Laboratory of Molecular Biology, National
Cancer Institute, Bethesda, Maryland 20892-4255
 |
INTRODUCTION |
Proteolysis of specific proteins
under particular conditions provides an important mechanism for
regulation of gene expression in all organisms (7, 19). An
important component of understanding the role of proteolysis in
regulatory cascades is understanding how particular substrates are
selected, and in particular, how they are selected under certain
environmental conditions and not others. In principle, regulated
degradation could reflect regulation of protease synthesis or activity
or regulation of substrate availability/susceptibility. For instance,
the caspases (or ICE proteases) implicated in eukaryotic cell death
appear to be activated (by a protein cleavage) at an early step in the
commitment to cell death (6). In prokaryotes, Bacillus
subtilis proteases involved in activation of developmental sigma
factors are made as part of a developmental cycle and therefore appear
and become active only under appropriate conditions (15). A
number of bacteriophages inactivate cellular proteases during infection. Bacteriophage T4 synthesizes an inhibitor of the Lon protease, PinA (13, 30, 31), and lambda RexB stabilizes lambda O protein, protecting it from degradation, probably by a general
inactivation of Clp proteases (5, 27). Lambda cIII appears
to inhibit the FtsH (HflB) protease, leading to stabilization of both
lambda cII and the heat shock sigma factor RpoH (11). The
most general case for selective modification of substrates for
degradation is the use, in eukaryotic cells, of ubiquitination to mark
proteins destined for rapid turnover (12, 14). For instance,
degradation of cyclins at given points in the cell cycle is due to
regulated ubiquitination; presumably, cell cycle signals are fed
through the ubiquitination machinery in ways that are still unclear
(24). No posttranslational tagging mechanism for degradation
equivalent to ubiquitination in prokaryotes has been described; the
only known tagging system is a cotranslational mechanism for degrading
certain protein fragments (17).
One of the most striking examples of regulated proteolysis in
Escherichia coli is degradation of the stationary-phase
sigma factor RpoS. RpoS is responsible for the transcription of a
variety of genes expressed after cells enter stationary phase and
during some sorts of starvation and stress (10). The
promoter recognition for the holoenzyme containing RpoS is similar to
that for the holoenzyme containing the major sigma factor of E. coli, RpoD, and some promoters can be transcribed by both
holoenzymes (34). Unlike RpoD-dependent promoters, however,
the activity of RpoS-dependent promoters appears to be regulated in
large part by changes in the accumulation of the RpoS protein, a result
of changes in rpoS transcription, translation, and, most
dramatically, degradation (18, 33). Under exponential growth
conditions at 37°C, RpoS is quite unstable, with a half-life of less
than 2 min. However, when cells enter stationary phase or under some
stress conditions, RpoS becomes stable, with a half-life of greater
than 30 min. The protease responsible for the rapid in vivo degradation
of RpoS has been shown to be the cytoplasmic ATP-dependent ClpXP protease (29).
Recently, a protein necessary for this rapid degradation of RpoS and an
excellent candidate to mediate environmental signalling has been
identified, named by various groups RssB or SprE (in E. coli), and MviA (in Salmonella typhimurium) (2,
23, 25). Null mutations in the rssB gene lead to RpoS
stabilization; overproduction of the protein leads to rapid degradation
of RpoS even in stationary phase (23, 25). The RssB N
terminus has homology to the family of response regulators, and
therefore its activity would be predicted to be subject to
phosphorylation, as these response regulators generally are. Regulation
by RssB of RpoS accumulation is dependent upon ClpXP; RpoS accumulates
in clpX and clpP mutants even when RssB is
overproduced (25). We began the work described here to ask
if RssB-mediated regulation of RpoS degradation reflected regulation of
the protease or of the substrate. We find that RssB acts in a
substrate-specific fashion and modulates RpoS activity as well as its
degradation.
| |
MATERIALS AND METHODS |
Bacterial strains.
For the protein turnover experiments, an
isogenic set of derivatives of MG1655 (1) was constructed by
P1 transduction, carrying clpP::Cat
(20) or rssB::Tet (23)
insertion mutations. Strains were lysogenized with 
cI857
and, in some cases, transformed with pUM-E, which overproduces RssB
(3) (received from T. Silhavy and L. Pratt). Note that pUM-E
also encodes a number of other proteins, including RssA (unknown
function, gene in operon with the rssB gene) and Tgs
(transient glycine starvation) (3, 23). Strains for assaying
RpoS activity were derived by P1 transduction or transformation with
the pUM-E plasmid from DDS1340, and all contain the
dsrB::lacZ fusion (32).
Protein turnover experiments.
Cells carrying the
heat-inducible cI857 prophage were grown in Luria-Bertani
(LB) broth with 50 µg of ampicillin per ml at 30°C to an optical
density at 600 nm (OD600) of 0.3 to 0.5 for logarithmic
growth and to an OD600 of 2.0 for stationary-phase samples,
transferred to 42°C for 8 min, and then transferred to 37°C and
treated with 100 µg of spectinomycin per ml. Samples were removed at
appropriate intervals and precipitated with 5% trichloroacetic acid in
the cold. Precipitated pellets were washed with 80% acetone and
resuspended in sodium dodecyl sulfate gel-loading buffer (Novex).
Samples were normalized by optical density, electrophoresed on 12%
sodium dodecyl sulfate gels, blotted to 0.2-µm-pore-size nitrocellulose, and probed with either anti-lambda O antibody (gift
from R. McMacken) or anti-RpoS monoclonal antibody (gift from R. Burgess), and Western blots were developed with the ECL system
(Amersham). Films were scanned with an Eagle Eye II scanner and
normalized to the density of the band after 8 min of induction, and the
half-life was estimated.
Quantitative Western blots.
To estimate the amounts of RpoS
in strains, cell extracts were analyzed by Western blots, using
anti-RpoS monoclonal antibody. Extracts were normalized for cell OD,
and serial dilutions were used for gel electrophoresis as described
above. Estimates of RpoS amounts were extrapolated from a series of
sample dilutions that showed a linear response on the film after
scanning with the Eagle Eye II scanner.
-Galactosidase assays.
Strains were grown in M63 salts
(21) with 0.2% glucose and vitamin B1
(0.0001%) at 32°C to an OD600 of approximately 0.45. One-tenth-milliliter samples of cells were permeabilized with 0.05 ml
of permeabilization buffer (100 mM Tris [pH 7.8], 32 mM NaPO4, 8 mM dithiothreitol, 8 mM
trans-1,2-diaminocyclohexane-N,N,N',N',tetraacetic acid, 4% Triton X-100, with 0.2 mg of polymyxin B per ml
[28]) in microtiter plate wells for at least 10 min
and assayed by adding 0.05 ml of
o-nitrophenyl--D-galactopyranoside (ONPG)
solution (4 mg of ONPG per ml in M63, 2 mM Na citrate) and measuring
absorption at 420 nM as a function of time in a SpectraMax 250 spectrophotometer. Specific activity was calculated by dividing the
slope of the line over time by the OD600 for the sample. In
other experiments, we find that units of activity calculated in this
manner are about 25-fold lower than Miller units.
| |
RESULTS |
RssB regulation of RpoS degradation is substrate specific.
Schweder and coworkers have shown that the protease ClpXP is present
both in exponential phase and stationary phase, suggesting that
regulation of the availability of the protease is unlikely (29). Therefore, to distinguish between protease-specific
and substrate-specific effects of RssB, we investigated the effect of
the switch from exponential to stationary phase and the effect of
rssB mutants and RssB overproduction on the stability of
another ClpXP substrate, lambda O protein. We have previously shown
that lambda O protein is degraded with a half-life of 1 to 2 min in wild-type cells; the half-life increases to more than 40 min in cells
carrying mutations in either clpX or clpP
(8). In vitro, ClpXP is also able to degrade lambda O
protein (35).
A set of isogenic strains was constructed, all carrying a
cI857 lysogen and a wild-type allele of rpoS,
and varying in the presence or absence of mutations in rssB
or clpP or of a plasmid overexpressing RssB. For each of
these strains, cells were grown in LB broth at 30°C, the temperature
was raised to 42°C for 8 min to induce lambda lytic growth and O
protein synthesis and then lowered to 37°C, and spectinomycin was
added to the culture to inhibit further protein synthesis. Culture
samples were removed before induction, at the start of the
spectinomycin treatment, and at various times after spectinomycin
addition; extracts were compared on Western blots for the presence of
RpoS and lambda O protein.
Figure 1A and B show the results of such
an experiment, with induction of the cI857 prophage when
cells are at an OD600 of 0.3 (during exponential growth).
Figure 1C and D show a similar experiment, with induction of the cells
at an OD600 of around 2.0 (during stationary phase). The
half-lives for lambda O protein and RpoS under each of these conditions
are summarized in Table 1. The band of
lambda O protein is visible after induction of lambda in wild-type
cells during logarithmic growth and disappears rapidly, as expected, in
the presence of spectinomycin (Fig. 1A, lanes 1 to 4). Neither a
mutation in rssB nor overproduction of RssB perturbed lambda
O protein turnover in exponentially growing cells (Fig. 1A, lanes 8 to
10 and 11 to 13). However, as previously seen, lambda O protein was
quite stable in cells with a mutation in clpP (Fig. 1A,
lanes 5 to 7) (8). In the same induced cultures, RpoS showed
the stability pattern previously seen (25); it was unstable
during exponential phase (Fig. 1B, lanes 2 to 4) and stable in
clpP (lanes 5 to 7) or rssB (lanes 8 to 10)
mutants. We could not detect RpoS in cells overproducing RssB (Fig. 1B, lanes 11 to 13). Overproduction of RssB in cells with a mutation in
clpP gave an easily detectable, stable band of RpoS (Fig.
2, lanes 10 to 12; see below), consistent
with the observations of Pratt and Silhavy that clpP is
epistatic to rssB (25) and supporting the idea
that the absence of an RpoS band in cells overproducing RssB reflects
accelerated RpoS degradation. We also noted an induction of RpoS after
the 8-min heat shock, compared to growth at 30°C (Fig. 1B, compare
lanes 1 and 2). This heat shock induction of RpoS has been reported
previously and attributed to decreased turnover (16, 22).
Our results would suggest that this decreased turnover is blocked by
RssB overproduction (Fig. 1B, lanes 11 to 13).
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FIG. 1.
Cells were grown to an OD600 of 0.3 to 0.5 for logarithmic growth (A and B) and to 2.0 for stationary-phase
samples (C and D) in LB broth with 50 µg of ampicillin per ml at
30°C, transferred to 42°C for 8 min, and then transferred to 37°C
and treated with 100 µg of spectinomycin per ml. Samples were removed
before the heat treatment (lanes 1) for the wild-type strain, after 8 min at 42°C (lanes 2, 5, 8, and 11), after 10 min of chase with
spectinomycin (lanes 3, 6, 9, and 12), and after 30 min of chase with
spectinomycin (lanes 4, 7, 10, and 13) and treated as described in
Materials and Methods. (A and C) Probed with anti-lambda O antibody; (B
and D) probed with anti-RpoS monoclonal antibody. All strains were
derivatives of MG1655; all carried a cI857 prophage. In
addition, they carried the following: YN186, wild-type (lanes 1 to 4); YN187 clpP1::Cat (lanes 5 to 7); YN188
rssB::Tet (23) (lanes 8 to 10); YN189,
plasmid pUM-E, overproducing RssB (3) (lanes 11 to 13).
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|
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FIG. 2.
RpoS levels in the absence of ClpXP. Strains grown for
assays (see Table 2) were sampled for RpoS levels. An equal amount of
cell extract was loaded in the first lane for each strain (lanes 1, 4, 7, 10, and 13). The amounts of RpoS in the second and third lanes for
each strain are twofold dilutions of the previous lane. Gels were
processed as described in Materials and Methods.
|
|
During stationary phase, RpoS was somewhat more stable, as expected
(Fig. 1D, lanes 2 to 4; Table 1). Overproduction of RssB again led to
barely detectable RpoS even during stationary phase (Fig. 1D, lanes 11 to 13). In stationary-phase cells, it was more difficult to detect
lambda O protein by Western blot unless the cells carried a
clpP mutation (Fig. 1C). This is consistent with our
previous observations that accumulation of lambda O protein after
induction of a prophage is primarily regulated by the half-life of the
protein (8) and with the observations that rssB
mutations do not interfere with rapid lambda O protein degradation in
exponentially growing cells. Presumably, the 8-min heat induction of
the lambda prophage leads to less lambda protein synthesis in cells in
stationary phase. To confirm that lambda O protein turnover was not
perturbed by rssB during stationary phase, we carried out a
parallel series of experiments with cells carrying a multicopy plasmid
(pRLM71) (26) expressing lambda O protein from the
pL promoter. In those cells, lambda O protein
was degraded in stationary-phase cells with a half-life of 3 min, and
turnover was not significantly changed in an rssB mutant or
when RssB was overproduced (data not shown). Therefore, these results
demonstrate that RssB and stationary phase change RpoS stability
without perturbing degradation of another ClpXP substrate, suggesting
that RssB acts on RpoS, not on the protease.
RssB regulates RpoS activity.
As seen above (Fig. 1B and D),
RpoS accumulates to significant extents in a clpP mutant
host, independent of the growth phase of the cells and therefore
presumably regardless of the presence or absence of RssB. We confirmed
this in a clpP mutant host by overproducing RssB; RpoS still
accumulates (Fig. 2, lanes 10 to 12). This allowed us to ask if RssB
modifies RpoS activity in the absence of degradation. RpoS activity was
monitored with a dsrB::lac fusion that
we have previously shown to be fully dependent on RpoS (32).
As shown in Table 2 for an isogenic set
of clpP::cat hosts, rssB mutant cells
have four to fivefold-higher RpoS activities than cells overproducing
RssB. Quantitative Western blots demonstrate that the amounts of RpoS
protein do not change more than twofold between these strains (Fig. 2).
Thus, no new proteolytic system attacks RpoS, but RssB can
down-regulate RpoS activity, an effect which is not easy to assess when
RpoS is rapidly degraded (in the presence of ClpXP).
| |
DISCUSSION |
The experiments described here strongly suggest that RssB
regulates RpoS degradation by a substrate-specific interaction. This
interaction also interferes with RpoS activity when the ClpXP protease
is not present. Given the similarity of RssB to response regulators, it
seems likely that RssB may be sensitive to environmental signalling via
reversible phosphorylation. Because increased RssB (made from the
plasmid in our experiments) is sufficient to increase RssB activity
even in stationary phase, either phosphorylation is not essential or it
can be provided in the absence of the normal signals. If RssB acts
directly on RpoS, it could in theory form a complex with RpoS, either
modifying RpoS to allow degradation or the complex itself may render
either RpoS itself or both RssB and RpoS subject to degradation.
Regions within RpoS necessary for rapid degradation by ClpXP have been
identified, primarily by examining the behavior of RpoS-LacZ fusion
proteins. Those fusions carrying the N terminus of RpoS up to amino
acid 160 did not show evidence of degradation; those carrying up to
amino acid 180 were degraded in a manner dependent upon growth phase,
ClpXP, and therefore presumably RssB (23, 29). This region
is just downstream of the region of sigma known to contact the 
10
region of target promoters and differs at relatively few positions from
the stable RpoD sigma factor. Whether this region includes recognition
sequences for RssB and/or recognition regions for the ClpXP protease,
only made accessible in the presence of RssB, remains to be seen. We
would predict that the same changes or complex that allows degradation also interferes with RpoS activity. Possibly both reflect a decrease in
either core or DNA binding; this region would be predicted to be
involved in DNA binding. It is interesting that RpoH, the heat shock
sigma factor of E. coli, is also subject to regulated degradation, that this degradation depends on a region of RpoH not far
from the one implicated in RpoS degradation, that accessory factors
(DnaJ, DnaK, and GrpE) are implicated in accessibility to degradation,
and finally, that these factors also participate in regulating activity
of RpoH (reviewed in reference 9).
The regulation of RpoS activity as well as degradation by
RssB-dependent phosphorylation would not have been detectable without the ability to specifically block degradation independently by mutations in the protease. It is not yet clear whether this modulation of RpoS activity has a physiological role, since normally the protease
will be available. However, it seems efficient to couple protein
behavior and susceptibility to degradation in this fashion. The
additional sensitivity to degradation provides both irreversibility and
multiplication of the effect of inactivation on protein
activity.
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Abstract
Introduction
