Previous Article | Next Article 
Journal of Bacteriology, September 1998, p. 4564-4570, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Promoter Substitution and Deletion Analysis of
Upstream Region Required for rpoS Translational
Regulation
Christofer
Cunning,
Larissa
Brown,
and
Thomas
Elliott*
Department of Microbiology and Immunology,
West Virginia University Health Sciences Center, Morgantown, West
Virginia 26506
Received 2 April 1998/Accepted 30 June 1998
 |
ABSTRACT |
The RpoS sigma factor of enteric bacteria is required for the
increased expression of a number of genes that are induced during nutrient limitation and growth into stationary phase and in response to
high osmolarity. RpoS is also a virulence factor for several pathogenic
species, including Salmonella typhimurium. The activity of
RpoS is regulated at both the level of synthesis and protein turnover.
Here we investigate the posttranscriptional control of RpoS synthesis
by using rpoS-lac protein and operon fusions. Substitution
of the native rpoS promoters with the tac or
lac UV5 promoters allowed essentially normal regulation
after growth into stationary phase in rich medium or after osmotic
challenge. Regulation of these fusions required the function of
hfq, encoding the RNA-binding protein host factor I (HF-I).
Short deletions from the 5' end of the rpoS transcript did
not affect regulation very much; however, a larger deletion mutation
that still retains 220 bp upstream of the rpoS ATG codon,
including a proposed antisense element inhibitory for rpoS
translation, was no longer regulated by HF-I. Several models for
regulation of rpoS expression by HF-I are discussed.
 |
INTRODUCTION |
In the enteric bacteria, including
Escherichia coli and Salmonella typhimurium,
the rpoS gene encodes an accessory sigma (specificity) factor for RNA polymerase (also called
S or
38 [36, 49]). RpoS is required for the
transcription of many genes expressed during the onset of stationary
phase. RpoS-dependent adaptations to nutrient limitation and starvation
identified so far in E. coli include not only shifts in
metabolic pathways but also resistance mechanisms protective against
life-threatening stresses, such as high osmolarity, low pH, heat shock,
elevated H2O2, and UV light (reviewed in
references 19 to 21 and
29). RpoS is also a virulence factor for
S. typhimurium (15) and other enteric
bacteria.
RpoS abundance can be increased in exponential-phase cells by a variety
of induction treatments, including osmotic challenge (22, 27,
34 [reviewed in reference 21]). High
levels of RpoS are also seen in cells grown to stationary phase in rich medium (reviewed in reference 19). It has been
demonstrated that RpoS abundance is increased by provoking an increase
in the level of the alarmone ppGpp (18), and this may
explain why so many treatments which induce RpoS also decrease the
growth rate, at least transiently. For osmotic challenge and
stationary phase, control of RpoS occurs both at the level of
synthesis and by regulated proteolysis. Genetic analysis of RpoS
regulation revealed a requirement for the energy-dependent ClpXP
protease (41), which promotes RpoS turnover with the help of
other factors (4, 32, 38). Both clpXP mutants and
hns mutants lacking the abundant DNA-binding protein H-NS
have an increased level of RpoS during the exponential phase (2,
53).
Comparative studies with rpoS-lac protein and operon
fusions have shown that control of RpoS synthesis occurs
mainly at a posttranscriptional level (27). Host factor
I (HF-I) is an RNA-binding protein that was discovered through
its role in the replication of Q
, an RNA bacteriophage that
infects E. coli (9, 16, 17, 23, 24,
42). The function of HF-I in uninfected cells has been
unknown, but hfq mutants are quite pleiotropic
(35, 51, 52). In recent work, it has been shown that
S. typhimurium and E. coli hfq mutants
lacking HF-I have substantially reduced expression of
rpoS (7, 33). The defect in rpoS
expression is posttranscriptional.
Some additional insight has been gained by analysis of
mutations that restore expression of rpoS in
hfq mutants (8; unpublished work cited in
reference 33). Most of these mutations disrupt a
predicted secondary structure that would sequester the rpoS ribosome binding site (RBS) (8). One attractive model is
that control of rpoS synthesis at the translational
level involves regulation of ribosome access by this inhibitory RNA
secondary structure. The RNA-binding protein HF-I may be
directly involved in relieving this inhibition, for example, as an RNA
chaperone which promotes equilibration between different RNA secondary
structures. However, other models of HF-I action are possible.
In this work, we tested two simple predictions of the translational
control model for rpoS. Two different promoters were
substituted for the native rpoS promoters: translational
regulation was retained in these constructs and was still dependent on
HF-I function. However, deletion analysis showed a requirement for
sequences well upstream of the proposed secondary structure, suggesting that interaction of HF-I with this structure is probably not sufficient for regulation.
 |
MATERIALS AND METHODS |
Bacterial strains and construction.
The strains used in this
study are derived from the wild-type S. typhimurium
strain LT-2 (originally obtained from J. Roth). S. typhimurium does not carry the lac operon. The LT-2
derivatives used here carry various lac fusions to the
rpoS gene of E. coli and the
hfq-1::Mud-Cam insertion (7), where
indicated, but no other mutations with respect to the parental LT-2
strain. Note that some strains of LT-2 have been shown to carry a
mutation in rpoS (a change of ATG
TTG in the
rpoS initiation codon [3]) as well as a
mutation inactivating mviA (the homolog of the E. coli gene rssB or sprE, which controls
turnover of RpoS [4]). The LT-2 strain used here
carries a mutation mapping in the mviA region that
stabilizes RpoS (data not shown). The lac fusions used in
this study do not encode the segment of the RpoS protein required for
turnover (41), and the hybrid RpoS-LacZ protein produced is
stable during exponential growth in minimal glucose medium
(7).
The high-frequency generalized transducing bacteriophage P22
mutant HT105/1 int-201 (39) was used for
transduction in S. typhimurium by standard methods
(12). Fusions of DNA fragments derived from the
E. coli rpoS gene to lac were
constructed as described below; these lac fusions were
transferred to the chromosome of an E. coli recD mutant
by linear transformation as described previously (14). P22
phage lysates were grown in E. coli (13, 14)
and used to transduce the fusions into the S. typhimurium chromosome. Each resulting strain carries a
lac fusion in single copy as an insertion of a
Kanr promoter-lac fragment in the put
operon.
Media and growth conditions.
Bacteria were grown at 37°C
in Luria-Bertani (LB) medium (43) or in minimal MOPS
(morpholinepropanesulfonic acid) medium (37), modified as
described in reference 6, with 0.2% glucose as the
carbon and energy source. For experiments employing continuous growth
in high-osmolarity medium, a modified version of the basal medium of
reference 25 was used. The modified medium contained 7 g of nutrient broth and 1 g of yeast extract (Difco) per
liter of NCE minimal salts medium (per reference 5,
but without MgSO4). This medium also contained 0.2%
glycerol, and where indicated, sucrose was added at 15%. Plates were
prepared with nutrient agar (Difco) with 5 g of NaCl per liter.
Antibiotics were added to the following final concentrations: sodium
ampicillin, 100 µg/ml; chloramphenicol, 20 µg/ml; kanamycin
sulfate, 50 µg/ml; and tetracycline hydrochloride, 20 µg/ml.
Construction of lac fusions.
The system we used
has been described previously (14). Fusions were made in the
pRS plasmid series of Simons et al. (44). Full-length
rpoS-lac fusions have been described previously
(7). Construct A was constructed by digesting pMMkatF2
(36) with ClaI and EagI, filling in
the ends with the Klenow fragment of DNA polymerase I, and cloning the
1.6-kb fragment into pRS552 that had been digested with
EcoRI and BamHI and filled in. To make construct
B, an rpoS-lac operon fusion, the same 1.6-kb
ClaI-EagI fragment of pMMkatF2 was filled in and
inserted into the SmaI site of pRS415. The fragment was
subsequently excised by digestion at the flanking EcoRI and
BamHI sites and inserted into pTE583 (7). These
parental lac protein and operon fusions to rpoS extend from the ClaI site upstream of nlpD to the
EagI site at codon 73 of the rpoS gene. The
operon fusion version includes an RNase III site directly upstream of
the lacZ RBS (28).
Construct C was made from construct A by deleting the
EcoRI-
KpnI fragment including the promoters
serving
rpoS; construct
D is similar, except that it is an
operon fusion and contains
extra nucleotides (specifying a
SacI site and a
KpnI site) at
the deletion joint
upstream of the
rpoS sequences. Construct E
is a fusion to
codon 8 of
rpoS (GTT CA
G GAT CCG). (Sequences
that
are not derived from
nlpD or
rpoS are
underlined here and below.)
This fusion joint was constructed by PCR.
The
tac promoter in construct F was generated by PCR to
create the junction
GAATT CTTGA CAATT AATCA TCGAC TAGTA TAATG
TATTT GGGTG AACAG AGTGC TAACA AAATG TTG. The
tac promoter in construct
G was derived from pTM30
(
31), yielding the
tac promoter and
lac operator followed by the junction sequence
AGGAG
TGTGA AATGC TGCAGGATCC GATAT CAAGC TTGGT
ACCAA CAGCA
AGCAC. This construct includes an RBS and ATG initiation
codon. The
BamHI site in this construct (in boldface) was filled
in
with DNA polymerase to produce the frameshifted version, construct
H. The
lac UV5 promoter in construct K was derived from pRS476
(
44) to create the junction
AGGAA ACAGG ATCCG ATATC
AAGCT TGGTA
CCAAC AGCAA GCAC. This version of the
lac UV5 promoter is not
equipped with an RBS. The
deletion derivatives I and L were made
from constructs G and K by
substitution of a PCR-generated fragment
which placed a
KpnI
site adjacent to bp 1 of the
rpoS sequence
numbered
according to reference
8 and below (the
deletion joint
lies 221 bp upstream of the
rpoS ATG codon).
Constructs J and
M are similar, but the deletion joint is 114 bp
upstream of the
rpoS ATG codon. This deletion removes
material up to the boundary
of the inhibitory secondary structure
referred to above, which
was described in detail previously
(
8). For deletions I and
J, the correct reading frame was
retained, so that ribosomes which
initiate translation at the RBS just
downstream of P
tac will terminate at the native
nlpD stop codon.
Construct N was made by substitution of an
XhoI-
EagI fragment from construct K, containing
the
lac UV5 promoter and
rpoS sequences,
into the
operon fusion vector pTE583. Construct O was made from
a plasmid that
is identical to pRS475 (
44), except that it contains
a
Kan
r marker from pUC4K upstream of the
lac UV5
promoter. Construct
P was made by substitution of an
XhoI-
BamHI fragment from construct
G, containing
the P
tac promoter, into the operon fusion
vector
pTE583. All PCR-derived fragments were sequenced completely;
all
constructs were sequenced to verify the predicted junctions.
-Galactosidase assays.
Cells were centrifuged and
resuspended in Z-buffer (100 mM NaPO4 [pH 7.0], 10 mM
KCl, 1 mM MgSO4) and then permeabilized by treatment with
sodium dodecyl sulfate and chloroform (7). Assays were
performed in Z-buffer containing 50 mM
-mercaptoethanol by a kinetic
method using a plate reader (Molecular Dynamics). Activities (change in
optical density at 420 nm [
OD420] per min) are
normalized to actual cell density (OD650) and were always compared to those of appropriate controls assayed at the same time. The
results shown are from a single experiment; each experiment was
repeated several times with similar results.
 |
RESULTS |
Deletion analysis of sequences required for translational control
of RpoS.
If HF-I or stimuli such as osmotic challenge
regulate RpoS synthesis at the translational level, then we expect
that the native rpoS promoters can be replaced by another
promoter without loss of regulation. A more restrictive
translational control model (discussed above) predicts that HF-I
interacts only with the segment of mRNA containing a putative secondary
structure that inhibits translation. The ultimate result of this
interaction is increased availability of the rpoS RBS for
binding to ribosomes. According to this model, transcript sequences
upstream of the secondary structure should not be required for HF-I
regulation of RpoS.
We constructed
rpoS-lac fusions containing deletions of the
native promoters and substituted new promoters to test these ideas.
In
the first set of experiments, expression of

-galactosidase
was
measured in cultures grown overnight to saturation in LB medium.
The
rpoS gene is highly expressed under these conditions.
Construct
A (Fig.
1) is the parental
lac fusion; it includes both of the
identified promoters
that are used to initiate transcripts of
rpoS. One of
the native promoters serves both the upstream
nlpD gene and
rpoS and is referred to here as
P
nlpD; the second
promoter lies within the
nlpD gene and is referred to as P
rpoS (
26,
48). The
lac fusion in construct A is a
protein or translational
fusion, and it is identical to the fusion used
in our previous
studies (
7,
8). Construct B is the same,
except that it
is an operon or transcriptional fusion. As shown
previously, expression
of the
rpoS-lac (protein) construct A
was stimulated in wild-type
cells (containing a functional
hfq gene) compared to that in an
otherwise isogenic strain
that carries a Mud-Cam insertion mutation
at codon 68 of
hfq
(
7). In contrast, the
rpoS-lac operon fusion
(construct B) containing the same sequences was expressed at a
similar
level in the presence or absence of HF-I.

View larger version (33K):
[in this window]
[in a new window]
|
FIG. 1.
rpoS-lac fusions. The top line shows a
restriction map of the nlpD and rpoS genes of
E. coli, including the restriction sites used to make
lac fusions in this study. The positions of two promoters
that serve rpoS are also shown by bent arrows. Each line
below the top one represents a different fusion; these are referred to
in the text as rpoS-lac fusions and are labeled (A to P).
The extent of the nlpD-rpoS sequence present in each fusion
is shown, together with the identity of the promoter substitution where
appropriate: Ptac or Plac
UV5. The nature of the lac fusion (protein [pr] or
operon [op]) is also indicated. ATG start codons (solid squares) and
their cognate stop codons (arrowheads) are shown. One-letter
abbreviations are used for restriction sites (the full name of each
site is on the top line). Additional sequence and construction details
are given in Materials and Methods. The lac region is not
drawn to scale. Expression of -galactosidase was measured in
overnight LB cultures of S. typhimurium strains bearing
each fusion in single copy in the bacterial chromosome and was compared
to the activity seen in an otherwise isogenic hfq mutant.
Values are in arbitrary units, normalized to that of construct A in a
wild-type background (set as 100). The ratio of the enzyme activity in
the wild type divided by that in the hfq mutant is also
given. N.D., not determined.
|
|
A
KpnI site 73 bp downstream of the
P
rpoS transcription initiation site (Fig.
1) was used to delete the native P
nlpD and
P
rpoS promoters. Loss of DNA upstream of this
site
in constructs C and D eliminated most
lac expression,
confirming
previous work by other groups showing that most
transcription
of
rpoS initiates upstream of this point. Two
different promoters
were substituted. The promoters used were the
tac and
lac UV5
promoters; the
sequences used are detailed in Materials and Methods.
Construct F contains the
tac promoter; this version of the
tac promoter does not include the
lac operator.
The promoter is placed
so that the resulting transcript should be
identical to the transcript
initiated from the
P
rpoS promoter that normally serves
rpoS. HF-I regulation of
rpoS expression from
this transcript
was the same as that for native
rpoS.
Although the absolute levels
of expression were elevated in both
wild-type and
hfq mutant hosts,
the stimulation ratio for
HF-I was similar to that seen for construct
A. Thus, as predicted by
all translational models, the native
rpoS promoters are not
required for regulation by HF-I. Control
experiments with operon
fusions to both the P
tac and
P
lac UV5 promoters (constructs N, O, and P)
showed no
HF-I regulation.
The transcript produced from construct F does not contain an identified
RBS to allow translation of the
nlpD gene fragment
upstream
of
rpoS. Constructs G and H differ from construct F in
two
respects: there is a further deletion of 73 bp extending to
the
KpnI site, and there is an added 5' leader sequence
which
includes a strong RBS. In construct G, translation initiated
at
the upstream RBS will terminate at the natural
nlpD
stop codon,
whereas construct H contains a frameshift in the leader
sequence
leading to termination of translation 346 nucleotides (nt)
upstream
of the natural stop (83 nt downstream of the
KpnI
site). Sites
of translation initiation and termination are indicated in
the
figure by solid squares and arrowheads, respectively. Both
constructs
G and H show substantial HF-I stimulation, suggesting that
the
small 73-bp deletion does not remove important sequences and also
that translation to the
nlpD stop codon does not affect
regulation
significantly. This result is surprising, given that the
nlpD stop codon lies between the two stems of the proposed
regulatory
secondary structure, and ribosomes translating
nlpD would be expected
to disrupt the structure at least
transiently. A similar construct
(K) with the
lac UV5
promoter placed at the
KpnI site also shows
normal
regulation by HF-I.
The effect of deletion of additional DNA from the region upstream of
rpoS was also investigated. Constructs I and L have
deletions
of DNA extending to a position 271 bp downstream of the
KpnI site
and 221 bp upstream of the
rpoS ATG
codon. Part of the sequence
of the
rpoS region which is
retained in these deletions is shown
in Fig.
2, including the postulated secondary
structure. The boundary
of the deletion in constructs I and L lies
about 115 bp upstream
of the postulated secondary structure (Fig.
2).
Therefore, if
HF-I interaction with the structure is all that is
needed, we
would predict that this deletion should be regulated
normally.
Instead, the fusion containing the
tac promoter
(construct I)
is partially defective in the HF-I response (Fig.
1), and
the
fusion to the
lac UV5 promoter (construct L) has nearly
identical
expression in the presence and absence of HF-I. Further
deletions
(to bp 111 in Fig.
2; constructs J and M in Fig.
1) extending
to a point immediately upstream of the secondary structure element
do
not change this result. Finally, the requirement for sequences
downstream of the
rpoS ATG codon was also tested. Much of
the
HF-I control was retained by a
lac fusion to codon 8 of
rpoS (construct
E), suggesting that the relevant target for
regulation lies upstream
of codon 8.

View larger version (17K):
[in this window]
[in a new window]
|
FIG. 2.
Model of the antisense-RBS structure near the
rpoS start codon. The structure presented here is that
suggested on the basis of genetic analysis (8), and the
previous numbering scheme is retained in this figure. Arrows point to
the nucleotides altered in the compensatory mutations which support the
structure (C126G and G206C, as well as a second pair not shown). The U
at nt 111 (also marked with an arrow and denoted 2) corresponds to
the first rpoS-specific nucleotide in constructs J and M;
material upstream of this point is deleted in these fusions. Not shown
are the additional 111 nt retained by 1 (constructs I and L of Fig.
1) or the additional 400 nt extending to the 5' end of the
PrpoS transcript. Two stems which pair the
nucleotides connecting the antisense and RBS elements have also been
added to this model. S.D., Shine-Dalgarno sequence.
|
|
Secondary structure in rpoS transcripts from deletion
constructs.
The lack of response to HF-I by the deletion
constructs, as described above, could have a simple explanation by
analogy to the behavior of RNA II, the primer for ColE1 plasmid
replication. RNA II folds into substantially different structures in
its downstream half, depending on either the presence of upstream
sequences or their sequestration in a complex with the antisense RNA,
RNA I (29a). Thus, if the inhibitory structure did not form
in an rpoS transcript which lacks important upstream
sequences, then a failure to respond to HF-I would be understandable
but not enlightening. As a test of this possibility, we prepared a set
of rpoS-lac fusions from construct L to use the method of
compensatory mutations as previously described (8). These
fusions carry either of two mutations, C126G (SD2) or G206C (SD3), as
well as the double mutant together with a wild-type control. Expression
of rpoS-lac in wild-type and hfq mutant
derivatives of these fusion strains is reported in Table
1. Since the elevated
-galactosidase
activity seen in the single mutants is restored nearly to wild-type
levels in the double mutant (measured in an hfq mutant
background), we conclude that at least the identified RNA secondary
structure forms in transcripts from this construct. None of the
constructs is significantly stimulated by the presence of HF-I, and the
lack of response to HF-I cannot be ascribed to loss of this structural
element.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Expression of rpoS-lac in derivatives of
fusion construct L that carry compensatory mutations in the
rpoS antisense-RBS region
|
|
Osmotic challenge.
We extended the analysis to include osmotic
challenge because it has been demonstrated that HF-I is required for
osmotic control of rpoS translation in E. coli (33). Several deletion constructs were
tested for their response to challenge with high salt. The first
experiment employed an osmotic challenge in which cultures growing in minimal MOPS glucose medium were treated with 0.3 M NaCl.
The wild-type rpoS-lac protein fusion (construct A) showed a
3.5- to 4-fold increase in lac expression over 40 min and
then reached a plateau by 60 min (Fig.
3A). The operon fusion (construct B) was
not induced by this treatment (Fig. 3F). When an hfq
mutation was present, the response of construct A to the osmotic
challenge was partially defective (Fig. 3D). The kinetics of the
response were changed, with an
2-fold increase at 40 min, but
lac expression continued to increase up to at least 90 min.

View larger version (23K):
[in this window]
[in a new window]
|
FIG. 3.
Induction of rpoS-lac after osmotic
challenge. Cultures of S. typhimurium strains carrying
a lac fusion (as identified in Fig. 1 and the text) and an
hfq mutation where indicated were grown in minimal MOPS
glucose medium to an OD600 of 0.4 and then challenged with
0.3 M NaCl. The activity of -galactosidase was determined at 20, 40, 60, and 90 min after challenge. Solid squares, cultures receiving NaCl;
open squares, control cultures. Data are reported as a percentage of
the initial value for each fusion. [pr], lac protein
fusion; [op], operon fusion.
|
|
Construct F, in which the
tac promoter expresses the normal
P
rpoS transcript, showed regulation similar to
that of
wild-type
rpoS-lac (Fig.
3B), and expression peaked
at a level
about threefold higher than that of the untreated control.
The
hfq mutant derivative of this fusion was also
defective. Similar
to the result for native
rpoS-lac, very
little increase in

-galactosidase
was seen at 20 min following the
addition of 0.3 M NaCl. Construct
I contains a deletion of 343 nt of
the P
rpoS transcript
leader, but retains 115 nt
upstream of the proposed secondary
structure. This fusion was defective
in osmotic stimulation of
lac expression (Fig.
3C). The
results are similar to those for
overnight growth in LB medium.
The
tac promoter gives normal regulation
of
rpoS-lac (protein fusion to
lac) but this
requires sequences
well upstream of the proposed secondary
structure. A larger set
of constructs was also examined in a
fixed-time assay (data not
shown). The results confirm the picture
from the kinetic assays.
In particular, since several constructs with
different upstream
sequences but retaining the secondary structure are
all defective
for response to osmotic challenge, this property of the
response
(as illustrated in Fig.
3C) is not due to inhibition by one
particular
leader sequence.
Continuous growth at high osmolarity.
In addition to changing
the osmotic strength of the medium, the osmotic challenge method also
changes the bacterial growth rate (at least transiently). Therefore, we
tested the effect of continuous growth in high-osmolarity medium. These
experiments (modeled on those described in reference
38) were carried out in a phosphate-buffered rich
medium including glycerol; sucrose was the solute used to vary the
osmolarity. Assays for
-galactosidase were performed with cells
sampled at three densities: OD600 of 0.15, OD600 of 0.6, and after growth overnight to stationary
phase. Similar to the observations with E. coli
(38), cells grown at high osmolarity showed an increase in
rpoS-lac expression in exponential phase (OD600
of 0.15 or 0.6), but not in stationary phase (data for
OD600 of 0.6 are shown in Table
2).
Expression from construct A (native
rpoS-lac) was increased

3-fold in high-osmolarity medium during exponential phase. Most
tac or
lac UV5 promoter constructs with
rpoS-lac protein fusions
(E, F, G, K, and L in Fig.
1)
showed an induction ratio of 2-
to 2.5-fold. The exception was
construct J, deleted to just upstream
of the proposed secondary
structure, with an induction ratio of
only about 1.5-fold. In contrast
to the protein fusions, operon
fusions (B, N, O, and P) were not
detectably induced by high osmolarity
(induction ratios of 0.9-1.1).
Surprisingly, the
hfq mutant derivatives
of the wild-type
fusion (construct A) and
tac promoter construct
(F) did not
show a significant defect in the response to high
osmolarity when
tested by this method. These results suggest that
continuous growth in high-osmolarity medium increases
rpoS expression
at a posttranscriptional level, but the
mechanism is independent
of HF-I. This contrasts with the result in
osmotic challenge experiments,
where the
hfq mutant was
partially defective, most severely in
the case in which the native
rpoS promoters were substituted with
the
tac
promoter.
 |
DISCUSSION |
Previous work with S. typhimurium and
E. coli has shown that the rate of synthesis of RpoS
protein is reduced four- to sixfold in hfq mutants which
lack HF-I (7, 33). Comparison of rpoS-lac protein
and operon fusions suggests that the defect in hfq mutants lies at a posttranscriptional step, but it is not established whether HF-I specifically increases the rate of translation initiation or affects mRNA stability instead. The lack of an HF-I requirement for
expression of rpoS-lac operon fusions is not incompatible with a role in mRNA stabilization, since the lacZ RBS of
operon fusions could be insulated from such effects (28).
There is as yet no in vitro system showing HF-I dependence of RpoS
expression, so it is not certain that HF-I acts directly.
Suppressor mutations that decrease the in vivo dependence of
rpoS-lac expression on HF-I function were found to map to a
region encompassing
100 bp near the rpoS ATG codon, and
genetic analysis of compensatory mutations suggests that an RNA
secondary structure formed in this region limits rpoS
expression (i.e., sequestration of the rpoS RBS by an
intramolecular antisense RNA [8]). Perhaps the
simplest model would be that HF-I binds to a site(s) in or near this
region and disrupts the antisense pairing to allow ribosomes access to the rpoS mRNA. But although suppressor mutations
may be suggestive of a potential mechanism, they do not necessarily recapitulate the role of HF-I for wild-type rpoS.
In this work, we tested whether such potential interactions of HF-I and
the rpoS RBS region are sufficient for HF-I function by
making promoter substitutions and by deletion of upstream segments from
rpoS-lac transcripts expressed from the
Ptac and Plac UV5
promoters. The results indicate that nonnative promoters still allow
correct regulation by HF-I during growth into stationary phase and
after osmotic challenge. However, in contrast to the prediction of the
simple model, some sequences required for HF-I regulation of
rpoS lie >100 nt upstream of the rpoS transcript
antisense element. Reapplication of the method of compensatory
mutations indicates that correct folding of the antisense-RBS structure
is likely to be preserved in these deletion variants. Thus, HF-I must
do more than simply melt this duplex.
We favor the idea that HF-I acts in the rpoS system very
similarly to its function in the replication of E. coli
RNA phage Q
. There, HF-I is specifically required for copying of
Q
plus strands (1, 17, 47). Electron microscopy of HF-I
protein bound to Q
RNA reveals doubly looped structures that
indicate specific and simultaneous interaction with two widely
separated internal sites (independently bound by the replicase) which
are then brought together with the RNA 3' end (30). Such
interactions are consistent with the multimeric nature of HF-I protein
(17, 24) and build on Senear and Steitz's demonstration of
site-specific RNA binding activity by HF-I directed at RNA targets from
phages R17 and Q
(42). Mutations that disrupt the
terminal RNA duplex of phage Q
overcome the requirement for HF-I
protein in phage replication (40), which suggests that HF-I
might facilitate melting of the phage RNA 3' end to allow initiation of
replication. This terminal stem of 5 bp is not by any means the most
stable element in the highly structured phage RNA, which suggests a
specific role for HF-I in replication initiation.
Thus, we could imagine that HF-I binds to a specific upstream site in
the rpoS mRNA and from that position interacts with downstream elements to melt the antisense-RBS duplex. This type of model could also accommodate recent genetic studies
showing that the DsrA RNA, a small untranslated RNA of
E. coli (45), acts directly to increase
rpoS expression by pairing with and sequestering the
upstream rpoS antisense element (18a, 46). DsrA-rpoS antisense RNA pairing requires HF-I, and
this suggests that HF-I might act like the Rop (Rom) protein, a
facilitator of the RNA-antisense RNA pairing that controls ColE1
plasmid copy number (50). Or perhaps, as suggested
previously, HF-I is actually a chaperone for RNA and promotes
structural rearrangements (35).
It seems less likely but still possible that HF-I has its primary
effect on rpoS mRNA stability. RNase E cleavage sites are rich in A and U residues (reviewed in reference 10);
these are also favored by HF-I. Another possibility is that bound HF-I
might direct rpoS mRNA down an alternative folding pathway
by preventing the formation of particular duplexes which are targets
for RNase E. Effects on mRNA turnover have been suggested to
explain the autoregulation of hfq gene expression in
E. coli (52). Consistent with this, we have
found that HF-I is much more promiscuous in its RNA binding activity
than predicted from earlier studies (unpublished data). However,
changes in mRNA turnover may also be a secondary consequence of changes
in the rate of initiation of translation (11), so only
a demonstration that rpoS mRNA turnover is not affected by
HF-I would be definitive. Finally, any model must explain why the
benefits of the postulated mRNA stabilization are not evident for an
mRNA having an unfettered, strong RBS or for a variety of other
rpoS single mutants that alter the antisense RNA element and
thereby have become completely independent of HF-I (unpublished data).
In summary, current evidence in this system still allows a number of
possible models including: (i) a looping interaction between HF-I
complexed to rpoS mRNA at far upstream sites and at sites
closer to the AUG codon; (ii) the existence of additional proteins, acting together with HF-I or indirectly under its control, which might bind to upstream sequences; (iii) a subtle influence of
upstream sequences on the ultimate folding pattern of rpoS mRNA in the region near the AUG initiation codon; and (iv) a
requirement for other components, such as small regulatory RNAs,
including DsrA RNA, to observe tight complex formation between HF-I and the rpoS mRNA.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, P.O. Box 9177, WVU Health Sciences Center, Morgantown, WV 26506-9177. Phone: (304) 293-8637. Fax: (304) 293-4667. E-mail: telliott{at}wvu.edu.
Present address: National Institutes of Health, NICHD Laboratory of
Molecular Genetics, Bethesda, MD 20892-2785.
 |
REFERENCES |
| 1.
|
August, J. T.,
A. K. Banerjee,
L. Eoyang,
M. T. Franze de Fernandez,
K. Hori,
C. H. Kuo,
U. Rensing, and L. Shapiro.
1968.
Synthesis of bacteriophage Q RNA.
Cold Spring Harbor Symp. Quant. Biol.
33:73-81[Abstract/Free Full Text].
|
| 2.
|
Barth, M.,
C. Marschall,
A. Muffler,
D. Fischer, and R. Hengge-Aronis.
1995.
Role for the histone-like protein H-NS in growth phase-dependent and osmotic regulation of S and many S-dependent genes in Escherichia coli.
J. Bacteriol.
177:3455-3464[Abstract/Free Full Text].
|
| 3.
|
Bearson, S. M. D.,
W. H. Benjamin, Jr.,
W. E. Swords, and J. W. Foster.
1996.
Acid shock induction of RpoS is mediated by the mouse virulence gene mviA of Salmonella typhimurium.
J. Bacteriol.
178:2572-2579[Abstract/Free Full Text].
|
| 4.
|
Benjamin, W. H., Jr.,
X. Wu, and W. E. Swords.
1996.
The predicted amino acid sequence of the Salmonella typhimurium virulence gene mviA+ strongly indicates that MviA is a regulator protein of a previously unknown S. typhimurium response regulator family.
Infect. Immun.
64:2365-2367[Abstract].
|
| 5.
|
Berkowitz, D.,
J. M. Hushon,
H. J. Whitfield, Jr.,
J. Roth, and B. N. Ames.
1968.
Procedure for identifying nonsense mutations.
J. Bacteriol.
96:215-220[Abstract/Free Full Text].
|
| 6.
|
Bochner, B. R., and B. N. Ames.
1982.
Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography.
J. Biol. Chem.
257:9759-9769[Abstract/Free Full Text].
|
| 7.
|
Brown, L., and T. Elliott.
1996.
Efficient translation of the RpoS sigma factor in Salmonella typhimurium requires host factor I, an RNA-binding protein encoded by the hfq gene.
J. Bacteriol.
178:3763-3770[Abstract/Free Full Text].
|
| 8.
|
Brown, L., and T. Elliott.
1997.
Mutations that increase expression of the rpoS gene and decrease its dependence on hfq function in Salmonella typhimurium.
J. Bacteriol.
179:656-662[Abstract/Free Full Text].
|
| 9.
|
Carmichael, G. G.,
K. Weber,
A. Niveleau, and A. J. Wahba.
1975.
The host factor required for RNA phage Q replication in vitro. Intracellular location, quantitation, and purification by polyadenylate-cellulose chromatography.
J. Biol. Chem.
250:3607-3612[Abstract/Free Full Text].
|
| 10.
|
Cohen, S. N., and K. J. McDowall.
1997.
RNase E: still a wonderfully mysterious enzyme.
Mol. Microbiol.
23:1099-1106[Medline].
|
| 11.
|
Cole, J. R., and M. Nomura.
1986.
Changes in the half-life of ribosomal protein messenger RNA caused by translational repression.
J. Mol. Biol.
188:383-392[Medline].
|
| 12.
|
Davis, R. W.,
D. Botstein, and J. R. Roth.
1980.
Advanced bacterial genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 13.
|
Elliott, T.
1989.
Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.
J. Bacteriol.
171:3948-3960[Abstract/Free Full Text].
|
| 14.
|
Elliott, T.
1992.
A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.
J. Bacteriol.
174:245-253[Abstract/Free Full Text].
|
| 15.
|
Fang, F. C.,
S. J. Libby,
N. A. Buchmeier,
P. C. Loewen,
J. Switala,
J. Harwood, and D. G. Guiney.
1992.
The alternative factor KatF (RpoS) regulates Salmonella virulence.
Proc. Natl. Acad. Sci. USA
89:11978-11982[Abstract/Free Full Text].
|
| 16.
|
Franze de Fernandez, M. T.,
L. Eoyang, and J. T. August.
1968.
Factor fraction required for the synthesis of bacteriophage Q RNA.
Nature
219:588-590[Medline].
|
| 17.
|
Franze de Fernandez, M. T.,
W. S. Hayward, and J. T. August.
1972.
Bacterial proteins required for replication of phage Q ribonucleic acid. Purification and properties of host factor I, a ribonucleic acid-binding protein.
J. Biol. Chem.
247:824-831[Abstract/Free Full Text].
|
| 18.
|
Gentry, D. R.,
V. J. Hernandez,
L. H. Nguyen,
D. B. Jensen, and M. Cashel.
1993.
Synthesis of the stationary-phase sigma factor S is positively regulated by ppGpp.
J. Bacteriol.
175:7982-7989[Abstract/Free Full Text].
|
| 18a.
| Gottesman, S. Personal communication.
|
| 19.
|
Hengge-Aronis, R.
1993.
Survival of hunger and stress: the role of rpoS in early stationary phase gene regulation in E. coli.
Cell
72:165-168[Medline].
|
| 20.
|
Hengge-Aronis, R.
1996.
Regulation of gene expression during entry into stationary phase, p. 1497-1512.
In
F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella. Cellular and molecular biology, 2nd ed., vol. 1. American Society for Microbiology, Washington, D.C.
|
| 21.
|
Hengge-Aronis, R.
1996.
Back to log phase: S as a global regulator in the osmotic control of gene expression in Escherichia coli.
Mol. Microbiol.
21:887-893[Medline].
|
| 22.
|
Hengge-Aronis, R.,
R. Lange,
N. Henneberg, and D. Fischer.
1993.
Osmotic regulation of rpoS-dependent genes in Escherichia coli.
J. Bacteriol.
175:259-265[Abstract/Free Full Text].
|
| 23.
|
Kajitani, M., and A. Ishihama.
1991.
Identification and sequence determination of the host factor gene for bacteriophage Q .
Nucleic Acids Res.
19:1063-1066[Abstract/Free Full Text].
|
| 24.
|
Kajitani, M.,
A. Kato,
A. Wada,
Y. Inokuchi, and A. Ishihama.
1994.
Regulation of the Escherichia coli hfq gene encoding the host factor for phage Q .
J. Bacteriol.
176:531-534[Abstract/Free Full Text].
|
| 25.
|
Kawaji, H.,
T. Mizuno, and S. Mizushima.
1979.
Influence of molecular size and osmolarity of sugars and dextrans on the synthesis of outer membrane proteins O-8 and O-9 of Escherichia coli K-12.
J. Bacteriol.
140:843-847[Abstract/Free Full Text].
|
| 26.
|
Lange, R.,
D. Fischer, and R. Hengge-Aronis.
1995.
Identification of transcriptional start sites and the role of ppGpp in the expression of rpoS, the structural gene for the S subunit of RNA polymerase in Escherichia coli.
J. Bacteriol.
177:4676-4680[Abstract/Free Full Text].
|
| 27.
|
Lange, R., and R. Hengge-Aronis.
1994.
The cellular concentration of the S subunit of RNA polymerase in Escherichia coli is controlled the at levels of transcription, translation and protein stability.
Genes Dev.
8:1600-1612[Abstract/Free Full Text].
|
| 28.
|
Linn, T., and R. St. Pierre.
1990.
Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ.
J. Bacteriol.
172:1077-1084[Abstract/Free Full Text].
|
| 29.
|
Loewen, P. C., and R. Hengge-Aronis.
1994.
The role of the sigma factor S (KatF) in bacterial global regulation.
Annu. Rev. Microbiol.
48:53-80[Medline].
|
| 29a.
|
Masukata, H., and J. Tomizawa.
1986.
Control of primer formation for ColE1 plasmid replication: conformational change of the primer transcript.
Cell
44:125-136[Medline].
|
| 30.
|
Miranda, G.,
D. Schuppli,
I. Barrera,
C. Hausherr,
J. M. Sogo, and H. Weber.
1997.
Recognition of bacteriophage Q plus strand RNA as a template by Q replicase: role of RNA interactions mediated by ribosomal proteins S1 and host factor.
J. Mol. Biol.
267:1089-1103[Medline].
|
| 31.
|
Morrison, T. B., and J. S. Parkinson.
1994.
Liberation of an interaction domain from the phosphotransfer region of CheA, a signaling kinase of Escherichia coli.
Proc. Natl. Acad. Sci. USA
91:5485-5489[Abstract/Free Full Text].
|
| 32.
|
Muffler, A.,
D. Fischer,
S. Altuvia,
G. Storz, and R. Hengge-Aronis.
1996.
The response regulator RssB controls stability of the S subunit of RNA polymerase in Escherichia coli.
EMBO J.
15:1333-1339[Medline].
|
| 33.
|
Muffler, A.,
D. Fischer, and R. Hengge-Aronis.
1996.
The RNA-binding protein HF-I, known as a host factor for phage Q RNA replication, is essential for rpoS translation in Escherichia coli.
Genes Dev.
10:1143-1151[Abstract/Free Full Text].
|
| 34.
|
Muffler, A.,
D. D. Traulsen,
R. Lange, and R. Hengge-Aronis.
1996.
Posttranscriptional osmotic regulation of the S subunit of RNA polymerase in Escherichia coli.
J. Bacteriol.
178:1607-1613[Abstract/Free Full Text].
|
| 35.
|
Muffler, A.,
D. D. Traulsen,
D. Fischer,
R. Lange, and R. Hengge-Aronis.
1997.
The RNA-binding protein HF-I plays a global regulatory role which is largely, but not exclusively, due to its role in expression of the S subunit of RNA polymerase in Escherichia coli.
J. Bacteriol.
179:297-300[Abstract/Free Full Text].
|
| 36.
|
Mulvey, M. R., and P. C. Loewen.
1989.
Nucleotide sequence of katF of Escherichia coli suggests KatF protein is a novel transcription factor.
Nucleic Acids Res.
17:9979-9991[Abstract/Free Full Text].
|
| 37.
|
Neidhardt, F. C.,
P. L. Bloch, and D. F. Smith.
1974.
Culture medium for enterobacteria.
J. Bacteriol.
119:736-747[Abstract/Free Full Text].
|
| 38.
|
Pratt, L. A., and T. J. Silhavy.
1996.
The response regulator SprE controls the stability of RpoS.
Proc. Natl. Acad. Sci. USA
93:2488-2492[Abstract/Free Full Text].
|
| 39.
|
Schmieger, H.
1972.
Phage P22 mutants with increased or decreased transductional abilities.
Mol. Gen. Genet.
119:75-88[Medline].
|
| 40.
|
Schuppli, D.,
G. Miranda,
H.-C. T. Tsui,
M. E. Winkler,
J. M. Sogo, and H. Weber.
1997.
Altered 3'-terminal RNA structure in phage Q adapted to host-factor-less Escherichia coli.
Proc. Natl. Acad. Sci. USA
94:10239-10242[Abstract/Free Full Text].
|
| 41.
|
Schweder, T.,
K.-H. Lee,
O. Lomovskaya, and A. Matin.
1996.
Regulation of Escherichia coli starvation sigma factor ( S) by ClpXP protease.
J. Bacteriol.
178:470-476[Abstract/Free Full Text].
|
| 42.
|
Senear, A. W., and J. A. Steitz.
1976.
Site-specific interaction of Q host factor and ribosomal protein S1 with Q and R17 bacteriophage RNAs.
J. Biol. Chem.
251:1902-1912[Abstract/Free Full Text].
|
| 43.
|
Silhavy, T. J.,
M. L. Berman, and L. W. Enquist.
1984.
Experiments with gene fusions.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 44.
|
Simons, R. W.,
F. Houman, and N. Kleckner.
1987.
Improved single and multicopy lac-based cloning vectors for protein and operon fusions.
Gene
53:85-96[Medline].
|
| 45.
|
Sledjeski, D., and S. Gottesman.
1995.
A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli.
Proc. Natl. Acad. Sci. USA
92:2003-2007[Abstract/Free Full Text].
|
| 46.
|
Sledjeski, D. D.,
A. Gupta, and S. Gottesman.
1996.
The small RNA, DsrA, is essential for the low temperature expression of RpoS during exponential growth in Escherichia coli.
EMBO J.
15:3993-4000[Medline].
|
| 47.
|
Su, Q.,
D. Schuppli,
H.-C. T. Tsui,
M. E. Winkler, and H. Weber.
1997.
Strongly reduced phage Q replication, but normal phage MS2 replication in an Escherichia coli K12 mutant with inactivated Q host factor (hfq) gene.
Virology
227:211-214[Medline].
|
| 48.
|
Takayanagi, Y.,
K. Tanaka, and H. Takahashi.
1994.
Structure of the 5' upstream region and the regulation of the rpoS gene of Escherichia coli.
Mol. Gen. Genet.
243:525-531[Medline].
|
| 49.
|
Tanaka, K.,
Y. Takayanagi,
N. Fujita,
A. Ishihama, and H. Takahashi.
1993.
Heterogeneity of the principal factor in Escherichia coli: the rpoS gene product, 38, is a second principal factor of RNA polymerase in stationary-phase Escherichia coli.
Proc. Natl. Acad. Sci. USA
90:3511-3515[Abstract/Free Full Text].
|
| 50.
|
Tomizawa, J., and T. Som.
1984.
Control of ColE1 plasmid replication: enhancement of binding of RNA I to the primer transcript by the Rom protein.
Cell
38:817-878.
|
| 51.
|
Tsui, H.-C. T.,
H.-C. E. Leung, and M. E. Winkler.
1994.
Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12.
Mol. Microbiol.
13:35-49[Medline].
|
| 52.
|
Tsui, H.-C. T.,
G. Feng, and M. E. Winkler.
1997.
Negative regulation of mutS and mutH repair gene expression by the Hfq and RpoS global regulators of Escherichia coli K-12.
J. Bacteriol.
179:7476-7487[Abstract/Free Full Text].
|
| 53.
|
Yamashino, T.,
C. Ueguchi, and T. Mizuno.
1995.
Quantitative control of the stationary phase-specific sigma factor, S, in Escherichia coli: involvement of the nucleoid protein H-NS.
EMBO J.
14:594-602[Medline].
|
Journal of Bacteriology, September 1998, p. 4564-4570, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Soper, T. J., Woodson, S. A.
(2008). The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA. RNA
14: 1907-1917
[Abstract]
[Full Text]
-
Bhagwat, A. A., Tan, J., Sharma, M., Kothary, M., Low, S., Tall, B. D., Bhagwat, M.
(2006). Functional Heterogeneity of RpoS in Stress Tolerance of Enterohemorrhagic Escherichia coli Strains.. Appl. Environ. Microbiol.
72: 4978-4986
[Abstract]
[Full Text]
-
Jones, A. M., Goodwill, A., Elliott, T.
(2006). Limited Role for the DsrA and RprA Regulatory RNAs in rpoS Regulation in Salmonella enterica.. J. Bacteriol.
188: 5077-5088
[Abstract]
[Full Text]
-
Hirsch, M., Elliott, T.
(2005). Stationary-Phase Regulation of RpoS Translation in Escherichia coli. J. Bacteriol.
187: 7204-7213
[Abstract]
[Full Text]
-
Hirsch, M., Elliott, T.
(2005). Fis Regulates Transcriptional Induction of RpoS in Salmonella enterica. J. Bacteriol.
187: 1568-1580
[Abstract]
[Full Text]
-
Chen, G., Patten, C. L., Schellhorn, H. E.
(2003). Controlled Expression of an rpoS Antisense RNA Can Inhibit RpoS Function in Escherichia coli. Antimicrob. Agents Chemother.
47: 3485-3493
[Abstract]
[Full Text]
-
Worhunsky, D. J., Godek, K., Litsch, S., Schlax, P. J.
(2003). Interactions of the Non-coding RNA DsrA and RpoS mRNA with the 30 S Ribosomal Subunit. J. Biol. Chem.
278: 15815-15824
[Abstract]
[Full Text]
-
Hirsch, M., Elliott, T.
(2002). Role of ppGpp in rpoS Stationary-Phase Regulation in Escherichia coli. J. Bacteriol.
184: 5077-5087
[Abstract]
[Full Text]
-
Hengge-Aronis, R.
(2002). Signal Transduction and Regulatory Mechanisms Involved in Control of the {sigma}S (RpoS) Subunit of RNA Polymerase. Microbiol. Mol. Biol. Rev.
66: 373-395
[Abstract]
[Full Text]
-
Brown, L., Gentry, D., Elliott, T., Cashel, M.
(2002). DksA Affects ppGpp Induction of RpoS at a Translational Level. J. Bacteriol.
184: 4455-4465
[Abstract]
[Full Text]
-
Kojic, M., Aguilar, C., Venturi, V.
(2002). TetR Family Member PsrA Directly Binds the Pseudomonas rpoS and psrA Promoters. J. Bacteriol.
184: 2324-2330
[Abstract]
[Full Text]
-
Sonnleitner, E., Moll, I., Blasi, U.
(2002). Functional replacement of the Escherichia coli hfq gene by the homologue of Pseudomonas aeruginosa. Microbiology
148: 883-891
[Abstract]
[Full Text]
-
Kojic, M., Venturi, V.
(2001). Regulation of rpoS Gene Expression in Pseudomonas: Involvement of a TetR Family Regulator. J. Bacteriol.
183: 3712-3720
[Abstract]
[Full Text]
-
Ueguchi, C., Misonou, N., Mizuno, T.
(2001). Negative Control of rpoS Expression by Phosphoenolpyruvate:Carbohydrate Phosphotransferase System in Escherichia coli. J. Bacteriol.
183: 520-527
[Abstract]
[Full Text]
-
Vytvytska, O., Moll, I., Kaberdin, V. R., von Gabain, A., Bläsi, U.
(2000). Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding. Genes Dev.
14: 1109-1118
[Abstract]
[Full Text]
-
Cunning, C., Elliott, T.
(1999). RpoS Synthesis Is Growth Rate Regulated in Salmonella typhimurium, but Its Turnover Is Not Dependent on Acetyl Phosphate Synthesis or PTS Function. J. Bacteriol.
181: 4853-4862
[Abstract]
[Full Text]