FEMS MicrobiologyLetters 9 (1980) 95-98
© Copyright Federation of European MicrobiologicalSocieties
Published by Elsevier/North-HollandBiomedicalPress
95
S T U D I E S O N T H E I N F L U E N C E O F T H E C Y C L I C AMP S Y S T E M ON M A J O R O U T E R
MEMBRANE PROTEINS OF ESCHERICHIA COLI K12
NIGEL W. SCOTT * and COLIN R. HARWOOD
Department of Microbiology, The MedicalSchool, Universityof Newcastle upon Tyne, NE1 7RU, U.K.
Received and accepted 14 July 1980
1. Introduction
2. Materials and Methods
Escherichia coli K12 strains deficient in either
adenylate cyclase (cya) or the cyclic AMP activator
protein CAP (crp) exhibit a number of cell envelopeassociated changes when compared with their adenylate cyclase and CAP proficient parents. These include
a shorter than normal bacillary shape ([1,2] and
Scott and Harwood, unpublished data), a reduction
in the number of flagella [3], increased sensitivity to
deoxycholate [2], resistance to various bacteriophages [2,4] and the overproduction of sex pili by
strains possessing certain derepressed conjugative plasmids [ 1]. In the case of cya mutants, but not crp mutants, these changes can to a large extent be phenotypically reversed by the addition of cyclic AMP to
the growth medium. We have been studying the protein composition of the cell envelope in an attempt to
understand the mechanisms underlying some of these
changes.
E. coli K12 has a number of major proteins in its
outer membrane in addition to the lipoprotein; these
include proteins a (3b), b (la), c (lb), d (3a) and III
(see [5] and [6] for nomenclature). We have studied
the levels of each of these proteins in the wild type
and cyclic AMP mutants after growth on a variety of
media and report that the cyclic AMP system appears
to influence markedly the levels of 3 of these proteins,
namely b, c and III.
2.1. Strains and growth conditions
* present address: Department of BiologicalSciences, University of Warwick, Coventry CV4 7AL, U.K.
The following strains were used: 1100 (HfrH, thiA,
cya ÷,crp ~) and its derivatives 5333 (cya ~, crp) and
5336 (cya, crp*) [7].
All strains were grown in batch culture with vigorous shaking at 37°C and were harvested at anA600nm
of 0.1 to 0.2, whilst still in exponential growth.
Media were either Lemco broth with or without
added glucose (0.2% w/v) or M9 salts medium [8],
with either glucose or glycerol (0.2% w/v) as carbon
and energy source, and supplemented with trace
elements and thiamine (1/ag/ml).
2.2. Isolation and electrophoresis of cell envelope
pro teins
50 ml cultures were harvested by centrifugation
and cell envelope proteins extracted by the method
of Lugtenberg [9]. The final extract was resuspended
in approx. 100/A of sample buffer, heated in a waterbath at 100°C and approx. 10/ag samples loaded onto
SDS-polyacrylamide gels. The composition of the gels
was identical with those of Lugtenberg but differed
in dimensions. Two gel sizes, with slightly differing
separation properties were used; one was 3 nun thick
and had a running distance of 60 mm, the other was
1.5 mm thick with a running distance of 90 mm. Gels
were electrophoresed at 25 mA constant current. The
proteins were visualized by staining with 0.5% (w/v)
Brilliant Blue G250 (Sigma Chemical Co.) in 50%
methanol/10% acetic acid for 2 4 - 3 6 h. Destaining
96
the levels of the proteins were less marked (data not
shown). The levels of proteins a and d remained substantially unaltered under all growth conditions
(11% + 2.3(40) and 38.4% -+ 3.7(40) respectively).
The relative amounts of proteins b, c and III in the
outer membrane have previously been shown to alter
in response to changes in the composition of the
growth medium [10,11 ] and our results indicate that
at least a part of this variation is effected via the cyclic
AMP system. The involvement of the crp ÷ gene as
well as the cya +gene suggests that the cyclic AMP
system exerts its influence at the level of transcription. Similar conclusions have been drawn for
III [12] and for another outer membrane protein, the
T6 receptor protein, tsx [4]. The differences in the
levels of these proteins after growth on simple and
complex media, even when cyclic AMP is present,
suggests that this nucleotide is only one component
in their regulation, a view reinforced by the promoterfusion studies Hall and Silhavy on protein c [13].
Genes at four loci have previously been shown to
influence the levels of proteins b and c in E. coli K12.
They are o m p C (par) and o m p F (tolF) thought to be
the structural genes for b and c respectively, perA
believed to be involved in secretory mechanisms [ 14]
and ornpB which appears to control the expression of
o m p C and ompF. The cyclic AMP system could act in
several ways. It might for example simultaneously
exert a positive controlling effect on the gene coding
for protein b (ompC) and a negative effect on the
genes coding for proteins c (omptO and III, the
explanation favoured by Malliak and Herrlich for the
control of protein III [12]. A statistical analysis of
was with 50% methanol/10% acetic acid. Bands were
intensified for photography by treating with 10%
acetic acid for 4 h. Densitometer traces were obtained
from photographs of gels with a Joyce Loebl Chromoscan 200 with a Scan 201 plate scanning attachment. Peak areas were determined by weighing.
3. Results and Discussion
The levels of proteins a, b, c, d and III obtained
are shown in Table 1. When grown on M9 medium
with glucose as the carbon source, the parental strain
(1100) shows a significantly reduced level of protein
b, compared with growth on glycerol as the carbon
source or in the presence of 3 mM cyclic AMP. Only
relatively small changes were observed in the levels
of proteins c and III. Much larger changes were observed in the mutants. The level of protein b was very
much lower, and levels of c and III correspondingly
higher when the cya strain (5336) was grown on M9
medium with glucose as the carbon source. This
response was reversed when cyclic AMP, but not 5'adenosine monophosphate (3 mM), was present in the
growth medium. Similar results were observed with
the crp strain (5333) except that in this case the
inclusion o f cyclic AMP in the growth medium had
no apparent effect. Neither mutant was able to utilize
glycerol as a carbon source.
Essentially similar results were obtained when the
parent and mutants were grown in nutrient media,
supplemented with various combinations of glucose,
cyclic AMP and 5' AMP, except that the changes in
TABLE I
Levels of outer membrane proteins as a % of a + b + c + d + III from densitometer traces of SDS-PAGE separation of whole
cell envelopes
Medium
Levels of outer membrane proteins as a % of a + b + c + d + III
1100 (cya+crp+)
a
M9 +Glucose
M9 +Glucose+cAMP
M9 + Glucose + 5'AMP
M9 + Glycerol
ND. not determined.
b
12.5 27
12
41
12
c
5336 (cya)
d
9.5 41
6
37
ND
39 10
33
III
a
10
4
8.5 4.5
9
47
8
5
6
b
5333 (crp)
c
d
III
a
b
34
5
27
ND
39
35
44
14
4
16
13.5 4.5
13
7
c
d
28
40
26.5 39
ND
ND
III
14
14.5
97
o
o
55
r 2 = 0.94
Slope = - 1,3
50
o
45
o °
4O
35-
25
30
2O
o
15
10
5
I
5
0
I
10
I
15
I
20
1
25
I
30
1
35
~t
40
1
45
5O
%c
60
%b
55
r2 = 0.97
o
o
('~
Slope = - 1 . 0 6
50
o
o °
°!
45
40
o
35
o
o o
30
o
25
o
20
15
10
°o " ~
I
5
l
10
l
15
l
20
l
25
I
30
I
35
I
40
I
45
¢:L'~
50
%C+111
Fig. 1. Relationship between protein b and other outer membrane proteins. (A) % protein b plotted against % protein c
only (as a % a + b + c + d). (B) % protein b plotted against %
protein c + III (as a % a + b + c + d + III). Data from cultures
of parent and mutants grown in nutrient and minimal media.
our data shown below, however, suggests another possibility, namely that the cyclic AMP system, either
directly or indirectly via control genes like ompB,
controls the synthesis of protein b only and that
changes in the levels of proteins c and III are simply
responses to changes in the levels of this protein.
It has been noted several times that changes in the
level of protein b appear to be compensated by reciprocal changes in protein c to maintain a relatively
constant proportion of matrix protein in the outer
membrane [10], although no systemic study of this
relationship has been published. This view is seemingly
strengthened by similarities in their structure and the
appearance of new membrane proteins in certain
strains [15,16]. In order to test this hypothesis we
have plotted values for protein b against protein c
(as a % of a + b + c + d) and have confirmed the
inverse correlation between the two. A linear regression analysis (by the "all-squares method") gives an
r 2 value of 0.94 and a slope of 1.3 (Fig. 1A). However,
when values for protein b were plotted against c + III
(as a % o f a + b + c + d + III) a significantly improved
fit was obtained and both the r: and slope values
were close to 1.0 (0.97 and 1.06 respectively; see
Fig. lb).
Although our data can be interpreted in a number
of ways, the possibility that we favour is that the
cyclic AMP system positively controls the synthesis
of protein b. Under catabolite repressing growth conditions, or when the cyclic AMP system is deficient,
the level of protein b is reduced and space is created
in the outer membrane. Our analysis leads us to believe
that this space is not necessarily filled simply by a
structural analogue (i.e. protein c) but by any protein
which is available and capable of being assimilated
into this membrane; the former presumably by de
novo synthesis since precursor pools have not been
detected for any of the major outer membrane proteins. One protein that fulfils both of these requirements is that of the sex pili of derepressed conjugative
plasmids. It has been shown that when sex pili are
removed by blending they can be regenerated in the
absence of protein synthesis [17], indicating the presence of a pool of precursor pilin. Recently, Achtman et al. have shown that at least part of this pool is
located in the outer membrane [ 18]. Cyclic AMPdeficient strains possessing depressed conjugative
plasmids show greatly increased number of sex pili
[1 ] and one possibility is that this is a consequence of
decreased levels of protein b allowing more pilus pre.
cursor to enter the outer membrane. This in turn
might be expected to shift the equilibrium between
pilin precursor and sex pilus in favour of the latter.
98
We are at present investigating the influence of ompB,
ompC, ompF and cya mutations on sex pilus production using miniceli techniques and electron microscopy.
Finally, by growing cultures of the cya mutant in a
chemostat under defined conditions supplemented
with various concentrations of cyclic AMP, it should
be possible to produce bacteria with differing proportions of proteins b and c for permeability studies.
Acknowledgement
N.W.S. was supported by a Studentship from the
Science Research Council.
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