation to the vector. A 3' PCR primer (5'-CTCGGATCCCTAGCATTTATGATTAC-3') that contains a
BamHI restriction endonuclease site was used for
amplification of both DNA fragments. After their ligation into pET3c,
both PCR inserts were sequenced in their entirety by double-stranded
DNA sequencing.
Measurement of chimeric protein activity levels. Strains E609L/pPR6821 and E609L/pPR6823 were grown overnight at 37°C in LB broth containing ampicillin and tetracycline. On the next day, cells were pelleted by centrifugation, washed with LB broth, and diluted in LB broth lacking antibiotics. Cultures were grown until the optical density at 600 nm (OD600) reached ~0.5, and then chimeric protein synthesis was induced by adding IPTG to 1 mM final concentration. Samples of the culture broths (cells plus supernatant), or the cell pellet and supernatant fractions isolated by 5-min centrifugation and filtration, were collected 3 h after IPTG induction. One portion of the samples was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis as described below to determine the relative synthesis levels of chimeric proteins. Another portion was boiled 10 min to inactivate the proteases, and aliquots were spotted onto TGE agar plates spread with Listeria innocua Lin11 to calculate the activity units (AU) per milliliter for each broth. Plates were incubated overnight at 30°C and examined to determine the smallest aliquot that produced a zone of growth inhibition in the lawn. The number of AU per milliliter of each broth was calculated based on the size of the smallest aliquot that formed a zone of inhibition (33). These values for the two broths were corrected for variations in the OD600 readings of the samples and the levels of chimeric proteins present. The MBP-prepediocin AcH chimeric protein also was tested for activity against the other pediocin AcH-sensitive strains listed in Table 1. In this case, activity titrations were not performed.
Effects of reducing agents on chimeric protein activity. Samples of E609L/pPR6821 and E609L/pPR6823 induced by a 3-h treatment with IPTG were prepared from cultures grown at 37°C in the absence of antibiotics. Culture broths were split into three portions. One portion served as a control, dithiothreitol (40 mM) was added to the second portion, and 2-mercaptoethanol (50 mM) was added to the third. The samples then were boiled for 10 min to reduce the pediocin AcH domains of the chimeric proteins and to inactivate proteases present in the broths. Control samples boiled with and without reducing agents were made by using LB medium alone and LB medium containing dissolved pediocin AcH from P. acidilactici LB42-923.
To determine the effects of reducing agents on activity, 1-ml aliquots of the samples were incubated for 1 h at 25°C with ~550 L. innocua Lin11 cells. After incubation, the mixtures were centrifuged, the supernatant fractions were removed, and the cells were resuspended and plated on TGE agar to determine the number of viable cells present. In addition, the supernatant fractions were boiled and tested for activity by being spotted onto a lawn of L. innocua Lin11. The numbers of AU per milliliter of the supernatant fractions were calculated and corrected for variations in culture ODs and chimeric protein synthesis levels, as described above.Chymotrypsin digestion of chimeric proteins. Boiled culture broths from strains E609L/pPR6821 and E609L/pPR6823 induced by a 3-h treatment with IPTG and grown at 37°C in the absence of antibiotics were incubated for up to 30 min at 25°C with 10 µg of chymotrypsin per ml. Aliquots of the reactions were withdrawn at specific intervals over the 30-min incubation period and were boiled to inactivate the enzyme. The digested samples and samples of the unincubated culture broths were spotted onto a lawn of L. innocua Lin11 to determine their AU values. These values were corrected based on the original OD600 readings of the broths and the levels of chimeric proteins present. The samples also were examined by SDS-PAGE and Western immunoblotting to assess the degradation of the chimeric proteins.
SDS-PAGE analysis of protein synthesis levels and activities. The synthesis levels of MBP chimeric proteins were determined by SDS-PAGE with 10% acrylamide-bisacrylamide-SDS gels (16). Gels were stained with Coomassie brilliant blue G-250 dye and scanned with a Bio-Rad Gel Dock laser densitometer to determine the relative levels of chimeric proteins present in samples. The same gel system was used for Western immunoblotting. In this case, chimeric proteins were electroblotted onto nitrocellulose membranes and were detected by staining with rabbit anti-MBP primary antiserum and goat anti-rabbit immunoglobulin G-alkaline phosphatase secondary-antibody complex.
The activities of MBP chimeric proteins and of the precursor and mature forms of pediocin AcH were analyzed by SDS-PAGE-gel overlay screening (18). Samples were obtained from chimeric protein expression strains grown at 37°C in LB-ampicillin-tetracycline medium and from T7 RNA polymerase expression strains grown at 37°C in LB-ampicillin medium. Proteins were separated on 20% acrylamide-bisacrylamide-SDS gels (25), and subsequently the gels were soaked in sterile water to remove SDS, placed on TGE agar plates, and overlaid with TGE top agar containing L. innocua Lin11 cells. Plates were incubated overnight at 25°C and examined for zones of growth inhibition caused by active proteins.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Properties of native and chimeric proteins. The sequences of the native mature and precursor forms of pediocin AcH are shown in Fig. 1A. The two species were produced in the respective E. coli T7 RNA polymerase expression strains BL21(DE3)/pT71 and BL21(DE3)/pT72. The BL21(DE3) strain lacks the leader peptide processing enzyme, PapD, and therefore can be used to obtain the unprocessed precursor. Both the precursor and mature forms of the bacteriocin accumulate in the cytoplasm of the strains.
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17 of the prepediocin AcH leader
peptide. The design enables, after MBP targeting to the cellular
sec machinery and signal sequence processing, the secretion
of the mature 682-prePapA protein into the periplasm of the E. coli host (18). Due to disruption of the outer membrane
protein structural gene lpp in this strain, about half of
the secreted chimeric protein is released into the culture medium. The
682-PapA protein produced by strain E609L/pPR6821 is similar to
682-prePapA except that it lacks the pediocin AcH leader peptide
sequence (18).
The native and chimeric forms of prepediocin AcH are biologically active. The activities of both native and chimeric prepediocin AcH molecules were examined. It was necessary to study chimeric molecules because of uncertainties in quantitating the levels of the native species produced by the T7 RNA polymerase expression strains and because native species could be obtained only by growing these strains in the presence of ampicillin (data not shown). The latter issue made it impossible to directly test culture broths from these strains against the pediocin AcH (and ampicillin)-sensitive bacteria listed in Table 1. On the other hand, significant levels of chimeric proteins were obtained from E609L strains grown in the absence of ampicillin, and therefore, culture broths from chimeric protein expression strains could be used directly in activity testing.
To verify that native prepediocin AcH is biologically active, SDS-PAGE-gel overlay screening analysis was performed. The presence of ampicillin in samples did not interfere with activity determination performed by this technique. BL21(DE3)/pT72 cells were grown in LB-ampicillin medium, and synthesis of prepediocin AcH was induced for 30 min by the addition of 1 mM IPTG to the culture. BL21(DE3)/pT71 cells were grown under identical conditions to obtain pediocin AcH as a positive control for activity. As shown in Fig. 2A, both samples formed zones of growth inhibition against L. innocua Lin11, indicating that prepediocin AcH is active. The zone of growth inhibition produced by the precursor appeared at a slightly higher molecular weight than the zone of growth inhibition produced by the mature form of the molecule.
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Specific activities of chimeric proteins.
The specific
activities of the 682-PapA and 682-prePapA chimeric proteins were
calculated to permit estimation of the relative activity of the
precursor. Cultures of E609L/pPR6821 and E609L/pPR6823 were grown in LB
medium in the absence of antibiotics, chimeric protein synthesis was
induced for 3 h with IPTG, and aliquots of the culture broths were
boiled and spotted onto lawns of L. innocua Lin11. The
numbers of AU per milliliter of the two broths were calculated and
corrected for variations in culture ODs and chimeric protein synthesis
levels determined by SDS-PAGE. As shown in Table
2, the corrected AU value of the
E609L/pPR6823 culture broth was 78% of that of the E609L/pPR6821
broth. Although breakdown products contributed to the calculated AU
values of the broths, the fractions of the total activities in the
strains attributable to the full-length chimeric proteins were about
the same (data not shown). Thus, the corrected activity levels
calculated for the broths are reflective of the specific activities of
the two chimeric proteins.
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Activity spectra of chimeric proteins. Culture broths from E609L/pPR6821 and E609L/pPR6823 induced for 3 h with IPTG and grown in the absence of ampicillin were tested against several pediocin AcH-sensitive strains. As has been reported for the 682-PapA protein (18, 19), the 682-prePapA protein was active against all five pediocin AcH-sensitive strains listed in Table 1 (data not shown). This suggests that the two molecules may have the same primary mode of action. In addition, both proteins were inactive against eight pediocin AcH producer strains that have been isolated in our laboratories (data not shown). All of the latter strains probably synthesize the PapB immunity protein (2), and therefore the results suggest that the activity of the precursor is blocked by the standard immunity mechanism.
Susceptibility of chimeric proteins to inactivation by reducing
agents.
The pediocin AcH precursor normally is found only in the
cytoplasm of producer cells (14). Experiments were performed
to determine whether the activity of the precursor could be decreased by exposing it to reducing conditions analogous to those that exist in
the cytoplasm. A culture broth of strain E609L/pPR6823 induced for
3 h with IPTG was prepared and treated with or without dithiothreitol or 2-mercaptoethanol to reduce the 682-prePapA protein.
An E609L/pPR6821 culture broth grown under identical conditions also
was prepared, as was a sample of native pediocin AcH from P. acidilactici LB42-923. The effects of the reducing agents on
activity were tested in two ways. Aliquots of treated samples were
incubated with L. innocua Lin11 cells, the cells were
pelleted by centrifugation, and the number of cells surviving the
incubation was determined by plating. In addition, the supernatant fractions from the centrifuged incubation mixtures were analyzed by
spot test assays to determine their AU values. As shown in Table
3, the activities of all three proteins
were substantially lowered by treatment with the reducing agents. The
number of cells that survived incubation increased for samples exposed
to reducing agents, and the numbers of AU per milliliter of treated
supernatants were lower than for untreated samples. Control experiments
performed with LB broth containing the reducing agents showed that
these compounds have no effects per se on the viability of the
indicator bacterium, at least at the concentrations used in the assays. The results indicate that the pediocin AcH precursor, like the mature
bacteriocin (14), is sensitive to reducing agents.
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Susceptibility of chimeric proteins to protease inactivation.
Experiments were performed to compare the susceptibilities of the
chimeric proteins to proteolytic attack. To assess protease sensitivity, culture broths of E609L/pPR6821 and E609L/pPR6823 induced
for 3 h with IPTG were incubated with chymotrypsin, and the
numbers of AU remaining in the samples after incubation were determined
(Table 4). Although the AU values of both
culture broths declined during the incubations, the number of AU per
milliliter for E609L/pPR6823 declined more rapidly and was nearly
eliminated after 15 min of incubation. In contrast, significant
activity remained in the E609L/pPR6821 broth even after 30 min of
incubation. It was confirmed by SDS-PAGE and Western immunoblotting
that both chimeric proteins were degraded by chymotrypsin and that the
682-prePapA protein was degraded more rapidly than 682-PapA (data not
shown). It also has been noted that the 682-prePapA protein is more
susceptible to degradation in unboiled culture broths than is the
682-PapA protein (data not shown). Taken together, the data suggest
that prepediocin AcH is more susceptible to both in vitro and in vivo proteolysis than is mature pediocin AcH.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Limited available information indicates that many of the prebacteriocins produced by gram-positive bacteria are virtually inactive. Such observations suggest that in many cases producer cells are protected from the action of precursors mainly because these molecules display little or no activity. It seems possible that leader peptides could neutralize the activities of prebacteriocins in several different ways (24, 26, 29). For example, leader peptides, which generally are polar, may increase the solubility of prebacteriocins in water (26) and therefore may cause them to partition more into the aqueous phase than into the membrane. In addition, leader peptides could interact directly with mature domains to reduce their affinity for membranes (24). In this regard, both leader peptides and mature regions are moderately amphipathic, and their nonpolar regions may interact in water and shield nonpolar membrane interaction sequences from contact with lipids.
In marked contrast, the pediocin AcH precursor appears to be ~80% as active as the mature form. This suggests that the leader peptide has little effect on the function of the mature domain. It has been demonstrated here and previously (18, 19) that fusion of the large MBP molecule to the N terminus of the mature domain also does not block activity. Apparently, the tertiary structure and membrane-binding properties of the mature domain are relatively insensitive to what is attached to the N terminus. Until the properties of more prebacteriocins have been investigated, it is impossible to conclude whether the activity displayed by prepediocin AcH is exceptional or not.
Based on these findings, it becomes necessary to consider alternative ways for how producer cells overcome the potentially lethal action of prepediocin AcH in the time between its translation and its secretion. One possible way in which binding of the precursor to the cytoplasmic membrane may be limited is that the binding site on the putative pediocin AcH receptor (4) may not be accessible to prepediocin AcH from inside the cell. However, pediocin PA-1 has been shown to display membrane permeabilization activity against phospholipid vesicles in the absence of a receptor (3), and therefore some damage may be caused even though a receptor is inaccessible. In addition, the membrane insertion activity of the precursor may be limited due to the reverse orientation of the membrane electrochemical potential that it should encounter from inside the cell. In this regard, the membrane permeabilization activity of pediocin PA-1 is stimulated by a membrane potential difference that is trans-side negative (3, 4).
It also is possible that prepediocin AcH may be neutralized by the cytoplasmic PapB immunity protein (2, 31), for which the mechanism of action currently is unknown. In most cases, immunity proteins protect cells from the bacteriocins they have secreted by preventing their interaction with the cytoplasmic membrane (1, 6). For example, the specific immunity proteins for nisin, subtilin, and epidermin are secreted and anchored to the outer surface of the cytoplasmic membrane and are believed to bind these lantibiotics before they can interact with membrane lipids. Similarly, the lactococcin A immunity protein is membrane bound, but in this case it may bind to and shield the receptor for this bacteriocin (1). In contrast, the carnobacteriocin B2 immunity protein is located predominantly in the cytoplasm and may instead plug pores after they have been formed in the membrane (23). If PapB can inhibit the activity of prepediocin AcH, then it may need to do so by binding to it in solution. It seems unlikely that PapB could plug pores formed by prepediocin AcH because the molecules within these pore complexes might have the reverse orientation within the bilayer compared to molecules that have inserted from outside.
Another possible way by which producer cells could control the activity of intracellular prepediocin AcH molecules is by using the cytoplasmic redox potential to maintain cysteine thiol groups in a reduced state (7). It has been demonstrated that the four cysteines within the native pediocin AcH molecule must be oxidized to two disulfide bonds for the bacteriocin to be active (4, 14). The substitution of serines and other residues for cysteines completely inactivates native pediocin AcH and MBP-pediocin AcH (19). We have shown here that cysteines within MBP-prepediocin AcH also must be oxidized to achieve maximal activity. These findings suggest that the activity of prepediocin AcH, and perhaps the activities of the precursors of other "cystibiotics" that require disulfide bonds (8, 14), could be controlled within producer cells by the reducing state of the cytoplasm. However, there are indications that natural, but not synthetic, leucocin A and also carnobacteriocin B2 exhibit activity when their two cysteines are reduced (8, 11, 22).
The finding that prepediocin AcH is active raises the question of why its leader peptide is removed during secretion. One possible reason is that the leader peptide makes the molecule more susceptible to proteases in the environment. In this regard, MBP-prepediocin AcH was found to be more susceptible than MBP-pediocin AcH to chymotrypsin and proteases in the culture broths of the producer strains. This suggests that prepediocin AcH would be highly susceptible to proteases produced by target cells and would be a less-effective antimicrobial agent than pediocin AcH. Furthermore, it may be important for producer cells to export the precursor before it can be cleaved by intracellular proteases. Rapid export also could limit damage caused to producer cells by cytoplasmic prepediocin AcH molecules.
In conclusion, the results show that the pediocin AcH precursor has significant biological activity. In cases where a precursor is active, producer cells must employ alternative schemes to avoid cytoplasmic toxicity. It is suggested that these mechanisms may include rapid and efficient translocation of prebacteriocins out of cells and, in the case of most cystibiotics, maintenance of cysteines in the reduced state. In addition, precursors that are not immediately secreted may be destroyed by intracellular proteases. Poor translocation efficiency and proteolysis could be contributing factors in cases where hybrid bacteriocins are produced in relatively small amounts when expressed using swapped leader peptides (5, 13, 29).
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ACKNOWLEDGMENTS |
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We thank the investigators for providing bacterial strains.
We acknowledge the financial support of the National Science Foundation, the State of Wyoming, and the Sustainable Directorate of the U.S. Army Natick Research, Development, and Engineering Center (contract number DAAK 60-97-R-9601).
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FOOTNOTES |
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* Corresponding author. Mailing address for Kurt W. Miller: Department of Molecular Biology, P.O. Box 3944, University of Wyoming, Laramie, WY 82071-3944. Phone: (307) 766-2037. Fax: (307) 766-5098. E-mail: kwmiller{at}uwyo.edu. Mailing address for Bibek Ray: Department of Animal Science, P.O. Box 3684, University of Wyoming, Laramie, WY 82071-3684. Phone: (307) 766-3140. Fax: (307) 766-2350. E-mail: labcin{at}uwyo.edu.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, June 1999, p. 2281-2286, Vol. 65, No. 6
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