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Journal of Bacteriology, November 2001, p. 6274-6281, Vol. 183, No. 21
Department of Molecular and Cell Biology,
Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku, Sendai
981-8555,1 and Department of
Microbiology, Miyazaki Medical College, 5200 Kiyotake, Miyazaki
899-1692,2 Japan
Received 16 April 2001/Accepted 31 May 2001
Carotovoricin Er is a phage-tail-like bacteriocin produced by
Erwinia carotovora subsp. carotovora
strain Er, a causative agent for soft rot disease in plants. Here we
studied binding and killing spectra of carotovoricin Er preparations
for various strains of the bacterium (strains 645Ar, EC-2, N786, and
P7) and found that the preparations contain two types of carotovoricin Er with different host specificities; carotovoricin Era possessing a
tail fiber protein of 68 kDa killed strains 645Ar and EC-2, while
carotovoricin Erb with a tail fiber protein of 76 kDa killed strains
N786 and P7. The tail fiber proteins of 68 and 76 kDa had identical
N-terminal amino acid sequences for at least 11 residues. A search of
the carotovoricin Er region in the chromosome of strain Er indicated
the occurrence of a DNA inversion system for the tail fiber protein
consisting of (i) two 26-bp inverted repeats inside and downstream of
the tail fiber gene that flank a 790-bp fragment and (ii) a putative
DNA invertase gene with a 90-bp recombinational enhancer sequence. In
fact, when a 1,400-bp region containing the 790-bp fragment was
amplified by a PCR using the chromosomal DNA of strain Er as the
template, both the forward and the reverse nucleotide sequences of the
790-bp fragment were detected. DNA inversion of the 790-bp fragment
also occurred in Escherichia coli DH5 Hamon and Peron first
described a substance(s) produced by Erwinia carotovora
subsp. carotovora, the causative agent of soft rot disease
in many plant species, that is bactericidal towards the same and
related species of bacteria (7). Because of the proteinaceous nature of this bacteriocide and its narrow bactericidal spectrum, they assumed it to be a bacteriocin and designated it carotovoricin (7). Previous reports from this laboratory
showed that E. carotovora subsp. carotovora Er
produced carotovoricin Er as well as pectin lyase when it was exposed
to nalidixic acid, mitomycin C, or bleomycin (27) and that
a phage-tail-like particle was observed in the partially purified
fraction of carotovoricin Er (16, 13). Recently, we
developed a simple and efficient procedure for purification of intact
carotovoricin Er and its major structural parts by use of sucrose
density gradient centrifugation in the presence of 10 to 20% (vol/vol)
ethanol and analyzed the highly purified carotovoricin Er for its
morphology and components (21). Electron microscopy showed
that carotovoricin Er consists of an antenna-like structure, a
sheath-and-core part, a base plate, and several tail fibers
(21). It was revealed that (i) carotovoricin Er has a
length of 184 nm (from the distal end of the sheath-and-core part to
the bottom of the base plate) and a diameter of 22 nm (at the
sheath-and-core part); (ii) the antenna-like structure at the distal
end of the sheath-and-core part is a rod structure with a length of 54 nm, which has never been reported for pyocin R (12) or
xenorhabdicin (26); (iii) the sheath is a contractile cylindrical structure surrounding an inner core, which is visible in
contracted carotovoricin Er; and (iv) the tail fibers have a length of
63 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified major parts of carotovoricin Er showed that
the sheath, core, and tail fiber consist of single major proteins of
50, 19, and 68 kDa, respectively (21). In addition to
these proteins, protein bands corresponding to 78, 76, 46, 44, 39, and
35 kDa were detected in the purified carotovoricin Er particle and were
analyzed for their N-terminal amino acid sequences (21).
Furthermore, we recently sequenced a 20-kbp chromosomal fragment of
E. carotovora subsp. carotovora Er which contained the open reading frames coding for the major sheath protein
(50 kDa), the major core protein (19 kDa), and the tail fiber protein
(68 kDa) (DDBJ nucleotide sequence accession no. AB017338). The
20-kbp fragment also contained the structural genes for the proteins of
46, 44, and 35 kDa, which might be the proteins for the antenna-like
and the base plate structures. Thus, carotovoricin Er is a
phage-tail-like bacteriocin consisting of at least six proteins.
A previous paper from our laboratory has shown that carotovoricin Er
binds to and kills several strains of E. carotovora subsp. carotovora, such as strains EC-2, P7, and 645Ar
(14). However, our recent experiments showed that (i) a
significant portion of the purified carotovoricin Er preparations
failed to bind to EC-2 and P7 but that almost all carotovoricin Er
particles bound to strain 645Ar under similar conditions and (ii) the
unbound fraction of carotovoricin Er recovered after the incubation
with EC-2 or P7 retained intact morphology and killing activity towards
P7 or EC-2, respectively (unpublished data). These results prompted us
to study whether or not the carotovoricin Er preparation contains multiple types of carotovoricin Er with different host specificities. In this paper, we show that E. carotovora subsp.
carotovora Er produces two types of carotovoricin Er with
different host specificities, which we named carotovoricin Era and Erb,
and that the different host specificities of carotovoricins Era and Erb
are due to alteration in the C-terminal part of the tail fiber protein
by a DNA inversion.
Bacterial strains, plasmids, and culture media.
Bacterial
strains and plasmids used in this study are listed in Table
1. Bacteria were cultured in LB medium
(10 g of polypeptone, 5 g of yeast extract, and 5 g of
NaCl in 1 liter, pH 7.2) or in a modified M9 medium (14.7 g of
Na2HPO412H2O,
3 g of KH2PO4, 5 g of NaCl, 1 g of NH4Cl, 0.0147 g of
CaCl22H2O, 0.203 g of
MgSO4, 0.0002 g of
FeCl34H2O, and 2 g of
glucose in 1 liter, pH 7.2) supplemented with casein acid hydrolysate
(2 g/liter).
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6274-6281.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
DNA Inversion in the Tail Fiber Gene Alters the Host Range
Specificity of Carotovoricin Er, a Phage-Tail-Like Bacteriocin of
Phytopathogenic Erwinia carotovora subsp.
carotovora Er
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
when two
compatible plasmids carrying either the 790-bp fragment or the
invertase gene were cotransformed into the bacterium. Furthermore,
hybrid carotovoricin CGE possessing the tail fiber protein of 68 or 76 kDa exhibited a host range specificity corresponding to that of
carotovoricin Era or Erb, respectively. Thus, a DNA inversion altered
the C-terminal part of the tail fiber protein of carotovoricin Er,
altering the host range specificity of the bacteriocin.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
Assay for carotovoricin activity. Test samples were serially diluted with 0.05 M sodium phosphate buffer, pH 7.2. A small portion (5 µl) of the dilutions were withdrawn and spotted on an LB soft agar plate (0.35% agar with a thickness of 2 mm) containing sensitive bacteria (2 × 107 CFU/ml), and the plate was incubated at 30°C for 8 to 12 h. The reciprocal of the highest dilution that formed a clear zone in the LB soft agar was defined as the relative killing titer (units) of carotovoricin.
Production and purification of carotovoricin Er and carotovoricin CGE. E. carotovora subsp. carotovora strain Er or CGE234 M403 was grown in LB medium at 30°C overnight. A portion (20 ml) of the culture was withdrawn, transferred into 600 ml of the modified M9 medium, and cultivated for several hours at 30°C. Mitomycin C (Wako Pure Chemical Industry, Osaka, Japan) was added at a final concentration of 0.4 µg/ml to an exponentially growing culture (with an optical density at 660 nm of 0.6), and the culture was incubated for a further 5 h at 30°C with shaking to induce carotovoricin production accompanied by cell lysis. Carotovoricin particles were purified from the cell lysates by a purification procedure including the use of 15 to 30% (wt/vol) sucrose linear gradient centrifugation in the presence of 20% (wt/vol) ethanol and 0.1 M NaCl, as described previously (21).
Fixation of bacterial cells with formaldehyde. Exponentially growing cells of E. carotovora subsp. carotovora in LB medium were collected by centrifugation at 2,500 × g at room temperature for 5 min. The cells were suspended in LB medium and were treated with 2% formaldehyde at room temperature for 60 min. After fixation, the cells were washed twice with LB medium, and were suspended in LB medium at a concentration of 1010 cells/ml.
Isolation of two types of carotovoricin Er. Carotovoricin Er particles were purified from the cell lysate of 2 liters of E. carotovora subsp. carotovora Er as described previously (21), and the carotovoricin Er preparation was divided into two parts. One-half of the carotovoricin Er preparation was mixed with 25 ml of formaldehyde-treated EC-2 cells, and the rest was mixed with 25 ml of formaldehyde-fixed N786 cells. The mixtures were rotated at room temperature for 30 min, followed by centrifugation at 2,500 × g at room temperature for 10 min. The supernatants obtained were centrifuged at 120,000 × g at 4°C for 2 h, and carotovoricin Er particles in the pellets were purified as described above (21).
Binding of carotovoricin Er to various strains of E. carotovora subsp. carotovora. Carotovoricin Er was incubated with formaldehyde-fixed cells (500 µl) of E. carotovora subsp. carotovora at 0°C for 1 h. The mixtures were centrifuged at 12,000 × g at 0°C for 5 min to collect bacterial cells. The supernatants obtained were centrifuged at 300,000 × g at 4°C for 2 h to collect unbound carotovoricin particles.
SDS-PAGE and Western blotting. SDS-PAGE was done as described by Laemmli (18) using 12.5% acrylamide gels. Proteins in the gel were electroblotted onto a polyvinylidene difluoride sheet, and the blotted protein bands were immunostained with specific antiserum raised against whole particles or the purified sheath of carotovoricin Er. The immunostained bands were visualized using alkaline phosphatase-conjugated anti-rabbit immunoglobulin G (Promega, Madison, Wis.), nitroblue tetrazolium (Wako Pure Chemicals), and 5-bromo-4-chloro-indolylphosphate (Wako Pure Chemicals). Antisera were raised in female New Zealand White rabbits, which were injected subcutaneously with the purified whole particle or sheath of carotovoricin Er (1 mg) emulsified in Freund's complete adjuvant (Difco Laboratories, Detroit, Mich.), followed by the injections of the purified whole particle or sheath of carotovoricin Er (0.5 mg) emulsified in Freund's incomplete adjuvant (Difco Laboratories) every 10 days for five times.
N-terminal amino acid sequences of proteins. N-terminal amino acid sequences of proteins were analyzed for the blotted protein bands on a polyvinylidene difluoride sheet (19) by using a model 491 protein sequencer (Perkin-Elmer Applied Biosystems, Foster City, Calif.).
DNA manipulations. All standard DNA manipulations were done as described by Sambrook et al. (23). PCR was done using Ex Taq polymerase (TaKaRa, Kyoto, Japan) for 30 cycles with the following temperature profile: 94°C for 30 s, 57°C for 30 s, and 72°C for 3 min. Six primers for PCR were synthesized according to the nucleotide sequence of the carotovoricin Er region of E. carotovora subsp. carotovora Er chromosome (DDBJ nucleotide sequence database accession no. AB017338).
Amplification of the DNA fragments containing invertible fragment E by PCR and analysis of the PCR products. The fragment Er16098-17502, which contains invertible fragment E and a part of the putative DNA invertase gene (Fig. 2), was amplified by PCR using chromosomal DNA of strain Er as the template and the following primers: 5'-AGCCGTGGGCGCGTATTTATAGCGATCAAG-3' (the forward primer), corresponding to the DNA fragment from nucleotides 16098 to 16127, and 5'-AGCAGCATCGAGTCCGGCCCGTGTCCGTTC-3' (the reverse primer), corresponding to the DNA fragment from nucleotides 17502 to 17473. Because a single NcoI site is contained in fragment E, cleavage of the amplified products at the NcoI site was used for diagnosis of the DNA inversion of fragment E. The amplified DNA fragments were digested with NcoI restriction endonuclease and were then separated on a Tris-acetate-EDTA agarose gel (1%). DNA fragments were extracted from the gel using Quantum Prep Freeze 'N Squeeze DNA gel extraction spin columns (Bio-Rad Laboratories, Hercules, Calif.). DNA sequencing was done using an ABI Prism 373 DNA sequencer (Perkin-Elmer Applied Biosystems) and an ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer Applied Biosystems).
DNA inversion of segment E in Escherichia
coli.
The invertible segment E and the invertase gene
ein were separately cloned in compatible plasmids and
transformed into E. coli DH5 to study DNA inversion
of segment E in E. coli cells. The DNA fragment
containing segment E (Er14307-17502) was amplified by PCR using the
chromosomal DNA of strain Er as the template. The following forward and
reverse primers were used:
5'-GATCTAGAATATTGGCCGCCTGAGCTACG-3' (the forward
primer, which corresponds to the DNA fragment from nucleotides 14307 to
14336 except in the italicized region, where 1 nucleotide was changed
to create an XbaI site) and
5'-AGAAGCTTCGAGTCCGGCCCGTGTCCGTTC-3' (the reverse
primer, which corresponds to the DNA fragment from nucleotides 17502 to
17473, where 2 nucleotides were changed to create a
HindIII site). The PCR product was digested with
XbaI and HindIII and inserted into plasmid
pACYC184 (3). The recombinant plasmid was transformed into
E. coli DH5 (8). The presence of Ea and
Eb was assayed for single colonies of the resultant transformants by a PCR amplification of the Er16098-17502 fragment, followed by NcoI digestion and agarose gel electrophoresis
as described above. The DNA fragment containing the ein gene
(Er16993-17739) was amplified by PCR using the chromosomal DNA of
strain Er as the template. The following forward and reverse primers
were used: 5'-CGGAGTTGCTTGCGGCCATGGTAA-3' (the
forward primer, which corresponds to the DNA fragment from nucleotides
16993 to 17016; the italicized region indicates the NcoI
site) and 5'-ACCAAGCTTCTGGTCACTCTGGCGCAGAGG-3' (the reverse primer, which corresponds to the DNA fragment
from nucleotides 17739 to 17710, where 2 nucleotides were changed to create a HindIII site). The PCR products containing the
ein gene were digested with NcoI and
HindIII and cloned into the pTrc99A plasmid
to construct plasmid pTrc99Aein.
pTrc99Aein was transformed into E. coli DH5 harboring either pACYC184Ea or pACYC184Eb. The presence of Ea and Eb in the resultant transformants was assayed as
described as above.
Expression of the tail fiber genes of carotovoricins Era and Erb in E. carotovora subsp. carotovora strain CGE234 M403. The DNA fragment containing the tail fiber gene of carotovoricin Era or Erb was amplified by PCR using the chromosomal DNA of strain Er as the template. To amplify the DNA fragment containing the tail fiber gene of carotovoricin Era (fibA), the following primers were used: 5'-GATCTAGAATATTGGCCGCCTGAGCTACG-3' (the forward primer, which is the same oligonucleotide as that described above for amplification of segment E) and 5'-ATAAGCTTACCATGGCCGCAAGCAACTCCG-3' (the reverse primer, which corresponds to the DNA fragment from nucleotides 17022 to 16993, where 2 nucleotides were changed to create a HindIII site). To amplify the DNA fragment containing the tail fiber gene of carotovoricin Erb (fibB), we used a reverse primer (5'-GGTAAGCTTTTGGGATTTAATCACTGTGCC-3', which corresponds to the DNA fragment from nucleotides 16586 to 16557 except in the italicized region, where 2 nucleotides were changed to create a HindIII site) in a combination with the same forward primer used for amplification of the tail fiber gene of carotovoricin Era (fibA). The PCR products were digested with XbaI and HindIII and were inserted into pTrc99A plasmids to construct plasmids pTrc99AfibA and pTrc99AfibB, respectively. pTrc99AfibA and pTrc99AfibB were transformed into strain CGE234 M403. Production of carotovoricin CGE by the transformants was induced with mitomycin C, and normal and hybrid carotovoricin CGE particles were purified as described previously (21).
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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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E. carotovora subsp. carotovora
Er produces two types of carotovoricin Er.
Carotovoricin Er was
purified from the lysate of mitomycin C-treated E. carotovora subsp. carotovora Er as described previously (21) and was assayed for its bactericidal activity towards
various strains of E. carotovora subsp.
carotovora (strains Er, 645Ar, EC-2, N786, and P7). The
carotovoricin Er preparation exhibited killing activity towards all of
the tested strains except towards the producing strain Er, although the
bactericidal titer of the carotovoricin Er preparation varied with
different strains (Table 2). To study
whether or not there exist multiple types of carotovoricin Er, the
killing spectrum of the carotovoricin Er preparation was assayed after
incubation with formaldehyde-treated cells of strain Er, 645Ar, EC-2,
N786, or P7 at 30°C for 30 min. When the carotovoricin Er preparation
was preincubated with the formaldehyde-fixed cells of strain EC-2, the
preparation lost the ability to kill 645Ar and EC-2 but it retained the
ability to kill N786 and P7 (Table 2). In contrast, the carotovoricin
Er preparation which was preincubated with fixed cells of N786 or P7
killed 645Ar and EC-2 but failed to kill N786 and P7 (Table 2). The
residual bactericidal activity of the carotovoricin Er preparation
which was recovered after incubation with fixed cells of EC-2 or N786
was abolished by further incubation with N786 or EC-2, respectively
(data not shown). These results suggested that the carotovoricin Er
preparations contained two types of carotovoricin Er with different
host range specificities.
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, not killed.
A DNA inversion in the tail fiber gene is responsible for the
production of two types of carotovoricin Er.
It has been
demonstrated that bacteriophages Mu and P1 alter the C-terminal regions
of tail fiber proteins by DNA inversion, leading to alteration of their
host range (5, 6, 10, 17, 24, 28). Since two properly
oriented recombination sites (inverted repeats), a DNA invertase, and a
recombinational enhancer are essential for a DNA inversion system
(15), we searched the corresponding sequences on the
20-kbp fragment from the E. carotovora subsp. carotovora Er chromosome (DDBJ nucleotide sequence database
accession no. AB017338), which includes the genes encoding the major proteins of the sheath, core, and tail fiber of carotovoricin Er. As
illustrated in Fig. 2, (i) the 20-kbp
fragment contains a pair of 26-bp inverted-repeat sequences which are
located inside and downstream of the gene encoding the tail fiber
protein of 68 kDa; (ii) the inverted repeats flank a 790-bp segment, or
the putative invertible segment, which covers the region corresponding to the C-terminal part of the tail fiber protein; (iii) a segment corresponding to a DNA invertase gene is located immediately
downstream of the 790-bp segment; and (iv) the putative DNA invertase
gene contains a recombinational-enhancer-like segment in its 5'
region (Fig. 2).
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with the recombinant
plasmid(s), the Ea and the Eb fragments in the transformants were
detected by an amplification of the Er16098-17502 fragments by PCR,
followed by NcoI digestion and agarose gel electrophoresis as described above. Single colonies (n = 20) of the
transformants harboring only recombinant pACYC184 gave either a
combination of 900- and 500-bp fragments or a combination of 1,200- and
200-bp fragments (data not shown). However, when E. coli DH5 carrying pACYC184Ea or pACYC184Eb (i.e.,
pACYC184 containing Ea or Eb) was further transformed with
pTrc99Aein (i.e., pTrc99A containing ein), the resultant transformants gave the bands
corresponding to 1,200, 900, 500, and 200 bp (data not shown). These
results indicated that the function of ein was necessary for
DNA inversion of segment E and that the DNA inversion took place in the
forward and backward directions.
Tail fiber proteins determine the host range specificity of
carotovoricin.
To study if the tail fiber proteins determine the
host specificity of carotovoricin Er, we expressed the tail fiber gene
fibA or fibB in E. carotovora
subsp. carotovora CGE234 M403 to produce hybrid
carotovoricin CGE possessing FibA or FibB and tested the host
specificity for the hybrid carotovoricins. E. carotovora subsp. carotovora CGE234 M403 is
nonpathogenic to plants but retains bactericidal activity towards the
same bacterial species, probably because of its ability to produce two
types of bacteriocin, high-molecular-weight and low-molecular-weight
bacteriocins (4, 25). In this study, we purified the
high-molecular-weight bacteriocin from the mitomycin C-treated cells of
strain CGE234 M403 according to the procedure for purification of
carotovoricin Er (21). Electron microscopy of the purified
high-molecular-weight bacteriocin showed the same phage-tail-like
morphology as that of carotovoricin Er (data not shown). SDS-PAGE for
the purified bacteriocin indicated major protein bands corresponding to
84, 70, 64, 55, 50, and 19 kDa (Fig. 4A).
The 20 N-terminal amino acid residues of the 50- and the 19-kDa
proteins were 100% identical to those of the sheath and the core
proteins of carotovoricin Er, respectively. Furthermore, the 15 N-terminal amino acid residues of the 64-kDa protein (i.e., Ala-Asn-Leu-Ser-Glu-Asn-Pro-Gln-Trp-Val-Asp-Ser-Ile-Tyr-Gln-) were identical
with those of the tail fiber protein of carotovoricin Er, except for
Ser in the 12th position (i.e., the tail fiber proteins of
carotovoricin Er, FibA and FibB, have Gly for Ser in the same
position). However, the 64-kDa protein was not immunostained with the
antiserum raised against carotovoricin Er (Fig. 4B), implying that the
C-terminal region of the 64-kDa protein is different from those of
carotovoricin Era and Erb. Based on these results, the
high-molecular-weight bacteriocin from strain CGE234 M403 was
designated carotovoricin CGE and we determined that carotovoricin CGE
has a structure similar to that of carotovoricin Er except for the tail
fiber protein, implying that the tail fiber protein of carotovoricin
CGE might be interchangeable with those of carotovoricin Er. Since
Western blotting of purified carotovoricin CGE using antiserum against
carotovoricin Er revealed a prominent band corresponding to 55 kDa
(Fig. 4B), we determined the amino acid sequence for the 11 N-terminal
residues of the 55-kDa protein:
Ala-Asn-Leu-Ser-Glu-Gln-Glu-Ser-Trp-Ile-Asp-. The 5 N-terminal amino
acid residues of the 55-kDa protein are identical to those of the tail
fiber protein of carotovoricin CGE, but the rest of the amino acid
residues are different from those of the tail fiber protein.
Furthermore, an open reading frame coding for the 64-kDa protein is
present in the 20-kbp carotovoricin CGE region of strain CGE234 M403,
while no open reading frame for the 55-kDa protein is present in the
same region (M. Hirota et al., unpublished results). Therefore,
the 64-kDa protein may be the major tail fiber protein of carotovoricin
CGE, but it remains unclear if the 55-kDa protein is a structural
component of carotovoricin CGE.
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, P1, Mu, and T4, the
N-terminal part of tail fiber proteins is bound to the base plate or to
the tail shaft and the C-terminal part is the distal part that binds to
a specific receptor (2, 20). In Mu and P1 phages, DNA inversion of segments G and C results in the formation of tail fiber
proteins with a constant N-terminal part and two alternative C-terminal
parts, alternating the host range specificities of the phages. With
these results taken together, it is quite conceivable that the host
range specificity of carotovoricin Er is determined by the C-terminal
part of the tail fiber protein. The production of carotovoricins aid
E. carotovora subsp. carotovora in surviving competition among the same and closely related species of bacteria in
nature, and alteration in the C-terminal part of tail fiber proteins by
DNA inversion is an effective way to furnish carotovoricin with a wider
bactericidal spectrum.
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ACKNOWLEDGMENTS |
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We thank Elisabeth Hofstad for her help in the preparation of the manuscript.
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan. H. A. Nguyen was the recipient of a Japanese government scholarship from the Ministry of Education, Science, Sports, and Culture of Japan.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan. Phone: 81(22)717-8779. Fax: 81(22)717-8780. E-mail: ykamio{at}biochem.tohoku.ac.jp.
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Journal of Bacteriology, November 2001, p. 6274-6281, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6274-6281.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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