xtracytoplasmic Function Sigma Factor Acts as a General Stress Response Regulator in
'
Sinorhizobium meliloti
Laurent Sauviac, Heinui Philippe, Kounthe´a Phok, and Claude Bruand*
Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR 2594-441 CNRS-INRA, BP52627, Castanet-Tolosan F-31320,
France
Received 2 February 2007/Accepted 19 March 2007
Sinorhizobium meliloti genes transcriptionally up-regulated after heat stress, as well as upon entry into
stationary phase, were identified by microarray analyses. Sixty stress response genes were thus found to be
up-regulated under both conditions. One of them, rpoE2 (smc01506), encodes a putative extracytoplasmic
function (ECF) sigma factor. We showed that this sigma factor controls its own transcription and is activated
by various stress conditions, including heat and salt, as well as entry into stationary phase after either carbon
or nitrogen starvation. We also present evidence that the product of the gene cotranscribed with rpoE2
negatively regulates RpoE2 activity, and we therefore propose that it plays the function of anti-sigma factor. By
combining transcriptomic, bioinformatic, and quantitative reverse transcription-PCR analyses, we identified 44
RpoE2-controlled genes and predicted the number of RpoE2 targets to be higher. Strikingly, more than
one-third of the 60 stress response genes identified in this study are RpoE2 targets. Interestingly, two genes
encoding proteins with known functions in stress responses, namely, katC and rpoH2, as well as a second
ECF-encoding gene, rpoE5, were found to be RpoE2 regulated. Altogether, these data suggest that RpoE2 is a
major global regulator of the general stress response in S. meliloti. Despite these observations, and although this
sigma factor is well conserved among alphaproteobacteria, no in vitro nor in planta phenotypic difference from
the wild-type strain could be detected for rpoE2 mutants. This therefore suggests that other important actors in
the general stress response have still to be identified in S. meliloti.
To survive the many stress conditions that they encounter in nature, bacteria have evolved rapid responses that result in the prevention
or repair of cellular damages caused by stresses. In addition to these usually stress-specific responses, more general stress responses take
place in reaction to a variety of insults as different as high osmolarity, heat or cold shock, pH variation, and nutrient starvation (which
results in stationary phase). One well-known consequence of these general stress responses is the ability of bacteria to resist stress better in
stationary phase than in exponential phase. This phenomenon, observed in most bacterial species studied so far, is considered a universal
way for these microorganisms to survive not only the stress that they currently experience, but also stress conditions that they could
potentially face in the future, and is therefore of primary importance in nature, where bacteria often are nutrient or oxygen limited and
where environmental conditions constantly change (28).
Much of our knowledge regarding the mechanisms of induction of general stress responses derives from studies of the model bacterium
Escherichia coli and its close relatives. In these bacteria, entry into stationary phase, as well as a number of different stress conditions,
leads to the activation of alternative sigma factors, which redirect the RNA polymerase holoenzyme to hundreds of genes, some of which
confer multiple-stress resistance on the cells. These alternative sigma factors
* Corresponding author. Mailing address: Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR 2594-441 CNRSINRA, BP52627, 31326
Castanet-Tolosan Cedex, France. Phone: (33) 5 61 28 53 20. Fax: (33) 5 61 28 50 61. E-mail: Claude.Bruand@toulouse .inra.fr.
'
Published ahead of print on 30 March 2007.
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are of two different classes. The first class is represented by E. coli u (encoded by rpoS), which is considered the master regulator of the
general stress response in this bacterium. The u content of the cell is regulated at multiple levels, including transcription, translation, and
proteolysis, with different stress conditions affecting different levels of control (for reviews, see references 29 and 52). Members of the u
regulon encode proteins with diverse functions, including stress response (for instance, katE, encoding a catalase) (72). rpoS mutants are
thus sensitive to multiple stresses and have a decreased capacity for survival in stationary phase (28). A second class of alternate sigma
factors is represented by u (encoded by rpoE), a sigma factor of the ECF family (for extracytoplasmic function) (27). The activity of u is
negatively controlled by a membrane-bound anti-sigma factor, which binds u and renders it inactive. u is activated by perturbations in the
cell envelope, like the accumulation of misfolded proteins in the periplasm. This signal triggers proteolysis of the anti-sigma factor, thus
releasing into the cytoplasm an active form of u , which can then interact with the core RNA polymerase (for recent reviews, see
references 1, 2, and 57). It now appears that u can also be activated upon entry into stationary phase through a different, incompletely
characterized mechanism (14). In addition to its own operon, targets of u include genes encoding proteins with functions in stress
response, such as the heat shock sigma factor u , as well as proteins involved in the synthesis, folding, or degradation of outer membrane
proteins (for reviews, see references 20 and 56). Inactivation of u in E. coli is lethal, and existing rpoE mutants contain suppressive
mutations (10, 16). In other bacteria, like Salmonella enterica serovar Typhimurium, the resistance to multiple stresses, as well as the
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Strain or plasmid Description
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Sinorhizobium meliloti Rm1021 Wild-type strain; Sm 43 Rm2011 Wild-type strain 13 CBT208 Rm1021 rpoE2::hph This work 2011mTn5STM.3.12.B10
Rm2011 rpoE2::mTn5 A. Becker (53)
Escherichia coli DH5c supE44 1lacU169 (<80 lacZ1M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1
58
r
Plasmids pRK2013 Helper plasmid for triparental matings 22 pGEM-T Cloning vector; Amp Promega pLS2.7 pGEM-T smc015051 smc01506 This
work pLS3.4 pGEM-T 'smc01504 smc015051 smc01506 This work pBBR1MCS-5 Cloning vector; Gm derivative of pBBR1 38 pCBT105
pBBR1MCS-5 smc01505 smc01506 This work pCBT109 pBBR1MCS-5 smc01505 This work pLS4.1 pBBR1MCS-5 smc015051
smc01506 This work pJQ200mp19 Gene replacement vector; Gm 54 pLS5.1 pJQ200mp19 'smc01504 smc015051 smc01506 This work pCBT104
pJQ200mp19 smc01505 smc01506 This work pCBT113 pJQ200mp19 smc01505 smc01506::hph This work pCZ750 lacZ expression probe vector; Tet
8 pLS6.20 pCZ750 Psmc01504-lacZ This work pLS6.32 pCZ750 Psmc01505-lacZ This work pVO205 Plasmid carrying the hph gene, conferring resistance
to hygromycin 5
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survival in stationary phase, of rpoE mutants is affected (32, 37, 66).
Two classes of sigma factors with similar functions in stress adaptation and/or stationary-phase survival were also identified in
gram-positive bacteria (26, 27, 42). Interestingly, in addition to the regulation of stress responses, both classes of sigma factors can also
control virulence genes in certain gram-negative and gram-positive pathogenic bacteria (28, 36, 42, 56).
Rhizobia are soil bacteria existing either in free-living forms or in symbiosis within nodules of legume plants, where they differentiate
into nitrogen-fixing bacteroids (23, 41, 48, 65). Both in the soil and in planta, rhizobia are subject to stress. For instance, rhizobia
experience oxidative stress during root hair infection and nodule invasion (33, 59, 60, 63), as well as oxygen limitation in the fixation zone
of the nodule (64). In the soil, variations of temperature, osmolarity, or pH, as well as nutrient starvation, are the stress conditions most
frequently faced by rhizobia (70, 71, 75). Therefore, general stress response must be important for the survival of rhizobia, both in the soil
and in planta. However, little is known about this response in rhizobia. In Rhizobium leguminosarum, stationary-phase bacteria were
found to be more resistant than exponentially growing bacteria to heat, salt, and oxidative and acidic stresses, suggesting that this kind of
response also exists in rhizobia (67). Nevertheless, little is known about the mechanisms of regulation of this response, although the
involvement of a quorum-sensing system has been reported in R. leguminosarum (68). Interestingly, most rhizobia belong to the alpha
subdivision of proteobacteria, and no u homologue could be found in the complete genomes of alphaproteobacteria, which include those
of six rhizobia. This suggests that other regulators could be involved in the general stress response of these bacteria. In contrast, many
ECF sigma factors are encoded by the genomes of rhizobia, but little is known about their functions (74).
As a first step toward understanding the rhizobial general stress response, we performed transcriptomic studies of the responses of
Sinorhizobium meliloti to heat shock and starvation. Among the genes up-regulated under both stress conditions (referred to as “stress
response genes”), we found rpoE2, which encodes a putative ECF sigma factor that is well conserved among alphaproteobacteria. We
show that this sigma factor controls its own transcription and is activated by various stress conditions, and we present evidence that the
product of the gene cotranscribed with rpoE2 could play the function of anti-sigma factor. We identified 44 genes under the control of
RpoE2, including katC and rpoH2, which encode proteins with predictable functions in stress responses (a catalase and a heat shock
sigma factor, respectively), as well as rpoE5, encoding another putative ECF sigma factor. Strikingly, more than one-third of the 60 stress
response genes identified here are under RpoE2 control, which suggests that RpoE2 is a major global regulator of the general stress
response in S. meliloti.
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MATERIALS AND METHODS
Bacterial strains and growth conditions. The strains and plasmids used in this study are listed in Table 1. S. meliloti strains, unless otherwise indicated, were grown at 28°C in Vincent
minimal medium (VMM) (1 mM MgSO 4, 18.7 mM NH4Cl, 10 mM Na2 succinate, 456 fM CaCl2,35 fM FeCl3,4 fM biotin, 48.5 fMH3BO3,10 fM MnSO4,1 fM ZnSO4, 0.5 fM CuSO4, 0.27 fM
CoCl2, 0.5 fM NaMoO4).
Strain constructions were performed in LB medium (58) supplemented with
-1
2.5 mM CaCl2 and 2.5 mM MgSO4 (LBMC). Viable cells in stress resistance assays were numbered on TY medium (5 g . liter tryptone,
3 g . liter yeast
-1
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extract, 15 g . liter agar) supplemented with 6 mM CaCl2 and
supplemented, or not, with streptomycin, depending on the strain background.
Antibiotics were added at the following concentrations: streptomycin, 100 to 300 fg .
-1
-1
-1
ml ; tetracycline, 10 fg . ml ; gentamicin (Gm), 40 fg . ml ;
-1
-1
hygromycin (Hyg), 50 fg . ml ; neomycin, 100 fg . ml .
Strain and plasmid constructions. All plasmid constructions were performed in
E. coli DH5c. DNA sequences of oligonucleotide primers used for PCR
amplifications are available in Table S1 (http://bioinfo.genopole-toulouse.prd.fr
/annotation/iANT/bacteria/rhime/DOC/Bruand2007/index.html).
For constructing plasmid pCBT104, a DNA fragment containing the smc01505 and
smc01506 open reading frames (ORFs) was generated by PCR using OCB529 and
OCB530 as primers and genomic DNA of strain Rm1021 as a template. This
fragment was then digested with BamHI-XbaI and ligated into BamHI-XbaI-cut
pJQ200-mp19. For constructing plasmid pCBT113, the smc01506 ORF in plasmid
pCBT104 was disrupted at the BssHII site by insertion of the hph gene, generated by
PCR using OCB461 and OCB462 as primers and plasmid pVO205 as a template.
Plasmid pCBT105 was constructed by subcloning the BamHI-XbaI DNA fragment
of pCBT104 into the similarly cut pBBR1MCS-5. For constructing plasmid
pCBT109, a DNA fragment carrying the smc01505 ORF was generated by PCR using
primers OCB529 and OCB531, digested with BamHI and XbaI, and inserted into the
similarly cut pBBR1MCS-5.
For constructing pLS4.1, a DNA fragment containing the smc01505 and smc01506
ORFs harboring a 129-bp deletion in smc01505 (bp 19 to 147 from the start of the
ORF) was generated in a two-step PCR. In the first step, the region upstream from the
deletion was amplified using OCB529 and OCB533 as primers, Rm1021 genomic
DNA as a template, and Pfu Ultra (Stratagene) as the DNA polymerase. OCB533
carries sequences -20 nucleotides upstream and +20 nucleotides downstream from the
deletion endpoints (i.e., bp -2to +18 and 148 to 167 of smc01505). In a second step,
the PCR fragment generated in the first step was used as a primer, together with
OCB530, to amplify by PCR a DNA fragment using the OCB539-OCB534 PCR
fragment as a template and GoTaq (Promega) as the DNA polymerase (the
OCB539-OCB534 PCR fragment was used as a template because only the
downstream part of OCB533 anneals in this region). The resulting PCR product was
then ligated into pGEM-T, generating plasmid pLS2.7. Finally, the
smc015051-smc01506 region was subcloned from pLS2.7 into pBBR1MCS-5 as an
ApaI-XbaI fragment to give pLS4.1.
Similarly, for constructing the plasmid pLS5.1, a first PCR fragment was generated
using OCB535 and OCB533 as primers, Rm1021 genomic DNA as a template, and
Pfu Ultra (Stratagene) as the DNA polymerase. This PCR fragment was used as a
primer, together with OCB536, to amplify a second PCR product using the
OCB539-OCB534 PCR fragment as a template and the GoTaq (Promega) DNA
polymerase. The resulting PCR product was then ligated into pGEM-T, generating
plasmid pLS3.4. Finally, the entire region was subcloned in pJQ200-mp19 as a
BamHI-XbaI fragment, giving plasmid pLS5.1.
For the construction of pLS6.20 and pLS6.32, a DNA fragment carrying the
smc01504-smc01505 intergenic region was generated by PCR using OCB529 and
OCB533 as primers, Rm1021 genomic DNA as a template, and Pfu Ultra (Pro-mega)
as the polymerase and ligated into pCZ750 digested with XbaI and blunted with Pfu
Ultra. The orientation of the insert in the resulting clones was screened by PCR and
checked by DNA sequencing. pLS6.20 and pLS6.32 correspond to plasmids in which
the lacZ gene is transcribed from the promoters of smc01504 and
smc01505-smc01506, respectively.
The S. meliloti DNA regions cloned into pCBT104, pCBT109, pLS2.7, pLS3.4,
pLS6.20, and pLS6.32 were verified by DNA sequencing.
The plasmids were introduced in S. meliloti by triparental mating using pRK2013
as a helper. The construction of S. meliloti strains is described in Results.
Preparation of samples for microarrays, qRT-PCR, and l-galactosidase assays.
For microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of the
entry into stationary phase after carbon starvation, Rm1021 cells were grown
exponentially at 28°C in 500 ml of VMM containing 2 mM sodium succinate. At an
optical density at 600 nm (OD600)of -0.12 (i.e.,1h30min before the first signs of slow
down of the culture), cells (25 ml) were harvested by filtration and immediately
frozen in liquid nitrogen (exponential-phase sample). Three hours later (the beginning
of the plateau; OD600, -0.25), cells were harvested again (stationary-phase sample).
For qRT-PCR analyses of the entry into stationary phase after nitrogen starvation,
Rm1021 cells were grown exponentially at 28°C in 500 ml of VMM containing 0.62
mM NH4Cl. At an OD600 of -0.1, cells (25 ml) were harvested by filtration as
described above (exponentialphase sample); 6 h later (OD600, -0.22), cells were
harvested again (stationaryphase sample).
For microarray and qRT-PCR analyses of heat shock, wild-type (Rm1021 or
Rm2011) or rpoE2 mutant (CBT208 or 2011mTn5STM.3.12.B10) cells, carrying or
not carrying plasmids, as indicated, were grown exponentially at 28°C in 100 ml
VMM. At an OD600 of -0.4, two 25-ml aliquots of the culture were transferred into
preheated flasks and incubated at 40°C, while the other half of the culture was kept at
28°C. After 30 min, cells were harvested as described above from each culture. For
qRT-PCR analyses of salt stress, Rm1021 cells were grown exponentially at 28°C in
100 ml of VMM. At an OD600 of -0.15, NaCl was added to half of the culture at a final
concentration of 250 mM, while the rest of culture was kept untreated. After 30 min
at 28°C, cells (25 ml) were harvested from treated and untreated cultures as described
above.
For microarrays or qRT-PCR, RNA was prepared from the collected samples as
previously described (11), followed by DNase I treatment (QIAGEN clean-up
procedure).
For l-galactosidase assays of heat shock, Rm1021 (wild-type) or CBT208 (rpoE2)
cells harboring the plasmid pCZ750, pLS6.20, or pLS6.32 were grown exponentially
at 28°C in 80 ml of VMM. At an OD600 of -0.2, two 25-ml aliquots of the culture were
transferred into preheated flasks and incubated at 40°C, while the other half of the
culture was kept at 28°C. After 1 h, a 50-fl sample of each culture was collected. For
l-galactosidase assays of the exponential-to stationary-phase transition, cells were
grown exponentially in the same medium at 28°C, and 50 fl of cells was collected at
an OD600 of -0.2 (exponential-phase sample). The culture was then incubated for an
additional 24 h at 28°C, and 50 fl of cells was collected at an OD 600 of -1.2
(stationary-phase sample). l-Galactosidase activity was assayed in the collected
samples as described previously (44).
Labeling of hybridization probes, microarray hybridizations, and analyses.
Cy3-and Cy5-labeled cDNAs were prepared according to the method of DeRisi et al.
(18) from 10 to 25 fg of RNA. For each experiment, RNA preparations from three
independent cultures were used. Sm6koligo microarrays were purchased from A.
Becker (University of Bielefeld, Bielefeld, Germany). They consisted of glass slides
carrying mainly 70-mer oligonucleotides representative of the 6,208 predicted ORFs
of S. meliloti spotted in triplicate, as well as a number of control spots (39).
Hybridizations were performed as described previously (6). Data were acquired on a
GenePix 4000 scanner (Axon Instruments), and quantifications of mean signal
intensities for each spot were performed using GenePix Pro 3.0 (Axon Instruments).
Data analyses were carried out using EMMA
2.2. software (CeBiTec; Bielefeld University, Bielefeld, Germany). Heat shock experiment data were normalized using the median of the signals. For experiments with
entry into stationary phase, since many genes were down-regulated in this condition
(>1,700) (12) (see Table S2 at http://bioinfo.genopole-toulouse.prd.fr
/annotation/iANT/bacteria/rhime/DOC/Bruand2007/index.html), data could not be
normalized using a global method. Instead, we first identified by microarrays and
qRT-PCR analysis three reference genes (of unknown function), which were
expressed at similar levels during exponential and stationary phases of growth
(sma2239, smb21413, and smc00817) (data not shown). The raw data were then
normalized (using Microsoft Excel) with the mean signal of these three reference
genes. For all experiments, M values (log2 experiment/control ratio) and P values (t
test) were calculated with EMMA. All data are available in Table S1 (http://bioinfo
.genopole-toulouse.prd.fr/annotation/iANT/bacteria/rhime/DOC/Bruand2007/index
.html).
Quantitative RT-PCR analyses. Reverse transcription was performed using
Superscript II reverse transcriptase (Invitrogen) with random hexamers as primers.
RNA samples isolated from at least three independent experiments were tested for
each condition. Real-time PCRs were run on a LightCycler system (Roche) using the
PLUS
FastStart DNA Master SYBRGreen I kit (Roche) according to the
manufacturer’s instructions. 16S rRNA was used as a reference for
normalization. The sequences of the primers used are available in
Table
S1
(http://bioinfo.genopole-toulouse.prd.fr/annotation/iANT/bacteria/
rhime/DOC /Bruand2007/index.html).
Stress resistance assays. To test resistance to salt stress, bacterial cultures in VMM
containing 2 or 10 mM Na2 succinate were grown to exponential or stationary phase
and treated with NaCl at a final concentration of 2.5 M, and cell viability was
measured by plating cells at 0, 30, 60, and 90 min after the salt shock.
Resistance to heat shock was tested in several ways. Exponential-or stationary-phase
cultures were grown at 28°C in rich (TY or LBMC) medium or minimal medium
(VMM) and exposed to high temperature (40°C, 45°C, 47°C, or 49°C), and the cell
viability was measured by plating cells at 0, 30, 60, and 90 min after the temperature
upshift. Alternatively, cultures were grown in VMM at various temperatures, and the
growth rates were compared.
Resistance to H2O2 was measured either by a disk inhibition assay (5 flof3% H2O2) or
by adding up to 4 mM H2O2 to exponential-or stationary-phase cultures grown at
28°C in VMM and measuring cell viability by plating them at 0, 15, 30, and 60 min
after exposure to stress.
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TABLE 2. General stress genes identified by microarray analysis
M valueb
Genea
Description
sma0612
sma0617
Stat vs. expoc
40 vs.
28°Cd
fixN3 cytochrome c oxidase subunit 1
fixP3 cytochrome c oxidase membrane-anchored subunit
2.60e
2.32
1.03e
1.30
sma0621
fixI2 E1 E2-type cation ATPase
1.52
1.07
sma1677
Hypothetical protein
1.74
1.06
sma1720
Putative LysR family transcriptional regulator
1.43
1.13
sma1765
Hypothetical protein
2.21
1.29
sma2071
Hypothetical protein
1.90
1.78
sma2253
Conserved hypothetical protein
1.14
1.50
smb20094
Putative phospholipase protein
1.26
1.43e
smb20204
pqqA putative pyrroloquinoline quinone synthesis protein A
3.71
1.19
smb20227
ndiA1 probable nutrient deprivation-induced protein
1.75e
1.32e
smb20228
ndiA2 putative nutrient deprivation-induced protein
1.92
1.78
smb20251
Hypothetical protein
1.07
1.21
smb20331
Hypothetical protein
3.81
1.45
smb20454
Conserved hypothetical protein
1.09
1.41
smb20879
Hypothetical protein
1.31
1.52
smb20933
exsG putative two-component sensor histidine kinase
1.45
1.67
smb20934
exsF putative two-component response regulator protein
1.09
1.22
smb21221
Putative sugar uptake ABC transporter periplasmic solute binding
1.76
1.28
protein precursor
smb21442
Hypothetical protein
1.61
2.05
smb21456
Hypothetical protein
5.14e
2.58e
smb21473
Conserved hypothetical protein
1.74
1.43
smb21481
Conserved hypothetical protein
1.30
1.51
smb21572
Putative amino acid uptake ABC transporter periplasmic solute binding
1.79
1.67
protein precursor
smb21640
paaA putative phenylacetic acid degradation protein
2.34
1.00
smb21673
Hypothetical protein
1.61
1.65
smc00048
Conserved hypothetical protein
1.65
1.04
smc00063
Hypothetical transmembrane protein
1.74
1.22
smc00371
Conserved hypothetical protein
1.83
3.97e
smc00506
Hypothetical transmembrane protein
4.57
1.23
smc00665
Hypothetical/unknown protein
2.69
1.18
smc00795
Conserved hypothetical protein
2.06
1.15
smc00796
Hypothetical transmembrane protein
2.24
1.98
smc00800
Hypothetical transmembrane protein
2.72
2.26
smc00885
Hypothetical transmembrane signal peptide protein
3.78e
3.57e
smc00931
Hypothetical/unknown protein
2.12
1.50
smc01140
Probable sigma 54 modulation protein
2.61
1.15e
smc01266
Conserved hypothetical protein
2.96
1.28
smc01267
Conserved hypothetical protein
2.85
1.57e
smc01504
Putative receiver domain protein
2.45e
1.77e
smc01505
smc01506
smc01758
Hypothetical protein rpoE2 putative RNA polymerase uE factor
4.17e 2.70e
groEL4 60-kDa chaperonin B (GroEL) protein
1.26
3.69e
1.78e
1.49
smc01814
Probable glutamate synthase small-chain protein
4.99
1.15e
smc01815
Putative oxidoreductase iron sulfur protein
4.50
1.23
smc01820
Putative N-carbamyl L-amino acid aminohydrolase protein
3.56
1.31
smc01933
Conserved hypothetical protein
1.11
1.08
smc01977
Putative sugar binding periplasmic ABC transporter protein
1.20
1.06
smc02171
Putative periplasmic binding ABC transporter protein
2.75
2.24
smc02172
Putative transcription regulator protein
2.22
1.48
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a
b
sma, smb, and smc indicate genes located on pSymA, pSymB, and the chromosome, respectively . log2 ratio of hybridization signals. Only genes with M
values of >1 and P values (t test) of <0.05 in both experiments are listed. The results shown are the averages
c
of three independent biological experiments. RNA samples from Rm1021 (wild-type) cells collected
in exponential phase (expo) or soon after entry into
stationary phase (stat). See Materials and Methods for details. RNA samples from Rm1021 (wild-type) cells collected in exponential phase at
28°C or after 30 min at 40°C. Gene validated by qRT-PCR (see Tables 3 and 5).
d
e
TABLE 3. qRT-PCR validation of some of the general stress genes identified by microarray analyses
a
Induction (n-fold ±SD)
Gene Stationary phase Stationary phase
+NaCl (0.25 M)
40°C vs. 28°C (C starvation) (N starvation)
vs. -NaCl
vs. log phase vs. log phase
sma0612 (fixN3) 4.8 (±2.5) 1.6 (±1.0) 14.8 (±5.6) 16.9 (±2.2) smb20227 (ndiA1) 16.7 (±14.2) 9.4 (±6.1) 28.5 (±5.6) 6.4 (±2.3) smb21456 8.4 (±5.4) 5.0
(±0.8) 142.0 (±25.3) 57.5 (±10.2) smc00885 153.6 (±93.5) 145.3 (±92.3) 93.7 (±23.1) 48.4 (±24.4) smc01504 106.7 (±72.1) 92.3 (±56.7) 80.8 (±26.3) 34.2
(±9.7) smc01505 30.7 (±7.0) 18.4 (±9.0) 58.2 (±21.8) 19.8 (±5.2) smc01506 (rpoE2) 39.5 (±23.1) 14.4 (±6.9) 32.4 (±7.9) 13.2 (±4.3)
a
Results are the average of at least three independent biological experiments.
Resistance to low pH was measured by diluting 100 fl of exponential-or stationary-phase cultures grown at 28°C in VMM in 900 fl of citrate-phosphate buffer, pH 3.5 (0.035
M citric acid, 0.03 M Na2HPO4), and measuring cell viability by plating cells at 0, 30, 60, and 90 min after low-pH exposure.
Plant assays. For plant assays of symbiotic phenotypes, seeds of Medicago sativa cv. Europe or Medicago truncatula Gaertn. cv. Jemalong A17 were surface sterilized,
4
germinated on agar plates, and allowed to grow on nitrogen-free Fahreus medium in test tubes for 3 days. Ten plants were inoculated with -5 X 10 bacteria/plant of the
mutants or the corresponding wild-type strains. The nodulation kinetics and aspect of the plant were followed for 30 days. The whole test
was performed at least twice independently on M. sativa.
Microarray data accession numbers. The entire set of microarray data has been deposited in the ArrayLims database (https://www.cebitec.uni-bielefeld.de
/groups/brf/software/arraylims/).
RESULTS
Identification of S. meliloti genes up-regulated in response to general stress. In order to identify genes transcriptionally induced
during the general stress response of S. meliloti,we looked for genes up-regulated in exponentially growing cells challenged by a stress, as
well as in cells entering stationary phase.
To identify genes induced in logarithmic phase under stress conditions, we exposed exponentially growing bacteria to high temperature
(40°C) for 30 min and compared, using microarrays, their transcription profiles to that of control, unstressed bacteria (28°C). A total of
169 genes were found to be up-regulated (M > 1; P < 0.05) at 40°C in comparison to 28°C, including expected genes, like the
chaperone-encoding groESL operons, as well as genes encoding putative heat shock proteins (smb21294, smb21295, smc02577, and
smc04040) (see Table S2 at (http://bioinfo.genopole-toulouse.prd.fr /annotation/iANT/bacteria/rhime/DOC/Bruand2007/index.html).
To determine the nature of genes induced upon entry into stationary phase, we compared the transcription profiles of bacteria growing
exponentially to that of bacteria taken -1.5 h after entry into stationary phase. This kind of experiment had been described previously (12)
but was repeated here using a minimal medium containing one-fifth of the original concentration of the carbon source (sodium succinate),
allowing the bacteria to enter stationary phase at a much lower density (OD 600, -0.25, in comparison to 1.2 in the previous study [12]). This
was done to ensure that the only limiting element in the medium was the carbon source and to rule out possible effects linked to
high-density cultures. A total of 374 genes were up-regulated (M > 1; P < 0.05) in bacteria entering stationary phase in comparison
to exponentially growing bacteria (see Table S2 at http://bioinfo.genopole-toulouse.prd.fr/annotation/iANT/bacteria
/rhime/DOC/Bruand2007/index.html).
Sixty genes were induced, both in exponential phase after a heat shock and upon entry into stationary phase (Table 2). The induction of
some of these genes under these conditions was confirmed by qRT-PCR (Table 3). The seven genes tested were also induced under two
additional conditions, i.e., upon entry into stationary phase following nitrogen starvation, as well as in exponential phase after a treatment
with 0.25 M NaCl (Table 3; note that fixN3 was hardly induced under the latter conditions). This confirmed that these genes are part of the
general stress response of S. meliloti.
Transcription of rpoE2 is induced by various stress conditions and is positively autoregulated. Among the 60 stress response genes
identified above, we paid particular attention to smc01506 (rpoE2), which encodes a putative ECF-type sigma factor. Using qRT-PCR
analysis, we confirmed the transcriptional induction of this gene upon heat shock and carbon starvation and additionally showed that it was
induced after saline stress or nitrogen starvation (Table 3). Interestingly, the two genes near rpoE2, named smc01504 and smc01505 (Fig.
1), were both up-regulated under the same conditions as rpoE2 (Table 3). The location of smc01505 upstream from rpoE2,as well as its
coinduction with rpoE2 under various conditions, suggests that the two ORFs are cotranscribed from a promoter located in the
smc01504-smc01505 intergenic region. The presence of promoters in this region was tested by cloning it in either orientation in front of the
promoterless lacZ reporter gene of plasmid pCZ750, giving plasmids pLS6.20 and
pLS6.32. The l-galactosidase activities of the resulting transcriptional fusions were
then measured in wild-type cells of S. meliloti. As shown in Table 4, the
l-galactosidase activities of both fusions were higher in exponentially growing
bacteria exposed to a 30-min heat shock at 40°C than in control bacteria grown at
28°C. Both fusions also displayed higher activities in cells collected in late stationary
phase than in cells growing
FIG. 1. Organization of the S. meliloti rpoE2 region. The arrows represent
ORFs and their directions of transcription.
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c
l-Galactosidase activity (Miller units) (±SD)
Promoter fused Strain to lacZa
(genotype)b Exponential Stationary
28°C 40°C
phase phase
Psmc01505
Rm1021 (wt) 160 (±15) 632 (±118) 162 (±25) 1153 (±172) CBT208 (rpoE2) 147 (±17) 148 (±14) 149 (±22) 161 (±11)
Psmc01504
Rm1021 (wt) 453 (±55) 923 (±165) 454 (±75) 1,121 (±123) CBT208 (rpoE2) 420 (±25) 445 (±34) 446 (±47) 432 (±20)
a
b
c
Promoter-lacZ fusions were carried by plasmids pLS6.32 (Psmc01505) and pLS6.20 (Psmc01504). wt, wild type. Results are the average of at least three independent
biological experiments.
exponentially (Table 4). These data therefore showed that two
divergent stress-inducible promoters are present in the smc01504smc01505 intergenic region.
ECF sigma factors often control their own transcription. To test
the possibility that RpoE2 is self-regulated, we constructed an
rpoE2 mutant. In E. coli, disruption of the rpoE gene is lethal,
and existing rpoE mutants actually contain one or several
suppressor mutations (10, 16). To minimize this problem during
the construction of the S. meliloti rpoE2 mutant, we performed a
two-step gene inactivation. For this, an -1-kb region containing the
rpoE2 gene disrupted with the hph gene was cloned into the
plasmid pJQ200mp19, which is nonreplicative in S. meliloti and
carries the Bacillus subtilis sacB gene, which is toxic in
gram-negative bacteria grown in the presence of saccharose (54).
The resulting construct (pCBT113) was introduced into S. meliloti
by triparental mating, and Hyg colonies were selected. In these
cells, the plasmid had been inserted into the chromosome by
single-crossover recombination. These cells therefore contained
both a wild-type and a mutated copy of rpoE2. Several
independent purified colonies were grown in LBMC medium in
the absence of antibiotic and then plated on the same medium
supplemented with 5% saccharose in order to select bacteria in
which the plasmid had been lost by homologous recombination;
among the resulting Sac Gm colonies, two kinds of recombinants
were theoretically expected, one Hyg , in which the recombination
had regenerated the wild-type situation, and one Hyg , in which the
rpoE2 gene was disrupted by the hph gene. If the rpoE2 gene
was nonessential, we expected -50% of each type, since the Hyg
gene was inserted near the middle of the DNA fragment cloned in
pCBT113. If rpoE2 was essential, we expected only Hyg colonies.
Of a total of 95 Sac Gm colonies isolated from three independent
initial integration events, 45 (47%) were Hyg , and the presence of
only one disrupted copy of rpoE2 was verified by PCR in 11 of
these colonies. One of these (CBT208) was kept for further
analyses. These observations suggested that the rpoE2 gene is not
essential. The presence of two rpoE2::Tn5 mutants in a library of
5,000 transposon mutants constructed by Pobigaylo and
collaborators (53) is in agreement with this observation.
As rpoE2 was disrupted at the 61st nucleotide of the ORF in
our rpoE2::hph mutant, rpoE2 transcription could not be easily
measured by qRT-PCR in this strain. We therefore measured only
the transcription of the upstream ORF, smc01505, which is likely
in an operon with rpoE2. To gain insight into other possible genes
r
r
controlled by RpoE2, we also quantified the transcripts of the
adjacent divergent gene smc01504. Whereas the basal levels of
expression of these genes at 28°C were similar in the wild type and
the rpoE2 mutant (not shown), the transcription of both smc01505
and smc01504 was no longer further elevated in the rpoE2 mutant
upon temperature upshift (Table 5) (the groEL5 gene, used as a
control, was induced by heat shock in both wild-type and mutant
strains). To check that this result was not specific to the genetic
background (Rm1021), we performed a similar experiment in a
rpoE2::Tn5 mutant isolated in the Rm2011 strain (53). In this
strain, the transposon was inserted further downstream in rpoE2,
allowing us to quantify rpoE2 transcripts as well. We could
confirm the data obtained as described above for smc01505 and
smc01504 in the Rm1021 background, and we additionally
verified that rpoE2 was no longer inducible in the mutant (n =
1; data not shown). We further confirmed these observations
using the
TABLE 5. Quantification of the induction by heat shock of selected
genes in wild-type and rpoE2 mutant cells of S. meliloti
Induction (n-fold) after heat shock
a
(±SD) in strain: Gene Rm1021
CBT208 (wild type) (rpoE2)
s
s
RpoE2-dependent sma0113 6.2 (±1.1) 1.5 (±0.5) sma0134 21.4 (±14.5)
1.2 (±0.4) smb20007 (katC) 29.5 (±13.9) 1.5 (±0.6) smb20094 36.7
(±23.7) 1.3 (±0.4) smb20227 (ndiA1) 16.7 (±14.2) 1.1 (±0.4) smb21484
(rpoE5) 59.7 (±17.7) 0.5 (±0.2) smc00371 130.5 (±100.1) 1.4 (±0.7)
smc00885 153.6 (±93.5) 1.2 (±0.3) smc01504 106.7 (±72.1) 1.6 (±0.6)
smc01505 30.7 (±7.0) 1.3 (±0.5) smc03873 (rpoH2) 21.2 (±9.7) 1.6
(±0.4)
r
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s
r
s
r
Independent or partially dependent on RpoE2 sma0612
(fixN3) 4.8 (±2.5) 4.3 (±1.4) smb21456 8.4 (±5.4) 4.6
(±1.3) smb21566 (groEL5) 124.7 (±123.5) 223.7
(±88.8) smc01140 3.3 (±1.5) 3.2 (±1.0) smc01267 10.9
(±4.3) 7.8 (±3.1) smc01814 5.3 (±3.9) 8.3 (±2.6)
smc02885 4.2 (±2.2) 3.6 (±2.7)
b
Measured by qRT-PCR as described in Materials and Methods.
The results are the averages of at least three independent biological
experiments.
Although designed to amplify groEL5, the primers used for PCR may also
a
b
amplify other groEL genes that are highly homologous to groEL5.
Downloaded from jb.asm.org at Albert R. Mann Library on August
24, 2009
plasmid-borne lacZ transcriptional fusions described above (Table 4), and additionally showed that both smc01504 and smc01505-rpoE2
promoters were no longer induced in the rpoE2 mutant in stationary
phase (Table 4). Note, however, that given the high background levels M valueb
Genea
Description
of l-galactosidase expression in nonstressed bacteria and in the rpoE2
mutant (Table 4), we cannot exclude the possibility that these genes sma0612
fixN3 cytochrome c oxidase subunit 1
may have two promoters, one recognized by RpoE2 and the other sma0617
fixP3 cytochrome c oxidase membrane-anchored subunit
recognized by an unknown sigma factor. We conclude from these data
sma0621
fixI2 E1 E2-type cation ATPase
that (i) RpoE2 is required for proper stress induction of the
sma1677
Hypothetical protein
transcription of its own operon, as well as that of the neighboring gene
smc01504, and (ii) RpoE2 is therefore activated by these stress sma1720
Putative LysR family transcriptional regulator
conditions.
sma1765
Hypothetical protein
RpoE2 controls a large regulon. We showed above that under
sma2071
Hypothetical protein
several stress conditions, RpoE2 controls the transcription of its own
sma2253
Conserved hypothetical protein
operon, as well as that of the adjacent gene smc01504. To determine
whether the RpoE2 regulon extends beyond these genes, we smb20094
Putative phospholipase protein
compared, using microarrays, the transcription profile of wild-type smb20204
pqqA putative pyrroloquinoline quinone synthesis protein A
(Rm1021) cells to that of rpoE2 (CBT208) mutant cells at 28°C and
smb20227
ndiA1 probable nutrient deprivation-induced protein
40°C. Whereas no difference could be detected between the wild-type
ndiA2 putative nutrient deprivation-induced protein
strain and the rpoE2 mutant at 28°C, 41 genes were at least twofold smb20228
(P < 0.05) more highly expressed in the wild-type strain than in the smb20251
Hypothetical protein
mutant at 40°C; reassuringly, these genes included smc01504 and smb20331
Hypothetical protein
smc01505 (Table 6) (rpoE2 also appeared to be more highly
smb20454
Conserved hypothetical protein
expressed in the wild type than in the mutant, but only because of its
Hypothetical protein
disruption). These data suggested that transcription of these genes was smb20879
induced by heat in an RpoE2-dependent manner. The organization of smb20933
exsG putative two-component sensor histidine kinase
these genes suggests that some of them are cotranscribed in operons, smb20934
exsF putative two-component response regulator protein
reducing the total of putative RpoE2-dependent transcription units to
smb21221
Putative sugar uptake ABC transporter periplasmic solute binding
34. In addition to smc01504 and smc01505, already described above,
protein precursor
seven of these genes were confirmed by qRT-PCR to be RpoE2
dependent (Table 5). Another (smb21456) was significantly induced smb21442
Hypothetical protein
in the mutant, although twofold less than in the wild-type strain, smb21456
Hypothetical protein
suggesting either that it was a false positive or that it is only partially
smb21473
Conserved hypothetical protein
dependent on RpoE2 (Table 5).
smb21481
Conserved hypothetical protein
As evidence to show that RpoE2 directly controls the 34
transcription units, we examined the nucleotide sequences of their smb21572
Putative amino acid uptake ABC transporter periplasmic solute binding
upstream regions in order to find possible RpoE2 binding sequences.
protein precursor
Interestingly, a motif was found to be conserved upstream in all of
paaA putative phenylacetic acid degradation protein
them, with the consensus GGAACNaN13-14 gcgTTt (Fig. 2). This smb21640
Hypothetical protein
sequence was located upstream from single ORFs, or from the first smb21673
ORF in putative operons, except in six instances, where it was located smc00048
Conserved hypothetical protein
in the predicted coding sequence (sma2071, smb21456, and smc00063
Hypothetical transmembrane protein
smb21673) or overlapping with the start codon (sma0541, smb20092,
smc00371
and smb21442). However, a wrong prediction of the start codon for these ORFs is Conserved
possible, hypothetical
as most of protein
them encode proteins with unknown
smc00506
Hypothetical
function and/or have no homologue in the NCBI database. Interestingly,
the sequence
GGAAC transmembrane
is often foundprotein
in the -35 region of promoters
recognized by other ECF sigma factors, and the AAC triplet seems to play
an importantHypothetical/unknown
role in the interactionprotein
of the sigma factor with the promoter
smc00665
DNA (40). This finding strongly suggests that RpoE2 directly controls the transcription of the identified genes by binding the conserved sequences,
smc00795
Conserved hypothetical protein
which likely represent the -35 and -10 elements of the promoters (Fig. 2). However, since another ECF-encoding gene (rpoE5) is among the
smc00796
Hypothetical transmembrane protein
RpoE2 targets, we cannot exJ. BACTERIOL.
TABLE 6.
a
smc00800
Hypothetical transmembrane protein
smc00885
Hypothetical transmembrane signal peptide protein
S. meliloti genes whose up-regulation by heat is
RpoE2
dependent
as identified by microarray
smc00931
Hypothetical/unknown
proteinanalysis
smc01140
Probable sigma 54 modulation protein
smc01266
Conserved hypothetical protein
log2 ratio (wild type/rpoE2) determined by microarray hybridization of RNA samples isolated from Rm1021 (wild type) and CBT208 (rpoE2) cells cultivated for 30 min at 40°C. Only
smc01267
Conserved hypothetical protein
genes with an M value of >1 and a P value (t test) of
<0.05 are listed. The results shown are the averages of three independent biological experiments. The smc01506 gene (rpoEZ) was omitted from the list, as its apparent lower expression in
smc01504
Putative receiver domain protein
the mutant strain was actually due to its disruption.
b
Genes validated by qRT-PCR (see Table 5).
smc01505
smc01506
smc01758
Hypothetical protein rpoE2 putative RNA polymerase uE factor
smc01814
Probable glutamate synthase small-chain protein
smc01933
Conserved hypothetical protein
smc01977
Putative sugar binding periplasmic ABC transporter protein
smc02171
Putative periplasmic binding ABC transporter protein
groEL4 60-kDa chaperonin B (GroEL) protein
clude the possibility that this sigma factor controls some of the putative RpoE2 targets by binding the same sequences.
smc01815
Putative oxidoreductase iron sulfur protein
A search for the sequence GGAACN16-17cgTT using PATSCAN software revealed that this sequence is highly represented in the S. meliloti
smc01820
N-carbamyl
acid
aminohydrolase
genome. Among the genes carrying this sequence in their upstream regions
was rpoH2 Putative
(smc03873),
whichL-amino
encodes
a heat
shock-typeprotein
sigma factor.
Although this gene was missing from our microarray analyses, we quantified its transcripts by qRT-PCR and showed that its transcription is
induced by heat shock in an RpoE2-dependent manner (Table 5). We conclude from this experiment that some RpoE2 targets may have been
missed in our microarray analysis. In agreement with this hypothesis, among the 72 genes whose M values were just below the threshold
considered significant in our wild-type versus mutant comparison (0.47 < M < 1; P <
Downloaded from jb.asm.org at Albert R. Mann Library on August 24, 2009
FIG. 2. Alignment of
nucleotide sequences located
upstream from S. meliloti
RpoE2-regulated genes. The
alignment was produced
using the VectorNTI 9.1
software (Invitrogen). The
consensus sequence shows
the nucleotides conserved in
at least 50% of the aligned
sequences (nucleotides 100%
conserved are indicated in
uppercase
letters).
The
position of the predicted
translation start codon is
indicated on the right;
asterisks indicate cases in
which the predicted start
codon is located either
upstream from or overlapping
with the conserved sequences
(see the
text).
0.05), 5 are possibly
transcribed in an operon
with, and 5 are close to,
genes shown above to
depend on RpoE2, and
many (>60%) carry a
putative RpoE2 binding
sequence
in
their
upstream regions (see Table S3 at http://bioinfo.genopole-toulouse.prd.fr /annotation/iANT/bacteria/rhime/DOC/Bruand2007/index.html).
Interestingly, among the 60 general stress genes identified in the present study, 20 were identified as RpoE2-dependent genes by
microarrays, and 2 could be predicted as RpoE2 targets because they are in operons with actual RpoE2 targets (smb21442 and smb20934).
Moreover, we found by qRT-PCR analysis that smb20094, located close to an RpoE2 target (smb20092), is also dependent on RpoE2
(Table 5). In contrast, five additional randomly chosen general stress genes showed RpoE2-independent induction (Table 5).
Altogether, these data show that RpoE2 controls a large regulon and is an important regulator of the general stress response, as more
than one-third (23/60) of the stress response genes identified in this study are controlled by RpoE2.
Phenotypic analysis of the rpoE2 mutants. RpoE2 appears to respond to several stress conditions by inducing a large regulon, including
genes involved in stress response. To exclude any strain-specific effect, the two types of rpoE2 mutants available to us (see above
and Table 1) were tested. We could not detect any apparent difference between the rpoE2 mutants and their wild-type counterparts.
Both strains exhibited the same doubling time in rich and in minimal media and showed comparable viabilities, both in exponential and
stationary phases of growth. We determined the capacities of rpoE2 mutant cells to resist various stresses and found that rpoE2 mutants
did not differ from the wild types in their resistance to heat, salt, acidic pH, or H 2O2, in both exponential and stationary phases of growth
(see Materials and Methods) (data not shown). The capacities of the rpoE2 mutants to establish symbiotic interactions with M. sativa and
M. truncatula plants were also tested. No difference could be observed between the mutants and the wild-type strains in the
kinetics of nodule formation or in the number and aspect of nodules, or in the final aspect of the plants (data not shown).
The smc01505 gene encodes a negative regulator of the RpoE2 regulon. To learn the possible function of smc01505, located
immediately upstream from and in the same operon as rpoE2, we decided to mutagenize it. In order not to perturb the
transcription-translation of rpoE2, we intended to introduce an in-frame deletion into smc01505. For this, we cloned in pJQ200mp19 an
-1-kb region surrounding smc01505-rpoE2 and containing a 129-bp in-frame deletion in smc01505 (see
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Materials and Methods). This plasmid (pLS5.1) was introduced
into S. meliloti Rm1021 by triparental mating, and Gm cells were
selected in which the plasmid was integrated in the genome
through single-crossover recombination. These cells therefore
contained both a wild-type and a deleted copy of smc01505. In a
second step, we selected for excision of the plasmid by growing
cells in the presence of 5% saccharose. If smc01505 was not
essential, we expected to get wild-type and mutant cells at
equivalent frequencies, as described above for the disruption of
rpoE2, since the deletion is located in the middle of the DNA
fragment cloned in pLS5.1. However, PCR analyses performed on
66 Sac Gm colonies, obtained from two independent events of
plasmid integration, showed that all of them contained the
wild-type structure, except one in which smc01505 contained the
desired deletion. Nevertheless, further analysis of the latter clone
revealed that the chromosomal region normally located
downstream from smc01505, including the rpoE2 coding
sequence, was not present in this strain (not shown). Altogether,
these observations indicated that the smc01505 ORF could not be
disrupted unless rpoE2 was absent from the strain.
The activities of ECF sigma factors are generally regulated by
anti-sigma factors encoded in the same operon as the sigma factor
itself (27). We therefore suspected that smc01505 could be the
RpoE2 anti-sigma factor and that disruption of smc01505 might
lead to permanent activation of RpoE2, which might be toxic for
the cells, as previously observed in other instances for ECF sigma
factors (31, 61). To test this hypothesis, we first constructed a
strain expressing RpoE2 from a multicopy plasmid. For this, we
inserted into pBBR1MCS-5 a DNA fragment containing the
smc01505-smc01506 region preceded by its own promoter but
carrying an in-frame deletion in smc01505. Whereas we could
easily introduce in S. meliloti the vector alone or derivatives
carrying either smc01505 (pCBT109) or the wild-type
smc01505-smc01506 region (pCBT105), the rpoE2-expressing
plasmid (pLS4.1) gave rise to tiny, slow-growing colonies. These
observations suggested that indeed, RpoE2 could be toxic if
overexpressed without simultaneous overexpression of SMc01505.
To test the possibility that smc01505 encodes a protein with
anti-RpoE2 activity, we introduced in S. meliloti a plasmid
expressing smc01505 under the control of its own promoter
sequences (pCBT109) and tested the expression of various genes
in this strain using qRT-PCR. smc01505 was strongly expressed at
28°C in this strain, i.e., 80-fold as much as in the wild-type strain
containing the empty vector at 28°C (not shown). This high basal
level of transcription was not dependent on RpoE2, as it was
similar in an rpoE2 mutant strain (not shown). This shows that
smc01505 is overexpressed in this strain, and we assume that the
multiple copy number of the plasmid associated with the basal
promoter activity of smc01505 and/or the presence of active
promoter sequences in the plasmid (for instance, the lacZ
promoter located next to the cloning site), is responsible for this
constitutive high expression of smc01505.
We then looked at the expression of six RpoE2-dependent genes
(i.e., rpoE2 itself, smc01504, katC, rpoH2, rpoE5, and
smc00885) in the strain carrying pCBT109 in comparison to the
control strain carrying the empty vector. Whereas the expression
levels of these genes at 28°C were similar in both
Induction (n-fold) after heat shock (±SD) in
a
wild-type strain harboring :
Gene pBBR1-MCS5 pCBT109 (empty vector)
(smc01505)
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TABLE 7. SMc01505 is a negative regulator of the RpoE2 regulon
smc01506 (rpoE2) 51.1 (±8.2) 1.5 (±0.3) smc01504 55.8 (±10.0) 2.7
(±0.6) smc03873 (rpoH2) 32.9 (±13.1) 2.2 (±0.5) smb21484 (rpoE5)
131.1 (±65.5) 1.2 (±0.4) smb20007 (katC) 92.9 (±12.1) 2.5 (±0.7)
smc00885 190.7 (±99.7) 3.4 (±0.8) smb21566 (groEL5) 304.1 (±153.2)
230.9 (±168.4)
b
a
Measured by qRT-PCR, as described in Materials and Methods.
The results are the averages of three independent biological
experiments.
Although designed to amplify groEL5, the primers used for PCR may also
b
amplify other groEL genes that are highly homologous to groEL5.
strains (not shown), all of them were induced at 40°C at a much
lower level (and were even sometimes no longer induced) in the
smc01505-overexpressing strain in comparison to the control
strain (Table 7) (the groEL5 gene used as a control was still fully
inducible in both strains). Therefore, this confirmed our hypothesis
that the smc01505 product is a negative regulator of RpoE2
activity and suggested that this polypeptide could have an
anti-sigma factor function.
DISCUSSION
In this study, we identified a list of 60 S. meliloti genes
up-regulated in exponentially growing bacteria after heat shock, as
well as in cells entering stationary phase following carbon
starvation. Among them, the ndiAB genes, whose functions are so
far unknown, had been previously described as up-regulated by
carbon and nitrogen deprivation, oxygen limitation, and osmotic
stress (15).
Among the 60 stress-induced genes, we characterized in more
detail rpoE2 (smc01506), encoding a putative ECF sigma factor.
We showed that stress induction of the rpoE2 gene is dependent
on RpoE2 itself, which indicates that this sigma factor is activated
by stress. We observed that several different stress conditions
could activate RpoE2: heat shock and salt shock, as well as entry
into stationary phase after carbon or nitrogen starvation. Using a
combination of transcriptomics, bioinformatics, and qRT-PCR, we
identified 44 genes under the control of RpoE2 and defined
putative promoter sequences recognized by this sigma factor.
Strikingly, all these genes were also induced by salt and/or osmotic
stress in a recent transcriptomic study by Domı´nguez-Ferreras et
al. (19), which confirms that RpoE2 is activated by salt stress.
Recently, Bobik et al. (9) showed that 74% (98/132) of the S.
meliloti genes induced by oxygen limitation are under the control
of the FixLJ two-component regulator (see Table S2 in reference
9). None of these genes was found to be dependent on RpoE2. In
contrast, 16 out of the remaining 34 genes induced by oxygen
limitation independently of FixJ were induced by heat shock in an
RpoE2-dependent manner in the present study. We therefore
conclude that RpoE2 likely controls the expression of these genes
under micro-oxic conditions. RpoE2 would thus be the second
most important regulator of gene expression under microaerobic
conditions in S. meliloti. Finally, more than oneDownloaded from jb.asm.org at Albert R. Mann Library on August
24
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third of the general stress genes identified in the present study
proved to be RpoE2 targets. Altogether, these data indicate that
RpoE2 is a major regulator of the general stress response in S.
meliloti.
Although most (>65%) of the RpoE2-regulated genes encode
proteins of unknown function, it is striking that eight of them
encode putative transcription regulators: three sigma factors
(rpoE2 itself, rpoE5, and rpoH2) and five members of
two-component systems (exsF-exsG, sma0113-sma0114, and
smc01504). Whether some of these regulators are responsible for
indirect control of RpoE2-dependent genes is presently not known.
Interestingly, several RpoE2 targets are known or supposed to be
related to stress. Some of them had previously been functionally
shown to be up-regulated under various stress conditions and in
stationary phase: katC, encoding a catalase homologous (48%
identity) to the RpoS-regulated E. coli catalase KatE (63), and
rpoH2, encoding a sigma factor of the heat shock family (49, 50).
smc01504 encodes a putative protein displaying significant
similarity (52% identity) to the recently described PhyR regulator
of Methylobacterium extorquens, involved in stress response
and
phyllosphere
colonization
(25).
An
additional
RpoE2-dependent gene encodes another ECF sigma factor
(rpoE5). In addition to katC, some other genes share homologies
with genes that are controlled by the master regulator of the
general stress response, RpoS, in other gram-negative bacteria:
smc00371, whose product is 40% identical to the E. coli YciF
protein of unknown function (30), as well as the glgA2 and glgX2
genes, possibly involved in glycogen synthesis. However, the
functions of these proteins in stress response are not known.
Interestingly, 55% of the RpoE2 targets map on pSymB. Recently,
Domı´nguez-Ferreras et al. have highlighted the important number
of pSymB genes that are upregulated in response to an increase in
external osmolarity (19). Our data confirm the importance of this
plasmid in S. meliloti stress response.
Despite the fact that the RpoE2 regulon is induced by stress
conditions and contains several stress response genes, no deficiency could be associated with rpoE2 mutations, either in vitro
or in planta. Although we cannot exclude the possibility that the
RpoE2 regulon is involved in resistance to a limited number of
unknown stresses, as observed for some other ECF sigma factors
in other bacteria (for instance, sigF in Caulobacter crescentus
[3]), the large number of genes regulated by RpoE2, as well as the
presence of genes clearly associated with stress responses, makes
us favor two other hypotheses. First, we cannot formally exclude
the existence of secondary mutations compensating for the absence
of rpoE2 in the mutant strains tested. Second, the lack of
phenotype of rpoE2 mutants could be due to the redundancy of
the S. meliloti genome (24). Thus, for instance, RpoE2 controls
the transcription of katC and rpoH2, but the S. meliloti genome
encodes two additional enzymes with catalase activities (KatA and
KatB) and another heat shock sigma factor (RpoH1). Although no
clear symbiotic phenotype could be associated with the lack of
katC (63) or rpoH2 (49, 50), the katA katC, katB katC, and
rpoH1 rpoH2 double mutants were clearly affected in their
symbiotic phenotypes (7, 33, 50, 63), showing that other
genes can compensate for the absence of some RpoE2
targets. Similarly, we can hypothesize that other regulators,
controlling the same genes as RpoE2, can compensate for
its absence. Although the heat induction of most genes tested in
the present study was completely dependent on RpoE2, one gene
(smb21456) was still significantly induced by heat in rpoE2
mutant cells, supporting the possibility that it is controlled by
another regulator in response to heat. If this gene, or other still
unknown similarly regulated genes, is involved in stress resistance,
it could explain the absence of phenotype of rpoE2 mutants.
Interestingly, the genome of S. meliloti contains nine genes
encoding putative ECF sigma factors, in addition to rpoE2, and
we cannot exclude the possibility that some of them complement
the absence of RpoE2, as observed in other systems (for instance,
the large overlaps between the regulons of B. subtilis ECF sigma
factors) (27). One of them (rpoE5) was found to be under the
control of RpoE2 and is therefore unlikely to do so. We are
presently testing whether other S. meliloti ECF sigma factors
are able to complement the lack of RpoE2.
The heat shock sigma factor RpoH1 has been previously
implicated in regulation of gene transcription after heat shock in S.
meliloti and could possibly be active in stationary phase (49).
Nevertheless, we could not find its consensus binding sequence
(CTTNAAN17CCANNT [46]) upstream from any of the 60 general
stress response genes identified here, although we found it
upstream from two genes specifically up-regulated by heat shock
(clpB and ibpA). This suggests that RpoH1 does not play a major
regulatory role in the S. meliloti general stress response.
We have identified smc01505, which is cotranscribed with rpoE2,
as encoding a possible anti-RpoE2 sigma factor, although this
small protein of 55 amino acids does not show any similarity to
known anti-sigma factors. As frequently observed for anti-sigma
factors, this protein is encoded in the same operon as the sigma
factor. However, its gene is located upstream from rpoE2 instead
of downstream, as is usually seen in other systems. The frequent
coexpression of the sigma factor with its anti-sigma factor can be
interpreted as a need for tight regulation of sigma factor activity.
Accordingly, we found that unbalanced expression of either one of
these two proteins leads to deregulation of the response: (i)
inactivation of smc01505 or overproduction of smc01506 both had
a toxic effect, presumably by excessive activation of RpoE2, and
(ii) overexpression of the smc01505 gene product hindered activation of RpoE2 by stress conditions. Similar effects were previously
described for other sigma–anti-sigma pairs: overexpression of E.
coli RpoE or its Pseudomonas aeruginosa homologue AlgU
was found to be toxic to E. coli cells (55, 61), and overexpression
of the corresponding anti-sigma factors was found to inhibit RpoE
or AlgU activity (45, 62). Interestingly, the SMc01505 peptide
does not carry any signal sequence or transmembrane domain,
which is unusual for anti-ECF sigma factors. If this protein is
indeed the anti-RpoE2, this would indicate that this sigma factor is
not activated in response to periplasmic signals, as generally
believed for this type of regulator, but rather by cytoplasmic
signals. The Streptomyces coelicolor ECF u is thus regulated
by a soluble anti-sigma factor that responds to redox change (34,
51). Although the molecular mechanisms leading to RpoE2
activation are still unknown, we can anticipate that at least two
signal transduction pathways exist, since RpoE2 is activated by
both physical/ chemical (heat and salt) and nutrient stresses, which
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TABLE 8. Conservation of the RpoE2 region in a selected group of
alphaproteobacteria
% Amino acid identity to S.
a
meliloti
Alphaproteobacterium SMc01504 SMc01505 SMc01506
Rhizobium etli CFN42 79 61 86 Rhizobium leguminosarum bv. viciae 3841 78 56
b
83 Brucella melitensis 16M 75 66 79 Bartonella quintana Toulouse 66 58 75
Agrobacterium tumefaciens C58 6853 71 Mesorhizobium loti MAFF303099 77
44 70 Rhodopseudomonas palustris CGA009 54 33 62 Nitrobacter winogradskyi
Nb-255 54 33 62 Bradyrhizobium japonicum USDA 110 54 33 62 Rhodobacter
sphaeroides 2.4.1 50 NS 50 Caulobacter crescentus CB15 55 NS 47
Rhodospirillum rubrum ATCC 11170 50 NS 42
Data from NCBI database. In addition to the similarity of their
products, the genes are also syntenic. NS, an ORF of similar size is
present, but the similarity with S. meliloti SMc01505 is not significant.
ORF not predicted/annotated in databases.
a
b
ally signaled in different ways (for example, the recent study of
E. coli u [14]).
The best RpoE2 homologues were found in almost all
free-living alphaproteobacteria, and a striking synteny of the
region was observed (Table 8). Thus, in bacteria phylogenetically
close to S. meliloti, a homologue of smc01505 was systematically
found upstream of the sigma factor-encoding ORF, reinforcing our
hypothesis that coexpression of these two proteins is important
(Table 8) (note that in Brucella species, this ORF had not been
previously detected/annotated in the published genome sequences).
Also, an ORF encoding a response regulator-like protein similar to
SMc01504 was systematically found next to the smc01505-like
genes. Examination of the intergenic regions between
smc01505-and smc01504-like genes revealed in every case the
presence of the putative -35 and -10 promoter sequences defined in
the present study, which further supports the hypothesis that they
are binding sites for these sigma factors (not shown). Phylogenetic
analysis revealed that the tree of RpoE2-like sigma factors is
congruent to the tree of alphaproteobacteria species, which
indicates that this class of sigma factors is of ancient origin (not
shown). Although little is known about these sigma factors, the R.
leguminosarum rpoE2 homologue (called rpoZ) had been
inactivated in a previous study and was suggested to be
autoregulated, although the inducing signal was not known (73).
More recently, the homologue of rpoE2 in Brucella (called
rpoE1 in that case) was disrupted, and the resulting mutant
showed attenuated virulence in mice (17).
Whereas some RpoE2 target genes are conserved in many
bacteria, others are specific to alphaproteobacteria closely related
to S. meliloti (not shown). The putative RpoE2 binding sequences
defined in the present study were found to be conserved upstream
of some of these genes, which suggests that they are similarly
controlled in these bacteria. The rpoH2 gene is an example of
these genes. Bradyrhizobium japonicum has three rpoH genes,
and one of them, called rpoH2 (although phylogenetically closer
to S. meliloti rpoH1) is transcriptionally up-regulated upon heat
shock (47), as is the S. meliloti rpoH2 (this work and reference
49). Its +1 transcription start site after a temperature upshift from
E
28 to 43 or 48°C has been mapped, and -10 and -35 sequences
similar to those defined in the present study were found upstream
of this start site (47). We also observed that the rpoH2
homologues of Rhizobium sp. strain TAL1145 and
Sinorhizobium sp. strain BL3 contain the putative RpoE2 binding
sites in their upstream sequences (data not shown and references
35 and 69), and rpoH2 from Sinorhizobium sp. strain BL3 was
described as being transcriptionally up-regulated upon entry into
stationary phase (69), as was the rpoH2 gene in S. meliloti 1021
(49). This suggests that in all these rhizobia, one of the rpoH genes
is under the control of the RpoE2 homologue in response to heat
shock and entry into stationary phase. Although the functions of
these genes remain elusive, rpoH2 from Rhizobium sp. strain
TAL1145 has been involved in exopolysaccharide biosynthesis
and nodulation (35), whereas rpoH2 from Sinorhizobium sp.
strain BL3 seems to be required for salt stress tolerance (69).
Interestingly, the rpoH2 homologue from Brucella, like the
rpoE2 homologue, is involved in the expression of some virulence
genes in this species (17). Transcription of an rpoH-like gene controlled by an ECF sigma factor has been observed in other
bacteria, including the alphaproteobacterium Rhodobacter
sphaeroides (4), as well as the more distant gammaproteobacterium E. coli (21).
Interestingly, some members of the RpoE2 regulon have been
observed to be up-regulated during the process of infection of M.
sativa by S. meliloti. Thus, using a transcriptional fusion to lacZ,
Jamet et al. observed preferential expression of the katC gene in
infection threads, as well as in the infection zone of the nodule
(33). With a transcriptional fusion to gus, Oke et al. showed
expression of rpoH2 at the apex of the nodule, which could
correspond to the infection zone (49). Using a transcriptomic
approach, we previously showed that 37 of the 44
RpoE2-dependent genes identified here are preferentially
expressed in bacteria isolated from infection threads (12).
Although it has still to be proven that expression of these genes
during infection depends on RpoE2, we can speculate that the
many stress conditions potentially encountered by the bacteria in
infection threads are signals for activation of RpoE2 and therefore
up-regulation of its regulon. However, we were unable to detect
any symbiotic defect of the rpoE2 mutant strain(s).
In summary, we have identified RpoE2 as an important regulator
of the general stress response in S. meliloti. However, the lack of
any phenotype of RpoE2 mutants suggests that other, unidentified
stress responses are activated in these strains. These are presently
being investigated by looking for regulators of the
RpoE2-independent general stress genes identified here.
ACKNOWLEDGMENTS
We thank Anke Becker and the University of Bielefeld for providing
microarrays and some S. meliloti strains, Claudine Zischek for providing
plasmid pCZ750, Lucie Palma for technical assistance during the search for
rpoE2 phenotypes, Marieta Hristozkova for production of some samples in
nitrogen starvation experiments, Se´bastien Carre`re and Ge´raldine Pascal for
help with bioinformatic analyses, and Eliane Meilhoc and Delphine Capela for
critical reading of the manuscript.
This work was supported in part by the De´partement Sante´ des Plantes et
Environnement of INRA.
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Methylobacterium extorquens reveals a response regulator essential for epi
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