Note: Descriptions are shown in the official language in which they were submitted.
CA 02322241 2000-10-23
ANTIBIOTIC PRODUCTON .
FIELD OF THE INVENTION
The present invention relates to methods and materials
for controlling antibiotic production in species of
Streptomyces, especially increasing antibiot~ production
in Streptomyces coelicolor and S. lividans.
INTRODUCTION
In addition to undergoing a complex process of
morphological differentiation, streptomycetes are
renowned for their ability to produce a vast array of
secondary metabolites, many of which possess antibiotic
or other pharmacologically useful activities. Most of
these secondary metabolites are the products of complex
biosynthetic pathways that are activated in a growth
phase-dependent manner. While the production oz
antibiotics in liquid culture is generally limited to
stationary phase, in surface-grown cultures it usually
coincides with the onset of morphological differentiation
(Chater and Bibb, 1997).
In several streptomycetes, y-butyrolactones (GBLs) have
been shown to play important, if not crucial, roles in
determining the onset of antibiotic production and
morphological differentiation (Horinouchi and Beppu,
1994; Yamada, 1999). The most characterised Y-
butyrolactone is A-factor (2-isocaryloyl-3R-
hydroxymethyl-y-butyrolactone), which is required for
both streptomycin production and sporulation in
Streptomyces griseus (Mori, 1983; Horinouchi and Beppu,
1994). Other well-studied Y-butyrolactones include
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CA 02322241 2000-10-23
virginiae butanolides (V8), which appears to control
virginiamycin production in Streptomyces virginiae
(Yamada et al., 1987; Kondo et al., 1989), and IM-2,
which elicits the production of showdomycin and minimycin
in Streptomyces lavendulae FRI-5 (Sato et al., 1989).
Although the details of A-factor synthesis have not been
elucidated, a putative A-factor biosynthetic gene, afsA,
was cloned from S. griseus and sequenced. Its predicted
translation product does not resemble any protein of
known function (Horinouchi et al., 1989). afsA mutants
of S. griseus are deficient in A-factor synthesis, and
hence in streptomycin production and sporulation.
Moreover, cloning of afsA in multiple copies leads to
precocious streptomycin production in S. griseus, and to
the production of a compound with A-factor activity in
other streptomycetes that normally do not make it
(Horinouchi et al, 1985). Culture supernatants of an
Escherichia coli strain over-expressing afsA restored
streptomycin production and sporulation in an A-factor
deficient mutant of S. griseus (Ando et al., 1997).
A-factor is detected in culture supernatants of S.
griseus just before the onset of streptomycin production.
It diffuses freely across the cytoplasmic membrane, and
binds with high affinity to a cytoplasmic A-factor-
binding protein, ArpA (Onaka et al, 1995). In the
absence of A-factor, ArpA acts as a negative regulator of
both streptomycin production and sporulation by
repressing transcription of the pleiotropic regulatory
gene adpA (Ohnishi et al., 1999). Homologues of afsA
and/or arpA have been isolated from several
streptomycetes, including S. virginiae (Okamoto et al.,
1995; Kinoshita et al., 1997), S. lavendulae (Waki et
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CA 02322241 2000-10-23
al., 1997), S. coelicolor (Onaka et al., 1998) and S.
fradiae (Fouces et al., 1999; Bate et al., 1999).
S. coelicolor is the most genetically characterised
streptomycete. It produces at least four chemically
distinct antibiotics. Two of these, actinorhodin (Act)
and undecylprodigiosin (Red), are pigmented. ~'he
stationary phase production of Act and Red results from
transcriptional activation of the pathway-specific
activator genes actII-ORF4 and redD, respectively
(Gramajo et al., 1993; Takano et al., 1992). Moreover,
production of Act and Red in exponential phase appears to
be prevented only by the absence of a threshold
concentration of the pathway specific activator
proteins.
Recently, four extracellular compounds were identified in
culture supernatants of Streptomyces coelicolor A3(2)
that elicited the precocious production of the
antibiotics actinorhodin (Act) and undecylprodigiosin
(Red) when added to the producing strain; none of the
compounds induced morphological differentiation. One of
these stimulatory factors, SCB1, was purified to
homogeneity and shown by structural elucidation to be a
Y-butyrolactone (Takano et al., 2000).
SUMMARY OF THE INVENTION AND DETAILED DESCRIPTION
The present inventors have identified genes of S.
coelicolor which are involved in the regulation of Act
and Red production. One gene, scbA, is a homologue of
afsA (A-factor synthetase of S. griseus), and the other,
scbR, encodes a Y-butyrolactone binding protein. By
analogy with the S. griseus system, ScbR was expected to
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CA 02322241 2002-O1-22
be a repressor of the pathway-specific activator genes
actII-ORF4 and redD. Release of such repression upon
binding of ScbR by the GBL SCB1 at high cell densities
would lead to antibiotic expression.
S
However, it was found that ScbR binds to the
transcription start sites of scbA and scbR, and is
released by addition of SCB1 from S. coelicolor. An in-
frame deletion mutant of scbA (a gene involved in GBL
synthesis) shows overproduction of Act and Red (when lack
of antibiotic production might have been expected) and an
in-frame deletion mutant of scbR shows delay in Red
production and earlier Act production (scbR - see Fig.
5A). These phenotypes therefore differ from what might
be expected by analogy to the S. griseus A-factor system.
Moreover, the inventors have found that S. lividans
strains carrying the same in-frame deletion mutant of
scbA, in place of the wild-type scbA gene usually present
in S. lividans, also overproduce Act and Red.
The inventors propose, therefore, that mutations to
homolgues of scbA and scbR in other Streptomyces species
may have similar effects.
Accordingly, in a first aspect, the present invention
provides a method of modifying an antibiotic-producing
strain of a Streptomyces species to increase antibiotic
production in said strain, the method comprising
functionally deleting in said strain a gene which is the
scbA gene of Streptomyces coelicolor or a homologue
thereof.
In a second aspect, the present invention provides a
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CA 02322241 2000-10-23
method of modifying an antibiotic-producing strain of a
Streptomyces species to alter the timing of antibiotic
production in said strain, the method comprising
functionally deleting in said strain a gene which is the
scbR gene of Streptomyces coelicolor or a homologue
thereof .
While it is appreciated that these effects will not be
found in S. griseus and some other Streptomyces species,
it is thought that the effects may not be confined to the
exemplified species of S. coelicolor and S. lividans. It
will be possible for the skilled person to repeat the
experimental disclosure presented herein on other
Streptomyces species, thereby to identify other species
in which similar effects occur. In particular, it will
be possible to identify in other species of Streptomyces
genes which are homologues of scbR and scbA in an
analogous way to the identification herein of scbR and
scbA. Following identification of the genes, it will be
possible to create strains in which these genes are
functionally deleted, and to compare the extent and/or
timing of antibiotic production in those modified strains
with the extent and/or timing of production in the parent
strain. Those modified strains in which similar effects
are found to those presented herein are regarded also to
be part of the invention.
Accordingly, in a third aspect, the present invention
provides a modified strain of a Streptomyces species, the
modified strain having a functional deletion of a gene
which is the scbA gene of S. coelicolor or a homologue
thereof, whereby production of at least one antibiotic in
said modified strain is increased compared to a wild-type
strain of said Streptomyces species.
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CA 02322241 2000-10-23
Similarly, in a fourth aspect, the present invention
provides a modified strain of a Streptomyces species, the
modified strain having a functional deletion of a gene
which the scbR gene of S. coelicolor or a homologue
thereof, whereby the timing of production of at least one
antibiotic in said modified strain is altered compared to
a wild-type strain of said Streptomyces species.
In a fifth aspect, the present invention provides a
method of producing an antibiotic, the method comprising
providing a modified Streptomyces strain of any preceding
aspect, and culturing said strain under conditions
suitable for production of antibiotic.
The method may also comprise the additional step of
purifying the antibiotic from the culture medium. It may
also comprise the further step of formulating the
antibiotic as a pharmaceutical.
The scbR and scbA genes are believed to be new, as is a
further gene, designated scbB, which is downstream of
scbR and which shows homology to the C-5 ketoreductase
gene of S. avermitilis. scbB is predicted to modify the
C-6 of SCB1 from keto to hydroxyl, and may therefore be
important in providing specificity of SCB1 as the cognate
GBL of scbR.
In a sixth aspect, the present invention provides a
nucleic acid comprising a nucleotide sequence having at
least about 80~ identity with a nucleic acid sequence
selected from the group consisting of (1) nucleotides
3032 to 3679, (2) nucleotides 2914 to 1970, and (3)
nucleotides 4529 to 3795, reading 5' to 3', of the
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CA 02322241 2002-O1-22
nucleic acid deposited as EMBL AJ007731, which may
alternatively be defined as (1) nucleotides 2261-2908,
(2) nucleotides 2142-1199 and (3) nucleotides 3758-3024,
respectively of Fig. 14.
As will be evident from Fig. 1, both strands of DNA in
this region encode polypeptides. Nucleotide.~umbering is
given in relation to the strand which runs from 5' to 3'
from right to left in Fig. 1. The sequence of part of
this strand is given in Fig. 14. However, the coding
sequences of scbA and scbB are on the complementary
strand. References to the nucleotide sequences in EMBL
AJ007731 and Fig. 14 which relate to these genes (i.e.
where the nucleotide numbering is shown as [higher
number)-[lower number]) should therefore be interpreted
as references, to the strand complementary to that shown.
Preferably the nucleic acid sequence identity is at least
85%, 90%, 95%, 98% or 99% or is 100%.
In a seventh aspect, the present invention provides a
nucleic acid comprising a nucleotide sequence which
encodes a polypeptide having at least about 70% amino
acid sequence identity with an amino acid sequence
selected from the group consisting of (1) the amino acid
sequence of ScbR, as shown in Fig. 9 , (2) the amino acid
sequence of ScbA, as shown in Fig. 10, and (3) the amino
acid sequence of ScbB, as shown in Fig. 11.
In further aspects, the present invention provides:
polypeptides encoded by the nucleic acid molecules of the
sixth and seventh aspects; vectors including the nucleic
acids of those aspects, optionally in operative
association with control sequences, e.g. promoter and/or
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enhancer sequences; host cells transfected with said
vectors; and methods of producing said polypeptides,
comprising culturing said host cells under conditions
suitable for polypeptide production and extracting said
polypeptides from the culture medium.
As used herein, the term "functional deletion" of a gene
may mean any alteration of the nucleic acid in a cell or
cells of the strain containing the functional deletion,
which alteration has the effect of preventing normal
expression of that gene. For example, the gene may
comprise a deletion in the coding sequence, leading to a
shortened transcript which is translated into a protein
lacking the normal function of the expression product of
the gene; or the transcriptional and/or translational
regulatory sites (e. g. promoter and/or enhancer
sequences) may be altered to prevent normal transcription
and/or translation of the gene; or the coding sequence
may contain an insertion or mutation (e. g. to introduce
or produce a stop codon or to cause a shift in reading
frame), leading to a non-functional expression product.
Alterations of the coding sequence may be in frame or may
cause a shift in reading frame. As a further
alternative, the cells) may be modified to produce
antisense mRNA, which prevents correct translation,
preventing gene expression even if the gene itself is
unmodified.
In using anti-sense genes or partial gene sequences to
down-regulate gene expression, a nucleotide sequence is
placed under the control of a promoter in a "reverse
orientation" such that transcription yields RNA which is
complementary to normal mRNA transcribed from the "sense"
strand of the target gene. See, for example, Rothstein
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et al, 1987; Smith et a1,(1988) Nature 334, 724-726;
Zhang et a1,(1992) The Plant Cell 4, 1575-1588, English
et al., (1996) The Plant Cell 8, 179-188. Antisense
technology is also reviewed in Bourque, (1995), Plant
S Science 105, 125-149, and Flavell, (1994) PNAS USA 91,
3490-3496.
An alternative is to use a copy of all or part of the
target gene inserted in sense, that is the same,
orientation as the target gene, to achieve reduction in
expression of the target gene by co-suppression. See,
for example, van der Krol et al., (1990) The Plant Cell
2, 291-299; Napoli et al., (1990) The Plant Cell 2, 279-
289; Zhang et al., (1992) The Plant Cell 4, 1575-1588,
and US-A-5,231,020.
The complete sequence corresponding to the coding
sequence (in reverse orientation for anti-sense) need not
be used. For example fragments of sufficient length may
be used. It is a routine matter for the person skilled
in the art to screen fragments of various sizes and from
various parts of the coding sequence to optimise the
level of anti-sense inhibition. It may be advantageous
to include the initiating methionine ATG codon, and
perhaps one or more nucleotides upstream of the
initiating codon. A further possibility is to target a
conserved sequence of a gene. Total complementarity or
similarity of sequence is not essential. The sequence
need not include an open reading frame or specify an RNA
that would be translatable. It may be preferred for
there to be sufficient homology for the respective anti-
sense and sense RNA molecules to hybridise. There may be
down regulation of gene expression even where there is
about 5%, 10%, 15% or 20% or more mismatch between the
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CA 02322241 2002-O1-22
sequence used and the target gene.
Preferred Streptomyces species for the practice of the
invention are species which possess adjacent and
divergent scbA and scbR genes of S. coelicolor or
adjacent and divergent homologues thereof, since it is
thought that this arrangement of genes may correlate with
the effects on amount and timing of antibiotic production
seen in S. coelicolor and S. lividans.
The closely related species S. coelicolor, S.
violaceoruber, S. lividans and S. parvulus are
particularly preferred. Strains of such species (i.e.
wild-type strains) are commonly available, e.g. from the
ATCC, for example under ATCC deposit numbers 12434 for S.
parvulus and 19832 for S. violaceoruber. S. coelicolor
A3(2) and S. lividans 66 are particularly preferred wild-
type strains and are available from the John Innes
Culture Collection (Norwich, UK) under JICC deposit
numbers 1147 and 1326, respectively. However, the
invention is not limited to such particular strains.
The present invention may exclude the modification of
barX and/or farX, the afsA homologues in S. virginiae and
S. fradiae, respectively.
A gene of a Streptomyces species or strain, which gene is
a "homologue" of or is "homologous" to the scbA gene of
S. coelicolor, may be the gene which shows greatest
deduced amino acid sequence identity to scbA of all genes
of said species or strain; alternatively or additionally,
it may be a gene which is capable of specific
hybridisation with the amplification product obtained
using the primers oligol c5'-GACCACGT(CG)CC(CG)GGCATG) (SEQ.ID.NO.:1)
CA 02322241 2002-O1-22
and O 1 i go2 (5'-GTCCTG(CG)TGGCC(CG)GT(CG)AC(CG)CG(CG)AC) (SEQ.ID.N0.:2)
to amplify total DNA of said species or strain (bracketed
nucleotides indicate positions of degeneracy);
alternatively or additionally, it may be a gene encoding
a polypeptide having at least about 35% sequence identity
with the deduced amino acid sequence of scbA as shown in
Fig. 10, preferably at least about 40% (which,~.s the
homology found between scbA and other homologues of the
afsA gene of S. griseus) more preferably about 50%, 60%,
65% (which is the homology found between scbA and afsA of
S. griseus), 70%, 80%, 90%, or 95%.
A gene of a Streptomyces species or strain, which gene is
a "homologue" of or is "homologous" to the scbR gene of
S. coelicolor, may be the gene which shows greatest
deduced amino~acid sequence identity to scbR of all genes
of said species or strain; alternatively or additionally,
it may be a gene which is adjacent to and divergent from
a gene which is capable of specific hybridisation with
the amplification product obtained using the primers
oligol (5'-GACCACGT(CG)CC(CG)GGCATG) and oligo2 (5'-
GTCCTG (CG) TGGCC (CG) GT (CG) AC (CG) CG (CG) AC) to amplify total
DNA of said species or strain (bracketed nucleotides
indicate positions of degeneracy); alternatively or
additionally, it may be a gene encoding a polypeptide
having at least about 35% sequence identity with the
deduced amino acid sequence of scbR as shown in Fig. 9,
preferably at least about 40%, more preferably about 45%
(which is the homology found between scbR and arpA of S.
griseus), 50%, 55% (which is the homology found between
scbR and the FarA gene of S. lavendulae) 60%, 65%, 70%,
80%, 90%, or 95%.
"Percent (%) amino acid sequence identity" is defined as
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CA 02322241 2000-10-23
the percentage of amino acid residues in a candidate
sequence that are identical with the amino acid residues
in the sequence with which it is being compared, after
aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence
identity, and not considering any conservative
substitutions as part of the sequence identity. The
identity values used herein are generated by WU-BLAST-2
which was obtained from Altschul et al. (1996);
http://blast.wustl/edu/blast/README.html. WU-BLAST-2
uses several search parameters, most of which are set to
the default values. The adjustable parameters are set
with the following values: overlap span =1, overlap
fraction = 0.125, word threshold (T) - 11. The HSPS and
HSPS2 parameters are dynamic values and are established
by the program itself depending upon the composition of
the particular sequence and composition of the particular
database against which the sequence of interest is being
searched; however, the values may be adjusted to increase
sensitivity. A ~ amino acid sequence identity value is
determined by the number of matching identical residues
divided by the total number of residues of the "longer"
sequence in the aligned region, multiplied by 100. The
"longer" sequence is the one having the most actual
residues in the aligned region (gaps introduced by WU-
BLAST-2 to maximize the alignment score are ignored).
"Percent (%) nucleic acid sequence identity" is defined
as the percentage of nucleotide residues in a candidate
sequence that are identical with the nucleotide residues
in the sequence under comparison. The identity values
used herein were generated by the BLASTN module of WU
BLAST-2 set to the default parameters, with overlap span
and overlap fraction set to 1 and 0.125, respectively.
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BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 Restriction map of 7.5kb DNA fragment isolated
S from S. coelicolor which includes scbA and scbR. The
positions of scbA, scbR, orfX (also referred to
herein as scbB) are indicated by shaded,~.oxes and
the other ORFs with no apparent homology to other
known streptomyces antibiotic regulatory genes (as
assessed by the BLAST program) are indicated by open
boxes. The ORFs are deduced from the FRAME program
(Bibb et al., 1983). pIJ6111 and pIJ6114 were used
for sequencing analysis of the 7.5kb fragment.
Restriction maps of the in-frame deletion mutant
constructs are also shown. Dotted lines indicate
the in-frame deletion of scbA for pIJ6120 or scbR
for pIJ6124. The PstI site in pIJ6124 was generated
by using a designed primer for PCR to allow ligation
with the corresponding PstI site. The BamHI site in
pIJ6140 was end filled and ligated with PvuII.
Single arrows (PrimersR) and double arrows
(PrimersA) denote the primers used to determine the
scbR and scbA mutation, respectively, after the
second crossover event. pIJ 6135 and pIJ6143 (insert
cloned into pSET152) were used to complement the
scbR and scbA mutant, respectively. pIJ6120 (insert
in pIJ2925) was used to express ScbR for gel
retardation and Dnase I footprinting experiments.
Fig. 2a S1 nuclease mapping of the transcriptional
start site of scbA and scbR. Asterisks indicate the
probable start points of the transcription; the
sequences given are those of the template strand.
Lanes T, G, C, and A are sequence ladders derived
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from the same primers as the probe generated by PCR
and using the Taqtrack kit along with the these
primers.
Fig. 2b S1 nuclease mapping of scbA, scbR, and the
major sigma factor hrdB, using RNA isolated from a
liquid time course of S. coelicolor M145 at the time
(hours) indicated. The EXP, TRANSITION and STAT
indicated the exponential, transition and stationary
phases of growth, respectively, and the shaded box
labelled RED denote the presence of
undecylprodigiosin in the mycelium; SM, end-labelled
HpaII-digested pBR322 size marker.
Fig. 3a Gel retardation experiment shown with crude
extract of E. coli JM101 harboring scbR. Various
crude extracts or unlabelled DNA fragments that were
used in the experiment are indicated as +.
Fig. 3b Gel retardation experiments using E. coli JM101
crude extract harboring scbR and various Y-
butyrolactones are indicated. CD denotes Circular
Dichroism positive (+) or negative (-).
Fig. 4a Dnase I footprinting experiment of ScbR.
Protection of the scbA and scbR promoter region by
ScbR from cleavage by Dnase I is shown by vertical
lines. No.l denotes ScbR binding site No.l and No.2
binding site No.2. Both DNA strands were tested for
protection by Dnase I by using two different 3zP
labelled oligonucleotides. The A and G sequence
ladder were used as size standards. Asterisk
indicates the oligonucleotide which has been
labelled. The presence or absence of crude extracts
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CA 02322241 2002-O1-22
from E. coli JM101 harboring scbR is indicated by +
or -. The numbers underneath the symbol > denote the
concentration gradient of the crude extract added to
the reaction.
Fig. 4b (sEQ.zD.rros.:l2-15) ScbR binding sites No.1 and No.2 from
Dnase I footprinting experiments. The protected sequences
are indicated by lines and the numbering is with
respect to the transcriptional start site of scbA
for binding site No.l and scbR for binding site
No.2. The arrows and pscbA, pscbR indicate the
transcriptional start site and direction of scbA and
scbR, respectively. [ScbA] and [ScbR] indicate the
coding sequence for scbA and scbR, respectively.
Fig. 5a Effect of deletion of scbA or scbR on
antibiotic production in solid grown S. coelicolor
M145. Confluent lawn of M145, M751 and M752 were
grown on SMMS at 30°C for 20h (top plates) or 40h
(bottom plates).
Fig. 5b Effect of deletion of scbA or scbR on response
to SCB1, a y-butyrolactone. Bioassay using spare
suspension of M145(top), M751(left bottom) and
M752(right bottom) as indicator strain and spotted
with 1 ug of SCB1. The plates were incubated at 30°C
for 20h (left panel) or 40h (right panel).
Fig. 6 Effect of deletion of scbA or scbR on the
production of Y-butyrolactones with antibiotic
stimulatory activity. Bioassay of ethyl acetate
extracts from SMMS solid cultures of M145 (pset152)
(top), M751 (pset152) (left side) and M751
complemented with scbA (right side), M752 (pset152)
CA 02322241 2002-O1-22
(right side) and M752 complemented with scbR (left
side). In each case the indicator lawn is M145 and
grown on SMMS at 30°C for 30h.
Fig. 7a S1 nuclease mapping of scbA, scbR, and the
major sigma factor hrdB, using RNA isolated from a
liquid time course of S. coelicolor M145, M751 and
M752 at the numbers indicated. The E, TRAM and S
indicates the exponential, transition and stationary
phases of growth, respectively, and the shaded box
labelled RED and A denote the presence of
undecylprodigiosin and actinorhodin in the mycelium.
The numbers in the box denotes the measurement of
antibiotic production, ACT for actinorhodin, and RED
for undecylprodigiosin, respectively. Numbers in
bold refer to the time points when the antibiotics
were measured, which corresponds to the time of RNA
isolation.
Fig. 7b Bioassay of supernatants isolated at the time
of RNA isolation from M145. The numbers denotes the
different time points indicated in Figure 7a.
Fig. 8 S1 nuclease mapping of scbA, scbR, and the
major sigma factor hrdB, using RNA isolated from
M571 grown on liquid media SMM, with 0 or 31.25ng
final concentration addition of SCB1.
Fig. 9 cssQ ID.NO.:ls) Deduced amino acid sequence of ScbR.
Fig. 10 csEQ iD.NO.:m~ Deduced amino acid sequence of ScbA.
Fig. 11 ~sEQ zD.NO.:se) Deduced amino acid sequence of ScbB.
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Fig. 12 csEQ zD.NO.:i9> Production of Act by S. lividans
strains carrying pIJ68.
Fig. 13 Production of Red by S. lividans strains
carrying pIJ6014.
Fig. 14 Nucleic acid sequence of region containing
scbA, scbR and scbB. M751 (OSCbA) is deleted from
nt position 1320 to 2021; M752 (~scbR) is deleted
from nt position 2359 to 2796 with five bases added;
pIJ6134 runs from nt position 2021 to 4346; and
pIJ6140 runs from nt position 1 to 3430.
The work on which the present invention is based will now
be described, by way of example only, with reference to
these figures,.
EXAMPLE 1 scbA - an S. coelicolor homologue of afsA
Alignment of the amino acid sequences of AfsA from S.
griseus and its homologue, BarX, from S. virginiae
(Kinoshita et al., 1997), revealed two highly conserved
regions (corresponding to amino acid residues 217-223 and
277-285 of AfsA). These sequences were used, with codon
usage data derived from 64 Streptomyces genes (Wright and
Bibb, 1992), to design degenerate oligonucleotides for
use as primers in PCR. BamHI sites were incorporated at
the 5' end of each primer to allow subsequent cloning of
the PCR product. An amplified fragment of the expected
size (189 by including the flanking BamHI sites) was
obtained using S. coelicolor M145 DNA as template. The
PCR product was cleaved with BamHI, and cloned in the
BamHI site of the pUCl9 derivative pIJ2925, yielding
pIJ6114. Sequencing using universal and reverse primers
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revealed an afsA homologue of S. coelicolor, which was
designated scbA.
The BamHI insert of pIJ6110 was isolated and labelled
with 32P by random oligonucleotide priming and used as a
hybridisation probe to isolate four cosmids from an
unaligned cosmid library of S. coelicolor M145 DNA. The
probe failed to hybridise to the ordered cosmid library
of Redenbach et al., 1996 (see below). Digestion of the
four cosmids with BamHI revealed several restriction
fragments of identical mobility, suggesting that each
cosmid respresented the same genetic locus. Southern
analysis of each of the cosmids using the same probe
identified a common 4.5 kb BamHI fragment and a smaller
hybridising fragment that ranged in size from 2.5 kb to
3.0 kb. The 4.5 kb fragment and the 3.0 kb BamHI
fragment from cosmid GB10 were cloned in the BamHI site
of pIJ2925 to yield pIJ6111 and pIJ6114, respectively.
The restriction map of the contiguous 7.5 kb region is
shown in Fig.l. All four hybridising cosmids from the
unaligned library were used as probes to identify their
position in the combined physical and genetic map of the
S. coelicolor chromosome. scbA was localised to the gap
that lies at approximately 5 o'clock in the ordered
cosmid library, in AseI fragment B, and next to cosmid
2H4 (H.M.Kieser personal communication; Kieser et al.,
1992) .
EXAMPLE 2: scbA lies adjacent to genes likely to be
involved in y-butyrolactone synthesis and binding
The nucleotide sequence of the 7.5 kb scbA region was
determined (the sequence has been deposited under EMBL
accession number AJ007731). Frame analysis (Bibb et al.,
18
CA 02322241 2000-10-23
1983) revealed open reading frames (ORFs) with predicted
translation products that showed homology to proteins
likely (by analogy to the S. griseus system above) to be
involved in both Y-butyrolactone synthesis and
perception. ScbA (corresponding to nucleotide positions
2914-1970 of EMBL AJ007731 and 2142-1199 of Fig. 14)
shares 64% amino acid sequence identity with ~.fsA, and
about 40% identity with other AfsA homologues. The
deduced amino acid sequence of ScbR, a protein encoded by
a divergent ORF (corresponding to nucleotides 3032-3679
of EMBL AJ007731 and 2261-2908 of Fig. 14) which is
adjacent to scbA, shows high levels of similarity to
several Y-butyrolactone binding proteins. It is 56%
identical to FarA of S. lavendulae FRI-5, and 45%
identical to ArpA. Each of these homologues possesses an
N-terminal DNA-binding domain that is also found in the
TetR family of transcriptional repressors. The C-
terminal regions of the ScbR family of proteins are
relatively poorly conserved, and the inventors propose
that this may reflect their ability to bind different Y-
butyrolactones.
Downstream of ScbR, and transcribed in the opposite
orientation, lies ScbB (nucleotides 3795-4529 of EMBL
AJ007731 and 3024-3758 of Fig. 14), whose predicted
product shows 50% amino acid identity to a C-5
ketoreductase from S. avermitilis (Ikeda et al., 1999);
based on BLAST search (Altschul et al., 1997).
EXAMPLE 3: Transcription of scbA and scbR occurs in a
growth-phase-dependent manner
S1 nuclease protection experiments were carried out to
determine the transcriptional start sites of scbA and
19
CA 02322241 2000-10-23
scbR using RNA isolated from S. coelicolor M145 cultured
in SMM to different stages of growth. A 259 by PCR
product (nucleotides 2786-3055 of EMBL, 2015-2284 of Fig.
14) labelled uniquely at the 5' end at position 2786/2015
was used as a probe for scbA transcripts, while a 280 by
PCR product (nucleotides 2894-3174 of EMBL, 2123-2403 of
Fig. 14) labelled uniquely at the 5' end at position
3174/2403, was used as a probe for scbR. Putative
transcriptional start sites were identified 46
nucleotides upstream of the likely translational start
site of scbA, and 123-124 nucleotides upstream of that
for scbR (Fig 2a), i.e. at nucleotides 2960 and 2909-8,
respectively (of the EMBL sequence, 2189 and 2138-7 of
Fig. 14). Transcription of scbA, which was undetectable
during exponential growth, increased markedly at late
transition phase, and fell quickly as the culture entered
stationary phase (Fig. 2b). The scbR transcript, while
detectable during exponential growth, increased markedly
in level in late transition phase, approximately one hour
after the increase in the level of the scbA transcript.
It also fell in stationary phase, after the decline in
the level of the scbA transcript. The transcript of the
major and essential sigma factor gene, hrdB, was used as
a control, and was present at essentially constant levels
through exponential growth, and fell gradually upon entry
into stationary phase.
EXAMPLE 4: Binding of ScbR to the promoter regions
of scbA and scbR is prevented by SCB1
scbR was expressed in E. coli JM101 by cloning the l.2kb
HincII-PvuII fragment containing scbR (Fig. 1) in
pIJ2925, yielding pIJ6120. Extracts of JM101/pIJ6120 were
then used in gel retardation assays with a 5' end-
CA 02322241 2000-10-23
labelled PCR product that contained the.scbA and scbR
promoter regions (Fig.3a). Retardation of the scbAR
promoter fragment was readily detected on addition of the
JM101/pIJ6120 extract (indicating binding of ScbR to the
promoter region); no retardation was observed if the
extract was first boiled, or with extract isolated from
JM101 (Fig.3b). Addition of an excess of unlabelled PCR
product resulted in a reduction in the proportion of the
labelled promoter-containing fragment that was retarded;
however, no competition was apparent when unlabelled
Streptomyces DNA (the plasmid pIJ922) was added,.
indicating a specific interaction between ScbR and the
promoter DNA.
SCB1 (which, like A-factor, is a GBL) and its three
chemically synthesized stereoisomers (Takano et al.,
2000) were added to the gel retardation assays to assess
their ability to influence the DNA-binding activity of
ScbR. Formation of the DNA-protein complex was markedly
reduced by addition of lug of SCB1, while addition of
equivalent amounts of each of the stereoisomers had
little or no effect (Fig 3c). Equivalent amounts of A-
factor, IM-2 and VB also failed to inhibit the DNA-
binding activity of ScbR (data not shown) indicating that
the specificity of ScbR for SCB1, its cognate y-
butyrolactone, is high.
DNase I footprinting was used to determine the location
of the DNA sites to which ScbR binds. Two protected
regions were identified (Fig 4a and b); one lies at
nucleotide position -4 to -33 with respect to the scbA
transcriptional start site (i.e. nucleotides 2964-2993 of
the EMBL sequence, 2193-2222 of Fig. 14), while the other
lies at nucleotide position -41 to -67 with respect to
21
CA 02322241 2000-10-23
the scbR transcriptional start site (i.e. nucleotides
2867/8-2841/2 of EMBL, 2096/7-2070/1 of Fig. 14).
Dilution of the JM101/pIJ6120 extract suggests that ScbR
has a stronger affinity for the binding site upstream of
scbA than for that upstream of scbR.
EXAMPLE 5: Deletion of scbA abolishes y-butyrolactone
synthesis, but results in overproduction of Act and
Red, while deletion of scbR also abolishes y-
butyrolactone synthesis, but causes delayed Red
production
To assess the role of scbA and scbR in antibiotic
production in S. coelicolor, in-frame deletions were made
in each gene. Mutant scbA and scbR alleles were
constructed in which most of the scbA and scbR coding
regions (amino acids 42-276 out of 315, and 33-178 out of
216, respectively) were deleted . The mutant scbA and
scbR alleles were cloned in the E. coli plasmid pKC1132,
yielding pIJ6140 and pIJ 6134, respectively (Fig. 1),
and introduced into S. coelicolor strain M145 by
conjugation; selection for apramycin resistance ensured
integration of the non-replicating plasmids into the
streptomycete chromosome by homologous recombination.
After three rounds of sporulation on non-selective
medium, apramycin-sensitive segregants were screened by
PCR, and putative scbA (M751) and scbR (M752) deletion
mutants further confirmed by Southern analysis.
Confluent lawns of the parental strain M145, M751
(~scbA) and M752 (~scbR) were grown on nitrogen-limited
SMMS agar plates to assess the affect of each deletion
(Fig. 5). After 20 h, Red production had just begun in
M145, while Act synthesis was undetectable. In contrast,
22
CA 02322241 2000-10-23
M751 had produced large amounts of both Red and Act
(detectable by exposing the agar plate to ammonia fumes,
which resulted in the blue pigmentation characteristic of
Act), and M752 had failed to produce either antibiotic.
S By 40 h, the overproduction of both Act and Red by M751
was very marked, while Red production was noticeably
delayed and Act production detected earlier ~.ig. 5A) in
M752 as compared to M145. The mutant phenotypes were
also observed on rich R5 agar and on phosphate-limited R2
agar, but both mutants resembled the parental strain on
rich SFM agar and minimal medium containing mannitol as
carbon source. Growth of the strains in SMM liquid
medium gave phenotypes that corresponded to those
observed with SMMS agar.
To assess thq ability of the mutants to respond to SCB1,
1 ug of chemically synthesised SCB1(Takano et al (2000)
supra) was spotted onto confluent lawns of M751, M752 and
M145 (Fig. 5b). While M145 responded in the expected way
to exogenous SCBl, M752 did not respond. Since M751
precociously overproduced both Act and Red, it was not
possible to determine whether it had retained the ability
to respond to the the Y-butyrolactone; however, the
inhibitory effect of high concentrations of SCB1 on
antibiotic production in M145 (the lighter halo
surrounding the point of application; Takano et al.,
2000) was also observed with M751, suggesting that it had
indeed retained the ability to sense SCB1.
The ability of the mutants to produce compounds,
including SCB1, with antibiotic stimulatory activity was
assessed using the standard bioassay (the ability to
induce precocious Act and Red production in a lawn of
M145). M751, M752 and M145 were grown on SMMS agar and
23
CA 02322241 2000-10-23
in SMM liquid medium, and samples of agar and culture
supernatant from different growth phases were extracted
with ethyl acetate. Neither mutants produced stimulatory
activity (Fig. 6) regardless of growth phase or medium .
To confirm that the mutant phenotypes reflected the
absence of a functional scbA or scbR, rather than a
mutation elsewhere in the genome, scbA or scbR were
reintroduced into M751 and M752, respectively. A 1194 by
PCR product containing scbA and its promoter (Fig. 1,
pIJ6143), and a 1.3 kb BglII fragment containing scbR and
its promoter (Fig.l, pIJ6135), were cloned in E. coli in
pSET152 yielding pIJ6147 and pIJ6135, respectively. The
plasmids were introduced into the corresponding S.
coelicolor mutant by conjugation and selection for
apramycin resistance, and integration at the 45C31
attachment site was confirmed by Southern hybridisation.
All of the mutant phenotypes were restored to those
observed in M145 (Fig. 6 for restoration of SCB1
synthesis).
EXAMPLE 6: scbR regulates the transcription of both
scbR and scbA, and scbA is required for the
transcription of scbA
Since ScbR binds in vitro to the scbA and scbR promoter
regions, and given that SCB1 is able to prevent such
binding, the effect of the scbA and scbR deletions on
transcription of each of the genes in vivo was assessed.
RNA was isolated from SMM-grown M145, M751 and M752
cultures at different stages of growth and subjected to
S1 nuclease protection experiments (Fig. 7a). While the
scbA transcript was readily detected in early transition
phase cultures of M145, it was absent in M751(~scbA) and
24
CA 02322241 2000-10-23
barely detectable in M752 (~scbR) regardless of growth
phase, suggesting that both scbA and scbR are required
for induction of scbA transcription. While the level of
the scbR transcript increased during late transition and
early stationary phase in M145, it was markedly
diminished in the scbA mutant, and highly abundant in
exponential and early transition phase in th~scbR
mutant. These observations suggest that scbR negatively
regulates its own transcription, and that relief of this
repression requires scbA. Transcription of hrdB, the
major and essential sigma factor of S. coeliclor, was
monitored as a control. Antibiotic production (Fig. 7a)
and production of antibiotic stimulatory factors (Fig.
7b) were also assessed at the times of RNA extraction.
The commencement of factor synthesis in M145 corresponded
well with the increase in the scbA transcription.
EXAMPLE 7: Addition of SCB1 to M751 (~scbA)
stimulates scbR transcription but fails to restore
scbA transcription
To assess the effect of addition of exogenous SCB1 on
scbA and scbR expression in the ascbA mutant, in which
transcription of both genes is markedly impaired,
chemically synthesised SCB1 was added at a final
concentration of 31ngm1-1 to a mid-exponential phase
(ODQSOnm= 0.5) culture of M751. While there was a marked
increase in the level of scbR transcription, scbA
transcription in the dscbA mutant was not restored (Fig.
8 ) .
EXAMPLE 8: Deletion of scbA in S. lividans 1326
abolishes gamma-butyrolactone synthesis and results
in increased production of Act and Red by strains
CA 02322241 2000-10-23
containing the multi-copy plasmids pIJ68 or pIJ6014.
The mutant scbA allelle from S. coelicolor (described in
example 5) was introduced into S. lividans 1326 using
pIJ6140. Integration of the non-replicating plasmid was
selected using apramycin. After three rounds of non
selective growth (on SFM agar) colonies were screened for
sensitivity to apramycin (indicating loss of the plasmid
due to a second homologous recombination event). 4
apramycin-sensitive colonies were identified among 3,000
colonies screened. PCR analysis of chromosomal DNA
produced amplified DNA fragments consistent with that
observed from wild type chromosomal DNA for three
colonies, whereas the fourth colony yielded a smaller DNA
fragment consistent with the in-frame deletion allelle.
Southern hybridisation experiments of chromosomal DNA
digested either with Ncol or a mixture of BglII and Pstl
produced hybridising bands consistent with the results
expected for the wild type arrangement for the first
three colonies and the mutant for the fourth colony,
which was designated S. lividans M707. When this strain
was grown on agar medium no ScbA was detected, whereas it
was demonstrably produced by the wild type S. lividans
1326 strain.
Spores of the M707 strain were inoculated into liquid
YEME medium (containing 0.5% glycine and 5mM MgCl2) and
grown with shaking at 30°C for 2 days. The mycelium was
collected by centrifugation and used to produce
protoplasts, which were transformed with pIJ68 (actII-
orf4) (Passantino R et al (1991) J. Gen Microbiol
137:2059 - 2064), pIJ6014 (redD) (Takano E et al (1992)
6(19): 2797 - 2804) or pIJ486 (vector control) (Ward J M
et al (1986) Mol Gen Genet 203:468 - 478). Thiostrepton-
26
CA 02322241 2000-10-23
resistant transformants were selected and tested in shake
flask fermentation experiments. Spores of the transformed
strains were streaked on SFM agar (containing 50 ~g/ml of
thiostrepton) and incubated at 30°C for 4-5 days. Spores
were harvested and inoculated into spring flasks with
50m1 YEME (containing 50 ~g/ml of thiostrepton). After
two days incubation at 30°C the mycelium was collected by
centrifugation and resuspended in fresh spring flasks
containing phosphate-limited Evans medium with 20 ~tg/ml
of thiostrepton. Incubation was continued at 30°C for a
further seven days with lml samples being removed for
assessment of antibiotic production. The Act or Red
production is shown in Figures 12 and 13 and confirmed
the findings observed for S. coelicolor that antibiotic
synthesis was precocious and elevated. Approximately
three to four' times the concentration of Red was observed
compared to the S. lividans 1326 strain carrying pIJ6014.
For Act the concentration difference was five to ten fold
for the pIJ68-containing strains. Moreover, when dry cell
weight (DCW) measurements were made it was noted that the
M707/pIJ68 strain produced less mycelial material than
the S. lividans 1326/pIJ68 under these conditions. Thus,
when expressed as concentration of Act produced per gram
DCW, the M707 strain produced 121 compared to the control
1.5.
(Plasmids pIJ68 / pIJ6014 were introduced into S.
lividans to supply the pathway specific transcriptional
activator genes for Act / Red production. No equivalent
plasmids are required in S. coelicolor.)
These strains were further tested for their ability to
produce Act in 1 litre liquid batch fermentations in
27
CA 02322241 2000-10-23
stirred tank bioreactors using a modified phosphate-
limited Evans medium (with NH4C1 instead of NaN03). The
M707/pIJ68 strain produced 10g/1 of act compared to the
S. lividans 1326/pIJ68 control, which made 5g/1.
DISCUSSION
Two genes, scbA and scbR, have been isolated from S.
coelicolor A3(2) and respectively show high homology to
the afsA and arpA genes of S. griseus, which encode A-
factor synthetase and A-factor binding protein. The in-
frame deletion mutant of scbA overproduces both
antibiotics, while the in-frame deletion mutant of scbR
is delayed in RED production and does not produce y-
butyrolactones which (in the wild-type strain) cause
precocious RED and ACT production. These phenotypes are
most surprising considering the high homology of the
genes to the A-factor system in S. griseus.
Onishi et al. (1999) reported the cascade for the
streptomycin production in S. griseus, triggered by A-
factor. ArpA (A-factor binding protein) binds to the
promoter region of adpA (a transcriptional activator for
streptomycin production) and represses the transcription
of adpA during early growth cultures. In transition
phase, A-factor is synthesised via AfsA and releases the
ArpA from the promoter region by binding to it. Thus adpA
is transcribed and activates the streptomycin
biosynthetic cluster via strR (streptomycin pathway-
specific activator) and the antibiotic is produced. To
corroborate their model, the afsA mutant (equivalent to
the scbA mutant of the present work) produces neither
streptomycin nor A-factor. Also the arpA mutant
(equivalent to the scbR mutant of the present work)
28
CA 02322241 2000-10-23
overproduces antibiotics; A-factor production is not
effected. These are the reverse phenotypes compared to
those of the in-frame deletion mutants of the present
work using S. coelicolor. The inventors propose that Y-
butyrolactones are involved in antibiotic production
differently in S. coelicolor, compared with the known GBL
model of S. griseus.
The two genes scbA and scbR are located next to each
other in the S. coelicolor genome, which is not the case
for the equivalent genes of S. griseus (afsA and arpA).
afsA is located at the end of the linear chromosome
(Lezhava et al., 1997) thus being easy to mutate to
obtain deletion mutants and arpA is located elsewhere on
the chromosome (Ohnishi et al., 1999). On the other
hand, like the arrangement in S. coelicolor, the
homologues of afsA in S. virginiae (barX) and S.fradiae
(farX) are located next to genes encoding Y-butyrolactone
binding proteins (barA and farA respectively) (Nakano et
al. , 1998; Waki et a1. , 1997) . In S. virginiae, a
mutation in barA (a homologue of arpA) results in
precocious virginiamycin production, consistent with the
role of arpA in S. griseus, yet it abolishes VB
production (Nakano et al., 1998). The inventors propose,
therefore, that the juxtaposition of streptomycete genes
encoding GBL synthetases and GBL binding proteins may be
reflective of a different antibiotic regulatory system
from that of the S. griseus model (possibly in some cases
additional to such a regulatory system), namely one in
which functional deletion of the gene encoding the GBL
binding protein leads not to overproduction of antibiotic
(as in the S. griseus system), but under- or delayed
production. Moreover, they suggest that in such systems,
functional deletion of the GBL synthetase leads to
29
CA 02322241 2000-10-23
overproduction of the antibiotic (in contrast to
abolition of streptomycin production in S. griseus).
MATERIALS AND METHODS
S
Bacterial strains, plasmids, and growth conditions
S coelicolor A3(2) strain M145 (Hopwood et a1.,1985) ,
M751 and M752 (this study) were manipulated as previously
described (Hopwood et al., 1985). E.coli K-12 strains
JM101( Sambrook et al., 1989) and ET12567 (MacNeil et
al., 1992) were grown and transformed according to
Sambrook et al., (1989). Vectors used were pIJ2925
(Janssen and Bibb., 1993), pKC1132 (Bierman et al.,
1992), pset152 (Bierman et al, 1992), pBluescript SK+
(Stratagene), pGEM-T vector (Promega). SMM is the
modified minimal medium of Takano et al., (1992); it
lacks (NHQ) 2S04 and has 0 .25 mM NaH2P04, 0 .25mM KZHP04
instead of 0.5mM each. SMMS is a modified solid version
of SMM, as described above. SFM medium was used to make
spore suspensions and for use in conjugation with E.coli
ET12567 containing the RP4 derivative pUZ8002 (Flett et
al., 1997).
PCR
The synthetic oligonucleotides oligol; 5'-
GACCACGT(CG)CC(CG)GGCATG and oligo2; 5'-
GTCCTG (CG) TGGCC (CG) GT (CG) AC (CG) CG (CG) AC (bracketed nt
indicate positions of degeneracy) were used in the PCR
(Erlich, 1989) to amplify the internal segment of scbA
from S. coelicolor M145 total DNA (Fig. 2a). The reaction
mixture contains: lOx reaction mixture supplied by
Boehringer Mannheim, 200uM final concentration of four
CA 02322241 2002-O1-22
dNTPs, 5% final concentration of DMSO, 50pmo1 of each
primer, SOng of chromosomal DNA in a final volume of
100u1. After denaturation by boiling 5 min, 2.5U of Taq
polymerase was added and subjected to 30 cycles of
denaturation at 94°C for 50 sec, annealing at 55°C for 40
sec and extension at 72°C for 40 sec, and then incubated
at 72°C for 10 min. PCR products were analyzerl-on a 2% w/v
agarose gel electrophoresis.
To complement M751, scbA coding sequence with its
promoter region was amplified by PCR from S. coelicolor
M145 cosmid GB10 DNA. Two synthetic oligonucleotides
5'-GCCAGCAGGTGGGCGACCTGAC,(SEQ.ID.N0.:3)~l~gSnt position) and'
5'-GATCGCCCGGTCCTGCTTGGCCATG (SEQ.ID.N0.:4)(3055nt position) were used.
The PCR conditions were as stated above except the High
Fidelity Kit ,(Beoringher Mannheim) was used and the PCR
cycle was reduced to 20. The PCR product was purified by
a Sephadex G-50 (Pharmacia) spin column then ligated to
the pGEM easy vector (Promega) and transformed to
JM101.The sequence of the transformant was confirmed by
using the ABI automated sequencer and Big Dye dye
terminator cycle sequencing kit (Perkin Elmer).
Nucleotide sequencing
The nucleotide sequencing of the 7.5kb scbA region was
sequenced by the ABI automated sequencer and using the
Big Dye dye terminator cycle sequencing kit (Perkin
Elmer) as recommended by the suppliers, except in the PCR
reaction, final concentration of 5% DMSO was added to the
reaction mixture. The sequence was submitted to the
databases (EMBL AJ007731) and sequenced on both strands.
S1 nuclease mapping
31
CA 02322241 2002-O1-22
For each S1 nuclease reaction, 30 or 90ug of RNA were
hybridized in NaTCA buffer (Murray, 1986; Solid
NaTCA(Aldrichi was dissolved to 3M in 50mM PIPES, 5mM
EDTA, pH7.0) 'o about 0.002pmol (approximately 104Cerenkov
counts min 10-') of the following probes. For scbA the
synthetic oligonulceotide 5'-TATCCAGCTGACCGGGAACGCGTC csEQ.iD.rro.:5),
corresponding to the region within the coding region of
scbA was labelled with ['2P]-ATP using T4 polynucleotide
kinase uniquely at the 5' end of the oligonucleotide,
then used in the PCR reaction with the unlabelled
oligonucleotide s'-ATCGCCCGGTCCTGCTTGGCCATG (SEQ.ID.b10.:6) which
corresponds to a region upstream of the scbA promoter
region to generate a 259bp probe. For scbR, the
synthetic oligonulceotide s'-AAGTAGAGGGCTCCCTTGGTCA (SEQ.ID.NO.:7),
corresponding to the region within the coding region of
scbR was labelled with ['2P]-ATP using T4 polynucleotide
kinase uniquely at the 5' end of the oligonucleotide,
then used in the PCR reaction with the unlabelled
oligonucleotide 5~-C~CTACTGCTTCGGGCATG cs~.ID.NO.:s> which
corresponds to a region upstream of the scbR promoter
region to generate a 280bp probe. Both PCR reactions were
done using M145 total DNA as template. For hrde, the
probe was made as previously described (Buttner et al.,
1990). Subsequent steps were as described by Strauch et
al. (1991) .
Gel retardation assays and Dnase 1 footprinting studies
32
CA 02322241 2002-O1-22
50pmo1 of the synthetic oligonucleotides 5'-CTGCACCCTGGTCCGGTGGACA
(SEQ.ID.N0.:9) arid 5'-ATCGCCCGGTCCTGCTTGGCCATG (SEQ.ID.NO.:10)
were both labelled with (''P]-ATP using T4 polynucleotide
kinase uniquely at the 5' end of the oligonucleotide,
then used in the PCR reaction with the unlabelled
synthetic oligonucleotide corresponding to the other
primer to generate a 244bp DNA fragment. The SCR
amplified fragment was further purified by Qiagen PCR
purification kit. The gel retardation assay reaction
mixture contains; 5x gelretardation buffer(125mM HEPES
pH7.5, 20mM DTT, lOmM ATP, 20% glycerol) 200mM KC1,
0.16ug/ul calf thymus DNA, and 0 to 15u1 of JM101 crude
extract containing ScbR protein in a final volume of 12.5
to 25 u1. The final concentration of DNA fragments used
was 2.5 ng/ml. The mixture was incubated at room
temperature for 10 min then 2 u1 of dye(50%(w/v) glycerol
with BPB in TE) was added to the mixture and 10u1 was
loaded to a 5%(w/v) non-denaturing polyacrylamide gel
buffered with TBE. SCB1 was added to the reaction
mixture either prior to incubation, or after 10 min of
incubation then incubated for further 10 min.
Dnase I footprinting studies were performed as described
by Drapal and Sawer, (1995). 25 ng/ml of DNA fragments
2~ were incubated in gel retardation assay mixture (final
total volume 25u1) with varying concentration of protein.
After incubation, 25u1 of lOmMMgCl and SmMCaClz was added.
After 1 min O.lunit of Dnase I (Boerhinger Mannheim) was
added and incubated for 45 sec then the reaction
terminated by adding 30u1 of stop solution (20mMEDTA,
200mM NaCl, 1% SDS(w/v), 250ug ml-' tRNA). The DNA
fragments were purified by phenol/chloroform extraction
and precipitated with three volumes of ethanol. The
precipitants were resuspended in loading buffer and ran
33
CA 02322241 2000-10-23
on a 6%(w/v) sequencing gel. Sequencing reactions were
performed using the synthesised oligonucleotides as
primers on double strand DNA and by using a dideoxy
sequencing kit (Taq Track, Progema).
S
Crude extract isolation
An overnight culture of E. coli JM101 harboring pIJ6120
was diluted 1/100 and innoculated into 25m1 LB media. The
culture was grown at 37°C for approximately 3.5 hr or
until the cultures were at 1.0 OD6oonm. The culture was
then induced with final concentration of 1mM IPTG. After
further 3 hr of growth, the cells were harvested by
centrifugation and the cell pellet was washed twice with
buffer (50mM Tris pH7.0, 1mM EDTA, 1mM DTT, 100mM PMSF),
resuspended in 500u1 of buffer and disrupted by
sonication. The cell lysate was then clarified by
centrifugation and the supernatant was used as crude
extracts.
Isolation of y-butyrolactones, bioassay and HPLC
analysis
y-butyrolactones were isolated from liquid or solid media
by extracting the culture supernant or the agar with
ethylacetate. The ethylacetate was evaporated and the
sample was resuspended in 100% methanol for use in a
bioassay or for HPLC analysis. Bioassay and HPLC
analysis were conducted as described previously(Takano et
al., 2000).
Construction of an in-frame deletion mutant of scbA and
scbR
34
CA 02322241 2002-O1-22
The in-frame deletion mutant of scbA was constructed by
digesting pIJ6136 which contains a l.4kb flanking DNA of
scbA in pIJ2925 (Fig. 1) with BamHI and end filled using
Klenow fragment and ligated with a l.lkb PvuII -HincII
fragment from pIJ6111. The transformants were analysed to
find the PwII -HincII fragment was inserted with the
internal PstI site at the EcoRI side of the multiple
cloning site of pIJ6136 and designated pIJ6137. The BgIII
fragment of pIJ6137 was inserted into the BamHI site of
pKC 1132 (Bierman et al . , 1992) to give pIJ6140 (Fig. 1) .
The in-frame deletion mutant of scbR was constructed by
PCR using the High Fidelity Kit (Beoringher Mannheim)
with a universal primer and s'-CATCTGCAGCGTGATCGTGGCAGCTTGGTAG
csEQ.zD.NO.:ii> (3130nt position) primer
designed to give a 1.059kb DNA fragment flanking scbA as
described earlier. A PstI site was designed into the end
of this fragment to enble ligation with a PstI site
internal of scbR. pIJ6111 was used as template for the
PCR reaction and the amplified product was cloned into
pGEM-T vector (Promega) to give pIJ6148. The sequence of
the PCR amplified insert of pIJ6148 was confirmed by ABI
automated sequencing. The BamHI-KpnI 3kb fragment of
pIJ6111 was cloned into pBluescript SK" (Stratagene) to
give pIJ6131. The 1.059kb BamHI-PstI fragment was
isolated from pIJ6148 and cloned into the BamHI-PstI
digested pIJ6131 to give pIJ6152. pIJ6152 was then
digested with KpnI and blunt ended then further digested
with BamHI. This 2.48kb DNA fragment was cloned into
pKC1132 digested with BamHI and EcoRV to give pIJ6134
(Fig. 1). Both plasmids were introduced into the
methylation deficient E. coli strain ET 12567 containing
the RP4 derivative pUZ8002 (Paget et al., 1999) and
transferred into S. coelicolor M145 by conjugation.
Single-crossover exconjugants were selected on SFM
CA 02322241 2000-10-23
containing apramycin. Three such single colonies were
then taken through three rounds of non-selective growth
on SFM to promote the second crossover. Spores were then
plated for single colonies which were scored for
S apramycin sensitivity. Deletions within scbA and scbR
were confirmed by PCR using primers correponding to
flanking sequences, and by Southern hybridisation. For
scbA, nine out of 20 apramycin sensitive colonies were
deleted for scbA while 11 had reverted to wildtype. For
scbR, 4 out of 20 apramycin sensitive colonies were
deleted for scbR while 16 reverted to wildtype. The scbA
and scbR deletion mutants were called M751 and M752,
respectively.
To complement the mutants, a 1194bp PCR product
(subsequently sequenced) containing the entire scbA
coding region with its promoter (pIJ6143) and a l.3kb
BglII fragment containing the entire region of scbR with
its promoter (pIJ6135) (Fig. 1) was cloned into a
conjugative vector pset152 (Bierman et al, 1992), which
integrates into the chromosome of S. coelicolor by site-
specific recombination at the bacteriophage ~C31
attachment site, attB (Kuhstoss, E. et al 1991). The
resulting plasmids, pIJ 6147 and pIJ6135 (Fig. 1),
respectively were transferred into S. coelicolor by
conjugation via the E. coli donor ET 12567 containing the
RP4 derivative pUZ8002 (Paget et al., 1999). Exconjugants
were purified by single-colony isolation, and the plasmid
integration were confirmed by southern hybridization.
Other methods
Antibiotic production was determined by extracting
actinorhodin and undecylprodigiosin as described
36
CA 02322241 2000-10-23
previously (Strauch et al., 1991). RNA was isolated as
described in Strauch et al., (1991). Southern
hybridisation was done as previously described (Hopwood
et al., 1985). Probes for southern hybridisation were
S made by labelling DNA fragments or PCR products with 32P
by random oligolabelling (Pharmacia).
Further protocols are performed according to standard
reference texts, such as Hopwood et al. (1985) and
Sambrook et al. (1989), or later editions thereof.
37
CA 02322241 2000-10-23
REFERENCES
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3389-3402
Ando, N., et al. (1997) J. Antibiot 50: 847-852.
Bate, N., et al. (1999) Chemistry & Biology 6: 617-624.
Bibb, M.J., et al. (1984) Gene 30: 157-166.
Bierman, M., et al. (1992) Gene 116: 43-49.
Buttner, M.J., et al. (1990) J Bacteriol 172:3367-3378.
Chater, K.F. and Bibb, M.J. (1997) Regulation of bacterial
antibiotic production. In Biotechnology, volume 7: Products of
Secondary Metabolism. Kleinkauf, H. and von Dohren, H. (eds).
Weinheim, VCH, Germany. pp 57-105.
Chater K.F., and Hopwood D.A. (1993) Streptomyces. In Bacillus
subtilis and other Gram-positive Bacteria: Biochemistry,
Physiology, and Molecular genetics. Sonenshein, A.L., Hoch,
J.A., and Losick, R. (eds.). Washington, D.C.: American
Society for Microbiology, pp. 83-89.
Chakraburtty, R., et al. (1996) Mol Microbiol 19:357-368.
Chakraburtty, R. and Bibb, M. (1997) J Bacteriol 179: 5854-
5861.
Drapal, N. and Gary, S. (1995) Mol Microbiol 16:597-607.
Erlich, H.A. (1989) PCR Technology. New York: Stockton Press.
Fouces, R. et al. (1999) Microbiol 145: 855-868.
Flett, F., et al. (1997) FEMS Microbiol Lett 155: 223-229
Gramajo, H.C., et al. (1993) Mol Microbiol 7:837-845.
Hara, O., et al. (1983) J Gen Microbiol 129:2939-2944.
Hopwood, D.A., et al. (1985) Genetic Manipulation of
Streptomyces: A Laboratory Manual. Norwich: John Innes
Foundation.
Hopwood, D.A., et al. (1995) Genetics of antibiotic production
in Streptomyces coelicolor A3(2). In: Genetics and
Biochemistry of Antibiotic Production. Vining, L. (ed)
Butterworth-Heinemann, Newton, MA, USA. pp. 65-102.
38
CA 02322241 2000-10-23
Horinouchi, S., and Beppu, T. (1994) Autoregulators. In:
Genetics and Biochemistry of Antibiotic Production. Vining, L.
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Horinouchi, S., et al. (1985) J Antibiot 36:636-641.
Horinouchi, S., et al. (1989) J Bacteriol 171:1206-1210.
Ikeda,H., et al. (1999) Proc. Natl. Acad. Sci. 17 ;9509-9514
Janssen, G.R., and Bibb,M.J. (1993) Gene 124: 133-134.
Kieser, H.M., et al. (1992) J Bacteriol 174:5496-5507.
Kinoshita, H., et al. (1997) J Bacteriol 179: 6986-93.
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Kondo, K., et al. (1989) J. Antibiot 42: 769-778.
Kuhstoss, S., and Rao, R.N. (1991) J Mol Bio1 222: 897-908.
Lezhava, A., et al. (1997) Mol Gen Genet 253: 478-483.
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Sambrook, J., et al. (1989) Molecular Cloning. A Laboratory
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Takano, E., et al. (1992) Mol Microbiol 6:2797-2804.
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CA 02322241 2000-10-23
Takano, E., et al. (2000) J.Biol.Chem. 275:11010-11016.
Waki, M., et al. (1997) J Bacteriol 16:5131-5137
Wright, F., and Bibb, M.J. (1992) Gene 113:55-65.
Yamada, Y. (1999) Auto regulatory factors and regulation of
antibiotic production in Streptomyces. In Microbial signalling
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Roberts, D. McL. (eds.) Cambridge: the Society for General
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Yamada, Y., et al. (1987) J Antibiot 40:496-504.
All of the above references (and any later editions thereof)
are hereby incorporated by reference in their entirety,
individually and for all purposes.
CA 02322241 2002-O1-22
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Plant Bioscience Limited
(B) STREET: Norwich Research Park, Colney Lane
(C) CITY: Norwich
(D) STATE: Norfolk
(E) COUNTRY: UK
(F) POSTAL CODE (ZIP): NR4 7UH
(ii) TITLE OF INVENTION: Antibiotic Production
(iii) NUMBER OF SEQUENCES: 19
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Bereskin & Parr
(B) STREET: Box 401, Scotia Plaza, 40 King Street West
(C) CITY: Toronto
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): M5H 3Y2
(v) COMPUTER READABLE FORM:
(A) COMPUTER: IBM PC compatible
(B) OPERATING SYSTEM: PC-DOS/MS-DOS
(C) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,322,241
(B) FILING DATE: 23-OCT-2000
(viii) PATENT AGENT INFORMATION:
(A) NAME: Micheline Gravelle
(B) REGISTRATION NUMBER: 4189
(C) REFERENCE NUMBER: 420-359
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
GACCACGTSC CSGGCATG 18
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GTCCTGSTGG CCSGTSACSC GSAC 24
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
41
CA 02322241 2002-O1-22
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GCCAGCAGGT GGGCGACCTG AC 22
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GATCGCCCGG TCCTGCTTGG CCATG 25
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
TATCCAGCTG ACCGGGAACG CGTC 24
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
ATCGCCCGGT CCTGCTTGGC CATG 24
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
AAGTAGAGGG CTCCCTTGGT CA 22
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
42
CA 02322241 2002-O1-22
CAAAACTACT GCTTCGGGCA TG 22
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CTGCACCCTG GTCCGGTGGA CA 22
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
ATCGCCCGGT CCTGCTTGGC CATG 24
(2) INFORMATION FOR SEQ ID N0: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CATCTGCAGC GTGATCGTGG CAGCTTGGTA G 31
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 401 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID NO:
12:
GGGCAGGACGGCGGTGACCGAGAACCGGTCACCGCCCTTCGGTATCCAGCTGACCGGGAA60
CGCGTCCTGCACCCTGGTCCGGTGGACAAGCGCCATCGGAACCGGCAATGCGGTTTGTTC120
GATCGAGTTGGCATCGGACGCAGAATTGATCAAAACTACTGCTTCGGGCATGGGTCCCCC180
CCAGGAATCATGTGATGCCGAGCTGTTCTGTATGCGCGAACGTTAAGATACAGACTGAGC240
GGTTTTTTTTCTATCCTTCCCGGGGGAGACATGAACAAGGAGGCAGGCATGGCCAAGCAG300
GACCGGGCGATCCGCACGCGGCAGACGATCCTGGACGCCGCGGCGCAGGTCTTCGAGAAG360
CAGGGCTACCAAGCTGCCACGATCACGGAGATCCTCAAGGT 401
(2) INFORMATION FOR SEQ ID NO: 13:
43
CA 02322241 2002-O1-22
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 401
base pairs
(B) TYPE: nucleic
acid
(C) STRANDEDNESS:
double
(D) TOPOLOGY:
circular
(xi) SEQUENCE DESCRIPTION:SEQ ID 13:
NO:
ACCTTGAGGATCTCCGTGAT CGTGGCAGCTTGGTAGCCCTGCTTCTCGAAGACCTGCGCC60
GCGGCGTCCAGGATCGTCTG CCGCGTGCGGATCGCCCGGTCCTGCTTGGCCATGCCTGCC120
TCCTTGTTCATGTCTCCCCC GGGAAGGATAGAAAAAAAACCGCTCAGTCTGTATCTTAAC180
GTTCGCGCATACAGAACAGC TCGGCATCACATGATTCCTGGGGGGGACCCATGCCCGAAG240
CAGTAGTTTTGATCAATTCT GCGTCCGATGCCAACTCGATCGAACAAACCGCATTGCCGG300
TTCCGATGGCGCTTGTCCAC CGGACCAGGGTGCAGGACGCGTTCCCGGTCAGCTGGATAC360
CGAAGGGCGGTGACCGGTTC TCGGTCACCGCCGTCCTGCCC 401
(2) INFORMATION FOR SEQ ID N0: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 14:
Met Pro Glu Ala Val Val Leu Ile Asn Ser Ala Ser Asp Ala Asn Ser
1 5 10 15
Ile Glu Gln Thr Ala Leu Pro Val Pro Met Ala Leu Val His Arg Thr
20 25 30
Arg Val Gln Asp Ala Phe Pro Val Ser Trp Ile Pro Lys Gly Gly Asp
35 40 45
Arg Phe Ser Val Thr Ala Val Leu Pro
50 55
(2) INFORMATION FOR SEQ ID N0: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 15:
Met Ala Lys Gln Asp Arg Ala Ile Arg Thr Arg Gln Thr Ile Leu Asp
1 5 10 15
Ala Ala Ala Gln Val Phe Glu Lys Gln Gly Tyr Gln Ala Ala Thr Ile
20 25 30
Thr Glu Ile Leu Lys
(2) INFORMATION FOR SEQ ID N0: 16:
44
CA 02322241 2002-O1-22
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Ala Lys Gln Asp Arg Ala Ile Arg Thr Arg Gln Thr Ile Leu Asp
1 5 10 15
Ala Ala Ala Gln Val Phe Glu Lys Gln Gly Tyr Gln Ala Ala Thr Ile
20 25 30
Thr Glu Ile Leu Lys Val Ala Gly Val Thr Lys Gly Ala Leu Tyr Phe
35 40 45
His Phe Gln Ser Lys Glu Glu Leu Ala Leu Gly Val Phe Asp Ala Gln
50 55 60
Glu Pro Pro Gln Ala Val Pro Glu Gln Pro Leu Arg Leu Gln Glu Leu
65 70 75 80
Ile Asp Met Gly Met Leu Phe Cys His Arg Leu Arg Thr Asn Val Val
85 90 95
Ala Arg Ala Gly Val Arg Leu Ser Met Asp Gln Gln Ala His Gly Leu
100 105 110
Asp Arg Arg Gly Pro Phe Arg Arg Trp His Glu Thr Leu Leu Lys Leu
115 120 125
Leu Asn Gln Ala Lys Glu Asn Gly Glu Leu Leu Pro His Val Val Thr
130 135 140
Thr Asp Ser Ala Asp Leu Tyr Val Gly Thr Phe Ala Gly Ile Gln Val
145 150 155 160
Val Ser Gln Thr Val Ser Asp Tyr Gln Asp Leu Glu His Arg Tyr Ala
165 170 175
Leu Leu Gln Lys His Ile Leu Pro Ala Ile Ala Val Pro Ser Val Leu
180 185 190
Ala Ala Leu Asp Leu Ser Glu Glu Arg Gly Ala Arg Leu Ala Ala Glu
195 200 205
Leu Ala Pro Thr Gly Lys Asp
210 215
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 313 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Met Pro Glu Ala Val Val Leu Ile Asn Ser Ala Ser Asp Ala Asn Ser
1 5 10 15
Ile Glu Gln Thr Ala Leu Pro Val Pro Met Ala Leu Val His Arg Thr
20 25 30
Arg Val Gln Asp Ala Phe Pro Val Ser Trp Ile Pro Lys Gly Gly Asp
CA 02322241 2002-O1-22
35 40 45
Arg Phe Ser Val Thr Ala Val Leu Pro His Asp His Pro Phe Phe Ala
50 55 60
Pro Val His Gly Asp Arg His Asp Pro Leu Leu Ile Ala Glu Thr Leu
65 70 75 80
Arg Gln Ala Ala Met Leu Val Phe His Ala Gly Tyr Gly Val Pro Val
85 90 95
Gly Tyr His Phe Leu Met Thr Leu Asp Tyr Thr Cys His Leu Asp His
100 105 110
Leu Gly Val Ser Gly Glu Val Ala Glu Leu Glu Val Glu Val Ala Cys
115 120 125
Ser Gln Leu Lys Phe Arg Gly Gly Gln Pro Val Gln Gly Gln Val Asp
130 135 140
Trp Ala Val Arg Arg Ala Gly Arg Leu Ala Ala Thr Gly Thr Ala Thr
145 150 155 160
Thr Arg Phe Thr Ser Pro Gln Val Tyr Arg Arg Met Arg Gly Asp Phe
165 170 175
Ala Thr Pro Thr Ala Ser Val Pro Gly Thr Ala Pro Val Pro Ala Ala
180 185 190
Arg Ala Gly Arg Thr Arg Asp Glu Asp Val Val Leu Ser Ala Ser Ser
195 200 205
Gln Gln Asp Thr Trp Arg Leu Arg Val Asp Thr Ser His Pro Thr Leu
210 215 220
Phe Gln Arg Pro Asn Asp His Val Pro Gly Met Leu Leu Leu Glu Ala
225 230 235 240
Ala Arg Gln Ala Ala Cys Leu Val Thr Gly Pro Ala Pro Phe Val Pro
245 250 255
Ser Ile Gly Gly Thr Arg Phe Val Arg Tyr Ala Glu Phe Asp Ser Pro
260 265 270
Cys Trp Ile Gln Ala Thr Val Arg Pro Gly Pro Ala Ala Gly Leu Thr
275 280 285
Thr Val Arg Val Thr Gly His Gln Asp Gly Ser Leu Val Phe Leu Thr
290 295 300
Thr Leu Ser Gly Pro Ala Phe Ser Gly
305 310
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Arg Ala His Gly Thr Arg Tyr Gly Arg Pro Leu Glu Gly Lys Thr
1 5 10 15
Ala Leu Val Thr Gly Gly Ser Arg Gly Ile Gly Arg Gly Ile Ala Leu
20 25 30
46
CA 02322241 2002-O1-22
Arg Leu Ala Ala Asp Gly Ala Leu Val Ala Val His Tyr Gly Ser Ser
35 40 45
Glu Ala Ala Ala Arg Glu Thr Val Glu Thr Ile Arg Ser Ser Gly Gly
50 55 60
Gln Ala Leu Ala Ile Arg Ala Glu Leu Gly Val Val Gly Asp Ala Ala
65 70 75 80
Ala Leu Tyr Ala Ala Phe Asp Ala Gly Met Gly Glu Phe Gly Val Pro
85 90 95
Pro Glu Phe Asp Ile Leu Val Asn Asn Ala Gly Val Ser Gly Ser Gly
100 105 110
Arg Ile Thr Glu Val Thr Glu Glu Val Phe Asp Arg Leu Val Ala Val
115 120 125
Asn Val Arg Ala Pro Leu Phe Leu Val Gln His Gly Leu Lys Arg Leu
130 135 140
Arg Asp Gly Gly Arg Ile Ile Asn Ile Ser Ser Ala Ala Thr Arg Arg
145 150 155 160
Ala Phe Pro Glu Ser Ile Gly Tyr Ala Met Thr Lys Gly Ala Val Asp
165 170 175
Thr Leu Thr Leu Ala Leu Ala Arg Gln Leu Gly Glu Arg Gly Ile Thr
180 185 190
Val Asn Ala Val Ala Pro Gly Phe Val Glu Thr Asp Met Asn Ala Arg
195 200 205
Arg Arg Gln Thr Pro Glu Ala Ala Ala Ala Leu Ala Ala Tyr Ser Val
210 215 220
Phe Asn Arg Ile Gly Arg Pro Asp Asp Ile Ala Asp Val Val Ala Phe
225 230 235 240
Leu Ala Ser Asp Asp Ser Arg Trp Ile Thr Gly Gln Tyr Val Asp Ala
245 250 255
Thr Gly Gly Thr Ile Leu
260
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4346 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: circular
(xi) SEQUENCE DESCRIPTION: SEQ 19:
ID NO:
GTCGACGACG GCGTCGGGTT CGACGCCGAC TTCCCGGCCACCGGGCACCG60
GCGGTACTCG
GGTCTGCGCT CGATGACCGA CCGCATCGAG GGCGGCTCCTGATAGTGAGC120
GACGTCGGCT
GGCCCCGCCG GCGGCACGCA CATCGACGTC TGCGCCCCCGGAAAGTGAGC180
CATCTCCCAC
ACCGCACCGC GGACGTGACG CCATGGGAGG CGGACGGATCACCCCTGGCT240
GCCACGTCCG
TCGGCCGAAG GCTTCCGCGT GGTCCGCCGC AACGGCCTGGCGGGCCGGCC300
CCAGATGCGG
CGTCACTTCC CGCACGGTCG GCACGACCTG CCCGCCCGCTGCCGCTCGGC360
CGCCTTGGCC
47
CA 02322241 2002-O1-22
GCTCTCCAGG CGACGGGCCTCGGATACTTC CGGAGCATCTGCTCGCGCGC420
AACGCGTCGG
CGCCTCCAGCCCCAGCTCCTCGAAACGCAGTGACCGCCCC AGCACCTCGGAGAGCCGCGC480
CGTCTGCTGCCTGGCGGTGATCGCCTCGGGCCCGGACAGC GCGTACGCCCGTCCCTCGTG540
GCCGGGCCGGGTCAGTGCCCTGACCGCCACTTCCGCGATG TCGCGCGGATCGACGCAGGC600
AACCGGGGACGTGCCGTACAGCGCGCGGACCACGCCGTCG GACCGGATGGCGGGCGCCCA660
GGACAGCGTGTTGGACATGAAGGTCCTGGCCCGCAGGAAG GTCCAGTCTAGCCCGGACTC720
GCGTACGGCCCGCTCGTTCTCGCGCTGCCGCCGCGTGATG AAGTCGTCCGCGCCCGGTTC780
CCCCACCGCGAGCATGGACAGCTTCACCAGGTGCCGGACG CCGGCCTCGCGCGCCGCCGC840
CGCGAAACGCTCGTCGTCCGGCTCGGTGGCACTGTTCGTG ACGAGGAACGCCGCCCGCAC900
CCCGTTGAGGGCCCGGTCCAGGCCCGGGCGGTCGGCGTAC TCGCCCGCGCAGACCTCGAC960
GTTCGGGCCGGTGACGGTCACCCGTTCCGGCCGCCGGGCG AGGACTCTGACGGGACCGGT1020
CCGGGCCAGCAGGTGGGCGACCTGACGGCCGACCACACCG GTCACGCCGGTCACAAGAAT1080
CACTCGGGGCTCCTCTCGGGCAGCGAGGCAGGGGCGCCTC CGAACATACATATGAGGGGA1140
AGGGCAGGATCTGCCCCGGGGCGCGAACCGGCGATGTTCG CGCCCCGGGGCCGGTGCTTC1200
AGCCGGAGAACGCGGGGCCGGACAGCGTGGTGAGGAAGAC GAGGCTGCCGTCCTGATGCC1260
CGGTGACCCGCACGGTGGTCAGCCCCGCCGCCGGCCCCGG CCGGACCGTCGCCTGGATCC1320
AGCACGGGCTGTCGAACTCCGCGTACCGGACGAACCGGGT GCCGCCGATCGACGGCACGA1380
AGGGCGCCGGACCGGTCACGAGGCACGCCGCCTGCCGTGC CGCCTCGAGCAGCAGCATGC1440
CCGGTACGTGGTCGTTGGGGCGCTGGAAGAGGGTCGGGTG ACTGGTGTCCACCCGCAGTC1500
GCCACGTGTCCTGCTGCGAACTCGCCGACAGGACCACGTC CTCGTCGCGGGTGCGACCGG1560
CGCGCGCCGCGGGCACGGGCGCGGTCCCGGGCACCGATGC GGTGGGAGTCGCGAAGTCGC1620
CGCGCATCCGCCGGTAGACTTGAGGACTGGTGAAGCGCGT CGTGGCAGTCCCCGTGGCAG1680
CGAGCCGTCCGGCGCGGCGCACGGCCCAGTCCACCTGTCC CTGTACGGGCTGCCCGCCGC1740
GGAACTTCAGCTGGGAACAGGCCACTTCCACCTCCAGCTC CGCGACCTCGCCCGACACGC1800
CGAGGTGGTCGAGGTGGCAGGTGTAGTCCAGCGTGGCCAT CAGGAAGTGGTAGCCCACCG1860
GCACGCCGTAGCCGGCGTGGAAGACGAGCATCGCCGCCTG ACGCAGGGTCTCGGCGATCA1920
GCAGCGGATCGTGTCGGTCCCCGTGGACCGGTGCGAAGAA CGGGTGGTCGTGGGGCAGGA1980
CGGCGGTGACCGAGAACCGGTCACCGCCCTTCGGTATCCA GCTGACCGGGAACGCGTCCT2040
GCACCCTGGTCCGGTGGACA GAACCGGCAA TGCGGTTTGTTCGATCGAGT2100
AGCGCCATCG
TGGCATCGGA CTGCTTCGGG CATGGGTCCCCCCCAGGAAT2160
CGCAGAATTG
ATCAAAACTA
CATGTGATGCCGAGCTGTTCTGTATGCGCG 2220
AACGTTAAGA
TACAGACTGA
GCGGTTTTTT
TTCTATCCTT 2280
CCCGGGGGAG
ACATGAACAA
GGAGGCAGGC
ATGGCCAAGC
AGGACCGGGC
GATCCGCACG 2340
CGGCAGACGA
TCCTGGACGC
CGCGGCGCAG
GTCTTCGAGA
AGCAGGGCTA
CCAAGCTGCC 2400
ACGATCACGG
AGATCCTCAA
GGTGGCCGGG
GTGACCAAGG
GAGCCCTCTA
CTTCCACTTC 2460
CAGTCCAAGG
AAGAACTGGC
GCTGGGCGTC
TTCGACGCCC
AGGAACCACC
48
CA 02322241 2002-O1-22
ACAGGCCGTTCCGGAGCAACCCCTCCGGCTGCAAGAACTCATCGACATGGGCATGTTGTT2520
CTGTCACCGCTTGCGCACGAACGTCGTGGCCCGGGCCGGCGTGCGCCTCTCCATGGACCA2580
GCAGGCGCACGGTCTCGATCGCCGAGGACCCTTCCGTCGCTGGCACGAGACACTCCTGAA2640
GCTGCTGAACCAGGCCAAGGAGAACGGTGAGTTGCTGCCCCATGTGGTCACCACCGACTC2700
GGCCGATCTCTACGTGGGCACGTTCGCCGGGATACAGGTCGTGTCCCAGACGGTCAGCGA2760
CTACCAGGACCTCGAACACCGCTACGCGCTGCTGCAGAAGCACATCCTGCCCGCCATCGC2820
GGTTCCCTCCGTGCTGGCCGCGCTCGATCTCTCCGAGGAGCGCGGAGCACGCCTCGCGGC2880
CGAACTGGCACCGACCGGGAAGGACTGACCGCCGAAGCGCCCGCACCGGATACCGACCCG2940
CCGTGCCCGAGCGGCCGACCGGGGCCGCCTACGGGCCCGGCGGCGGGCCCGTAGGTCTGC3000
CCTGCGTACCGAAGCGTGGCGGGTCAGAGAATCGTTCCGCCTGTGGCATCGACGTACTGG3060
CCGGTGATCCACCGTGAGTCGTCGGAGGCCAGAAAGGCCACCACGTCGGCGATGTCGTCG3120
GGTCTGCCGATGCGGTTGAACACGGAGTTGGCGGCCAGTGCCGCGGCCGCCTCGGGGGTC3180
TGCCGCCGCCGTGCGTTCATGTCCGTCTCCACGAAACCCGGCGCCACCGCGTTGACCGTG3240
ATCCCCCGTTCCCCCAGTTGCCTGGCCAGGGCGAGCGTGAGCGTGTCCACCGCACCCTTG3300
GTCATCGCGTATCCGATGGACTCGGGGAACGCGCGCCGGGTCGCGGCAGACGAGATGTTG3360
ATGATCCGCCCGCCGTCGCGCAGTCGTTTCAGTCCGTGCTGGACCAGGAACAGCGGTGCC3420
CGGACGTTGACGGCGACCAGTCGGTCGAAGACCTCCTCGGTGACTTCCGTGATCCGTCCC3480
GAGCCGCTGACGCCCGCGTTGTTCACCAGGATGTCGAACTCGGGCGGCACTCCGAACTCG3540
CCCATCCCGGCGTCGAACGCCGCGTAGAGCGCGGCCGCGTCACCCACGACGCCGAGTTCG3600
GCCCGGATGGCCAACGCCTGTCCGCCGCTGCTCCGGATGGTCTCGACGGTCTCTCGCGCC3660
GCCGCCTCGCTGCTGCCGTAGTGGACTGCCACGAGCGCCCCGTCCGCGGCCAGCCGCAGG3720
GCGATACCGCGTCCGATGCCCCGGCTTCCCCCGGTCACCAGGGCGGTCTTGCCCTCCAGC3780
GGTCTTCCATACCTCGTCCCATGTGCACGCATATCAGCCCCCGCCGTGCGTGAGCGACCC3840
ATGGCGGCCGCTCGGCCGTTCGAATCGACGGTCACAGCCTACCTGTGACCGCGTCAGACG3900
GGGCCGGAGTGGCCCGGTTGGACGGCTGGGGCCAGATCGGGCGGCGCGCACGGGGAACCG3960
GCGCCGGTCAGGGGTCAGGGGTCGCCGGGACCGCCCAGGCCGGTCAGGGCACCGACCGGA4020
TCGAGGTCGGGCGTGCCACGCGGCCACCAGTCCTCGCGGCCCAGCTCCGACTCGTACGCG4080
TACCAGAGCCCGGTCCGGCCGAGTCTGAGCTGGACGTGGCCGCGCGGGTGGGTGAGGCGG4140
TTGCGCCAGGGGCGGAAGGCGGGGAGGTCGGCGGCGAGCATCATGGGGCGGGCGCGGTCG4200
AAACGGCCGGCCGGCGGGTCCCAGGGCTCCTCCAGGACGTCTAGACCCGCCAACCCGCCC4260
TGCCGCCAGGCGGCGACGGCCCGCGCCAGCTCCGCCGTGTCGCGTCCGGCGGCCGAGGCG4320
AGCGACGCGTAGAGCGCGCGGGTACC 4346
49
