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Journal of Bacteriology, March 2000, p. 1423-1426, Vol. 182, No. 5
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cpx Two-Component Signal Transduction in
Escherichia coli: Excessive CpxR-P Levels Underlie
CpxA* Phenotypes
Peter
De Wulf and
E. C. C.
Lin*
Department of Microbiology and Molecular
Genetics, Harvard Medical School, Boston, Massachusetts 02115
Received 27 October 1999/Accepted 13 December 1999
 |
ABSTRACT |
In Escherichia coli, the CpxA-CpxR two-component signal
transduction system and the 
E and 32
response pathways jointly regulate gene expression in adaptation to
adverse conditions. These include envelope protein distress, heat
shock, oxidative stress, high pH, and entry into stationary phase.
Certain mutant versions of the CpxA sensor protein (CpxA* proteins)
exhibit an elevated ratio of kinase to phosphatase activity on CpxR,
the cognate response regulator. As a result, CpxA* strains display
numerous phenotypes, many of which cannot be easily related to
currently known functions of the CpxA-CpxR pathway. It is unclear whether CpxA* phenotypes are caused solely by hyperphosphorylation of
CpxR. We here report that all of the tested CpxA*
phenotypes depend on elevated levels of CpxR-P and not on
cross-signalling of CpxA* to noncognate response regulators.
| |
TEXT |
A typical sensor protein of a
two-component signal transduction system catalyzes both the
phosphorylation and dephosphorylation of the cognate response
regulator. The ratio between the kinase and the phosphatase activities
of the sensor determines the steady-state level of the phosphorylated
or functional form of the response regulator (32). Thus, any
mutation in the sensor protein that significantly affects its
kinase/phosphatase activity ratio should have important physiological
consequences. An example is provided by the Cpx system of
Escherichia coli, comprising the CpxA sensor kinase/phosphatase and the CpxR response regulator. Certain mutant CpxA
proteins, CpxA* proteins, appear to possess autokinase activity but are
deficient in CpxR-P phosphatase activity, leading to an overaccumulation of CpxR-P (25). The elevated level of
CpxR-P in turn hyperactivates the expression of the cpxRA
operon (6, 27). Cells synthesizing CpxA* show numerous
phenotypes, which include an impaired donor conjugative ability
(11, 12, 28, 29); a deficiency in murein lipoprotein and
OmpF in the cell envelope (14, 15); an anomalous positioning
of the FtsZ ring during cell division (22); a decreased
swarming ability (6); impaired abilities to grow on
succinate (24), L-lactose (21), and
L-proline (20); an acquired ability to utilize
L-serine as the sole carbon source (17, 18, 33);
partial auxotrophies for isoleucine and valine (13,
34); a growth sensitivity to high temperature (11) and
sodium dodecyl sulfate (SDS) (1); and an enhanced tolerance
to high pH (3), CuCl2 (6), colicins A and K (19), amikacin, and kanamycin (24,
35).
It is known that the Cpx system and the E and
32 response pathways cooperatively manage envelope
protein distress by activating the expression of ppiA and
ppiD (encoding periplasmic peptidyl-prolyl cis-trans isomerases), dsbA (encoding a
periplasmic disulfide oxidoreductase), degP
(encoding a periplasmic protease), and cpxP (encoding a periplasmic protein) (2-5, 23, 26). The
Cpx system has also been implicated in conjugation (11, 12, 28,
29), invasion of host cells, and virulence (8, 9). An
even broader role for this two-component system is indicated by recent
findings that the expression of the cpxRA operon increases
at the onset of stationary growth in an RpoS-dependent manner and that
operons involved in motility and chemotaxis (motAB cheAW and
tsr) are under direct negative control of CpxR-P
(6). In view of such an extensive role of the Cpx system, it
is not surprising that a multitude of physiological anomalies are
exhibited by CpxA* strains.
To test whether the CpxA* phenotypes are exclusively attributable to
exaggerated levels of CpxR-P, we compared the phenotypes of strain
ECL3501 (
cpxRA) (Table 1)
(6) expressing four different cpxRA operons: (i)
wild-type cpxR+A+, (ii)
cpxR+A*, (iii)
cpxR
A+; and (iv)
cpxRA*. The low-copy-number plasmid pRS415
(30) was used to express the various cpx operons,
since cpxRA is autogenously activated by CpxR-P
(6, 27). Consequently, in the absence of CpxR, a single
copy of the operon may not supply an adequate amount of CpxA* for
phenotypic manifestation. The missense mutation in CpxA* consists of a
Leu38-to-Phe substitution (22). The deletion in
CpxR extends from amino acid residues 28 to 166 (6), a region that includes the conserved phosphoryl
acceptor Asp51 (25). The plasmidborne operons carried the
cpx promoter region that stretches 309 bp upstream of the
cpxR start codon and includes the CpxR-P boxes (6). To monitor the transcription of the cpx
constructs, a promoterless lacZYA operon was placed
downstream of cpxA or cpxA* (Fig.
1). The synthesis of CpxA, CpxA*, CpxR,
and CpxR by the transformants was confirmed by
SDS-polyacrylamide (12.5 and 15%) gel electrophoresis. No Cpx proteins
were synthesized by ECL3501 bearing vector pRS415 without a
cpx insert (data not shown). The levels of expression of the
various cpx operons were quantified by 
-galactosidase
activities (Miller units) during growth of the transformants in glucose
(0.2%) minimal medium. The -galactosidase activity levels obtained
from pRS415/R+A* (Fig.
2A) exceeded those obtained from
pRS415/R+A+ (Fig. 2B). This result
was expected because the increased kinase/phosphatase ratio of CpxA*
should cause elevated CpxR-P levels, which in turn should enhance
cpxR+A* expression by autoactivation. Likewise,
the -galactosidase activity levels obtained from
pRS415/RA* (Fig. 2C) and
pRS415/RA+ (Fig. 2D) were lower
than those obtained from
pRS415/R+A+, due to the absence of
autoactivation. It should be mentioned, however, that even in the
absence of CpxR, the expression of cpxA and cpxA*
(from 1,500 to 4,500 Miller units) (Fig. 2B and C) exceeded that from
single-copy cpxR+A* (from 900 to 1,800 Miller
units [6]). The -galactosidase activity levels in
the control transformant, bearing vector pRS415, which contains a
promoterless lacZYA, varied from 15 to 35 Miller units (data
not shown). Thus, the levels of plasmid-specified CpxA and CpxA* should
be sufficient to give the appropriate phenotypes in the absence of
CpxR-P.
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FIG. 1.
Plasmid constructs for expressing
cpxR+A+,
cpxRA+,
cpxR+A*, and cpxRA* in
strain ECL3501 (cpxRA). The dark boxes depict the deleted
regions within the chromosomal cpxRA and lacZYA
operons. The hatched boxes depict the in-frame deletion (418 bp)
within cpxR. The cpx operon constructs contain
309 bp upstream from the cpxR start codon, including the
cpx promoter region (PcpxRA).
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FIG. 2.
Growth and specific -galactosidase activity
profiles of E. coli ECL3501 expressing various
cpx operon constructs. The cells were grown in 20 ml of
glucose (0.2%)-containing minimal medium containing 50 µg of
ampicillin per ml (300 rpm, 37°C) and assayed for their
-galactosidase activity as described by Miller (16).
OD600, optical density at 600 nm.
|
|
ECL3501 strains bearing pRS415 expressing different cpx
alleles were then scored for CpxA* anomalies: temperature sensitivity (11); inability to grow on succinate (24);
resistance to amikacin (24, 35), high pH
(3), CuCl2 (6), the redox dye
toluidine blue (J. M. Dong, unpublished data),
H2O2 (P. De Wulf, unpublished data), and SDS
(1); sensitivity to EDTA (P. De Wulf, unpublished data);
diminished swarming ability (6); and aberrant
positioning of the division septum (22). The conditions and
results of the analyses are shown in Table
2. None of the 11 CpxA* phenotypes was
expressed in the absence of CpxR, indicating that the anomalies result
exclusively from excessive levels of CpxR-P caused by CpxA*. To confirm
this conclusion, a 1.65-kb segment containing cpxA* was
excised from pRS415/RA* with SmaI and
BamHI. The fragment was then treated with DraI (a
site is present 43 bp upstream of the CpxR start codon), to remove the
cpx promoter region, and cloned into
SmaI/BamHI-restricted single-copy plasmid
pEXT20 (7), yielding pEXTA*. The expression of
cpxA* from the tac promoter in pEXTA* is IPTG
(isopropyl--D-thiogalactopyranoside) inducible. Because
cpxA* is dominant over the wild-type allele (23),
we transformed pEXTA* into strains ECL3501 (cpxRA) and BW21355 (cpxR+A+ parent of ECL3501)
(Table 1) to test whether cpxA* phenotypes strictly depend
on the presence of CpxR. The resistance of the transformants to
amikacin, CuCl2, and pH 9 in the presence of 1 mM IPTG was
scored under conditions described in Table 2. All three CpxA*
phenotypes were observed only in BW21355 (data not shown).
The pleiotropy of cpxA* mutations is reminiscent of the
envZ* mutations, whose manifestations are also dependent on
the cognate response regulator (31). In principle, a
structural alteration of CpxA may entail a specificity change that
leads to an aberrant phenotype by phosphorylating or dephosphorylating
a noncognate response regulator. Cross-phosphorylation between
different two-component systems has been observed and was postulated to
integrate adaptive responses by separate control networks. For
instance, PhoB, the cognate response regulator of the
Pi-sensing PhoR, can also be phosphorylated by the
catabolite sensor CreC (36). OmpR, the cognate response
regulator of the osmo-sensing EnvZ, can be cross-phosphorylated by an
as-yet-unidentified histidine sensor kinase (10). However, it should be emphasized that in both cases the cross-phosphorylation of
the noncognate response regulators was observed in mutant backgrounds that lack the cognate sensor kinase. In a wild-type situation, such
cross-phosphorylation may not have a chance to occur to a significant
extent. Theoretically, cross-talk at the level of dephosphorylation of
response regulators by noncognate sensor kinases may also occur, but to
the best of our knowledge, no such phenomenon has yet been reported. In
view of the above discussion, it is not surprising that the CpxA*
phenotypes did not apparently involve cross-phosphorylation of
noncognate response regulators.
It should be recognized, however, that the excessively high
levels of CpxR-P in CpxA* mutants might recruit nonphysiological target operons whose promoter regions possess a sequence(s) that resembles the recognition consensus for CpxR-P. The occurrence of
such "cross-regulation" may contribute to the complexity of CpxA*
phenotypes and cannot be excluded by the approach of this study.
| |
ACKNOWLEDGMENTS |
P.D.W. is a postdoctoral D. Collen Fellow of the Belgian American
Educational Foundation. This work was financed by Public Health Service
grants GM40993 and GM39693 from the National Institute of General
Medical Sciences.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1925. Fax: (617)
738-7446. E-mail: elin{at}hms.harvard.edu.
| |
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Journal of Bacteriology, March 2000, p. 1423-1426, Vol. 182, No. 5
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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