Reactive Oxygen Species-Inducible ECF σ Factors of Bradyrhizobium japonicum
et al. (2012) Reactive Oxygen Species-Inducible ECF s Factors of Bradyrhizobium
japonicum. PLoS ONE 7(8): e43421. doi:10.1371/journal.pone.0043421
Reactive Oxygen Species-Inducible ECF s Factors of Bradyrhizobium japonicum
Nadezda Masloboeva 0 1
Luzia Reutimann 0 1
Philipp Stiefel 0 1
Rainer Follador 0 1
Nadja Leimer 0 1
Hauke Hennecke 0 1
Socorro Mesa 0 1
Hans-Martin Fischer 0 1
Pierre Cornelis, Vrije Universiteit Brussel, Belgium
0 Current address: Department of Soil Microbiology and Symbiotic Systems, Estacio n Experimental del Zaid n, Consejo Superior de Investigaciones Cient ficas , Granada , Spain
1 ETH, Institute of Microbiology , Zurich , Switzerland
Extracytoplasmic function (ECF) s factors control the transcription of genes involved in different cellular functions, such as stress responses, metal homeostasis, virulence-related traits, and cell envelope structure. The genome of Bradyrhizobium japonicum, the nitrogen-fixing soybean endosymbiont, encodes 17 putative ECF s factors belonging to nine different ECF s factor families. The genes for two of them, ecfQ (bll1028) and ecfF (blr3038), are highly induced in response to the reactive oxygen species hydrogen peroxide (H2O2) and singlet oxygen (1O2). The ecfF gene is followed by the predicted anti-s factor gene osrA (blr3039). Mutants lacking EcfQ, EcfF plus OsrA, OsrA alone, or both s factors plus OsrA were phenotypically characterized. While the symbiotic properties of all mutants were indistinguishable from the wild type, they showed increased sensitivity to singlet oxygen under free-living conditions. Possible target genes of EcfQ and EcfF were determined by microarray analyses, and candidate genes were compared with the H2O2-responsive regulon. These experiments disclosed that the two s factors control rather small and, for the most part, distinct sets of genes, with about half of the genes representing 13% of the members of H2O2-responsive regulon. To get more insight into transcriptional regulation of both s factors, the 59 ends of ecfQ and ecfF mRNA were determined. The presence of conserved sequence motifs in the promoter region of ecfQ and genes encoding EcfQ-like s factors in related a-proteobacteria suggests regulation via a yet unknown transcription factor. By contrast, we have evidence that ecfF is autoregulated by transcription from an EcfFdependent consensus promoter, and its product is negatively regulated via protein-protein interaction with OsrA. Conserved cysteine residues 129 and 179 of OsrA are required for normal function of OsrA. Cysteine 179 is essential for release of EcfF from an EcfF-OsrA complex upon H2O2 stress while cysteine 129 is possibly needed for EcfF-OsrA interaction.
Funding: This work was supported by grants from ETH Zu rich and from the Swiss National Foundation for Scientific Research (grant no. 31003A-122371). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Extracytoplasmic function (ECF) s factors are alternative
bacterial RNA polymerase s factors that play key roles in the
response and adaptation of bacteria to different stresses and
environments (for reviews and a comprehensive classification, see
[1,2]). ECF s factors are members of the s70 family, which is
divided into four groups. Primary s factors of group 1, to which
the housekeeping s factors belong, contain four conserved
domains 1 to 4 and some of them also comprise an additional
non-conserved region. They usually recognize promoters with the
sequence TTGaca (235) and TAtaaT (210) . In contrast, ECF
s factors belong to group 4 of the s70 family and contain only the
conserved domains 2 and 4. Many of them are thought to respond
to environmental signals, they are often associated with an anti-s
factor, and usually auto-regulate their own expression [1,2,4].
Among the environmental cues are reactive oxygen species (ROS)
which almost all bacteria encounter and against which even
anaerobes have evolved defense mechanisms . In aerobic
organisms, ROS are generated also endogenously, e.g. by
incomplete reduction of oxygen during respiration. The term
ROS is generic, embracing not only free radicals such as
superoxide anion (O22) and hydroxyl radicals (OHN) but also
hydrogen peroxide (H2O2) and singlet oxygen (1O2) (for reviews,
Generation of ROS occurs via different routes (for reviews, see
). Briefly, the best studied enzymatic generation of
superoxide, and consequently hydrogen peroxide, originates from
NADPH oxidases that catalyze the production of superoxide by
the one-electron reduction of molecular oxygen using NADPH as
an electron donor [11,13]. The main source of singlet oxygen is
the photosynthetic apparatus where it is generated in photosystem
II as a side product by energy transfer from excited triplet-state
chlorophyll pigments to O2 . Energy can also be transferred to
molecular oxygen by excited photosensitizers such as phytoalexins
which are produced by plants in response to pathogens . Apart
from plant-derived sources, singlet oxygen is also produced in
natural waters by the exposure of chromophoric dissolved organic
matter to light .
Several ECF s factors have been described to play a role in the
response of bacteria to oxidative stress. Examples are Streptomyces
coelicolor SigR which responds to disulfide stress produced by
superoxide and diamide , Caulobacter crescentus SigT which is
necessary for survival under osmotic and oxidative stress , and
SigF of the same organism mediating the response to oxidative
stress in stationary phase . In the photosynthetic bacterium
Rhodobacter sphaeroides, transcription of rpoE is increased upon singlet
oxygen stress ( ; for review, see ) while RpoE activity is
controlled by the anti-s factor ChrR . Orthologs of the
RpoEChrR system are present in various bacterial species  including
C. crescentus  and Myxococcus xanthus .
Rhizobia, soil bacteria that fix nitrogen in symbiosis with
leguminous plants, are exposed to a wide range of environmental
stimuli, including ROS, both in their free-living state in the soil
and in the interaction with host plants, i.e., during infection and
establishment of symbiosis, during nitrogen fixation in root
nodules, and during senescence of these nodules ( [26,27]; for
reviews, see [28,29]). Accordingly, rhizobia use a set of
transcription regulators to reprogram gene expression in order to cope with
these stresses. Notably, during symbiosis ROS act as signaling
molecules and are needed for an efficient Rhizobium-legume
The soybean endosymbiont Bradyrhizobium japonicum encodes a
total of 23 predicted s factor-coding genes in its genome [30,31].
Whereas two of them are s54-type factors, 21 belong to the s70
family. The latter category includes the housekeeping s factor
SigA (group 1), three RpoH s factors (group 3), and 17 ECF s
factors (group 4) whose relationship is depicted in the phylogenetic
tree shown in Figure 1. With SigA as an outgroup, the tree
subdivides the ECF s factors into two groups of 12 and 5 members
documenting substantial diversity among them. In the larger
group, three pairs of similar s factors are found: EcfS (Blr4928)
and Blr3038, Bll1028 and Blr3042, and Bll6484 and Bll2628,
which show 35%, 55%, and 45% amino acid sequence identity,
respectively. Up to now, only two members of the B. japonicum ECF
s family have been functionally studied in more detail. Most
recently, it was described that EcfS (Blr4928) plays a critical role in
the establishment of a functional symbiosis with soybean .
Previously, we have shown that EcfG (Blr7797) is involved in
tolerance to heat and desiccation as well as in the symbiotic
interaction with soybean and mungbean . Other functionally
studied EcfG orthologs in rhizobia include RpoE2 of Sinorhizobium
meliloti  and RpoE4 of Rhizobium etli  which control
regulons typical of general stress response as does sT of C. crescentus
. Similarly, ECF s factor RpoE of the plant-associated
bacterium Azospirillum brasilense is involved in tolerance to singlet
oxygen and other abiotic stresses . Yet another rhizobial ECF
s factor, RpoI of Rhizobium leguminosarum, is required for synthesis
of the siderophore vicibactin and iron uptake [37,38].
The transcriptome analysis of H2O2-stressed B. japonicum cells,
which is presented here, revealed that the expression of two
predicted ECF s factors is induced by H2O2 and also in response
to treatment with other ROS: Bll1028 (hereafter named EcfQ in
accordance with its ECF s factor function and annotation as
CarQ in Rhizobase (http://genome-legacy.kazusa.or.jp/
rhizobase/Bradyrhizobium) and Blr3038 (hereafter termed EcfF
according to the SigF prototype ECF s factor of this class ). We
have determined the regulons of both s factors, and demonstrated
that mutant strains lacking either one or both ECF s factor(s)
show increased sensitivity to singlet oxygen. Furthermore, we have
analyzed the distinct regulatory mechanisms controlling synthesis
and activity of EcfQ and EcfF. While expression of both genes is
controlled at the transcriptional level, activity of EcfF is
additionally regulated by protein-protein interaction with its
cognate anti-s factor Blr3039 (hereafter termed OsrA for
oxidative stress-response anti-s factor). Conserved cysteine
residues of OsrA are involved in H2O2 responsiveness and
inhibition of EcfF activity under non-stressed conditions.
Transcriptional Profile of B. Japonicum in Response to
H2O2-mediated Oxidative Stress
In order to identify B. japonicum genes involved in oxidative stress
response, global transcriptome analyses were performed with
wildFigure 1. Phylogenetic relationship of 17 predicted ECF s-factors in B. japonicum. The tree (generated by the UPGMA method ) is drawn
(N) or 50% (o) are indicated. The primary s factor (SigA) sequence of B. japonicum is included as an outgroup (dashed branch).
to scale with respect to evolutionary distances. Bootstrap values were obtained after 1,000 repeats, and nodes with a confidence of greater than 90%
type cells that had been treated with 2 mM H2O2 for 10 min and
with untreated wild-type cells (control). To mimic the symbiotic
environment we used micro-oxic conditions as standard condition
for all microarray analysis throughout this study. A total of 225
genes were differentially expressed in response to H2O2 (144
upregulated, 81 downregulated; see Table S1), with 56% of them
encoding proteins of unknown functions. Several genes known to
be involved in the oxidative stress response were upregulated, such
as catalase (blr0778), hydroperoxide resistance proteins (bll4012,
bll0735) and methionine sulfoxide (MetSO) reductases (bll5855,
blr7043). Notably, almost one third (29 genes) of the
H2O2regulated genes that encode proteins of known or predicted
function are transcriptional regulators including five MarR-, four
TetR-, and three LysR-type proteins. Furthermore, transcription
of genes for three s factors was affected by H2O2 exposure. While
blr1883 encoding one of two s54-type s factors of B. japonicum was
slightly down-regulated, the genes for two ECF s factors ecfQ and
ecfF were strongly induced (34.8 and 14.4 fold, respectively). The
latter s factors are the primary focus of this study, and some of
their characteristic features are summarized in Table 1.
Response of EcfQ and EcfF to Different ROS
To validate microarray data obtained for ecfQ and ecfF, and to
gain insight into the expression of ecfQ and ecfF upon treatment
with other sources of ROS, quantitative, cDNA-based real-time
PCR (qRT-PCR) analyses were performed. Besides treatment with
H2O2, the following two reagents were used: paraquat
(methylviologen) generating superoxide, and rose bengal in combination
with light, generating singlet oxygen (1O2). The results shown in
Table 2 document induction of ecfQ and ecfF not only in response
to H2O2 but also to singlet oxygen. Expression of ecfQ, but not ecfF,
is also elevated under treatment with paraquat.
Phenotypic Characterization of Deletion Mutants DecfQ,
D(ecfF-osrA), DosrA, and D(ecfQ, ecfF-osrA)
To further elucidate the role of ECF s factors EcfQ and EcfF in
oxidative stress response, mutant strains DecfQ, D(ecfF-osrA) and
D(ecfQ, ecfF-osrA) were constructed (Figure 2). In addition, strain
DosrA was generated to study the predicted function of OsrA as an
anti-s factor of EcfF. Finally, a deletion strain lacking Blr3042 was
constructed to elucidate the function of this EcfQ paralog
Paradigm of ECF group
Genetically linked anti-s factora
RNA polymerase s factor
s70 region 2 s70 region 4
2 mM H2O2, 10 min
0.2 mM paraquat, 5 min
0.2 mM paraquat, 10 min
0.5 mM rose bengal,
20,000 lux, 10 min
0.5 mM rose bengal,
20,000 lux, 180 min
Light only 20,000 lux, 60 min 1.160.5
aMicro-oxically grown wild-type cells exposed to H2O2 (experiment 1) or to
paraquat (2, 3) for the indicated time were compared to untreated cells.
Similarly, cells exposed to rose bengal plus light were compared to cells
exposed to rose-bengal in the dark (4,5). In the control experiment (6),
lightexposed cells were compared to cells grown in the dark. For details, see
Materials and Methods.
(Figure 1). As the latter strain was indistinguishable from the wild
type in all phenotypic tests, it will not be further discussed in this
Growth kinetics of DecfQ, D(ecfF-osrA), DosrA and D(ecfQ,
ecfFosrA) strains were determined under oxic, micro-oxic and anoxic
conditions. Growth of the mutant strains followed a similar trend
as seen with the wild type under micro-oxic conditions (data not
shown). In oxic and anoxic conditions, growth of strains
D(ecfFosrA), DosrA and D(ecfQ, ecfF-osrA) but not of DecfQ was retarded
compared to the wild type (Figure 3A,B). In anoxic conditions
doubling time of strain DosrA was approximately twice that of the
wild type, and the final optical density reached by this mutant was
lower (Figure 3B).
All mutant strains were symbiotically proficient and
indistinguishable from the wild type when tested on two different soybean
varieties (Glycine max cultivar Williams 82 and cultivar Green
Butterbean), on mungbean (Vigna radiata) and on cowpea (Vigna
unguiculata) (data not shown).
s70 region 2 s70 region 4
DUF1109 (six transmembrane domains)
Further, the sensitivity of the mutants towards different ROS
was tested in filter disk assays, on gradient plates and in spot tests.
All four mutant strains showed increased sensitivity towards
oxidative stress caused by singlet oxygen both on gradient plates
(data not shown) and when spotted on PSY plates containing rose
bengal (Figure 4). In filter disk assays, the mutants showed a
wildtype phenotype with respect to their sensitivity to the following
compounds: (i) H2O2; (ii) diamide, a reactive electrophilic species
which affects the thiol redox balance; (iii) FeSO4 that can generate
oxidative stress via the Fenton reaction; (iv) NO-generating agents
such as S-nitroso-N-acetylpenicillamine and S-nitrosoglutathione;
(v) methylglyoxal, a toxic, electrophilic compound; (vi) paraquat
(methylviologen) which causes the formation of superoxide (data
Figure 3. Growth characteristics of B. japonicum wild type and mutant strains. Bacterial cultures of B. japonicum wild type (N) and mutant
strains D(ecfF-osrA) (#), DosrA (m), DecfQ (D), and D(ecfQ, ecfF-osrA) (%) were grown aerobically (A; PSY medium) or anaerobically (B; YEM medium).
Data points are means of three cultures grown in parallel with bars representing standard errors of the means.
Figure 4. Singlet oxygen sensitivity test. Cultures of B. japonicum wild type and mutant strains DecfQ, D(ecfF-osrA), DosrA and D(ecfQ, ecfF-osrA)
were pre-grown to early stationary phase, and aliquots of serial dilutions were spotted on plates containing 0.1 mM rose bengal (two independent
dilution series per strain). The control plate shown in the upper panel was incubated in the dark while the plate shown in the lower panel was light
exposed (2,000 lux) for 1 h to allow generation of singlet oxygen (for more details, see Materials and Methods).
The Regulon of EcfQ
Microarray analysis was used to identify potential target genes
of EcfQ. To this end RNA was isolated from the wild type and the
DecfQ strain, both grown unstressed or stressed by exposure to
H2O2. Expression of nine genes differed between the wild type and
the DecfQ mutant under non-stressed conditions (four
up-regulated, five down-regulated; Table S2A). In H2O2-stressed cells, the
number of differentially expressed genes increased to 34 with seven
genes up-regulated and 27 genes down-regulated (Table S2B). The
latter category might include direct targets of EcfQ given the
positive regulation mode exerted by s factors. However,
inspection of the DNA regions (200 bp) upstream of these genes
did not reveal common motifs that might function as recognition
site of EcfQ. Notably, two thirds of the differentially regulated
genes encode hypothetical or functionally unknown proteins.
Among genes with predicted functions are blr0337 and blr3534
which code for a subunit of putative carbon monoxide
dehydrogenases and are both down-regulated in the mutant.
The Promoter Region of ecfQ and of other Genes Coding
for Class 33 ECF s Factors are Conserved
When we had a closer look at the 13 a-proteobacterial genes
representing the class 33 of ECF s factors to which EcfQ belongs
 we made several observations: (i) in almost every organism of
this group, except Mesorhizobium loti, there are two genes coding for
this type of ECF s factor; (ii) consistently, one of them has a
predicted anti-s factor gene in its proximity, but for the other, a
predicted anti-s factor gene is absent (EcfQ together with five
other class 33 s factors belongs to the latter category); (iii) by
aligning the upstream regions of the six genes of this second group,
a striking pattern of sequence conservation was observed
(Figure 5A). To obtain information on the relative position of
these elements in the promoter, the 59 end of ecfQ mRNA was
determined by primer extension, using RNA isolated from the
B. japonicum wild type grown under different conditions (Figure 5B).
The results of reverse transcription revealed the ecfQ transcription
start point at a C located 44 nucleotides upstream of the annotated
ecfQ start codon (Figure 5B). In agreement with the microarray and
qRT-PCR analyses, the amount of cDNA derived from RNA in
H2O2-treated cells (lane 2) was higher than the amount derived
from untreated cells (lane 1). Putative 235 and 210 promoter
boxes were identified, forming the consensus GCAGAC and
TAACAAT, respectively, however, the spacing between the motifs
is unusually long (20 nt).
Several additional stretches of nucleotides are also conserved in
the upstream region of ecfQ and the other five s factor-coding
genes which belong to the same group. A stretch reading GAAAC
is repeated several times in the upstream region (boxes labeled
226, 259, and 280 in Figure 5A). At box 280, the GAAAC
sequence is part of the inverted repeat TGTTTC-N172GAAACA
(Figure 5A). Database searches with the virtual footprint tool
Prodoric to find regulators that might bind to this region revealed
no obvious candidates. Also, the identified region does not
resemble any described binding sites for several B. japonicum
regulators such as Irr, Fur, FixK2, and RegR .
The Regulon of EcfF
To identify genes possibly controlled by s factor EcfF,
microarray analyses were performed with the D(ecfF-osrA) mutant
strain. In micro-oxically grown, unstressed mutant cells, expression
of only three genes was slightly up-regulated apart from the
obvious decrease of expression of the two deleted genes (Table
S3A). This indicated that in the wild type EcfF is mainly inactive
under these growth conditions. Upon H2O2 treatment, expression
of 22 genes (including ecfF and osrA) differed between mutant and
wild-type cells confirming that EcfF-dependent transcription is
activated by H2O2 exposure (Table S3B). Notably, all regulated
genes had negative fold-change values, which is in line with the
role of EcfF as a positive regulator and suggests that there are
direct target genes in this group. Other than ecfF and osrA no genes
were common to the lists of differentially expressed genes in
unstressed and stressed cells.
To examine the predicted function of OsrA as an anti-s factor,
we also performed microarray experiments with the DosrA mutant
strain. We used unstressed, micro-oxically grown cells in these
experiments because we assumed that even in unstressed cells the
absence of the anti-s factor OsrA should result in up-regulation of
EcfF-dependent genes if the function of OsrA in the wild type were
to inhibit the activity of EcfF under these conditions. Expression of
39 B. japonicum genes (including ecfF and osrA) was altered in the
DosrA strain, with 24 genes (more than 60%) encoding hypothetical
or unknown proteins (Table S4). Only 3 genes had negative
foldchange values (one of them being the deleted osrA gene) while the
large majority of 36 genes was up-regulated, likely due to
hyperactivity of EcfF caused by the absence of its anti-s factor.
Elevated expression of ecfF (18.8 fold) in the DosrA background
suggested that the ecfF-osrA operon is autoregulated. Interestingly,
not only ecfF but also its paralog ecfS (blr4928) was more highly
expressed (3.8 fold) in the mutant pointing to potential cross-talk
between the two s factor2anti-s factor systems.
When the expression data generated with stressed cells of the
D(ecfF-osrA) mutant was compared with that of unstressed DosrA
cells, nine genes (apart from the mutated genes) showed a
regulatory pattern that is expected for EcfF-OsrA being a cognate
s factor2anti-s factor pair, i.e., down-regulation in the
D(ecfFosrA) mutant and up-regulation in the DosrA mutant (Table 3).
Except for blr7044, all genes of this group belong to the
H2O2inducible genes. Notably, genes bll5855, blr7043, blr7044 all
encode predicted peptide MetSO reductases.
Taking into account the predicted operon structure for
bll102726, ecfF-osrA and blr7043-45, the genes listed in Table 3 comprise a
total of seven transcription units with promoters that are primary
candidates for being direct EcfF targets. When we searched in a
200-bp window upstream of the respective start codons for
common putative promoter elements we could indeed identify a
conserved GTAAC(g,a)N14-15(c,t)CG(t,a) motif (Figure 6A,B).
This element is remarkably similar to the tGTAACcN16CGAA
promoter sequence that was proposed for group 16 of ECF s
factors  to which EcfF belongs. The predicted EcfF target
promoter preceding ecfF-osrA was confirmed by primer extension
experiments (Figure 6C). Indeed, a transcript starting at a C
located 6 bp downstream of the predicted 10 box of ecfF was
detected in cells exposed to H2O2 but not in untreated cells. The
experimentally detected transcription start site overlaps the ecfF
ATG start codon annotated in Rhizobase  which argues for
the more distal translational start codon as indicated in Figure 6C.
Likewise, the annotated GTG start codon of bll5855 might be
incorrect because the predicted 210 box of the respective
promoter is located only 8 bp upstream of this start codon
(Figure 6A). When the EcfF consensus motif (GTAAC(g,a)N14
15(t,c)CG(t,a); Fig. 6B) was used as a query for a genome-wide in
silico search (for details, see Materials and Methods) a total of 18
hits were identified of which 7 are associated with the genes or
operons listed in Table 3. The remaining 11 motifs precede genes
that did not fulfill the selection criteria applied to the genes
included in Table 3. Those 11 hits either represent false positives,
or EcfF-mediated regulation of the associated genes is masked by
other unknown regulatory effects.
In Vivo Interaction of EcfF and OsrA
If EcfF and OsrA functioned as a typical cognate s
factorantis factor pair they ought to interact directly at the protein level. We
have used a bacterial two-hybrid system (BACTH system; [42,43])
to further evaluate this model. Plasmids pRJ9746 and pRJ9744
encoding protein fusions of EcfF and OsrA to adenylate cyclase
Cya subdomains T18 and T25, respectively, were constructed
(Figure 7A). Cotransformation of E. coli BTH101 cells with these
plasmids resulted in strain 1 which showed significant
bgalactosidase activity (Figure 7B). By contrast, no b-galactosidase
activity above background was detected in E. coli BTH101 cells
that contained either of the fusion plasmid in combination with the
empty vector of the other hybrid plasmid (data not shown). This
indicated that interaction of EcfF with OsrA enabled functional
complementation of the T18 and T25 adenylate cyclase domains.
Conserved Cysteine 129 of OsrA Might be Required for
Interaction with EcfF
Amino acid sequence alignment of OsrA with orthologous
putative anti-s factors associated with group-16 s factors in other
proteobacteria revealed two highly conserved cysteine residues.
These residues are located at positions 129 and 179 of OsrA, and
they are the only cysteines present in this protein (Figure 8, Figure
To probe the function(s) of the conserved cysteines, mutant
variants of OsrA (C129S, C179S, C129S+C179S) were fused to
T25 (Figure 7A) and tested for two-hybrid interaction in
combination with the T18-EcfF fusion protein. In strain 3
harboring the T25-OsrA C179S fusion, b-galactosidase activity
reached about 70% of the reference strain 1 (T25-OsrA) whereas
in strains 2 (T25-OsrA C129S, C179S) and 4 (T25-OsrA C129S)
only background activity was detected (Figure 7B). Assuming that
the point mutations did not drastically alter protein expression
levels or stability, these results imply that cysteine 129 of OsrA, but
not cysteine 179, is required for interaction with EcfF.
Cysteine 179 of OsrA is Required for the H2O2 Response
of EcfF in B. Japonicum
To validate the data obtained with the E. coli-based two-hybrid
system in B. japonicum and for further functional analysis of the
Gene no.b Fold change
aCells were grown micro-oxically and those of strain D(ecfF-osrA) were exposed
to 2 mM H2O2 for 10 min prior to harvest. Wild-type cells grown under the
respective conditions served as reference in both experiments. Listed are genes
with an absolute fold-change value of .3 in at least one of the mutants and .2
in the other mutant.
bNomenclature according to . Putative operons are shown in italics with
cotranscribed promoter-distal genes indented to the right.
cFold-change values from the comparison of micro-oxically grown wild-type
cells exposed to 2 mM H2O2 for 10 min with untreated cells (see Table S1).
dGene description according to  with modifications.
conserved cysteines of OsrA, expression of the autoregulated ecfF
gene was monitored in derivatives of the DosrA strain
complemented with wild-type or mutant variants of OsrA. To this end,
single copies of wild-type osrA and mutant variants (present on
pSUP202pol4-based plasmids) were chromosomally integrated
into the DosrA strain. The resulting strains (DosrA complemented
with wild-type OsrA, OsrA C129S+C179S, OsrA C179S, or OsrA
C129S) and the control strain DosrA containing the pSUP202pol4
vector integrated in the chromosome (Figure 2B), were grown
under micro-oxic conditions without or with stress exposure
(2 mM H2O2 10 min) prior to cell harvest. RNA was isolated and
reverse-transcribed into cDNA which was used for quantitative
real-time PCR. From the results shown in Table 4 we conclude
that (i) the complementation strategy is effective because wild-type
OsrA restores the normal ecfF expression pattern (cf. Table 2); (ii)
OsrA C129S and OsrA C129S+C179S are not functional because
the respective strains showed a very similar ecfF expression pattern
as the control strain which lacks OsrA; (iii) C179 of OsrA is crucial
for H2O2 responsiveness because ecfF expression in the strains
complemented with OsrA C179S or wild-type OsrA was very
similar under non-stressed conditions; yet in the former strain, no
induction occurred after H2O2 exposure.
Rhizobia are exposed to oxidative stress originating from ROS
that are generated either intrinsically in aerobic metabolism or by
legume host plants during rhizobial infection . Here we have
analyzed the transcriptional response of B. japonicum cells to
oxidative stress. Special emphasis was given to the two ECF s
factors EcfQ and EcfF because (i) their transcription was strongly
induced upon exposure to ROS, and (ii) ECF s factors are typical
regulators in the bacterial stress response. Hydrogen peroxide
exposure of free-living cells which were grown micro-oxically to
mimic symbiotic conditions resulted in altered transcription of
more than 200 genes. Many of them are functionally
uncharacterized, others are related to oxidative stress or encode
transcriptional regulators. Among the latter category are five
MarR-type and three LysR-type regulators which are involved in
the oxidative stress response in various other bacteria .
In recent studies, effects of H2O2 and paraquat exposure on
transcription in oxically grown B. japonicum cells were described
[49,50]. A comparison of that study with our own results revealed
that genes induced by H2O2 in both oxic and micro-oxic cells
comprise those encoding hydroperoxide resistance proteins
(bll4012, bll0735), putative epoxide hydrolase 1 (bll3418), a
putative glutathione S-transferase (bll7849), and the ECF s
factors mentioned above (ecfQ, ecfF). Fold-change values differed
substantially between the two studies which is likely due to
different growth conditions and different microarray platforms.
Remarkably, the gene for catalase KatG (blr0778), whose role in
protection from oxidative stress in B. japonicum is well documented
, appeared to be induced by hydrogen peroxide treatment
only in micro-oxically but not in oxically grown cells. We speculate
that blr0778 is induced even in untreated aerobic cells due to
endogenous ROS production that might be higher in oxic cells
than in micro-oxic cells.
ECF s factors EcfQ and EcfF were functionally characterized
by phenotypic analysis of respective mutants and microarray
analyses. Deletion mutants DecfQ, D(ecfF-osrA), DosrA and D(ecfQ,
ecfF-osrA) were more sensitive to singlet oxygen, and thus
confirmed that both s factors are indeed involved in
oxidativestress tolerance. Singlet oxygen sensitivity of the mutants was
moderately increased and restricted to this type of ROS. This
might be due to an intrinsic tolerance of B. japonicum and/or the
existence of functionally redundant, EcfQ2/EcfF-independent
ROS-protective systems. This hypothesis is in line with the finding
that the regulons of EcfQ and EcfF showed only limited overlap
with the large group of H2O2-responsive genes (Figure S2), and it
also could explain the symbiotic proficiency of the mutants.
In the absence of stress, both s factors are probably inactive
because under these conditions regulation of only few genes was
altered in the deletion mutants, possibly by indirect means as none
of them was differentially expressed in stressed cells (Tables S2 and
S3). Nevertheless, growth of the mutant lacking OsrA was
impaired even without externally applied oxidative stress,
particularly under anoxic conditions, which indicates that hyperactivity
of EcfF might be deleterious.
Transcriptional control of ecfQ and ecfF is likely to occur via
different mechanisms. The presence of conserved motifs within the
ecfQ promoter region points to the involvement of a yet
unidentified transcriptional regulator, a model that is compatible
with the absence of an anti-s factor gene associated with ecfQ. By
contrast, the ecfF-osrA operon appears to be autoregulated, which is
typical for cognate s and anti-s factors genes organized in an
operon. The difference in the regulatory mode may also be
responsible for the differential response of these s factor genes to
treatment with paraquat.
EcfQ and EcfF control rather small and largely distinct groups
of genes. Common to both regulons are only four clustered genes
(bll03310333; blr0337) of which bll0333 encodes a precursor of a
putative alcohol dehydrogenase and blr0337 a subunit of a
predicted carbon monoxide dehydrogenase. Notably, genes
blr0335 and blr0336 encoding two additional subunits of the
latter enzyme are also controlled by EcfF. The regulon of EcfQ is
functionally rather undefined because almost 70% of its members
are hypothetical or unknown proteins. By contrast, more than
70% of the proteins encoded by the genes belonging to the EcfF
regulon have a (predicted) functional annotation. Strikingly, half of
them are oxidoreductases including MetSO reductases Bll5855
and Blr7043 whose genes were induced by H2O2 treatment. A
third MetSO reductase, Blr7044, that is induced 2.9-fold by H2O2
exposure, is yet another member of the EcfF regulon as its
expression was inversely affected in the D(ecfF-osrA) and DosrA
strains (Table 3). Thus, at least three of five MetSO reductases
encoded in the B. japonicum genome are H2O2 responsive and
controlled by EcfF/OsrA (Blr0834 and Bll6260 being the
remaining two). Neisseria gonorrhoeae  and Neisseria meningitidis
 represent two other bacterial species where genes encoding
MetSO reductases are controlled by an ECF s factoranti-s
For many bacterial species the function of MetSO reductases as
antioxidant repair enzyme is well documented ; for review,
see ). Repair of oxidized methionines by MetSO reductases
depends on protein electron donors such a thioredoxin (for review,
see ). Based on its putative signal sequence Blr7043 is
predicted to localize to the periplasm. Thus, for Blr7043 to
function additional components are required which transfer
electrons across the cytoplasmic membrane and deliver them to
this enzyme. DsbDC of E. coli is a well characterized example
which is needed for reduction or isomerization of disulfide bonds
in the periplasm . Based on the predicted topology and
domain structure of Bll1026 (membrane-anchored periplasmic
thioredoxin) and Bll1027 (membrane protein with a DsbD b core
domain), we speculate that Blr7043 in B. japonicum may receive
electrons via these proteins whose genes are co-regulated with
blr7043 by EcfF/OsrA.
In vivo interaction of EcfF with OsrA was demonstrated with a
bacterial two-hybrid system in E. coli. These experiments revealed
that a conserved cysteine at position 129 of the anti-s factor OsrA
is crucial for interaction with EcfF, and this result was further
substantiated by the finding that replacement of this residue led to
constitutive EcfF activity in B. japonicum. The predicted localization
of C129 in the cytoplasmic membrane argues against this amino
acid making direct contact with EcfF. However, it is possible that
OsrA variant tested for complementation of DosrA
aCells were grown micro-oxically without stress or were exposed to 2 mM H2O2 for 10 min prior to harvest. Expression levels of ecfF in different backgrounds were
determined by qRT-PCR and expressed as fold-change values 6 standard errors relative to the expression detected in the pseudo wild-type strain 92-29 under
nonstress conditions. Data are based on three technical replicates of a representative experiment which was repeated in three biological replicates. For details, see Materials
bControl strain containing vector pSUP202pol4 chromosomally integrated downstream of the DosrA::aphII locus (see Figure 2B).
C129 is crucial for keeping OsrA in an interaction-competent
conformation and that its replacement with serine interfered with
The second conserved cysteine of OsrA, C179, is probably
involved in sensing and/or transducing the stress signal because
inhibition of EcfF by the OsrA C179S variant was not released
when B. japonicum cells were stressed with hydrogen peroxide.
Taking into account our data and the predicted OsrA topology
(Figure 8) we propose that oxidative stress detected via
periplasmexposed C179 is signaled to the cytoplasmic portion of OsrA
where it leads to the release of bound EcfF. Although cysteines are
redox-active amino acids and thus well suited to monitor oxidative
stress, C179 of OsrA is not necessarily the primary signal input
site. Inspection of the OsrA amino acid sequence revealed a
striking accumulation of eight methionine residues in two
predicted periplasmic loops (Figure 8), with only two of them
being conserved in the closest B. japonicum paralog TmrS (Blr4929),
the anti-s factor of EcfS (Blr4928) (Figure 1, Figure S1; ).
Given that three MetSO reductases are controlled by EcfF/OsrA
it is tempting to speculate that the presence of multiple
methionines in OsrA makes this regulator intrinsically responsive
to molecules that elicit methionine oxidation.
Amino acids that are critical for oxidative stress signaling were
identified previously in other anti-s factors which, however, are
not homologs of OsrA. Specifically, conserved histidine or cysteine
residues in the ChrR anti-s factor proteins of C. crescentus, R.
sphaeroides, and N. meningitidis are required for proper regulation of
the respective sE proteins in response to organic hydroperoxide
[24,53,63]. Likewise, in M. xanthus blue-light responsiveness of the
membrane-bound CarQ anti-s factor is controlled by another
membrane-associated protein, CarF, whose anti-anti-s factor
activity depends on several histidine residues [64,65].
Our study contributes to the characterization of an ECF s
factor family found in B. japonicum. With EcfQ and EcfF studied
here and the previously described s factors EcfG  and EcfS
, functional information is now available for a total of four
ECF s factors of this bacterium. While the general stress response
regulator EcfG contributes to both free-living and symbiotic traits,
functions of EcfS are largely confined to symbiosis and those of
EcfQ and EcfF to the free-living state. The function of the EcfQ
paralog Blr3042 remains enigmatic as it turned out to be
dispensable under all tested conditions. Challenging goals for
future studies include the characterization of signals, mechanisms
of their transduction, and functions of target genes of EcfQ and
Materials and Methods
Bacterial Strains and Growth Conditions
Bacterial strains used in this work are listed in Table S5.
Escherichia coli strains were grown in Luria-Bertani medium at 37uC
 containing these concentrations of antibiotics for plasmid
selection (mg ml21): ampicillin, 200; kanamycin, 30; tetracycline,
10. B. japonicum strains were cultivated at 30uC aerobically (21%
O2 in the gas phase) or micro-oxically (0.5% O2) in
peptone-saltsyeast extract (PSY) medium supplemented with 0.1% arabinose
, or anaerobically (100% N2) in yeast extract-mannitol (YEM)
medium containing 10 mM KNO3 [67,68]. Where appropriate,
antibiotics were used at these concentrations (mg ml21):
spectinomycin, 100; kanamycin, 100; streptomycin, 100 (solid media) and
50 (liquid media); tetracycline, 50 (solid media) and 25 (liquid
media). Aerobic cultures were grown in vigorously shaken
(160 rpm) Erlenmeyer flasks containing one-fifth of their total
volume of PSY medium. In oxidative stress experiments, cells were
exposed to 2 mM H2O2 for 10 min, conditions that do not inhibit
growth as shown previously .
Mutant strains 0202 (DecfQ), 9688 (D[ecfF-osrA]) and 9692
(DosrA) were constructed by marker-exchange mutagenesis. Briefly,
the 59- and 39-flanking regions of the genes to be deleted were
amplified by PCR using primer pairs listed in Table S6, cloned in
the pGEM-T Easy vector (Promega Corp., Madison, WI, USA),
verified by sequencing, and finally cloned in tandem in vector
pSUP202pol4. A 1.2-kb kanamycin resistance cassette (aphII)
derived from pBSL86  was inserted between the up- and
downstream regions to generate plasmids pRJ0202 (for deletion of
ecfQ), pRJ9688 (for deletion of ecfF plus osrA), and pRJ9692 (for
deletion of osrA). The resulting plasmids were transformed into E.
coli S17-1 and then mobilized by conjugation into B. japonicum
wild-type strain 110spc4 as previously described . The correct
genomic structure of the resulting deletion mutants 0202 (DecfQ),
9688 (D[ecfF-osrA]), and 9692 (DosrA) was verified by PCR. In
strains 9688 and 9692, the cassette was inserted in the same
orientation as the deleted gene(s) while in strain 0202 the cassette
was oriented opposite to the deleted ecfQ gene (Figure 2). The
deletion in strains 0202, 9688 and 9692 spans the genomic regions
from position 191349763 to 191359446, 393559445 to 393569598
and 393569040 to 393569598, respectively.
Strain 15-02 (D[ecfQ, ecfF-osrA]) was constructed as follows: first,
the kanamycin resistance cassette in strain 9688 was replaced by a
spectinomycin/streptomycin resistance cassette (V) resulting in
strain 9715. The cassette exchange was performed by conjugation
into strain 9688 plasmid pRJ9715 whose insert corresponds to that
of pRJ9688 with the kanamycin resistance cassette replaced by the
V cassette inserted between the up- and downstream regions of the
ecfF-osrA genes. Mutant strain 15-02 which is deleted for ecfQ and
ecfF-osrA was obtained by using plasmid pRJ0202 to introduce the
ecfQ deletion into strain 9715 via marker-exchange mutagenesis
(Figure 2). The resulting deletions in strain 15-02 span the same
genomic regions as in the individual mutants described above.
The Dblr3042 strain 0203 was constructed by a markerless
inframe deletion mutagenesis. This approach was chosen because
tiling analysis of microarray data indicated that blr3042 is the
promoter-proximal gene of a tricistronic operon consisting of
blr3042, blr3043 and blr3044. Flanking regions of blr3042 were
cloned into the suicide plasmid pK18mobsacB to yield plasmid
pRJ0203. Plasmid pRJ0203 was transferred by conjugation from
E. coli S17-1 to B. japonicum 110spc4. Kanamycin resistant
exconjugants were selected and grown in the presence of 5%
sucrose to force loss of the vector-encoded sacB gene. Resulting
colonies were checked for kanamycin sensitivity, and the desired
deletion was confirmed by PCR. In the resulting strain 0203 the
genomic region from position 393599337 to 393599926 is deleted.
For complementation of strain 9692 (DosrA) with wild-type OsrA
(resulting strain: 92-29) or mutant variants of OsrA (OsrA C129S:
strain 92-36; OsrA C179S: strain 92-37; OsrA C129S: strain
9238) respective plasmids were chromosomally integrated (see Table
S5). Briefly, a 19116-bp fragment containing the 39 end of ecfF plus
osrA (genome coordinates 393559542 to 393569646) was amplified
by PCR using primer pairs listed in Table S6, cloned in the
pGEM-T Easy vector, verified by sequencing and re-cloned into
vector pSUP202pol4 to yield plasmid pRJ9729. To generate
mutant versions of osrA, we used natural restriction sites within osrA
(NcoI, AscI) and a PstI site at the 39 end of osrA, which was
incorporated via PCR. A 569-bp NcoI-PstI DNA fragment
corresponding to the 39 portion of osrA yet with osrA cysteine
codons 129 and 179 mutated to TCC serine codons was
synthesized (Eurofins MWG Operon, Ebersberg, Gemany).
NcoIPstI, AscI-PstI, or NcoI-AscI restriction fragments of the synthetic
sequence were used to replace corresponding fragments in
pRJ9729 resulting in plasmids pRJ9736 (OsrA C129S, C179S),
pRJ9737 (OsrA C179S) and pRJ9738 (OsrA C129S).
Strain 92-30 served a control and contains vector pSUP202pol4
chromosomally inserted between osrA and bll3040. For its
construction, a 451-bp fragment containing the 39 end of
bll3040 (genome coordinates 393569640 to 393579075) was PCR
amplified using the primer pair listed in Table S6, cloned in the
pGEM-T Easy vector, verified by sequencing and re-cloned in
pSUP202pol4 resulting in plasmid pRJ9729. Plasmids pRJ9729,
pRJ9736, pRJ9737, pRJ9738 and pRJ9730 were transformed into
E. coli S17-1 and then mobilized by conjugation into B. japonicum
strain 9692 as previously described  resulting in mutant strains
92-29, 92-36, 92-37, 92-38 and 92-30, respectively. The correct
genomic structure (Figure 2) of the resulting strains was verified by
Recombinant DNA work was performed according to standard
protocols . B. japonicum chromosomal DNA was isolated as
Analyses of Stress Sensitivity
Zone inhibition assays were performed as described in . The
following compounds were tested at the indicated concentrations:
H2O2 (10 mM, 100 mM, 1 M), diamide (10 mM, 100 mM, 1 M),
FeSO4 (1 mM, 10 mM, 100 mM), S-nitroso-N-acetylpenicillamine
(100 mM), S-nitrosoglutathione (100 mM), methylglyoxal (10 mM,
50 mM). Sensitivity to rose bengal was tested by spotting serial
dilutions of bacteria from late exponential-phase cultures onto 1%
PSY agar containing rose bengal (0.1 mM, 0.2 mM, 0.5 mM). Plates
were illuminated with a tungsten light bulb (100 W, distance 95 cm,
2,000 lux) for 1 or 2 h and incubated in the dark four days at 30uC.
Control plates were not exposed to light.
Plant Growth Conditions and Inoculation.
Soybean (Glycine max [L.] Merr. cv. Williams and cultivar
Green Butterbean), mungbean (Vigna radiata) and cowpea (Vigna
unguiculata [L.] Walp. cv. Red Caloona) seedlings were
surfacesterilized as described [31,71,73,74]. Determination of nitrogenase
activity in bacteroids were performed as described previously .
RNA Extraction and CDNA Synthesis
Harvest and storage of cells, RNA extraction and cDNA
synthesis were done as previously described .
Quantitative Real-time PCR
Expression of genes ecfQ and ecfF was analyzed by reverse
transcription-based quantitative real-time PCR as previously
described . RNA was isolated from micro-oxically grown
mid-log phase wild-type cells that were either untreated or exposed
prior to harvest to one of the following treatments: 2 mM H2O2
for 10 min; 0.2 mM paraquat for 5 or 10 min; 0.5 mM rose bengal
plus light exposure (20,000 lux) for 10 or 180 min; exposure to
light for 60 min (control). Expression of the ecfF gene was analyzed
in strains 92-29, 92-36, 92-37, 92-28 and 92-30 grown
microoxically to mid-log phase, either untreated or exposed to 2 mM
H2O2 for 10 min prior harvesting. cDNA (0.2 to 20 ng) in
combination with 2.5 mM of primers pairs 1028-RT-F and
1028RT-R or 3038-RT-F/3038-RT-R (Table S6) were used for
monitoring expression of ecfQ and ecfF, respectively. The primary
s factor gene sigA was used as a reference for normalization
(primers SigA-1155R and SigA-1069F; ). Data were evaluated
by the method of Pfaffl .
The transcription start site of ecfQ and ecfF were determined as
previously described [76,77]. RNA was extracted from
microoxically grown wild-type cells either non-stressed or treated with
2 mM H2O2 for 10 min. To determine transcription start site of
ecfQ cDNAs were synthesized with primers pe-1028-1 or pe-1028-2
(Table S6). The same primers were used to obtain sequencing
ladders from plasmid pRJ0211 (Table S5), containing the
promoter and part of the ecfQ coding region. Likewise, primers
pe-3038-1 and 3038-RT-R and plasmid pRJ9724 were used for
determination of the transcription start site of ecfF-osrA.
Global transcription levels were determined as described
previously using a custom-designed Affymetrix chip [68,69].
RNA template for cDNA synthesis was isolated from
microoxically grown cells of the wild type and mutant strains 0202, 9688
and 9692, and also of H2O2-treated cells (2 mM, 10 min) for the
wild type and two mutant strains (0202, 9688). For each strain and
condition, a minimum of three biological replicates was prepared.
RNA extraction, cDNA synthesis, fragmentation and labeling
were done as described previously [68,78]. GeneChip data
analysis was performed using GeneSpring GX 7.3.1 software
(Agilent). After filtering for probe sets which were called present or
marginal in at least two out of three replicas, a statistical student
ttest with a P-value threshold of 0.01 was applied. Genes were
considered as differentially expressed if the fold-change value was
,3 or .+3 when comparing two strains or conditions. Data sets
generated in this work are deposited in the GEO database under
record number GSE39165.
Phylogenetic analysis of B. japonicum ECF s factors was
conducted using MEGA version 4 . For alignment of
nucleotide and amino acid sequences, the T-COFFEE program
was used (http://www.ebi.ac.uk/Tools/msa/tcoffee/[80,81]).
Results were visualized with GeneDoc  and BioEdit .
Database searches for regulators that might bind to the up-stream
region of ecfQ were done with the virtual footprint tool Prodoric
(http://www.prodoric.de; ). Search for consensus motifs in
EcfQ- and EcfF-target promoters was performed using the
BioProspector suite (http://ai.stanford.edu/ xsliu/BioProspector/;
). DNA sequences corresponding to 200 bp located upstream
of the promoter-proximal genes listed in Table 3 were used as
input for the analysis. Parameters were set to search for a
twoblock motif with 5 nucleotides per block and a gap of 13 to 16
nucleotides between the blocks. Identified sequence motifs were
aligned and visualized using the WebLogo tool (http://weblogo.
berkeley.edu/; ). Genome-wide searches for putative EcfF
target promoters focused on 200 bp regions upstream of genes or
operons and were performed with the genome-scale DNA pattern
search program from the RSAT collection of sequence analysis
tools (http://rsat.ulb.ac.be/; ) Searches for amino acid
sequence similarities were performed with BlastP (http://blast.
ncbi.nlm.nih.gov/Blast.cgi?PAGE = Proteins). Topology
prediction for OsrA was done with TOPCONS (http://topcons.cbr.su.
se/; ). Protein localization prediction via a signal peptide
search was performed using SignalP 4.0 (http://www.cbs.dtu.dk/
Bacterial Two-hybrid System
For analysis of EcfF-OsrA interactions the BATCH system was
used (Euromedex, Souffelweyersheim, France). Translational
fusions of wild-type and mutant versions of OsrA to the
Cterminal end of the T25 fragment of Bordetella pertussis adenylate
cyclase (Cya) were generated by cloning of PCR-generated
PstIEcoRI fragments into vector pKT25 (resulting in plasmids
pRJ9744, pRJ9752, pRJ9753, and pRJ9754; Table S5). Primers
are listed in Table S6. For amplification of wild-type osrA,
genomic DNA of B. japonicum 110spc4 was used while mutated
osrA versions were amplified with plasmids pRJ9736, pRJ9737, or
pRJ9738 as templates. In parallel, a translational fusion of EcfF
to the C-terminal end of the Cya T18 fragment was generated.
To do so, a PstI-XbaI fragment containing the wild-type ecfF
gene was amplified (primer pair listed in Table S6, genomic B.
japonicum DNA as template) and cloned into vector pUT18C
yielding plasmid pRJ9746. All constructed plasmids were verified
by sequencing. To study interaction of EcfF with different
versions of OsrA, E. coli strain BTH101 was co-transformed with
pRJ9746 and one of the plasmids expressing a T25-OsrA fusion.
For b-galactosidase activity assays, co-transformed clones were
inoculated into 6 ml LB medium containing appropriate
antibiotics and 0.5 mM IPTG (isopropyl
b-D-1-thiogalactopyranonoside). Cultures were grown for 18 h at 30uC, and aliquot(s)
from 50 ml to 200 ml were used to determine b-galactosidase
activity as described elsewhere .
Figure S1 Alignment of OsrA-homologs. Numbers on the
right of each line refer to the position of the last amino acid within
the corresponding protein sequence. Shaded in black, dark grey,
and light grey are nucleotides which are identical in all, 80%, and
60% of the sequences, respectively. Arrowheads indicate
conserved cysteines. GI numbers of the proteins are as follows:
Bradyrhizobium japonicum USDA 110 (BJ) OsrA - 81738347 and
TmrS (Blr4929) - 81736761, Mesorhizobium loti MAFF303099 (ML)
81779508, Agrobacterium tumefaciens str. C58 (AT) 15889542,
Rhizobium etli CFN 42 (RE) 123508957, Sinorhizobium meliloti 1021
(SM) 81813033, Burkholderia pseudomallei (BP) 81379776, Pseudomonas
putida KT2440 (PP) 81442010, Dechloromonas aromatica RCB (DA)
Daro_152171907153 and Daro_258971908203.
Figure S2 Venn diagram of H2O2-responsive genes in
the B. japonicum wild-type strain and the regulons of
ECF s factors EcfQ and EcfF. Hydrogen peroxide-responsive
genes were identified by transcriptome analyses of untreated
wildtype cells with cells exposed to 2 mM H2O2 for 10 min. Similarly,
regulons of EcfQ and EcfF were determined by comparing the
transcriptome of DecfQ and D(ecfF-osrA) mutant strains,
respectively, both treated with 2 mM H2O2 for 10 min, with identically
stressed wild-type cells. All strains we grown micro-oxically. Size
and overlap of the regulons are drawn to scale with numbers of
differentially expressed genes (3-fold change cut-off) indicated in
the respective segments. Total number and numbers of down- (Q)
and up-regulated genes (q) are shown next to individual regulons.
Table S3 List of B. japonicum genes differentially
expressed in the D(ecfF-osrA) strain 9688 compared to
the wild type. Cells were grown micro-oxically and harvested
after no further treatment (A) or after exposure to 2 mM H2O2 for
10 min (B).
Table S4 List of B. japonicum genes differentially
expressed in micro-oxically grown cells of the DosrA
mutant strain 9692 compared to the wild type.
Bacterial strains and plasmids used in this
Primers used in this study.
We thank Gabriella Pessi for advice and assistance in the microarray
experiments. We are grateful to Anne Francez-Charlot and Julia Vorholt
for scientific expertise and helpful discussions. The staff of the Functional
Genomics Center Zurich is acknowledged for the help with the microarray
Conceived and designed the experiments: NM LR SM HMF. Performed
the experiments: NM LR PS RF NL. Analyzed the data: NM LR SM
HMF. Wrote the paper: NM LR HH SM HMF.
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