Note: Descriptions are shown in the official language in which they were submitted.
CA 02449601 2003-12-05
1
TITLE OF THE INVENTION
[0001] Geldanamycin-producing strains, uses thereof and methods of
producing same
FIELD OF THE INVENTION
[0002] The present invention relates to geldanamycin-producing strains,
uses thereof and methods of producing same. More specifically, the present
invention is concerned with these strains and uses thereof as biocontrol
agents
against common scab.
BACKGROUND OF THE INVENTION
[0003] Common scab is a disease widely distributed in potato-growing
areas. Superficial or deep corky lesions on potato tubers characterise the
disease. Streptomyces scabies (Lambert and Loria, 1989) is the main causal
agent of the disease (Goyer et al., 1996). Methods used to control common
scab include chemical treatments of seed potato tubers (Davis et al., 1976),
irrigation (Adams and Lapwood, 1978), soil amendments (Weinhold and Brown,
1968), cultivar choice (Bouchek-Mechiche et at., 2000) and rotation strategies
(Li et at., 1999).
[0004] Biological control of potato scab by nonpathogenic
streptomycetes was also reported (Doumbou et at., 2001b; Doumbou et at.,
1998; Liu et at., 1995a; Ryan and Kinkel, 1997). For example, introduction
into
an infested soil of some Streptomyces diastatochromogenes and S.
albogriseolus strains isolated from suppressive soils decreased common scab
symptoms on potato tubers (Eckwall and Schottel, 1997; Liu et at., 1995a;
Lorang et al., 1995). Common scab severity was also decreased by an
CA 02449601 2003-12-05
2
amendment of an antibiotic biofertilizer produced from swine feces containing
S. albidoflavus strain CH-33 (Hayashida et at., 1989). These strains are not
known to produce geldanamycin.
[0005]
Geldanamycin is known to inhibit the growth of several plant
pathogenic fungi (Toussaint et at., 1997) and geldanamycin-producing
streptomycetes were shown to protect crops against several fungal diseases
(Rothrock and Gottlieb, 1984; Valois et at., 1996). This antibiotic is also
active
against some Gram-positive bacteria (Toussaint et at., 1997) such as S.
scabies (Agbessi, 2002) but the efficiency of this bioagent to control common
scab has not yet been determined. It cannot be predicted whether a strain
known to be antagonistic against a microbiological pathogen will be able to
compete in the rhizosphere against this pathogen and against other soil
microbiological pathogens. It cannot therefore be predicted whether this
strain
would be effective in controlling a plant disease caused by this
microbiological
pathogen.
[0006] Certain
references have shown that specific chitin waste-based
composts suppress common scab (COte et al., 2001; Vruggink (1970)). It was
never demonstrated however whether the little amount of chitosan contained in
these wastes participated in controlling the disease. Some success of
biocontrol have been reported by the combined use of a biocontrol agent and of
chitosan without however demonstrating the role played by chitosan itself
(Cuero et at., 1991; Singh et al., 1999). Chitosan is known to be toxic
against
most microorganisms. There thus remains a need to demonstrate whether
chitosan may help control common scab and whether it may be used in
combination with microorganisms antagonistic to common scab.
[0007] There
thus remains a need for new effective biocontrols of
-
CA 02449601 2013-06-05
3
common scab.
SUMMARY OF THE INVENTION
[0008] The present invention demonstrates for the first time the
biocontrol efficiency of geldanamycin-producing strains against a bacterial
plant
disease and more particularly against common scab.
[0009] More specifically, in accordance with the present invention, there
is provided a use of an inoculum of a geldanamycin-producing strain able to
survive in a plant rhizosphere as a biocontrol of common scab affecting the
plant. In a specific embodiment, the strain encodes a protein having the
sequence of SEQ ID NO: 6 or a homologous sequence having geldanamycin
activity. In another embodiment, the strain comprises the nucleotide sequence
of SEQ ID NO: 7 or a homologous sequence contributing to geldanamycin
activity. In another embodiment, the strain comprises a nucleotide sequence
able to hybridize under high stringency conditions to : the nucleotide
sequence
of SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 2, the nucleotide
sequence of SEQ ID NO: 3, the complementary sequence of the nucleotide
sequence of SEQ ID NO: 5, or the complementary sequence of the nucleotide
sequence of SEQ ID NO: 7. According to specific embodiments, the strain is a
Streptomyces strain. In more specific embodiments, the strain is selected from
the group consisting of Streptomyces violaceusniger, Streptomyces
hygroscopicus and Streptomyces melanosporafasciens strains. In more
specific embodiments, the strain is deposited at the American Type Culture
Collection (ATCC) under Accession number BAA-668. According to specific
embodiment, the biocontrol comprises a reduction of the severity of common
scab or a reduction of the incidence of common scab.
CA 02449601 2013-06-05
4
[0010] According to another aspect of the invention, there is provided a
biologically pure culture of a Streptomyces strain deposited at the American
Type Culture Collection (ATCC) Accession number BAA-668, or a variant
thereof. This strain is a Streptomyces melanosporofasciens and was deposited
at the American Type Culture Collection (ATCC) at P.O. Box 1549 Manassas,
VA 20108 USA under Accession number BAA-668 on December 9, 2002.
[0011] According to another aspect of the invention, there is provided a
composition comprising an inoculum of a Streptomyces strain deposited at the
American Type Culture Collection (ATCC) Accession number BAA-668 and a
carrier. In a specific embodiment, the carrier comprises chitosan.
[0012] According to another aspect of the invention, there is provided a
method of biocontrolling common scab comprising applying on a plant an
effective amount of an inoculum of a geldanamycin-producing strain able to
survive in the plant rhizosphere. In a specific embodiment, the strain encodes
a
protein having the sequence of SEQ ID NO: 6 or an homologous sequence
contributing to geldanamycin activity. In another embodiment, the strain
comprises the nucleotide sequence of SEQ ID NO: 7 or a homologous
sequence contributing to geldanamycin activity. In another embodiment, the
strain comprises a nucleotide sequence able to hybridize under high stringency
conditions to : the nucleotide sequence of SEQ ID NO: 1, the nucleotide
sequence of SEQ ID NO: 2, the nucleotide sequence of SEQ ID NO: 3, the
complementary sequence of the nucleotide sequence of SEQ ID NO: 5, or the
complementary sequence of the nucleotide sequence of SEQ ID NO: 7.
According to specific embodiments, the strain is a Streptomyces strain. In
more
specific embodiments, the strain is selected from the group consisting of
Streptomyces violaceusniger, Streptomyces hygroscopicus and Streptomyces
melanosporafasciens strains. In more specific embodiments, the strain is
CA 02449601 2013-06-05
4a
deposited at the American Type Culture Collection (ATCC) under Accession
number BAA-668. According to specific embodiment, the biocontrol comprises
CA 02449601 2013-06-05
a reduction of the severity of common scab or a reduction of the incidence of
common scab.
[0013] According to another aspect of the present invention, there is
provided a method for modifying the biocontrol efficiency of a bacterial
strain
comprising intraspecific protoplasm fusion of the bacterial strain with
another
strain having a desirable biocontrol property against common scab.
[0014] According to another aspect of the present invention, there is
provided a method for making a biocontrol agent against common scab, which
comprises the steps of: obtaining a microbial strain susceptible of producing
geldanamycin; contacting the nucleic acids or the proteins of said strain with
a
ligand specific to a geldanamycin-producing protein or to a nucleic acid
encoding a geldanamycin-producing protein; detecting the formation of a
complex as an indication of the synthesis of geldanamycin or of a nucleic acid
encoding a geldanamycin-producing protein in the strain, whereby said strain
or
a geldanamycin-producing part of said strain may be used as a biocontrol
agent against common scab. In a specific embodiment, the ligand is a nucleic
acid having at least 12 nucleotides in length hybridizing with a) the
complementary sequence of the nucleotide sequence of SEQ ID NO: 1; b) the
complementary sequence of the nucleotide sequence of SEQ ID NO: 2; c) the
complementary sequence of the nucleotide sequence of SEQ ID NO: 3; d) the
nucleotide sequence of SEQ ID NO: 5; and the nucleotide sequence of SEQ ID
NO: 7. The ligand may also bind to the geldanamycin-producing protein. Such
ligand may include an antibody specific to the geldanamycin-producing protein.
[0015] Robinson et al. (1981) showed that it was possible to increase the
level of antibiotic production by protoplast fusion. Biosynthesis of new
compounds by recombinant strains obtained by intraspecific protoplast fusion
(Fujimoto et al. 1990) as well as by interspecific protoplast fusion (Xiufen
and
CA 02449601 2013-06-05
6
Qi 1989) has been previously reported. The present invention also therefore
concerns a method for modifying the biocontrol efficiency of a Streptomyces
strain comprising protoplast fusion. The Applicant was the first to use such
method for modifying the biocontrol efficency of a microorganism.
[0016] As used herein, the terminology "geldanamycin-producing strain"
refers to any bacterial strain producing geldanamycin including strains
naturally
found in the nature such as EF-76, Streptomyces hygroscopicus var. geldanus
ATCC 55256, Streptomyces violaceusniger YCED9, Streptomyces
hygroscopicus strain NRRL 3602. It also refers to any synthetic strain
producing geldanamycin such as a recombinant strain produced according to
means known by persons of ordinary skill in the art and strains obtained from
protoplasm fusion including FP-54. These means include methods of cloning
genes of a geldanamycin-producing strain into a non-producing strain.
[0017] The terminology "geldanamycin-producing strain" also includes
strains containing a sequence encoding a protein identified in Rasher et al.
"Cloning and characterization of a gene cluster for geldanamycin production in
Streptomyces hygroscopicus" Microbiol. Lett. 218 (2), 223-230 (2003) as a
secreted protein (SEQ ID NO : 6). This sequence is also available in NCB'
database under no AY179507. It is reasonably predictable that this secreted
protein contributes to the synthesis of geldanamycin. The definition therefore
also includes strains containing a nucleotide sequence encoding this protein,
namely nucleotide sequence (SEQ ID NO: 7). The definition also includes
strains producing a protein having this sequence (SEQ ID NO: 6) or an
homologous protein contributing to a geldanamycin activity. Homologous >> is
intended to mean a protein similar or identical to any protein contributing to
geldanamycin synthesis which is produced by a variant S.
melanosporofasciens strain, another Streptomyces species, another microbial
species (e.g. fungus or bacteria) of a natural or synthetic origin. Such
CA 02449601 2011-09-22
7
homologous or corresponding protein shares amino acid and nucleotide
sequences susceptible to encode a protein involved in the biosynthesis of a
molecule having the same activity profile as geldanamycin. This activity is
monitored on sensitive strains like S. scabies. The definition also includes
strains containing nucleotide sequences able to hybridize under stringent
conditions to a sequence complementary to sequences involved in
geldanamycin biosynthesis. For instance, FP-60, a strain shown not to produce
geldanamycin does not hybridize to probes Bm27, Bm3 and BS15. This
definition also includes strains possessing a gene encoding an amino DHQ
synthase. Indeed, EF-76 (data not shown) and Streptomyces hygroscopicus
strain NRRL 3602 (see NCBI AY179507) possess a gene encoding an amino
DHQ involved in geldanamycin biosynthesis.
[0018] As used herein, the terms "EF-76" and "Streptomyces
melanosporofasciens" are used interchangeably.
[0019] As used herein, the terminology "biologically pure" strain is
intended to mean a strain separated from materials with which it is normally
associated in nature. Note that a strain associated with other strains, or
with
compounds or materials that it is not normally found with in nature, is still
defined as "biologically pure." A monoculture of a particular strain is, of
course,
"biologically pure."
[0020] For the methods and uses of the present invention, it is not
necessary that the whole broth culture of the strains of the invention be
used.
Indeed, the present invention encompasses the use of a whole broth culture of
a strain of the present invention, spores produced by such strain, dried
biomass
of the strains and lyophilized strains. As used herein therefore, the
terminology
"inoculum of a strain" refers to any form or part of the strain of the present
invention or a combination thereof that possesses the desired ability to
control
CA 02449601 2003-12-05
8
common scab.
[0021] There is
also provided a combination of an inoculum of a strain
according to the present invention and of a carrier.
[0022] In order
to achieve good dispersion, adhesion and
conservation/stability of compositions within the present invention, it may be
advantageous to formulate the whole broth culture or supernatant with
components that aid dispersion, adhesion and conservation/stability or even
assist in the biocontrol of the plant disease. These components are referred
to
herein individually or collectively as "carrier". Suitable formulations for
this
carrier will be known to those skilled in the art (wettable powders, granules
and
the like, or carriers within which the inoculum can be microencapsulated in a
suitable medium and the like, liquids such as aqueous flowables and aqueous
suspensions, and emulsifiable concentrates). Other suitable formulations will
be known to those skilled in the art. The carrier may include components such
as chitosan, vermiculite, compost, talc, milk powder, gel, etc.
[0023]
Chitosan, a chitin deacetylated derivative, is another
product that was shown effective to control several fungal diseases (Benhamou
and Theriault, 1992; Sathiyabama and Balasubramanian, 1998). Protection
conferred to plants by chitosan depends on both the elicitation of plant
defense
mechanisms (Kauss et al., 1989; Pearce and Ride, 1982; Walker-Simmons et
al., 1983) and the fungicidal property of chitosan oligomers (Allan and
Hadwiger, 1979; Hirano and Nagao, 1989). Chitosan is known to exhibit
bacteriostatic activity towards Gram-negative and Gram-positive human
pathogens (Allan et al., 1984), foodborne pathogens (Wang, 1992) and lactic
bacteria (Savard et al., 2002). Chitosan has usually no toxic effect on
microorganisms producing chitosanases. Chitosanolytic organisms would
CA 02449601 2003-12-05
9
benefit from the presence of chitosan in their environment as carbon and
nitrogen sources. Chitosanolytic activities have been reported for strains of
different bacterial genera including Streptomyces (Fukamizo and Brzezinski,
1997).
[0024] As used
herein, the terms "mutant" and "variant" are used
interchangeably. A variant of the EF-76 strain deposited at the ATCC under
access no BAA-668 may or may not have the same identifying biological
characteristics of the EF-76 strain, as long as the variant possesses
biocontrol
efficiency against common scab. Illustrative examples of suitable methods for
preparing variants of the inventive microorganism strain include, but are not
limited to: intraspecific protoplast fusion, mutagenesis by irradiation with
ultraviolet light or X-rays, or by treatment with a chemical mutagen such as
nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine),
methylmethane
sulfonate, nitrogen mustard and the like; gene integration techniques, such as
those mediated by insertional elements or transposons or by homologous
recombination of transforming linear or circular DNA molecules; and
transduction mediated by bacteriophages such as P1. These methods are well
known in the art and are described, for example, in J. H. Miller, Experiments
in
Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1972); J. H. Miller, A Short Course in Bacterial Genetics, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1992); M. Singer and P.
Berg, Genes & Genomes, University Science Books, Mill Valley, CA (1991); J.
Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory
Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989); P. B. Kaufman et al., Handbook of Molecular and Cellular Methods
in Biology and Medicine, CRC Press, Boca Raton, FL (1995); Methods in Plant
Molecular Biology and Biotechnology, B. R. Glick and J. E. Thompson, eds.,
CRC Press, Boca Raton, FL (1993); and P. F. Smith-Keary, Molecular Genetics
CA 02449601 2003-12-05
of Escherichia coil, The Guilford Press, New York, N.Y. (1989).
[0025] Variant strains derived from the EF-76 strain using known
methods are then preferably selected or screened for biocontrol efficiency
against common scab. In a specific embodiment, fusant cells are selected on
the basis of their ability to control common scab. Strain FP-54 described
herein
constitutes a variant as defined herein. It is a strain obtained from the
protoplasm fusion of EF-76 and possesses biocontrol efficiency against
common scab.
[0026] As used herein, the terminology "biocontrol" is meant herein to
refer to reduction of severity or reduction of incidence a plant disease.
[0027] As used herein, the terminology "biocontrol efficiency" of a
strain
refers to the strain's ability to ameliorate or stabilize the state of a plant
affected
by a disease or reverse, slow or delay progression of the plant disease. It
may
be assessed though a number of parameters including: antibiotic production,
decrease of the plant disease severity index, antagonistic activity against a
plant pathogen, antibiosis, lysis, phytotoxin degradation and rhizocompetence.
When the biocontrol efficiency assessed is that of strains of the present
invention against common scab, the parameters used include antagonistic
activity against S. scabies, geldanamycin production, disease severity index
of
common scab, disease incidence of common scab. As used herein, the term
"disease incidence" refers to the percentage of tubers or roots displaying
visible
signs of common scab lesions.
[0028] As used herein, the terminology "common scab" is meant to
include any Streptomyces-induced common scab including those affecting root
and tuber food crops such as red and sugar beets, carrots, parsnips, radishes,
CA 02449601 2003-12-05
11
rutabagas and turnips (Goyer et al. 1997). Geldanamycin was shown to be
active against other common scab causing bacterial species including
acidiscabies and caviscabies (data not shown).
[0029] As used herein, the terminology "severity of common scab" is
meant to refer to the scope of common scab symptoms on vegetal tissues. It
may be assessed through a number of means including the determination of
the extent of the surface of the plant covered by scab lesions.
[0030] As used herein, the terminology "effective amount" is meant to
refer to an amount sufficient to effect beneficial or desired results. An
effective
amount can be provided in one or more administrations. In terms of treatment
of and protection against common scab, an "effective amount" is an amount
sufficient to ameliorate, stabilize, reverse, slow or delay progression of the
plant
disease state. In specific embodiments, an "effective amount" is an amount
sufficient to ameliorate, stabilize, reverse, slow or delay progression of the
plant
disease state by at least 5% with regard to a non-treated diseased control
plant
In other specific embodiments, the effective amount may be comprised
between about 103 and 109 spores/g of carrier.
[0031] According to the methods of the present invention for applying the
inoculum of a strain according to the present invention, the inoculum may be
applied on various parts of the plant affected by common scab including the
tubers and the root and any part thereof. It may be applied at plantation or
later
during the season.
[0032] Other objects, advantages and features of the present invention
will become more apparent upon reading of the following non-restrictive
description of preferred embodiments thereof, given by way of example only
CA 02449601 2011-09-22
12
with reference to the accompanying drawings.
[0033] The present description refers to a number of documents.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] In the appended drawings:
[0035] Figure 1 shows the growth of Streptomyces scabies EF-35 in the
presence of Streptomyces melanosporofaciens strains FP-60 (A), EF- 76 (B),
and FP-54 (C);
[0036] Figure 2 presents a southern blot hybridization between BamHI-
digested genomic DNAs of S. melanosporofaciens strains EF-76, FP-54 and
FP-60 and probe Kp38 (SEQ ID NO: 1);
[0037] Figure 3 presents a southern blot hybridization of Barn- HI-
digested genomic DNAs and probes Bm27 (SEQ ID NO: 2) (A), Bm3 (SEQ ID
NO: 3) (B), and BS15 (C);
[0038] Figure 4 presents the effect of S. melanosporofaciens EF-76 and
of two fusant strains on common scab of potato. Panel (A) and B) presents
assays in a growth chamber and in the field, respectively. Bars accompanied
by the same letter indicate that the corresponding treatments did not
significantly differ (p<0.05)*;
[0039] Figure 5 shows the growth of S. scabies strain EF-35 (left) and of
S. melanosporofaciens strain EF-76 (right) on a chitosanase detection medium;
CA 02449601 2011-09-22
13
[0040] Figure 6 shows the DNA sequence of probe Kp38 containing
type-I polyketide synthase (PKS) genes (SEQ ID NO: 1);
[0041] Figure 7 shows the DNA sequence of probe Bm27 (SEQ ID NO:
2);
[0042] Figure 8 shows the DNA sequence of Bm3 (SEQ ID NO: 3);
[0043] Figure 9 shows the amino acid sequence of the putative
aminoDHQ synthase of Streptomyces hygroscopicus strain NRRL 3602 (SEQ
ID NO: 4);
[0044] Figure 10 shows the DNA sequence encoding the putative
aminoDHQ synthase of Streptomyces hygroscopicus strain NRRL 3602 (SEQ
ID NO: 5);
[0045] Figure 11 shows the amino acid sequence of a putative
geldanamycin-producing protein of Streptomyces hygroscopicus strain NRRL
3602 (SEQ ID NO: 6); and
[0046] Figure 12 shows the DNA sequence encoding a putative
geldanannycin-producing protein of Streptomyces hygroscopicus strain NRRL
3602 (SEQ ID NO: 7).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
EXAMPLE 1
Strains used
CA 02449601 2003-12-05
14
[0047] Streptomyces scabies strain EF-35 and S. melanosporofaciens
strain EF-76 were isolated from common scab lesions on potato tubers
(Faucher et al., 1992). Streptomyces melanosporofaciens EF-76 (Doumbou et
al., 2001a), formely S. hygroscopicus sbsp. geldanus, was selected after
screening for the ability to inhibit Phytophthora fragariae growth causing
raspberry root rot (Valois et al., 1996). Strain EF-76 produces geldanamycin
(Toussaint et al., 1997), a polyketide exhibiting antimicrobial activity
(DeBoer et
al., 1970).
EXAMPLE 2
Detection medium for chitosanase activity
[0048] Chitosanase detection medium was prepared according to
Brzezinski et al. (1997) with the following modifications. The detection
medium
was prepared by adding successively to a sterile base medium (1.74 g of
peptone and 3.6 g of agar in 170 ml of distilled water) 90 mL of chitosan
solution, 30 mL of solution A, 10 mL of 0.25 M K2HPO4 and 1 mL of 5N KOH.
Chitosan solution was prepared by dissolving 1 g of chitosan (Sigma-Aldrich,
St-Louis, MO) in 100 mL of 0.1 N HCI. Solution A consisted of 10 g L-1 MgSO4,
g L-1 NaCl, 1 g L-1 K2HPO4, 100 mg L-1 FeSO4, 100 mg L-1 CaCl2, 66 mg
L-1 MnCl2 and 7 mg L-1 ZnC12. Strains were inoculated on the chitosanase
detection medium and incubated at 30 .0 for 24 ¨ 48 h. Chitosanase activity
was detected by the formation of a clear zone of degradation around the
growing colonies.
[0049] Figure 5 showed that strain EF-76 had the property to hydrolyze
chitosan. A clearing zone was observed around EF-76 colony indicating that
the insoluble chitosan contained in the growth medium was hydrolyzed. S.
scabies strain EF-35 produced melanoid pigment on this medium but no
chitosanase activity was detected. Treatments of seed pieces did not appear to
affect the yield at harvest. Each year, the yield values of the four
treatments did
CA 02449601 2003-12-05
not significantly differ (P< 0.05) (Table 2).
EXAMPLE 3
Greenhouse assay
[0050] The pathogenicity test was carried out using potato tubers
(Solanum tuberosum cv. Shepody) obtained from glasshouse grown plants
derived from tissue culture with no visible sign of disease. Potato tubers
were
planted in 250-mm diameter pots containing sand and vermiculite (2:1). S.
scabies inoculum was prepared as previously described by Faucher et al.
(1992). S. scabies strain EF-35 was grown for 2 weeks at 30 C, in 50 mL
tubes containing 3 g vermiculite saturated with Say-solution (Labruyere,
1971).
This inoculum was mixed with the plant growth substrate before planting. At
planting, talc or chitosan (0.5 g) with or without S. melanosporofaciens
strain
EF-76 was sprinkled on the top of each tuber. Spores were previously collected
from a 10-day-old culture on YME agar and then mixed with talc or chitosan
(108 spores/g of carrier). The pathogenicity test was carried out in five
replicates. Pots were randomly dispersed in a greenhouse and their contents
were watered every three days. A soluble fertilizer (20-20-20) was added every
2 weeks. The progeny tubers were harvested after three months and were then
examined for common scab (Goyer et al., 1998). Disease severity was visually
estimated by attributing a disease severity index (1 ¨ 10) to each tuber in
regard of the extent of their surfaces covered with scab lesions. A disease
index of 1 was associated to healthy tubers while a disease index of 10
represented tubers covered by scab lesions on more than 90% of their surface.
[0051] The highest common scab incidence (43%) was associated with
the talc only treatment. When compared to talc only, chitosan only reduced
common scab incidence from 43 to 27% but this difference was not statistically
different (P < 0.05). When progeny tubers were grown in the presence of S.
CA 02449601 2003-12-05
16
melanosporofaciens strain EF-76 independently of the carrier used (talc or
chitosan), disease incidence was significantly reduced as compared to that
obtained to the talc only treatment and was reduced, although not
significantly,
as compared to that obtained with the chitosan only treatment (Table 1).
Table I. Effect of chitosan and S. melanosporofaciens EF-76 on common scab
incidence under controlled conditions
Treatment Common scab incidence (%)
Talc (control) 43a'
Chi tosan 27ab
Spores of S. melanosporofaciens EF-76 in talc 1 lb
Spores of S. melanosporofaciens EF-76 in chitosan 14b
'Numbers accompanied by the same letters are not significantly different (P
<0.05, 2 test).
EXAMPLE 4
Field assay
[0052] Trials were performed in a naturally infested field at
L'Assomption
(Quebec, Canada) in 2000 and in 2001. At planting, a formulation powder (0.5
g) was added on the top of each tuber (Solanum tuberosum cv. Shepody).
Formulations consisted of one of two carriers (talc or chitosan) with or
without
dried biomass of S. melanosporofaciens EF-76 (1/300 w:w inoculum/carrier).
EF-76 biomass was prepared as follows. The bacteria were grown 7 ¨ 10 days
in a 5 L reactor, in YME liquid medium supplemented with 10 mM CaCO3. The
cultures were centrifuged and the pellets were freeze-dried for 18 h. An
experimental plot consisted of 4 rows planted with 26 seed tubers. The plots
were arranged as randomized complete blocks with 4 replicates. Common scab
symptoms were estimated visually on 100 tubers harvested from each plot. The
disease incidence as well as the disease severity was scored for each
treatment. A disease severity index (1 ¨ 10) was attributed to each tuber in
regard of the extent of their surfaces covered with scab lesions. A disease
index of 1 was associated to healthy tubers while a disease index of 10
represented tubers covered by scab lesions on more than 90% of their surface.
Yield was determined by the weight (kg) of progeny tubers harvested from each
plot.
CA 02449601 2003-12-05
17
[0053] The four treatments [talc (control treatment), chitosan, talc
supplemented with S. melanosporofaciens spores, and chitosan supplemented
with S. melanosporofaciens spores] were compared. Treatments of seed
pieces did not appear to affect the yield at harvest. Each year, the yield
values
of the four treatments did not significantly differ (P< 0.05) (Table 2).
[0054] Common scab was more severe in 2000 than in 2001 as shown
by both a higher average disease severity index (an average severity index of
2.43 for 2000 versus 1.43 for 2001) and a higher level of disease incidence
(an
average disease incidence of 80.3 for 2000 versus 35.8% for 2000) as seen in
Table 2.
[0055] Each year, the highest common scab disease severity index was
associated with the talc only treatment. Common scab severity was
significantly
lower for all other treatments except for the chitosan treatment in 2001. The
level of protection against the disease incidence conferred by chitosan and S.
melanosporofaciens spores into talc was equivalent since the disease severity
index value for both treatments did not significantly differ in 2000.
[0056] If most treatments reduced common scab severity when
compared to the talc treatment, only some treatments significantly reduced
common scab incidence. In 2000, chitosan with or without S.
melanosporofaciens spores reduced the disease incidence of 35 and 8%,
respectively. In 2001, S. melanosporofaciens spores in talc or in chitosan
reduced the disease incidence of 21 and 23%, respectively. The combination of
chitosan and of S. melanosporofaciens spores offered in 2000 a protective
effect against common scab that was higher than those conferred by chitosan
or by S. melanosporofaciens spores in talc. This additive effect was not
observed in 2001.
CA 02449601 2003-12-05
18
Table 2. Effect of chitosan and of S. melanosporofaciens EF-76 on common scab
of potato under field conditions
Treatment Field assay 2000 Field assay 2001
Disease Disease Yield (kg/plot) Disease Disease Yield
(kg/plot)
severity index incidence (%) severity index incidence
Talc (control) 2.83a' 91a2 53.6a1 1.61a' 47a2 61.9a'
Chitosan 2.36b 83b 45.8a 1.53a 46a 60.6a
Strain EF-76 in 2.49b 91a 49.3a I .26b 26b 58.8a
talc
Strain EF-76 in 2.05c 56c 49.2a 1.31b 24b 56.6a
chitosan
I Numbers of the column that are accompanied by a same letter did not
significantly differ (P <0.05, ANOVA test).
2 Numbers of the column that are accompanied by a same letter did not
significantly differ (P <0.05, 2 test).
Effect of a combination of EF-76 and chitosan
[0057] EF-76 appears to be an efficient biocontrol agent against
common scab of potato. For the two consecutive years in the field trial, this
strain, when applied with talc, reduced disease severity as compared to that
obtained with to the control treatment. EF-76 also reduced disease incidence
in
the 2001 field assay but not in 2000. The level of pathogenic inoculum
possibly
affected the ability of EF-76 to reduce common scab incidence. The disease
incidence was reduced when the pathogen inoculum was moderate (the
greenhouse assay and the 2001 field trial) but not when the inoculum was high
(the 2000 field assay). Inoculation of potato seeds with EF-76 reduced the
common scab index both in growth-chamber and field conditions.
[0058] The present invention presents the first demonstration of
biocontrol by a geldanamycin-producing strain against a bacterial disease.
Effect of a combination of EF-76 and chitosan
[0059] As may be seen in Figure 1, EF-76 exhibited chitosanolytic
activity and thus could be protected against the toxic effect of chitosan.
[0060] Combination of chitosan and EF-76 ensured in both controlled
and field conditions a significant decrease of disease severity and of disease
incidence when compared to the control treatment. Even better, the combined
CA 02449601 2013-06-05
19
use of strain EF-76 and of chitosan provided in some cases a higher protection
than did strain EF-76 in talc or chitosan by itself. Even though this additive
effect was not always observed, combined application of chitosan and EF-76
was always one of the best treatments. As the effectiveness of chitosan and of
EF-76 in the control of common scab seems to be differentially influenced by
environmental conditions such as the level of pathogen inoculum, the combined
use of both products would ensure in most conditions a significant level of
protection.
EXAMPLE 5
Intraspecific Protoplastic fusion of the wild-type strain EF-76
[0061] Protoplasts of the strain EF-76 were made according to the
procedure described by Hopwood et al. (1985). A solution of 50% PEG 1000 in
P buffer (Hopwood et al. 1985) was added to 108 protoplasts. These
protoplasts were resuspended and kept 5 min at room temperature. Samples of
this suspension were plated on R2YE medium (Hopwood et al. 1985) and
incubated at 30 C. After their regeneration on R2YE plates, colonies were
streaked on YME medium for the screening of strains improved or deficient in
antibiotic production.
[0062] Several hundreds colonies were recovered from the protoplast
fusion experiment. Among these colonies, 100 were streaked on YME medium
and tested for their ability to inhibit the growth of B. cereus ATCC 14579.
Seven
isolates were positively or negatively affected in their inhibition power
against
Bacillus when compared to the wild-type strain EF-76.
[0063] Two fusants were studied in more detail, namely FP-54 (ATCC
BAA-669) and FP-60 (ATCC BAA-670). These strains were deposited at the
American Type Culture Collection (ATCC) at P.O. Box 1549 Manassas, VA
20108 USA on December 9, 2002.
CA 02449601 2003-12-05
Prototrophv of FP-54 and FP-60
[0064] The nutritional requirements of strains FP-54 and FP-60 was
verified by inoculating them on minimal medium [(NH4)2 SO4 2 WI, K2HPO4 0.5
g/I, MgSO4-7 H20 0.2 g/I, FeSO4=7 H20 0.01 g/I, glucose 5 g/I and agarose 15
g/1]. Bacterial growth was observed after 5 days of incubation at 30 C. The
ability of the two fusant strains to produce b-1,3-glucanase, b-1,4-glucanase
and b-1,6-glucanase was tested in the same minimal medium containing,
instead of glucose, 0.4% laminarin (Sigma-Aldrich, St. Louis, Mo., USA), 1%
carboxymethylcellulose (Fluka, Buchs, Switzerland) and 1% pustulan
(Calbiochem, San Diego, Calif., USA), respectively. The plates were incubated
5 days at 30 C and then overlaid with a solution of Congo red (0.2%) for 10
min
followed by two washes with a 1 M NaCI solution. A clear zone appeared
around colonies when the carbohydrate had been degraded.
[0065] Like the wild-type strain EF-76, strains FP-54 and FP-60 were
able to grow on a minimal medium. These strains retained the ability to
catabolize laminarin, cellulose, and pustulan. Their growth patterns were
different from that of the wild-type strain. At stationary phase, the biomass
reached by strains FP-54 and FP-60 was smaller than that of strain EF-76
(data not shown).
Growth curves of EF-76, FP-54 and FP-60
[0066] Growth curves of strains EF-76, FP-54 and FP-60 were
established as follows. Two-day-old cultures were used after standardisation
to
inoculate 100 ml of a fresh YME medium. The flasks were incubated at 30 C.
Ten-ml samples were then withdrawn periodically and the dry weight of cells
recovered from these samples was determined. This experiment was carried
out in triplicate.
CA 02449601 2003-12-05
21
Southern blot analysis
[0067] BamHI-digested genomic DNA was transferred onto Hybond
NTM
nylon membrane (Amersham Pharmacia Biotech, Baie d'Urfe, Canada).
Genomic DNAs were hybridized with four probes (Kp38, Bm27, Bm3, BS15).
These probes all came from a contiguous 40-kb DNA fragment of strain EF-76
genome (Agbessi 2002). Probes were labeled with digoxigenin-dUTP using a
kit (Roche Molecular Biochemicals, Laval, Canada). The samples were
prehybridized (150 min) and hybridized (overnight) at 68 C. After the
incubation, the membranes were washed twice in 2x SSC (150 mM NaCl, 15
mM sodium citrate) containing 0.1% SDS for 7 min at 68 C and twice in 0.1x
SSC containing 0.1% SDS for 20 min at 68 C. As used herein, these
conditions qualify as "high stringency conditions". Blots were developed with
a
colorimetric development kit according to manufacturer specifications (Roche
Molecular Biochemicals).
Genetic characterization of strains FP-54 and FP-60
[0068] The taxonomic identity of two selected fusant strains
(FP-54 and
FP-60) was determined by partial sequencing of the 16S rRNA gene (Doumbou
et al. 2001a).
[0069] Strains FP-54 and FP-60 exhibited a 16S rDNA sequence
identical to that of strain EF-76 over the 600-bp region sequenced for the
three
strains. Probes Kp38, Bm27, Bm3 and BS15 were hybridized with the genomic
DNA of strains FP-54 and FP-60 to detect genetic modifications.
[0070] Figure 2 presents the hybridization between BamHI-
digested
genomic DNAs of S. melanosporofaciens strains EF-76, FP-54 and FP-60 and
probe Kp38. This probe contains regions of the ketoacylsynthase and
acyltransferase domains of type-I polyketide synthase (PKS) genes (SEQ ID
_
CA 02449601 2003-12-05
22
NO: 1). Since type-I PKSs multifunctional enzymes have repetitive domains,
hybridization between S. melanosporofaciens genomic DNA and probe Kp38
gives several signals. The two most intense signals that correspond to the 3.8
and 5.5 BamHI fragments of strain EF-76 were absent from the hybridization
pattern of strain FP-60.
[0071] Figure 3 presents a southern blot hybridization of Barn- HI-
digested genomic DNAs of S. melanosporofaciens strains EF-76, FP-54 and
FP-60 and probes Bm27 (A), Bm3 (B), and BS15 (C). Probe Bm27 contains a
sequence encoding a putative formaldehyde dehydrogenase. Probe Bm3
contains some sequences encoding a putative transporter, a chitosanase, and
a glycosyl transferase. Probe BS15 contains some sequences encoding a
putative alcohol dehydrogenase and a methyl malonate- semialdehyde
dehydrogenase.
[0072] No modification was detected in the genome of strain FP-54. The
hybridization pattern of strain FP-60 with probe Kp38 was different from those
of strains EF-76 and FP 54. Strain FP-60 was the only strain displaying no
hybridization signal with probes Bm3, Bm27 and BS15.
EXAMPLE 6
In vitro antibiosis assays
Crossed resistance between EF-76, FP-54 and FP-60
[0073] Crossed resistance tests between strain EF-76 and FP-54 and
FP-60 strains were carried out as described above except that YME was used
instead of TSA and the plates were incubated 48 h following the overlay. Each
of the three strains was individually plated on YME and their antagonistic
properties against the two other strains were recorded.
CA 02449601 2003-12-05
23
[0074] FP-60's
growth was inhibited in the presence of strains EF-76 and
FP-54 (data not shown). Growth of strains FP-54 and EF-76 was not affected
by the presence of each other or by the presence of strain FP-60 (data not
shown). Since a strain that produces an antibiotic is generally resistant to
it and
one that is sensitive to this antibiotic does not produce it, these results
suggest
that FP-60 does not produce geldanamycin while EF-76 and FP-54 do.
Antibiosis of EF-76 against B. cereus and S. scabies
[0075] The
ability of S. melanosporofaciens strains to inhibit the growth
of B. cereus ATCC 14579 and S. scabies EF-35 was tested on YME as follows.
S. melanosporofaciens strains (108 spores) were streaked in the center of YME
plates. The plates were incubated 5 days at 30 C and then covered with an
overlay of soft TSA (0.3% agar) containing B. cereus ATCC 14579 or S.
scabies EF-35 (500 pl of an overnight culture in 4 ml of soft TSA). After 24 h
at
30 C, the diameter of the antibiosis zones around the Streptomyces inoculum
was recorded. This experiment was carried out in five replicates. Strain EF-76
was shown to inhibit the growth of S. scabies.
Antibiosis of FP-54 and FP-60 against P. fragariae, Bacillus cereus, and S.
scabies EF-35
[0076] The
antagonistic property of the fusants was tested on Bacillus
cereus ATCC 14579, Phytophthora fragariae var. rubi 390 and S. scabies EF-
35. The Streptomyces strains, B. cereus and P. fragariae var. rubi were grown
on yeast malt extract (YME) broth (4 g L-1yeast extract, 4 g L-1 glucose, 10 g
L-1 malt extract, 15 g L-1 agar) or agar (15 g/I) (Pridham et al. 1956-1957),
on
trypticase soy broth (TSB), and on potato dextrose agar (PDA) (Difco
Laboratories, Montreal, Canada), respectively.
[0077] The
ability of fusants to inhibit the growth of B. cereus ATCC 14579
CA 02449601 2003-12-05
=
24
and of S. scabies EF-35 was tested on YME plates as follows. Streptomyces
strains (108 spores) were streaked in the center of YME plates. The plates
were
incubated 5 days at 30 C and were then covered with an overlay of soft
trypticase soy agar medium (TSA, 0.3 (Yo agar) containing B. cereus ATCC
14579 or S. scabies EF-35 (500 pl of an overnight culture in 4 ml of inoculum
soft TSA). After 24 h at 30 C, the diameter of the antibiosis zones around the
Streptomyces inoculum was recorded. This experiment was carried out in five
replicates.
[0078] The
ability of the fusant strains to inhibit P. fragariae var. rubi was
tested as follows. Fusant strains were streaked (108 spores) in the center of
PDA plates and incubated for 2 days at 30 C. A piece of PDA medium (8-mm
diameter) from a 7-day-old culture of P. fragariae var. rubi 390 was then
placed
1 cm from the border of the Streptomyces inoculum. Phytophthora growth
inhibition was recorded after 5-7 days of incubation at 15 C.
[0079] Table
1 below shows the growth inhibition achieved by EF-
76, FP-54 and FP-60 against certain strains and their retardation factor (Rf),
namely the ratio of the compound migration distance on distance traveled by
the solvent front. As may be seen in Table 1, strain FP-60 lost the ability to
inhibit the growth of B. cereus ATCC 14579, P. fragariae var. rubi 390 and S.
scabies EF-35 (Fig. 1). Strain FP-54 exhibited higher antagonistic activities,
defined herein as the growth inhibition zone, against these three
microorganisms than did the wildtype strain EF-76 (Fig. 1). The higher
antagonistic property of strain FP-54 might be the consequence of a cumulative
effect of various secondary metabolites. Indeed, strain FP-54 was shown to
produce, in addition to geldanamycin, two other antimicrobial compounds that
were absent in culture supernatants of strain EF-76.
CA 02449601 2003-12-05
Table 1 Antagonistic properties of EF-76 and fusant strains FP-54 and FP-60. _
No growth
inhibition, + growth inhibition, ++ higher level of growth inhibition
Strains Antagonistic
properties Retardation factor (Rf) of
against Bacillus cereus,
products inhibiting the growth
Streptomyces scabies and of Bacillus cereus
Phytophtora fragariae var.
rubia
EF-76 0.51b, 0.44
FP-54 ++ 0.85, 0.72, 0.51b, 0.44
FP-60 no compound found
a Compounds were separated by thin layer chromatography on silica gel 60E-250
b Rf corresponding to geldanamycin (Toussaint et al. 1997)
Antibiosis of supernatant
[0080]
Antibiotics were isolated from 96-h-old YME cultures. Culture
supernatants (500 ml) were filtered through paper (OsmonicsTM, Minnetonka,
Minn., USA) and the filtrates were extracted three times with one-third volume
of chloroform. The chloroform fractions were evaporated on a BuChi Rotavapor
R-14 TM (13iichi Laboratoriums, Flawil, Switzerland). The resulting material
was
then dissolved in chloroform and separated by thin-layer chromatography on
glass plates precoated with 0.5 mm silica gel 60E-250 using
chloroform:methanol (95:5, v/v). After migration, the dried TLC was overlaid
with soft TSA containing B. cereus ATCC 14579. The TLC plate was then
incubated overnight at 30 C and the presence of growth inhibition zones was
recorded.
[0081] In
addition to geldanamycin, strain FP-54 produced antibiotics
that were absent in strain EF-76 supernatant (Table 1).
[0082] Strain
FP-60 lost the ability to synthesize geldanamycin (Table 1).
[0083] This
example shows that intraspecific protoplast fusion can be
CA 02449601 2013-06-05
26
used to modify the biocontrol agent's efficiency of the strains of the present
invention.
EXAMPLE 7
Effect on potato scab of seed inoculation with two fusants of EF-76
[0084] The ability of the fusant strains to reduce common scab
symptoms was tested both in controlled and field conditions.
Growth chamber
[0085] lnoculum for the growth chamber assay were prepared by
growing S. scabies EF-35 for 2 weeks at 30 C in 50-ml tubes containing
vermiculite saturated with Say-solution (Faucher et al. 1992). The
antagonistic
strain EF-76 and the fusant strains FP-54 and FP-60 were grown on YME agar
for 10 days at 30 C. Their spores were collected with a glass beads and then
mixed with talc (108 spores/g talc). Scab-free potato tubers cv. Green
Mountain
were planted in 25-cm-diameter pots containing sterile sand and vermiculite
(2:1, w/w) mixed with the pathogenic inoculum. At plantation, 0.5 g of talc
with
or without an actinomycete strain was sprinkled on the top of each tuber.
Potatoes were grown at 25 C with a 16-h photoperiod. Progeny tubers were
harvested after 12 weeks and examined visually for common scab symptoms.
A disease index (1-10) corresponding to the surface of coverage by common
scab lesions was assigned to each infected tuber. On this scale, 1 means no
disease while 10 means 100% surface coverage. The experiment was carried
out in five replicates.
Field
[0086] The field experiment was carried out in a field naturally infested
by S. scabies (L'Assomption, Canada). Plots consisting of four rows of 0.5m x
4m were planted with 104 potato seeds cv. Shepody. Plots were arranged as a
CA 02449601 2013-06-05
27
completely randomized block with four replicates. The inoculum to be applied
on the tubers was prepared as follows. Strains EF-76, FP-54 and FP-60 were
grown in YME supplemented with 10 mM CaCO3 for 7-10 days in a 5-1
bioreactor. The cultures were centrifuged and the pellets were freeze-dried
for
18 h. Dried biomass (1 g) prepared as described before was mixed with talc
(300 g). Talc containing lyophilized bacteria (dried biomass) (0.5 g) was
sprinkled on the top of each tuber at plantation. In control plots, talc
without
bacteria was applied on potato seeds. At harvest, tubers were examined for
common scab symptoms as described above.
[0087] S. melanosporofaciens EF-76 had the capacity to reduce
common scab both in controlled and field conditions. The disease index was
reduced from 6.30 to 4.81 and from 2.83 to 2.49 in growth chamber and field
experiments, respectively (Fig. 4). Strain FP-54 also reduced common scab
severity on potato tuber, but no significant difference was observed between
the disease index attributed to tubers treated with strain EF-76 or with
strain
FP-54 (Fig. 4).
[0088] It was observed that FP-54 strain survived in soil and
significantly
reduced the common scab disease incidence as compared to the reduction
achieved with the control and EF-76. The disease incidence of tubers treated
with FP-54 was of 56%, those treated with the control was of 72%, and those
treated with EF-76 was of 70%.
[0089] Strain FP-60 showed no protective effect against common scab;
moreover, the disease index of tubers treated with this recombinant was higher
than the index associated with potato tubers from the control treatment (Fig.
4).
This suggests that geldanamycin biosynthesis is a mechanism associated with
biocontrol. Strain FP-60 is not only ineffective as a biocontrol tool but
potatoes
CA 02449601 2013-06-05
28
treated with this fusant had a disease index higher than potatoes from control
treatment.
[0090] It also appears that the strains capable of producing
geldanamycin, for instance strains EF-76 and FP-54, are effective against the
scab causative agent.
[0091] It has been shown in Example 5 under subtitle "Genetic
characterization of strains FP-54 and FP-60" that EF-76 and FP-54 possess
sequences complementary to probes Bm3, Bm27 and BS15 while FP-60 does
not. These results therefore suggest that the production of geldanamycin by
EF-76 and FP-54 coincides with the presence of each or all sequences
complementary to probes Bm3, Bm27 and BS15 in these strains.
EXAMPLE 8
In vitro antibiosis assays with other geldanamycin-producing strain
[0092]The ability of Streptomyces hygroscopicus var. geldanus ATCC 55256 to
inhibit the growth of B. cereus ATCC 14579 and of S. scabies EF-35 was
tested on YME plates as follows. Streptomyces strains (108 spores) were
streaked in the centre of YME plates. The plates were incubated 5 days at
30 C and were then covered with an overlay of soft trypticase soy agar medium
(TSA, 0.3 % agar) containing B. cereus ATCC 14579 or S. scabies EF-35 (500
pl of an overnight culture in 4 ml of inoculum soft TSA). After 24 h at 30 C,
the
diameter of the antibiosis zones around the Streptomyces hygroscopicus var.
geldanus inoculum was recorded. This experiment was carried out in three
replicates. The antibiosis capacity of ATCC 5256 was similar to that of EF-76.
[0093] It is therefore submitted that geldanamycin-producing strains may
be used as biocontrol agents against common scab.
CA 02449601 2011-09-22
29
[0094] Any
probe or primer of at least 12 nucleotides in length derived
from Bm3, Bm27, BS15, the amino DHQ synthase and a geldanamycin-
producing protein encoding sequence (SEQ ID NO: 7) will be used to screen
and select a strain useful as a biocontrol agent against common scab. In the
alternative, a ligand to a geldanamycin-producing protein may be used for the
same purpose.
CA 02449601 2003-12-05
REFERENCES
Agbessi, S, Beausejour, J and Beaulieu, C. 2003. Antagonistic Properties of
Two Recombinant Strains of Streptomyces melanosporofaciens
Obtained by Intraspecific Protoplast Fusion. J. Appl. Microbiol.
Biotechnol. 62 : 233-238.
Allan C R and Hadwiger L A 1979 The fungicidal effect of chitosan on fungi of
varying cell wall composition. Exp. Mycol. 3,285-287.
Benhamou N and Theriault G 1992 Treatment with chitosan
enhancesresistance of tomato plants to the crown and root rot
pathogen, Fusarium oxysporum f. sp. radicis-lycopersici.Physiol.
Mol. Plant. Pathol. 41,33-52.
Benhamou N, Lafontaine P J and Nicole M 1994 Induction of systemic
resistance to Fusarium crown and root rot in tomato plantsby
seed treatment with chitosan. Phytopathology 84,1432-1444.
Brzezinski R, Boucher I, Dupuy A and Plouffe B 1997 Actinomycetes as model
organisms for the study of chitosanases. In Chitin Handbook. Eds.
R A A Muzzarelli and M G Peter. pp 291-295.European Chitin
Society, Torrette.
Cote N, Hogue R, Beaulieu C and Brzezinski R 2001 Suppressive effect of
chitin waste-based composts on common scab of potato. In Chitin
Enzymology. Ed. R A A Muzzarelli. pp 155-161. Atec Edizioni,
Italy.
Cuero R G, Duffus E, Osuji G and Pettit R 1991 Aflatoxin control in
preharvested maize: effects of chitosan and two microbial
agents.J. Agric. Sci. 117,165-169.
DeBoer C, Meulman P A, Wnuk R J and Peterson D H 1970Geldanamycin, a
new antibiotic. J. Antibiotics 23,442-447.
Doumbou C L, Akimov V and Beaulieu C 1998 Selection and characterization
of microorganisms utilizing thaxtomin A, phytotoxin produced by
Streptomyces scabies. Appl. Environ. Microbiol. 64,4313-4316.
Doumbou CL, Akimov VV, Cote M, Charest PM, Beaulieu (2001a) Taxonomic
study on non pathogenic streptomycetes isolated from common
scab lesions on potato tubers. Syst Appl Microbiol 24:451-456.
Doumbou CL, Hamby Salove MK, Crawford DL, and Beaulieu C (2001b)
Actinomycetes, promising tools to control plant diseases and to
promote plant growth. Phytoprotection 82:85¨ 102
Eckwall E C and Schottel J L 1997 Isolation and characterization of an
CA 02449601 2003-12-05
31
antibiotic produced by the scab disease-suppressive
Streptomyces diastatochromogenes strain PonSSII. J. Ind.
Microbiol.Biotechnol. 19,220-225.
Faucher E, Savard T, Beaulieu C (1992) Characterization of actinomycetes
isolated from common scab lesions on potato tubers. Can J Plant
Pathol 14:197-202
Fujimoto Y, lmamura A, lyeiri C, Shoji S, Kubota Y, Shibata M (1990) Features
of regenerated clones with or without fusion treatment between
auxotrophic mutants of Streptomyces antibioticus and their
antibiotic productivity. Agric Biol Chem 54:2855-2861
Fukamizo T and Brzezinski R 1997 Chitosanase from Streptomycessp. strain
N174: A comparative review of its structure and function.
Biochem. Cell Bio1.75,687-696.
Goyer C, Otrysko B and Beaulieu C 1996 Taxonomic studies on
streptomycetes causing potato common scab: A review. Can.
J.Plant Pathol. 18,107-113.
Goyer C, Vachon J and Beaulieu C 1998 Pathogenicity of Streptomyces
scabies mutants altered in thaxtomin A production.
Phytopathology 88,442-445.
Hayashida S, ChoiMY, Nanri N, Yokoyama M and Uematsu T 1989Control of
potato common scab with an antibiotic biofertilizer produced from
swine feces containing Streptomyces albidoflavusCH-33. Agric.
Biol. Chem. 53,349-354.
Hirano S and Nagao N 1989 Effects of chitosan, peptic acid, lysosyme and
chitinase on the growth of several phytopathogens. Agric. Biol.
Chem. 53,3065-3066.
Hopwood DA, Bibb MJ, Chater KF, Keiser T, Bruton CJ, Kieser HM, Lydiate
DJ, Smith CP, Ward JM, Schrempf H (1985) Genetic
manipulation of Streptomyces: a Laboratory Manual, The John
Innes Foundation, Norwich
Hopwood DA, Wright HM, Bibb MJ (1977) Genetic recombination through
protoplast fusion in Streptomyces. Nature 168:171¨ 174
Labrie C, Leclerc P, COte N, Roy S, Brzezinski R, Hogue R and Beaulieu C
2001 Effect of chitin waste-based composts producedby two-
phase composting on two oomycete plant pathogens.Plant Soil
235,27-34.
Liu D, Anderson N A and Kinkel L L 1995a Biological control of potato scab in
the field with antagonistic Streptomyces scabies. Phytopathology
85,827-831.
CA 02449601 2003-12-05
32
Lorang J M, Liu D, Anderson N A and Schottel J L 1995 Identification of potato
scab inducing and suppressive species ofStreptomyces.
Phytopathology 85,261-268.
Pridham TG, Anderson P, Foley C, Lindenfelser LA, Hessetime CW, Benedict
RG (1956-1957) A selection of media for maintenance and
taxonomic study of streptomycetes. Antibiot Annu 1956-
1957:947-953
Robinson M, Lewis E, Napier E (1981) Occurrence of reiterated DNA
sequences in strains of Streptomyces produced by an
interspecific protoplast fusion. Mol Gen Genet 182:336-340
Rothrock GS, Gottlieb D (1984) Role of antibiosis of Streptomyces
hygroscopicus var. geldanus to Rhizoctonia solani in soil. Can J
Microbiol 30:1440-1447
Ryan A D and Kinkel L L 1997 lnoculum density and population dynamics of
suppressive and pathogenic Streptomyces strains andtheir
relationship to bacterial control of potato scab. Biol. Control 10,
180-186.
Savard T, Beaulieu C, Boucher I and Champagne C P 2002 Antimicrobial
action of hydrolyzed chitosan against spoilage yeasts andlactic
acid bacteria of fermented vegetables. J. Food Protection 65,
828-833.
Toussaint V, Valois D, Dodier M, Faucher E, Dory C, Brzezinski R, Ruest L,
Beaulieu C (1997) Characterization of actinomycetes 237
antagonistic to Phytophthora fragariae var. rubi, the causal agent
of raspberry root rot. Phytoprotection 78:43-51
Trejo-Estrada SR, Sepulveds IR and Crawford DL 1998 In vitro ans in vivo
antagonism of Streptomyces violaceusniger YCED9 against
fungal pathogens of turfgrass W J Microbiol Biotechnol 14 :865-
872
Valois D, Fayad K, Barasubiye T, Garon M, Dery C, Brzezinski R, Beaulieu C
(1996) Glucanolytic actinomycetes antagonistic to Phytophthora
fragariae var. rubi, the causal agent of raspberry root rot. Appl
Environ Microbiol 62:1630-1635
Vruggink H 1970 The effect of chitin amendment on actinomycetes in soil and
on the infection of potato tubers by Streptomyce scabies. Neth. J.
Plant Pathol. 76,293-295.
Wang G H 1992 Inhibition and inactivation of five species of foodborne
pathogens by chitosan. J. Food Protection 55,916-919.
CA 02449601 2011-09-22
33
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Beaulieu, Carole
Agbessi, Sonya
Beausejour, Julie
Universite de Sherbrooke
(ii) TITLE OF INVENTION: GELDANAMYCIN-PRODUCING STRAINS, USES
THEREOF AND METHODS OF PRODUCING SAME
(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONDENCE ADDRESS:
(7) ADDRESSEE: GOUDREAU GAGE DUBUC
(B) STREET: 800 Place Victoria, P.O. Box 242
(C) CITY: Montreal
(D) STATE: Quebec
(E) COUNTRY: Canada
(F) ZIP: H4Z 1E9
=
(v) COMPUTER READABLE FORM:
00 MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,449,601
(B) FILING DATE: 05-DEC-2003
(C) CLASSIFICATION: Not assigned
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gauvreau, Julie
(B) REGISTRATION NUMBER: 11127
(C) REFERENCE/DOCKET NUMBER: 10857.368
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (514) 397-4374
(B) TELEFAX: (514) 397-4382
(C) TELEX: N/A
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2700 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ARTIFICIAL SEQUENCE - PROBE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CA 02449601 2011-09-22
34
GGTCTCGAGG GCCCGGCCGT GACCATCGAC ACCGCCTGCT CGTCGTCGCT GGTGGCGCTG 60
CACCTTGCCG CGCAGGCGCT GCGGCAGGGT GAATGCTCGC TGGCGCTGGC GGGCGGGGTG 120
GCCGTGATGT CCACCCCCGG CACCTTCGTG GAGTTCAGCC GTCAGCGGGG TCTTGCGCCG 180
GACGGCCGGT GCAAGGCGTT CGCGGCGGCA GCGGACGGTA CGGGCTGGGG CGAGGGTGTG 240
GGCATGCTGC TGCTGGAGCG GCTGTCGGAC GCGCGGCGCA ACGGACACCA GATCCTCGCG 300
GTGGTACGCG GCTCCGCCGT CAACCAGGAC GGTGCGAGCA ACGGGCTCAC CGCGGCCCAA 360
TGGCCCTCGC AACAGCGGGT GATCCGGGCG GCGCTGGCCA ACGCGCGGCT GTCGGCGGCC 420
GAGGTGGACG TGGTCGAGGC GCATGGTACG GGTACCACGC TGGGCGACCC GATCGAGGCG 480
CAGGCGCTTC TTGCCACGTA CGGCCGTGAA CACACCGACG ACCAGCCCCT GTGGCTCGGC 540
TCGATCAAGT CCAACATCGG GCACACCCAG GCCGCGGCCG GTGTCGCGGG CATTATGAAG 600
ATGGTGCTTG CCATGCGGCA TGGTCTGTTG CCGCAGACGC TGGGCGTCGA CGAACCGTCG 660
CCGCACATCG ACTGGACGGC GGGAGCCTCG AAGCTGCTCA CCGAGGCCAG GGCCTGGCCC 720
GAGACCGACC GCCCACGGCG GGCGGGCGTC TCGTCCTTCG GCCTCAGCGG CACCAACGGC 780
CACATCATTC TCGAACAGGA GCCGCCGACC GAGGCCGACG AGGAAACCTC CCAGGAGGAC 840
GCGCAACTTC CTCCCGCCGT CGTGCCATGG GTGCTGTCGG CGAAGTCCGA TGCCGGTGTG 900
CGGGGGCAGG CCGCGCGACT GCAGTCGGCG GTGGCCGGGG ATACCAGCCC GGGGATGACG 960
GACATCGGTC TGTCGCTGGT CACCACGCGT GCGGCGTTCG AGCGGCGGGC GGTGGTACTG 1020
GGTGGTGACC GTGCCGCGCT CGTCAGTGGC CTGACCGCGC TGACCGAGGG CCGGGAGGCG 1080
ACGCGCGTGG TGCGGGGGGC CGTGGTCGGC TCCGATGCCC GAGTGGCCTT TGTCTTTCCT 1140
GGTCGAGGGG TCGCAGTGGG TGGGGATGGC GGCTGGGTTG CTGGAGTCTT CGCCGGTGTT 1200
CGCGGAGCGA TTGGTGAGTG TGCGGCGGCT TCGGCGCCGT TCGTCGACTG GTCGCTCGGG 1260
GATGTGTTGC GGGGTGGGAA GGGTGCTGCG GAGGCGTTGG AGCGGGTGGA TGTGGTGCAG 1320
CCGGTGTTGT GGGCGGTGAT GGTGTCGTTG GCGGAGCTGT GGCGTTCGTA CGGTGTGGAG 1380
CCTGCGGCCG TTATCGGTCA TTCGCAGGGT GAGATCGCGG CGGCGTGTGT GGCGGGTGCG 1440
TTGTCGCTGG AGGACGCCGC GCGCGTGGTG GCGTTGCGAA GCCAAGCACT GCGGGCGTTG 1500
TCCGGCGGTG GTGGCATGGT GTCGGTATCA CTGCCCGTGA AGGCGGTACG AGAGCGGCTG 1560
GTCCGGTGGG GTGAGCGGCT GTCGGTGGCA GCGGTGAACG GGCCCTCGGC GGTTGTTGTC 1620
TCGGGTGACG CGGACGCGTT GGACGAGCTG CTGGCGGTGT GCGAGGGCGA GGAGATCCGG 1680
GCCCGTCGCA TCCCCGTGGA CTACGCCTCG CACTGCGCCC ATGTGGAGGA AATCGAGGAG 1740
ACGTTGTTGC GGGAGCTGGC GGATATCGCT CCCCGGGCGT CGTCGGTGCC GTTCTACTCC 1800
CA 02449601 2011-09-22
AGGGTCACGG CAGGCGTGCT CGATACGACC GGACTGGACG CCGGGTACTG GTACCGGAAT 1860
CTGCGTCAGA CGGTCCGCTT CGATGAGACC GTACGCACCC TCCTGGCCGA CGGCTTCCAG 1920
GTGTTCATCG AGGCCAGCGC CCACCCCGTC CTGACGATGG GAGTGGAGCA GACGGCCGAG 1980
GACCACGGCA CCCGCGTCAC CGCCGTCGGT TCCCTGCGCC GCGACGATGG CGGTCCCGAC 2040
CGGTTCGCCA CCTCCCTCGC CGAGGCGTAT GTCGGCGGCG CGCCCGTCGA CTGGGCGAGG 2100
ATGTTCGCCG GAACGGGCGC GGAGCGGGCC GATCTGCCGA CGTATGCCTT CCAGCGCACG 2160
CACTTCTGGC TGGAGTCCGA GACGGTCGAG GCCGGTGATG TGCCGTCGGT GGGGCTGGAC 2220
TCGGCCGGGC ATCCGTTGCT GGGTGCCGCC GTGCCGCTGC CCGACTCCGA CGGCTTCCTG 2280
CTCACCGGCC GGCTGTCGCT CCGTACCCAT CCCTGGGTCG CCGACCACGC GGTGGCGGAT 2340
GTAACGCTGC TGCCCGGGAC GGCCTTCGTG GAGCTGCTGA TGCGGGCCGG TGACGCGGTC 2400
GGCTGCGACC GGGTGGACGA ACTGACCCTG GGAGCACCGC TTGTGCTGCC CGAGCAGGGC 2460
ACGGTCCGGC TGCAGGTCGC CGTCGGCGGC CCCGACGAGG CGGGGCGGCG CTCGGTCGGT 2520
GTGTACGCGC AGACGGAGGA CGGCCCCTGG ACGCAGCACG CGACCGGTGT GCTCGGCGGC 2580
GGTGTGCTCG GCGGCGGCAG CAGCTCGGCC GATCGCGTCA CTGAGACGGA GGCATGGCCA 2640
CCGACTGGTG CGGAGGCCGT TGATGTCGCC GGGCTCTACG AGAGGTTCGC CCGGACCGGA 2700
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1114 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ARTIFICIAL SEQUENCE - PROBE
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 29
(D) OTHER INFORMATION: /note= "n is a, c, g, or t"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 393
(D) OTHER INFORMATION: /note= "n is a, c, g, or t"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 422
(D) OTHER INFORMATION: /note= "n is a, c, g, or t"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
CA 02449601 2011-09-22
36
(B) LOCATION: 439
(D) OTHER INFORMATION: /note= "n is a, c, g, or t"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 447
(D) OTHER INFORMATION: /note= "n is a, c, g, or t"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 519
(D) OTHER INFORMATION: /note= "n is a, c, g, or t"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 525
(D) OTHER INFORMATION: /note= "n is a, c, g, or t"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 611
(D) OTHER INFORMATION: /note= "n is a, c, g, or t"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TACCCGGGTG GCGCTGGCCT TCGTCAGTNG CTATCTCTTC GGGCGCGGCC ACAGGGGGAC 60
CTTGGTCCTC GGCCTCGCGG TCTCGCCGCG GGCAAGCGGC TCTCCGGTAT CAGCCCACCG 120
GGCGCCCAGG CACTGAAGTC CTCCGAGCTC GGCAAGCTGG GCCAGGAGAT CGGCAGCCGG 180
CTGGTCTCCG CGGGTCGGGA GGCGGCCATG TCGGCGGCCA GTAGCCGCAT CGACGGCCTG 240
AGCGACCCGC TGGAGCACCG CGCCACGGCG CMCGCACCG GCGGCGCGGG GGGCGGGGAG 300
CCGGAGSCGG GGAGGACGAG GAGCCGGACG AGCAGGAGGA GCGCGAGCCA CGCGAGCCCA 360
CCGGCAAGCA GCAGCRCAAG CCGGCCGACC GTNCGGCGCA GGCCAGGAAG CGGACGGYTC 420
CNAAGGGCTC GCGCGACGNA GCCAGANGTG AGGGCCATGG CTGAGGAAAC CCCCAAGGGG 480
CGGGCCSGTG GGCGCSGCGC CTGCCCACCG ACCACCTGNC GAAGNAAGCA CAGGGCCTGC 540
TGATGGCGCT GGSCGAAAAG GCTCTGGAAT CGGTCACCCA CCTGGGGGGC AACCCCGGGA 600
CGGGCGCGCT NAAGGGCGGA CTGGACCAGG TCAAGGGCAA GGTCGTCGAC ACGGCCAAGG 660
AGCAGATCAA GGAGAAGGTC AAGGACCAGG TCAAGGAAAA GGTCGTCGAC AAGGCCAAGA 720
GCTTCATCCC GGGCCTCGGC GGTGGCGGCG ACGGTGGCGG CAAGGGCGGC AAGAAGCTCA 780
AGGTCACCAA CATCGTGGAG CAGATCGACG TGGGCGCGCC CCTCTCCCTC ACCTACAACC 840
TCTGGACGGA GTGGGAGAAC TTCCCCTCGT ACATGAAGAA GGTCGAGGAC GTCCAGAACC 900
AGGGCGAGGA GGAGGGTGAG GACGAGGACG GGHCCGGGAC GGAATCCGAG TGGAAGGCCC 960
AGGTCTTCTG GTCGCACCGC AAGTGGCAGG CAGAGGTCGT CGAGCAGGTG CCCGAACAAG 1020
SCGGATCATC TGGAGCGTCT VCGGGCCGAD CAAGGGMCCA TGTSCGACRG VACGATCACC 1080
CA 02449601 2011-09-22
37
TTCCATGAGC TGGCCCCCGA GCTCACCCGG ATCC 1114
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1326 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: ARTIFICIAL SEQUENCE - PROBE
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 924
(D) OTHER INFORMATION: /note= "n is a, c, g, or t"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GCATGCGSGC GGGCGGCGAA SSGCGTTCGT CGGTCACGGG GGCGTAGGCG TAGGCCCSGC 60
CACGCGGGGT GCGGGTGAGC AACTGCTTGG TGTGCATACG GGTGAGGATC GTCACCACGC 120
TGTTGTAGGC CAGTTCGCCG CCGAGGCGTT CGGTGACCTC CCGGGGGGTC AGCGCGCCGT 180
CGGCCCGCTG CAGCAGTTCG AGGATCTCGG CCTCGCGGGC ACCGTTGGGC CGCTTGGGCC 240
CGTGTCCTGG CGTGCGGATG CCCATGCGGT TGGCCTCCCT CATCTCATCA CCCBACACTC 300
GTGTAGGACT CCTACACCGG TGTAAAAGCT GTTGTCTCCC AAGTGTGCCA TGCACGCGTG 360
CCTGGGCAGA ACTCTGCCTT GGGCGGGCAC GGSCGGAAAG GACCCTGCGG TGCCTTTGGC 420
CCTCAACCCG CTGGATGCCG CCGAACTGCT GACAGCGTTC GGCACGGCGG GTGTCTTCGT 480
GGTGCTGTTC GCCGAGACCG GACTTCTGAT CGGCTTCTTC CTGCCCGGTG ACTCCCTGTT 540
GGTCACCGCC GGCCTKCTGT GTACGGCCTC GGGCAGGGGC GTCCACCTGT CGGTGCCCGG 600
TGTGCCGGTC CGTACCCGCG GCCGGCACCT GATCGACGGT GTTAGGCGTG CGGAGGAGCT 660
TCTYGGCCGV TACGACCGCT ACCGGTTCCT CTGTTCGCGC TGATCGACGC GTYTYCCCGC 720
TGCCGCTCGC CCTGGGAGCG CTCAGGTCCC GCCGGGCCCG CACCGCCGSG GAGCCGACCG 780
TGGGGAGCGC CAAGTGAAGA CACCTTCCCC TGATCCACAA CGTGTACGCG ATCGGCGGVA 840
CGATCCGCAC CAAGCTAAAT CTCGCCGCCG GGCTCGCCGA CCGGCACRAG GTGACGAYCG 900
TATCGATGCT CCGCCACCGC ACCNACCCGC GATTCCGTCA TCGATCCACG GGTGACGGTC 960
GTGCCCCTGG TTGACATACA CGCGGACGCC GCCGACCCCC TGCTGCATCA GCCGGCCGAG 1020
GTCTTCCCCA CCGCCGAGAA GCGGTACAGG CAGTACAGCC GCCTCACCGA CCAGSGGGCG 1080
CGCGAGTACC TGCGGAAGCT GCGACGCGGA CGTGATCATC GGCACGCGGC CGGGCATCAA 1140
CA 02449601 2011-09-22
38
TGTGTACCTG GCCCCTTCGC ACCGCCCCGG GCACTGCGCA TCGCCCAGGA ACACCTCACC 1200
CACGASACGC ACACCAAGAG CTGCGCGCCC AGCTCGSSSG CCAGTACCGC GACCTGGATG 1260
CCGTGGTCAC CACGACCGAA GCCGACGCGG CCGTCTACCG GGCGAGATGC GGCTGCCGGG 1320
CGGGGG 1326
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptomyces hygroscopicus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Thr Thr Ser Thr Ser Ser Pro Ala Ala Ser Ser Ala Ser Pro Ala
1 5 10 15
Arg Gln Val Val Val Gly Leu Ala Glu Arg Ser Tyr Thr Val His Ile
20 25 30
Gly His Gly Val Gin Arg Leu Leu Pro Gin Val Val Ala Ala Leu Gly
35 40 45
Ala Arg Arg Ala Val Val Val Thr Ala Arg Pro Ala Glu Gin Thr Pro
50 55 60
Asp Pro Gly Val Pro Ser Leu Val Val Pro Ala Arg Asp Gly Glu Ala
65 70 75 80
Ala Lys Asp Leu Ala Ala Val Thr Asp Leu Cys Arg Arg Phe Val Gly
85 90 95
Phe Gly Leu Thr Arg Ser Asp Val Val Val Ser Cys Gly Gly Gly Thr
100 105 110
Thr Thr Asp Thr Val Gly Leu Ala Ala Ala Leu Tyr His Arg Gly Thr
115 120 125
Pro Val Val His Val Pro Thr Ser Leu Leu Ala Gin Val Asp Ala Ser
130 135 140
Val Gly Gly Lys Thr Ala Val Asn Leu Pro Glu Gly Lys Asn Leu Val
145 150 155 160
Gly Ala Tyr Trp Gin Pro Ala Ala Val Leu Cys Asp Leu Glu His Leu
165 170 175
Lys Thr Leu Pro Glu Arg Glu Trp Arg Asn Gly Leu Gly Glu Ile Ala
180 185 190
CA 02449601 2011-09-22
39
Arg Cys His Phe Ile Gly Ala Pro Asp Leu Asp Gly Leu Pro Leu Leu
195 200 205
Asp Gin Ile Ser Ala Ser Val Thr Leu Lys Ala Gly Ile Val Ala Ala
210 215 220
Asp Glu Arg Asp Ser Gly Leu Arg His Leu Leu Asn Tyr Gly His Thr
225 230 235 240
Leu Gly His Ala Leu Glu Arg Ala Thr Gly Phe Ala Leu Arg His Gly
245 250 255
Glu Gly Val Ala Ile Gly Thr Val Phe Ala Gly Arg Leu Ala Gly Ala
260 265 270
Leu Gly Arg Ile Gly Pro Glu Arg Val Ala Glu His His Asp Val Val
275 280 285
Ala Arg Tyr Gly Leu Pro Thr Ala Leu Pro Pro His Val Ser Val Ser
290 295 300
Glu Leu Val Glu Leu Met Arg Leu Asp Lys Lys Ala Thr Asp Gly Leu
305 310 315 320
Thr Phe Val Leu Asp Ser Pro Ala Gly Pro Gly Leu Val Arg Gly Ile
325 330 335
Ala Glu Asp Thr Val Gly Ala Thr Leu Ala Ala Met Pro Arg Ala Pro
340 345 350
Ala Trp
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1064 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptomyces hygroscopicus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CACCATGCGG GCGCGCGGGG CATCGCCGCG AGGGTGGCGC CGACGGTGTC CTCGGCGATC 60
CCGCGCACCA GTCCGGGCCC CGCGGGGCTA TCCAGGACGA ACGTCAGCCC GTCGGTGGCC 120
TTCTTGTCCA GGCGCATCAG CTCCACCAGC TCGGACACGG AGACATGCGG GGGCAGCGCG 180
GTCGGCAGGC CGTAGCGGGC GACCACGTCA TGATGCTCGG CCACGCGCTC CGGGCCGATG 240
CGCCCCAGCG CGCCGGCGAG CCGGCCGGCG AAAACCGTGC CGATGGCCAC TCCCTCCCCG 300
CA 02449601 2011-09-22
TGCCGCAGCG CGAACCCGGT GGCACGTTCC AGCGCATGCC CCAACGTGTG TCCGTAGTTG 360
AGGAGGTGGC GCAGGCCCGA GTCGCGCTCG TCCGCGGCGA CGATGCCCGC CTTGAGCGTC 420
ACACTGGCCG AGATCTGGTC GAGCAGCGGC AGCCCGTCGA GATCGGGCGC GCCGATGAAG 480
TGGCAGCGGG CGATCTCACC GAGGCCGTTG CGCCATTCCC GTTCGGGCAG GGTCTTCAGA 540
TGTTCGAGGT CGCAGAGCAC GGCCGCGGGC TGCCAGTAGG CGCCGACCAG ATTCTTGCCC 600
TCGGGCAGAT TCACCGCGGT CTTCCCGCCG ACGCTCGCGT CCACCTGGGC GAGCAGCGAG 660
GTCGGCACGT GTACGACCGG GGTGCCCCGG TGGTAGAGGG CGGCGGCCAG GCCCACCGTG 720
TCGGTCGTGG TGCCGCCGCC ACAGGACACC ACCACATCCG AGCGGGTCAG TCCGAATCCG 780
ACGAACCGGC GGCACAGATC GGTCACGGCG GCCAGGTCCT TGGCCGCCTC CCCGTCGCGG 840
GCGGGTACGA CGAGCGAGGG CACTCCTGGG TCGGGGGTCT GCTCGGCGGG CCGCGCGGTG 900
ACCACCACCG CCCTGCGCGC GCCCAGGGCG GCCACCACCT GTGGGAGCAG CCGCTGCACA 960
CCGTGTCCGA TGTGCACGGT GTAGGAGCGT TCGGCCAGCC CGACGACGAC CTGTCGGGCG 1020
GGGGAAGCGG AACTGGCGGC CGGACTGGAA GTCGACGTGG TCAA 1064
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(IQ LENGTH: 300 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptomyces hygroscopicus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Asp Lys Arg Thr Met Gly Arg His Arg Arg Ile Thr Gin Pro Pro
1 5 10 15
Arg Thr Thr Leu Ala Thr Arg Ala Val Leu Ala Ala Gly Val Leu Val
20 25 30
Pro Thr Ile Ala Ser Ala Gly Ser Ala His Ala Ala Thr Pro Gin Ala
35 40 45
Ala Ile Cys Thr Ser Asp Arg Pro Glu Leu Ala Asp Lys Leu Ser Glu
55 60
Asp Ile Asn Ser Ala Leu Glu Gly Ser Ala Ala Thr Thr Ala Ile Ser
65 70 75 80
Leu His Asp Arg Thr Thr Asn Thr Thr Cys Thr Leu Asp Ala Asp Arg
85 90 95
CA 02449601 2011-09-22
41
His Phe Asp Ser Ala Ser Thr Val Lys Val Thr Val Leu Ser Thr Leu
100 105 110
Leu Trp Asp Ala Gin Lys Asp Asn Arg Ala Leu Thr Gin Glu Glu Lys
115 120 125
Asp His Ala Thr Ala Met Ile Thr Glu Ser Asp Asn Asp Ala Thr Thr
130 135 140
Ala Leu Trp Lys Gin Leu Gly Ala Asp Lys Ile Asn Gly Phe Leu Gin
145 150 155 160
Ala Ala Gly Met Thr Asn Thr Thr Leu Asp Ser Glu Gly His Trp Gly
165 170 175
Leu Thr Gin Ile Thr Ala Asn Asp Glu Glu Lys Leu Leu Gin Leu Val
180 185 190
Thr His Thr Asn Pro Val Leu Ser Asp Asp Ser Arg Ala Tyr Ile Leu
195 200 205
Lys Leu Thr Ala Glu Val Ile Pro Ser Gin Arg Trp Gly Thr Pro Ala
210 215 220
Gly Ala Pro Ser Asp Ala Gin Val His Val Lys Asn Gly Trp Leu Glu
225 230 235 240
Arg Ala Thr Asn Gly Trp Arg Val His Ser Leu Gly Ala Phe Thr Gly
245 250 255
Gly Asp His Asp Tyr Thr Ile Thr Val Leu Ser Gin Asp Asn Ala Thr
260 265 270
Met Asp Asp Gly Ile Ala Asn Ile Glu Gly Ile Ala Arg Ala Val His
275 280 285
Glu Asn Leu Asn Ala Pro Val Ser Ser Ala Gin Ser
290 295 300
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 903 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptomyces hygroscopicus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
TTACGACTGA GCGCTGGACA CGGGCGCGTT GAGGTTCTCG TGGACCGCGC GGGCGATGCC 60
CTCGATGTTG GCGATGCCGT CGTCCATCGT GGCGTTGTCC TGCGAGAGCA CCGTGATCGT 120
GTAGTCGTGG TCGCCGCCGG TGAAGGCGCC GAGGCTGTGC ACCCGCCAGC CGTTCGTGGC 180
CA 02449601 2011-09-22
42
CCGCTCCAGC CAGCCGTTCT TCACATGCAC CTGGGCGTCG CTCGGCGCGC CGGCCGGGGT 240
GCCCCAGCGC TGCGAGGGGA TGACCTCGGC CGTCAGCTTC AGGATGTAGG CGCGGGAGTC 300
ATCGCTGAGC ACCGGGTTGG TGTGGGTCAC CAGTTGGAGG AGCTTTTCCT CATCGTTCGC 360
GGTGATCTGG GTGAGCCCCC AGTGGCCCTC GCTGTCGAGG GTGGTGTTGG TCATTCCCGC 420
GGCCTGCAGG AACCCGTTGA TCTTGTCCGC CCCGAGCTGC TTCCACAGCG CGGTGGTGGC 480
GTCGTTGTCG GACTCTGTGA TCATGGCGGT GGCATGGTCC TTCTCCTCCT GTGTCAGGGC 540
GCGATTGTCC TTCTGCGCGT CCCACAGCAG GGTGCTGAGC ACGGTCACCT TGACCGTGCT 600
CGCGGAGTCG AAGTGCCGGT CCGCATCCAG AGTGCAGGTG GTGTTCGTGG TGCGGTCGTG 660
GAGGCTGATC GCCGTGGTGG CGGCGGAGCC CTCCAGCGCC GAATTGATGT CCTCGGAGAG 720
CTTGTCGGCG AGTTCCGGCC GGTCCGAGGT GCAGATCGCC GCCTGCGGGG TGGCCGCGTG 780
AGCCGACCCC GCCGAGGCGA TCGTCGGCAC GAGCACCCCG GCGGCCAGCA CCGCTCTTGT 840
CGCCAGGGTG GTACGGGGAG GCTGGGTTAT TCGTCGGTGT CGACCCATGG TGCGCTTGTC 900
CAT 903
