Note: Descriptions are shown in the official language in which they were submitted.
~3~''`5~9
This invention relates to a new process of fa-
cilitating genetic exchange within the genus Streptomyces by
protoplast ~usion.
Genetic exchange, in conjunction with spontaneous
mutation, is a natural method by which microorganisms
maintain the variability needed to adapt to specific en-
vironments. Genetic exchange apparentlv occurs in nature,
at least between members of the same species. Classical
techniques used in the laboratory to effect genetic exchange
between microorganisms include conjugation, DNA transfor-
mation and phage-mediated transduction. This exchange can
involve either chromosomal or extrachromosomal genetic
material. Genetic exchange can lead to hybrid strain
formation by genetic recombination, plasmid transfer,
heterokaryon formation and merodiploid formation. Finding
laborator~ conditions for efficient genetic e~change for
microorganisms which do not possess efficient natural mating
systems, however, can be very time consuming and in some
cases fruitless. Difficulties encountered in D~A trans-
formation include physical and enzymatic barriers to DNAuptake, such as cell walls and nucleases. In phage-
mediated transduction, a major difficulty encountered in
developing a transducing system is isolation and identi-
fication of viruses with transducing properties for the
specific microorganisms in question. The paucity of general
principles and lack of a universal method to effect genetic
exchange in microorganisms has hindered the development or-
efficlent techniques for genetic exchange in many micro-
organlsms .
3~
~-4830 -2-
Nonetneless, genetic exchange remalns a very
impor~ant tool to increase variability within species which
produce economically and therapeutically important meta-
bolites, such as antibiotics. Industrial applications of
this tool include constructing strains which produce high
levels of specific metabolites such as antibiotics, anti-
tumor agents, enzymes and other microbial products having
useful properties and constructing hybrid species which
produce novel metabolites with useful properties.
A more recent method of effecting exchange of
genetic material by forcing cell fusion has been used
successfully in genetic studies with some eucaryotic or-
ganisms. Genetic exchange mediated by cell fusion had not
been demonstrated with procaryotic microorganisms until
Schaeffer et al. and Fodor and Alfodi devised a technique
involving fusion of protoplasts and regeneration of cells of
the genus Bacillus [P. Schaeffer _ al., Proc. Nat. Acad.
Sci. 73, 2151-2155 (1976) and K. Fodor and L. Alfoldi,
; Proc. Nat. Acad. Sci. 73, 2147-2150 (1976)].
:;
We have now discovered that it is possible to
:,
accomplish protoplast-fusion-induced genetic exchange in the
procaryotic genus Streptomyces. This discovery permits the
use of a general and thus extremely important technique for
facilitating genetic exchange within the same or different
species of the economically important genus Streptomyces.
We have also discovered that it is possible to accomplish
protoplast-fusion-induced genetic exchange between the genus
Streptomyces and the closPly related genus Nocardia. Proto-
plast-fusion-induced genetic exchange ~etween other genera
X-4830 -3-
.~
within the family Actinomycetales is also contemplated as
part of our invention.
We have discovered that it is possible to facilitate
genetic exchange within the genus Streptomyces ~y protoplast
fusion. The process of this invention involves a multistep
procedure consisting of 1) protoplast formation and sta-
bilization, 2) genetic exchange by protoplast fusion, and 3)
cell regeneration from fused protoplasts. The regenerated
hybrid, including recombinant, strains produced are then
1~ screened for a specifically-desired property. Examples of
desired properties include production of a known metabolite,
such as an antibiotic or antitumor agent, in greater yield
or in a strain from which it is more easily recovered. In
the case of hybrid species, desired properties can include
production of new, useful metabolites or improved production
of known metabolites.
This invention provides a process of facilitating
genetic exchange within the genera Streptomvces and Nocardia
which comprises using the techni~ue of protoplast fusion.
The new process, is a multistep procedure consisting of 1)
protoplast formation and stabilization, 2) genetic exchange
by protoplast fusion, and 3) cell regeneration from the
fused protoplasts.
Protoplast formatlon and stabilization are ac-
complished by a) growing cells under conditions that sensitize
; them to lysozyme and b) treating these cells with lysozyme
in a hypertonic buffer to remove cell walls and form proto-
plasts. Cell-wall removal may be accomplished by growing
the cells for several generations in a medium containing
X-4830 -4-
subinhibitory concentrations of glycine. Growth in the
presence of glycine renders the streptomycete cell walls
susceptible to the enzyme lysozyme [.~. Okanishi, et al.,
. Gen. Microbiol. 80, 389-400 (1974)]. Although in many
_
cases cells grown in the absence of glycine will form
protoplasts, the pr~toplasts are formed more slowly and less
efficiently.
The medium can be any suitable liquid medium, such
as nutrient broth or trypticase soy broth (TSB). After
growth in the presence of glycine, the cells are treated to
form protoplasts. This is typically done by washing the
mycelia and resuspending in a hypertonic medium. A suitable
hypertonic medium contains, for example, sucrose, magnesium
ions, calcium ions, and phosphate. Medium P described by
Okanishi, et al., supra, is an example of a suitable hyper-
tonic medium. Lysozyme (1 to 2 mg/ml) is added to the cell
suspension, and the resulting suspension is incubated at
about 30 37C until protoplast formation is complete
(0.5 to 2.0 hours). Completion of protoplast formation can
be monitored by phase-contrast microscopy.
Protoplast fusion is achieved by mixing the
protoplasts from the two parental organisms to induce the
fusion of the parental protoplasts. The parental organisms
may be strains of the same species (intraspecies) or dif-
ferent species (interspecies). The protoplast mixture is
centrifuged, and the protoplast pellet formed is resuspended
in a small volume of hypertonic buffer. Fusion is enhanced
by treating the parental protoplasts with polyethylene
glycol (PEG). For example, a solution of PEG in hypertonic
X-4830 ~5~
59
buffer (preferably a 40~ solution) can be added to the
resuspended protoplasts. The resulting rused protoplasts
are plated on a hypertonic agar medium (e.g., medium R2 of
Okanlshi, et al., supra, or variations thereof).
Cell regeneration from the fused protoplasts is
achieved by incubating the fused protoplasts on a hypertonic
agar medium at a suitable temperature. A suitable tem-
perature can be determined from the optimum temperaturcs at
which the parental strains grow. To confirm genetic exchange
it is preferable to use species containing suitable genetic
markers such as auxotrophy or antibiotic resistance.
At this stage it is preferable, particularly in
interspecies genetic crosses, to determine first the phys-
iological growth state of the cells which is optimum for
protoplast regeneration. The efficiency of protoplast
regeneration can vary from <10 5 to 5 x 10 1, depending upon
the physiological state of the cells prior to protoplast
fusion. A copending patent application of Baltz titled
METHOD OF OBTAINING STREPTOMYCES PROTOPLASTS CAPABLE OF
EFFICIENT CELL REGENERATION (Doc]cct ~lo. ~ 447~), Serial No.
~O~3 ~ filed this even date, relates to a process of
obtaining Streptomyces protoplasts which are capable of
regenerating viable cells efficiently. This process in-
volves determining the optimum state for protoplast re-
version. The optimum state, the most competent state, is
the transition phase between the exponential- and station-
ary-growth phases.
X-4830 -6-
The most competent state can be determined by
monitoring the Stre~tomyces growth cycle. This is con-
veniently done usina a turbidometric assay and measuring
change in optical density (OD) at an absorbancy of 600 nm
(A600). In general, Streptomyces species undergo fairly
rapid exponential growth with cell-doubiing times ranging
from about 1.5 hours to several hours at low cell density
(A600 less than l.S) in soluble complex media. As cell
growth reaches A600 readings of 1.5 to 4.0, cells enter a
transition phase which precedes the stationary growth phase.
The transition phase may last from 2 to 24 hours, and cell
mass may increase from 50% to 6-fold during thls growth
phase, depending on the species in question.
Putative hybrid, including recombinant, strains
are then recloned and tested genetically by standard methods
to conflrm that they contain genes derived from both parents.
True hybrid, including recombinant, strains are then tested
for the desired beneficial property.
The process of this invention is useful for
species within the genera Streptomyces and Nocardia which
are normally inefficient or incapable of genetic exchange
because of physical incompatibility. This method permits
exchange and recombination of deoxyribonucleic acid (DNA) at
very high frequencies. The method is especially useful for
genetic exchange between mutant organisms of the same
- species, but also facllitates interspecies gene transfer.
The method is particularly valuable for strain development
~ of the antibiotic-producing Streptomyces.
i 30
X-483Q -7-
For example, application of this method to Strepto-
myces fradiae strains readily gave recombinant yields of 10
'o 10~ per ml or frequencies of l0 4 to 10 3 per viable
protoplast. In our laboratories previous attempts to obtain
genetic recombinants of Stre~tomyces fradiae by standard
methods [see D. A. Hopwood, Bact. Rev. 31, 373-403 (1967)]
were unsuccessful: recombinant clones were not detected
(i.e., less than 1 in 107). Thus, the technique of our
invention increases the probability of detecting a specific
S. ~radiae recombinational event by at least 104-fold.
Our invention is also sui-table for transmission of
extrachromosomal DNA (plasmids), normally non-autotrans-
missible, from one Streptomyces species to another. Thus,
under suitable conditions plasmid-encoded genes for anti-
biotic synthesis or regulation can be moved from a strain
with poor metabolic potential for antibiotic synthesis into
a more favorable strain.
In addition, the process of our invention extends
to the closely related genus Nocardia. We have found that
it is possible to accomplish protoplast-fusion-induced gene
transfer within the genus Nocardia and between the genus
Streptomyces and the genus Nocardia.
Our invention will be especially useful for
Stre~tomyces and Nocardla species of economic importance.
Preferred Streptomyces and Nocardia species are those which
produce antibiotics, such as aminoglycoside, macrolide,
beta-lactam, polyether and glycopeptide antibiotics. Use of
our method to effect genetic exchange when at least one
parental organism is an antibiotic producing species within
X-4830 -8-
the genera Streptomyces and Nocardia is, therefore, an
example of an especiall~ preferred application of our
lnvention. For example, effecting genetic exchange ~hen one
parental organism is a macrolide-antibiotic-producing. an
amino-glycoside-antibiotic-producing, a beta-lactam-
antibiotic-producing, a polyether-antibiotic-producing, or
a glycopeptide-antibiotic-producing S eptomyces or Nocardia
strain is a pre~erred application of our invention.
Streptomyces species which are known to produce
aminoglycoside antibiotics include, ~or example: S. kana-
mvceticus (kanamycins), S. chrestomycetlcus (aminosidine),
S. griseoflavus (antibiotic MA 1267), S. microsporeus
(antibiotic SF-767), _, r _osidificus (antibiotic SF733),
S. flavopersicus (spectinomycin), S. spectabilis (actino-
spectacin), S. rimosus forma paromomycinus (paromomycins,
catenulin), _. fradiae var. italicus (aminosidine), _.
bluensis var. bluensis (bluensomycin), _. catenulae
(catenulin), S. olivoreticuli var. cellulophilus (destomycin
A), S. tenebrarius (tobramycin, apramycin), S. lavendulae
(neomycin), S. albogriseolus (neomycins), _. albus var.
metamycinus (metamycin), _. hygroscopicus var. sagamiensis
(spectinomycin), _. blkiniensis (streptomycin), _. griseus
(streptomycin), _. erythrochromogenes var. narutoensis
(streptomycin), S. poolensis (streptomycin), S. galbus
(streptomycin), S. rameus (streptomycin), S. olivaceus
(streptomycin), S. mashuensis (streptomycin), S. hygro-
scoDicus var. limoneus (validamycins), S. rimofaciens
(destomycins), _. h~groscopicus orma glebosus (glebomycin),
S. fradiae (hybrimycins neomycins), S. eurocidicus (anti-
X-4830 -9-
.
biotic A16316-C), S. aquacanus (N-methyl hygromycin B), S.
crystallinus (hygromycin A), S. noboritoensis (hygromycin),
_. hygroscopicus (hygromycins), S. atrofaciens (hygromycin),
S. kasu~aspinus (kasugamycins), S. kasugaensis (kasugamycins),
S. netropsis (antibiotic L~-AM31), S. lividus (lividomycins),
_. hofuensis (seldomycin complex), and S. canus (ribosyl
paromamine).
Streptomyces and Nocardia species which are l~nown
to produce macrolide antibiotics include, for example: N.
gardneri (proactinomycin), N. mesenterica (mesenterin), S.
caelestis (antibiotic M188), S. platensis (platenomycin), S.
rochei var. volubilis (antibiotic T2636), _. venezuelae
(methymycins), S. griseofuscus (bundlin), S. narbonensis
(josamycin, narbomycin), S. fungicidicus (antibiotic
NA-181), S. griseofaciens (antibiotic PA133A, B), S.
roseocitreus (albocycline), S. bruneogriseus (albocycline),
S. roseochromogenes (albocycline), S cinerochromo~enes
--
(cineromycin B), S. albus (albomycetin), S. felleus
(argomycin, picromycin), S. rochei (lankacidin, borrelidin),
S. violaceoniger (lankacidin), S. griseus (borrelidin), S.
maizeus (ingramycin), S. albus var. coilmyceticus (coleimycin),
S. mycarofaciens (acetyl-leukomycin, espinomycin), S.
hygroscoplcus (turimycin, relomycin, maridomycin, tylosin,
carbomycin), S. griseospiralis (relomycin), S. lavendulae
(aldgamycin), S. rimosus (neutramycin), S. deltae (delta-
mycins), S. fungicidicus var. espinomyceticus (espinomycins),
S. furdicidicus (mydecamycin), S. ambofaciens (foromacidin
` D), S. eurocidicus (methymycin), S. griseolus (griseomycin),
S. flavochromogenes (amaromycin, shin~omycins), S. fimbriatus
X-4830 -10-
~ ~ 5~
(amaromycin), ~. fasciculus (amaromycin), S. erythreus
(erythromycins), S. antibioticus (oleandomycin), S.
oli~ochromogenes (oleandomycin), S. spinlchromogenes var.
suragaoensis (kujimycins), S. kitasatoensis (leucomycin), S.
narbonensis var. josamYceticus (leucomycin A3, josamycin),
S. alboariseolus (mikonomycin), S. bikiniensis (chalcomycin),
S. cirratus (cirramycin), S. dja~artensis (niddamycin), S.
eurythermus (angolamycin), _. fradiae (tylosin, lactenocin,
macrocin), S. goshikiensis (bandamycin), S. griseoflavus
(acumycin), S. halstedii (carbomycin), S. tendae (carbomycin),
S. macrosporeus (carbomycin), S. thermotolerans (carbomycin),
and S. albireticuli (carbomycin).
_
Streptomyces and Nocardia species which are known
to produce beta-lactam antibiotics include, for example: S.
lipmanii (A16884, MM 4550, MM 13902), N. uniformis (nocar-
dicin), S. clavuligerus (A16886B, clavulanic acid), S.
lactamdurans (cephamycin C), S. griseus (cephamycin A, B),
-
S. hygroscopicus (deacetoxycephalosporin C), S. wadayamensis
(WS-3442-D), S. chartreusis (SF 1623), S. heteromorphus and
S. panayensis (C2081X); S. cinnamonensis, S. fimbriatus, S.
halstedii, S. rochei and S. viridochromogenes (cephamycins
A, B); S. cattleya (thienamycin); and S. olivaceus, S.
flavovirens, S. flavus, S. fulvoviridis, S. argenteolus, and
. S. sioYaensis (MM 4550 and MM 13902).
-
Streptomyces species which are known to producepolyether antihiotics include, for example; S. albus (A204,
A28695A and B, salinomycin), S. hygroscopicus (A218, emericid,
DE3936), A120A, A28695A and B, etheromycin, dianemycin), S.
griseus (grisorixin), S. conglobatus (ionomycin), ~.
X-4830 -11-
eurocidicus var. asterocidicus tlaidlomycin), S. lasaliensis
(lasalocid), S. ribosidificus (lonomycin), S. cacaoi var.
asoensis (lvsocellin), S. cinnamonensis (monensln), S.
aureofaciens (narasin), S. gallinarius (RP 30504), S.
longwoodensis (lysocellin), S. flaveolus (CP38936), S.
mutabilis (S-11743a), and _. violaceoniger (nigericln).
Streptomvces and Nocardia species which are known
to produce glycopeptide antibiotics include, for example:
N. fructiferi (ristomycin), N. lurida (ristocetin), N.
acti~oides (actinoidin), S. orientalis and S. haranomachiensis
(vancomycin); S. candidus (A-35512, avoparcin), and _.
eburosporeus (LL-AM 374).
In order to illustrate more fully the operation of
this invention, the following specific examples are provided:
EXAMPLE 1
Streptomyces fradiae auxotrophic mutants Al(leu) and
D6(met) were used. Liquid nitrogen suspensions of vegetative
cells (0.5 ml) were inoculated separately into 50 ml of
trypticase soy broth (TSB) containing 0.4% glycine and 0.4
maltose. The cultures were incubated at 37C for 18 hours
wlth aeration (250 RPM; 2.5-cm stroke). The mycelia were
washed by centrifugation and then resuspended in 20 ml of
medium P (M. Okanishi, et al., supra) containing lysozyme (1
mg/ml). The suspended mycelial cells were incu~ated for 2
hours at 30C. The resulting protoplasts were mixed,
centrifuged, and then resuspended in 1 ml of medium P.
Solutions of PEG 6000 in medium P (0.9 ml) at various
concentrations were each added to 0.1 ml of the protoplast
suspension to induce cell-membrane fusion. The fused proto-
X-4830 -12-
,
.
~r ~
?lasts were immedlately diluted in medium P and plated on amodlfied R2 medium (Okanishi, et al., supra, supplemented
~i'h 20~ sucrose ~nd containlng no casamino aclds). The
results are summarized in Table 1.
~-483~ -13-
, I
o ~o l
v
~ ~ ~ o ~ ~ ~ ~
o ~ ~ ~u~ o
u a~
u~ l
~ v
r~ ~ ~ ~ ~
o o o o o o
r~ ~ ~ ~ ~ ~ ~
~ ~ x x x x x ~:
h g ~a~
~ ~ ~r 1-'1 ~1
o
U~
C o
.,, ~ ~ ~ h
O ~ ~ ~ ~ ,1
o\ o\o~O p~
~ ~ o o o U~
p~ I O ~r
O
1 0~O ~
oJ~ O O U~
_~ ~o~ ~ ~ ~ ~ o O
O O ~,~ _~ ~ ~ ~ O
.~U~
a,
O O o o
+ + + + ~, ~ ~
O U~ N
~C ~
2 0 ~ u~ O
? r~ 1 r-~ r~ ~ a
~J --~ ~ r~
u~ Q~ O
~ ~ O ~ O ~ S~
O~1 ~r~ O O h
IJO 1:) ~ CL
CJO ~ r-l.--1 r~ r~ h
S . . . ~ ~ ~ 0 5
O o o O O o ;
1 .
O
O ~
.
.,1 _i ~ ~ ~ U~
~ : '
,
X--4830 -14--
The above mating was repeated using a 40~ solution
of filter-sterllized PEG. A recombination frequency of 3.5
~ 10~ recombinants per ml was obtained. The recombinant
colonies were recloned on nonhypertonic selective medium and
tested for stability. Of the ten recombinants tested, all
were prototrophic and stable ~did not lose selected marker
on extensive subculture under nonselective conditions).
EXAMPLE 2
Streptomvces fradiae auxotrophic mutants were
used. At least one parent strain contained two auxotrophic
markers and a spectinomycin resistance (spc) marker. Each
of the genetically-marked _. fradiae strains was grown in
TSB containing 0.4% glycine. When growth reached an optical
density of 1.5 to 5, as measured at 600 nm in a colorimeter
(Baush and Lomb), the mycelia were washed twice by centri-
fugation and then were resuspended in medium P (M. Okanishi,
et al., supra). Lysozyme (l to 2 mg/ml) was added to the
suspension. The suspended mycelial cells were incubated for
0.5 to 2 hours at 30~ or 34C. The resulting protoplasts
were mixed (0.5 ml of each parent suspension). The mixture
was washed several times by centrifugation, resuspending in
medium P and finally resuspending in 0.1 ml of medium ?. A
solution of 40~ PEG 6000 in medium P (0.9 ml) was added to
the final suspension to induce cell membrane fusion.
Protoplast fusion was confirmed by phase-contrast micro-
scopy. The fused protoplasts were immediately diluted into
one of the following media: medium P containing 40% PEG,
medium P, or distilled water. The dilutions were plated on
medium R2 (Okanishi, et al., supra) to allow detection of
X-4830 -15-
recombir.ation and regeneration of prototrophic recombinants.
The R2 medium used contained asparagine instead of proline
as nitroaen source. Recombinants were counted after 10 to
24 days incubation at 34C. In many of the crosses the
prototrophic recombinants were further tested for the
presence of an unselected marker (spectinomycin resistance)
to eliminate single mutant reversion artifacts. Additional
controls were carried out to confirm recombination. Total
recombinants are based on original volumes of mixed proto-
plasts which generally contained from 108 to 109 proto-
plasts/ml, as determined by direct counting in a hemocyto-
meter.
A summary of several genetic crosses by protoplast
fusion is given in Table 2.
X-4830 -16-
5~
I
ri ~
~ _
J- Q
o U~ o
--1 h ~
~` CO ~ O O O
O ~ ~ ~!
C) ^' ~ ~D X ~ O O O
'1) 0
3 h h
U~ O ~
~ '.~
er r~ ,~
.,~ ~ ~ o o o
o ~ ~ X X X
~1 ~ ~
R ~ ~ ~D o o o o
oe~ . . . ~
J- O ~ ~ ~ I` V '/ V
o t,
h
.` O ~ ~ O ~ O
~-1 '3 ~ ~ N ~, ~4 N
~1 ~ 3:~
+ +
a)
. a
a)
~ ~ + I I +
P~
N E-
a)
R N
u~ ~ I ~q l m l I I I
h
.
~' ~
: ~ QQ, Q Q Q, Q,
nu~ ~ u~ 01 ta
s~ C ~ ~ ~ 4 5 1 s~
E~~
o
,/
~1 ~ ~ ~ ~ U~ ~o
Co
.
3 0
X-4830 -17-
c
~1 ~
;~ c ~n
~ v ~ ~ l
o ~n o ~ ~n
C ~ _ ~
n J ~ `~D c o IC r~) ~n
O ~ ~ \ ~ ~ ~ l_
r~ ~J o o o 3 z z r 5
a~ ol ~
r~
c~, o ~1 a)
I
O
C
` r~ r~ ~
~ rn -- Ul j_
_, ~\ o
S c ~n
',1, fS JJ O O O
O C :: ~ ~ ~ X !` ~
rr~ ~ ~ ~ ~ r~ '.1)
X X r~ o o X
O ~ ~ . ,~ ~ . ~,~
o a~ ,.~, ,.~, ,.
o U ~ a
~ a) 'IJ :c
. ~.
~ U7
~ u a
C
o ~1 ~ o --E~
,~ 3 ~ ~.'1 ~4 ~ P~
I +
~ ~ ,~
,~ a
,~
'~l) ,,,~,
,~ o
,," ~ ` ~,
, ,~
u o o
+ I I + + + 3
P~
O ,~ -o
E~
.
O
~n ,~
o ~ o
~n ~
ml ~ql a I al al ~ ~ o
C ~ ~ ~ ~7 U~ I ~ ~ ~
D a) a~ a~ a~ ~ ~
~,, e e e u u e ~ ~
u~ ~ ~ u
.~ r~ ~ rI~
~ ~ a
Y ~ ,n~ ~ ~n
~ r.~ r~ r~ 1:
,~ u I u I
~ ~ C~ '1) C ~ t~
JJ ,~ ~n ~n _, o ~ '~
.,~ ~1 ra O
c I I I s~ I s~
~ ~ I I I ~ I ~ ~ _ ~ r~ O
p~ ~ ~ r~
~ ,~ ~ a~ ~ ~ a
P~ ~ ~ ,~ ~ ~ e ~
~ ~ ~ ~ U
e e
a ~ s
O ~t C D
.
r` co r~ O
t~
O
X-4830 -18-
.
~ s was the case in Example 1, a low~r, ~ut sig-
nificant, level of recombination was obtained by cen-tri-
fuglng the protoplasts and resuspending in medium P without
?~G. This le~el of protoplast fusion is presuma~ly due to
the presence of Ca in the buffer. Dilution of the proto-
plasts in distilled water reduced the number of recombinants
by 100-fold. Virtually all of the genetic recombinants
.ested contained the spc marker from the strain carrying the
metA arg markers, ruling out the possiblity that reversion
of the metB strain might account for the data. The doubly
.
marked auxotrophic strain has never been shown to revert to
prototropy, thus eliminating reversion of this strain as an
explanation of the results. The other controls in Table 2
give additional evidence that recombination does indeed take
place after protoplast fusion. Upon recloning, all putative
;~ recombinants were shown to be stable. The S. fradiae strain
used is one which produces the antibiotic tylosin. Many
genetic recombinants of this S. fradiae were shown to be
tylosin producers.
EXAMPLE 3
Streptomyces griseofuscus was used in these
genetic crosses. The procedures were the same as those used
in Example 2 except that: 1) the TSB was supplemented with
0.8~ glycine and 2) recombinant colonies were counted after
7 days incubation at 34C. Results are summarized in Table
3. In all six conditions protoplasts were treated with PEG,
diluted in medium P and plated on medium R2. In all cases,
the frequency of genetic recombinants was from 103 to
104-fold higher than background prototrophic revertants.
X-4830 -19-
~s~
I
o
~rr~ ~ ~
ri ~ o o o o
S ~ ~n ~ ~ ~ ~
o ~ ~ x x x x
~' R ~~-1 ~ ~o o o
O E~ ~ . . `. ~
1 0 ~ ~r~ r v
O
a)
,,
, ~1
~ ~ Q
U~ ~ S~ 5.1 S~
a
5~ P~
~:
~1
E~
s~ ~
"~ O a~ 5~ ~ h
P~
O
'~
,~ ~ ~ ~ ~ U~ ~o
~ .,
O
~-4830 -20-
3~
EX~MPLE 4
Streptomyces fradiae (met) and Streptomyces
iniensis (nic ade) T~ere used. Each culture was incubated
according to the procedure described in Example 1. Crosses
made between the strains are listed in Table 4 where con-
ventional mating techniques are compared to the protoplast
fusion technique. Table 4 demonstrates that protoplast
fusion gives at least a 200-fold increase in recombination
frequency compared to conventional mating procedures.
~-4830 -21-
~L~L4~;S~
u~ h ~
~_/ 1~ Q,a ~!
aJ cr~ :~ ~ ~ u~
~ l ~
as 0 ~ ~ CJ~ O ~1:! O
~J O t~ 1 ~1 0 ~1
10s.~ o r-l X I t~
V ~X ~ I ~
J
~/V
~1 :::)
~ rl CL U~
O JJ O
3 1~5
O '
rl
~o
~ ~ ~ ~ JJ S~ .L + +
er O ~ I ,1 C~
C ~ ~X X I
1 a) ,~ 11
O 0 ,, ~ tJ~ :~
~ 1 ~ ~n
v
0 ~ ~
o
P~ z
0 0 0
1 ~
O~ ~
C S-
2'~ 0 a~ I I u~ r~l
'' U~-,l-,l ::~ I I ~~1 D
rl ~ U c~
h CJ a) O u~ ¦ :~ O
Q Q~ ,-1
4 0 ~ ~J
1 X r
o
~,) ~ ~ tn o
a) u~ u~
-1 ~ Q.~ r~
~:: U~ C
1 .~ ~ .
~ o '1 .~, ~a o
) ~ ~ ~ r~)
r-l r-l h ~J
Q ~1 P~:
~ . . .
: r~
X-4830 -22-
g
~ I
~1 I l; :~
a
-l ~r
o~
u~ u~ s:: O 4 Q R ~ ~ ~ R ~1 .4
L~ u~ O S U~
a) I I I a~
~: o o o ~ O S~
S~ ~ ,_1 ,1 )~ C) O
~ ral XXX~ ~ ~
r~
a) . . . a~ a) + +
O ~ ~
1 0 a
, u~ ~ r` co U~ t~
, ~
o o o :~ ~:
XXX I
'~ 1 u~ I
1 ~
V V
~a ,, ~ h
~1 u a)
.a a~ ~ ~I
0 ~ ~ + + + + + I + + + +
8 ~
+ I ~ I
~a
a
0
E~ ~ ~ U ~ ~ aJ a~ a~ ~ ~ a) ~ r~ c
0 0 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ .~ .
~, ~ h a
2 0 ~
~ 'I 'I u
O U~ C
~: C ~1 X X ~ ~:
:~ 3 3 ~ ~ u~ u~ ~ ~ z
u~ u~ u~ 0 o
a) .~ ~;
.~ .~ 0
0 0 r~ ~1 .~ ~ O
C~ ~ X ~ ~ ~,
~_1 rl S~
~ X ~ ~ ~ ~ X
m
X-4830 -23-
~ u~~ ~
~;
~' - ~ o
!~ ; 0 C:~
~ ~1 U~
.
O C~
h 3 N
.ri 0 ,-1 ~1
O
O
O ~ ~S
a) o
3 ~
r;l 0 0 ~-1
0 ~ S O
1 0 ~ 0
O C
O ~-1 0 O
O O
t) ;l)
IV ~ 1 11
O
rl 0
rl ~ ~
Ll S ~ ~1
O O
3 oP ~,
,~ O ~ ~ ~ ~r 0
U ~ 0 O :~ h
~10 ~ rl rl O N
Qrl ~ ~ Q U~
rl ~ O
~ O ~ C~
2 ~ O ~ 1:~ h h~ ~ ~
V t~ ,C 3 a) ~1 h
O ~ O
h ~:: O
~V~ ~1 o Ul ~ ~1
O C ~ C OX O
~1~ O ~1 (~ 1 ~ ~1
R u~ O
~ ~r m ,l + ~ u~l ~ Q
~1 1a) E~ O ~ 11
0-~,1 Q ~1
,
, X-483() 24-
~' ' .
EX~IPLE 5
Several interspecies crosses were studied using
the protoplast-fusion technique. Strains were incubated
overnlght under optimal cultural conditions with growth-
limiting concentrations of glycine. Protoplasts were
isolated from each strain by treatment with lysozyme (1
mg/ml) at 30C for 1 to 4 hours. The resulting protoplasts
were mi~ed and resuspended in medium P (see E~ample 1). A
solution of 40% PEG 6000 in medium P (0.9 ml) was added to
the finaL suspension to induce cell-membrane fusion. As a
control, 0.1 ml of the protoplast suspension was resuspended
in 0.9 ml of medium P. The treated and nontreated proto-
plasts were immedlately diluted in medium P and plated on
selective medium (R2 without casamino acid supplementation).
The following strains were used:
~-4830 -25-
a
u~
x
.
s
l `
v~
~ vl rvl o
o ~1 3 ~
~:: ~ 3 ~ u l a~ l
.,~ I a) ~ I ~ ~ a ~ a ~ ~ .
P~s I ~ ~, I s ~ ~ C V
a
,¢ a) o
~¢ ~ er ~ U
I ~ o U
~ ~ ~ U~ I I ~ o 'l
o ~ ~ ~ ~ ~ ~ ~ S
I` u~ o~ n u~ o
, ~1 ~ ~1 ~ ~r 0 .~ u~ u~ u~ ~ U
~ ~ r¢ ~ U~ ~ C ~ u~ ~
~ z ~D C ~ ~ ~ s~ C a) a~ ,¢ r~ ~ ~ ~ o
o c~ a~ ,~ a) ~ .~, -,~ u~ ~ ~ X
.~ ~ .,, ~ ~ ~ ~ Ul
O ~ ~ O ~ ~ ~ r~ ~ O ~ ~ ~ Q~ a~
~ o ~o ~ ~ ~ ~ ~ ~ ~ ~o ~o ~ 'c ~ o~
u~ ~1 Cl~ ~ ~ Ei aJ E~ ~ C O O ~:1 .~ ~ J h
~1 ~ O tl~ ~ Q. ~ Q~ 11~ ~: Ll 5-1 ~ .Y t~ O
~ O S-l S.l ~,~ ~_1 O ~1 _1 ~1 ~ ~ (1~ ~1 S l 4 U~
1~ O ~S ~1 t) ~1 IJ ~1 t~ t) ~5 ~ t) Q ~1
~1 U~ ~ U~ 01 01 Ul O~ C) C
o a) ~, o a) ~ a.) a~ I a~ ~ a) I a~ I a~ I ~ I O I a~ I ~-1 I 'J~ ri a
~ ~ ~ O O C) C)I~ UlOl ~ s
~ ~ 1 ~ ~ ~ ~ ~ l e l ~ o~ c
o ' 1 o o o 1 1 1 1 1 1 1 l~1 ~ h
Q~ 5~ I Q ~ ~ Ql Q.l ~1 ~I P~ 1 1 0 11
,~la) a) ~ o ola)la)la~la)lolola)l 31X
C~ h S-l ~1 ~ l h
~ 01~ ~J ~ ~ ~JI~JI~JIJ~I~I~I~JI~JI~ql 01
u~ ZIcl~ u~ u~ u~ u~
~,
'',
.
X-4830 -~6-
u~ l
2~
v ~ o ~ o~ ~ ~ ~ ~ o ~ o ~ o
O o o ~ o3 o oo o O o O O o
X ~ X X X X X X X X ~ X X X
O o o U~ o o o ~ ~ o o o o o o
.. .... .. .. .. ..
~ _ V `/ `./ iJ
Z ~
es~ .~
+ + ~a ~
V Q~ O
O ~
+ +
~a + + t~
~,~
~1 U~
+ I + I+ I + I+ I + I + I h
~ . ~r
Lr) ~
Q I I I I
z z l l~J7 ~ ~ ~ ~ ~
~ ~ 1~ 1~0 0 ~ ~ ~ ~ I
0 0 ~ ~u~ u~ ~ ~ l l ~ ~ ~l
.,, .,,, cO 0~ ~ ~ u, 0 a) a~ ~ ~ tn u~
r-~ ,-~ ~ ~~,1 .,J, ~ 3 .,, ~1,
O O ~ ~ h ~C~ V u3 tn ~ O
Q~ ~ U~ rrl tr5 ~ a) ~ ~d ~ 5
'~/ ~1 ~-I ~ ~ ~:J ~ ~1 ~:5 ~ ~1 ''~ ~ U~
5~ s~ 0 u~ ~ Q ~ ,~ O O .~ ,~
2 0 ,s ~:: 1:: ~:: ~ a) ~ ~ a) a) ~ ~ ~ ~ o ,
al ~ :: :: 3 :) ~ S~ ~ ~ S~
:~ ~ ~ ~~ a
O O~ JJ 0 ~~ ~ U C~ ~
a~ ~ ~ ~ ,~ . . 0 o
~ ~ u~
ælæl ~ ~ N O
u~ U V X X U~ I X X X X X X ,_~
a~ X X
U~ I er er X X er ~
C~ O O ~ CO ~i?. ~, r~ -1 ~ ~ ~ Ql O
e o ~
X X~ ~O ~ ~U~ U~ ~ ~
_~ ~ ~ C er~l ~, U) ~ ~ ~ ,, Ul
0 U~ ~ ~ 0 0 ~ o ~ ~
~D ~~ ~ ~ ~ ~ ~ a) ~ ,, ,, ~ ~ ~ rl
,~ o a a~ ,~ .,1l~ ~ , . ~ 0 ., ~ ~ 0
O O ~ 1 ~ 1 ,1 .~,~ O O ~ ~ ~ ~ ~ ~ ~
o ~ 0 1 ~ I ~ c~ I ~ I ~ ~ ~ .,~ ,~ 11 o
~1 ~1 ~ ~ 0 1 ~ I ~ 0 O O ~ _
(IJ -1 ~ Q Q, eQI ~ ~ ~:: a) ~ .~ .y 0~ 0Q' ~ ~
O O ~ ~ ~ 1 ~1 ~ ~ ~ ~1 ~ C)
~ ~1 r-l 1~ t~ ~ :~ q a 1~
O
X-483~ -27-
