Note: Descriptions are shown in the official language in which they were submitted.
' 3~ J
VACCINE AGAINST SEPTICEMIC BACTERIA, SEPTICEMIC
BACTERIA ANTIGEN PREPARATIONS, NEW BACTERIA AND
VECTORS FOR THE PREPARATION OF THESE ANTIGENS OR VACCINES
This invention relates to bacteria belonging to
genera including pathogenic bacteria and expressing
large amounts of external membrane antigenic proteins
regulated by iron.
It also relates to processes for the
production of these bacteria.
It furthermore relates to vaccines
against septicemic bacteria containing as an active
principle bacteria which express in large amounts
external membrane antigenic proteins regulated
by iron, or fragments of these bacteria or
antigens expressed by these bacteria.
It furthermore relates to other bacterial,
viral or other vectors allowing the expression
of these antigenic proteins, as well as to vaccines
containing these proteins as their active principle~
It furthermore relates to recombinant
live vaccines, notably bacterial or viral vaccines
. .
. ~
c~ 3
expressing these antigenic proteins in the vaccinated
organism.
It is known that apart from some lactobacilli
iron is a necessary nutr~ment for all living
forms including bacteria. These need iron to be
able to multiply in a host cell. The capacity
of the bacterium to multiply in vivo is an essential
factor of its virulence.
Although iron is present in large amounts
in a human body, the bacterium only has a
very small amount of free iron atitsdisposal in
order to multiply.
Indeed by far the greater part of an
animal host iron is intracellular (in the form
of ferritin , hae~osiderin r heme) and therefore
its access is difficult. The small amount of iron
which is present in the body fluids only exists
in the form of extremely stable complexes, which
are principa~ly made up of two iron chelating glycopro-
teins : transferrin in the plasma and lactoferrin
in the secretions. The existence of these glyco-
proteins strongly, but in a reversible manner,
linking the iron, is necessary in order to allow
its use by the cells, preventing its precipitation
in the form of ferric hydroxide.
The plasma contains iron complexes
in the form of haptoglobin-heme, ceruloplasmine,
ferritin , lactoferrin and transferrin.
The major part of the iron is transported
by transferrin s. Three major classes of transferrin5
may be recognized : seric transferrin , lactoferrin
and ovotransferrin .
The transferrin captures about 9s%of the
iron in the plasma and its saturation rate
is only about 35~ in the healthy individual.
3 . . f; i
3 1 ~ . s
Lactoferrin has a very small iron
saturation rate and keeps its chelating properties
in a wide pH range ; its presence in all secretions
of the organism, that is to say at the level of
potential microbial invasion sites, impos~ a
wider restriction for iron in these places than
elsewhere in the organism.
The complexation of iron to glycoproteins
results in that only a very small concentration
o of free ferric iron (10 9M) remains, this being
quite insufficient to allow a normal growth of
bacteria.
In order to acquire the iron they need
to multiply in the host, bacteria have a number
of means available.
It seems that some microorganisms may
obtain their iron by a mechanism impl ying direct
interaction between the bacterial cell surface
and the protein linking the iron in the host.
However, this direct acquisition mode only affects
a very limited number of species. Most bacteria,
pathogenous or not, react to the lack of availability
of the iron in a hosL/in some aerobic environments,
by producing iron chelating compounds called siderophores.
Siderophores are made up by molecules
having a small molecular weight forming specific
complexes with a very high affinity for the ferric
ion. Their biosynthesis is regulated by the iron
and their function is to supply the bacterial
cell with iron.
These siderophores possess an extremely
high affinity for the ferric ion (their association
constant is about 1030M 1) which allowsthem to
displace the iron associated with the host protein
or to solubilize the ferric iron which is precipitated
in the form of hydroxide.
Most of previously identified siderophores
belong to two chemical classes : phenolates-
catecholates (deriving from 2,3-dihydroxybenzoic
acid) and hydroxamates (deriving from hydroxamic
acia).
The better known among siderophores
o belonging to the class of phenolates- is the entero-
bactin which is excreted by the bacteria belonging
to the genera Escherichia, Klebsiella, Salmonella
and Shiqella. This enterobactin is made up of
a cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-
serine and is the chemical compound having thehighest known affinity with the ferric ion (Ka =
105 M ).
Several enteric species synthetise
another hydroxamate siderophore~aei-obactin, This
2u siderophoreis particularly synthetised by septicemic
ox invasive Es~herichia coli strains having a
._
type Col V plasmid, or by Salmonella typhymurium
and Shi~ella.
This biosynthesis of siderophoresby
bacteria is associated with the production of
proteins at the outer membrane, some of these
proteins behaving as receptors for siderophoxes,
as well as mechanisms allowing iron transportation
and release inside the bacterium.
The common characteristic of these proteins
which are formed in the outer membrane, and are
often called "IROMP" meaning Iron Regulated Outer
Membrane Protein, is a size between 70 kDa and
90 kDa, and their synthesis as well ln vitro
~ - ~ r ~j 3
in a restricted iron environment as in vivo during
infec-tion.
The outer membrane proteins, or siderophore
receptors, are therefore the second element of
systems characterized as having a high affinity
for the bacterial intake of iron (the first
element being made up by siderophor es)~
Apart fr~ these high affinity systems,
many bacteria possess low affinity transportation
systems which allow them to use ferric hydroxide
in polymerized forms.
The absorption mechanisms for iron
have been particularly studied with Escherichia
coli which is the bestgenetically known micro-
organism.
The high affinity iron transportation
endogenous system in E. coli uses the siderophore
enterobactin. Enterobactin is synthetised and
excreted in the medium when E. coli is placed
in a restricted iron environment. The ferric entero-
bactin complexes are then taken up by the outer
membrane (81 kDa Fep A protein) and transported
to the cytoplasm. When internalized the iron is
freed by erric enterobactin hydrolysis, then
reduced to ferrous iron.
E. coli's enterobactin system comprises
at least thirteen genes. Seven genes (ent) are
involved in the biosynthesis of the siderophore,
and five genes (fep) code for transportation
~0 proteins.
Apart from the enterobactin system,
E. coli septicemic strains excrete and carry an
hydroxamate siderophore,aerobactin.
It has been discovered in 1979 by P.H.
WILLIAMS (37) that some Col V type plasmids carried
genes for the aerobactin siderophore and its
s receptor located in the outer membrane and called
Iut A protein (74 kDa protein).
Although the aerobactin has an association
constant with the ferric ion which is lower than
that of enterobactin, it however has structural
properties increasing its capacity to take up
the iron linked to the transferrin or to the
lactoferrin.
After this was demonstrated in 1979
by P.H. WILLIAMS, many studies have shown that
the aerobactin's iron transportation system played
a major part in the virulence of pathogenic strains
of E. coli and many other bacteria (GRIFFITH
and al. (13)).
The presence of the aerobactin siderophore
~u strongly favours the virulence of pathogenic strains.
Although aerobactin is less powerfulthan
enterobactin as a chelating agent, it is
active in much more varied environment conditions
(enterobactin is very sensitive to oxidation
~5 and pH variations). Aerobactin therefore confers a
higher degree of adaptation to the bacterium.
Besides, aerobactin is a better bacterial
growth stimulator, and it seems that it is much
more quickly excreted than enterobactin , probably
3~ because of a preferential genetic induction when
E. coli is grown in the presence of a chelating
agent.
7 '~
With the aerobactin operon, the bacterium
acquires an extremely efficient iron transportation
system with a minimum number of additional genes,
or only four genes for the synthesis of aerobactin
which is a small simple siderophore,and one gene
which codes for the outer membrane receptor. Indeed
the other genes necessary for the transportation
of hydroxamates are inherently present in all
Escherichia coli.
The expression of all genes coding
for membrane proteins, side;-ophorereceptors and
corresponding siderophoresis regulated by a single
protein, Fur, which acts as a repressor when the
iron is available in sufficient amounts. The central
regulation is superimposed on an individual modulation
which regulates the expression of each system
according to the state of the environment.
Some authors have grown bacteria,
so as to increase the expression of IROMPs in
environment with an iron deficiency with the help
of chemical chelators, such as ~, ~' dipyridyl (A.
BINDEREIF et al~ The cloacin receptor of Col V-
bearing Escherichia coli is part of the Fe 3+
aerobactin system, J. Bacteriol., 1982, 150, 1472-
475 ; C. MAROLDA et al. : Flanking and internal
regions of chromosomal genes mediating aerobactin
iron uptake system in enteroinvasive Escherichia
coli and Shigella flexneri, J. General Microbiology,
1987, 133, 2269-2278 ; A. BINDEREIF et- al. :
Cloning of the aerobactin-mediated iron assimilation
system of plasmid col V, J. Bacteriol., 1983,
153, ~ _1113 ; De LORENZO et al. : Aerobactin
biosynthesis and transport genes of plasmid col V -
K 30 in Escherichia coli K 12, J. Bacteriol. 1986,
r ~ r ;)
165, 570-578 ; P. WARNER et al. : col V - plasmid-
specified aerobactin synthesis by invasive strains
of Escherichia coli, Infection and Immunity, 1981,
33, 540-545 ~. E. F . GRIFFITHS et al. have shown
in : Synthesis of aerobactin and a 76000 Daltons
iron-regulated outer membrane protein by Escherichia
coli K-12 - Shiqella flexneri hybrids and by
enteroinvasive strains of Escherichia coli, Infection
and Immunity, 1985, 49, 67-71, that enteroinvasive
lo strains of E. coli produce aerobactin and a 76
K outer membrane protein when grown in a reduced
iron environment in the presence of ovotransferrin.
The recently acquired knowledge on
the iron absorption systems of bacteria has allowed
one to explore new ways of fighting pat~ genic
bacteria.
It has been suggested to synthetize
siderophoreanalogs which are toxic for the bacterium
and may deceive the iron transportation systems
~0 in order to penetrate into the bacterial cell.
But these synthetic chelators have a lower affinity
for the iron (III) than natural siderophoreS,and
they are unable to displace iron in transferrins.
- ROGERS has suggested to form complexes
between aerobactin and trivalent metal ions in
order to use them as antimetabolites towards the
enterobactin -Fe natural complex. Only complexes
formed with scandium (Sc3 ) and indium (In3 )
have some antibacterial activity (ROGERS et al.
(26) : ROGERS (27)).
It has also been suggested to adsorb
phenolate type siderophores,which are aromatic
molecules, on some seric proteins, which then
play the part of carrier molecules, this allowing
the induction of specific antibodies against
the siderophore.
Thus, BYERS (5) has described a vaccine
against/phenolate siderophorewhich is produced
by Aeromonas hydrophila (a bacteri~which is responsible
for human and fish septicemia), which has been
assayed with fish. The siderophore is covalently
coupled with human or bovine albumine. Fish~which
are immunized with these preparations generate
antibodies reacting against the siderophore It
is, however not specified if the antibodies which
are formed are able to neutralize the siderophore5.
One has also tried to prevent the bacteria
from taking up siderophoreswith antibodies that
would be specifically directed against the siderophore
receptors.
BOLLIN eb al. (4) report the results
of a study indicating some passive immunization
with antibodies against the outer membrane proteins
regulated by iron, thus protecting turkeys from
an Escherichia coli septicemia.
However, all efforts towards the elaboration
of an efficient vaccine have, until now, failed
because of the difficulties in having a bacterium
express membrane proteins which are regulated
by iron at a sufficient level in the bacterial
culture.
This invention allows one to overcome
this difficulty by suggesting the use in a vaccine
as an active principle, of bacteria expressing
in large amounts membrane proteins which are
10 ~ ~ ~ ~ r~ ~ r~
regulated by iron and more particularly, siderophore
receptors at a sufficient level to induce the development
of antibodies which prevent the specific recognition
of siderophoresby their receptors.
An aim of the invention is to supply
bacteria expressing outer membrane proteins regulated
by iron (IROMPs), which may be used as protective
antigens.
Another aim of the invention is to
lo suggest the synthesis of outer membrane proteins
regulated by iron of septicemic bacteria by genetic
recombination.
Another aim of the invention is to
supply large amounts of IROMPs, notably Iut A
and Fep A proteins, siderophore receptors, aerobactin
and enterobactin in Escherichia coli and other
families, through synthesis of these proteins
by a genetic recombination.
Another aim of the invention is to
supply vaccines containing as an active principle,
bacteria or fragments of these bacteria having
in their outer membrane large amounts of IROMPs
and notably Iut A and Fep A proteins, as obtained
by genetic recombination or by other processes.
Another aim of the invention is to
supply vaccines contai~ing as an active principle
IROMPs, for example, Iut A and/or Eep A proteins
or antigenic preparations incorporating these
proteins.
This invention therefore uses bacteria
expressing outer membrane proteins regulated by
iron and some of which are sideropho~ receptors,
which may be used in a vaccine preparation.
c ` s ~
1 1 ' ~ i
sacteria according to the invention
are characterized in that they express larger
amounts of these outer membrane proteins, and
more particularly, transferrin receptors, notably
siderophore receptors, to induce, when these proteins
are used in a vaccine, the generation of antibodies
preventing the specific recognition function by
the receptors, and thus putting an end to the
iron supply of the pathogenic bacterium.
Bacteria which are used are preferably
enterobacteria and they preferably excrete enterobactin
and/or aerobactin siderophores.
Bacteria are preferably chosen among
the group made up by Escherichia coli, Klebsiella,
Salmonella thyphimurium, Shiqella.
Bacteria preferably excrete together
the aerobactin and enterobactin siderophores.
According to the invention and according
to a first embodiment thereof, bacteria are obtained
by growing naturally existing strains or strains
that may be found in laboratories or collections
in a minimal medium wherein the availability o~
iron is reduced to a level allowing a satisfying
higher expression of/~brane proteins.The culture
is preferably grown in presence of a strong iron
(III)chelating protein such as lactoferrins, these
being che]ators which advantageously establish
an iron shortage with the same characteristics
as those that are to be found _ vivo.
Object of the invention is also a process
for producing such bacteria to be used for the
preparation of vaccines, characterized in that
' ~ ,
.. ..
~2
said bacteria are grown in a culture medium containing
an iron (III) chelating protein such as transferrins,
and notably lactoferrins.
As a minimum medium, one can use that
which is described for instance by SIMON and
TESSMAN (30).
However, this embodiment remains difflcult
to apply, because with the iron chelators which
are generally used one must sufficiently reduce
the iron content in the culture medium so as
to obtain a sufficient expression of the outer
membrane proteins. Often, small amounts of iron
remain, preventing the expression of membrane
proteins (iron from the fermentor, for instance
or from pipes which are, generally, made of stainless
steel). One must then resort to comparatively
complex process~in order to lower the iron content
to a level allowing the expression of the external
membrane proteins of bacteria, and their application
is costly.
According to a second embodiment of
the invention, which is also the preferred embodiment,
bacteria expressing in large amounts the outer
membrane proteins, sideropho~e receptors, are
transformed by recombinant plasmids.
Indeed, the advantages of synthetizing
the outer membrane proteins, sideropho~e/ or tPransferrin,
through genetic recombination, are many :
- it allows one to generate an important
expression of these proteins, whatever the iron
concentration in the culture medium,
- it allows one to study immune reactions
directly aiming at these proteins, while excluding
13
any other constituent of the original strain,
- it represents the cheaper solution
for the expression of membrane proteins in an
environment wherein iron is always present (fermentors,
pipes and sundry stainless steel equipment).
If the applicant more particularly
aims at the synthesis of Iut A and Fep A proteins,
aerobactin and enterobactin siderophore receptors
of E. Coli, it can be understood that the below
described genetic recombination methods - will
apply by an ~ogy with the synthesis of membrane
proteins (IROMPs), siderophore receptors, aerobactin
and enterobactin or tranferrins from pathogenic
bacteria other than E. coli.
The invention therefore also relates
to the preparation of E. coli Iut A and/or Fep A
proteins by genetic recombination.
The Iut A protein may be synthetized
by a process according to which, in particular :
- one isolates the plasmid or chromosomefrom
Salmonella, Shigella or Klebsiella pathogenic
E. coli strains, bearing the~aerobactin operon,
- one separates from t.he plasmid or
chromosome a fragment containing the iut A gene,
- one links said fragments with a
cloning vector,
- one inserts the clones having inte grated
the iut A gene in an expression vector (for example
GTI 001 plasmid),
- one then expresses the Iut A protein
by growing the clones.
14 ~ r J ~ ,J
The Fep A protein is obtained :
- by isolating from a plasmid (for
example pMS 101 plasmid built by LAIRD and YOUNG
(19)) or from a bacterial chromosome, E. coli,
Salmonellae or Klebsiellae, a fragment bearing
the f~e_~ gene,
- by cloning said fragment in a cloning
vector,
- by inserting the fep A gene in an
expression vector, preferably in a vector which
is used for the expression of Iut A protein (GTI
001 plasmid),
- by the expression of the Fep A protein
by growing the clones.
The expression vectors for Iut A and/or
Fep A proteins may be bacteria and one prefers
to use E. coli whose expression systems are best
known. One may however also use other vectors,
notably viral vectors or vectors made up of yeast,
and which can be built by specialists.
Bacterial clones expressing Iut A and/
or Fep A proteins may be multiplied in an appropriate
medium at a sufficiently low temperature so as
to prevent or limit the expression, generally
below 32C. The expression is then induced by
rising the temperature, for example to 42C
during about 4 hours, so as to induce the expression
of iut A and fep A genes.
One therefore obtains bacteria integrating
Iut A and Fep A proteins as well as their proIut
A and proEep A precursors, these having the
shape of large size cytoplasmic inclusions.
15 ¢.
These bacteria as used in a vaccine,
as an active principle, induce the generation
of antibodies directed against Iut A and Fep
A proteins, preventing recognition by these proteins
of their respective siderophores aerobactin and
enterobactin, this therefore strongly reducing
the iron supply to the bacterium and blocking
its multiplication.
The invention therefore also relates
to vaccines containing as an active principle
recombinant bacteria expressing external membrane
proteins regulated by iron.
The invention more particularly relates
to vaccines containing as an active principle :
recombinant bacteria or fragments of these bacteria,
notably fragments of membranes integrating Iut
A and/or Fep A proteins or their precursors, proIut
A and/or proFep A ; or again Iut A and/or Fep
A proteins and/or their precursors, for example,
extracted from cytoplasm or eX-tracted from the outer
membrane of recombinant bacteria.
In another embodiment, the invention
relates to vaccines containing as an active principle :
bacteria which are homologous to septicemic bacteria,
or fragments of these bacteria, grown in a medium
with restricted iron supply, and which integrate
ln larger amounts Iut A and/ror Fep A proteins
and/or their precursors; /I~t A and/or Fep A proteins
(and/or their precursors) being suitably extracted
The latter vaccines are preferably
prepared from bacteria which are grown in a medium
containing a strong protein type iron (III) chelator,
16
notably transferrin, lactoferrin, or ovotransferrin.
The invention would be better understood
on reading the following specification , referring
to the appended drawings, wherein :
Figure 1 is the protein profile as
obtained by electrophoresis of a clone expressing
Iut A protein,
Figure 2 is a protein profile as obtained
by electrophoresis of a clone expressing Fep
A protein.
Theabbreviations used in the following
specification have the following meanings :
Ampr ampicillin resist2nt
Clos cloacin sensitive
dATP deoxy-adenosin-triphosphate
EDTA ethylene-diamine-tetraacetic acid.
Ent enterobactin
E.O~P.S. without specific pathogenic organisms
IPTG isopropyl-~-thiogalactopyranosid
kpb bases kilopair
LB luria broth
OMP Outer-membrane protein
PAGE polyacrylamide gel electrophoresis
pb bases pair~
PBS phosphate buffered saline
SDS Sodium dodecyl sulfate
5T Simon and Tessman
TEMED N,N,N',Ii'-tetramethylene diamine
Tris tris-hydroxy-aminomethyl-methane
tetr tetracyclin resistant.
17
MATERIALS AND METHODS
I. MATERIALS
1. Stralns
Table I recapitulates the various strains.
The pathogenic strains used are septicemic strains
from calves otchicks and are to be found in the RHONE-
MERIEUX strain collection.
The host strains _ used for cloningr
sequencing and expression are all derived from
o Escherichia coli K 12.
These strains may be easily replaced
by other wild septicemic strains or laboratory
strains;
2. Plasmids
Origin and characteristics of plasmids
used for cloning and expression are presented
ln Table II.
3. Media
SIMON and TESSMAN mlnimum medium (30)
Contents :
NaCl 5.8
XC1 3,7 g
cacl2~ 2H2 0 15
MgC12, 2 0.10 g
NH4C1 1.10 9
Na2S4 0,142 g
KH2~04 0.272
Tris 11 20 9
H20 ~5p loOO ml pH 7~4
The only carbon source is sodium succinate
added to a final concentration of 10 g/l.
18
In order to establish a limitation
to the iron in this medium, one adds ovotransferrln
(SIGMA) at a final concentration of 250 ~g/ml
(One may have concentrations abo~e 500 ~g/ml).
The iron-rich control medium is obtained
by adding FeC13, 6 H20 (MERCK) at a final concentratlon
of 40 uM.
M9 MANIATIS minimum medium (29)
Na2HP0~ 6.0 g
KH2P04g
NaCl0.5 g
NH4C11.0 g
H20 ~sp1000 ml pH 7,4
to this basic medium are added :
M~ S04 1M 2 ml/1000 ml
glucose 20 ~O 10 ml/1000 ml
CaC12 1M 0.1 m1/1000 ml
- LB rich medium (MANIATIS and al. (23))
bactotryptone 10 g
Yeast extract 5 g
NaCl 5 g
H20 q.s.p. 1000 ml pH 7~4
- BTS rich medium (BIO MERIEUX)
Biotryptase17 g
Biosoyase 3
NaC1 5 g
K2HP04 2.5
glucose 2.5 g
H20 l~sp1000 ml pH 7~3
- BHI rich medium (heart-brain BIO MERIEUX)
Calf brain infusion 200 g
ox heart infusion 250 g
~lo~ lytone 10
NaCl 5 g
Na2HPO4 2,5
glucose 2 0 ~
1~2() ~s~ 1000 ~1pH 7,4
- "M9 SP" rich medium for the expression medium :
SP (tryptone 3.2~ : yeast 2~) 100 ml
M9 (6.6 x concentrate filtered on 0~22 ym
filter (Millipore) 15 ml
MgSO4 100 mM 1.5 ml
FeC13 0.1 mM 1.5 ml
vitamin B1 (5% solution) 1.5 ml
'l'he solid media have the same composition
as that of the corresponding llquid media and
o contain 12 g agar per liter of the medium.
Antibiotics are used in solid and
liquid media at the following final concentratlons :
Ampicillin 25 ~g/ml
tetracyclin 12.5 yg/ml
isopropyl-~-D-thiogalactopyranoside (IPTG) is optionally
added at a final concentration from 0.05 mM to
0,4 mM.
Sterilization of liquid and solid media
is made by autoclave at 120C during 20 minutes.
Antibiotics, vitamin Bl, sodium succinate
M9 6.6X, MgSO4, FeC13, IPTG and ovotransferrin
solutions are made in the form of concentrated
stock solutions, and sterilized by ~iltration
on a filter having 0.22 ym porosity (Millipore).
After sterilization, the growth media
are kept at room temperature.
Antibiotics, IPTG and ovotransferrin
solutions are kept at -20C.
Other solutions are kept at +4C
II. METHODS
1. 3acterial cultures
Apart from clones with structures built
in the pGTI 001 expression vector, which are grown
at +30C, all cultures are made at +37C, while
stirring, during 18 hours.
Whenever necessary, bacterial growth
is estimated by measuringthe suspension turbidity
at600 nm, with the help of a BECKMAN DU 40 spectro-
photometer.
Cultures are usually made in a volume
of 2 ml after seeding with a colony. Cultures
in a more important volume (20 ml to 1000 ml)
are made by seeding to the l/lOOth with a preculture
in stationary phase~
2. Sensitivity toward bacterocins
Productions of cloacin DF 13 and colicin
B are made with Enterobacter cloacae DF 13 and
Escherichia coli 1300 strains, respectively, according
to the process described by DE GRAAF (DE GRAAF
et al. (8) and (9)).
These strains are grown at +37C, in
a BHI medium, until the optical density reaches
0.5 (1 cm, 600 nm).Mitomycin C is then added to
the growth medium, so as to reach a final concentration
of 1 ~g/ml, which allows one to induce the synthesis
of bacteriocins. This culture is prolonged during
6 hours, at +37C, until lysis phase. Bacterial
bo-~ies are centrifugated (8000 g, 30 nm, +4C)
and the supernatant is harvested. Ammonium sulfate
is then slowly added at +4C, until the concentration
reaches 365 g/litre.
The supernatant is taken up in 0.05 M
phosphate buffered pH 7.0 and dialysed against
several succeeding baths of this buffer. The dialysate
is filtrated over 0.22 ~m (Millipore) and kept
at -20C.
About 10 bacteria of the clone under
study are spread on LB agarose containing the
appropriate selection antibiotic. When the deposition
liquid is comple~lyabsorbed, one places at the
center of the Petri dish 75 ~1 of the bacteriocin
solution. When this drop has itself dried, the
Petri dish is placed in an incubator t+30C or
+37C, as the case may be) during 18 heures. Clones
which present a growth inhibition around this
deposition have became bacteriocin-sensitive.
Clones which resist the toxic effect of bacteriocin
on the contrary exhibit a uniform bacterial mat.
3. Preparation of antisera directed a~ainst
the outer membrane proteins regulated by iron
The protocol which is used is a repetition
of that which is described by BOLIN and JENSEN
(4).
The outer membrane proteins regulated
by iron are separated by polyacrylamide gel
preparative electrophoresis with sodium dodecyl
sulfate added.
When the gels are coloured, the strip
containing the IRCMP to be used for the immunlzation
is cut offJthen comminuted with distilled water,
by passing through several needles havin~ sinaller
and smaller diameters.
This ground product is injected to E.O.P.S.
22
Ne~ Zealand White rabbits, from the RHONE MERIEUX
Company Farm, according to the protocol presented
in table III.
To eliminate antibodies against other
outer membrane proteins, lipopolysaccharids and
other Escherichia coli antigens, sera harvested
in immunized rabbits are adsorbed with an Escherichia
coli strain which does not express IROMPs.
To each milliliter serum are added
about 101 bacterial bodies which have been inactivated
by heat during 30 minutes at 100C and the mixture
is then incubated at +37C during 1 hour.
After centrifugation, the surpernatant
serum is recovered and filtrated on a 0.22 ~m
porositty filter (Millipore).
It is kept at -20C.
5. Outer membrane proieins extraction
The method which is used to extract
proteins from the outer membrane derives from
me-thods described by VAN TIEL-MENXVELD et al.
(36) and by FISS et al. (12).
After centrifugation, the bacterial pellet
T is resuspended in Tris-Hcl 0.2M pH 8.0 buffer
so as to obtain an optical density of about 10.
~acterial cells are then ruptured by sonication
using ultrasoundS 3 x 3 minutes while keeping
the bacterial suspension on anice-ethanol bath.
Cellular debris and untouched bacteria,
are eliminated by centrifugation at 5000 g during
1.0 minute at ~4C.
Bacterial membranes suspended in the
supernatant are collected by ultracentrifugation
at 110 000 g during 60 minutes at +4C.
The membrane pelletistaken off with
5 ml extraction buffer described by SCHNAITMAN
23
(29) : Triton X-100 2% ; MgC12 10 mM ; Tris/HCl
50 mM pH 8Ø
Incubation is carried out during 30
minutes at room temperature, while shaking every
five minutes. During incubation the cytoplasmic
membrane proteins are preferentially solubilized
by Triton X-100. Outer membranes are collected
through a new ultracentrifugation (111,000 g,
60 minutes, +4C). The obtained pellet is thrice
washed in distilled water, finally resuspended
in 1 ml distilled watex and frozen at -20C for
storage.
6. Proteins dosaqe
The membrane extract protein concentration
is measured by a colorimetric method derived from
that published ~y LOWRY et al (20).
To 0.5 ml protein solution to be dosed
are added 2.5 ml of the following solution :
1~ CuSO4 solution 1 ml
2~ sodium tartrate
solution 1 ml
2~ sodium carbonate
solution in 0.lN NaOH q.s.p.
After incubating 10 minutes at room
temperature, 0.25 ml 50~ Folin reactant (Merck) is
added.
Incubation is carried out for 30 minutes
at room temperature, and the optical density of
the blue color which has evolved is measured
at 779 nm.
Protein concentration of samples is
determined with a standard interval prepared with
bovine serum albumin .
24
The optical density is proportional
to the proteln concentration in an interval
of 5 to 200 ~m/ml.
7. Techniques for the analysis of the
outer membrane protein composition : polyacrylamide
qel electrophoresis under denaturatin~conditions
Polyacrylamide gels are prepared according
to the characteristics described by LUGTENBERG
et al. (21).
lo The align~ment gel has the following
composition :
acrylamide-bisacrylamide (30/0,8 p/p)
5% ; ~ris/HCl 130 nm pH 6,8 ; SDS 3,5 mM ; ammonium
persulfate 44 mM TEMED 8 mM.
The separation gel has the same composition
as the align~ent gel, except for the acrylamide/bis-
acrylamide concentration (8 or 10%) and the Tris/HCl
buffer 380 mM pH 8.8 concentration.
The migration buffer used has the following
20 composition :
glycine 14.4 g
Tris3.0 g
SDS 1.0 g
H2O q.s.p. 1000 ml pH 8.3
Extracts to be analyzed or purified
by electrophoresis are diluted in at least an
equal volume of the following dissociation buffer :
Tris/HCl 100 mM pH 6.8 ; glycerol 20~ ; SDS 70 mM,
~-mercapto-ethanol 100 mM ; bromophenol blue 75 ~m.,
The thus diluted extracts are heated
to 100C during 5 minutes.
In order to analyze the outer membrane
protein composition 30 to 50 ~g proteins are deposited
in each well.
For preparative electrophoreses, as
much as 2 mg proteins are deposited in the sole
preparative well.
Migration is carried out at +14C during
5 hours at 160 V or 14 hours at 60 V (Vertical
gel LKB apparatus). In order to increase the resolution
of the outer membrane proteins regulated by iron,
some electrophoreses have been under a voltage
of 100 V during 16 hours. At the end of the electrophoreses,
proteins are fixated and coloured during 30 minutes
at room temperature with 1.2 mM Coomassie blue
in a (50:10:50 v/v/v) methanol/acetic acid/water
mixture. The unfixated colour is eliminated with
several succeeding (40:15:145 v/v/v) methanol/acetic
acid/water baths at 37C. Once decolorated, the
gel is photographed, and dried.
The outer membrane protein profiles
for each strain may then be analyzed by densitometry
(LKB ULTROSCAN laser Densitometer).
8. Detection and analysis of anti-
IROMPs antibodies
The presence of specific anti-IROMPs
antibodies is lnvestigated with the microplate
~LISA technique (ENGVALL and PERLMANN (10) ; COULTON
(7)). Antigens which are coupled to the solid
phase are fractions which are very much enriched
in proFep A protein or proIut A protein.
The revelation of anti-IROMPs antibodies
is made with a rabbit anti IgG conjugate (or chicken
anti IgG) coupled to peroxidase (Nordic). The
substrate used is orthophenylene-diamine. Readings
of optical densities are made at 492 nm.
.
~ . r~ r~
26
-"Western-blotting" technique
Proteins which are separated by polyacrylamide
jel e1ectrophoresiswith SDS are transferred on
a polyvinylidene fluoride membrane (PVDF 0.45 ~m
Millipore) according to the method described by
TOWBIN et al. (34).
Transfer is made under 24 V during
one hour with a RIOLYON apparatus using the following
anodic and cathodic buffers :
10 anodic buffer cathodic buffer
Tris 0.3 g Tris 0.3 g
glycine 1.44 g glycine 1.44 g
methanol 100 ml SDS 0.1 g
H~O qsp 500 ml H2O qsp 500 ml
After transfer, the PVDF membrane is
saturated during one hOur at +37C in PBS buffer
containing 1% S'cimmed milk.
The membrane is then cut into strips
corresponding to electrophoresis tracks.
Sera to be studied are diluted into
PBS buffer containing 1% ~'cim.~e~ milk, then contacted
with membrane strips, 4ml diluted serum per strip.
After incubation for one hour at +37C,
while gently stirring, three 20 minutes washings
are made in PBS buffer containing 2% skimmed
milk at room temperature.
An anti IgG conjugate coupled to peroxIdase,
diluted to the 1/1000 in PBS containing 1% skimmed
milk is added at a rate of 3 ml per strip.
After incubating one hour at +37C
while gently stirring, 3 20 minutes washings
are made at room temperature in PBS buffer.
27
The diaminobenzidine substrate, diluted
to 0.1~ in a physiological water at pH 7.15 to
which 30 volumes 0.1% H2O2 have been added,
is then added to the whole. One then notices
(after 5 to 20 minutes) brown coloured strips
around proteins which are recognized by the antibodies
in the serum under study.
The membrane is then washed in distilled
water and dried up.
9. Plasmid DNA preparation method
Large plasmids contained in pathogenic
strains are extracted according to the method
published by KADO et al. (16).
Plasmids obtained during the various
stages of cloning, undercloning, and construction
in the expression vector are extracted according
to the method of BIRNBOIM (BIRNBOIM and DOLY)
(3). Plasmid DNA obtained after preparative extractionS
made with one/~ese methodsis purified on caesiurn
chloride gradient (MANIATIS et al. (23)).
Whence purified, the plasmids are taken
up in Tris 10 mM ; EDTA 1 m~1 pH 8.0 buffer so
as to reach a final concentration of l~g DNA/~l,
and frozen at -20C for storage.
10. DNA analysis and modification methods
All methods used for cloning, DMA digesting,
restriction fragment analysis in agarose gel,
modification of the ends of restriction
fragments, hybridation with a radioactive probe
after tranfer of DNA on nitrocellulose membrane,
are described by MANIATIS (MANIATIS et al. (23)).
DNA has been sequenced and oligonucleotides have
been synthetized according to special methods.
r~ r~ f i ~
28
11. DNA sequenclnq
Genes to be sequenced are subcloned
in M13 mp 18 and mp 19 vectors (YANISCH-PERRON
et al. (38)) and sequenced according to the
chain endmethod by dideoxynucleotides (SANGER
et al. (28)). Labelling of the various chains
is made with 5S dATP (AMERSHAM).
Sequencing proper is made with reactants
and sequencing kits enzymes Amersham and Sequenase
lo (USB). Electrophoreses in a polyacrylamide gel
with urea are made in a Sequi-Gen (BioRad) apparatus.
Sequencing data are processed with
Microgénie (Beckman) software.
12. Oliqonucleotides synthesis
The various oligonucleotides which
are necessary to theconstructions/the expressing
vectors or to the mutageneses are synthetised
according to the cyano-ethyl-phosphoramidites
method on anApplied Systems 381-A apparatus.
These oligonucleotides are directly
used after deprotection and precipitation with
ethanol.
13. Directed mutagenesis method
Mutagenesis is made according to the
method described by ECKSTEIN (TAYLOR et al. (33) ;
NAKAMAYE and ECKSTEIN (24)) with the directed
mutagenesis kit sold by the Amersham Company.
RESULTS
1. ISOLATION AND ANALYSIS OF iut A
GENES
The presence of the aerobactin operon
has been searched on plasmids born by 15393,
15972 and 16003.
29
After purification on caesium chloride
gradient, these plasmids are digested by Bam HI,
Hind III, Pvu and Sal I restriction enzymes (Boehringer).
The various restriction fragments of each plasmid
are separated on 0.8% agarose gel and transferred
on a nitrocellulose membrane according to the
"Southern blot" (SOUTHERN (31)) method. These
fragments are then hybridated with two radioactive
probes (labelled with 32p by displacement of cut)
prepared from pABNl plasmid restriction fragments
bearing the aerobactin operon of pCol V-K30,
(BINDEREIF and NEILANDS (2)). It has been found
that the gene coding for the IutA receptor exists
on all plasmids on a 6.6 kb Bam HI-Bam Hi restriction
fragment. These results are a confirmation of
the authors' work of undercloning this gene from
an equivalent PCol V-K30 fragment (KRONE et
al. (17)).
- Cloninq of plasmid DNA fragments
bearing the iut A gene
Plasmids of 15393, 15972 et 16003
strains are digested by Bam HI restriction enzyme.
After electrophoresis on 0.8~ agarose gel, the
gel strip containing the 6.6 kb fragment of each
plasmid is cut and the DNA is electroeluted.
The three 6.6 kb Bam HI-Bam HI fragments
are ligated separately and the paT 153 vector digested
by Bam HI. The three ligation mixtures are used
to transform competent HB 101 bacteria (table
IV)~
The DNA of ampicillin-resistant, tetra-
cycline sensitive clones is extracted (by the
method of BIRNBOIM and DOLY) and digested by Bam
HI in order to check the size of the insert.
3 ~
Clones having integrated the 6.6 kb fragment are
selected on the basis of their sensitivity to
cloacine DF 13.
For each starting plasmid a cloacine
sensitive clone is kept for analyses and further
build ups. They are the following clones :
Strain 15393 clone HB 101 p 5 - 15
Strain 15972 clone HB 101 p P - 13
Strain 16003 clone HB 101 p 4 - 18
lo - Clones analysis
A study of the expression of Iut A
receptor.
Having been able to select the desired
clones with a cloacine sensitivity test shows
15 that there is an expression of the receptor to
cloacine and aerobactin. Expression of the iut
A gene born by the 6.6 kb Bam HI-Bam HI fragment
probably depends from a weak promoter situated on this
fragment (KRONE et al. (17)) and does not depend
20 on the iron concentration as does the main promoter
of the operon aerobactin. Indeed, the sensitivity
to cloacine is demonstrated with a culture on
iron-rich ampicillin-LB ~elose.
In order to study the expression level
25 of the Iut A receptor, the various clones are
grown in LB-ampicillin medium (for one night at
+37C) with or without ovotransferrin (500 ~g/ml).
The outer membrane proteins of each
of the three clones are extracted and analyzed
30 on polyacrylamide gel with SDS.
Whatever the growth conditions, it
is impossible to note a 76 kDa supernumerary protein
expression in cloacine sensitive clones.
- Restriction ma~s
Plasmid DNA of clones 4-18, 5-15 and
P-13 is extracted in large amounts and purified
on caesium chloride gradient.
Restriction mapshaving the three 6.6
fra~ments
kb Bam HI-Bam HI/whlch have been cloned are drawn
with Bgl II, Bst E II, Cla I, Eco RI, Kpn I,
Pst I, Pvu II and Sma I.
These three maps are totally identical
and correspond to the restriction map of the 6.6
kb Bam HI-Bam HI fragment of p ColV-K30 aerobactin
operon deduced from maps published by BINDEREIF
and NEIEANDS (1,2) and KRONE et al. (17).
These four maps are represented in
table V.
- Sequencing of iut A qenes
When comparing restriction maps for
the three 6.6 kb Bam HI-Bam HI fragments with
the restriction map as deduced from the sequence
of the iut A gene (KRONE et al. (18)), it appears
that the iut A gene is entirely to be found
on a 3.2 kb Bst E II-Bst E II restriction fragment
(table VI).
This fragment is isolated by electro-
elution, religated upon itself with T4 phage ligase
(Boehringer) and digested with several restriction
enzyme systems. The various fragments thus obtained
(size 150-600 pb) are isolated by Geneclean (Bio
101) and sub~cloned in the M13 mp 18 and mp 19
vectors previously digested with the appropriate
enzymes. The sequence of each su~clone is then
~ F
32
determined according to SANGER's method with
the use of Amersham and Sequenase ~USB) sequencing
kits.
Only the 3.2 kb Bst E II-Bst E II
fragment of clone P-13 has been entirely sequenced.
The two remaining iut A genes have been sequenced
between the Bgl II (1) site and the Eco RV (2396)
site.
The Bst E II-Bgl II region of clone
P-13 has been compared with the sequence of the
iuc D gene 51Ocated just upstream of iut A
(HERRERO et al. (14)) and the Pvu II-Bst E II
region of this clone has been compared with the
sequence/ISl element (OHTSUBO and OHTSUBO (25)).
These comparisons as well as the
comparisonS of the three
iut A genes with the sequence of the iut A gene
of pColV-K30 are presented in table VII.
Analysis of these four sequences reveals
that the iut A gene was extremely well kept at
the molecular level. Apart from one or two bases,
the three iut A genes isolated from E. Coli strains
of animal origins are identical. Differences observed
with the sequence published by KRONE et al. (18)
are minimal.
The three iut A gene under study are
99.77% homologous to the iut A gene of pColV-K30.
Regions where differences have been
demonstrated are represented in table VIII. (Numbers
relate to the position of bases in sequences presented
in table VII).
,~
,~
V ~ 9i;
33
Two important regions are totally preserved :
the sequence coding for the signal peptide and
the consensus sequence (~Ton B box" ) typical
of the outer membrane protein receptors whose
function depends from Ton B.
The existence of four inserts in relation
to gene iut A of ColV-K30 has been demonstrated
on each of the three sequenced genes. These inserts
trigger limited changes in the reading frame.
Thus, the primary structure of iut A proteins
coded by isolated plasmids of the strains under
study is a little larger (+ 8 amino acids) than
that of protein Iut A of p ColV-K30. The sequence
of isolated iut A genes of the strains under study
codes for a 733 amino acid polypeptide comprising
a 25 amino acids signal peptide which is identical
to that of the Iut A polypeptide of strain ColV-
K30. The calculated mass of the mature protein
is 78097 daltons, which differs slightly from
the size observed on gel (76 kDa). However, the
changes in the primary structure are not sufficiently
important to alter the secondary structure and
the hydrophilicity profile.
~. EXPRESSION OF CLONED IUT A AND fep A
GENES
- Characteristics of the vector under
study
The expression vector used is the
GTI 001 plasmid built up by the Mérieux Institute
3 genetics engineering laboratory.
The genes it bears and its restriction
map are presented in table IX.
34
This plasmid may be built up as follows .
Plasmid pBRTac, made up by pBR322
(Bolivar F. et al. Gene 2, 95-113 (1977)) propagating
between HindIII and BamHI~promotor Tac (Ammann
E. et al., Gene 25, 167, (1983)), has its XhoI
site destroyed by the Kleenow polymerase (also
called "the kleenow") to give plasmid pBRTacX .
This plasmid is digested by NcoI, treated by
the kleenow, then digested by AvaI, and its smallex
fragment is ligated to the pMC9 fragment (Casadaban
M.J. et al., Journal of Bacteriology, 143, 971-
980 (1980)) digested by MstII, treated with the
kleenow and then digested by AvaI bearing gene
Lac i and the replication origin of pBR322.
The resulting plasmid (named pBRLaciX )
is digested by HindIII, treated with the kleenow
and then digested by PstI, and the 2350 bases
pair (or "pb") fragment is ligated to the 2300
pb'fragment of pBRTac digested by EcoRI, and then
treated with the kleenow, and digested by PstI,
thus creating plasmid pBRTaci. The latter's 4406
pb fragment, obtained by digested with EcoRI and
PstI is ligated with a 1688 pb fragment digested
by EcoRI and PstI and derived from pBR322 sequences
and bearing the pBR322's tetracycline resistance
gene.
The obtained plasmid is called pBRTaciTet.
The latest replication origin is separated by
digestion with BamHI, and the remaining 2096
pb fragment is ligated to the 2033 pb fragment
of pATI53 (Twigg A.J. & Sherratt, D. Nature, 283,
216-218 (1980)) digested by XhoI, thus creating
plasmid pATTaciOri.
C ? ,' ~ ? !~
~, ., .' ., / )
This plasmid is digested by EcoRI,
then treated with the kleenow, and digested by
AvaI and the 2704 pb fragment is ligated with a
fragment bearing promoter Pr and its thermosensitive
CI857 repressor of the 3076 pb Lambda bacterio-
phagederived from pCQV2 ~Queen C. et al., J. Mol.
Appl. Genèt. 2, 1-10 (1983)) by digestion by PstI,
treatment with the Mung Bean nuclease, and partial
digestion with AvaI. The resulting plasmid is
lo called pGTI001.
The replication origin of this plasmid
is under control of promoter tac, which allows
one to regulate the number of copies by growing
the bacteria in a medium containing various IPTG
(gene lac i inductor) concentration.
The gene to be expressed is placed
under control of phage CI 857 strong "Pr" promoter
(the phage's repressor being thermosensitive).
The ATG of the gene to be expressed is replaced
by the ATG of gene cro. This ATG is created by
partial Bam HI digestion of p GTI 001 followed
by digestion with Mung Bean nuclease so as to
obtain a~ blunt end.
The gene to be expressed is inserted
in phase (starting from codon number 2) between
the blunt ATG end and the XhoI site. An
ending signal for the transcription, placed just
downstream from the XhoI site, avoids the
production of too long messenger RNAs.
Other plasmids of this kind able to
express gene iut A (or fep A), are easy to obtain
or to build and one l~nows such piasmids wherein the gene
to be expressed is controlled by a promoter whose
repressor is thermosensitive.
- Construction_of the expression vector of gene _ut A
~; '': ~'' l'l " '
36
iut A genes isolated from strains 15972
and 16003 are cloned with their .signal sequence at the level
of the expression site Bam HI (899) of p GTI 001.
To obtain a blunt 5' end, starting
with the ,~econd amino acid of the signal sequence
(this time methionine), a two strands synthetic
oligonucl.eotide is used to replace the region
comprised between the initi.ation ATG and the only
Acc I site s:Ltuated in 5' of the coding sequence.
,o The sequence of this oligonucleotide is presented
in table X.
The two complementary s-trands of this
oligonucleotide are .synthetised and the double
strands form is obtained by heating to 90C
15 in a NaCl 50 mM, Tris 10 mM, MgCl 10 mM, pH 7.5
buffer, of an equimolar mixture of the two simple
strands, ol.1.owed by a slow cooling down to room
temperature,
The strategy which is followed in order
20 to insert gene iut ~ of strain 15972 (clone p
P-13) is presented in table XI.
With a not very satisEyi.nc, yield, a modified strategy
is adopted to put in phase gene iut A of strain,
16003. This new strategy uses a" sub-cloning
25 of the E:co RI-Eco RI region of the intermediary
construct ~step 4, table XII~ in vector pSB 118.
This vector is a pVC 18 derivative. It has a
"polylinker" bet~Jeell two Eco RI sites. Sub-cloning of
the Eco RI-Eco RI fragment in this vector has thus
30 allowed o~e to pu-t in phase gene iu-t A while avoiding
partial digestions. (Table XII).
r~
~, J .
37
Restriction maps of the
GTI P-2 (gene iut A of strain 16003) and GTI
B-5 (gene iut A of strain 16003) expression
plasmids thUs obtained are presented in table
XIII.
Clones obtained after renewed ligation
with the double strand "iut" oligonucleotide
are selected on the basis of the preservation
of site Acc I in gene iut A and the disappearance
o f site Bam HI in p GTI 001. All clones presenting
this restriction profile have been controlled
at the expression of protein Iut A.
- Control of the expression of Iut A
Qualitative control
The selected clones are grown in M9
SP tetracycline medium with 0.4 mM IPTG at 32C
(start of induction). The sensitivity of these
clones towards cloacine is examined by the above
described method.
Two clones out of 25 are positive for
the constructions GTI-iut 15972.
Three clones out of 6 are positive
for the constructions GTI-iut 16003.
The cloacine sensitive clones are
grown in 50 ml tetracyclin M9 SP medium containing
0.4 mM IPTG.
The culture is made at a temperature
of 30C tillthe optical density reaches 1. Induction
of gene iutA expression is then made by continui
the culture for four hours at +42C. The bacteria
are centrifugated and their total proteins are
analyzed by polyacrylamide gel-SDS electrophoresis.
This allows one to directly appreciate the importance
38
of Iut A protein production (figure 1). The analysis
of clone CMK 603 GTI P-2 reveals that protein
Iut A and its precursor represent, after induction,
25% of the bacterium's total proteins. The sole
Iut A protein represents 30% of the outer membrane
proteins.
- ~ of the expression vector
for gene fep A.
Characteristics of the initial clone
p MS 101
Following the results of CODERE and
EARHART (6) indicating that gene fep a is located
on a 6.3 kb Bam HI-Bam-HI fragment of plasmid
pMS 101 constructed by LAIRD and YOUNG (19), this
fragment is sub-cloned and vector pBR 322 digested
by Bam HI. The restriction map of the obtained
plasmid (F-l) appears similar to that of plasmid
pITS 1 (FLEMING et al. (11) -table XIV).
The publication of the sequence of
fep A (LUNDRIGAN and KADNER) (22) has enabled
one to precisely locate this gene on the restriction
fragment Ssp I-Stu I 2530 pb of plasmid F-l.
The following strategy is used to insert
gene fep A into p GTI 001.
An oriented mutagenesis is made in
terminal 5' region of the coding region in order
to transform sequence :
5'ATGAACAAG 3'
into a HpaI restriction site
MET ASN LYS
G T T A A C A A G
V. ' ~ ~ .' ', 1
39
This site is cut into blunt ended ends
in the following manner GTT AA C. This allows
direct ligation of gene ~ with the ATG end
created in p GTI 001.
The mutagenesis is made according to
the ECKSTEIN method from7Oligonucleotide (table
XV), after sub-cloning of 800 pb Ssp I-Eco
RI fragment in the replicative form of phage M13
mp 19.
The mutated fragment is entirely sequenced
in order to check that the sequence had not been
modified elsewhere than at the desired site.
Details of integration of gene fep
A are summarized in tables XVI and X~II.
The various clones obtained after ligating
fragment HpaI-XhoI 2350 pb in plasmid GTI 001
are selected by the presence of a 1700 pb
Eco RI-Eco RI fragment.
- Control of the expression of Fep A
Qualitative control.
The ligationmixture between the 2350
pb Hpa I- Xho I fragment and plasmid GTI 001 is
used to transform competent bacteria RWB 18. This
strain being-fep A, it is colicine B resistant.
Clones having the desired restriction
map (table XVII) are tested upon their sensitivity
to colicine B.
One clone (RWB 18 GTI F-12) appears
sensitive to the action of colicine B.
Quantitative control.
The expression of protein Fep A and
its precursor is analyzed on polyacrylamide-SDS
gel (figure 2). Clone CMK 603 GTI F-12 expresses
;
Fep A and its precursor in a very large amount
(20~ of total proteins).
Protein Fep A represents 32% of the
outer membrane proteins.
- Physioloqical and morpholoqical study
of clones expressinq Iut A and Fep A
Growth potential
The growth potential of obtained clones
is tested at various growing temperatures in LB
tetracycline IPTG medium.
When seeded as a layer on LB tetracyclin
IPTG agarose 0.1 mM, all clones form bacterial
maps which are sensitive to bacteriocins when
grown at temperatures between 30C and 34C.
Above 34C, bacterial mat~ no longer
form. This has also been observed in liquid LB
medium.
Sensitivity to bacteriocins is also
found for IPTG concentrations of only 0.05 mM
and at a temperature of 30C. Therefore, there
exists a level of expression for genes iut A and
fep A, in the absence of an induction of vector
p GTI 001.
Morphological study.
The various clones undergo morphological
changes following overexpression of Iut A and
Fep A.
Bacteria, when observed with a phase
contrast optical microscope after induction at
+ 42C during 4 hours, show a notable elongation
(up to 10 times the average length of a normal
K 12 Escherichia coli). However, the more striking
.........
s~ s l ~-
41
characteristic is the presence of one to several
intracytoplasmic inclusions inside each bacterial
body.
Inclusions as observed with the optical
5 microscope may be found again when one observes
with an electronic microscope after negative
coloring of bacterial sections grown at ~42C
during 4 hours.
These inclusions are peripherical and
adjacent to the inner face of the cytoplasmic
membrane.
3.Immunological properties of proteins
I_ A and FeP A
Immunogenicity of Iut A and FeP A
Iut A proteins extracted from outer
membranes of strains E. coli 15022, 15393, 16003
and recombinant strain Escherichia coli CMK 603
GTI P-2 are isolated by preparative polyacrylamide
gels and injected to rabbits according to the
above described protocol.
Changes in the title of anti-Iut A
antibodies secreted by each rabbit/assessed with
the ELISA method, taking as an antigen a "granules"
(precipitated proIut A protein) solution extracted
from a culture of the strain E. coli CMK 603 GTI
P-2.
The positive reference serum used
is a rabbit anti-Iut A protein serum of E. coli
ColV-K30, supplied by B. OUDEGA.
The same protocol is followed for proteins
Fep A extracted from membranes of strains E. coli
150022 and E. coli RWB 18 GTI F12.
42
In all cases, the rabbits react to
the injection of proteins Iut A and Fep A by producing
a high titer of antibodies specifically directed
against these proteins.
- Antiqenic properties of proteins
Iut A and Fep A
The specificity of the various obta ined
antibodies is studied towards several outer membranes
preparations (Strains E.coli 15022, 15393, CMK
603 GTI P-2, CMK 603 GTI B-5 and RWB 18 GTI F-
12). This study is made using the "Western blot"
method.
The four anti-Iut A sera
which are prepared, as well as the standard anti-
Iut A colV-K30 serum specifically recognize
a 76 kDa protein in all outer~membrane preparations
from strains expressing the aerobactin system.
Whenever the (wild or recombinant) Iut A protein
used for their induction, the antibodies of one
serum specifically recognize the Iut A protein
expressed by bird and ox E. coli proteins and
the two Iut A proteins synthetized by the recombinant
strains E. coli CMK 603 GTI P-2 and CMK 603 GTI
B-5.
The precursor of the Iut A protein,
protein proIut A is also specifically recognized
by all anti-Iut A sera. (Immunoblots made with
purified "granules" generated by strains CMK 603
GTI P-2 and CMK 603 GTI B-S).
Protein Fep 2 is not recognized by
anti-Iut A sera and, conversely, anti-Fep A sera
do not recognize Iut A proteins.
43
Cloning of genes iut A and fepA in
expression vector GTI 001 allows one to generate
large amounts of proteins Iut A and Fep A as well
as their respective precursors. Proteins Iut
A or Fep A and their precursors which are
synthetized following the induction of the trans-
cription by the growth of bacteria at 42C,
rapidly accumulate in the form of large cytoplasmic
inclusions (granules) which are visible with
lo a phase contrast optical microscope. Observation
with the electronic microscope of section of
induced bacteria reveals that these granules
are closely joined to the inner face of cytoplasmic
membrane.
One must obser~ the importance of the
expression level of Iut A and Fep A precursors
(an average of 25% of total proteins under non-
optimized conditions). Mature proteins make up
as much as 35% of the protein content of the outer
membrane. This percentage may be considered as
the upper limit for the integration of this type
of protein in the outer membrane. As a comparison,
proteins Iut A and Fep A expressed by the wild
strains Escherichia coli 15022 represent together
30~ of the outer membrane proteins. It thus
appears that the total expression of proteins
Iut A and Fep A, and their precursors, by recombinant
strains according to the invention is much higher
than their natural expression.
The characterization of a sensitivity
towards cloacine DF 13 in clones expressing the
Iut A proteins, and of a sensitivity to colicine
B in those which express protein Fep A shows that
! f ~
~4
the synthesis and integration of these proteins
in the outer membrane take place normally.
The identity of proteins obtained by
genetic recombination with wild proteins is also
5 demonstrated by the recognition of these proteins
by antibodies directed against natural Iut A and
Fep A proteins. One will note that these antibodies
also recognize, with the same specificity, precursors
proIut A and proFep A into intracytoplasmic inclusions.
Antibodies induced by proteins Iut
A and Fep A as obtained by genetic recombination
specifically recognize in the same way proteins
Iut A and Fep A as expressed by various strains
of pathogenic Escherichia coli.
Overexpression of receptors Iut A and
Fep A by cloning of their genes on an expression
vector, allows one to obtain in an iron-rich medium
external membrane proteins which are functionnally
and antigenically identical to proteins expressed
by pathogenic bacteria during their ln vivo multiplication.
The synthesis of proteins Iut A and
Fep A through genetic recombination thus has
many advantages :
- it allows one to obtain proteins
Iut A and Fep A in very large amounts, while
freeing from regulation by iron,
- the synthetised proteins are functionnally
and antigenically identical to the proteins as
expressed by pathogenic bacteria in their ln vivo
multiplication and they induce the production
of antibodies,
-when used as an active principle in
a vaccine, they induce the production of antibodies
f r, .-~
preventing the specific recogni~ion by membrane
proteins regulated by iron,o the siderophores,
thus stopping the supply of iron and blocking
their multiplication in a host ; thus they allow,
one to prepare very useful vaccines to prevent
or fight infections including septicemiae.
Vaccines may also be preparated simply
from inactivated recombinant clones or from membrane
fragments obtained by lysis of recombinant clones
followed by purification, according to usual methods
for the preparation of vaccines based on surface
or wall antigens.
IV. PREPARATION OF IROMPS BY GROWING
IN IRON-RESTRICTED MEDIUM.
1) Strain : E. Coli 078 reference 15022 :
origine : chicken
2~ culture :
- Medium :
ST medium + succinate (Simon E.H. and
Z Tesmann (1963) Proc. Natl. Acad. Sci. USA 50,
526-532)
. addition of lactoferrin (250 ~g/ml)
to obtain a medium with an iron deficiency,
. or addition of FeC13, 6H2~ (40 ~M) :
for an iron-rich medium.
- Culture
- passing 3 times the strain in a
iron-rich medium, this being followed by an adaptating
stage in a deficient medium before final culture
in a deficient medium,
- simultaneously, one proceeds to grow
the same strain in an iron-rich medium,
46
- the cultures are made at 37C during
24 heures.
3) Analysis
At end of growth, in each medium, one
proceeds to the followlng operations :
- harvesting by centrifugation,
- collecting~pellet in 0.2 M Tris HCl Ph 8, and
ultrasonication,
- centrifugation (5000 g 30 minutes)
- recentrifugation of supernatant (100 000 g,
1 hour),
- taking up ofthe pelletin Tris HCl (50 mM, pH
8), MgC12(10 mM), EDTA 1 mM, Triton 100 (2~) and
agitating during 20 minutes at 37C,
- centrifugating one hour at 100 000 g then reextracting
the pellet,
- washing the pe/letn demineralised water,
- analysing thepellet in polyacrylamide gel (PAGE
SDS) under denaturating conditions (mercaptoethanol,
SDS)-
4. Result :
- Membranes of bacteria grown with lactoferrin :
presence of two proteins, in large amounts, having
apparent molecular weights80000 da (enterobactin
receptor Fep A) and 76000 da (aerobactin receptor
Iut A),
- membranes of bacteria grown in iron-rich medium :
no 76000 and 80000 da band.
V - PREPARATION OF VACCINES
Preferably, the active principles according
to the invention will be associated, in the vaccines,
with a conventional antigenic preparation in
known human or animal antibacterial vaccines,
and notably in the case when one uses purified
proteins.
These vaccines may show the active
inventive principles in usual liquid vehicles
for parenteral administratlon. They may include
conventional , for example oily adjuvants.
The legends of figures 1 and 2, hereabove refer-
red to, follow :
For figure 1 :
Control of the expression of the Iut A protein
and of its precursor on two CMK 603 clones containing
GTI-Iut A constructs.
PAGE-SDS pattern of total proteins of the clones:
1 E coli CMK 603 GTI-001
.
3. E. coli CMK 603 GTI-Iut A (strain 16003)
4. E. coli CMK 603 GTI-Iut A (strain 15972)
2. Molecular weight references.
For figure 2 :
Control of the expression of the Fep A protein
and of its precursor.
PAGE-SDS pattern of total proteins of the clones.
1. E. coli CMK 603 GTI 001
2. E. coli CMK 603 GTI-Fep A.
48
TABLE I
PATHOGENIC STRAINS SEROTYP~ ORIGINAL ANIMAL REFERENCE
E. eoli 15 022 O 78 chieken souchothèque RM
15 393 0 86 calf
" 15 972 O 2 chicken "
" 16 000 0138 calf "
" 16 003 0138 calf "
10E. eoli KM 576 (p ColV-K30)Man B. O~DEGA
Host strains E. coli _ K12 genotype origin or reference
. C 600 F- thr, thi, leu, lac Y, fhu A, sup E ~ ~ r~ e~
. ~3 101 F- hsd (r~~, m~~), recA, ara, Pro, lacY, ( ~ P~ie~ ~31
8~1. r~5, xYl, mtl, suPE
~WB 18 F- thi, ProG, leu, ~ , en~, ~?A ~H-LL ~ D e~
NEn~ 4)
. CMK 603 F~ thr, thi, leu, suPE, re~ BC, fhu A, Instl~u~ ~er.eux
lac Y, r~~, m~
. 15~25 F- Souchothèque RM
Bacteriocines-producing strains
E. coli 1300 Colicin B produeing R. PORTALIER
Enterobacter clocae DF 13 S458
Cloacin DF 13 producing
B. OUDEGA
4~
'rABLE II
Name Size Characteristics Reference or origin
(bases pairs)
pB~ 322 ~363 ~mpr Te~ ~. BOLIV~R
cloning vector
10 pAT 153 3600 Ampr Tetr A. TWIGG
cloning vector
pSB 118 2692 ~mp' derived from P. STRAGIER
pUCl8 cloning vector It.Pasteur Par1s
15 pGTI 001 5780 Tetr expressing vector P. BRUNEAU
I t Mérieux
pABN 1 18300 Ampr vector p Plac + E~E~F ~
16.3 kb Hind III frag- N~IN~ (2)
ment of ColV-K30 bearing
operon aerobactin
pMS 101 15300 Ampr vector pBr 322 + L~RDe~
11 kb HInd III fragment
of E. coli chro~osome bearing
genes entD, fepA, r'es et
entF
.:
.
ç ~ ~ r~
TABLE III
Day Day 14 Day 28 Day 35
intradermic intramuscular intramuscular intramuscular
injection injection injection injection
~.25~g 125~g 125~g 250u~
Blood5 ml Blood 10 ml
sampling sampling
Day 42Day 49 Day 56
intramuscular intramuscular
injection injection
250~8250~
Blood50 ml Blood 60 ml
sampling sampling
r,. -
TABLE IV f
f ~ Eco R I
(WILD PLASMID ) ~ ~am
kbDigestion Bam HI ~ / A~R TETR
~3 ~7 pAT 153
- - - \ ~ ,
g, ~
6,7 ~~~ ~ectroelution
,~,3 ~ D1gestion eam HI
LIGATION
BAM HI
AM~ R
~ ~ TETg
\\ /
>~
Bam hI
Selection. with cloacine
CLONES AMPR TETg CLOg
CLONING STRATEGY OF iut A GENES
52 f,, ~ , rj
w ~ 8
~ O= -C$9~ _ _~ ~ ~ '~
~z h~) ~ ~ q ~ _~ ~
W
~O ~ ~ ~/~ ~ ~t~9~
~ r5 '~ -~r9" -~ _~
O~q ~ _, o~ ~
W ~
C ~ 7[ ~ 1 t
5 3 f
TAE~LE VI
~- ]~
Z~
~i
U~
5 4 s~ r~ r.
TABLE. VII
Comparison between nucleotide sequences of Bst~ BstE II
(3.2 kpb) restriction fragments ~of aerobacti~e operons
of p Col.V-K30 et p 15972
C~lV~ GGTTACCGTTCTGCGTTGCCACAAATACTTCCCTCACTGATGCCCCT5ATCACCATGCAC
15972 GGTTACCGTTCTGCGT,GCCACAAATACTTCCClCACTGATGCCCCIuArCACCATGCAC
61 GATAAGAAcAccTTTAAAGTGcGTGATGAcTTcAcTcTuGAATGGAGTrùGcccGAAAGAG
61 GATAAGAAcAcclTTAAAGTGcGTGATGdcTTcAcTcTGGAAT5GAGT5GcrcGAAAGAG
121 AAcAAcATcTTcGTGGTcAAcGccAGTATGcAAAcccATGGcATcGcc5AAccccAGcTc
171 AACAACATCTTCGTGGTCAACGCCPGTATGCAAACCCATGGCATCGCCGAACCCCAGCTC
!81 AGccTGATGGcATGGAGATcTGcAcGTATTcTTAATcGcGTAATGGGAcJTGATTlATTr
181 AGCCtGATGGCATGGAGATCTGCACGTATTCTTAATCGCGTAATGGGACGrGATTTATTC
15 241 GATcTcAGTATGcrGcccGcccTGATTcAGTGGcGcAGGcAccT~GGAAAAcGcAGccG
241 GATCTCAGTATGCCSCCCGCCCTGATTCAGTGGCGCAGGCACCIAGGGAAAACGCAGCCG
END 1 uc D
301 GACTTGTCTTTAACTCGCTACACAGCATCTTTGGGCTGATTTTTTCCGCCCGTATGGAGG
301 GACTTGTC TTAAcTcGcTAcAcAGcATcTTTGGGclGArlTTTTccGcc;GlATGGAGG
20 '61 AATAA-~uATGATAAGCAAAAAGTATACGCTTTGuGGCTCTCAACCCAC',CrTCTTACCA
~6; AATAArl-ATGATAAGcAAAAAGlAlAcGcTTTGGûc-l`-cAAcccAc-Gc -c -Al:CA'
~2' GATGGcuccAGcAGlcrucTcAAcAAAcc5AlTGATGAAAc5TTcùTGG-GlciTuccAAccG
1^' GATûGCGCCAGCAGTC5CTCAACAAACCGArGAT5AAACGTTCGT5G;'GTîT5CCAACC5
481 CAGcAATfGcAccGTAGcGGAGAT5GcGcAAAccAccTGGGTTATcGAAAAcGccGAAcT
48: CAGcAATcrucAccGTAGcGGAGATGGcGcAAAccAccTGGGTTATcGAAAAcGccGAAcT
54i GGhAcAGcAGATTrAGGGcGGcAAAGAGcTTAAAGAcGcAcTGGc-cAGcT5AlrcccrGG
541 GGAACAGCAGATTCAGGGCGGCAAAGAGCTTAAAGACGCACTGGCTCAGCTGATCCCTGû
601 CcTTGAcGTcAGcAGccGGAGccGcAccAAcTAcGGTATGAATGTGcGTGGccGcccGcT
601 CCTTGACGTCAGCAGCCGGAGCCGCACCAACTACGGTATGAATGTGCGTGGCCGCCCGCT
661 GGTCGTGCTGGTTGACGGCGTGCGTCTCAACTCTTCACGTPCCGACAGCCGACAACTGGA
661 GGTCGTGCTGGTTGACGGCGTGCGTCTCAACTCTTCACGTACCGACAGCCGACAACTGGA
721 CTCTATQGATCCTTTTAATATûCACCATATTGAAGTGATCTTCGGTGCGACGTCCCTGTA
721 CTcTATAGATccTTTTAATATGcAccATATTGAAGTGATcTccGGTGcGAcGTcccTGTA
781 CGGCGGCGGCAGTACCGGTGGCCTGATCAACATCGTGACCAAAAAAGGCCAGCCGGAAAC
~81 CGGcGGcGGcAGTAccGGTGGccTGATcAAcATcGTGAccAAAAAAGGccAGccGGAAAc
841 CATGATGGAGTTTGAGGcTGGcAccAAAAGTGGcTTTAGcAGcAGTAAAGATcAcGATGA
841 CATGATGGAGTTTGAGGcTGGcAccAAAAGTGGcTTTAGcAGcAGTAAAGATcAcGATGA
__ .
,r~
J ~ .
TABLE. VII (continued)
~()i AcGcATTGccGGAGcTGTcTccGGcGGAAATciAGcATATcTc~GGAC5TC-TTCC5TGGC
~rJ; AcGcATTGccGGAGcTGTcTccGGcGGAAArGAGcATATrTccsGAcsTcTTTcci-àlGGc
ATATcAGAAArTTGGcGGcTGGTTTGAcGGTAAcGGcGArGccAcc--GcTTGArAAcAc
'~61 ATATcAGAAATrTGGcGGcTGGTTTGAcGGTAAcGûi5`ATc~l-cAccTTlJcrrGATAAcAc
! ~2: CCAGACCGGCCTI .AG T AC-CCGATCGGCTGGACATCATGG iAAC-. G i T ACGC T G;~rAT
: ~2: CcAGAcc5Gcc~G;AGTAclccGATcGGc~GGAcATcATvGGAAcTG~iTAcGcTGAAcAT
081 CGATGAATCCC ii;CAGCT CAGTTGATCACACAGTAC ATAAAAGCCAûûGCGAC5A(:SA
CGATGAATC'`CGGCAGCTTCAGTTGATCACACAGTACrATAAAAGCCAGGGCGACGAC5h
;!Gl TTAcGGGcl-AATcTcGGGAAAGGcTTcTcTGccATrAGAGGGAccAGcAcGcrAl-cGT
I l 4 l rTAcGGGcTrAArcTcGGGQAAGGcTTcTcTGccATcAGAGGGAccAGcAcGccATTcGT
.201 cAGTAAcGGGcrGAArTccGAccGTATTcccGGcAcT5AGcGGcATrTGArcAGcc-GcA
CAGTAQCGGû(`IGAATTCCGACCGTATTCCCGGCACTGAGCGGCATT~GArCAGCCTGCA
i2~i GTAcTi:r5AcAGcGcTTTlcTGGGAcAGGAGcTGGTcsGlcAGGT TAcrAccGcGAT~A
:26i GTACTC~.ACAGCGC,~TTC,ûGGACAGGAGCTGGTCGGlCAGG,TlACTACCGCGATGA
: ?2: GTCGTTGCGArTC ACCCG T TCCCGPCGGTAAArGCGAACAAACAG -GACGGCTT C C
.32; GTCGTTGCGAr T CTACCC5T T CCCGACGulAAATGC5AACAAPCAGGTûACGGCTT crc
38 I TTcGTcAcAGcAGGAcAccGAccAGTAcGGcATGAAAcrsAcTcrGAAcAGcAAAccGAr
~8~ TTCGTcAcAGcAGûAcAccGAccAGTAcGGcATGAAAc,'SAclcTGAAcAGCAAAccsAT
;aa~ GGACGGCTGGCAAATCACCTGGGGGCTGGATGCTGATCATGAGCGCTTTACC-CCAACCA
aal GGAcGGcTGGcAAATcAccTGGGGGcTGGATGcTGArcATGAGcucTTTAcclccAAccA
!501 GATGTTCTTCGACCTGGCTCAGGCAAGCGCTTCCGGAGGGCTGAACAACAAGAAGATTTA
i 50 I GATGTTCTTCGACCTGGCTCAGGCAAGCGCTTCCGGAGGGCTGAACAACAAGAAGATTTA
1561 CACCACCGGGCGCTATCCGTCGTATGACATCACCAACCTGGCGGCC~--CTGCAATCAGG
:561 CACCACCGGGCGCTATCCGTCGTATGACATCACCAACCTGGCGGCCTTCCTGCAATCAGG
' 621 CTATGAcATcAATAATcTcTTTAcccTcAAcGGTGGcGTAcGcTATcAGTAcAcTGAAAA
! 62 i CTATGACATCAATAATCTCTTTACCCTCAACGGTGGCGTACGCTATCAGTACACTGAAAA
i S8 I CAAGATTGATGATTTCATCGGCTACGCGCAGCAACGGCAGATTGGCGCCGGGAAGGCTAC
168I CAAGATTGATGATTTCATCGGCTACGCGCAGCAACGGCAGATTGGCGCCGGGAAGGCTAC
,7al ATccGccGAcG CATT CTGGCGGCTCAGTCGATTACSA~: AcTTccTGTTcAAcGccGG
174 1 ATCCGCCGACGCCATTCCTGGCGGCTCAGTCGATTACGACAACTTCCTGTTCAACGCCGG
r~ ' ' J '~
56
TABLE VI I ~( continued )
?~8 rCTGCTGATGCACATCACCGAACCGCAGCAGGCATGGCTrAACT-C,CCCAGGGI-.GIGGA
~01 rCTGCTGArGCAChTCACCGAACGCCAGCAGGCA i-iGC.^AAC~TCT~CCAGGGC5rGrih
'858 5CTGCCGGACCCGGGTAAA r AcTATGGTcGcGGcATcTATGGTGcTGcAGTGAAcG5ccA
!861 GCTGCCGGACCC5GGIAAArACTATGGTCGCGGCATCTATGGTGCTGCAGTGAACGGCCA
19!8 TCTTCCICl~ACAAAGAGTGT5AACGTCAGcGACAGcAA5C,5GAAGGC~TGAAA51-5A
,,,,,,..,..,,,.,,,,,,,,,,,,,,,,,,,,.,...,. .,~,.. .... ...
:~,?: TcTTccTcrAAcAAAGAGTGTGAAcGlcAGcGhcAGcAAG!~lGGAAGGcGlGAAAGTcGA
i~7~ TTCTTATGAGCTGGGC~,GGcGCTTTAcTGGcAATAArc-GcG,AccCAAArcGCGGccTA
~ l TTCTTATGhGCTGGGCTGGCGCTTTACTGGCAATAATCTGCGTACCCAAATCGCGGCCTA
2038 CTATTCGATTTCTGATAAGAGCGTGGTGGCGAATAAAGhTCTGACCATCAGCGTGGIGGA
20Gl cTATTcGArlTc-GATAAGAGcGTGGTGGcGAATAAAGATcTGAccArcAGcGTGG-GGA
2098 CGACAAAI:GCC5-,ATT-ACGGCGTGGAAGGTGCGGTGGACTAcrTGAT-CCTGATACTGA
2~ CGAcAAAcG~-5TATTTAcGGcGTGGAAGGTGcGGlGGAcTAccTGAlTrcTGATAcT5A
'!58 CTGGAGTAC;5GA~ iAaCTTCAACGTGcTGAAAAcTGAG,cG~i~AGTliA~c5~~A,rl,i
.?16. CTGGAG~ .GAii G~ACT-CAACGTGCTGAAAAI`-GAG C'iA~G,GAACGGTAC'IG
2218 GCAGAAATACGArGIGAAGACAGCAAGCr_ATCAAAAGCGACAGCr,ACATTGGCT5iGC
'22! GCAGAAArAC'iATGT5AAGACAGCAAGCCCATCAAAAGC;iACAGCC-.ACATTGGC-, UliiiC
,?~7`8 QccGiiAccciTGGAGlcTGcGcGTGcAGAGcAccAccl-cTTTGAcGT5AGcGAcGcGcA
`281 ACCGGACCCiiTGGAGICTGCGCGTGCAGAGCACCACCT.--Trl-iAC'i~GAGCGACûCGCA
2338 GGGCTAC~AG5TCGATGGCTATACCACCGTGGAT~TGCTCGG,AG,TATCAGCTTCCGGT
........................... ....... .... .... ...
2341 GGGCTACAAGGICGATGGCTATACCACCGTGGATCliiCTCGiiCAGTlATCAGC-TCCiGl
23~ GGGTACACTCAGCT'CAGCATTGAAAACCTCTTCGACCGTGACTACACCACT5TCTGGG5
2aol GGGTACACTCAGCTTCAGCATTGAAAACCTTTTCGACCGli'iACTACAC_ACTGTCTGGGii
2G58 GCAGCGTGCACCACTGTACTACAGCCCGGGTTACGGCCCAGCGTCACTGTACGACTACAA
2461 GcAGcGTGcAccAcTGTAcTAcAGcccGGGTTAcGGcccAGcGTcAcTGTAcGAcTAcAA
1~ Il D
25~Q AGGCAGGGGCC CACCTTTGGTCT~eACTACTCTGTGCT5TTCTGACCGGTATTCCTTTA
'52! AGGcA5GGGccGAAccrTTGGTcTGAAcTAcTcTGTliclGTTcT~ GGTArTccTrTA
2577 CAACAAAGGTACGCTGQTATCAACATGGCCGCTGACAGCCAAGTTGATATCArATAAlAC
'5~1 CAACAAAGGTACGCTGATATCAACATGGCCGCTGACAGCCAAGTTGATATCATATAArAC
2637 AcGAcATAATcTGTAGTcAGGGAGGATAGAcTcTTTAcTGAcTAcAGATTATGTccTGTT
264i ACGACATAATCTGTAGTCAGGGAGGATAGACTCTTTACTGACTACAGATTATGTCGTGTT
57
TABLE VI I ( continued )
2697 CCGTGCTCArTTCCTCAAAAAAATACAAGAAAAGAAT,~GTAr-CT. AACAAAAAGIGAAA
. 7'-)1 CCGTGCrCArTTCCTCl;AAAAAArACAAGAAAA~ ,AL\AA~ JI~A~.
7~ , AAATTGTATCAAACT~ CcT-TT ~ TAATcc ~ G ~ TGA(~ rAAA l -A~ T l I TGCAATî~GiJAr
~76 i TAAATTGTATCAAACTCCCTCT, TTAATccTGrTGAGTpAArcAGcT~-rTGcAAThGGAT
281, TGAAAGAGTGTAAGTGGAATcTcrrccGGATAcrcGlrAccAccGrGGcrAGAArArcTA
'821 TGAAAGAGrGrAAGTGGAATcTcTTccGGArAcTcrirT~i^.PccG~CGCrAGAAIAr^TA
BEGINNING I
28-- CGGC ,GcGGGGliTGArGcrGccAAcT ~Cr~ArrrAG'G-~ GiiT,'i, TT-G;~
~88 ' cGGcrGcGGGGGTGArGcTGccAAcrrAcTGATT TAGTG-,ArGATGGIGuT-. TT GAGGTG
2937 CTCCAGTGGCTTCTGrTTC T ATCAGCTGTCCCrCCrGTTCAGC-ACTGACGGGGTGGTGC
2941 CTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGT,CAGCTACTGACGGGGTGGTGC
2~ 7 GTAAcGGcAAAAGcAccGccGGAcA-cAGcGcTA~c r~ -cAcrGcc~ TAAAA(AT
300! GTAAcGGcAAAAGcAccGccGGAcArcAGcGc-ATcT~ crc l cAcrGccGTAAAAcAT
3057 GGCAACrGCAGTTCACTTACACCGCTTCTCAACCi-GGTACGCPCCAGAAAATCATTGATA
3061 GGCAACTGCAGTT cAcrTAcAccGcT~TcTcAAcc~-GG-Ac.iiArc~GpApATrATTlip~rp
3; ! ~ TGGccATGAATGGci~TTGGArGc~GGGcAAccGccc~ A r-~-^;iGC;, -Gc~i`c-^A~cA
31 ~ TGGccArGAATGGcGlTGGATGccGGGTAAcTGcccGcAT-AlLiûGcsTTGGcL^-cAAcA
317, cGATlrTTccGccATTTAAAAAAcTcAGGccGcAGTcGGrAAcL
3181 cûArTTTAcGTcAcTTAAAAAAcrcAGGccccAGTcGGTAAcc
Matc~.es = 3207 Mismatches = !2 Unmatched = '
Leng~n - 3223 ~atches/length = 99.5 Percent
~ .
.` . ., { . "
58
TABLE. VIII
7~5
Col~-K30 GTG ATC TTC QGT GCG
15393
15972 C
16003 ~ ~ ~ - -~~~~~~~~
~ 7411
ColV-K30 GAC GCA TTC TGG CGG CTC AGr CGA TTA CGA CAC TTC
16393 ~ -C ArT CCT GGC ~GC TCA GTC ~Ar TAC GAC AAC ~TC
15972 -- - -C ATT CCT G~C GGC rcA GTC GAT TAC GAC AAC rTc _T~
16003 --C ATT CCT G~C ~C TCA GTC GAT TAC GAC AAC TTC CTG
al4
ColV-~30 ACC GAA CCG CAG CAG
15393 ~ -GC - -
1597~ GC
16003 -- ~- - -GC - --
2421
ColV-~30 GAA AAC CTC TTC GAC
15393 - T
15972 T
1~03 - - _ __ _ _
252~
ColV-~30 G~C C~A CCT TTG GTC TGA
Gly Pro Pro Leu Va 1 E~O
15393 G~C CGA ACC m GGT CTG AAC TAC TCT GTG CTG TTC TGA
15972 -----GA ACC m GGT CTG M C TAC rcT GTG CTG TTC TG~
~6003 -----GA ACC 1ll GGT CTG AAC TAC TCT GTG CTG T~C rGA
Gly Arg Thr Phe Gly Leu Asn Tyr Ser Val Leu Phe ENC
Differences between the sequence of the three clones iut A
_ and the sequence ofqene iut A DE p ColV-K30
59
~ABLE IX
Eco RI C~57
~ \
\ \ Shine and
~ oR2 / Delgarno
5401 Ec~ ~ RI ~ CR1/ de Cro
5125 E~n HI I ~/
Acc ~ ~ ~n HI
A T G G A T C C
\ \ B~n Hl 899
\~Ter" ATG ~e Cro
p Gn 001
_ 5780 pb
Bam HI 2388
"Ter"
Creation of ATG
Diges.tion BamHi ... A T G G A T C C..
T A C C T A G G..
Digestion ~u~ B~Yn 5~ A T G 3' ~>~ 5' A T G 3'
3'... T A C C T A G 5' > 3' T A C 5'
PHYSICAL AND GENETIC MAP of p GTI 001
,,, ~.
TABLE X
ACC I
A T G A T G A T A A G C A A A A A G r A T A C G C T T
MET MET ILE SER LYS LYS TYR THR LEU
1) 5' A T G A T A A G C A A A A A G T 3'
2) 3' T A C T A T T C G T T T T T C A T A 5'
5' A T G A T A A G C A A A A A G T 3' oligo "iut"
3~ T A C T A T T C G T T T T T C A T A 5'
Start of gene iut A and 9equence of double strand
oligonucleotide used for the phasing of gene iut A in p GTI 00
TABLE XI
Insertion strategy of B~m HI
gene iut A in p GTI OOl ~
1st part pAT 153 /~ \ Bst E Il
10200 pb
~T HI
ve~:tc~r pAT 153 Bst E II
Dige6tion Bst E II- Removal band 3225 pb
~ Bsm HI Bam HI
Digestion Fco RV KU~W ~GC I ~ AcC I
~560 pb ~
LIGATION ~~~
~(ECD ~V) ¦ ~
. iut A partial digestion 3am HI
6500 pb
Eco R V
. ~ (Eoo RV) I partial digestiOnAcc I
Di~ion Eoo RV / ligation wit~ oligo iut
~ I double strand
Ligation by 1inker XhoI
~nm H ~ ~V~ / AoC~T)AI~
/ 5 ¦ GTI P-2 j ~ ~ Eco
8010 pb--~
Digestion by Bam HI and XhoI and ¦ \
removal band 2780 pb /
Ligation with pGTI OOlfpartial 9~m HI
Bam ~I/XhoI
~ ~ .
, ~ ~
' ' ~ ~ PO 1 Y I i~e,-
62
Bam llI Eco RI ABLE XII ~ RI
Step ~ Eco RI ' Eco ~1
~ i560p~
~~ `~ I .
BalT HI l Digsstlol Eco RI
D i8est ion Eco RI
Fra~nt 2~00 pD ~
~~ Ba~n Hl ~ LI~TICN
- Fra~r~3nt 6260 pb
Eco RI ~ 4990 pb ~_
D i gestic~ B~T HI
(E3
Di~e5ti~n ~c I E~ HI ECO RI (t~n
~ ~Eco RI LigatlC~ with /~ ~;c
EC~ ~ ol igo iut
Di g~tion Eco RI "~
Removal f~ 1750 P~ / ~
,l / ~o I
LIG~nCN
~, ~ ` t'~
63
TABLE XIII
(Bam HI~
,1 20
~ ~ ~ Eco RI
276 ~ ~
Acc I y / "1~1" \\
V i~t A ~\ 1380
p ~TI ~2 ~ ~
P GTI ~-6
79gO pb ~ Xho I
2737 \
/1489
Bam HI
RESTRICTION MAP OF PLASMID.S GTI P-2 and GTI B-5
: ' ~
;:
~AB L ~ X I V 6~, F~l
" ~ ,, ,,J,~ )
Hind III
3~ 1~ ~n HI
/ ~
// ~ Stu I
Bam HI ~
Hind III _ p MS 101 --Eco RI
15 500 p~ ~
/~ Ssp I
-- -.- Ban HI Eco RI Ssp I
digested by
Di~e5~icn Bam HI
~~ LIG~TICN
Ban HI
\~ ~' Sgp I
B~n HI /~/
E~ RI Ssp I
RESTRICTION MAP OF p MS l0l and CONSTRUCTION OF p F--1
~.,.,p~l4i,;~
~ TABLE XV
start of the sequence of gene fep A
11 11 11 11
5' ~ G A A T A A A A C A A T G A A C A A G A A G A T T 3'
MET ASN LYS LYS ILE
~- sequence to be obtained after mutagenesis :
5' G G A A T A A A A C A G T T A A C A A G A A G A T T 3'
3' C C T T A T T T T G T C A A T T G T T C T T C T A A 5'
site Hpa I
oligonucleotide(27 mer)
used
5' C T T C T T G T T A A C T G T T T T A T T C C 3'
SEQUENCE OF SYNTHETIC OLIGONUCLEOTIDE USED FOR THE
MUTAGENESIS OF fep A
`
,:~ .1,"
66
TABLE XVI
Bam HI INSERTION STRATEGY OF GENE FEP A
IN p GTI 00l
Eco RI I 1100
~ Stu I 1st part
/~ ~ 1720
/ l ~., \ Dige~tion S~u I
pBR 322 p F-1 ,~.
10600 pb ~ ~ Eco RI
Soh I. ! ~ ~J 810 Addition 1inker Xho I
Bam HI \ ~ Ssp I
LIGATIO~
3Qo ~ o0 Eco RI Bam HI ~
Eco RI Ssp I ~ Xho I
!~ P FX~
ll 1o6oopb ~
~ ~ Eco RI
B3m H ~ ~O I
Eoo RI
Sph I
- l
partial digestion Eco RI
linear 10600 pb
Di~esticn Sph I
Dig~tion Ss~ I
r ~
removal fragment 810 pO
Removal ; 6gO0 pb
fragment
Liyation in M13 mp 19~Eco RI ~ H1nc II
~r rlLJ~AC~
~ `
6 7
TABLE XVII
. Dlgestion Eco RI-~pn I du of
; utated fragment (810 p~)
~ ' ..
LIGATION
Bam HI
Eoo RI
pBq 322
lSPh _ site Hpa I cr~
Digestion Xho I-~Ph I - Removal fragment 2539 pb
I, .
partial diges- ,~sa I - Removal fragmen~ Hpa I-Xho I 2350 P4
tion
LIGATION with~GTI 001/ partial Bam HI/Nucl~ase Mung ~/Xho I
~Eoo RI (Bam HI) ATG
Eco RI
~,.\
GTI F-12
~110 pb ,~ feo A
. . Xho I
~?
~., . , ~3 .
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