RESEARCH LETTER
The role of the small regulatory RNA GcvB in GcvB/mRNA
posttranscriptional regulation of oppA and dppA in Escherichia coli
Sarah C. Pulvermacher, Lorraine T. Stauffer & George V. Stauffer
Department of Microbiology, University of Iowa, Iowa City, IA, USA
Correspondence: George V. Stauffer,
Department of Microbiology, University of
Iowa, Iowa City, IA, USA 52242. Tel.: 1319
335 7791; fax: 1319 335 9006;
e-mail: [email protected]
Received 5 October 2007; accepted
21 December 2007.
First published online February 2008.
DOI:10.1111/j.1574-6968.2008.01068.x
Editor: Roger Buxton
Keywords
GcvB; sRNA; dppA-lacZ ; oppA-phoA .
Abstract
The gcvB gene encodes two small, nontranslated RNAs that regulate OppA and DppA,
periplasmic binding proteins for the oligopeptide and dipeptide transport systems.
Analysis of the gcvB sequence identified a region of complementarity near the
ribosome-binding sites of dppA and oppA mRNAs. Several changes in gcvB predicted
to reduce complementarity of GcvB with dppA-lacZ and oppA-phoA reduced the ability
of GcvB to repress the target RNAs while other changes had no effect or resulted in
stronger repression of the target mRNAs. Mutations in dppA-lacZ and oppA-phoA that
restored complementarity to GcvB restored the ability of GcvB to repress dppA-lacZ
but not oppA-phoA. Additionally, a change that reduced complementarity of GcvB to
dppA-lacZ reduced GcvB repression of dppA-lacZ with no effect on oppA-phoA. The
results suggest that different regions of GcvB have different roles in regulating dppA and
oppA mRNA, and although pairing between GcvB and dppA mRNA is likely part of the
regulatory mechanism, the results do not support a simple base pairing interaction
between GcvB and its target mRNAs as the complete mechanism of repression.
Introduction
The Escherichia coli gcvTHP (gcv) operon encodes the
glycine cleavage enzyme complex that provides one-carbon
units for cellular methylation reactions (Kikuchi, 1973). The
gcvB gene encodes two small, nontranslated RNAs (sRNAs)
of 130 and 206 nucleotides (nts) (referred to as GcvB) that
negatively regulate OppA and DppA, the oligopeptide and
dipeptide periplasmic binding proteins (Urbanowski et al.,
2000). Both gcv and gcvB are activated by GcvA in response
to glycine and repressed by GcvA1GcvR in the absence of
glycine (Wilson et al., 1993; Stauffer & Stauffer, 1994;
Urbanowski et al., 2000). Because the expression pattern of
gcvB is qualitatively similar to the expression pattern of the
gcv operon, it is hypothesized that GcvB plays a role in an
integrated cellular response to regulate other genes in
conjunction with the glycine cleavage enzyme system.
The E. coli chromosome encodes 50–100 sRNAs that
function in different cellular processes (Wassarman et al.,
1999, 2001; Argaman et al., 2001; Wagner & Flardh, 2002;
Wassarman, 2002; Gottesman, 2004). Many sRNAs posttranscriptionally regulate expression of target genes positively or negatively by forming base pairs (bps) with target
mRNAs (Storz et al., 2004). Often sRNAs, including MicF,
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DicF, OxyS, and Spot42, negatively regulate target mRNAs
by base pairing and preventing the ribosome accessibility
with the target mRNA (Chen et al., 2004). A region in GcvB
was identified complementary to both dppA and oppA
mRNAs near their ribosome-binding sites. The nucleotides
in GcvB predicted to base pair with dppA and oppA mRNAs
were changed and their effects on dppA-lacZ and oppA-phoA
regulation were determined. Compensatory mutations in
either dppA-lacZ or oppA-phoA were then created to restore
base pairing to determine whether regulation was restored.
These results suggest that although complementary pairing
of GcvB and its target sequences is important, GcvBmediated regulation of the DppA and OppA proteins is
more complex than simply pairing of GcvB to the mRNAs.
Materials and methods
Bacterial strains, plasmids, and phage
The E. coli strains and plasmids used are listed in Table 1.
The ldppA-lacZ translational fusion includes 300 bp upstream of the translation initiation site and the first 14
codons of dppA fused in frame with the eighth codon of the
lacZYA genes in phage lgt2 (Urbanowski et al., 2000). The
FEMS Microbiol Lett 281 (2008) 42–50
43
GcvB regulation of OppA and DppA
Table 1. Strains and plasmids
Strain or plasmid
Strain
GS162
GS1144
Plasmid
pGS311
pGS341
pGS571
pGS594
pGS595
pGS596
pGS597
pGS602
pGS603
pGS605
pGS618
pGS619
Relevant genotype
Sources or references
WT
GS162DgcvB<OCMr
This lab
This study
Single-copy vector
Single-copy vector1WT gcvA
Multicopy vector1WT gcvB
Single-copy vector1WT gcvB
pGS594 with a 7 bp change in gcvB Con (gcvBCon–7)
pGS594 with a –TGT– to –CCC– change of 171 to 173 bp in gcvB (gcvB171CCC)
pGS594 with a deletion of –G– at 175 bp in gcvB (gcvB175DG)
pGS594 with a –TGT– to –AAA– change of 176 to 178 bp in gcvB (gcvB176AAA)
pGS594 with a deletion of –GTGTTTGCAAT– from 183 to 193 bp in gcvB (gcvB183D11bp)
pGS594 with a –TT– to –AA– change of 165 and 166 bp in gcvB (gcvB165AA)
pGS594 with a –T– to –C– change at 173 bp in gcvB (gcvB173C)
pGS594 with a –TG– to –CC– change of 171 and 172 bp in gcvB (gcvB171CC)
This lab
Jourdan & Stauffer (1998)
This study
This lab
This study
This study
This study
This study
This study
This study
This study
This study
All strains also carry the pheA905 thi araD129 rpsL150 relA1 deoC1 flbB5301 ptsF25 rbsR mutations.
loppA-phoA translational fusion includes 210 bp upstream
of the translation initiation site and the first 37 codons of
oppA, including the leader peptide, fused in frame to the
leaderless phoA gene in phage lgt2 (Brickman & Beckwith,
1975). Plasmid pGS571 carries the gcvB gene on a 406-bp
EcoRI–HindIII fragment in plasmid pGS272 (Jourdan &
Stauffer, 1999). Plasmids pGS571 (gcvB), pA55 (dppA-lacZ
translational fusion) (Urbanowski et al., 2000), and pA32
(oppA-phoA translational fusion) (Urbanowski et al., 2000)
were used as templates to create nucleotide changes in gcvB
(Fig. 1a and b), dppA-lacZ (Fig. 1a), and oppA-phoA (Fig. 1b)
by PCR ‘megaprimer’ mutagenesis (Sarkar & Sommer,
1990) or by QuikChanges II site-directed mutagenesis
(Stratagene, Cedar Creek, TX). Mutations were verified by
DNA sequence analysis at the DNA Core Facility of the
University of Iowa. The gcvB mutations were subcloned into
the single-copy plasmid pGS341 (Jourdan & Stauffer, 1998),
replacing the wild-type (WT) gcvA gene. The 5947-bp
EcoRI–MfeI fragments carrying the dppA-lacZ mutations
and the 3768-bp EcoRI–MfeI fragments carrying the oppAphoA mutations were ligated into the EcoRI site of phage
lgt2 (Panasenko et al., 1977). These phages were used to
lysogenize various E. coli strains in single copy as described
(Shimada et al., 1972; Urbanowski & Stauffer, 1986). All
lysogens were grown at 30 1C because all fusion phages carry
the lcI857 mutation, resulting in a temperature-sensitive
lcI repressor (Panasenko et al., 1977).
Media
The complex medium used was Luria–Bertani broth (LB)
(Miller, 1972). Agar was added at 1.5% (w/v) to make solid
media. Ampicillin was added at 50 mg mL 1.
FEMS Microbiol Lett 281 (2008) 42–50
Enzyme assays
Cells were grown in LB or LB1ampicillin to mid-log phase
(OD600 nm c. 0.5) and assayed for either b-galactosidase
activity using the chloroform/sodium dodecylsulfate (SDS)
lysis procedure (Miller, 1972) or alkaline phosphatase activity
(Brickman & Beckwith, 1975). Results are the averages of two
or more assays with each sample carried out in triplicate.
RNA extraction and Northern blot analysis
Escherichia coli strains were grown in 10 mL LB or LB1ampicillin to an OD600 nm c. 0.5. Rifampicin (250 mg mL 1) was
added to each sample, and cells were immediately added to
5% (v/v) acidic phenol/95% (v/v) ethanol, centrifuged for
5 min at 2000 g, and pellets were frozen at 80 1C. RNA
was isolated by phenol extraction (Ledeboer et al., 2006). An
8 M urea 8% polyacrylamide gel was run with 10 mg of each
RNA sample and was electroblotted to a positively charged
nylon membrane (Roche, Mannheim, Germany). The blot
was hybridized with a DNA probe specific to 11 to 1134 of
the E. coli gcvB gene labeled using the PCR-digoxigenin
Probe synthesis kit (Roche, Mannheim, Germany). Hybridization was performed at 42 1C as described (Engler-Blum
et al., 1993), and the membrane was exposed to a film and
imaged using the FUJIFILM LAS-1000 camera and Intelligent Dark Box. Quantification of RNA was performed using
IMAGE GAUGE 3.12 software. The membrane was subsequently
stripped using 0.1% (w/v) SDS/2x SSC heated to c. 95 1C
and rehybridized using a digoxigenin-labeled DNA probe
specific for 5S RNA gene from 12 to 1112. The relative
amount of GcvB expressed from each mutant gcvB allele was
determined by taking the ratio of GcvB to 5S RNA gene
detected for each sample.
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44
S.C. Pulvermacher et al.
Fig. 1. Comparison of GcvB between 160 and 1100 nt with the dppA (a) and oppA (b) mRNAs. The AUG translation initiation sites for dppA and oppA
mRNAs are underlined and potential Shine–Dalgarno sequences are overlined and labeled SD (Igarashi et al., 1997; Sharma et al., 2007). A 13-base region
of GcvB from 179 to 191 nt (Con) conserved in GcvB homologs from the genera Escherichia, Salmonella, Yersinia, Haemophilus, Vibrio, Pasteurella,
Shigella, Klebsiella, and Photorhabdus is underlined (see Fig. 3). Regions of complementarity are indicated with dots between the sequences. GU base pairs
are indicated by a line. Changes made to GcvB are indicated below the sequence. A 1 and an 11 bp deletion are indicated with Ds. A 7 bp substitution in
the Con region is shown in brown. The –AAA– mutation made to GcvB, GcvB176AAA, is shown in pink, the –CCC– mutation, GcvB171CCC, is shown in red,
the –CC– mutation, GcvB171CC, is shown in blue, the –C– mutation, GcvB173C, is shown in purple, and the –AA– mutation, GcvB165AA, is shown in green.
Mutations made in dppA-lacZ and oppA-phoA that restore complementarity to the GcvB mutations are shown above the sequences and are color coded to
correspond with the mutations made in GcvB. The –A– nucleotide of the translation initiation codon in dppA and oppA mRNAs is designated 11.
Mutations upstream of the translation initiation sites are defined using negative numbers and mutations downstream are defined with positive numbers.
(c) Proposed secondary structure of Escherichia coli GcvB using M-FOLD (Zuker, 2003). The nucleotides changed in GcvB are shown to the left of the WT GcvB
secondary structure in the respective colors designated in (a) and (b). The single nucleotide deletion is designated by D. The bases between the arrows
designate the nucleotides deleted by the 11 bp deletion. Conserved predicted stem-loop structures among E. coli, Shigella dysenteriae, Salmonella
typhimurium, Yersinia pestis, Haemophilus influenzae, and Vibrio cholerae are designated by letters A–F.
DNA manipulation
The procedures for plasmid DNA purification, restriction
enzyme digestion, etc. were as described (Sambrook et al.,
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c
1989). PCR amplifications were carried out under standard
reaction conditions using Vent DNA polymerase (New England
BioLabs Inc., Beverly, MA). Restriction enzymes and other
DNA-modifying enzymes were from New England Biolabs Inc.
FEMS Microbiol Lett 281 (2008) 42–50
45
GcvB regulation of OppA and DppA
Results and discussion
Effects of mutations in gcvB on dppA-lacZ and
oppA-phoA expression
A G–U rich region was identified in GcvB complementary to
dppA and oppA mRNAs near the translation start sites (Fig.
1a and b). Sharma et al. (2007). report that this same region
of Salmonella’s GcvB base pairs with dppA and oppA mRNA
in Salmonella. In E. coli changes were made in gcvB in the
region of complementarity with dppA and oppA mRNAs and
their effects on dppA-lacZ and oppA-phoA expression were
tested. All changes are predicted by the M-FOLD program to
leave the GcvB secondary structure intact (Mathews et al.,
1999; Zuker, 2003). Mutations made include a –CCC–
change of 171 to 173 bp and an –AAA– change of 176 to
178 bp that disrupt complementarity between GcvB and the
dppA and oppA mRNAs, an –AA– change of 165 and
166 bp that primarily decreases complementarity to the
dppA mRNA, and a deletion of 175 bp that increases
complementarity with the dppA mRNA but decreases complementarity with oppA mRNA (Fig. 1a and b). Single-copy
plasmids carrying either WT gcvB or a gcvB mutation were
transformed into the DgcvB strain, GS1144, lysogenized
with either ldppA-lacZ or loppA-phoA, and assayed for
either b-galactosidase or alkaline phosphatase activity. As
expected, expression of dppA-lacZ and oppA-phoA in the
WT lysogens were nine- and fourfold lower than in the
DgcvB lysogens (Fig. 2a and b). Transformation of either
DgcvB lysogen with WT gcvB restored repression to WT
levels, and transformation with the empty vector alone
(pGS311) showed expression levels similar to the untransformed DgcvB lysogens (Fig. 2a and b). To determine the
effects of mutations in gcvB, activity from DgcvB lysogens
transformed with gcvB mutant alleles was always compared
with WT and DgcvB lysogens transformed with WT gcvB.
When the DgcvB ldppA-lacZ or DgcvB loppA-phoA strains
carried the gcvB171CCC allele, b-galactosidase activity was
4.5-fold higher (Fig. 2a) and alkaline phosphatase activity
was twofold higher (Fig. 2b) compared with the controls.
The gcvB176AAA allele did not significantly affect repression
of dppA-lacZ or oppA-phoA in the DgcvB strain (Fig. 2a and
b), suggesting that these nucleotides are not necessary for
GcvB regulation of either target. The gcvB165AA allele
resulted in a reduced ability of GcvB to repress dppA-lacZ,
as the b-galactosidase level in DgcvB ldppA-lacZ carrying
gcvB165AA was more than threefold higher (Fig. 2a). In
contrast, the gcvB165AA allele resulted in an increased ability
of GcvB to repress oppA-phoA, as the alkaline phosphatase
level in DgcvB loppA-phoA carrying gcvB165AA was twofold
lower (Fig. 2b). The –AA– change disrupts complementarity
between GcvB and dppA mRNA, and the reduced repression
of dppA-lacZ is possibly due to the lack of complementary
FEMS Microbiol Lett 281 (2008) 42–50
base pairing between GcvB and dppA-lacZ mRNA. However,
the changes do not significantly disrupt complementarity
between GcvB and oppA mRNA (Fig. 1a and 1b), it is
unclear why these changes result in increased repression of
oppA-phoA. The gcvB175DG allele, which results in a better
match of GcvB to dppA mRNA but reduces complementarity to oppA mRNA (Fig. 1a and 1b), did not affect expression
of either fusion (Fig. 2a and b). These results show that
the region in GcvB between base 165 and 178 is important
for GcvB regulation of both dppA-lacZ and oppA-phoA.
However, certain changes affect regulation of the two
targets differently. In addition, changing bases that
decrease or increase complementarity with the target
mRNAs does not necessarily correlate with decreased or
increased repression.
A comparison of the gcvB gene from E. coli with presumptive gcvB genes in the genera Shigella, Salmonella,
Klebsiella, Photorhabdus, Yersinia, Haemophilus, Pasterurella,
and Vibrio shows extensive homology (Fig. 3). One 13 bp
region of gcvB, designated Con, is highly conserved (Fig. 3)
and forms part of the predicted stem loop B in GcvB (Fig.
1c). It is not entirely clear whether the predicted stem loop B
forms as part of the secondary structure because the E. coli
and S. typhimurium GcvB sRNAs are c. 95% identical and
stem loop B was not observed in Salmonella by in vitro
structure mapping (Sharma et al., 2007). It was tested
whether the Con sequence or the stem loop B is required
for GcvB regulation of either dppA-lacZ or oppA-phoA
expression. Seven base pairs in the gcvB Con sequence were
changed and an 11 bp deletion from 183 to 193 was also
made that removes the last 9 bp of the Con sequence and the
predicted stem loop B, designated pGS595 and pGS603 (Fig.
1a and b). The deletion of predicted stem loop B disrupts
significant complementarity between GcvB and oppA
mRNA, with six predicted base pairing interactions destroyed compared with GcvB and dppA mRNA, with only
one predicted base pairing interaction destroyed (Fig. 1).
The gcvBCon–7 allele resulted in an inability of GcvB to fully
repress dppA-lacZ or oppA-phoA, as the b-galactosidase level
was fourfold higher (Fig. 2a) and the alkaline phosphatase
level was twofold higher (Fig. 2b) compared with the
controls. Thus, the conserved region is important for GcvB
regulation of both dppA and oppA mRNA. Sharma et al.
(2007) also found that a deletion of 166 to 189 nt in gcvB
in Salmonella results in an inability to repress either dppAgfp or oppA-gfp. Because of the extensive number of changes
in the gcvBCon–7 allele, it is unknown if the effect of the
mutation is due to a decrease in complementarity, or
whether it plays some additional role in GcvB regulation.
In contrast, the gcvB183D11 bp allele showed increased repression of both dppA-lacZ and oppA-phoA (Fig. 2a and b)
despite the loss of a region of GcvB predicted to be involved
in base pairing. Thus, part of the Con sequence or stem-loop
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c
46
S.C. Pulvermacher et al.
(a)
600
Miller units
500
400
300
200
100
vB
gc
/
vB
∆
+7
C
vB
gc
B/
v
gc
c
∆g
5∆
1C
1C
+7
∆g cvB
/g
cv
cv
B/
BC
gc
vB + on7
83
∆g
∆1
cv
1b
B/
p
gc
∆g
v
cv
B +7
B/
3
C
gc
vB +
7
gc
∆g
vB
cv
B/
gc
∆g
v
cv B +76
A
B/
A
gc
vB + A
∆
G
65
A
A
CC
gc
∆g
.
e.v
/
vB
∆g
cv
B/
W
T
∆g
cv
B
0
WT λdppA-lacZ
(b)
(c)
140
70
120
60
100
50
80
40
60
30
40
20
20
10
0
71
CC
3C
+7
vB +
vB
c
B/
cv
∆g
/g
vB
gc
T
p
1b
7
83
gc
B/
cv
B
cv
∆g
c
∆g
cv
∆g
∆1
on
vB C
gc
B
B
cv
cv
/g
/g
B
B
cv
cv
∆g
∆g
vB +
5
+7
B/
5A
+6
∆G
∆g
A
A
B +7
B/
g
∆g
cv
∆g
A
cv
B
cv
g
B/
6A
vB
+7
cv
∆g
gc
/
B
cv
CC
1C
B/
c
∆g
.
e.v
cv
vB
∆g
W
T
0
W
Alkaline phosphatase activity
160
WT λoppA-phoA
Fig. 2. b-Galactosidase (a) or alkaline phosphatase (b and c) levels of ldppA-lacZ or loppA-phoA from WT, DgcvB, or the DgcvB strain transformed
with the single copy empty vector (e.v.) or a single copy plasmid carrying either the WT gcvB or a mutated gcvB allele. The respective gcvB allele in the
DgcvB background strain is designated below the bar graph after the slash mark. The lysogens and transformants were grown in LB or LB1ampicillin to
an OD600 nm c. 0.5 and assayed for either b-galactosidase activity (Miller, 1972) (a) or alkaline phosphatase activity (Brickman & Beckwith, 1975) (b and
c). Results are the averages of two or more assays, with each assay performed in triplicate.
B normally functions to prevent repression of dppA and
oppA mRNA by GcvB.
Effects of mutations made to gcvB on GcvB RNA
production
It is possible the increased repression and derepression of
dppA-lacZ and oppA-phoA observed for the different gcvB
alleles is a result of over- or underexpression of the mutant
RNAs. To determine whether each gcvB allele produces
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c
comparable amounts of GcvB, Northern blots were performed. Although there are small changes in the levels of
GcvB observed in the Northern blot (less than twofold)
(Fig. 4), these differences do not appear to be responsible for
the observed effects on regulation. The gcvB176AAA and
gcvB183D11 bp alleles, which show the lowest levels of GcvB
(Fig. 4), result in essentially normal or increased repression
of dppA-lacZ and oppA-phoA rather than a loss of repression
(Fig. 2a and b). The gcvBCon–7 allele, which has the highest
level of RNA, shows a loss of repression of both dppA-lacZ
FEMS Microbiol Lett 281 (2008) 42–50
47
GcvB regulation of OppA and DppA
Fig. 3. Comparison of the Escherichia coli gcvB sequence with homologs from other organisms. Regions of high consensus are boxed in gray. The
consensus is shown below the sequence with high consensus bases in capital letters. One 13 bp region conserved in these homologs is overlined and
designated Con. Numbering is based on the E. coli sequence. The region of complementarity between GcvB and the target mRNAs dppA and oppA
occurs between 160 and 1100 nt in GcvB. Ec, E. coli; Sd, Shigella dysenteriae; St, Salmonella typhimurium; Kp, Klebsiella pneumoniae; Pl,
Photorhabdus luminescens; Yp, Yersinia pestis; Hi, Haemophilus influenzae; Pm, Pasteurella multocida; Vc, Vibrio cholerae; Con, conserved sequence.
and oppA-phoA (Fig. 2a and b). These results suggest that
the loss of or increased repression observed for dppA-lacZ
and oppA-phoA is likely not due to changes in the synthesis
or stability of GcvB, but are direct effects on GcvB’s role in
regulation of dppA and oppA mRNA.
Effects of mutations in dppA-lacZ or oppAphoA that restore base pairing to GcvB on GcvB
repression
The above results identified a region of GcvB complementary to dppA and oppA mRNAs required for normal regulation. To determine whether GcvB regulates dppA and oppA
FEMS Microbiol Lett 281 (2008) 42–50
mRNA by base pairing with the mRNAs, three nucleotides
were changed in either dppA-lacZ or oppA-phoA to restore
base pairing with the gcvB171CCC mutation. However, these
mutations in dppA-lacZ and oppA-phoA had dramatic effects
on b-galactosidase and alkaline phosphatase activity (data
not shown). Instead, a –GG– change at 20 and 21 nts
and a –G– change at
22 nts in dppA-lacZ and a –GG–
change at 19 and 110 nts and a –G– change at 18 nts in
oppA-phoA were made (Fig. 1a and b). The activity levels of
the DgcvB strain lysogenized with these four fusions were
high enough compared with the WT fusions to test whether
complementary pairing between GcvB and the mRNAs is
part of the regulatory mechanism (Fig. 5a and c). Thus, we
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c
48
CC
gc
vB
+
71
gc
vB
C
23562
21 000
29180
31700
27940
38570
18960
28580
21130
Counts
70540
85490
93900
182600
126 000
122400
114300
97630
100500
73120
GcvB/5S ratio
0.344
0.276
0.224
0.160
0.252
0.228
0.337
0.194
0.284
0.289
1
2
3
4
5
6
7
8
9
10
gc
vB
+
gc
24260
gc
vB
+
gc
vB
+
73
C
83
∆1
1b
p
-7
gc
vB
+
Counts
vB
gc
vB
+
on
+7
5∆
G
A
A
gc
vB
+
65
W
T
71
CC
C
76
A
A
A
S.C. Pulvermacher et al.
GcvB
5S
Lane
Fig. 4. Northern blot analysis of GcvB expressed from plasmids carrying either a WT or a mutant gcvB allele. Total cell RNA was isolated from each
strain, run on an 8 M urea–8% polyacrylamide gel, and probed with either a digoxigenin-labeled GcvB or a 5S-specific DNA probe. Total RNA extracts
are as follows: lane 1, WT (GS162); lane 2, gcvB1 (GS1144 pGS594); lane 3, gcvB171CCC (GS1144 pGS596); lane 4, gcvB176AAA (GS1144 pGS602); lane
5, gcvB165AA (GS1144 pGS605); lane 6, gcvB175DG (GS1144 pGS597); lane 7, gcvBCon–7 (GS1144 pGS595); lane 8, gcvB183D11 bp (GS1144 pGS603);
lane 9, gcvB173C (GS1144 pGS618); lane 10, gcvB171CC (GS1144 pGS619). RNA was quantified using IMAGE GAUGE 3.12 software, and the counts
determined for each sample are shown below each lane. The bottom row shows the ratio of GcvB RNA compared with the 5S RNA gene for each
sample. The ratio observed for lane 1 (WT GcvB expressed in WT cells) was considered to be the WT amount of GcvB expressed in LB.
constructed a –C– change at 173 nts and a –CC– change at
171 and 172 nts in gcvB, designated pGS618 and pGS619
(Fig. 1), transformed the DgcvB strain lysogenized with
ldppA-lacZ, ldppA 22G-lacZ, and ldppA 20GG-lacZ, and
assayed for b-galactosidase activity. The b-galactosidase
levels in DgcvB ldppA-lacZ carrying the gcvB173C or
gcvB171CC allele were 1.3- and 4.5-fold higher than the levels
in the WT ldppA-lacZ lysogen, demonstrating that the
gcvB173C and gcvB171CC alleles fail to repress fully when
complementarity is disrupted (Fig. 2a). The gcvB173C and
gcvB171CC alleles produce essentially equivalent amounts of
RNA compared with WT gcvB (Fig. 4), suggesting that their
failure to repress dppA-lacZ is not due to low levels of GcvB.
The b-galactosidase levels in DgcvB ldppA 22G-lacZ carrying
WT gcvB or the gcvB173C allele were reduced 4- and 5.2-fold
compared with DgcvB ldppA 22G-lacZ (Fig. 5a, compare bar
3 with bars 4 and 5). The b-galactosidase levels in DgcvB
ldppA 20GG-lacZ carrying WT gcvB or the gcvB171CC allele
were reduced 2.9- and 9-fold (Fig. 5a, compare bar 6 with
bars 7 and 8). Thus, compensatory mutations in gcvB
increased the ability of GcvB to repress the dppA 22G-lacZ
and dppA 20GG-lacZ lysogens compared with WT GcvB,
consistent with base pairing between GcvB and dppA-lacZ
mRNA as part of the regulatory mechanism.
To test the effect of gcvB173C and gcvB171CC alleles on
oppA-phoA expression, plasmids pGS618 and pGS619 were
transformed into DgcvB lysogenized with loppA-phoA,
loppA18G-phoA, and loppA19GG-phoA, and assayed for
alkaline phosphatase activity. These strains were assayed
with a different batch of reagents compared with the other
alkaline phosphatase results, and probably explain the
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
slightly lower expression levels for the WT and DgcvB
loppA-phoA control strains in Fig. 2c. The alkaline phosphatase level in DgcvB loppA-phoA carrying the gcvB173C
allele was essentially the same as the WT loppA-phoA
lysogen, demonstrating that the gcvB173C allele is able to
repress when complementarity is disrupted at this position
(Fig. 2c). The alkaline phosphatase level in DgcvB loppAphoA carrying the gcvB171CC allele was twofold higher than
WT loppA-phoA, demonstrating that the gcvB171CC allele
fails to fully repress when complementarity is disrupted
(Fig. 2c). The alkaline phosphatase level in DgcvB loppA18G-phoA carrying WT gcvB or the gcvB173 allele was
reduced 1.5- and 2.4-fold compared with DgcvB loppA18GphoA (Fig. 5c, compare bar 3 with bars 4 and 5). The alkaline
phosphatase level in DgcvB loppA19GG-phoA carrying WT
gcvB or the gcvB171CC allele was reduced 1.7- and 1.1-fold
compared with DgcvB loppA19GG-phoA (Fig. 5c, compare
bar 6 with bars 7 and 8). Surprisingly, there is better
repression of oppA19GG-phoA with the WT gcvB allele than
with the complementary gcvB171CC allele. It is unclear why
restoring complementarity between the gcvB173C allele and
oppA18G-phoA represses, but restoring complementarity
between the gcvB171CC allele and oppA19GG-phoA does
not. It is possible that regulation of oppA-phoA by GcvB
occurs by a different mechanism than for dppA-lacZ. Previous results suggested that GcvB-mediated repression of
dppA-lacZ and oppA-phoA is different (Urbanowski et al.,
2000). The TARGETRNA program (Tjaden et al., 2006)
predicts other small regions of possible base pairing between
GcvB and oppA mRNA; therefore, it is possible that
other nucleotides in oppA mRNA are more essential for
FEMS Microbiol Lett 281 (2008) 42–50
49
GcvB regulation of OppA and DppA
regulation by GcvB and base pairing is part of the regulatory
mechanism.
Additionally, it was tested whether loss of gcvB165AA
repression of dppA-lacZ is due to loss of base pairing.
Nucleotides were changed in dppA-lacZ to –UU– at
14
and
15 bps to restore pairing with the gcvB165AA allele.
Expression of b-galactosidase activity from DgcvB
ldppA 14UU-lacZ was 5.3-fold higher than WT ldppA-lacZ
(Fig. 5b, compare bar 3 with bar 1). The b-galactosidase level
in DgcvB ldppA 14UU-lacZ carrying WT gcvB or the
gcvB165AA allele was 2.8-or 3.4-fold lower compared with the
control DgcvB ldppA 14UU-lacZ (Fig. 5b, compare bar 3 with
bars 4 and 5). Thus, the compensatory mutation in gcvB
increased the ability of GcvB165AA to repress dppA 14UU-lacZ
better than WT GcvB. Because the gcvB165AA mutation
showed a threefold loss of repression compared with WT
ldppA-lacZ (Fig. 2a), these results are consistent with RNA/
RNA pairing as part of the mechanism for GcvB regulation of
dppA-lacZ. Additionally, the gcvB165AA allele shows that
certain sequences of GcvB play different roles in regulating
dppA-lacZ and oppA-phoA (Fig. 2a and b).
The largest family of sRNAs acts by base pairing with target
mRNAs to alter mRNA translation or stability (Majdalani
et al., 2005). Although these results suggest that complementary RNA/RNA pairing between GcvB and dppA-lacZ mRNA
is likely part of the regulatory mechanism, the regulation for
both dppA and oppA mRNA is more complex than simply
base pairing between GcvB and the target mRNAs. It was
predicted that the binding of GcvB with dppA or oppA mRNA
blocks ribosomal binding, preventing translation of both
dppA and oppA mRNAs. Sharma et al. reported that WT
GcvB in Salmonella blocks translation of both dppA and oppA
mRNAs, and when 166 to 189 nts were deleted, the nucleotides that are predicted to be involved in base pairing in
E. coli, translation was no longer blocked (Sharma et al.,
2007). Additionally, all E. coli sRNAs that regulate by RNA/
RNA base pairing with target mRNAs bind the RNA chaperone protein Hfq (Chen et al., 2004), and Hfq has been shown
to interact directly with some mRNAs (Geissmann & Touati,
2004; Vytvytska et al., 1998). Recently, it was shown GcvB is a
target for Hfq binding (Zhang et al., 2003), and it has been
found that Hfq is required for the ability of GcvB to regulate
both dppA-lacZ and oppA-phoA negatively (unpublished
results). It is possible one or more of the gcvB mutant alleles
that fail to fully repress dppA-lacZ or oppA-phoA or that result
in increased repression fails to interact with Hfq, or binds Hfq
more tightly, possibly explaining the altered regulation. We
Fig. 5. b-Galactosidase levels of WT ldppA-lacZ, ldppA 22G-lacZ, or
ldppA 20GG-lacZ (a) or alkaline phosphatase activity of WT loppAphoA, loppA18G-phoA, or loppA19GG-phoA (c) from either WT, DgcvB,
or the DgcvB strain transformed with WT gcvB or the mutant gcvB173C
or gcvB171CC alleles. b-Galactosidase levels of either ldppA-lacZ or
ldppA 14UU-lacZ (b) from WT, DgcvB, or the DgcvB strain transformed
either WT gcvB or the mutant gcvB165AA allele. The fusion assayed in
each strain is designated below the bar graph. The lysogens and
transformants were grown in LB or LB1ampicillin to an OD600 nm c. 0.5
and assayed for either b-galactosidase activity (Miller, 1972) (a and b) or
alkaline phosphatase activity (Brickman & Beckwith, 1975) (c). Results
are the average of two or more assays with each assay performed in
triplicate.
FEMS Microbiol Lett 281 (2008) 42–50
2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
50
are in the process of determining how Hfq is involved in GcvB
regulation of dppA and oppA mRNA as well as where Hfq
binds with GcvB. These studies should clarify whether RNA/
RNA pairing is necessary for GcvB regulation of dppA and
oppA mRNA.
Acknowledgements
This work was supported by Public Health Service Grant
GM069506 from the National Institute of General Medical
Sciences.
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