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Mol Gen Genet (1984) 195:190494
© Springer-Verlag 1984
Cloning of phoM, a gene involved in regulation
of the synthesis of phosphate limitation
inducible proteins in Escherichia coli K12
Jan Tommassen, Pieter Hiemstra, Piet Overduin, and Ben Lugtenberg
Institute for Molecular Biology and Department of Molecular Cell Biology, State University, Transitorium 3,
Padualaan 8, 3584 CH Utrecht, The Netherlands
Summary. Like the synthesis of alkaline phosphatase, the
synthesis of outer membrane PhoE protein is shown to be
dependent on the phoM gene product in phoR mutants of
E. coli K12. This phoM gene has been cloned into the multicopy vector pACYC184 using selection for alkaline phosphatase constitutive synthesis in a phoR background. The
gene was localized on the hybrid plasmids by analysis of
deletion plasmids constructed in vitro and of mutant plasmids generated by 76 insertions. Interestingly, two of the
selected hybrid plasmids contained the entire phoA-phoBphoR region of the chromosome, as a multiple copy state
of these genes results in the constitutive synthesis of alkaline
phosphatase. The presence of multiple copies of the phoM
gene hardly influences the level of expression of alkaline
phosphatase and PhoE protein in a pho + strain, but significantly increases the levels of these proteins in a phoR mutant
strain.
Introduction
PhoE protein of Escherichia coli K12 is involved in the
formation of aqueous pores in the outer membrane through
which small hydrophilic solutes can permeate. The protein
is induced by growth at limiting concentrations of inorganic
phosphate (Overbeeke and Lugtenberg 1980) and is coregulated with alkaline phosphatase and a number of other
phosphate limitation inducible proteins (Tommassen and
Lugtenberg 1980). In comparison with the other pore proteins, OmpC and OmpF, PhoE protein forms more efficient
pores for the uptake of phosphate and phosphate-containing compounds (Korteland et al. 1982).
Several genes are involved in the regulation of the expression of PhoE protein and alkaline phosphatase. Mutations in the regulatory gene phoR lead to the constitutive
synthesis of alkaline phosphatase and of PhoE protein (Torriani and Rothman 1961; Tommassen and Lugtenberg
1980) at 25% of the fully induced level (Echols et al. 1961 ;
Tommassen and Lugtenberg 1980). The phoR gene product
is presumed to be a repressor in the presence of phosphate,
but an activator in the absence of phosphate (Garen and
Echols 1962; Wanner and Latterell 1980). Strains carrying
mutations in phoB are uninducible for alkaline phosphatase
and PhoE protein (Bracha and Yagil 1973; Tommassen
and Lugtenberg 1980). The phoB product is presumed to
Offprint requests to: J. Tommassen
be an activator protein (Brickman and Beckwith 1975; Yagil et al. 1975). The genes phoB and phoR are located at
rain 9 on the chromosomal map (Bachmann 1983).
Recently, a new regulatory gene for the synthesis of
alkaline phosphatase, phoM, has been described (Wanner
and Latterell 1980). Mutants in the phoM gene, which is
located at rain 100 on the chromosomal map, are indistinguishable from wild-type strains. However, whereas phoR
mutants produce alkaline phosphatase constitutively, phoR
phoM double mutants are uninducible for alkaline phosphatase (Wanner and Latterell 1980). It has been proposed
that the phoM product is an activator which can partially
replace the activator function of the phoR product (Wanner
and Latterell 1980). Whereas the synthesis of alkaline phosphatase in phoR mutants is dependent on the phoM gene
product, it has been reported that this is not the case for
some other phosphate limitation inducible proteins (Wanner 1983).
To study the regulation of the expression of PhoE protein, we have cloned the regulatory genes. The cloning of
phoB and phoR has been described previously and we observed that the phoR product represses the synthesis ofphoB
product in minicells (Tommassen et al. 1982a). In the present paper, we show that the synthesis of PhoE protein in
phoR mutants is dependent on a phoM + allele and we describe the cloning ofphoM.
Materials and methods
Strains and growth conditions. All bacterial strains used are
derivatives of E. coli K12. Their sources and relevant characteristics are listed in Table 1.
Except where noted, cells were grown overnight in Lbroth (Tommassen et al. 1983) at 37 ° C under aeration. To
Table l. Bacterial strains and their characteristics
Strain
Characteristics
Source
BW256
BW255
BW705
BW89
thi rpsL
thi rpsLphoR68
thi rpsLphoR68phoM453 lacproC: ."Tn5
thirpsLphoR68phoM453phoA458
lacZ524
reeA56pro + derivative of BW705
F'lae+, thr thi recA171 lacZ22 lacI
rpsL rpoBsupE
B.L. Wanner
B.L. Wanner
B.L. Wanner
B.L. Wanner
CE1225
CEt304
This study
de Geus et al.
(1983)
191
select for chloramphenicol (Cm) or tetracycline (Tc) resistant strains, concentrations of 25 and 10 ~tg/ml, respectively, of these antibiotics were applied.
Pho E
~ OmpT
OmpF
-"OmpC
~OmpA
Cell envelope preparation and minicell techniques. Cell envelopes were prepared as described by Lugtenberg et al.
(1975). Labeling of plasmid-coded proteins in minicells was
carried out as described previously (Tommassen et al.
1982 b). Protein patterns of cell envelopes and minicell preparations were analyzed by sodium dodecyl sulfate (SDS)
polyacrylamide gel electrophoresis (Lugtenberg et al. 1975).
Genetic techniques. Transformation was carried out as described by Brown et al. (1979). Sensitivity to bacteriophage
TC45 was determined by cross-streaking. This phage uses
PhoE protein as part of its receptor (Chai and Foulds 1978).
Thus, only strains that produce PhoE protein constitutively
are sensitive to this phage.
DNA techniques. Chromosomal D N A was isolated as described by Cosloy and Oishi (1973). Plasmid D N A was
isolated by the alkaline extraction procedure of Birnboim
and Doly (1979). This procedure was followed by CsC1/
ethidium bromide isopycnic centrifugation if plasmid D N A
was needed for further plasmid constructions.
The conditions for restriction endonuclease reactions
were those proposed by the manufactures. Analysis of plasmid D N A fragments was performed by electrophoresis in
a 0.6% agarose slab gel (van den Hondel et al. 1979).
HindIII-generated fragments of bacteriophage 2 D N A were
used as molecular weight standards in gels. Ligation was
performed as described by Tanaka and Weisblum (1975).
Assays for alkaline phosphatase. For screening clones which
produce alkaline phosphatase constitutively, the indicator
dye 5-bromo-4-chloro-3-indolyl phosphate (XP) was used
as described by Brickman and Beckwith (1975). Quantitative assays for alkaline phosphatase activity using para-nitrophenyl phosphate as the substrate were carried out as
described previously (Tommassen and Lugtenberg 1980).
Isolation of plasmids carrying an inserted y5 element. Insertions of the transposable element )~5 (Guyer 1978, Reed
1981) in plasmids pJP80 and pJP86 were obtained as follows. Plasmids pJP80 and pJP86 were transformed into the
F' lac+-containing strain CE1304, selecting for Cm or Tc
resistance, respectively. Transformants were subsequently
conjugated with strain CE1225, selecting for Cm or Tc resistance, respectively, and for leu + colonies on plates containing XP. Transconjugants with an intact phoM + allele on
the plasmid produce alkaline phosphatase constitutively,
resulting in blue colonies, whereas transconjugants with a
73 insertion in phoM yield white colonies.
Results
Expression of PhoE protein in phoR mutants
is dependent on a phoM + allele
To investigate whether the synthesis of PhoE protein in
phoR mutants is dependent on aphoM + allele, cell envelope
proteins from thepho + strain BW256, phoR mutant BW255
and phoR phoM double mutant BW705 were analyzed by
polyacrylamide gel electrophoresis. The results (Fig. 1)
a
b
c
d
e
f
g
Fig. l. SDS polyacrylamide gel electrophoresis patterns of the cell
envelope proteins of pho ÷ strain BW256 (a), phoR strain BW255
(b), phoR phoM strains BW705 (c) and CE1225 (d) and derivatives
of CE1225 containing pJP72 (e), pJP71 (f) and pJP77 (g), respectively. Only the relevant part of the gel is shown
show that the phoR mutant strain (lane b) produces PhoE
protein constitutively in contrast to the pho ÷ strain (lane a)
and to the phoR phoM double mutant (lane c). Thus, as
for alkaline phosphatase (Wanner and Latterell 1980), the
constitutive synthesis of PhoE protein in phoR mutants is
dependent on a phoM + allele. This conclusion was confirmed by the observation that the phoR mutant strain, in
contrast to the phoR phoM double mutant, is sensitive to
the PhoE protein specific phage, TC45.
Cloning of the phoM gene
Since the constitutive synthesis of alkaline phosphatase in
phoR mutants is dependent on a phoM + allele, we attempted to clone the phoM gene by selecting for hybrid plasmids
conferring constitutive synthesis of alkaline phosphatase to
a phoR phoM double mutant.
Purified chromosomal D N A of the prototrophic strain
PC0031 was used as the source of phoM + D N A and the
miniplasmid pACYC184 as the cloning vector. This vector
renders cells resistant to chloramphenicol and tetracycline
and has unique sites for the restriction endonucleases
HindIII, BamHI and SalI in the Tc resistance gene (Chang
and Cohen 1978). In independent cloning experiments combinations of two of these enzymes were used to prevent
recircularization of the vector DNA. Chromosomal D N A
and plasmid D N A were mixed and digested with two restriction enzymes. In two independent cloning experiments
the combinations of restriction enzymes HindlII-SalI and
HindIII-BamHI were used. After inactivation of the enzymes at 65 ° C and ligation with T4 D N A ligase, the D N A
mixtures were used to transform the phoM phoR strain
CE1225, selecting for Cm-resistant colonies on plates containing XP as an indicator dye for alkaline phosphatase
activity. In both experiments approximately 10,000 colonies
per gg vector D N A were obtained of which 0.5% displayed
a blue-green colour. Plasmid D N A was extracted from six
Cm-resistant blue-green transformants and reintroduced
into strain CE1225. Three plasmids did not render the cells
constitutive for alkaline phosphatase, showing that the original transformants did not contain a cloned phoM ÷ gene,
but more probably contained a reversion of the chromosomal phoM- mutation. The three other plasmids, designated pJP71, pJP72 and pJP77, did render the cells constitutive for alkaline phosphatase and are described below.
Characterization of pJP72
Introduction of pJP72 into strain CE1225 results in the
constitutive synthesis of both alkaline phosphatase (Ta-
192
Table 2. Levels of alkaline phosphatase in strains carrying hybrid
plasmids
Strain
BW256
BW255
BW705
CE1225
CE1225
BW255
BW256
CE1225
CE1225
BW256
BW256
Medium"
LB
LB
LB
LB
LB
LB
LB
LB
LB
HPi
LPi
Genetic
background
Plasmid
pho +
phoR
phoR phoM
phoR phoM
phoR phoM
phoR
pho +
phoRphoM
phoR phoM
pho +
pho +
pJP72
pJP72
pJP72
pJP71
pJP77
-
ClaI H i n d ~
11~
Alkaline
phosphatase
activity b
0.3
30.8
0.2
0.2
92.0
89.4
0.1
18.2
16.4
0.3
121.5
" Cells were grown overnight in L-broth (LB), except that strain
BW256 was also grown in high (HPi) and low (LPi) phosphate
containing minimal medium (Tommassen and Lugtenberg 1980).
b Alkaline phosphatase activity is expressed as nanomol para-nitrophenol released per rain reaction time per mg cells (dry
weight)
ble 2) and of PhoE protein as appeared from the analysis
of cell envelope protein patterns on gels (Fig. 1, lane e)
and from sensitivity to phage TC45. Introduction of the
plasmid into the phoR ÷ strain BW256 neither resulted in
the constitutive synthesis of alkaline phosphatase (Table 2)
nor of PhoE protein (not shown). Thus, pJP72 completely
complements phoM mutations and therefore probably contains a phoM + allele.
To obtain a physical map, the purified pJP72 was subjected to single and double digestions with several restriction enzymes and the resulting fragments were analyzed
on agarose gels. The position of the pACYC184 vector in
the map (Fig. 2) was deduced from its known restriction
map (Chang and Cohen 1978). The size of the entire plasmid is 11.1 kilobase pairs (kb). The plasmid contains two
HindIII sites as well as two SalI sites (Fig. 2). Since the
plasmid was derived from a cloning experiment in which
the combination of the restriction enzymes HindIII and SalI
was used, this means that either the digestion with the restriction enzymes was incomplete or that the cloned D N A
in pJP72 consists of several fragments that are not contiguous on the chromosome. A total of two ClaI sites were
mapped on pJP72. Since the D N A used for the construction
of the map did not pass through a dam strain, additional
ClaI sites may be present.
Physical localization of phoM
To localize the phoM gene, deletion mutant plasmids of
pJP72 were constructed in vitro by digesting purified pJP72
D N A with HindIII or ClaI. After subsequent ligation, the
D N A was transformed into strain CE1225, selecting Cmresistant colonies. Plasmid D N A was isolated from the
transformants and analyzed on agarose gels. Plasmids
pJP80 and pJP82 were selected for further characterization.
The HindIII-generated plasmid pJP80, which is depicted
in Fig. 3, complements the phoM mutation of CE1225,
whereas the ClaI-generated plasmid pJP82 (Fig. 3) does
not. F r o m these results it is concluded that phoM is at
least partially located on the 3.6 kb D N A fragment between
~
Pvul
E c / ' l O
pal
pdP72
oR7
Sal
Fig. 2. Restriction enzyme cleavage map of pJP72. The pACYC184
vector is indicated by the heavy line; the chromosomal E. coli
DNA is represented by a thin line. Map units are in kb. The restriction enzymes BamHI, BgIII, NdeI, PstI, SmaI and J(hoI do not
cleave the DNA
HindT~lCla]1 EcoRI
pde72
I(
0kb
pdP80 0
I
cm
Hind]]I C/a] EcoRI
L(
Sail Pvu]I 3 ClaI2
I
~
,,I
PvulI 4
HindN 2
I,
I
I
Clal,
Pvul]
Hind~
i I
SalI Pvu 11
cml
~
i:
I,bob
r,',
I
i
H.,nd'~1
I
11.1kb
I
~9.3kb
i
i
i
i
pJP82
Clal
EcoRI
I
0kb
cmI
Sail
Pvun
I
Ii~i
,,,,
,,,
c/'al'
ii',
5.8 ~b
ii
,,
i,
Pvug EcoRI
0oPt°
2°.
SalI Clal Pvu~
I I
Tc
I!
!',~
i
ClaI PvulI
J:',
I
, "k6.3kb
Fig. 3. Localization of phoM on hybrid plasmids. The restriction
enzyme cleavage maps of pJP72 and several sublcones are shown
as well as the location of 76 insertions in pJP80 and pJP86. Circles
and squares represent insertions which do or do not inactivate
phoM, respectively, pACYC184 DNA is indicated by the heavy
lines, pJP72 contains six PvuII sites; only those sites, which were
important for the construction of pJP86, are depicted
the restriction sites ClaI-2 and HindIII-2 of pJP72
(Fig. 3).
To further localize phoM, PvuII fragments of pJP80 were
subcloned into pACYC184 by exchanging the 0.4 kb PvuII
fragment in the Cm resistance gene of pACYC184 for PvuII
fragments of pJP80. Plasmid pJP86, depicted in Fig. 3, is
one of the resulting plasmids. This plasmid complements
the phoM mutation of CE1225, showing that phoM is located completely on this 2.7 kb PvuII fragment.
The 5.6 kb 76 transposable element of the F plasmid
was used to generate insertional mutations in pJP80 and
193
pJP86 as described in Materials and methods. The locations
of the insertions on the plasmids were mapped using ClaI,
SalI and BamHI-generated restriction fragments. ClaI and
SalI cleave the 75 element approximately in the middle,
whereas BamHI cleaves at 0.2 kb from one end. The results
(Fig. 3) show that the insertions in phoM are clustered in
a region of approximately 1.4 kb.
The phoM gene product
To identify the phoM product the synthesis of plasmidcoded proteins was studied in a minicell system. Compared
with minicells isolated from strains carrying the cloning
vector pACYC184, minicells from strains carrying the hybrid plasmid pJP80 produced one additional labeled polypeptide with an apparent molecular weight of 30,000 (not
shown). However, as this band was also found in minicells
from strains carrying plasmids with 75 insertions in phoM,
this polypeptide cannot be the phoM product. In some,
but not all, experiments with an intact phoM gene, a very
weak band with an apparent molecular weight of 60,000
was found. This band was never found when phoM was
inactivated by 75 insertions. However, because the band
was not always found, we cannot definitively conclude that
this is the phoM product.
From the location of the 75 insertions in pJP80 and
in pJP86 (Fig. 3), it can be deduced that the phoM gene
covers at the most a region of 1.8 kb and, if the insertions
that inactivate phoM are indeed located in phoM and are
not polar insertions in an unknown gene in the same operon
as phoM, at least 1.4 kb. Therefore, the phoM product is
probably a polypeptide with a molecular weight between
approximately 51,000 and 66,000.
]
pJP91
[
t I "1~ It
0kb
II
pJP92
I
0kb
pPBI01
]
Okb
J
Okb
[ --
~J
I, II,
I
I
,
A
A
I
II
'
~
"11 [
~
~PI
~1
h,I
~[
/[
116.2 kb
't
Okb
pJP50
I ]
',
I
15.4kb
I
8.3kb
,lO,7kb
I
,I
,
B
R
l&7kb
Fig. 4. Restriction enzyme cleavage maps of pJP91 and pJP92 and
comparison with the maps ofphoA +plasmids pPB101 (Berg 1981)
and pHI-l (Inouye et al. 1981) and ofphoB+phoR+plasmid pJP50
(Tommassen et al. 1982a). The positions ofphoA, phoB and phoR
on pPB101, pill-1 and pJP50 are indicated by A, B and R, respectively
some. Figure 4 shows the restriction maps of plasmids
pJP91 and pJP92, which are derivatives of plasmids pJP71
and pJP77, respectively, from which a 10 kb PstI D N A
fragment has been deleted. As shown in Fig. 4, both plasmids contain a region that corresponds to the phoA region
of plasmids pPB101 and pHI-l, and also a region that corresponds to the phoR phoB region of plasmid pJP50. Thus,
the presence ofphoA, phoR, and phoB on a multicopy plasmid apparently results in the constitutive synthesis of alkaline phosphatase.
Characterization of pJP71 and pJP77
Introduction of either pJP71 or pJP77 into strain CE1225
resulted in the constitutive synthesis of alkaline phosphatase, although not at the same level as the phoR phoM +
strain BW255 or the pJP72-containing derivative of CE1225
(Table 2). In addition, constitutive synthesis of PhoE protein could not be detected in the pJP71 and pJP77-containing derivatives of CE1225 (Fig. 1, lanes f and g). Therefore,
these plasmids do not contain a phoM + allele. As introduction of the plasmids into the phoR phoM strain BW89,
which also carries a mutation in phoA, the strctural gene
for alkaline phosphatase, resulted in the constitutive synthesis of this enzyme (not shown), these plasmids must contain
a cloned phoA gene. As expression of alkaline phosphatase
is dependent on the presence of either a phoM + or a phoR +
allele, the plasmids are likely to contain also the phoR gene,
which is located close to phoA (Bachmann ~983). This was
confirmed by analysis of the restriction enzyme cleavage
patterns of pJP71 and pJP77 and comparison of the maps
with those of the phoA containing plasmids pPB101 (Berg
1981) and p H I - l (Inouye et al. 1981) and with the phoB
and phoR-contalning plasmid pJP50 (Tommassen et al.
1982a). Plasmid pJP71 is 26.2 kb long and derived from
a cloning experiment in which a combination of the restriction enzymes HindIII and SalI was used. Plasmid pJP77
is 25.4 kb long and derived from a cloning experiment in
which the restriction enzymes HindIlI and BamHI were
used. Since both plasmids contain multiple HindIII and
BamHI sites, this means that the cloned fragments on the
plasmids are not necessarily contiguous on the chromo-
Discussion
Growth of E. coli K12 under phosphate limitation leads
to the induction of many proteins involved in the uptake
and metabolism of phosphate (for a review, see Tommassen
and Lugtenberg 1982). By fusing lacZ to phosphate starvation inducible (psi) promoters, Wanner and McSharry
(1982) showed that E. coli K12 contains at least 18 psi
promoters, some of which are regulated like alkaline phosphatase, i.e., in a phoB, phoR and phoM dependent way,
whereas others are regulated only in part or not at all by
the products of these regulatory genes. We previously
showed that PhoE protein is coregulated with alkaline
phosphatase with respect to its expression in strains with
mutations in the regulatory genes phoB and phoR (Tommassen and Lugtenberg 1980). We have now demonstrated that
expression of PhoE protein is influenced in a similar way
to alkaline phosphatase by mutations in the recently discovered phoM gene.
We have cloned the phoM gene on a multicopy plasmid
and located the gene in a region of at least 1.4 kb and
at the most 1.8 kb, corresponding to a protein with a molecular weight between approximately 51,000 and 66,000. We
were not able to identify definitively the phoM product in
minicell experiments, but a weak band with an apparent
molecular weight of 60,000, which could well be the phoM
product, was sometimes observed. Apparently, the phoM
gene has a very weak promoter. Thus, for the definitive
identification and for the purification of the phoM product,
which is necessary for studying the exact role of the protein
194
in the regulation o f the synthesis o f P h o E protein and alkaline phosphatase, it is necessary to clone phoM under a
stronger promoter.
In the course o f these studies, we also cloned the phoAphoB-phoR region of the chromosome. Apparently, the
presence o f these genes on a multicopy plasmid results in
the constitutive synthesis o f alkaline phosphatase. This m a y
be explained either by the presence of phoA, the structural
gene for alkaline phosphatase, or by the presence o f the
regulatory genes phoB and phoR in a multicopy state, or
by the c o m b i n a t i o n of both. It has been reported that the
presence ofphoA on a multicopy plasmid results in a slight
derepression of alkaline p h o s p h a t a s e synthesis in media
containing excess o f p h o s p h a t e (Berg 1981; Inouye et al.
1981). It has also been reported that the presence o f a multicopy plasmid that contains phoB and p r o b a b l y also phoR,
results in a certain derepression of alkaline phosphatase
synthesis ( M a k i n o et al. 1982). Apparently, a combination
of these effects results in strongly derepressed synthesis o f
alkaline phosphatase in media containing excess phosphate
(Table 2).
In contrast to the effect ofphoB andphoR in a multicopy
state on the synthesis of alkaline phosphatase ( M a k i n o
et al. 1982), multiple copies of phoM did not result in a
derepression of alkaline phosphatase (Table 2) and PhoE
protein synthesis in a pho + strain. Apparently, the repressor
form o f the phoR p r o d u c t either completely abolishes the
effect o f an increased n u m b e r of copies o f the activator
phoM product, or, alternatively, the phoR p r o d u c t represses
efficiently the synthesis o f p h o M product, even if the p h o M
gene is present on a multicopy plasmid. On the other hand,
in a phoR m u t a n t strain where the synthesis o f alkaline
phosphatase and PhoE protein is dependent on the p h o M
gene product, an increased copy number o f phoM results
in increased levels of alkaline phosphatase (Table 2) and
PhoE protein (Fig. 1).
Acknowledgements. We thank B.L. Wanner for supplying strains
and John Verbakel for assistance in some of the experiments.
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C o m m u n i c a t e d by A. B6ck
Received September 19, 1983 / February 16, 1984