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Antonie van Leeuwenhoek 47 (1981) 325-337
325
Genetic and biochemical characterization of an Escherichia coli
K-l 2 mutant with an altered outer membrane protein
JAN TOMMASSEN, PETER VAN DER LEY AND
BEN LUGTENBERG
Department of Molecular Cell Biology and Institute for Molecular Biology,
State University of Utrecht, Transitorium 3, Padualaan 8, 3584 CH Utrecht,
The Netherlands
TOMMASSEN, J., VAN DER LEY, P. and LUGTENBERG, B. 1981. Genetic and
biochemical characterization of an Escherichia coli K-12 mutant with an altered
outer membrane protein. Antonie van Leeuwenhoek 47: 325-337.
The properties of an Escherichia coli K-12 mutant are described which seemingly produces a "new" major outer membrane protein with an apparent molecular weight of 40 000. This 40K protein was purified and its cyanogen bromide
(CNBr) fragments were compared with those of several known major outer
membrane proteins. A similarity was found between the CNBr fragments of the
40K protein and those of the OmpF protein (molecular weight 37000). In
addition, the 40K protein was found to be regulated exactly like the OmpF
protein, and the mutation which causes the production of the 40K protein has
been localized in (or very close to) the ompF gene. It is concluded that the 40K
protein is a mutant form of the OmpF protein. The results provide additional
evidence that the ompF gene at minute 21 is the structural gene for the OmpF
protein.
INTRODUCTION
The outer membrane of Escherichia coti K-12 contains a number of proteins
which form channels or pores through which different, and unrelated, hydrophilic solutes of molecular weight up to about 700 can pass this membrane
(Nakae, 1976; Beacham et al., 1977; Lutkenhaus, 1977; van Alphen et al., 1978a,
van Alphen et al., 1978b). Two of these so called porins, the OmpF protein and
the OmpC protein, are constitutively formed. Mutants which lack these two
proteins are sensitive to 3 % sodium dodecyl sulphate (SDS). They are often
unstable and easily revert to SDS-resistant strains which have either regained
one or both of the two porins or contain one of the so called "new major outer
membrane proteins" (Henning et al., 1977; Foulds and Chai, 1978a; Lugtenberg
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J. TOMMASSEN, P. VAN DER LEY AND B. LUGTENBERG
et al., 1978; Pugsley and Schnaitman, 1978). These new membrane proteins have
also pore properties (van Alphen et al., 1978b; Lugtenberg et al., 1978; Pugsley
and Schnaitman, 1978). Three loci are involved in the appearance of these new
proteins, namely nmpA at minute 83, nmpB at minute 9 and nmpC at minute 12
(Foulds and Chai, 1978b; Pugsley and Schnaitman, 1978). Mutations at nmpA or
nrnpB lead to the production of a new protein which has been designated as
protein Ic (Henning et al., 1977), e (Lugtenberg et al., 1978), E (Foulds and Chai,
I978a) or NmpAB protein (Pugsley et al., 1980). Recently we showed that this
protein is co-regulated with alkaline phosphatase and that the nmpA gene is
identical to phoS, phoT or pst, whereas nmpB is identical to phoR (Tommassen
and Lugtenberg, 1980). The structural gene for protein e is localized at minute 6
at a locus designated as phoE (Tommassen and Lugtenberg, 1981). In accordance with the way in which other outer membrane proteins with known structural genes are designated, this protein will now be referred to as PhoE protein.
Mutations at nrnpC lead to the production of a new outer membrane protein
which is not identical to the PhoE protein (Lee et al., 1979).
In this paper the properties of a mutant which seemingly produces another
"new" outer membrane protein are described. The results show that this protein
is a mutant form of the OmpF protein.
MATERIALS AND METHODS
Strains, phages and growth conditions
All bacterial strains are derivatives of E. coli K- 12. Their sources and relevant
characteristics are listed in Table 1. A heptose-deficient lipopolysaccharide
(LPS) mutant of strain CE t 193, strain CE 1212, was isolated as described earlier
with the aid of the LPS-specific phages T3, T4 and T7 (van Alphen et al., 1976).
Strain CE 1213 was obtained after conjugation of strain CE 1170 with Hfr KL 16
and selection for galK +, rpsL transconjugants. The presence of ompF +, ompA +,
his- and ompC- alleles was determined in this strain by analyzing cell envelope protein patterns and by testing for growth on minimal medium with
or without histidine. Strain CE1214 is a his +, rpsL transconjugant from a
cross of strain CEll70 with HfrH. The presence of the ompF-, ompA + and
ompC- alleles in this strain was determined by analysis of the cell envelope
protein pattern.
Marker positions, origin and direction of transfer of the donor strains are
given in Fig. 1. Except where noted otherwise, cells were grown overnight in yeast
broth (Lugtenberg et al., 1976) under vigorous aeration at 37°C. Laboratory
stocks of phages TuIa (Datta et al., 1977), Mel (Verhoef et al., 1977), K3
(Skurray et al., 1974), TC45 (Chai and Foulds, 1978) and P1 (Lennox, 1955) were
used.
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MUTANT OUTER MEMBRANE PROTEIN IN E. COLI K-12
Table 1. Origin and characteristics of bacterial strains ~
Strain
Characteristics
Source2, reference
CE1163
F - , thi, argE, his, proA2, thr, leu, mtl, xyl, galK, lacY,
rpsL, supE, pldA
Burnell et al., 1980
CEll70
F-, ompA460, ompC468, ompF482 derivative of strain
CE1163, producing 40K protein
This study
CE1161
F-, pyrD34, rpsL
Verhoef et al., I979
CE1189
F , maITderivative ofCE1161
This study
CEl193
F-,pyrD+,ompF482transductantofstrainCEl189, pro ducing 40K protein but not OmpF protein
This study
CE1212
F - , T3, T4, T7 resistant, pro derivative ofCE1193
This study
CE1108
F - , thr, leu, thi, pyrF, thy, ilvA, his, lacY, argG, tonA, tsx,
rpsL, cod, dra, vtr, glpR, ompB471, phoS200
CE1213
CE1214
Lugtenberg et al., 1978
F-,galK+,ompA+,ompF+ derivativeofCEl170produc ing OmpF protein but not 40K protein
This study
F - , his +, ompA + derivative of CEl170 producing 40K
protein but not OmpF protein
This study
HfrH
PC
HfrKL16
PC
Genotype descriptions follow the recommendations of Bachmann and Lo w (1980).
z PC, Phabagen collection, Dept. of Molecular Cell Biology, Section Microbiology, State University
of Utrecht, Utrecht, The Netherlands.
Genetic techniques
P1 transductions (Willetts et al., 1969) were carried out as described. Conjugations with an ompA mutant as recipient strain were performed on filters to
stabilize mating aggregates (Havekes et al., 1976). Sensitivity to bacteriophages
was determined by cross-streaking.
KL16
b
ompF
'
'
'
'
I
'11
ompB
'
~
galK pyrD
80
90
100//0
10
20
I
'
'
his
30
40
' I IL
rpsL malt
50
60
70
80
Fig. 1. Schematic representation of the E. coli K- 12 chromosome. Relevant markers used in acceptor
strains, as well as the origin and direction of transfer (indicated by arrows) of the Hfr strains used, are
indicated.
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J. TOMMASSEN, P. VAN DER LEY AND B. LUGTENBERG
Isolation and characterization of membrane fractions
Procedures for the isolation of cell envelopes (Lugtenberg et al., 1975),
protein-peptidoglycan complexes and peptidoglycan-associated proteins (Lugtenberg et al., 1977) have been described previously. Procedures used for the
purification of the OmpC protein and OmpF protein (Verhoef et al., 1979)
and of the PhoE protein (Lugtenberg et al., 1978) have been published. Cyanogen bromide (CNBr) fragmentation was carried out in either 7 0 ~ formic
acid or 70 ~ trifluoracetic acid as the solvent, resulting in incomplete or complete
cleavage, respectively (Schroeder et al., 1969). Separation of proteins by SDSpolyacrylamide gel electrophoresis was performed as described previously (Lugtenberg et al., 1975). Convex exponential gradient gels (Verhoef et al., 1979)
containing 11-15 ~ aerylamide were used for the analysis of CNBr fragments.
Assay for pore activity for ampicillin
To measure the rate of diffusion of ampicillin across the outer membrane of
intact cells, the rate of hydrolysis of this antibiotic was measured essentially as
described by van Alphen et al. (1978a). The rates of hydrolysis are expressed in
nmol per rain and per mg (equivalent) of dry weight cells.
RESULTS
Outer membrane proteins of strain CE1170
In the course of our genetic studies on the PhoE porin of the outer membrane
of E. coli K-12 we observed that mutant strain CEl170 contained an outer
membrane protein which, although it had the same electrophoretic mobility as
the PhoE protein, could not be identical with this protein. Mutant strain CE 1170
was derived from strain CE1163 in three steps by selecting for clones which had
become spontaneously resistant to the OmpA protein specific phage K3, the
OmpC protein specific phage Mel and the OmpF protein specific phage TuIa.
Comparison of the cell envelope protein patterns of the strains showed that
mutant strain CE1170 (Fig. 2b) in contrast to its parental strain (Fig. 2a) lacks
the OmpF protein, the OmpC protein and the OmpA protein. In addition a
strong increase in the amount of protein in the electrophoretic position of PhoE
protein and protein a was observed in the mutant. In contrast to protein a this
new protein, preliminary designated as "40K protein" because of its apparent
molecular weight, was peptidoglycan-associated (Fig. 2c). The seemingly obvious conclusion, namely that the 40K protein is identical to the PhoE protein,
which has the same electrophoretic mobility (Fig. 2d) and which is also
peptidoglycan-associated (Lugtenberg et al., 1978), is very unlikely for the following reasons: (i) strain CE1170 is resistant to the PhoE protein specific phage
TC45, (ii) strain CE 1170 does not produce alkaline phosphatase constitutively
(as measured according to Tommassen and Lugtenberg, 1980), whereas the
PhoE protein is co-regulated with this enzyme (Tommassen and Lugtenberg,
MUTANT OUTER MEMBRANE PROTEIN IN E. COLI K-12
329
Fig. 2. SDS-polyacrylamide get eleetrophoresis patterns of the cell envelope proteins of strain
CE1163 (a) and its mutant strain CEl170 (b), of the peptidoglycan-associated proteins of strain
CE1170 (c) and of purified PhoE protein (d). Only the relevant part of the gel is shown.
1980), (iii) strain CE 1170 contains the mutationproA2, which is a deletion which
includes thes genes proA, gpt (Hoekstra and Vis, 1977) and the structural gene for
the PhoE protein (Tommassen and Lugtenberg, 1981 and unpublished observation). We, therefore, assumed that the 40K protein was either another "new"
outer membrane protein or a mutant form or precursor form of one of the other
peptidoglycan-associated proteins with similar apparent molecular weights namely the OmpF protein and OmpC protein.
Purification of the 40Kprotein and comparison ofits cyanogen bromide fragments
with those of other porins.
The 40K protein of strain CE 1170 was purified by using the property that it is
peptidoglycan-associated (Verhoef et al., 1979). Since the resulting preparation
consisted for over ninety percent of 40K protein, further purification steps by
column chromatography were not necessary for our purpose. To see whether the
40K protein could be related to one of the proteins OmpF protein, OmpC
protein or PhoE protein, the four proteins were individually treated with cyanogen bromide using formic acid as the solvent, a procedure which often results
in incomplete cleavage (Schroeder et al., 1969). Analysis of the resulting peptides
(Fig. 3) shows that the fragments of the 40K protein (slot d) differ from those of
the PhoE protein (slot a) and of the OmpC protein (slot b), whereas a similarity
with the OmpF protein (slot c) is suggested. Cleavage of the OmpF protein yields
5 bands, named, a, b, c, d 3 and d z (nomenclature according to Schemitges and
Henning, 1976, and Verhoef et al., 1979). CNBr fragmentation of the 40K
protein yields two fragments with the same electrophoretic mobility as the
fragments c and d 2 of the OmpF protein, and two fragments with a slightly lower
electrophoretic mobility as the fragments a and b. If one takes into account that
fragment a of the OmpF protein is an incomplete cleavage product which
includes fragment b (Schmitges and Henning, 1976; Chen et al., 1978 ; Verhoef et
330
s. TOMMASSEN, P. VAN DER LEY AND B. LUGTENBERG
Fig. 3. Comparison of cyanogen bromide fragments of the PhoE protein (a), the OmpC protein (b),
the OmpF protein (c and e) and the 40K protein of strain CEll70 (d and f). Fragmentation was
performed in formic acid (slots a, b, c and d) or trifluoracetic acid (slots e and f). The positions of the
CNBr fragments a, b, c, d3, d 2 and d 1 of the OmpF protein are indicated.
al., 1979) and that fragment d 3 is not always visible, due to difficult staining and
that it also can be lost from gels during the destaining procedure (Schmitges and
Henning, 1976), these results suggest that the 40K protein is a structurally altered
OmpF protein, with an alteration in the CNBr fragment b, which results in a
different electrophoretic mobility of this fragment. Since fragment a includes
fragment b, also this fragment would have a different electrophoretic mobility.
Much more complete cleavage of the OmpF protein is achieved if CNBr
fragmentation is carried out in trifluoracetic acid as the solvent (Schmitges and
Henning, 1976). Complete cleavage of fragment a yields b and d 1, whereas
fragment c yields d2 and d 3 (Chen et al., 1978). The CNBr fragments of the
OmpF protein after cleavage in trifluoracetic acid are shown in Fig. 3 (slot e). As
compared with the incomplete cleavage fragments (slot c), fragment a has almost
completely disappeared, the amount of fragment c is diminished, the amount of
fragment d 2 is increased, and a new band in the position ofd 1has appeared. If the
40K protein is indeed an altered OmpF protein, similar results would be expected
after cleavage of the 40K protein in trifluoracetic acid. As is shown in slot f, the
largest CNBr fragment of the 40K protein has disappeared after complete
cleavage, the amount of the fragment in the position of fragment c is diminished,
the amount of the fragment in the position ofd 2 has increased, and a new band in
the position of d 1 has appeared. These results suggest that the 40K protein is
indeed an altered OmpF protein.
MUTANTOUTERMEMBRANEPROTEININ E. COLI K-12
331
Localization of the mutation which leads to the production of the 40K protein
Analysis of his+rpsL transconjugants and of gal+rpsL transconjugants of
crosses between strain CE1170 and the donor strains H f r H and K L I 6 respectively (see Fig. 1) showed that (i) transconjugants contained either the 40K
protein (e.g. strain CE 1214) or the O m p F protein (e.g. strain CE 1213) and (ii) the
mutation causing the appearance of the 40K protein is located between minutes
17 and 44 on the chromosomal map (Bachmann and Low, 1980), a region which
contains the ompF gene (Fig. 1). In order to see whether the mutation could be
located in the ompF gene at minute 21, P1 transductions were performed using
phage P1 grown on strain CE1170 as the donor and the pyrD strain CE1189 as
the acceptor strain. Fifty three out of ninety six pyrD + transductants were
resistant to the O m p F protein specific phage Tula whereas the cell envelopes of
twelve representative examples contained the 40K protein but lacked the OmpF
protein (e.g. strain CE 1193, Fig 4a). The other fourty three pyrD +transductants
were sensitive to phage TuIa whereas the cell envelopes of twelve representative
examples contained the O m p F protein but lacked the 40K protein. These results
show that the mutation causing the appearance of the 40K protein has the same
cotransduction percentage (55 ~ ) with the pyrD gene as gene ompF (Foulds,
1976; Verhoef et al., 1979). This result, together with the observation that transductants contain either the 40K protein or the OmpF protein provides further
evidence for the assumption that the 40K protein is an altered O m p F protein.
regulation of the 40K protein
Further proof for the idea that the 40K protein is an altered OmpF protein can
be provided if it can be shown that the 40K protein is subject to the same
regulation as the O m p F protein. The ompB gene is involved in the regulation of
both the OmpF protein and the OmpC protein (Hall and Silhavy, 1979; Ichihara
and Mizushima, 1978; Verhoef et al., 1979). In order to see whether the 40K
Fig. 4. SDS-polyacrylamidegel electrophoresispatterns of the cell envelope proteins of strain
CE1193(a), a malT+ompBderivativeofCE1193(b), strain CEt 189grownin yeastbroth (c), in yeast
broth supplementedwith 300mMNaC1(d), in yeastbroth supplementedwith600 m~ sucrose(e) and
strain CE1193 grown in yeast broth (f), in yeast broth supplementedwith 300 m~ NaC1 (g) and in
yeast broth supplementedwith 600 mN sucrose(h). Only the relevantpart of the gel is shown.
332
J. TOMMASSEN, P. VAN DER LEY AND B. LUGTENBERG
protein is synthesized under control of the ompB gene, we transferred the
ompB471 mutation of strain CE1108 to strain CE1193 using P1 transduction and
selection for malT + transductants. Resistance to the OmpC protein specific
phage Mel was used to check for cotransduction of the ompB gene. The cell
envelope protein patterns of strain CE1193 (slot a) and of a representative
example of a malT + Mel resistant (and therefore ompB) transductant of this
strain (slot b) are shown in Fig. 4. The results show that in the ompB transductant
not only the OmpC protein but also the 40K protein is absent (Fig. 4b). Therefore it can be concluded that the synthesis of the 40K protein, like those of the
OmpF protein and OmpC protein, requires the ompB gene product.
A second wellknown regulation phenomenon of the OmpF protein is that its
amount depends on the composition of the growth medium. For instance,
supplementation of the growth medium with high concentrations of NaC1 or
sucrose causes a drastic decrease in the amount of OmpF protein (van Alphen and
Lugtenberg, 1977; Hasegawa et al., 1976). As is shown in Fig 4, the synthesis of
the 40K protein in strain CE 1193 (slots f, g and h) and the OmpF protein in strain
CE1189 (slots c, d and e) are sensitive to the osmolarity of the growth medium in
a similar way. The amount of OmpF protein in the outer membrane is also
dependent on the structure of the LPS; mutants with a heptose-deficient LPS
have strongly decreased amounts of OmpF protein (Havekes et at., 1976). To
determine whether the amount of 40K protein in the outer membrane is also
dependent on the structure of the LPS, heptose-deficient LPS mutants were
isolated from strain CE1193, as mutants simultaneously resistant to the LPS
phages T3, T4 and T7. Strain CE1212 is such a mutant and, in contrast to its
parental strain CE1193, this strain is dependent on proline in the growth medium, indicating that an IpcA-pro deletion mutant has been isolated. Analysis of
cell envelopes of strain CE1212 on SDS polyacrylamide gels revealed that only
approximately 15 ~ of the 40K protein was present as compared with strain
CE1193 (not shown). Therefore, by using the three described criteria, it appears
that the syntheses of the OmpF protein and of the 40K protein are regulated in
the same way.
Pore activity of the 40K protein for ampicillin
It has been shown that the OmpF protein and the OmpC protein, but not the
OmpA protein play a role in the formation of pores through which the [3-1actam
antibuitics ampicillin and cephaloridine can pass the outer membrane (van
Alphen et al., 1978a). In order to determine whether the 40K protein can serve as a
pore for ampicillin, plasmid Rldrd-19 (Havekes et al., 1977) which codes for the
periplasmic enzyme 13-1actamase (Richmond and Sykes, 1973) was introduced
into strains CE 1213, which contains the OmpF protein and the OmpA protein as
only major outer membrane proteins, and CE1214 which contains the 40K
protein and the OmpA protein as the only major outer membrane proteins. To
measure the rate of diffusion of ampicillin across the outer membrane, the rate of
MUTANT OUTER MEMBRANE PROTEIN IN E. COLI
K-12
333
hydrolysis of this antibiotic was measured in intact cells according to the procedure described by van Alphen et al. (1978a), The rates of hydrolysis of ampicillin
in the R I drd-19 containing cells of strains CE 1213 and CE 1214 were found to be
8.8 and 9.6 nmol per min and per mg equivalent dry weight cells, respectively. To
determine the total J3-1actamase activity of the cells, the rate of hydrolysis of
ampicillin by preparations of spheroplasted cells was measured, and values of
536 and 562 nmol per rain and per mg cells were found for Rldrd-19 containing
derivatives of strains CE1213 and CE1214, respectively. These results show that
permeation through the outer membrane is the rate-limiting factor for ampicillin
hydrolysis in both strains and that the 40K protein is an equally good pore for
ampicillin as the OmpF protein.
DISCUSSION
In this paper we describe a mutant which produces an outer membrane protein
with an apparent molecular weight of 40000. Several lines of evidence show that
this 40K protein is not a '°new" outer membrane protein but represents an
altered form of the OmpF protein: (i) like the OmpF protein (Rosenbusch,
1974; Lugtenberg et al., 1976; Schmitges and Henning, 1976) it is peptidoglycan-associated (Fig. 2c), (ii) the CNBr fragments of both proteins are
very similar (Fig. 3), (iii) the mutation responsible for the production of the 40K
protein is located at minute 21 in (or very close to) the ompF gene, (iv) neither
protein is produced in ompB mutants (Fig. 4), (v) the amounts of both proteins
are dependent on the osmolarity of the growth medium (Fig. 4), (vi) like the
OmpF protein (van Alphen et al., 1978a) the 40K protein forms an active pore
for ampicillin and (vii) like the amount of the OmpF protein (Havekes et al.,
1976) the amount of the 40K protein in the cell envelope is dependent on the
structure of the lipopolysaccharide (LPS), since mutants with a heptoseless LPS
have strongly decreased amounts of this protein. From these combined results
we conclude that the 40K protein of strain CE1170 is apparently an altered
OmpF protein.
With respect to the nature of the mutation which leads to an altered OmpF
protein, it can be noted that the altered protein migrates more slowly in SDS
polyacrylamide gels than the wild type OmpF protein, as if its molecular weight
were higher by as much as 2000-3000. One might speculate that the 40K protein
is an unprocessed precursor of the OmpF protein, since precursors of exported
proteins contain an additional signal peptide of approximately 15 to 30 amino
acids at the amino terminus (Silhavy et al., 1979). However, this explanation
seems unlikely, since the largest CNBr fragment of the OmpF protein, which is
the only altered fragment in the 40K protein (Fig. 3), is not the aminoterminal
fragment (Chen et al., 1978). In addition, as is known for the mature OmpF
protein (Henning et al., 1977), the aminoterminal amino acid of the 40K protein
334
J. TOMMASSEN~ P. VAN DER LEY AND B. LUGTENBERG
is also alanine (B. Verhey, unpublished observation), whereas methionine would
be expected for the precursor form. Since the altered CNBr fragment can also not
be the carboxy-terminal fragment (Chen et al., 1978), the mutation is probably
not the result of an amino acid extension at the carboxy-terminus of the protein.
On the other hand, it has been reported that a single amino acid substitution can
be sufficient to produce a change in electrophoretic mobility similar to that
observed in the 40K protein (Noel et al., 1979). This might explain why one
property of the protein has been lost (phage TuIa receptor activity) whereas
others are retained (pore activity for ampicillin, association to the peptidogtycan, interaction with LPS).
At least two genes are involved in the expression of the OmpF protein, namely
ompFand ompB. Strong experimental evidence for a role of the ompB gene in the
regulation of both the OmpC protein and the OmpF protein has been published
(Ichihara and Mizushima, 1978; Hall and Silhavy, 1979; Verhoef et al., 1979).
Evidence for ompF being the structural gene for the OmpF protein has been
reported by Sato and Yura (1979) who transferred the 21 minute region of the
chromosome of Salmonella typhimurium to E. coli K-12 by transduction. They
found two classes of transductants, containing either the OmpF protein or two
proteins which seem to be structurally related to, but not identical with, the 35 K
protein of S. typhimurium. In addition, Sato and Yura transferred an E. coli F'
plasmid carrying the ompF gene to S. typhimurium. The resulting strain produced, in addition to the Salmonella porins, an outer membrane protein indistinguishable from the OmpF protein of E. coli K-12 (Sato and Yura, 1979).
However, as this plasmid covers a chromosome region of at least 7 minutes
between the markers pyrD and trp, these results only allow the conclusion that
the structural gene is located between minute 21 and minute 28 of the chromosome. Since we localized the mutation which causes an altered OmpF protein by
transduction in (or very close to) ompFat minute 21, and since mutations which
cause an altered protein are assumed to be localized in the structural gene for this
protein (van Alphen et al., 1979; Pugsley et al., 1980; Tommassen and Lugtenberg, 1981), our results show that indeed ompFat minute 21 (or a gene extremely
close to it) is the structural gene for the OmpF protein. This result is in agreement
with recent results of Mutoh et al. (1981) who cloned the ompF gene as part of a
10.5 megadalton fragment in a specialized transducing lambda phage and showed that the OmpF protein is synthesized in UV-irradiated cells infected with the
)~ompF transducing phage.
We thank Ria van Boxtel for technical assistance. This work was supported by
the Netherlands Foundation for Chemical Research (SON) with financial aid
from the Netherlands Organization for the Advancement of Pure Research
(ZWO).
Received t5 April 1981
MUTANT OUTER MEMBRANE PROTEIN IN E. COLI K-12
335
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Note added in p r o o f
W e r e c e n t l y r e c e i v e d a n e w O m p F p r o t e i n specific p h a g e , K 2 0 , f r o m P.
M a n n i n g a n d P. R e e v e s . S t r a i n C E 1170 t u r n e d o u t to b e sensitive to this
p h a g e . T h i s r e s u l t (i) a g a i n i n d i c a t e s t h a t t h e 4 0 K p r o t e i n is a n a l t e r e d O m p F
p r o t e i n , a n d (ii) it s u g g e s t s t h a t t h e r e c o g n i t i o n sites f o r t h e p h a g e s T u I a a n d K 2 0
o n the O m p F p r o t e i n a r e d i f f e r e n t f r o m e a c h o t h e r .