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Mol Gen Genet (1984) 197:503-508
© Springer-Verlag 1984
Gene encoding a hybrid OmpF-PhoE pore protein
in the outer membrane of Escherichia coli K12
Jan Tommassen 1, 2, Anthony P. Pugsley 3, Jaap Korteland 1, 2, John Verbakel 2, and Ben Lugtenberg 1, 2.
1 Institute of Molecular Biology and
2 Department of Molecular Cell Biology, State University of Utrecht, Transitorium 3, Padualaan 8, 3584 CH Utrecht,
The Netherlands, and
3 Unit~ de G~n~tique Mol~culaire, Institut Pasteur, F-75724 Paris, France
Summary. To study the structure-function relationship of
outer membrane pore proteins of E. coli K12, a hybrid
gene was constructed in which the D N A encoding amino
acid residues 2-73 of the mature PhoE protein is replaced
by the homologous part of the related ompF gene. The
product of this gene is incorporated normally into the outer
membrane. It was characterized with respect to its pore
activity and its phage receptor and colicin receptor properties. It is concluded (i) that the preference of the PhoE
protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, (ii) that part
of the receptor site of PhoE protein for phage TC45 is
located in this part of the protein, (iii) that colicin N uses
OmpF protein as (part of) its receptor, (iv) that the specificity of OmpF protein as a colicin N receptor is completely
located within the 80 amino terminal amino acid residues,
whereas the specificity of this protein as a colicin A receptor
is completely located within the 260 carboxy terminal amino acid residues, and (v) that the amino terminal 73 amino
acid residues of PhoE protein span the membrane at least
once.
Introduction
The outer membrane of Escherichia coli functions as a barrier for harmful compounds. The membrane contains a
number of proteins, designated as porins or pore proteins,
that allow the influx of nutrients. These pore proteins function as nonspecific diffusion channels through which small
hydrophilic solutes with a molecular weight up to about
650 can pass (Nikaido 1979). In addition, the porins function as (part of) the receptor for certain phages (Chai and
Foulds 1978; Datta et al. 1977; Verhoef et al. 1977) and
they are required for the efficient killing action of a number
of colicins (Foulds and Chai 1978; Mock and Pugsley 1982;
Pugsley and Schnaitman 1978).
Under standard laboratory conditions, E. coli K12 produces two distinct pore proteins, namely OmpC protein
and OmpF protein (Ichihara and Mizushima 1978; Verhoef
et al. 1979). The synthesis of another pore protein, desig-
Offprint requests to: J, Tommassen at the Institute of Molecular
Biology
* Present address: Department of Plant Molecular Biology, State
University of Leiden, Botanical Laboratory, Nonnensteeg 3,
2311 VJ Leiden, The Netherlands
hated PhoE protein (Tommassen and Lugtenberg 1981),
is induced when cells are grown under phosphate limitation
(Overbeeke and Lugtenberg 1980; Tommassen and Lugtenberg 1980). The latter protein is produced constitutively
in strains carrying phoR, phoS, phoT, and pst mutations
(Tommassen and Lugtenberg 1980). The structural genes
for these proteins, ompC, ompF, and phoE, have been
cloned (Mizuno et al. 1983a; Mutoh et al. 1981; Tommassen et al. 1982a, b) and sequenced (Inokuchi et al. 1982;
Mizuno et al. 1983b; Overbeeke et al. 1983) and the genes
appeared to be very similar. In the predicted primary structures of the proteins, approximately 60% of their amino
acid residues are identical (Mizuno et al. 1983 a; Overbeeke
et al. 1983).
We are interested in the following questions: (i) how
porins are embedded in the membrane, (ii) how these proteins exert their functions, and (iii) which amino acid residues are involved in a particular function. To approach
these questions, we have chosen to construct genes in which
part of the coding sequence for one porin is replaced by
the homologous part of the structural gene for another
porin. Subsequent characterization of the hybrid gene products will enable us to determine distinct domains within
the native porins.
In this paper, the construction of a hybrid gene is described in which the D N A encoding amino acid residues
~ 7 3 of the mature PhoE protein is replaced by the homologous part of ompF and the product of the resulting hybrid
gene is characterized.
Materials and methods
Strains, phages and growth conditions. All bacterial strains
were derivatives of E. coli KI2. The sources and relevant
characteristics of most strains are listed in Table 1.
ompR: :Tn5-886 and ompF888 mutants of BZBI011 were
selected as colicin N insensitive and the ompC889 mutant
was selected as resistant to the OmpC protein-specific phage
Mel. Plasmids were introduced into strains by transformation according to Brown et al. (1979) with selection for
chloramphenicol (25 gg/ml) or ampicillin (50 pg/ml) resistance.
Laboratory stocks of the PhoE protein-specific phage
TC45 (Chai and Foulds 1978) and the OmpF protein specific phage TuIa (Datta et al. 1977) were used. The OmpF
protein-specific phage K20 was a gift from P. Reeves and
P. Manning. The host range mutant phage TC45 hrN3 was
504
Table 1. Characteristics of bacterial strains"
Strain
Characteristics
Source/Reference
CE1265
F , thi pyrF thy ilvA argG rpsL cod dra vtr glpR ompR471 phoR18 recA56 A
(phoE-proA,B)
F-, thi pyrF thy ilvA his argG rpsL cod dra vtr glpR ompR471 phoR69 phoA8
phoE proA, B derivative of CE 1237
F , thr leu thipyrF thy ilvA his lacYargG tonA tsx rpsL cod dra vtr
glpR ompR472
F-, gyrA
Overbeeke
CE1237
CE1238
CEtll0
BZBI011
Korteland et al. (1982)
Korteland et al. (1982)
Verhoef et al. (1979)
Pugsley et al. (1983)
" Genotype descriptions follow the recommendations of Bachmann (1983)
a gift from N. Overbeeke. This phage recognizes PhoE protein and is also able to grow on the TC45-resistant strain
CE1202, which produces an electrophoretically altered
PhoE protein (Tommassen and Lugtenberg 1981).
Cells were grown at 37°C under aeration in L-broth,
which contains 1% tryptone, 0.5% yeast extract, 0.5%
NaC1, 0.002% thymine, pH 7.0.
DNA techniques. All D N A techniques used were as described previously (Tommassen et al. 1982a).
Isolation and characterization of cell fractions. Cell envelopes were isolated by differential centrifugation after disintegration of ceils by ultrasonic treatment (Lugtenberg et al.
1975). Protein-peptidoglycan complexes were isolated by
ultracentrifugation after incubation of cell envelopes at
60 ° C or 70 ° C in a buffer containing 2% sodium dodecyl
sulfate (Rosenbusch 1974). The protein patterns of cell fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously (Lugtenberg et al. 1975).
Rate of permeation of fl-lactam antibiotics. The rate of permeation of fl-lactam antibiotics through the outer membrane and the inhibition of this permeation by polyphosphate was measured using a method originally described
by Zimmerman and Rosselet (1977) and modified by Overbeeke and Lugtenberg (1982), except that the final concentrations of the antibiotics cefsulodin and cephaloridine in
the suspensions were 0.8 mM instead of 0.7 and 0.9 raM,
respectively.
Phage sensitivity and adsorption. The sensitivity of strains
to phages was determined by cross-streaking. Irreversible
phage adsorption to whole cells was measured by the method of Van Boxtel, as described by Van der Ley et al. (submitted for publication).
CoHcin sensitivity and adsorption. Colicins A, K, L, and
N were prepared as described by Pugsley (submitted for
publication). Strains used for the preparation of the colicins
were BZB1011 pColA-CA31, BZB1011 pColK-K235, Serratia marcescens JF246 and BZB1011 ompF888 pAPIP401
(ColN), respectively. The sensitivity of strains to colicins
was determined as described previously (Pugsley and
Schnaitman 1978).
Radioactive colicin N was prepared as described for
radioactive colicin E2 by Pugsley et ah (1983). Autoradiographs showed that colicin N accounted for 60%-70% of
the radioactivity in the preparation. Disodium ethylene di-
aminetetraacetic acid (2 mM) was added to the concentrated colicin to reduce proteolysis. Adsorption of radioactive colicin N to cells was determined as described (Pugsley
et al. 1983), except that the cells were harvested and washed
by centrifugation rather than by membrane filtration. Triton X-100 was not used in the reaction mixtures.
Results
Construction of an ompF-phoE hybrid gene
Comparison of the nucleotide sequence ofphoE (Overbeeke
et al. 1983) and ompF (Inokuchi et al. 1982) reveals that
these genes contain unique restriction enzyme cleavage sites
for PstI and MluI at identical locations within the genes
(Fig. 1). The PstI sites are located immediately behind the
signal peptidase cleavage sites in the proteins and were used
in an earlier study to construct plasmid pJP38 (Tommassen
et ah 1983). This plasmid consists ofphoE + plasmid pJP12
(Tommassen et al. 1982a) with a 1.6 kilobase pair D N A
fragment containing the ompF structural sequence, cloned
into the PstI site within phoE (Fig. 2). As shown in Fig. 2,
Pstl
!
phoE
ompF
GCT GCA~GAA ATA
GCT ,,GCA GAA AJ.C~
,I1
1 /
35
PhoE AE IYNKDGNKLDVYGKVKAMHYMSDNASKD** ***GDQSY
OmpF .......... V-L---AVGL--F-KGNGENSYGGN--MTI
40
36
75
PhoE IRFGFKGETQ INDQLTGYGRWEAEFAGNKAE SDTAQQ* *KTR
OmpF A-L ......... SD ..... Q--YN-E--NS-GAD--TG~--I
41
,/~
82
phoE ~AA
ompF
ACG CGT
Ml~I~CGCGT
Fig. 1. Comparison of the amino-terminal 75 amino acid residues
of mature PhoE protein with the homologous region of OmpF
protein according to Overbeeke et al. (]983). The complete proteins
are 330 and 340 amino acids long, respectively. The amino acid
sequence of PhoE protein is shown in the upper line and is denoted
in the one letter notation according to 1UPAC-IUB Commission
on Biochemical Nomenclature (1968). Only the amino acids on
OmpF protein that differ from PhoE protein are indicated by the
one letter code, while breaks in the proteins are indicated by an
asterisk (*). The positions of the PstI and MluI sites in the genes
phoE and ompF are indicated
505
Pstl MluI
pJP38
Pstl MluI
LI
,,,,~
~
J
t
LI
~,vw,/v,,v~
I
,
L
I
I,
I
1
1I
Mlu I digest
I T4 DNA ligase
~
Transformation
Pstl MluI
pJP47
I[
Fig. 2. Construction of the phoE-ompFhybrid gene. phoE sequences
are indicated by a zigzag line, whereas ompF sequences are indicated by a double line. Plasmid pJP38 is schematically represented
as linearized. This plasmid contains two regions containing porin
gene sequences. Region I consists of DNA up to the PstI site
in the phoE gene (see Fig. 1), followed by ompF DNA from the
PstI site in the gene to the end of the gene. Region II consists
of phoE DNA starting from the PstI site to the end of the gene.
Plasmid pJP47 contains the desired hybrid gene and was constructed by deleting an MluI fragment of pJP38
PhoE~_
OmpF
OmpA--a
b
c d
e
f
g
h
i
Fig. 3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis
patterns of cell envelope proteins of strain CE1265 containing plasmids pJP38 (a), pJP12 (b) or pJP47 (c), the peptidoglycan-associated proteins of the same strains after extraction of the cell envelope preparations in 2% sodium dodecyl sulfate at 60°C (lanes
d, e and f, respectively) or at 70° C (lanes g, h and i, respectively)
removal of an MluI fragment of pJP38 and subsequent ligation results in a phoE gene in which the PstI to MluI region
is replaced by the homologous part of ompF. The resulting
plasmid is designated pJP47.
Expression of the ompF-phoE hybrid gene
To study the expression of the hybrid gene as compared
to phoE and ompF, plasmids pJP47, pJP12 and pJP38 were
transformed to the porin-less phoR strain CE1265 and cell
envelope protein patterns of transformants were analyzed
on gels. As shown in Fig. 3, the pJP47-containing cells
(lane c) produce a cell envelope protein in approximately
the same amounts as PhoE protein in the pJP12-containing
cells (lane b) and OmpF protein in the pJP38-containing
cells (lane a). This protein migrates slightly slower in the
gel than PhoE protein, which is consistent with the fact
that the hybrid gene product is seven amino acid residues
larger than PhoE protein (Fig. 1).
To determine whether the hybrid gene product is normally incorporated into the outer membrane, protein-peptidoglycan complexes were prepared. Like OmpF protein
(Fig. 3, lane d) and PhoE protein (lane e), the hybrid gene
product can be isolated complexed to the peptidoglycan
(lane f).
Whereas OmpF protein dissociates from the peptidoglycan at 700-80 ° C, PhoE protein-peptidoglycan complexes
dissociates at 600-70 ° C (Lugtenberg et al. 1978). As shown
in Fig. 3, the hybrid gene product behaves like PhoE protein
in this respect.
Pore function of the hybrid gene product
It has been reported that PhoE protein pores have a preference for anionic solutes (Korteland et al. 1982; Nikaido
et al. 1983; Overbeeke and Lugtenberg 1982), in contrast
to OmpF and OmpC protein pores (Korteland et al. 1982;
Nikaido et al. 1983). This anion selectivity can be demonstrated by measuring the rate of permeation of the fi-lactam
antibiotics cephaloridine and cefsulodin in strains containing
only one pore protein species (Nikaido et al. 1983; Overbeeke and Lugtenberg 1982). The latter antibiotic is closely
related to the former one, but contains an additional negative charge and its molecular weight is higher (Overbeeke
and Lugtenberg 1982). Consistent with the results described
by Overbeeke and Lugtenberg (1982) and Nikaido et al.
(1983), we observed that cephaloridine permeates more
than ten times faster through OmpF protein pores than
through PhoE protein pores (Table 2). In addition, cefsulodin permeates about 25 times slower through OmpF pores
than cephaloridine, whereas both cefsulodin and cephaloridine permeate equally fast through PhoE protein pores (Table 2). The results for the OmpF-PhoE hybrid protein pores
are intermediate (Table 2).
The anion specificity of the PhoE protein pores has been
explained in terms of a recognition site on the pore for
negatively charged compounds (Overbeeke and Lugtenberg
1982). Proof for this assumption was obtained in experiments showing competition for this site. Thus, permeation
of fl-lactam antibiotics through PhoE protein pores, but
not through OmpF protein pores is inhibited by negatively
charged compounds, e.g., polyphosphates (Overbeeke and
Lugtenberg 1982). To determine whether this recognition
site is present on the hybrid protein, the influence of
0.2 mM polyphosphate, type P15, on the rate of permeation
of cephaloridine and cefsulodin was measured (Table 2).
Table 2. Influence of polyphosphate on the rate of permeation of/%lactam antibiotics through pores in the outer membrane
fl-lactam antibiotic
(0.8 raM)
Cephaloridine
Cephaloridine
Cefsulodin
Cefsulodin
Polyphosphate
P15 (0.2 raM)
+
+
Rate of uptake by intact cells"
CE1110 (pBR322)
(OmpF protein)
CE1237 (pBR322)
(PhoE protein)
CE1238 (pJP47) (pBR322)
(Hybrid OmpF-PhoE protein)
157
150 (< 5)
5.5
5.2 (5)
8.0
2.2 (71)
9.1
2.5 (72)
42
30 (29)
7.0
4.8 (31)
a Rate of uptake in the presence or absence of polyphosphate is expressed as nmol/min, gg pore protein. Percentages of inhibition
are given in parentheses
506
Table 3. Sensitivity of strains producing different porins to colicins and adsorption of radioactive colicin N to these strains a
Strain
BZB1011
BZBI011 ompF
BZB1011 ompB
BZB1011 ompC
BZB1011 ompBphoS
CE1265 pJP38
CE1265 pJP12
CE1265 pJP47
Porins produced
OmpC, OmpF
OmpC
OmpF
PhoE
OmpF
PhoE
Hybrid OmpF-PhoE
Colicin sensitivity
A
K
L
N
Adsorption of
radioactive
colicin N b
1
<10 -4
<10 4
2
<10 -4
1
< 10 -4
<10 4
1
0.03
0.03
2
0.06
1
5.10 -4
0.015
1
0.03
0.03
2
0.03
i
8.10- 3
8.10 -3
1
<10 -4
<10 -4
2
<10 .-4
1
< 10-4
0.25
1.0
0.023
0.028
1.15
0.019
1.0
0.043
0.86
" Colicin sensitivities and receptor activity are expressed relative to strain BZB1011 or to strain CE1265pJP38
b An average of three determinations is shown for a given colicin N concentration
The results show that the permeation of the antibiotics
through the hybrid protein pores is significantly inhibited
by polyphosphate, but not to the same extent as that
through PhoE protein pores. Thus, by exchanging amino
acid residues 2-73 of PhoE protein by the homologous part
of OmpF protein, the pores lose part of their anion specificity.
Phage receptor activity
PhoE protein serves as (part of) the receptor for phage
TC45 and its host range derivative TC45hrN3, and OmpF
protein functions as (part of) the receptor for phages TuIa
and K20. Strain CE1265 containing pJP47 turned out to
be resistant to phages TC45, TuIa and K20 but sensitive
to TC45hrN3. Phage adsorption experiments were performed to determine whether strain CE1265 containing
pJP47 can bind irreversibly the phages TC45, TuIa or K20.
However, none of these phages was bound by the cells producing the hybrid porin. Thus, amino acids 2-73 of PhoE
protein are involved in the TC45 receptor function of the
protein, whereas the homologous part of the OmpF protein
does not contain sufficient information for the TuIa and
K20 receptor sites. However, the specificity of TC45hrN3
for PhoE protein is not determined by this part of the protein.
Sensitivity to colicins
Porins are required for the efficient action of a number
of colicins, namely A, E2-E7, K, L, $4, and X, against
E. coli K12 (Foulds and Chai 1978; Mock and Pugsley
1982; Pugsley and Schnaitman 1978). In all cases, OmpF
protein seems to be preferred, but other porins can often
be substituted with only a slight reduction in killing efficiency (Foulds and Chai 1978; Mock and Pugsley 1982;
Pugsley and Schnaitman 1978). However, of these colicins,
A, K, and L show substantially reduced killing efficiency
when the indicator cells only produce porins other than
OmpF protein (Foulds and Chai 1978; Mock and Pugsley
1982; Pugsley and Schnaitman 1978; see also Table 3).
The reason for the uniquely high activity of colicins
K and L against OmpF protein-producing cells is unknown.
OmpF protein is unlikely to play a direct role in colicinreceptor interaction, since the receptor for colicin K has
been identified as Tsx protein (Manning and Reeves 1978),
whereas the receptor for colicin L has not been identified
(Foulds and Barrett 1973). The absolute requirement for
OmpF protein in the action of colicin A can be attributed
to direct participation in the colicin receptor (Cavard and
Lazdunski 1981 ; Chai et al. 1982).
As shown in Table 3, OmpF protein is also required
specifically for the activity of colicin N. To determine
whether OmpF protein is directly involved in the colicin
N receptor site, the binding of radioactively labeled colicin
N to isogenic E. coli K12 strains was studied. Strains producing no porins or only porins other than OmpF protein
adsorbed less than 3% of the normal level of radioactive
colicin N (Table 3). The residual binding activity is probably caused by nonspecific adsorption of the colicin to the
cell surface. Thus, since mutants that do not produce OmpF
protein are insensitive to colicin N and do not adsorb the
colicin, OmpF protein is probably (part of) the receptor
for colicin N.
To determine whether the hybrid OmpF-PhoE protein
can replace OmpF protein as (part of) the receptor for colicins A and N and in its unknown role in the action of
colicins K and L, the sensitivity of a pJP47-containing derivative of strain CE1265 was tested and compared with
the sensitivity of pJP38 and pJP12-containing strains. As
shown in Table 3, the pJP47-containing strain is almost
as sensitive to colicin N as the ompF protein-producing
strain, whereas the sensitivity of this strain to colicins A,
K, and L is only slightly or not at all enhanced as compared
to the PhoE protein-producing strain. In addition, the
amounts of 14C-labeled colicin N bound by this strain and
by the OmpF protein-producing strain were almost identical (Table 3). These results show that the specific requirement for OmpF protein as receptor for colicin N is (almost)
completely located in the amino-terminal 80 amino acid residues, whereas the requirement in the action of colicins A,
K, and L is (almost) completely located in the carboxy
terminal 260 amino acids of OmpF protein.
Discussion
The genes phoE, ompF, and ompC, encoding porins of E.
coli K12, are very similar in primary structure (Mizuno
et al. 1983b; Overbeeke et al. 1983), and it has been suggested that they have been derived from a common ancestral gene (Ichihara and Mizushima 1978 ; Tommassen et al.
1982b; Verhoef et al. 1979). Although functional differences between the porins exist, it may be assumed that the
overall conformation of the proteins is identical and that
507
those sequences which are involved in the folding of the
proteins in the membrane are mainly conserved. Therefore,
it is unlikely that the exchange of homologous parts of
the proteins will result in gross conformational distortion.
Thus, if replacement of part of one protein by the homologous part of one of the other porins results in a functional
change, this part of the protein is probably directly involved
in this function. In contrast, a mutation that results in a
functional change in one of the porins might only indirectly
be involved in this function. Therefore, we have chosen
for the approach of making hybrid proteins to study the
structure-function relationship of the porins.
In this paper, a hybrid gene is described in which the
D N A encoding amino acid residues 2-73 of mature PhoE
protein is replaced by the homologous part of ompF. This
replacement results in the exchange of 29 amino acid residues, and the addition of two fragments of 5 and 2 amino
acids, respectively (Fig. 1). Apparently, the hybrid gene
product is normally folded in the membrane, since (i) normal amounts of protein are found in the cell envelope fraction (Fig. 3), (ii) the protein is peptidoglycan-associated
(Fig. 3), and (iii) the protein functions as a pore (Table 2).
The pore activity of the hybrid porin was characterized
by measuring the rates of permeation of fl-lactam antibiotics and the inhibition of this permeation by polyphosphate.
The characteristics of the hybrid protein pore were intermediate between PhoE protein pores and OmpF protein pores
(Table 2). Thus, the hybrid protein has lost much but not
all of the preference of PhoE protein for negatively charged
solutes. This shows that this preference of the PhoE protein
pore is determined by at least two regions of the gene.
Thus, assuming that the distinct porin genes were derived
from a common ancestral gene, it is clear that the preference
for negatively charged solutes has evolved by multiple steps.
The hybrid protein does not function as the receptor
for phage TC45. Apparently, part of the exchanged fragment is directly involved in the TC45 receptor site of the
protein. A total of five independently isolated mutations
has been sequenced which result in the synthesis of an altered PhoE protein that does not function as the receptor
for TC45 (Korteland et al. in preparation). All these mutations result in the exchange of the amino acid residue at
position 158 of the mature PhoE protein from an arginine
to a histidine. Thus, if this arginine residue is also directly
involved in the TC45 receptor site of the protein, this receptor site is not simply determined by a short stretch of amino
acids in the primary structure of the protein, but is created
by the secondary folding of the protein, which brings together the important amino acids at the outside surface.
OmpF protein serves as (part of) the receptor for colicin
A (Cavard and Lazdunski 1981; Chai et al. 1982), and,
as shown in this paper, also for colicin N. PhoE protein
cannot replace the receptor function of OmpF protein but
the hybrid protein can function as the receptor for colicin
N but not for colicin A. This shows that the receptor part
of OmpF protein for colicin N is at least partly located
within the first 80 amino acids. It should be noted that
it does not necessarily mean that the complete receptor
function is located within these 80 amino acid residues.
However, it does mean that if some sequences in the rest
of the protein are involved in this receptor function, these
sequences are also present on PhoE protein.
The recognition site for negatively charged solutes, the
phage receptor sites and the colicin receptor sites must be
located on the outside surface of the membrane. As replacement of amino acids 2-73 of PhoE protein by the homologous part of OmpF protein results in changes in these sites,
some of these amino acid residues must be located on the
outside surface. We previously showed that the extreme
N-terminus of PhoE protein is located at the periplasmic
side of the membrane (Tommassen and Lugtenberg 1984).
Therefore, the first 73 amino acid residues span the membrane at least once.
Van der Ley et al. (submitted for publication) have isolated six monoclonal antibodies against PhoE protein which
recognize determinants on PhoE protein located on the outside surface of the cell. All these antibodies are also adsorbed by cells producing the OmpF-PhoE hybrid porin
(Van der Ley et al. submitted for publication), indicating
that their determinants are located in the C-terminal
257 amino acid residues of PhoE protein.
The results presented in this paper demonstrate the usefulness of hybrid proteins in the study of the structurefunction relationship of the porins. The hybrid gene described here was constructed with the aid of identical restriction enzyme cleavage sites on phoE and ompF. However, the number of identical restriction sites within the
porin genes is limited. Therefore, we have planned to isolate
further hybrid genes by a method that is independent of
restriction sites, based on recombination between these homologous genes.
Acknowledgements. We thank Pier Overduin and Egbert Van der
Waal for technical assistance in some of the experiments.
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C o m m u n i c a t e d by H. B6hme
Received May 29, 1984