IE83441B1 - Process for the genetic manipulation of myxobacteria - Google Patents
Process for the genetic manipulation of myxobacteriaInfo
- Publication number
- IE83441B1 IE83441B1 IE1992/0654A IE920654A IE83441B1 IE 83441 B1 IE83441 B1 IE 83441B1 IE 1992/0654 A IE1992/0654 A IE 1992/0654A IE 920654 A IE920654 A IE 920654A IE 83441 B1 IE83441 B1 IE 83441B1
- Authority
- IE
- Ireland
- Prior art keywords
- dna
- homologous
- myxobacterial
- sections
- plasmid
- Prior art date
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
Description
PATENTS ACT l992
920654
PROCESS FOR THE GENETIC MANIPULATION OF MYXOBACTERIA
NOVARTIS AG
The present invention relates to a novel process for
the genetic manipulation of myxobacteria of the
Sorangium/Polyangium group, which makes it possible for
the first time specifically to apply recombinant DNA
techniques to this group of organisms.
invention relates to a
In particular, present
process for insertion of sequences of
homologous or heterologous origin or a combination of
DNA sequences of homologous or heterologous origin into
the chromosome of said myxobacteria via homologous
recombination, and to genetically modified myxobacteria
prepared with the aid of this process.
DNA
plasmids and vectors which are particularly suited for
the the
Likewise embraced are recombinant molecules,
use in process according to invention,
genetically modified myxobacteria of
Sorangium/Polyangium group containing exogenous DNA of
homologous and/or heterologous origin.
The myxobacteria of the Sorangium/Polyangium group are
highly specialised organisms which are commonly
detectable in soil samples, dead plant material or in
animal dung. Characteristic of this group of
microorganisms is their ability to utilise cellulose or
cellulose—containing degradation products as sole C
source. Another characteristic feature of this group is
their ability to highly
metabolites.
produce active secondary
A large number of strains from this group which, for
example, are able to synthesise plant—microbicidal
compounds have now been described. Particularly
important in this connection are the so—called
soraphens, macrocyclic compounds which have a
beneficial biocidal spectrum against phytopathogenic
microorganisms, but especially against phytopathogenic
fungi. These compounds have very advantageous curative,
systemic and ix) particular, preventive properties and
can be employed to protect numerous crop plants
[EP 0 358 606].
It is also known of other representatives of the group
of myxobacteria that they are able to synthesise highly
active compounds with antibiotic potency [Reichenbach
et al (l988)]. Because of the importance of these
compounds, there is a great interest in understanding
the genetic bases of their synthesis in order thus to
provide the possibility of being able specifically to
influence these where appropriate.
The precondition for this is the provision of a process
which makes possible direct, and preferably targeted,
manipulation of these organisms using recombinant DNA
techniques, for example by the targeted incorporation
DNA
into the genome of
of novel genes or gene fragments or other
sequences, including whole plasmids,
the myxobacteria.
EP 0 372 230 describes processes for
conjugative transfer of mobilisable vectors from
E.coli into Gram—positive bacteria, in particular
coryne bacteria.
A few representatives of the group of myxobacteria have
also already been the subject of investigations in this
direction. Special interest in this connection was
primarily directed at .MyXococcus xanthus, which is a
myxobacterium which has now been extensively researched
and for which various gene transfer processes have
already been described. Thus, for example, the coli
phage P1 has been used very intensively, initially for
the insertion of transposon Tn5 into the Myxococcus
xanthus chromosome [Kaiser (1984); Kuner and Kaiser
(l98l)] and then later for the transfer of genes cloned
in Myxococcus xanthus back into the original E.coli
host [O’Conner and Zusman (1983); Shimkets et al
(1983)). Another example relates to the genetic
manipulation of Myxococcus
(l988)].
xanthus by transduction
[Shimkets and Asher,
Another process for gene transfer is based on the use
of the plasmid RP4 which has a very wide host range.
(1985)
transferred via
Breton et al were able to show that this plasmid
can be conjugation from E.coli into
Myxococcus xanthus, and there is stably integrated into
the chromosome. Based on these properties, Breton et al
(1986) (1987)
and Breton and Guespin-Michel were able
to integrate foreign genes into the chromosome of
Myxococcus xanthus. Investigations by Jaoua et al
(1987; 1989) revealed that the observed integration is
based, with a high degree of probability, on a so—
called site specific recombination. The latter is
confined to particular sites, which have a narrow
spatial restriction, within the Myxococcus Xanthus
chromosome and is mediated by one or more so—called hot
spots on the RP4 plasmid. In addition, it has emerged
during the investigations carried out within the scope
of the present invention that the previously known
Myxococcus system discovered here cannot be applied to
bacteria of the Sorangium/Polyangium group. It is
assumed that these organisms lack the specific
structural elements which are necessary for site
specific recombination on their chromosomes. In
addition, it has been found that no stable
transposition takes place with these organisms either,
for example on use of transposon Tn5.
the
the
the
the
the
The object which it was intended to achieve within
scope of this invention thus related primarily to
provision of a universally applicable process for
genetic manipulation of myxobacteria of
Sorangium/Polyangium group, which is free of
abovementioned restrictions of the known processes and
thus permits undirected or else, preferably, targeted
insertion of genetic material into myxobacteria,
independent of structural elements present on the
myxobacterial chromosome or of specific transposition
GVGITCS .
This can be achieved according to the invention, for
example, by preparing genetic constructs, especially
plasmid vectors, which by reason of their specific
structure, can be inserted at the desired sites within
the chromosome and which are not linked, as is the case
in the previously known processes, to
the
organisation of the myxobacterium chromosome or of the
(hot
particular
integration sites predetermined by functional
plasmid used spots), or else dependent on
transpositions of integrated transposons.
The present invention thus relates primarily to a
process for the genetic manipulation of myxobacteria of
the Sorangium/ Polyangium group, which is characterised
in that genetic material of homologous or heterologous
origin or a combination of genetic material of
homologous and heterologous origin is inserted into the
cell
recombination at
myxobacterial and integrated, via homologous
random or else, when there is
appropriate knowledge of the structural and functional
organisation of the bacterial genome, specifically at a
site, which is accurately defined on the basis of the
homology present between the inserted DNA and DNA
intrinsic to the bacteria, into the chromosome of said
myxobacteria, independent of structural elements
present on the myxobacterial chromosome or of specific
transposition events.
The process according to the invention for the genetic
manipulation of myxobacteria of the Sorangium/
Polyangium group is particularly characterised in that
(a) genetic material of homologous or heterologous
origin or a combination of genetic material of
homologous and heterologous origin, which is homologous
with or else at least essentially homologous with a
corresponding region on the myxobacterial chromosome;
or else
(b) genetic material which naturally contains no
sections which are homologous with or else at least
essentially homologous with a corresponding region on
myxobacterial chromosome and which is
therefore
artificially linked with the aid of rDNA techniques
known per se to such homologous or
DNA
genetic material to be
else essentially
of which the
flanked by said
homologous or else essentially homologous DNA sections,
the cell
via homologous recombination, at a
homologous
sections, as a result
inserted is
is inserted into myxobacterial and there
integrated, site,
which is accurately defined by reason of the homologies
present between inserted DNA and DNA which is intrinsic
to the bacteria, into the chromosome of said
myxobacteria.
The integration of the genetic material into the
bacterial chromosome via homologous recombination is
mediated by one or more DNA sections within the DNA to
be inserted, which are homologous with or else at least
essentially homologous with a corresponding region on
the myxobacterial chromosome. It is thus not linked to
particular chromosomal structures and can in principle
take place at any desired site within the bacterial
chromosome.
The homologous DNA sections which can be used within
the scope of the process according to the invention do
identity with the
corresponding sections on the myxobacterial chromosome
able to the
not necessarily have to have lOO%
in order to be bring about required
recombination event. On the contrary, it suffices for
these DNA sections to be essentially homologous with
the corresponding regions on the bacterial genome, that
is to say when these have a
and 100%
between 90% and 100%.
degree of homology of
between 80% and Very particularly preferably
Thus, “homologous” DNA sections are intended also to
mean hereinafter those sections which do not have lOO%
with the the
myxobacterial chromosome but are at least “essentially
identity corresponding regions on
homologous” therewith.
It is possible in this connection for the homologous
DNA to be
organism itself or else
sections isolated either from the
target
from related organisms, for
example after fragmentation of the particular genome.
When the DNA sequence of the DNA sections on the
myxobacterial chromosome which are intended in each
case for integration is known, the corresponding DNA
fragments which are homologous with or else at least
essentially homologous with these chromosomal sections
can, of course, also be prepared by synthesis.
The DNA sections of homologous origin which can be used
within the scope of the present invention can moreover
be either DNA sections of known sequence or else DNA
obtainable at random, for after
fragments example
restriction digestion of homologous DNA.
When the structural and functional organisation of the
appropriate parts of the bacterial genome is known, the
process according to the invention thus makes possible
for the first time a targeted, predictable modification
of the genes present on the myxobacterial genome (in
situ modification) by exchange of natural or
artificially modified genes, gene fragments or other
useful DNA sequences with homologous DNA sections
within the bacterial genome. The process according to
the invention furthermore makes possible a specific
identification of the function of individual genes
within the complete genome of myxobacteria of the
Sorangium/Polyangium group by specific switching off of
genes [gene disruption] or by complementation of genes
which have previously been inactivated by application
of other processes, especially of mutation processes.
Besides the targeted and thus very efficient
the
in situ
modification of genes intrinsic to bacteria
invention has a number of other possible applications
(directed mutagenesis), the process according to
such as, for example, the incorporation of additional
gene copies in regions known to have a high expression
rate, and elimination and thus switching off of
unwanted genes. It is furthermore also possible now to
think about the following further possible
applications.
- Incorporation of strong or controllable promoters in
front of genes, intrinsic to the bacteria, with
interesting functions.
- Cloning of genes of various origin into myxobacteria
of the Sorangium/Polyangium group.
— Cloning and expression of genes of various origin.
— Transfer of the inserted DNA back into another
microorganisn1 such as, for example, into E.coli for
further processing.
— Use of the heterologous DNA of an inserted vector
firstly as radioactive probe for identifying the
myxobacterium fragments adjacent to the site of
integration, and secondly as pattern [template] for
amplification within the
[PCR].
scope of a. polymerase chain
reaction
The present invention further relates to a process for
the preparation of genetically modified bacteria of the
Sorangium/Polyangium group, which is characterised in
that
(al) genetic material of homologous or heterologous
origin or a combination of genetic material of
homologous and heterologous origin, which is homologous
with or else at least essentially homologous with a
corresponding region on the myxobacterial chromosome;
or else
(a2) genetic material which
sections
naturally contains no
which are homologous with or else at least
essentially homologous with a corresponding region on
the
artificially linked with the
and is therefore
aid of
such homologous or
myxobacterial chromosome
rDNA techniques
se to
DNA
known per else essentially
which the
flanked by
homologous or else essentially homologous DNA sections;
the cell
recombination, at a
homologous
sections, as a result of
genetic material to be inserted is
said
is inserted into
myxobacterial and there
integrated, via homologous site,
which is accurately defined by reason of the homologies
present between inserted DNA and DNA which is intrinsic
to the the chromosome of said
bacteria, into
myxobacteria; and
(b) positive transformants are selected with the aid of
selection processes known per se and cultivated as pure
culture.
The present invention thus makes possible for the first
time a targeted genetic manipulation of the genome of
myxobacteria of the Sorangium/Polyangium group, where
the integration of the genetic material can be
mediated, depending on the particular aim of the
planned procedure,
DNA
alternatively either by homologous
sections of known sequence or else by randomly
selected homologous sections of unknown sequence.
The present invention likewise embraces recombinant DNA
molecules which, by reason of their specific
construction, are able to integrate genetic material
such as, for example, genes or gene fragments or other
useful DNA sequences which, where appropriate, code for
novel and desirable properties, with the aid of
homologous recombination at randoH1 or else, when the
structural and functional organisation of the bacterial
genome is known, also targeted at sites, which are
accurately defined by reason of the homologies present
between inserted DNA and DNA intrinsic to the bacteria,
within the bacterial genome, as well as processes for
the preparation of said recombinant DNA molecules.
The present invention furthermore embraces genetically
modified myxobacteria of the Sorangium/Polyangium group
with, where appropriate, novel and/or improved
properties, which have been prepared by insertion of
said recombinant DNA molecules.
The present invention additionally relates to the
offspring of said modified myxobacteria and to mutants
and variants thereof which still contain said
recombinant DNA molecule.
A number of terms which are customary in recombinant
DNA technology and in bacterial genetics are used in
the following description.
In order to ensure clear and uniform understanding of
the description and of the claims, as well as of the
scope intended to apply to said terms, the following
definitions are stated:
Gene(s) or DNA of heterologous origin: A DNA sequence
which codes for a specific product or products or
fulfils a biological function and which originates from
a species other than that into which the said gene is
inserted; said DNA sequence is also called foreign gene
or foreign DNA, or exogenous DNA.
Gene(s) or DNA of homologous origin: A DNA sequence
which codes for a specific product or products or
fulfils a biological function and which originates from
the same species into which the said gene is inserted.
This DNA is also called exogenous DNA.
DNA homology: Degree of agreement between two or more
DNA sequences.
_ 10 _
fulfils a
prepared by a
for a specific product or products or
biological function and which is
synthetic route.
Promoter: A control sequence of DNA expression which
ensures the transcription of any desired homologous or
heterologous DNA gene sequence in a host cell, as long
as said gene sequence is linked in an operable manner
to a promoter of this type and the latter is active in
said host cell.
Termination DNA
sequence: at the
sequence end of a
transcription unit which signals end of the
transcription process.
Promoter which is able to
the
Overproducing promoter (OPP):
host cell
sequence(s)
bring about in a expression of any
functional gene linked in an operable
manner to an extent (measured in the form of the RNA or
of the polypeptide amount) which is distinctly higher
than is naturally observed in host cells which are not
transformed with said OPP.
//5/
located downstream/upstrean1 of the
non—trans1ated region: DNA sections which are
coding region and
which, although transcribed in mRNA,
This
are not translated
into a polypeptide. region contains regulatory
sequences
(5')-
such as, for example, the ribosome binding
site
DNA expression vector: for
Cloning vehicle such as,
example, which contains
all the
expression of an inserted DNA in a suitable host cell.
a plasmid or a bacteriophage,
signal sequences which are necessary for
which
DNA transfer vector: Transfer vehicle such as,
example, a plasmid or a bacteriophage vector,
_.ll..
makes it possible to insert genetic material into a
suitable host cell.
Homologous recombination: Reciprocal exchange of DNA
pieces between homologous DNA molecules.
Mutants, variants: Spontaneously or else artificially,
by application of known process measures such as,
UV treatment,
for
example, treatment with mutagenic agents
which
still has the features and properties, essential to the
etc , produced derivative of a microorganism
invention, of the initial strain which has acquired the
latter by reason of the transformation with exogenous
DNA.
It has now been possible for the first time within the
scope of the present invention to provide a process
which makes possible a
preferably targeted genetic
the
in that it is now possible
manipulation of genome of myxobacteria of
Sorangium/Polyangium group,
for genetic material to be integrated, and also to be
expressed therein, at randonx or else, when there is
appropriate knowledge of the structural and functional
organisation of the bacterial genome,
defined
predetermined in
specifically at
which
the
independent of structural elements
accurately
positions, can be
some cases, within bacterial
genome,
the
present on
myxobacterial chromosome or of specific
transposition events.
The process according to the invention is, moreover,
the that it is
possible to incorporate exogenous genetic material with
essentially based on recognition
the aid of homologous recombination into the genome of
myxobacteria, it being possible to use, besides
natural, also artificially modified and/or synthetic
genes or gene fragments or other DNA sequences
including whole plasmids, as long as they have DNA
sections or are flanked by DNA sections which have a
homology, which is sufficient for recombination, with
corresponding sections on the myxobacterial genome.
The homologous DNA sections which can be used within
the of the this
connection necessarily have to have 100% identity with
scope present invention do not in
sections on the
to be able
recombination event. On the
the corresponding myxobacterial
about the
contrary, it
chromosome in order to bring
required
sections are
suffices if these
with the
essentially
homologous corresponding regions on the
bacterial genome, that is to say if these have a degree
100%
particularly preferably between 90% and 100%.
of homology of between 80% and and very
The size of said homologous regions can vary, but ought
to be at least 100 Bp. of homology
comprise between 0.3 Kb and 4 Kb,
between 1 Kb and 3 Kb,
Regions which
but preferably
are preferred within the scope
of this invention.
Recombinant DNA molecules which, by reason of their
specific construction, make possible the specific
incorporation of genes or gene fragments or other
interesting DNA sequences, including whole
cell with the
plasmids,
into the genome of a target aid of
homologous recombination in the abovementioned manner
form an essential component of the present invention.
invention relates in
DNA
The present particular to
recombinant molecules which make possible a
targeted integration of genetic material such as, for
example, genes, gene fragments or other DNA fragments
at a defined site within the genome of myxobacteria of
which are
the Sorangium/Polyangium and
group,
characterised in that they contain the DNA which is to
be integrated, and in that said DNA has homologies with
DNA regions within the
else is flanked by
corresponding myxobacterial
genome, or such homologous DNA
_l3_.
sequences, to an extent such that, on transformation of
the myxobacterial cell containing the homologous DNA
region, there is undirected or else, preferably,
targeted integration of said DNA, which is to be
integrated, at a site, which is exactly defined by
reason of the homology present between the inserted DNA
and the DNA the the
myxobacterial genome via homologous recombination.
intrinsic to bacteria, within
Said recombinant DNA molecules can be prepared very
straightforwardly in such a way that the DNA which is
to be which has the
and abovementioned
integrated
properties
(a) is isolated from a suitable source; or
(b) when said DNA which is to be integrated naturally
contains no sections which are homologous with or else
at least with a
essentially homologous corresponding
region on the myxobacterial this DNA is
artificially linked with the
chromosome,
aid of rDNA techniques
known per se to corresponding homologous or else
DNA
which the genetic material to be inserted is flanked by
DNA
essentially homologous sections, as a result of
said homologous or else essentially homologous
sections.
If the DNA which is to be integrated is an expressable
DNA sequence it is advantageous for the latter to be
linked in an operable manner to expression signals
capable of functioning in the bacterial cell, and,
where appropriate, to be flanked by DNA sections which
have homologies with a particular region within the
bacterial genome. These flanking, homologous DNA
sections are present, preferably fused together‘ to a
unit, as component of DNA molecules which are closed in
the form of a ring.
The latter can be dispensed with if said expressable
DNA itself sufficiently great
sequence
with
already has
homology corresponding DNA regions within
_l4_
sequence for said homologous genomic DNA can take place
bacterial genome so that direct exchange of this
by means of homologous recombination.
Besides double—stranded DNA, it is also possible to
the
single—stranded DNA and partially single—stranded DNA.
employ in the process according to invention
Suitable for use in the the
invention are both homologous and heterologous gene(s)
or DNA DNA
sequences complying with the definition made within the
process according to
sequences, and synthetic gene(s) or
scope of the present invention.
The DNA which
be constructed exclusively from genomic,
cDNA or DNA. Another possibility comprises
the of hybrid DNA
both of cDNA and of genomic DNA and/or synthetic DNA.
sequences are to be integrated can,
moreover, from
synthetic
construction sequences consisting
In this case, the cDNA can originate from the same gene
or DNA section as the genomic DNA or else both the CDNA
and the genomic DNA can originate from different genes
or DNA sections. In each case, however, it is possible
for both the genomic DNA and/or the cDNA, each on its
own, to be prepared fronm the same or front different
genes or DNA sections.
If the DNA sequence contains portions of more than one
gene or DNA section, these can derive either from one
and the same organism, from a plurality of organisms,
which belong to various strains, or varieties of the
same species or different species of the same genus, or
else from organisms which belong to more than one genus
thereof or to another taxonomic unit.
In order to ensure the expression of a structural gene
in the bacterial cell, it is possible, where
appropriate, for the coding gene sequences initially to
_l5_
be linked in an operable manner to expression sequences
able to function in the myxobacterial cell.
present
the
the
which
and,
expressable hybrid constructions of
thus
gene
invention usually contain, besides
structural gene(s), also expression signals
include both promoter and terminator sequences
preferably, further regulatory sequences of the 3’ and
’ non—translated regions.
Every promoter and every terminator which is able to
bring about induction of expression of an expressable
DNA sequence in the
myxobacteria of Sorangium/
Polyangium group can be used as a component of the
hybrid gene construction.
Examples of promoters suitable for use in the process
according to the invention are
— the light—inducible
[EP 310 619];
— other Myxococcus xanthus promoters;
promoter of Myxococcus xanthus
— promoters of Actinomycetes, especially of Strepto-
mycetes;
— E.coli promoters such as, for example, the
Tac(hybrid), PL or Trp promoter.
Suitable termination sequences which can be used within
the scope of this invention are described, for example,
by Rosenber and Court (1979) and by Gentz et al (1981).
The functional unit which has been formed in this way
and consists of a gene and of expression signals active
in the myxobacterial cells can subsequently, where
appropriate, be flanked by one or more DNA sections
which have homologies with corresponding DNA regions
within the myxobacterial genome to an extent such that,
on transformation of the myxobacterial cell containing
said homologous region, there is random or else,
preferably, specific integration of a gene sequence
which is flanked by homologous DNA sequences at a site,
which is defined on the basis of the homologies present
between inserted DNA and DNA intrinsic to the bacteria,
the bacterial
The
within
within genome by homologous
flanking, DNA
the this
preferably fused together to a
recombination.
homologous sections
are, moreover, scope of invention
present, unit, as a
component of a DNA molecule which is closed in the form
of a ring.
In a preferred embodiment,
to be
moreover, the DNA which is
inserted is integrated into a plasmid which
either already contains homologous DNA sections or else
acquires the latter cloned in at a later time.
Thus, if the intention is to integrate not just single
genes or gene fragments but the complete plasmid DNA,
which may contain said genes or gene fragments, into
the myxobacterial genome, it is sufficient to clone
said homologous DNA sequences into the plasmid DNA at a
required site, although, where possible, the genes
intended for expression should be functionally
retained. Thus, it is also possible in this case too
for the DNA which is to be integrated (plasmid DNA) to
be regarded in principle as flanked by homologous DNA
sequences because these homologous DNA sections can be
thought of as fused together to a unit within the DNA
molecule which is closed in the form of a ring.
Besides structural genes, it is also possible to use
any other desirable genes or gene fragments or other
useful DNA sequences such as, for example, binding
sites of regulator molecules, promoters, terminator
sequences etc.
It is now possible for the first time, by the choice of
suitable DNA sequences of homologous or heterologous
origin, which have sufficiently great homologies with
corresponding sections within the bacterial genome and
thus allow exchange of genetic material via homologous
recombination, for genes or other DNA sequences to be
integrated specifically at predetermined sites in the
myxobacterial genome and to be expressed there where
appropriate.
The extent of the homology, which is necessary for
exchange via homologous recombination, between the
homologous DNA sections and the corresponding genomic
DNA region depends on a variety of parameters and must
therefore be adapted to the appropriate needs in each
the DNA used. It is
that a
least 100 Bp is
case, depending on sequences
assumed on the present state of knowledge
homologous region comprising at
sufficient to bring about the required recombination
event.
A homologous region which extends over a range of 0.3
to 4 Kb, but preferably over a range of 1 to 3 Kb, is
preferred within the scope of this invention.
Suitable for use as homologous DNA sections within the
scope of this invention are primarily DNA sequences of
homologous origin which can be obtained by isolation of
the complete myxobacterial DNA and subsequent digestion
the DNA
sequence of said homologous DNA fragments is known they
with suitable restriction enzymes. Where
can, of course, also be prepared by synthesis.
However, it is furthermore also possible to use
homologous DNA sections of heterologous origin, which
have been isolated not directly from the genome of the
but, for related
organisms and which thus do not necessarily have 100%
identity with the
target organism example, from
corresponding DNA regions on the
genome of the target organism but are only essentially
homologous with the latter, that is to say have a
degree of homology between 80% and 100%. and very
particularly preferably between 90% and l00%.
An essential the
therefore formed by a process for the specific genetic
the
characterised in
component of
present invention is
manipulation of myxobacteria of Sorangium/
group; that
genetic material of homologous origin or a combination
Polyangium which is
of genetic material of homologous and heterologous
origin is inserted into the myxobacterial cell and
integrated there via homologous recombination
specifically at a site, which is accurately defined by
reason of the homologies present, into the chromosome
of said myxobacteria.
It is thus now possible for the first time within the
scope of this invention, by preparing appropriate
hybrid gene constructions in the manner described
above, to carry out specific modifications of bacteria-
intrinsic genes within the myxobacterial genome or else
to incorporate additional genes or other DNA fragments
into the myxobacterial genome. If the integration takes
place within a functional gene or operon, this usually
leads to inactivation thereof and, as a consequence, to
a phenotypically observable defect.
The specific procedure for this can be such that the
of the
with the
myxobacterial cells are transformed with one
recombinant DNA molecules described above,
genes, hybrid gene constructions or other DNA fragments
contained in said recombinant DNA molecule being
integrated by homologous recombination randomly or
else, preferably, specifically at a site, which is
defined by reason of the homologies present and thus
can be predetermined, into the bacterial genome.
In a specific embodiment of the present invention, the
insertion of the genetic material into myxobacteria of
the Sorangium/Polyangium group, takes place in an
undirected manner via a donor organism capable of
conjugation—like information exchange with the
_19_.
myxobacterium. target organism from the
donor to the
myxobacterium recipient.
The procedure for the preparation of suitable plasmids
which have a homology with the myxobacterial chromosome
which is sufficient for integration via homologous
recombination can be, for example, such that the
complete DNA is initially isolated from. myxobacteria
and subsequently fragmented. This fragmentation can be
carried out either mechanically by the action of shear
forces or else, preferably, by
using suitable
restriction enzymes.
It is then possible to isolate from the large number of
resulting fragments those of suitable size and
subsequently clone thent into a suitable plasmid. The
ligation of homologous DNA fragments and of DNA
fragments of homologous and heterologous origin into a
suitable cloning vector is carried out with the aid of
standard methods as for
.
are described, example, by
Maniatis et al,
This usually entails the vector and the DNA sequence
which is to be integrated initially being cut with
suitable restriction enzymes. Examples of suitable
restriction enzymes are those which provide fragments
with blunt ends, such as, for example, Smal, Hpal and
EcoRV, or else enzymes which form cohesive ends, such
as, for example, EcoRI, Sacl, BamHI, Sall, Pvul etc.
Both fragments with blunt ends and those with cohesive
ends, which are complementary with one another, can be
linked again, with the aid of suitable DNA ligases, to
give a single continuous DNA molecule.
Blunt ends can also be prepared. by treatment of DNA
fragments which have protruding cohesive ends with the
Klenow fragment of E.coli DNA polymerase by filling in
the gaps with the appropriate
nucleotides.
complementary
On the other hand, cohesive ends can also be prepared
artificially, for example by attaching complementary
homopolymer tails to the ends of a required DNA
sequence and of the cut vector molecule using a
terminal deoxynucleotidyl transferase or else by
attaching synthetic oligonucleotide sequences (linkers)
which carry a restriction cleavage site, and subsequent
cutting with the appropriate enzyme.
It is possible in principle to use for the preparation
and multiplication. of the constructs which have been
described above and which contain DNA fragments of
homologous or else a combination of DNA fragments of
all
example,
homologous and heterologous origin conventional
cloning vectors such as, for plasmid or
bacteriophage vectors as long as they have replication
which
which are compatible with the host cell.
and control sequences originate from species
replication,
cloning vector usually carries an origin of
in addition specific genes which lead to
phenotypical selection features in the transformed host
cell,
transformed vectors
especially to resistance against antibiotics. The
can be selected on the basis of
these phenotypical markers after a transformation into
a host cell.
Selectable
the
markers which can be used
this
phenotypical
within scope of invention comprise, for
example, without this representing a limitation on the
the
tetracycline,
subject—matter of invention, resistances to
ampicillin,
G418,
chloramphenicol, hygromycin,
kanamycin, neomycin and bleomycin. A prototrophy
particular amino acids can function as further
selectable marker, for example.
Preferred within the scope of the present invention are
the
plasmid pSUP202l used within the scope of the present
primarily E.coli plasmids such as, for example,
invention.
Suitable host cells for the cloning described above
which are within the scope of this invention are
primarily prokaryotes, including bacterial hosts such
as, for example, A.tumefaciens, E.coli, S.typhimurium
and Serratia marcescens, furthermore pseudomonads,
actinomycetes, salmonellae and myxobacteria themselves.
E.coli hosts such as, the E.coli
for example, strain
HBlOl are particularly preferred.
Competent cells of the E.coli strain HBlOl are in this
with the aid of the
customarily used for the transformation of E.coli
connection prepared processes
[seez
“General recombinant DNA techniques”].
Transformation and subsequent incubation on a suitable
medium are followed by the resulting colonies being
subjected to differential screening by plating out on
selective media. It is then subsequently possible to
isolate the appropriate plasmid DNA from those colonies
which contain plasmids with DNA fragments cloned in.
Recombinant plasmids of different size are obtained in
this After then
possible for plasmids of suitable size to be selected
for the
way. restriction analysis it is
subsequent insertion of the plasmid DNA into
the myxobacterial cell. This DNA transfer can moreover
take place either directly or else, preferably, via an
intermediate host (donor cell) capable of conjugation-
like information exchange with the myxobacterium target
organism within the scope of a conjugal transfer.
the invention
An essential component of
present
therefore relates to construction of plasmids
_22_
which, besides homologous
sections, can also contain
one or more gene constructions consisting’ of one or
more structural genes or other desirable genes or gene
linked
signals able to function
The
homologous DNA fragments can in this connection either
fragments which are, where appropriate, in an
operable manner to expression
in bacterial cells, or other useful DNA sequences.
consist entirely of genome 4 which are intrinsic to the
the
and thus are completely
bacteria (myxobacteria of Sorangium/Polyangium
group)
or else
of homologous origin,
they can, besides homologous also
sections,
contain more or less expressed portions of heterologous
origin. The use of homologous DNA sections of purely
heterologous origin is also conceivable.
These plasmids can be used in a further process step
for insertion of the genetic constructions which have
which contain,
which
into the myxobacterial cell and
been described above and where
appropriate, a structural gene codes for a
required gene product,
integration there into the bacterial genome.
The transfer of the genetic constructions according to
the application into the myxobacterial cells can be
carried out in a variety of ways. Preferred within the
of this
cell
scope invention is conjugal transfer fron1 a
donor capable of conjugation—like information
exchange with the myxobacterium target organism to the
myxobacterial recipient.
It is possible within the scope of this conjugal
transfer for the DNA which is to be transferred
moreover to be either initially cloned, as described
above, in one of the cloning vectors customarily used,
suitable
and subsequently transformed into a
donor cell.
host
intermediate host which functions as
the
avoided by using a host strain which is suitable both
roundabout route via intermediate can be
-23..
for the cloning of DNA and for the use as donor cell
within the scope of the conjugation.
Intermediate hosts which can be used within the scope
of this invention as donor cells are essentially
prokaryotic cells selected from the group consisting of
E.coli, pseudomonads,
actinomycetes, salmonellae and
myxobacteria themselves.
The precondition for conjugal transfer of plasmid DNA
from a. donor cell to a. recipient is
(tra)
the
least the transfer origin
the presence of
transfer and mobilisation functions (mob).
Moreover mobilisation function must contain at
(oriT) and be located on the
plasmid to be transferred.
(tra)
By contrast, the transfer
function can be either located on the plasmid or
on a helper plasmid or else be present integrated into
the chromosome of the donor cell.
Plasmids which meet the abovementioned precondition and
are therefore preferred within the scope of this
invention essentially fall into incompatibility groups
P, Q, T, N, W and Coll. The prototype of the P group
plasmids is the plasmid RP4.
the scope of this
pSUP202I which
plasmid RP4, which has as component of the mob function
(RP4mob) the origin Other
with the mob function (RP4mob), for example,
pSUPlOl, pSUP30l, pSUP401, pSUP20I, pSUP202, pSUP203 or
pSUP205, and the derivatives derived therefrom
et al (l988)]
the process according to the invention.
Particularly preferred
the
1.9 Kb fragment from the
within invention is plasmid
contains a
(oriT).
transfer plasmids
such as,
[Simon
can likewise be used within the scope of
During the course of the experiments carried out within
the scope of this invention it has emerged that it is
the
exposed to a brief heat treatment during the course of
advantageous when myxobacterial recipient is
the conjugal transfer‘ before the incubation with the
donor strain. A preincubation of the recipient cell at
a temperature of 35°C to 60°C, preferably at a
temperature of 42°C to 55°C and very particularly
preferably at a temperature of 48°C to 52°C for one to
minutes, but in particular 5 to 20 minutes, is
preferred.
Used in a preferred embodiment of the present invention
is an E.coli donor strain which contains the transfer
genes (tra) of plasmid RP4 incorporated into the
chromosomal DNA. Preferred within the scope of this
invention is the E.coli donor strain W3lOl(pME305)
which contains the helper plasmid pME305 which has the
transfer function (tra) of RP4.
Particularly interesting for the process technique, and
thus particularly preferred within the scope of this
invention, are bacterial strains which are suitable
both as hosts for cloning of vectors with integrated
DNA sequences and for use as donor cell within the
scope of the conjugal transfer. Likewise particularly
preferred are bacterial strains which are restriction-
negative and thus do not degrade inserted
Both of the
E.coli strain ED8767(pUZ8)
foreign DNA.
met by the
but this
abovementioned criteria are
in an ideal manner,
is nentioned at this point only as representative of
other suitable bacterial strains and is not intended to
limit the application in any way.
Besides
the conjugal gene transfer, described above,
frmn a donor cell into a myxobacterial recipient, it
is, of course, also possible to use other suitable gene
transfer processes for inserting genetic material into
myxobacteria of the Sorangium/Polyangium group. Mention
may be made here primarily of gene transfer via
electroporation, within the scope of which the
myxobacterial cells are briefly exposed to high
electric field strengths [Kuspa and Kaiser (l989)]. The
general outline conditions for electroporation of
_25_
prokaryotic cells described in detail in US-
P 4,910,140.
NON-LIMITING EXEMPLARY EMBODIMENTS
General recombinant DNA techniques
Since many of the recombinant DNA techniques used in
this invention are routine for the person skilled in
the art, a brief description of these generally used
techniques is to be given below. All these processes
A the reference of Maniatis et al
are described in
(1982), unless separate reference is made thereto.
A. Cutting with restriction endonucleases
about 50 to
500 ug/ml DNA in the buffer solution recommended by the
Typically, the reaction. mixture contains
manufacturer, primarily New England Biolabs, Beverly,
(FRG). 2 to 5 units of
restriction endonucleases are added for each pg of DNA
the the
temperature recommended by the manufacturer for one to
MA. and Bohringer, Mannheim
and reaction mixture is incubated at
three hours. The reaction is stopped by heating at 65°C
followed
ethanol. This
technique is also described on pages lO4 to 106 of the
(1982)
for lO minutes or by extraction with phenol,
by precipitation of the DNA with
Maniatis et al reference.
B. Treatment of the DNA with polymerase in order to
generate blunt ends
to 500 pg/ml DNA fragments are added to a reaction
mixture in the buffer recommended by the manufacturer,
primarily New England Biolabs, Beverly, MA. and
Bohringer, Mannheim (FRG). The reaction mixture
contains all four deoxynucleotide triphosphates in
concentrations of 0.2 mM. The reaction is carried out
at 15°C for 30 minutes and is then stopped by heating
at 65°C for 10
obtained by
minutes. For which
with
fragments are
endonucleases
EcoRI
cutting restriction
and
of DNA
polymerase is used. For fragments which are obtained by
which generate 5’-protruding ends, such as
BamHI, the large fragment, or Klenow fragment,
endonucleases which generate 3’—protruding ends, such
as PstI and Sacl, T4 DNA polymerase is used. The use of
these two enzymes is described on pages 113 to 121 of
the Maniatis et al (1982) reference.
C. Agarose gel electrophoresis and purification of DNA
fragments from gels
The agarose gel electrophoresis is carried out in a
horizontal apparatus as described on pages 150 to 163
The buffer used is the
The DNA
ethidium. bromide
gel or tank buffer
added the
The DNA is visualised by illumination
of the Maniatis et al reference.
buffer
stained by 0.5 pg/ml
tris—acetate described therein.
fragments are
either in the
which is present
during electrophoresis or after
electrophoresis.
with long-wavelength ultraviolet light.
When the fragments are to be removed from the gel, the
agarose used is one which gels at low temperature and
can be obtained from Sigma Chemical, St Louis,
Missouri. After the electrophoresis, the required
fragment is cut out, placed in a plastic tube, heated
at 65°C for about 15 minutes, extracted three times
with phenol and precipitated twice with ethanol. This
process is a slight modification of that described by
Maniatis et al (1982) on page 170.
As alternative, the DNA can be isolated from the
agarose with the aid of the Geneclean kit (Bio 101
Inc , La Jolla, CA, USA).
D. Addition of synthetic linker fragments onto DNA ends
-27..
If it is required to attach a new endonuclease cleavage
site onto the end of a DNA molecule, the molecule is,
treated with DNA
generate blunt ends as described
About 0.1 to 1.0 ug of the
about 10 ng of phosphorylated
been obtained from New England
where appropriate, initially
polymerase in order to
section.
added to
which has
in the above
fragment is
linker DNA,
Biolabs, in a volume of 20 to 30 pl with 2 pl of T4 DNA
and l mM ATP in the
buffer recommended by the manufacturer.
ligase from New England Biolabs,
After the
stopped by heating at 65°C for l0 minutes.
incubation at 15°C overnight, reaction is
The reaction
mixture is diluted to about lOO ul in a buffer which is
correct for the restriction endonuclease which cuts the
synthetic linker sequence. Approximately 50 to 200
units of this endonuclease are added. The ndxture is
incubated at the appropriate temperature for 2 to 6
hours, and then the fragment is subjected to an agarose
gel electrophoresis and purified as described above.
The resulting fragment will now have ends with endings
which with the
restriction
have been generated by
These
cutting
endonuclease. ends are usually
cohesive so that the resulting fragment can now easily
with the cohesive
be linked to other fragments same
ends.
E. Removal of 5’-terminal phosphates from DNA fragments
the
the
During the plasmid cloning treatment of
with
steps,
vector plasmid phosphatase reduces
(discussed on page l3
After the DNA has
recircularisation of the vector
of the Maniatis et al reference).
been cut with the correct restriction endonuclease, one
unit of alkaline phosphatase from the intestine of
calves, which has been obtained from Boehringer—
Mannheim, Mannheim, is added. The DNA is incubated at
°C for one hour and subsequently twice extracted with
phenol and precipitated with ethanol.
_28_
F. Linkage of the DNA fragments
When fragments with complementary cohesive ends are to
be linked together, about 100 ng of each fragment are
incubated ixu a reaction nuxture of 20 to 40 ul with
about 0.2 of T4 DNA ligase
Biolabs in the buffer recommended by the manufacturer.
units from New England
The incubation is carried out at 15°C for 1 to 20
hours. When DNA fragments with blunt ends are to be
linked, they are incubated as above apart from the
amount of T4 DNA ligase being increased from 2 to 4
units.
G. Transformation of DNA into E.coli
The E.coli strains HBlOl, W3lOl and ED8767 are used for
most of the experiments. DNA is introduced into E.coli
by the calcium chloride process as has been described
by Maniatis et al (1982), pages 250 to 251.
H. Screening of E.coli for plasmids
After the
the colonies of
of the
transformation,
tested for
resulting
E.coli the presence required
plasmid by a rapid plasmid isolation process. Two usual
366 to 369 of the
processes are described. on pages
Maniatis et al (1982) reference.
I. Isolation of plasmid DNA on a large scale
Processes for the isolation of plasmids from E.coli on
a large scale are described on pages 88 to 94 of the
(1982)
Maniatis et al reference.
Examples:
Example 1: Cultivation conditions for Sorangium
in a G5lb liquid
Sorangium cellulosum is brought up
-29..
“Media
The
It is also possible to use a G52c
section and
°C.
shaking at 180 rpm.
medium [see buffers”] at a
temperature of cultures are aerated by
medium as alternative medium.
“Media
cultivation on
The SolE mediun1 described in the section and
buffers” the
medium. The incubation temperature is 30°C in this case
can be used for solid
too.
Example 2: Cultivation conditions for E.coli
E.coli cells are cultivated in an LB medium [Miller
(1972)] at a temperature of 37°C.
Example 3: Preparation of a streptomycin—resistant
spontaneous mutant of Sorangium cellulosum
pl of a three—day old Sorangium cellulosum culture
which has been raised in
(So1E
pg/ml
(wild—type strain So ce 26]
plated
which is
The incubation
°C. The
spontaneous
solid medium
with 300
liquid medium is out on
medium] supplemented
streptomycin. time is 14 days at a
temperature of colonies growing on this
medium are streptomycin-resistant mutants
which are cultivated once more on the same medium (with
streptomycin) for further concentration and
purification.
One of these streptomycin—resistant colonies is
selected and is called SJ3. A sample of this mutated
Sorangium cellulosum So ce 26 strain was deposited on
.01.1991 at the
Mikroorganismen und Zellkulturen GmbH”
FRG],
provisions
“Deutsche Sammlung Von
[Braunschweig,
which is recognised in accordance with the
of the
under deposit number DSM 6380.
Budapest Treaty as international
depository,
Example 4: Preparation of the complete DNA of Sorangium
To isolate the complete DNA,
a Sorangium culture in the
stationary phase is centrifuged at 10,000 rpm for
-30..
minutes. The cells are removed and resuspended in STE
buffer [see section “Media and buffers”]
to a cell density of about 109 cells/ml.
and adjusted
Subsequently 450 pl of this suspension are mixed with
200 pl of RLM buffer [see section “Media and buffers”]
and 2 pl of diethyl pyrocarbonate. Thorough and uniform
mixing are ensured by using suitable equipment such as,
After incubation in
for example, a Vortex or the like.
an incubator at 70°C for 30 minutes, 100 pl of
potassium acetate [5 M] are added. This mixture is
incubated on ice for 15 ndnutes and thoroughly mixed
[Vortex] every 5 minutes. After centrifugation [15
minutes at 10,000 rpm] the supernatant is subsequently
mixed with 500 pl of phenol/chloroform/isoamyl alcohol
[25/24/1] for extraction of the proteins. After renewed
centrifugation [15 minutes at 10,000 rpm] the upper
phase which contains the DNA fraction is removed, and
any phenol content still present therein is removed
with diethyl ether [1 ml]. The DNA is then precipitated
by adding 1 ml of ethanol. After incubation at -70°C
for 30 minutes, the complete mixture is centrifuged at
,000 rpm for 15 nunutes,
70% ethanol and dried in vacuo.
dissolved in TER buffer.
the pellet is washed with
Finally, the DNA is
Example 5: Conjugative transfer of pSJB55 into
Sorangiumcellulosum SJ3
.1 Two-stage process
.1.1 Cloning of Sorangium DNA into plasmid pSUP202l
The chromosome isolated from the Sorangium SJ3 strain
is cut with the restriction enzyme Pvul.
this into the
[Simon R et al This entails 0.2 pg of
The fragments
obtainable in are cloned
pSUP2021 (1983)).
plasmid DNA and 1 pg of chromosomal DNA being initially
way plasmid
digested with Pvul and subsequently precipitated with
ethanol. The precipitate is removed, dried and the
dried pellet is suspended in 14 pl of double-distilled
water. Then 2.5 pl of a ten—fold concentrated ligation
buffer “Media and buffers”], 2.5 pl of
bovine serum albumin [O.1%], 2.5 pl of ATP [10 mM], 2.5
pl of DTT [O.2 M], 8 pl of H20 and 1 pl of T4 DNA ligase
added. The
incubated at a temperature of about 8°C overnight.
[see section
are complete ligation mixture is then
pl of this ligation mixture are transformed into the
strain HBlO1 for the
this, competent
E.coli recombinant
the
strain HB10l are prepared with the aid of the processes
cloning of
plasmids. For cells of E.coli
normally used for the transformation of E.coli (see:
“General recombinant DNA techniques”].
After transformation and subsequent incubation for 24
[25 pg/ml]
the resulting colonies
hours on LB agar supplemented with kanamycin
[25 Hg/ml],
are subjected to a differential screening by parallel
[60 pg/ml]
It is subsequently possible to
and chloramphenicol
plating out on ampicillin-containing and
ampicillin—free medium.
isolate those colonies which have lost their ampicillin
resistance due to the integration of the Sorangium DNA
are then isolated. from these
fragments. The plasmids
ampicillin—sensitive colonies.
Recombinant plasmids of different size are obtained in
this way. After restriction analysis, three of these
plasmids are selected for further experiments. These
plasmids, called pSJB50, pSJB55 and pSJB58, contain
Sorangium DNA inserts of 1 Kb, 3.5 Kb and 4 Kb
respectively.
.1.2. Conjugative transfer of plasmid pSJB55 into
Sorangium cellulosum
The transfer of plasmid pSJB55 into Sorangium
cellulosum takes of E.coli
strain W3101(pME305)
place with the mediation
(1987)],
capable of a conjugation-like information exchange with
[Jaoua S et al which is
_32_
Sorangium. The E.coli plasmid pME305 [Rella (l984)] is
in this case used as helper plasmid for the
mobilisation of pSJB55.
Initially, competent cells of the E.coli strain
W3l0l(pME305) are transformed, with the aid of the
processes normally used for the transformation of
E.coli, with 5 ;H_ of the previously isolated pSJB55
plasmid DNA. The transformed E.coli cells thereby
become the donor for the plasmid pSJB55.
For the actual transfer, 15 ml of a Sorangium
cellulosum SJ3 culture (4 x lOB cells/ml to 1-4 x lO9
cells/ml] in the stationary phase are mixed with 10 ml
of a late log phase culture of E.coli donor cells which
contain a comparable content of cells. These are then
centrifuged together at 4000 to 8000 rpm for 10 minutes
and resuspended in 500 pl of a G5lb or G5lt medium.
It has proved advantageous for the Sorangium recipient
cells to be exposed briefly to a heat treatment in a
waterbath before the conjugation with E.coli. The best
transfer results with the Sorangium cellulosum strain
SJ3 can be achieved with a heat treatment at a
temperature of 50°C for l0 minutes. Under these
conditions transfer frequencies of 1-5 x l0% can be
achieved, which corresponds to an increase by a factor
of 10 compared with. a process without previous heat
treatment.
Transfer to plates with So1E solid medium is followed
by a two—day incubation at 30°C. The cells are then
harvested and resuspended in 1 ml of G5lb or G51t
medium. 100 pl of this bacterial suspension are plated
out on a selective SolE medium which, besides kanamycin
[25 mg/l] [20 to 35 mg/l]
streptomycin Counter-
also contains phleomycin and
[300 mg/l] as
selection of the donor strain
selective agents.
[E.coli W3l0l(pME305)] is
carried out with the aid of streptomycin.
after an
colonies this selective So1E medium
time of 10 to 14
growing on
incubation days
which
conjugative
transconjugants of Sorangium cellulosum have
acquired phleomycin resistance owing to
transfer of the plasmid pSJB55. These phleomycin-
resistant colonies can be used for the subsequent
molecular biological investigations. The transformation
averages 3 x
transfer of
1o'6
frequency for plasmid
pSJB55 to
Sorangium based on recipient
strain SJ3.
The plasmids pSJB5O and pSJB58 can be transferred to
Sorangium in an analogous manner.
.2 One—stage process
.2.1 Cloning of Sorangium DNA into plasmid pSUP2021
The cloning of Sorangium DNA into plasmid pSUP2021 can
be carried out as described in Example 5.1.1.
Owing to the helper plasmid pME305 used in 5.1.1 being
exchanged for the plasmid pUZ8 [Hedges and Matthew
(1979)] which, in contrast to the abovementioned
plasmid, carries no ampicillin—resistance gene, the
cloning step in the E.coli intermediate host HB101 can
be dispensed with because direct cloning in the E.coli
donor strain ED8767 which is intended for the conjugal
transfer is now possible.
The plasmid pUZ8 is a derivative of the plasmid RP4
which covers a wide host range and is described by
Datta et al (1971). The modifications compared with the
initial plasmid RP4 relate essentially to the
ampicillin—resistance gene and to the insertion element
IS21, both of which are deleted, and to the
incorporation of an additional gene which confers
resistance to mercury ions [see Jaoua et al (1987)].
_34_
The ligation mixture prepared as in Example 5.1.1 can
therefore now be transformed directly into the E.coli
strain ED8767. For this, competent cells of the E.coli
strain ED8767 are prepared with the aid of the
processes customarily used for the transformation of
E.coli [seez “General recombinant DNA techniques”].
After transformation and subsequent incubation on LB
agar supplemented with tetracycline [10 ug/ml] and
chloramphenicol [25 pg/ml] for 24 hours, the resulting
colonies are subjected to a differential screening by
parallel plating out on ampicillin—containing [60
ug/ml] and ampicillin—free medium. It is subsequently
possible to isolate those colonies which have lost
their ampicillin resistance owing to the integration of
the Sorangium DNA fragments. The cultures obtainable in
this way can then be employed directly as donor cells
for the conjugative transfer of recombinant plasmids
into Sorangium cellulosum cells.
In place of the abovementioned ligation mixture, it is,
case to clone the
pSJB55 or pSJB58, prepared
into the E.coli strain ED8767.
of course, also possible in this
recombinant plasmids pSJB50,
as in Example 5.1.1,
.2.2 Conjugative transfer of the recombinant plasmids
into Sorangium cellulosum
For the
cellulosum SJ3
actual transfer, 15 ml of a
[1—4 x 109
stationary phase are Hdxed with 10 nfl. of a late log
Sorangium
culture cells/ml] in the
phase culture of E.coli donor cells which contain a
comparable content of cells. These are then centrifuged
together at 4000 rpm for 10 minutes and resuspended in
500 pl of a G51b or G51t medium.
It proves advantageous in this case too for the
Sorangium recipient cells to be exposed briefly to a
heat treatment
with E.coli.
in a waterbath before the conjugation
The best transfer results can be achieved
_35_
with the Sorangium cellulosum strain SJ3 with a heat
of 50°C for
transfer frequencies of l—5 x
treatment at a
temperature lO minutes.
Under these conditions,
1o‘5
by a
can be achieved,
of 10
previous heat treatment.
which corresponds to an increase
factor
compared with a process without
Further cultivation of the transformed Sorangium cells
is carried out in analogy to the procedure described in
Example 5.1.2.
Owing to the use of a restriction—negative E.coli
strain as donor strain, such as, for example, E.coli
ED8767 [Murray et al (l977)], the transformation
frequency can be drastically increased by comparison
with the process described above
103).
(up to a factor of
Molecular genetic analysis
(A) Detection of the integration of the plasmid pSJB55
into the chromosome of Sorangium cellulosum SJ3
The DNA which has
complete been isolated from the
transformed Sorangium cells as described above [compare
is digested. with Sml and SalI and loaded
[40 mM tris—HCl, 20 mM
2 mM EDTANa2, pH 7.8]
After the electrophoresis the placed
[l.5 M NaCl, 0.5 M
NaOH] for 30 minutes and subsequently in a neutralising
solution [l.5 M NaCl, 0.5 M tris—HCl, 1 mM EDTANa2, pH
7.2]. The DNA is transferred, by means of a Southern
capillary blotting,
buffer
Example 4]
onto a horizontal tris—acetate
sodium
[O.9%].
initially in a denaturing solution
acetate, agarose gel
gel is
using a 20-fold concentrated SSC
[see section “Media and buffers”] onto a nylon
membrane [for example an Amersham Hybond nylon
membrane; Amersham. International plc, Amershanl Place,
Amersham, England HP7 9NA] and fixed there by UV
treatment for 6 minutes. Further details of this
process are described in the Amersham International
_36_
handbook
(1985).
“Membrane Transfer and Detection Methods”,
The DNA intended as hybridisation probe is labelled by
means of a nick translation [Rigby DWJ et al (l977)].
This the form of the ”P—labelled Pvul insert
comprising 3.5 Kb from the plasmid pSJB55. The actual
takes
out
of Denhardt
hybridisation is carried using a slight
modification of the process [Denhardt DT
(1976)]. The buffer used for the prehybridisation and
hybridisation has the 6 x SSC
(l982)] [Maniatis et al
+ 0.5% SDS + 0.2 mg/ml denatured salmon sperm
following composition:
[Maniatis et a1 + 5 x Denhardt
(l982)]
DNA.
The prehybridisation is carried out at 65°C and takes 3
the reaction is
P__
hours, while actual hybridisation
complete after 20 hours. For the hybridisation, a
labelled [lO5 cpm per cm2 of filter], denatured Pvul
fragment comprising 3.5 Kb from plasmid pSJB55 is
added. After the hybridisation the filter is washed
first for 2 X 15 minutes in 2-fold concentrated SSC at
a temperature of 65°C, subsequently in 2 x SSC + 0.1%
SDS likewise at 65°C for 30 nunutes and finally once
more in 0.5 x SSC [15 minutes at 65°C].
The subsequent autoradiography is carried out using an
film FUJY X film]. The
autoradiographs show no bands
X—ray [for example Rays
of the transconjugants
which correspond to free pSJB55 plasmid DNA which is in
over—spiralised form. By contrast, however, a positive
signal is found in the chromosomal region of the filter
membrane.
bands of
The hybridisation pattern of the
The SmaI—digested plasmid pSJB55 provides
8.9,6.7 and 1.6 Kb.
parent strain SJ3 after Smal digestion likewise shows
three bands, one of which corresponds to an internal
fragment, comprising 1.6 Kb, of the Sorangium insert
-37..
cloned into the plasmid pSJB55. The
pattern of the SmaI—digested DNA of the transconjugants
[8.9 and 6.7 Kb band of plasmid pSJB55
.6 Kb band which is
hybridisation
shows 5 bands
and SJ3 bands]
common to all three.
including the
After Sall digestion,
plasmid pSJB55 (the other Sall fragment comprising 3.1
Kb of pSJB55 does not hybridise with the probe). The
the SalI—digested SJ3 DNA
a band of 14.1 Kb is found for
hybridisation pattern of
likewise shows a band of 5 Kb. In the transconjugants
the 14.1 Kb fragment of plasmid. pSJB55 and the 5 Kb
fragment of SJ3 disappear after Sall digestion.
These are replaced by two new bands of 11.5 Kb and 7.7
The Smal data show that all the pSJB55 fragments are
intact in the genome of the transconjugants. This rules
out the possibility of a site—specific recombination
because in this case at least one of the Smal fragments
would have had to disappear. the
of the Sall digestion make it clear that the
Furthermore, results
plasmid
pSJB55 has been integrated into the Sorangium genome,
specifically at the site where the DNA region
homologous with pSJB55 is located (= 3.5 Kb Pvul
fragment). This integration takes place in the course
of a homologous recombination between a Sorangium
insert comprising 3.5 Kb from pSJB55 and the same
insert within the Sorangium genome.
MEDIA AND BUFFER SOLUTIONS
G51b medium (pH 7.4)
Glucose 0.2%
Starch 0.5%
[potato starch,
Italy]
[DIFCO Laboratories,
Noredux type; CERESTAR ITALIA S.p.a.,
Milan,
Peptone USA]
Q C)
|—‘ (\)
o\0 o\0
Probion S
_38_
[Single Cell Protein; HOCHST AG, Frankfurt, FRG]
CaCl2 X 2 H20 0.05%
MgS04 x 7 H20 0.05%
HEPES [FLUKA] 1.2%
G51t medium (pH 7.4)
Glucose 0.2%
Starch 0.5%
[potato starch, Noredux type; CERESTAR ITALIA S.p.a.,
Milan, Italy]
Tryptone [MARCO, Hackensack, NJ USA] 0.2%
Probion S 0.1%
[Single Cell Protein; HOCHST AG, Frankfurt, FRG]
CaCl2 X 2 H20 0.05%
MgS04 x 7 H20 0.05%
HEPES [FLUKA] l. %
G52c medium (pH 7.4)
Glucose 2.0 g/l
Starch 8.0 g/l
[potato starch, Noredux type; CERESTAR ITALIA S.p.a.,
Milan, Italy]
Soya meal defatted 2.0 g/l
[MUCEDOLA S.r.l., Settimo Milanese, Italy]
Yeast extract 2.0 g/l
[FOULD & SPRINGER, Maison Alfort, France]
CaCl2 X 2 H20 1.0 g/l
MgS04 x 7 H20 1.0 g/l
Fe-EDTA (8 g/l stock solution] 1.0 ml
HEPES [FLUKA] 2.0 g/l
Distilled water ad 1000 ml
pH is
[20 minutes at 120°C].
adjusted to 7.4 with NaOH before sterilisation
pH after sterilisation: 7.4
SolE medium (pH 7.4)
Glucose* 0.35%
Tryptone [MARCO, Hackensack, NJ USA] 0.05%
MgS04 X 7 H20 0.15%
Ammonium sulphate*
CaCl2 X 2 H2O*
K2HPO4*
Sodium dithionite*
Fe—EDTA*
HEPES [FLUKA]
Supernatant of a sterilised,
stationary S. cellulosum culture
Agar
l—'C)©CD©©
U707
o\0
.05%
.l%
.006%
.0l%
.O0O8%
(V/V)
*Addition takes place only after sterilisation
pH is adjusted to 7.4 with NaOH before sterilisation
[20 minutes at 120°C].
LB medium
Tryptone
Yeast extract
NaCl
STE buffer (pH 8.0)
Sucrose
EDTANa2
Tris~HCl
RLM buffer (pH 7.6)
SDS
EDTANa2
Tris—HCl
TER buffer
Tris-HCl (pH 8.0)
1 mM EDTANa2
RNAse
Ligation buffer
Mgclz
Tris—HCl (pH 7.8)
.0 g/1
.0 g/l
.0 g/l
%
1 mM
l0 mM
%
l25 mM
0.5 mM
mM
1 mM
pg/ml
.1 M
0.5 M
TABLES
Table 1: Bacterial strains and plasmids
Strain
Relevant characteristics
Escherichia coli
W310lNal
HBlOl
ED8767
Sorangium cellulosum
So Ce 26
So ce 26/SJ3
RecAl3, trpE, NalR
F—, hsds2O (r-, m—), recAl3, ara
14, proA2, lacYl, galK2, rpsL20
(SmR), xyl—5, mtl—l, sup E 44,
lambda-
recA, supE, supF, hsds
Wild-type strain
SmR spontaneous mutant
Plasmid
pSUP202l Ap, Cm, Km, Ph
pSJB5O Cm, Km, Ph
pSJB55 Cm, Km, Ph
pSJB58 Cm, Km, Ph
pME305 Ap, Tc
pUZ8 Tc, Km, Hg
DEPOSITION
Within the scope of the present application, the
following microorganisms and plasmids have been
deposited at the “Deutsche Sammlung Von Mikroorganismen
und Zellkulturen GmbH” in Braunschweig (FRG), which is
recognised in accordance with the Budapest Treaty as
international depository, to comply with the
requirements for the international recognition of the
deposit of microorganisms for the purpose of patenting.
Microorganism/ Date of Deposit Date of viability
plasmid deposit number certificate
pSJB55 25.01.1991 DSM 6321 25.01.1991
(cloned into
E.coli)
Sorangium 25.01.1991 DSM 6380 14.02.1991
cellulosum
So ce 26/SJ3
LIST OF REFERENCES
Datta N et al, J Bacteriol 108: 1244-1249 (1971)
Denhardt DT, Biochem Biophys Res Commun, 23: 641-646
(1976)
Hedges RW and Matthew M, Plasmid 2: 269-278 (1979)
Gentz R et al, Proc Natl Acad Sci, USA 18: 4926-4940
(1981)
Jaoua S et al, Plasmid 18: 111-1l9(1987)
Jaoua S et al, Plasmid 13: 183-193(1990)
Kaiser D, Genetics of Myxobacteria, in: “Myxobacteria:
Development and Cell Interactions”, ed E Rosenberg, pp
163-184, Springer Verlag, Berlin/New York (1984)
Kuner JM and Kaiser D, Proc Natl Acad Sci USA, 18:425-
429 (1981)
Kuspa A and Kaiser D, J Bacteriol 111: 2762-2772 (1989)
Maniatis T et al, “Molecular Cloning”, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY (1982)
Miller JH, “Experiments in Molecular Genetics”, Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY (1972)
Murray NE et al, Mol Gen Genet 150: 53 (1977)
O’Conner KA and Zusman DR, J Bacteriol 155: 317-329
(1983) '
Rella M, Dissertation ETH Zurich, No. 7601, SEITZ
Reichenbach H et al, Trends in Biotechnology, 6: 115-
121 (1988)
Rigby DWJ et al, J Mol Biol, 113: 237-251 (1977)
Rosenberg M and Court D, Ann Rev Genetics 13: 319-353
(1979)
Shimkets LJ and Asher SJ, Mol Gen Genet 111: 63-71
(1988)
Shimkets LJ et al, Proc Natl Acad Sci USA 80: 1406-
1410(1983)
Simon R et al Bio/Technol, November 1983: 784-791
(1983)
EP 0 310 619
EP 0 358 606
EP 0 372 230
US-P 4,910,140
Claims (30)
1. Process for the genetic manipulation of myxobacteria from the Sorangium/Polyangium group, characterised in that (a) genetic material of homologous or heterologous origin or a combination of genetic material of homologous and heterologous origin, which is homologous with or else at least essentially homologous with a corresponding region on the myxobacterial chromosome; or else (b) genetic material which naturally contains no sections which are homologous with or else at least essentially homologous with a corresponding region on the artificially linked with the and is therefore aid of myxobacterial chromosome rDNA techniques known per se DNA to such homologous or else essentially result of which the flanked by homologous or else essentially homologous DNA sections; the cell recombination, at a homologous sections, as a genetic material to be inserted is said is inserted into myxobacterial and there integrated, via homologous site, which is accurately defined by reason of the homologies present between inserted DNA and DNA which is intrinsic to the bacteria, into the chromosome of said myxobacteria.
2. Process according to Claim l, characterised in that said genetic material to be inserted is an expressable DNA which is linked in an operable manner to expression sequences able to function in the myxobacterial cell.
3. Process according to Claim 1, characterised in that the insertion of the genetic material takes place via a donor organism with capable of the conjugation—like information exchange myxobacterium target organism from the donor to the myxobacterium recipient. the the
4. Process for genetic manipulation of myxobacteria from Sorangium/Polyangium group according to Claim 1, which is characterised by the following process steps: the DNA of the myxobacterium target organism or of a related organism; (a) preparation of complete (b) fragmentation of the complete DNA isolated as in (a); (c) cloning of the fragments as prepared in (b) into suitable plasmid vectors and transformation of said vectors into one of the host organisms normally used for cloning purposes; (d) selection of those colonies which contain a plasmid with integrated myxobacterium DNA fragment and isolation of said plasmids; (e) transformation of the plasmid DNA isolated as in (d) into a donor organism capable of conjugation—like information exchange with the myxobacterium target organism; (f) conjugal transfer of the recombinant plasmid DNA to the myxobacterium target organism; (g) cultivation of the transformed myxobacterium cells and selection of positive transformants.
5. Process according to either of Claims that the l or 4, characterised in genetic material to be inserted. is a plasmid. which may contain one or more expressable DNA sections and which contains the homologous or else the essentially homologous DNA regions cloned in.
6. Process according to either of Claims 1 or 4, characterised in that the degree of homology between said homologous DNA sections and the corresponding regions within the myxobacterial chromosome is between 80% and 100%.
7. Process according to that either of Claims DNA 1 or 4, characterised in said homologous sections comprise at least 100 Bp. either of Claims 1 or 4, DNA
8. Process according to characterised in that said homologous sections comprise between 0.3 and 4 Kb.
9. Process according to Claim 4, characterised in that a prokaryote selected from the group consisting of E. coli, pseudomonads, actinomycetes, salmonellae and myxobacteria themselves is used as host organism for the cloning of the myxobacterium DNA fragments or as donor organism: within the scope of the conjugal DNA transfer.
‘ 10. Process according to Claim 4, characterised in that a restriction—negative or restriction-deficient bacterial strain is used as host organism for the cloning of the myxobacterium DNA fragments or as donor organism within the scope of the conjugal DNA transfer.
11. Process according to Claim 10, characterised in that the cloning of the myxobacterium DNA fragments is carried out directly in a donor organism which is capable of conjugation-like information exchange with the myxobacterium target organism.
12. Process according to Claim 4, characterised in that the myxobacterium cells are subjected to a brief heat treatment immediately before the conjugal DNA transfer.
13. Process according to Claim 12, characterised in that the heat treatment is carried out at a temperature of 35°C to 60°C over a period of 1 to 120 minutes.
14. Recombinant DNA molecule which makes possible integration of genetic material at a defined site within the genome of myxobacteria from the Sorangium/Polyangium group, characterised in that said recombinant DNA molecule contains the DNA which is to be integrated, and in that said DNA has homologies with corresponding DNA regions within the myxobacterial genome, or else is flanked by one or more such homologous DNA sequences, to an extent such that, on transformation of the myxobacterial cell containing the homologous DNA region, there is integration of said DNA, which is to be integrated, at a site, which is exactly defined by reason of the homology present between the inserted DNA and the DNA intrinsic to the within bacteria, the myxobacterial genome via homologous recombination.
15. Recombinant DNA molecule according to Claim 14, characterised. in that the degree of homology between DNA the regions within the myxobacterial chromosome is between 80 and 100%.
16. Recombinant DNA molecule according to Claim 14, that DNA said homologous sections and corresponding characterised in sections said homologous comprise a region of at least 100 Bp.
17. Recombinant DNA molecule according to Claim 14, characterised in that said DNA itself with sequence to be has DNA integrated already sufficiently great the DNA homology corresponding regions within bacterial genome so that direct exchange of this sequence for said homologous genomic DNA can take place by means of homologous recombination.
18. Recombinant DNA molecule according to Claim 14, characterised in that said DNA to be double—stranded DNA.
19. Recombinant DNA molecule according inserted is to Claim 14, characterised in that said DNA to be inserted is single—stranded DNA.
20. Recombinant DNA molecule according to Claim 14, characterised in that said DNA to be inserted is an expressable DNA which is linked in an operable manner to expression sequences able to function in the myxobacterial cell.
21. Recombinant DNA molecule according to Claim 14, characterised in that said flanking DNA sections are fused together to a unit, as component of a DNA molecule which is closed in the form of a ring.
22. Recombinant DNA molecule according to Claim 14, characterised in that said homologous DNA sections originate from the myxobacterium genome itself.
23. Cloning vector containing a recombinant DNA molecule according to any of Claims 14 to 22.
24. Plasmid DNA for the conjugal transfer from a donor organism to the myxobacterium recipient from the Sorangium/Polyangium group, characterised in that said plasmid DNA contains, besides the DNA to be inserted, homologous or else essentially homologous DNA sections, and transfer (tra) and mobilisation functions (mob) suitable for transfer into myxobacterium cells.
25. Plasmid DNA according to Claim 24 containing a recombinant DNA molecule according to any of Claims 14 to 22.
26. Process for the preparation of a recombinant DNA Inolecule according to Claint 14, characterised in that the DNA which is to be integrated, which has homologies with corresponding DNA regions within the myxobacterial genome to an extent such that, on transformation of the myxobacterial genome containing the said homologous DNA region, said DNA at a site, there is integration of which is defined by reason of the homology present between the inserted DNA and the DNA intrinsic to the bacteria, within the bacterial genome via homologous recombination, (a) is isolated from a source which contains homologous or else essentially homologous DNA sections; or (b) when said DNA which is to be integrated naturally contains no sections which are homologous with or else at least essentially homologous with a corresponding region on the myxobacterial chromosome, this DNA is artificially linked with the aid of rDNA techniques known per se to corresponding homologous or else essentially homologous DNA sections.
27. Process according to Claim 26, characterised in that said DNA to be inserted is located on a plasmid, and the homologous DNA sequences are cloned into the plasmid DNA at any desired site without, however, thereby destroying the functional integrity of the DNA to be inserted.
28. Process for the preparation of the characterised in that genetically modified myxobacteria from Sorangium/Polyangium group according to Claim 1, (a1) genetic material of homologous or heterologous origin or a combination of genetic material of homologous and heterologous origin which is homologous with or else at least essentially homologous with a corresponding region on the myxobacterial chromosome; or else (a2) genetic material which naturally contains no sections which are homologous with or else at least essentially homologous with a corresponding region on the myxobacterial chromosome and is therefore artificially linked with the aid of rDNA techniques known per se to such homologous or else essentially homologous DNA sections, as a result of which the genetic material to be inserted is flanked by said homologous or else the essentially homologous DNA sections; is inserted into the myxobacterial cell and there integrated, via homologous recombination, at a site, which is accurately defined by reason of the homologies present between inserted DNA and DNA which is intrinsic to the the chromosome of said bacteria, into myxobacteria; and (b) positive transformants are selected with the aid of selection processes known per se and cultivated as pure culture. cell prepared by a process according to any of Claims l to 13 and 28.
29. Genetically modified myxobacterial
30. Genetically modified myxobacterial cell from the Sorangium/Polyangium group containing an exogenous DNA of homologous and/or heterologous origin integrated into the myxobacterial via genome homologous recombination. F. R. KELLY & co., AGENTS FOR THE APPLICANTS.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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CHSWITZERLAND01/03/1991626/91-1 | |||
CH62691 | 1991-03-01 |
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IE920654A1 IE920654A1 (en) | 1992-09-09 |
Family
ID=4191394
Family Applications (1)
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US (1) | US5686295A (en) |
EP (1) | EP0501921B1 (en) |
JP (1) | JP3932517B2 (en) |
KR (1) | KR100229152B1 (en) |
AT (1) | ATE203275T1 (en) |
AU (1) | AU655797B2 (en) |
CA (1) | CA2062095C (en) |
DE (1) | DE59209908D1 (en) |
DK (1) | DK0501921T3 (en) |
ES (1) | ES2161213T3 (en) |
GR (1) | GR3036885T3 (en) |
HU (1) | HU218035B (en) |
IE (1) | IE920654A1 (en) |
IL (1) | IL101097A (en) |
NZ (1) | NZ241786A (en) |
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JP3613614B2 (en) * | 1992-08-31 | 2005-01-26 | シンジェンタ パーティシペーションズ アクチエンゲゼルシャフト | DNA sequences related to soraphen biosynthesis by myxobacteria |
US6121029A (en) * | 1998-06-18 | 2000-09-19 | Novartis Ag | Genes for the biosynthesis of epothilones |
AU768220B2 (en) | 1998-11-20 | 2003-12-04 | Kosan Biosciences, Inc. | Recombinant methods and materials for producing epothilone and epothilone derivatives |
US6410301B1 (en) | 1998-11-20 | 2002-06-25 | Kosan Biosciences, Inc. | Myxococcus host cells for the production of epothilones |
GB0003753D0 (en) * | 2000-02-17 | 2000-04-05 | Biochemie Gmbh | Organic compounds |
JP4454934B2 (en) * | 2001-01-26 | 2010-04-21 | コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガニゼイション | Methods and means for producing efficient silencing constructs using recombinant cloning |
WO2004015088A2 (en) * | 2002-08-13 | 2004-02-19 | Kosan Biosciences, Inc. | Transposon-based transformation system |
WO2004053065A2 (en) * | 2002-12-06 | 2004-06-24 | Kosan Biosciences, Inc. | Disorazole polyketide synthase encoding polynucleotides |
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US4910140A (en) * | 1988-04-18 | 1990-03-20 | Bio-Rad Laboratories, Inc. | Electroporation of prokaryotic cells |
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DE3841453A1 (en) * | 1988-12-09 | 1990-06-13 | Degussa | PROCESS FOR CONJUGATIVELY TRANSFERRING E.COLI MOBILIZABLE VECTORS TO GRAM-POSITIVE BACTERIA AND VECTORS SUITABLE THEREOF |
-
1992
- 1992-02-21 DE DE59209908T patent/DE59209908D1/en not_active Expired - Fee Related
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