Convergence of the Transcriptional Responses to Heat
Shock and Singlet Oxygen Stresses
Yann S. Dufour1,2¤, Saheed Imam1,2,3, Byoung-Mo Koo4, Heather A. Green1, Timothy J. Donohue1,2*
1 Department of Bacteriology, University of Wisconsin–Madison, Madison, Wisconsin, United States of America, 2 BACTER Institute, University of Wisconsin–Madison,
Madison, Wisconsin, United States of America, 3 Program in Cellular and Molecular Biology, University of Wisconsin–Madison, Madison, Wisconsin, United States of
America, 4 Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California, United States of America
Abstract
Cells often mount transcriptional responses and activate specific sets of genes in response to stress-inducing signals such as
heat or reactive oxygen species. Transcription factors in the RpoH family of bacterial alternative s factors usually control
gene expression during a heat shock response. Interestingly, several a-proteobacteria possess two or more paralogs of
RpoH, suggesting some functional distinction. We investigated the target promoters of Rhodobacter sphaeroides RpoHI and
RpoHII using genome-scale data derived from gene expression profiling and the direct interactions of each protein with
DNA in vivo. We found that the RpoHI and RpoHII regulons have both distinct and overlapping gene sets. We predicted DNA
sequence elements that dictate promoter recognition specificity by each RpoH paralog. We found that several bases in the
highly conserved TTG in the 235 element are important for activity with both RpoH homologs; that the T-9 position, which
is over-represented in the RpoHI promoter sequence logo, is critical for RpoHI–dependent transcription; and that several
bases in the predicted 210 element were important for activity with either RpoHII or both RpoH homologs. Genes that are
transcribed by both RpoHI and RpoHII are predicted to encode for functions involved in general cell maintenance. The
functions specific to the RpoHI regulon are associated with a classic heat shock response, while those specific to RpoHII are
associated with the response to the reactive oxygen species, singlet oxygen. We propose that a gene duplication event
followed by changes in promoter recognition by RpoHI and RpoHII allowed convergence of the transcriptional responses to
heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria.
Citation: Dufour YS, Imam S, Koo B-M, Green HA, Donohue TJ (2012) Convergence of the Transcriptional Responses to Heat Shock and Singlet Oxygen
Stresses. PLoS Genet 8(9): e1002929. doi:10.1371/journal.pgen.1002929
Editor: William F. Burkholder, Agency for Science, Technology, and Research, Singapore
Received October 10, 2011; Accepted July 16, 2012; Published September 13, 2012
Copyright: ß 2012 Dufour et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIGMS grants GM075273 to TJD and GM057755 to Carol A. Gross (University of California at San Francisco). YSD and SI
were each fellows on the BACTER Grant from the U.S. Department of Energy Genomics: GTL and SciDAC Programs (DE-FG02-04ER25627). YSD was a recipient of a
Wisconsin Distinguished Graduate Fellowship from the University of Wisconsin–Madison College of Agricultural and Life Sciences and of the William H. Peterson
Predoctoral Fellowship from the University of Wisconsin–Madison Department of Bacteriology. 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.
* E-mail: tdonohue@bact.wisc.edu
¤ Current address: Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, United States of America
However, the definition of functional promoters for this family of
alternative s factor using only the presence or the extent of
sequence identity for the predicted 210 and 235 binding regions
is not a sufficient predictor of transcription activity [7].
While bacteria often possess many alternative s factors, they
usually possess only one member of the RpoH family. However,
several a-proteobacteria, including Brucella melitensis [8], Sinorhizobium meliloti [9,10], Bradyrhizobium japonicum [11,12], Rhizobium elti
[13] and Rhodobacter sphaeroides [14], possess two or more RpoH
homologs. In some cases, one or more of these RpoH homologs
completely or partially complement the phenotypes of E. coli DrpoH
mutants, suggesting that these proteins can functionally interact
with RNA polymerase and recognize similar promoter elements [8–
11,14,15]. However, in the nitrogen-fixing plant symbiont Rhizobium
elti, the DrpoH1 mutant was sensitive to heat and oxidative stress
while the DrpoH2 mutant was sensitive to osmotic stress [13].
Therefore, the additional members of the RpoH family in aproteobacteria may have roles in other stress responses.
Previous work demonstrated that either R. sphaeroides RpoHI or
RpoHII can complement the temperature sensitive phenotype of
Introduction
Transcriptional responses to stress are critical to cell growth and
survival. In bacteria, stress responses are often controlled by
alternative s factors that direct RNA polymerase to transcribe
promoters different from those recognized by the primary s factor
[1,2]. Therefore, identifying the target genes for a particular
alternative s factor can help identify the functions necessary to
respond to a given stress. For example, the transcriptional response
to heat shock in Escherichia coli uses the alternative s factor s32 to
increase synthesis of gene products involved in protein homeostasis
or membrane integrity [3]. From available genome sequences,
proteins related to E. coli s32 are conserved across virtually all
proteobacteria. This so-called RpoH family of alternative s factors
is characterized by a conserved amino acid sequence (the ‘‘RpoH
box’’) that is involved in RNA polymerase interactions [4,5].
RpoH family members also possess conserved amino acid
sequences in s factor regions 2.4 and 4.2 that interact with
promoter sequences situated approximately 210 and 235 base
pairs upstream of the transcriptional start sites, respectively [6].
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Convergent Transcriptional Stress Responses
alternative s factor and subsequent changes in promoter
recognition could have allowed convergence of transcriptional
responses to separate signals. In the case of R. sphaeroides, we
predict that these events allowed convergence of the transcriptional responses to heat shock and singlet oxygen stresses to be
under control of these two RpoH paralogs.
Author Summary
An important property of living systems is their ability to
survive under conditions of stress such as increased
temperature or the presence of reactive oxygen species.
Central to the function of these stress responses are
transcription factors that activate specific sets of genes
needed for this response. Despite the central role of stress
responses across all forms of life, the processes driving
their organization and evolution across organisms are
poorly understood. This paper uses genomic, computational, and mutational analyses to dissect stress responses
controlled by two proteins that are each members of the
RpoH family of alternative s factors. RpoH family members
usually control gene expression during a heat shock
response. However, the photosynthetic bacterium Rhodobacter sphaeroides and several other a-proteobacteria
possess two or more paralogs of RpoH, suggesting some
functional distinction. Our findings predict that a gene
duplication event followed by changes in DNA recognition
by RpoHI and RpoHII allowed convergence of the
transcriptional responses to heat and singlet oxygen stress
in R. sphaeroides and possibly other bacteria. Our approach
and findings should interest those studying the evolution
of transcription factors or the signal transduction pathways
that control stress responses.
Results
Defining the distinct and overlapping regulons of R.
sphaeroides RpoHI and RpoHII
To define members of the RpoHI and RpoHII regulons, we
monitored transcript levels and protein-DNA interactions in R.
sphaeroides strains ectopically expressing either RpoHI or RpoHII. To
generate these strains, we constructed low copy plasmids carrying
rpoHI or rpoHII under the control of an IPTG-inducible promoter
[22] and conjugated them into R. sphaeroides mutant strains lacking
rpoHI [16] or rpoHII [15], respectively. To induce target gene
expression, we exposed exponentially growing aerobic cultures to
IPTG for one generation before cells were either harvested to
extract total RNA for analysis of transcript levels or treated with
formaldehyde to prepare samples for chromatin immunoprecipitation on a chip (ChIP-chip) assays. The Western blot analysis used to
measure levels of these alternative s factors demonstrates that cells
ectopically expressing RpoHI and RpoHII contained each protein at
levels comparable to those following either heat shock or singlet
oxygen stress (Figure 1). Thus, these strains can be used to
characterize members of the RpoHI and RpoHII regulons.
As controls for this experiment, we measured the abundance of
individual RpoH proteins and a control transcription factor (PrrA)
[23], which is not known to be dependent on either alternative s
factor for its expression, when wild type cells were exposed to
either heat or singlet oxygen stress. This analysis showed that
RpoHI is detectable prior to heat stress, but its levels increase 10
and 20 minutes after the shift to increased temperature (Figure 1A).
RpoHI levels remain elevated after the temperature shift but they
decline within 60 minutes after heat shock, suggesting that as in
the case of E. coli s32, there is an initial rise in RpoHI levels
immediately on heat shock before they return to a new steady state
level at elevated temperature [24]. RpoHII was also detected prior
to exposure to singlet oxygen and within 10 minutes of exposure to
this reactive oxygen species, levels of this protein were increased
(Figure 1B). Levels of RpoHII found within 20 minutes after
exposure to singlet oxygen remained relatively constant over the
time course of this experiment, suggesting a continuous requirement for RpoHII during this stress response (Figure 1B). The
abundance of the control transcription factor PrrA did not follow
these same trends, suggesting that the observed increases in
individual RpoH proteins was associated with these stress
responses. In addition, the abundance of individual RpoH proteins
did not increase significantly to both stress responses, as expected if
these increases were not due to a general increase in protein levels
in response to different signals.
To identify transcripts that were increased in abundance as a
result of RpoHI or RpoHII activity, we compared mRNA levels of
cells expressing RpoHI or RpoHII ectopically to those of control
cells lacking either rpoHI or rpoHII. We selected differentially
expressed genes with a significance level set for a false discovery
rate #5% and that displayed at least 1.5-fold higher transcript
levels in cells expressing either RpoH family member. This
analysis revealed that transcripts from 241 and 186 genes were
increased by expression of RpoHI and RpoHII, respectively
(Figure 2). These two sets of differentially expressed genes have 60
genes in common.
an E. coli DrpoH mutant; that singly mutant R. sphaeroides strains
lacking either rpoHI or rpoHII are able to mount a heat shock
response; and that RNA polymerase containing either RpoHI or
RpoHII can initiate transcription from a common set of promoters
in vitro [14–16]. Combined, these observations suggest that RpoHI
and RpoHII have some overlapping functions in R. sphaeroides. On
the other hand, in vitro transcription assays identified promoters
that were selectively transcribed by either RpoHI or RpoHII
[14,15]. Moreover, rpoHII is under direct transcriptional control of
RpoE, a Group IV alternative s factor that acts as the master
regulator of the response of R. sphaeroides to singlet oxygen stress
[17–19]. These later results and the recent observation that a
DrpoHII mutant is more sensitive to singlet oxygen stress than the
wild-type strain [15,17] suggest that RpoHI and RpoHII also have
distinct functions in R. sphaeroides. Finally, global protein profiles of
R. sphaeroides mutants lacking rpoHI, rpoHII, or both genes,
suggested that RpoHI and RpoHII have distinct and overlapping
regulons [15,17,20]. However, the extent of genes that are direct
targets for RpoHI and RpoHII is still unknown because past
studies have been unable to distinguish direct from indirect effects
on gene expression or identify all the direct targets for either of
these s factors.
In this study, we characterized the RpoHI and RpoHII regulons
using a combination of expression microarrays, chromatin
immunoprecipitation and computational methods which have
been previously been shown to predict correctly direct targets for
other alternative s factors or DNA binding proteins [19,21]. We
found that the genes predicted to be common to the RpoHI and
RpoHII regulons function in protein repair or turnover, membrane maintenance, and DNA repair. Genes specific to the RpoHI
regulon encode other proteins involved in protein maintenance
and DNA repair, whereas genes specific to the RpoHII regulon
include proteins involved in maintaining the oxidation-reduction
state of the cytoplasmic thiol pool. We used information on the
members of each regulon to generate and test hypotheses about
DNA sequences that determine promoter specificity of these two
RpoH homologs. The observed properties of these two R.
sphaeroides RpoH homologs illustrate how duplication of an
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Convergent Transcriptional Stress Responses
Figure 2. Overlap between the RpoHI and RpoHII regulons.
Venn diagram representing the overlaps between genes that were
significantly induced by the expression of RpoHI or RpoHII, and genes
whose promoters were bound by RpoHI or RpoHII containing RNA
polymerase holoenzyme in vivo. The total numbers of genes identified
in each study are indicated in the parentheses. The RpoHI (solid outline)
and RpoHII (dashed outline) regulons, as defined in this study, are
identified by the emphasized outlines. The total numbers of genes
contained in each regulon are indicated below the arrows.
doi:10.1371/journal.pgen.1002929.g002
Figure 1. RpoHI and RpoHII accumulation following heat and
singlet oxygen stresses. Western blots illustrating the levels of RpoHI
and RpoHII in wild-type R. sphaeroides (WT) at different times following
(A) a shift of temperature from 30uC to 42uC (heat shock) or (B) addition
of the photosensitizer methylene blue in the presence of oxygen
(singlet oxygen stress). On the same western blots, the levels of FLAGRpoHI and RpoHII obtained from ectopic expression vectors used in the
expression profiling and ChIP-chip experiments under normal conditions. Note that because of the addition of the FLAG polypeptide,
RpoHI-FLAG migrates slower than the wild-type RpoHI. The abundance
of RpoHI and RpoHII in wild-type cells in the absence of added stress are
shown in the first lane. As a gel loading control, the membranes were
also subsequently treated polyclonal antibodies against the response
regulator PrrA, a control transcription factor who’s expression is not
known to be dependent on either of the RpoH homologs. The
experiment was designed to analyze changes in levels of RpoHI, RpoHII
and PrrA before and after a stress, so the differences between panels
reflect different exposure times used when developing the Western
blots.
doi:10.1371/journal.pgen.1002929.g001
against RpoHI and RpoHII, respectively, using a significance level
set for a false discovery rate #5%. Because the signal from a single
s factor binding site extends on average over a 1 kb region, some
enriched regions may contain multiple binding sites. To increase
the resolution of the putative RpoHI and RpoHII binding sites, we
identified the modes of the ChIP-chip signal distributions within
each enriched region. This adjustment increased the number of
putative binding sites for RpoHI and RpoHII to 1085 and 1765,
respectively.
We then identified all the annotated genes that contained a
ChIP-chip peak within 300 base pairs upstream of their start
codons as a way to define candidate genes or operons in the
RpoHI or RpoHII regulons. Included in this list of potential
regulon members were genes that are predicted to be cotranscribed using a previous computational analysis of R. sphaeroides
operon organization (http://www.microbesonline.org/operons/)
[26]. Therefore, by these criteria, the upper limits of the total
numbers of genes potentially regulated by RpoHI or RpoHII are
1120 and 1616, respectively (Figure 2). We recognized that a
significant number of the putative RpoHI or RpoHII promoters
may not be assigned from the ChIP-chip dataset alone, especially
because promoter orientation needs to be considered and that
because s factor or RNA polymerase binding events do not always
promote transcription. Therefore, we refined the respective
RpoHI and RpoHII regulons by intersecting the lists of target
genes identified from the ChIP-chip analysis with the lists of
candidate genes identified from the expression profiling analysis.
After this intersection, we predict that the RpoHI regulon contains
175 genes and the RpoHII regulon contains 144 genes with 45
genes common to both regulons (Figure 2).
Upon examining the annotations of these predicted target
genes, the 45 genes that are members of both the RpoHI and
RpoHII regulons are predicted to encode mainly for functions
related to the electron transport chain, protein homeostasis, and
DNA repair (Table 1 and Table S1). The 130 predicted members
of the RpoHI regulon also encode functions in these three groups,
We recognize that some of these differentially-expressed
transcripts might be not be direct targets for RpoHI and RpoHII.
Therefore, to determine which of the above genes were directly
transcribed by RNA polymerase holoenzyme containing either
RpoHI or RpoHII, we performed ChIP-chip assays from
comparable cultures to map direct interactions of RpoHI or
RpoHII with genomic DNA. We were able to raise specific
antibodies against RpoHII that performed well for the ChIP-chip
assay, but repeated attempts to raise suitable antibodies against
RpoHI failed. Therefore, we placed a FLAG polypeptide tag [25]
at the N-terminus of the RpoHI protein sequence and used antiFLAG monoclonal antibodies to perform the ChIP-chip assay. As
a control we tested and showed that addition of the polypeptide
tag did not alter the activity and specificity of RpoHI by
comparing the mRNA level profiles of cells expressing the tagged
version of RpoHI with cells expressing wild-type RpoHI (Figure
S1). In addition, other control experiments showed there was no
detectable cross-reaction between FLAG-RpoHI and the antibody
used to precipitate RpoHII, and vice versa (data not shown). From
the ChIP-chip analysis we identified 812 and 1353 genomic
regions enriched after immunoprecipitation with antibodies
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Table 1. Compositions of the RpoHI and RpoHII regulons.
Mainrole1
Subrole{
Locus{
Energy metabolism
Electron transport
RSP_0100, RSP_0101, RSP_0102, RSP_0103, RSP_0104,
RSP_0105, RSP_0106, RSP_0107, RSP_2805
Other
RSP_0472
Protein synthesis/fate
Degradation of proteins, peptides, and glycopeptides
RSP_0665, RSP_1076, RSP_1174, RSP_2710, RSP_2806
RpoHI/II regulon (45 genes)
Fatty acid and cell envelope
Other
RSP_1825
Protein folding and stabilization
RSP_1207
Ribosomal proteins: synthesis and modification
RSP_0570
Serine family
RSP_2481
Biosynthesis
RSP_0473
Biosynthesis and degradation of surface
polysaccharides and lipopolysaccharides
RSP_2569
Other
RSP_3601
DNA metabolism
DNA replication, recombination, and repair
RSP_2388, RSP_2966
Central intermediary metabolism
Phosphorus compounds
RSP_0013
Cellular processes
Adaptations to atypical conditions
RSP_2617
Signal transduction
Two-component systems
RSP_0847
Unknown function
Unknown function
RSP_2718,
RSP_1760,
RSP_3327,
RSP_1840,
Amino acid biosynthesis
RSP_0244, RSP_0377, RSP_1475
Degradation of proteins, peptides, and glycopeptides
RSP_0357, RSP_0554, RSP_1408, RSP_1531, RSP_1742,
RSP_2412, RSP_2649
Protein and peptide secretion and trafficking
RSP_1169, RSP_1797, RSP_1798, RSP_1799, RSP_1843,
RSP_2540, RSP_2541
Protein folding and stabilization
RSP_1016, RSP_1173, RSP_1532, RSP_1572, RSP_1805,
RSP_4043
RSP_0011,
RSP_2218,
RSP_3599,
RSP_2261,
RSP_0370, RSP_1025, RSP_1239,
RSP_2421, RSP_2625, RSP_2973,
RSP_0870, RSP_1026, RSP_1421,
RSP_2265
RpoHI regulon (130 genes)
Protein synthesis/fate
Energy metabolism
Fatty acid and cell envelope
Biosynthesis of cofactors,
prosthetic groups, and carriers
Regulatory functions
Transport and binding proteins
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Protein modification and repair
RSP_0559, RSP_0872, RSP_0873, RSP_0874, RSP_0923
tRNA aminoacylation
RSP_0875
Amino acids and amines
RSP_3957
Electron transport
RSP_0296, RSP_0610, RSP_1194, RSP_1489, RSP_1529,
RSP_1576, RSP_2375, RSP_2685, RSP_2945
Glycolysis/gluconeogenesis
RSP_0361
Biosynthesis
RSP_0720, RSP_0929, RSP_2776
Biosynthesis and degradation of murein sacculus and
peptidoglycan
RSP_1240
Biosynthesis and degradation of surface
polysaccharides and lipopolysaccharides
RSP_0125, RSP_3187
Degradation
RSP_0409
Other
RSP_1889
Folic acid
RSP_0930
Lipoate
RSP_2783
Molybdopterin
RSP_0235, RSP_1071, RSP_1072
Other
RSP_2658
Pyridoxine
RSP_1672
DNA interactions
RSP_0014, RSP_2200
Other
RSP_2236
Protein interactions
RSP_4193
Transcription factors
RSP_2410
Amino acids, peptides and amines
RSP_1564
Cations and iron carrying compounds
RSP_2542, RSP_2891
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Table 1. Cont.
Mainrole1
DNA metabolism
Cellular processes
Central intermediary metabolism
Subrole{
Locus{
Unknown substrate
RSP_2696, RSP_2897
DNA replication, recombination, and repair
RSP_1074, RSP_2815, RSP_4199
Purine ribonucleotide biosynthesis
RSP_2454
Adaptations to atypical conditions
RSP_4198
Detoxification
RSP_0890, RSP_1058
Other
RSP_1196, RSP_1949
Sulfur metabolism
RSP_2738
Signal transduction
Two-component systems
RSP_2130, RSP_3105
Mobile and extra-chromosomal element
functions
Transposon functions
RSP_3007
Unknown function
Unknown function
RSP_0126,
RSP_0999,
RSP_1241,
RSP_1573,
RSP_1743,
RSP_2219,
RSP_2739,
RSP_2953,
RSP_3552,
RSP_3810,
Biosynthesis and degradation of polysaccharides
RSP_0482
Electron transport
RSP_0108, RSP_0109, RSP_0110, RSP_0112, RSP_0474,
RSP_2785, RSP_3212, RSP_3305, RSP_3537
Entner-Doudoroff
RSP_2646
Fermentation
RSP_3164
RSP_0362,
RSP_1104,
RSP_1360,
RSP_1581,
RSP_1852,
RSP_2387,
RSP_2763,
RSP_3067,
RSP_3597,
RSP_4244,
RSP_0363,
RSP_1193,
RSP_1406,
RSP_1615,
RSP_2121,
RSP_2638,
RSP_2764,
RSP_3068,
RSP_3598,
RSP_4245,
RSP_0408,
RSP_1204,
RSP_1549,
RSP_1671,
RSP_2125,
RSP_2640,
RSP_2816,
RSP_3378,
RSP_3634,
RSP_4248,
RSP_0719,
RSP_1238,
RSP_1563,
RSP_1684,
RSP_2214,
RSP_2641,
RSP_2952,
RSP_3426,
RSP_3809,
RSP_4305
RpoHII regulon (99 genes)
Energy metabolism
Biosynthesis of cofactors,
prosthetic groups, and carriers
Transport and binding proteins
Glycolysis/gluconeogenesis
RSP_2736, RSP_4045, RSP_4211
Other
RSP_0392, RSP_2294
Pentose phosphate pathway
RSP_2734, RSP_2735
Sugars
RSP_2937, RSP_3138
Glutathione and analogs
RSP_3272
Heme, porphyrin, and cobalamin
RSP_1197, RSP_1692, RSP_2831
Menaquinone and ubiquinone
RSP_1175, RSP_1338, RSP_1492, RSP_1869
Other
RSP_0750, RSP_0898, RSP_2314
Amino acids, peptides and amines
RSP_1542, RSP_3274
Carbohydrates, organic alcohols, and acids
RSP_0149, RSP_0150
Cations and iron carrying compounds
RSP_1546, RSP_2608
Unknown substrate
RSP_1895, RSP_2802, RSP_3160
DNA metabolism
DNA replication, recombination, and repair
RSP_1466, RSP_2083, RSP_2414, RSP_2850, RSP_3077,
RSP_3423
Pyrimidine ribonucleotide biosynthesis
RSP_3722
Fatty acid and cell envelope
Biosynthesis and degradation of surface
polysaccharides and lipopolysaccharides
RSP_1491, RSP_2163, RSP_3721
Regulatory functions
Degradation
RSP_0119
Other
RSP_0422, RSP_0595, RSP_0855
DNA interactions
RSP_1083, RSP_4210
Other
RSP_0148, RSP_2631, RSP_3430, RSP_3431
Transcription factors
RSP_0601
Cellular processes
Detoxification
RSP_1057, RSP_2389, RSP_2693, RSP_3263
Toxin production and resistance
RSP_2803
Central intermediary metabolism
Other
RSP_0897, RSP_1258, RSP_1397, RSP_3072
Phosphorus compounds
RSP_0782
Protein synthesis/fate
Amino acid biosynthesis
RSP_0398
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Table 1. Cont.
Mainrole1
Unknown function
Subrole{
Locus{
Degradation of proteins, peptides, and glycopeptides
RSP_0686, RSP_1490
Protein folding and stabilization
RSP_1219
tRNA and rRNA base modification
RSP_2971
Unknown function
RSP_0151,
RSP_0799,
RSP_2225,
RSP_3310,
RSP_0152,
RSP_0896,
RSP_2268,
RSP_3329,
RSP_0269,
RSP_1591,
RSP_3075,
RSP_4144,
RSP_0423, RSP_0557,
RSP_1956, RSP_1985,
RSP_3076, RSP_3089,
RSP_4209
Summary of the functional annotations of members of the RpoHI and RpoHII regulons in R. sphaeroides defined by the intersections of the results from the expression
profiling and chromatin immunoprecipitation experiments.
1
Classification of the functional main categories according to the JCVI-CRM database (http://cmr.jcvi.org/).
{
Classification of the functional sub-categories according to the JCVI-CRM database.
{
Unique locus identifiers for R. sphaeroides 2.4.1.
doi:10.1371/journal.pgen.1002929.t001
were sorted into three groups according to the expression profiling
and ChIP-chip data sets and converted into sequence logos
(Figure 3, Table S2). The sequence logos derived from the three
groups include: two groups that are preferentially or selectively
bound and transcribed by either RpoHI or RpoHII and one group
that is bound and transcribed by both s factors. As noted above,
some promoters appear to be bound by RpoHI or RpoHII without
inducing detectable changes in transcript levels. We aligned these
promoters separately to determine if they possessed unique
characteristics, but no significant differences were detected (data
not shown).
The conservation of a TTG motif in the 235 region in all three
logos is consistent with the importance of this triplet in a previous
analysis of at least one promoter known to be recognized by both
RpoHI and RpoHII [27]. However, there was also evidence for
sequence-specific elements in the logos for each RpoH paralog. In
the logo for the RpoHI-dependent promoters, a cytosine is
overrepresented at position 237 and a thymine is overrepresented
at position 29. In the logo for RpoHII-dependent promoters,
cytosine and thymine are overrepresented at positions 214 and
213, respectively.
Overall, the comparison between RpoHI and RpoHII-specific
promoter logos allowed us to identify significant differences in the
promoter sequences that may be used to adjust promoter
selectivity and strength for RpoHI or RpoHII. In addition, the
predicted sequence elements for RpoHI or RpoHII promoters are
not mutually exclusive. Rather, it appears that promoter
specificities for RpoHI or RpoHII are distributed along a gradient
using a combination of specific bases at various positions of the
235 or 210 promoter elements.
but with a larger representation for functions associated with
protein homeostasis. The 99 predicted members of the RpoHII
regulon include fewer proteins predicted to play a role in protein
homeostasis and a larger number of proteins predicted to help
maintain the oxidation-reduction state of the cytoplasmic thiol
pool. However, a large number of genes in both the unique and
overlapping RpoHI and RpoHII regulons are annotated as having
no predicted functions. Overall, this analysis revealed that RpoHI
or RpoHII activate a large set of distinct and overlapping sets of
target genes.
Predicted differences in promoter sequences recognized
by RpoHI or RpoHII
Previous work indicated that RpoHI and RpoHII can recognize
and initiate transcription from similar promoter sequences
[14,15,20]. The characterization of their respective regulons also
suggests that some promoters can be transcribed by both s factors
while others are specific to either RpoHI or RpoHII. Therefore, we
hypothesized that while the promoter sequences of the two s
factors may be similar, different sequence-specific interactions of
RpoHI or RpoHII with promoter elements are the basis of
promoter specificity for transcription initiation by RNA polymerase.
To overcome the limited resolution of the ChIP-chip experiment and predict determinants of promoter specificity for RpoHI
or RpoHII, we searched the regions upstream of genes in each
regulon for conserved sequence elements (137 sequences for
RpoHI and 120 sequences for RpoHII). The conserved sequence
elements we identified mapped to putative promoter elements that
were within 100 bp of the coordinates of the modes of the
distributions of the ChIP-chip signal. Thus, the predictions of these
searches identified conserved sequence elements that were in
agreement with the experimental data. In addition, even though
we analyzed the individual RpoHI and RpoHII regulons
independently for these motifs, the sequence alignment algorithm
converged to the same sequence elements for promoters that were
predicted to be recognized by both RpoHI and RpoHII. This
result is not surprising given that both s factors have similar amino
acid sequences in their DNA recognition regions and are thus
expected to recognize similar promoter sequences. However, this
observation supports the hypothesis that RpoHI and RpoHII
recognize common promoter sequences in their respective target
genes as opposed to distinct promoters.
To predict specificity sequence determinants for each RpoH
paralog, the putative distinct and overlapping promoter sequences
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Degrees of promoter specificity of RpoHI and RpoHII
To test predictions about specificity determinants derived from
these logos, we cloned several putative promoters upstream of a
lacZ reporter gene and integrated these into the genome of a R.
sphaeroides DrpoHI DrpoHII mutant [15] via homologous recombination. The activity of each promoter was measured by assaying bgalactosidase activity in these R. sphaeroides reporter strains
ectopically expressing either RpoHI or RpoHII (Figure 4) at levels
comparable to those found during a stress response (see above and
Figure 1). The RSP_1173, RSP_1408, and RSP_1531 promoters
(which were either predicted to be members of the RpoHI regulon
or, in the case of RSP_1173, known to be heat inducible and
transcribed by RpoHI [16], had significant activity in the strain
expressing RpoHI, but not when the same strain expressed RpoHII
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Convergent Transcriptional Stress Responses
Figure 4. Relative activities of selected RpoHI- and RpoHIIdependent promoters. b-galactosidase activity of lacZ operon
fusions with selected R. sphaeroides promoter regions monitored in
tester strains expressing RpoHI (black), RpoHII (grey), or neither proteins.
Genes are grouped according to the gene expression profiles displayed
in the gene expression experiments: genes whose expressions were
affected only by RpoHI, only by RpoHII, and by both RpoHI and RpoHII.
Error bars represents the standard error of the mean from three
independent replicates.
doi:10.1371/journal.pgen.1002929.g004
data from this analysis revealed that base substitutions in the TTG
motif of the 235 region of this RpoH-dependent promoter
(positions 236, 235, and 234) reduced its activity by at least 80%
with RpoHI (Figure 5A), as expected from the predictions of
promoter logo. We also found a slight increase in promoter activity
when position 232 was changed to a cytosine, even though the C32 is not conserved in RpoHI promoters. This observation is
consistent with the results of a previous mutational analysis
showing that E. coli s32 prefers a cytosine at position 232 when
the alanine at position 264 of its amino acid sequence is substituted
to an arginine (corresponding to R267 of RpoHI) [28], but also
suggests that the 232 position is not utilized to distinguish between
RpoHI- and RpoHII-specific promoters. In the 210 region of the
groE promoter, substitutions of the cytosine at position 214 for an
adenine or guanine, the cytosine at position 213 for an adenine,
or substitution of the thymine at position 211 for a cytosine, each
reduced RpoHI-dependent promoter activity. In addition, a
substitution of the adenine at position 212 for a cytosine or
changing the thymine at position 29 for any other base reduced
RpoHI-dependent activity by .90%. These observations are
consistent with the conservation of a thymine at position 29 of the
derived RpoHI promoter logo (Figure 3).
To test the predicted requirement of RpoHI for a thymine at
position 29, we also analyzed the properties of two R. sphaeroides
promoters in this E. coli tester strain. Activity of the RpoHIdependent RSP_1531 promoter was reduced by 90% when the
thymine at position 29 was changed to a cytosine, whereas the
RpoHII-dependent RSP_2314 promoter had higher RpoHIdependent activity when a thymine was placed at position 29
(Figure 5B). Therefore, this analysis confirmed that position 29
plays a critical role in promoter specificity for RpoHI. In
conclusion, the measured effects of mutations in the E. coli groE
promoter on RpoHI-dependent transcription confirmed that our
Figure 3. Conserved promoter sequences recognized by RpoHI
and RpoHII. The logos were constructed from promoter sequences
alignments sorted into three categories according to their predicted
specificity. The consensus sequence for s32-dependent promoters in E.
coli,as determined by Nomaka et al. [37], is shown as a reference. The
heights of the letters represent the degree of conservation across
sequences (information in bits, logos generated using WebLogo: http://
weblogo.berkeley.edu/). The coordinates on the x-axes represent the
positions relative to the predicted transcription start site. The numbers
of promoter sequences used to create the logos are indicated in
parentheses on the left of the logos. Below the logos are the sequence
alignments of selected promoters that were used for direct experimental validation.
doi:10.1371/journal.pgen.1002929.g003
(Figure 4). In contrast, the RSP_2314, RSP_2389, and RSP_3274
promoters (which were either predicted to be members of the
RpoHII regulon by our analysis or known to be induced by
conditions that generate singlet oxygen [17,18,20]) showed activity
in the presence of RpoHII but not RpoHI (Figure 4). Finally, the
RSP_1207 and RSP_2617 promoters (which were predicted to be
transcribed by both RpoH proteins and, in the case of RSP_1207,
known to be transcribed by RNA polymerase holoenzyme
containing either RpoH homolog [15] showed activity in cells
containing either RpoHI or RpoHII (Figure 4). Overall, these
results support predictions about members of the RpoHI or
RpoHII regulons derived by combining the transcription profiling,
ChIP-chip and computational analyses.
To test the predictions about the contributions of individual
bases to promoter recognition, we measured the activity of R.
sphaeroides RpoHI with an existing library of mutant E. coli groE
promoters fused to a lacZ reporter in an E. coli tester strain [7]. The
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Convergent Transcriptional Stress Responses
Figure 5. Activities of selected mutant promoters when transcribed by RpoHI or RpoHII. b-galactosidase activity of lacZ operon fusions
with selected mutant E. coli (A) or R. sphaeroides (B) promoters monitored in an E. coli tester strain expressing R. sphaeroides RpoHI. The original
promoter and specific base substitutions are indicated below the x-axis. (C) b-galatosidase activity of lacZ operon fusions integrated into the genome
containing either the wild type of indicated mutant R. sphaeroides cycA P1 promoter in a tester strain expressing the indicated RpoH homolog. Most
of the promoter mutations were made in the G-36T cycA P1 background, as this promoter had activity with RpoHI or RpoHII than its wild type (WT)
counterpart. Base substitutions are indicated on the x-axis. Error bars represents the standard error of the mean from three independent replicates.
doi:10.1371/journal.pgen.1002929.g005
essential for RpoHII activity but not RpoHI activity. The T at
position 29 of cycA P1 is also predicted to have significantly higher
information content for RpoHI than RpoHII, while a C at this
position should have more information content for RpoHII than
RpoHI (Figure 3). As predicted, we found RpoHII retained
significant activity after placing a T-9C mutation in the context of
the G-36T cycA P1 promoter. Furthermore, we found that this
mutation completely abolished its activity with RpoHI, illustrating
the high information content of a T at this position for
transcription by this RpoH homolog. The importance for a T at
the analogous position was also observed when testing activity of
mutant E. coli groE promoters with RpoHI (T-9C mutation
Figure 5A) or assaying function of the R. sphaeroides RSP_1531
promoter (which contains a T, Figure 5B) that is only transcribed
to a detectable level by RpoHI (Figure 4). Finally, we also replaced
the A at position 210 of the cycA P1 promoter with a G, as the
sequence logo suggests there to be little information content at this
position for either RpoHI or RpoHII (Figure 3). As predicted, there
is little impact of the A-10G mutation on promoter function,
though activity with RpoHII is more significantly reduced than
that with RpoHI activity (Figure 5C).
models captured elements that are critical for promoter recognition by RpoHI.
We were unable to test activity of R. sphaeroides RpoHII against
this groEL promoter library in the same E. coli tester strain (data not
shown). Instead, we generated a small set of point mutations in the
P1 promoter of the R. sphaeroides cycA promoter (Figure 3) which
was previously shown to be transcribed by both RpoHI and
RpoHII [27] and measured activity from single-copy fusions of
these mutant promoters to lacZ in cells that either lacked both
RpoH homologs or that contained a single rpoH gene under
control of an IPTG-inducible promoter (Materials and Methods).
By analyzing this promoter library, we found that a G to T
mutation at position 236 of cycA P1 (G-36T) increased its
transcription by both RpoHI and RpoHII (Figure 5C). This result
is consistent with the high predicted information content for T at
this position for both RpoHI and HII (Figure 3), as well as the
previous observation that the overall increase in activity of cycA P1
is caused by the G-36T mutation [27]. While our RpoHI and
RpoHII promoter models (Figure 3) predict that a C could be
allowed at position 236, a G-36C mutation lowered activity with
RpoHII and had no positive impact on transcription by RpoHI
(Figure 5C). Due to the significantly increased in activity from the
G-36T mutation in cycA P1, all of the other promoter mutations we
tested were generated in this background. Mutations we tested in
the 235 region, T-35C and G-34C, resulted in virtually complete
loss of cycA P1 activity with either RpoHI and RpoHII when
compared to their G-36T parent promoter (Figure 5C), indicating
that these bases are essential for transcription initiation by both
RpoH homologs. Based on the relatively low information content
predicted by our models for other positions in the 235 element
(Figure 3), we did not test the effects of other mutations in this
region on promoter selectivity by RpoH homologs.
In the predicted 210 region, A-12 has very high information
content for both RpoHI and RpoHII, but the sequence logo
suggests a T at this position might allow selective recognition by
RpoHI (Figure 3). Indeed, a promoter containing a T at position
212 is still active only with RpoHI, suggesting that A-12 is
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Discussion
When organisms encounter environmental or internal stress
they often increase the transcription of genes encoding proteins
that help mitigate damage to cellular components. Therefore,
identifying functions that are involved in transcriptional stress
responses is critical to understand both the nature of the damage
caused to cellular components and how organisms respond to
these challenges. Singlet oxygen and increased temperature are
very different phenomena, but in R. sphaeroides the transcriptional
responses to these two stresses involve two alternative s factors,
RpoHI and RpoHII, that each belong to the RpoH family
[15,16,18]. Several other a-proteobacteria contain two or more
members of the RpoH family that appear to control different stress
responses [13,29,30]. However, as it is the case in R. sphaeroides,
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Convergent Transcriptional Stress Responses
accumulation of ,25 proteins was dependent on RpoHII [17], our
data indicate that some 150 genes are directly controlled by each
R. sphaeroides RpoH paralog.
Genes in the direct but RpoHI-specific regulon encode functions
that are involved in protein homeostasis, maintaining membrane
integrity, and DNA repair, as is found for the E. coli s32 regulon
[3] (Table 1) The RpoHI specific regulon is also predicted to
encode cation transporters and proteins in the thioredoxindependent reduction system (Table 1). Ion transporters can aid
the heat shock stress response since exporting cations like iron,
which may be released by thermal denaturation of damaged ironsulfur or other metalloproteins, decreases secondary effects caused
by formation of toxic reactive oxygen species [34]. The
thioredoxin-dependent reduction system reduces disulfide bonds
and peroxides, which are created by protein oxidation, and
thereby helps maintain cytoplasmic proteins in a reduced state
[35]. Inclusion of these functions in the RpoHI regulon suggests
that oxidative damage may be an important secondary effect of
heat shock, perhaps caused by protein denaturation or permeabilization of the cell envelope. Overall, these results support the
hypothesis that the function of RpoHI in R. sphaeroides is similar to
that of s32 in E. coli for the response to heat shock stress. In
addition, it is also possible that RpoHI plays a role in the R.
sphaeroides response to other forms of stress. There is precedent for
roles of s32 homologs in other stress responses by other bacteria
since the activity of RpoH in Caulobacter crescentus is increased by
heavy metal stress [36].
In contrast, rpoHII transcription is under direct control of a
Group IV alternative s factor (RpoE) that serves as the master
regulator of the singlet oxygen stress response [18]. In addition, an
R. sphaeroides DrpoHII mutant is more sensitive to singlet oxygen
than a wild-type or DrpoHI strain [15,17]. Therefore, members of
the direct RpoHII-specific regulon might be expected to play an
important role in the response to singlet oxygen stress. Among the
genes in the RpoHII-specific regulon are others predicted to
function in maintaining membrane integrity and performing DNA
repair, both potential targets for damage by singlet oxygen.
However, the RpoHII–specific regulon contains fewer genes
encoding functions related to protein homeostasis than found in
the RpoHI regulon (Table 1). Other functions apparently unique
to the RpoHII regulon include the glutathione-dependent reduction system, which like the thioredoxin-dependent system repair
oxidized protein residues and maintain a reduced cytoplasm
(Table 1). Even though the thioredoxin- and gluthationedependent reduction systems serve similar cellular functions, they
are apparently under the control of different RpoH-dependent
transcriptional networks in R. sphaeroides. Thus, it is possible that
the thioredoxin- and gluthatione-dependent reduction systems
preferentially function on different oxidized substrates. Glutathione-dependent reduction systems are known to function on lipids
or other types of protein oxidative damage that might be
experienced by the cell following singlet oxygen damage [35].
We also found that the RpoHII-specific regulon includes the multisubunit NADH:quinone oxidoreductase and genes encoding
enzymes in heme and quinone biosynthesis (Table 1). Each of
these functions are critical for the respiratory and photosynthetic
electron transport chains of R. sphaeroides and are known or
predicted to contain one or more oxidant-sensitive metal centers.
Thus, placement of these genes in the RpoHII-specific regulon
suggests that these membrane or bioenergetic functions are
damaged by and need to be replaced in the presence of singlet
oxygen. Overall, our data indicates that the RpoHII-specific
regulon controls expression of functions in the repair of oxidized
proteins and replacement or assembly of critical electron transport
little is known about the target genes for these multiple RpoH
homologs. In this work, we characterized genes that are directly
transcribed by R. sphaeroides RpoHI and RpoHII to gain a better
understanding of the biological response to heat shock and singlet
oxygen stresses. We found that each of these RpoH paralogs
control transcription of over 100 genes, suggesting that each of
these phenomena lead to large changes in gene expression.
However, we also found that there is significant overlap in the
RpoHI and RpoHII regulons, creating an unexpectedly extensive
connection between the transcriptional responses to these two
signals. In addition, we investigated the characteristics of RpoHIand RpoHII-dependent promoters. This effort allowed us to
identify sequence elements that define promoter specificity for
each s factor, thereby allowing cells to selectively partition target
genes for each RpoH paralog into different stress responses.
R. sphaeroides RpoHI and RpoHII control the expression of
a common set of functions
This work revealed a surprisingly extensive overlap of the
RpoHI and RpoHII regulons even though these two homologs
activate transcriptional responses to different signals in R.
sphaeroides. This suggests that genes activated by these two
pathways of the transcriptional regulation network play a role in
the physiological response to both these, and even possibly, other
stresses. Indeed, the genes regulated by both RpoHI and RpoHII
encode known or annotated functions involved in protein
homeostasis, DNA repair, and maintenance of cell membrane
integrity (Table 1). These types of functions are central to cell
viability and may be relevant for the physiological responses to
multiple stresses that can have broad primary and secondary
effects on cells. Indeed, the predicted functions of the overlapping
members of the RpoHI and RpoHII regulons encode functions
that are also part of the general stress response regulons for sS in
E. coli or sB in Bacillus subtilis [31,32]. Interestingly, sS homologs
are mostly present in b- and c-proteobacteria, but to date absent
from sequenced genomes of a-proteobacteria like R. sphaeroides
(http://img.jgi.doe.gov/) [33]. Thus, it is possible that the set of
genes controlled by both RpoHI and RpoHII is part of a general
stress response that is common to the heat shock, singlet oxygen
and possibly other uncharacterized signals in R. sphaeroides
[14,15,17,18,20]. This hypothesis is supported by the observation
that R. sphaeroides and R. elti strains lacking both RpoHI and
RpoHII are more sensitive to several conditions than strains
lacking only one of these proteins [13,15,20].
In considering the scope of functions that are regulated by both
RpoHI and RpoHII, it is also important to note that this set of
genes may be larger than the one we characterized because some
promoters known to be transcribed by both s factors were only
marginally affected by ectopic expression of either RpoHI or
RpoHII. For example, the RSP_2310 (groES) promoter was shown
to be transcribed by both RpoHI and RpoHII in previous in vitro
experiments [14] and was detected by our ChIP-chip experiment
to be bound by both RpoHI and RpoHII, but did not meet all the
criteria of our analysis. Thus, the groES promoter, like other
promoters, may be subject to complex regulation in vivo.
RpoHI and RpoHII each control functions specific to heat
shock or singlet oxygen stresses, respectively
Our data also significantly extend the number and types of
functions that are specifically controlled by RpoHI or RpoHII
(Table 1). We expected to find specific sets of target genes because
strains lacking either RpoHI or RpoHII displayed different
phenotypes [14,15,17,20]. While previous results indicated that
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Convergent Transcriptional Stress Responses
chain components. Furthermore, the different types of repair
functions found in the RpoHII regulon predict that singlet oxygen
can damage numerous cellular components.
examine other examples of such convergence across bacteria and
other organisms that possess multiple homologs of RpoH or other
transcription factors.
RpoHI and RpoHII recognize different but compatible
promoter sequence elements
Materials and Methods
Bacterial strains and growth conditions
Our global gene expression data, results from analysis of gene
fusions, as well as previously reported in vitro experiments [14,15]
all indicate that RNA polymerase containing either RpoHI or
RpoHII can recognize some promoters in common. This
observation is not surprising considering that RpoHI and RpoHII
have similar amino acid sequences in their respective promoter
recognition regions and are each able to rescue growth of E. coli
s32 mutants [14–16]. Likewise, the sequence logos derived here
revealed that the promoter sequences recognized by each of the R.
sphaeroides RpoH homologs are similar to both each other and to
that recognized by E. coli s32 [37].
Our experiments provide definitive evidence that some
promoters are transcribed either exclusively or predominantly by
RpoHI or by RpoHII. We were also able to predict and confirm
the importance of bases for activity with individual RpoH
homologs (particularly those in the 235 element). We have
computational and experimental observations that can explain
some aspects of promoter selectivity by RpoHI and RpoHII. For
example, our experiments identify T-9 and other positions in the
210 element as potential candidates in this discrimination, as one
or more substitutions have larger effects on activity with individual
RpoHI homologs. Mutation of T-9 to any other base reduced
RpoHI-driven expression of GroE promoter by more than 90%,
and this same effect was observed using an authentic RpoHI
promoter from R. sphaeroides. Importantly, changing the 29
position of an RpoHII R. sphaeroides promoter to T permitted
expression by RpoHI. Together, these data suggest that T-9 is
either required for or significantly enhances expression of RpoHI
promoters, but is likely to be less important for expression of
RpoHII promoters, as there is only weak conservation of -9T in
RpoHII promoters. Our data also predict that other bases, which
are overrepresented in the RpoHII promoters, could be critical for
expression by that s factor. As is the case with E. coli s32 there are
likely to be specificity determinants that lie outside the canonical
235 and 210 elements [7,37]. Thus, additional in vivo and in vitro
experiments with a larger suite of mutant promoters and a library
of mutant RpoH proteins are needed to better define the
determinants of promoter selectivity by RpoHI and RpoHII.
In conclusion, our results suggest that, at least in R. sphaeroides,
RpoHI controls functions that are necessary for maintenance of
protein homeostasis and membrane integrity after temperature
increase and other cytoplasmic stress, similar to the wellcharacterized role of E. coli s32 in the heat shock response [3].
However, we propose that, in R. sphaeroides, some RpoHI-regulated
functions are also useful for survival in the presence of other forms
of stress because these target genes also contain promoters that are
recognized by RpoHII. We propose that the duplication of an
ancestral RpoH protein to create a second homolog of this
alterative s factor provided R. sphaeroides the opportunity to
connect stress response functions to another stimulus. In this
model, rpoHII was placed under the control of the master regulator
of the singlet oxygen stress response and the two RpoH proteins
evolved to recognize somewhat different but compatible promoter
elements to assure the optimal regulation of distinct but
overlapping stress regulons. As a result of these events, the
transcriptional responses of R. sphaeroides to heat shock and singlet
oxygen stress were separable but allowed to converge and contain
a common set of functions. It will be interesting to identify and
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E. coli strains were grown in Luria-Bertani medium [38] at 30uC
or 37uC. R. sphaeroides strains were grown at 30uC in Sistrom’s
succinate-based medium [39]. E. coli DH5a was used as a plasmid
host, and E. coli S17-1 was used as a donor for plasmid conjugation
into R. sphaeroides. The media were supplemented with kanamycin
(25 mg/ml), ampicillin (100 mg/ml), chloramphenicol (30 mg/ml),
spectinomycin (50 mg/ml), tetracycline (10 mg/ml for E. coli and
1 mg/ml for R. sphaeroides), trimethoprim (30 mg/ml), or 0.1% of
L-(+)-arabinose when required. Unless noted, all reagents were
used according to the manufacturer’s specifications. The list of
bacterial strains and plasmids used in this study are summarized in
Table S3.
Construction of plasmids for controlled expression of
RpoHI and RpoHII in R. sphaeroides
Plasmids for ectopically expressing RpoHI or RpoHII were
constructed by separately cloning the rpoHI or rpoHII genes
downstream of the IPTG-inducible promoter in pIND4 [22].
DNA fragments containing rpoHI or rpoHII were amplified from R.
sphaeroides 2.4.1 genomic DNA using oligonucleotides containing
BsrDI and BglII restriction sites (for RpoHII, RSP_0601_BsrDI_F
GTAGCAATGCATGGCACTGGACGGATATACCGATC, RSP
_0601_BglII_R GTAAGATCTTCATAGGAGGAAGTGATGCACCTCC, and for RpoHI, RSP_2410_BsrDI_F GTAGCAATGCATGAGCACTTACACCAGCCTTC, and RSP_2410
_BglII_R GTAAGATCTTCAGGCGGGGATCGTCATGCC).
These resulting fragments were digested with BsrDI and BglII
and ligated into pIND4 that was digested with BseRI and BglII to
create pYSD40 (rpoHI) and pYSD41 (rpoHII), respectively. The
pYSD42 plasmid expressing the FLAG-tagged version of RpoHI
was constructed following the same procedure but with an
oligonucleotide primer containing a sequence encoding for three
consecutive copies of the FLAG epitope (DYKDDDDK) at the Nterminus (RSP_2410_3FLAG_BsrDI GTAGCAATGCATGGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGAGCACTTACACCAGCCTTCCCGCTC). pYSD40, pYSD41, and pYSD42 were
conjugated into R. sphaeroides DrpoHI [16] and R. sphaeroides DrpoHII
respectively.
Western blot analysis for the expression of RpoHI and
RpoHII
To monitor levels of RpoHI and RpoHII after heat shock,
exponential phase aerobic cultures (69% nitrogen, 30% oxygen
and 1% carbon dioxide) of wild type R. sphaeroides strain 2.4.1
grown at 30uC, were transferred to a 42uC warm bath with
samples collected before heat treatment and at 10 min time
intervals after heat shock, up to 60 min. To assess induction
resulting from singlet oxygen stress, similarly grown wild type cells
were treated with 1 mM methylene blue and exposed to 10 W/m2
incandescent light with samples collected before treatment and at
10 min time intervals after treatment, up to 60 min. Exponentially
growing aerobic cultures of R. sphaeroides DrpoHI and DrpoHII
mutants carrying the pYSD40 or pYSD42 plasmids respectively,
were treated with 100 mM IPTG for one generation and
harvested. All cell samples were resuspended in 3 M urea
containing 16 protease inhibitor cocktail (Thermo Scientific,
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Convergent Transcriptional Stress Responses
Bioprospector [48] set to search for bi-partite conserved sequence
motifs. The promoter sequence alignments were refined using
HMMER 1.8.5 [49]. The logo representations of the promoter
sequence alignment were generated using WebLogo (http://
weblogo.berkeley.edu/) [50,51].
Rockford, IL) and sonicated. Samples were centrifuged to remove
debris and total protein concentration of the samples determined
with a protein assay kit following the manufacturer protocol (BioRad, Hercules, CA). An equal amount of total protein for each
sample was loaded onto a NuPAGE acrylamide gel (Invitrogen,
Carlsbad, CA) and run in 16 4-morpholineethanesulfonic acid
running buffer at 150 V for ,90 min. Proteins were transferred to
Invitrolon PVDF membranes (Invitrogen, Carlsbad, CA), which
were subsequently incubated for 1 hr in 16 Tris-buffered saline,
0.1% Triton-X, and 5% milk protein. The membranes were
incubated with rabbit polyclonal antibodies raised against either
RpoHI, RpoHII or PrrA. Horseradish-peroxidase-conjugated goat
anti-Rabbit IgG antibody (Thermo Scientific, Rockford, IL) was
used as secondary antibody for detection with Super Signal West
Dura extended duration substrate (Thermo Scientific, Rockford, IL).
Construction of plasmid vectors, lacZ reporter promoter
fusions, and b-galactosidase assays to assay promoter
activity in vivo
To assay the in vivo activity of RpoHI and RpoHII at target
promoters, b-galactosidase assays were conducted with R.
sphaeroides DrpoHI DrpoHII mutant strains containing individual
reporter gene fusions. To construct this set of reporter strains
,350 base pair regions upstream of putative target genes:
RSP_1173, RSP_1408, RSP_1531, RSP_2314, RSP_2389,
RSP_3274, RSP_1207 and RSP_2617, were amplified from
genomic DNA using sequence specific primers, with NcoI and
XbaI restriction sites at the ends of the upstream and downstream
primers respectively. The amplified DNA fragments were purified,
digested with NcoI-XbaI and then cloned in a pSUP202 suicide
vector containing a promoterless lacZ gene (pYSD51). These Tcr
plasmids were then conjugated into an R. sphaeroides DrpoHI
DrpoHII mutant [15], generating single copy promoter-lacZ fusions
integrated in the genome. pYSD40, pYSD41 or pIND4 (empty
vector) were then conjugated into each of these reporter strains.
Exponential phase cultures of these strains, grown by shaking
10 mL in 125 mL conical flasks, were then treated with 100 mM
IPTG for one generation and samples analyzed for b-galactosidase
activity. b-galactosidase assays were performed as previously
described [52]. The data, presented in Miller units, represents
the average of three independent replicates.
To test bases that contribute to RpoHI and RpoHII promoter
specificity, b-galactosidase assays were conducted in R. sphaeroides
tester strains containing reporter gene fusions of the cycA
(RSP_0296) P1 promoter with a variety of point mutations (see
Results). These reporter strains were constructed as described
above, with individual point mutations being generated by overlap
extension PCR [53]. b-galactosidase assays were conducted as
described above and the data represents the average of three
independent replicates. Background LacZ activity from control
strains for each promoter fusion containing only the empty pIND4
plasmid (i.e. not expressing either RpoHI or RpoHII) was subtracted
from the measured LacZ activity for each mutant promoter.
The construction of the E. coli CAG57102 mutant strain, the
promoter library, and the b-galactosidase assay used to test the
activity of R. sphaeroides RpoHI in vivo on mutant promoters were
described previously [7]. To express R. sphaeroides RpoHI the E. coli
rpoH gene of pSAKT32 [7] was replaced with the R. sphaeroides
rpoHI gene. At least triplicate assays for b-galactosidase activity
were performed on all strains.
Gene expression microarrays
Triplicate 500 ml cultures were grown aerobically with
bubbling (30%O2, 69% N2, 1% CO2) until they reached early
exponential phase (OD at 600 nm of 0.15). At this point IPTG
(Isopropyl b-D-1-thiogalactopyranoside) was added to a final
concentration of 100 mM to induce gene expression from the
pIND4 derivatives. After 3 hours incubation (OD at 600 nm of
0.30), 44 ml of cell culture were collected and 6 ml of 5% v/v
phenol in ethanol was immediately added. Cells were collected by
centrifugation at 6,000 g and frozen at 280uC until sample
preparation. RNA extraction, cDNA synthesis, labeling, and
hybridization were performed as previously described on Genechip Custom Express microarrays (Affymetrix, Santa Clara, CA)
[40]. Processing, normalization, and statistical analysis of the
expression profile data were performed in the R statistical software
environment (http://www.r-project.org/) [41]. Data were normalized using the affyPLM package with default settings [42–44].
The expression microarray data have been deposited in the
NCBI’s Gene Expression Omnibus [45] and are accessible
through GEO Series accession number GSE39806 (http://www.
ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc = GSE39806).
Chromatin immunoprecipitation on a chip
Cells were harvested at mid-exponential growth (OD at 600 nm of
0.30) from the same cell cultures used for the expression microarray
experiment to prepare samples for a ChIP-chip assay [19]. RpoHIFLAG was immunoprecipitated using commercial monoclonal
antibodies against the FLAG polypeptide (DYKDDDDK ) (Sigma
Aldrich, St Louis MO). RpoHII was immunoprecipitated with anti-R.
sphaeroides RpoHII rabbit serum. Labeled DNA was hybridized on a
custom-made tiling microarray, synthesized by NimbleGen (Roche
NimbleGen Inc, Madison, WI), covering the genome of R. sphaeroides
2.4.1 [19]. Before data analysis, dye intensity bias and array-to-array
absolute intensity variations were corrected using quantile normalization across replicates (limma package in the R environment) [46].
Regions of the genome enriched for occupancy by RpoHI or RpoHII
were identified using CMARRT with a false-discovery rate #0.05
[47]. The ChIP-chip data have been deposited in the NCBI’s Gene
Expression Omnibus [45] and are accessible through GEO Series
accession number GSE39806 (http://www.ncbi.nlm.nih.gov/
projects/geo/query/acc.cgi?acc = GSE39806).
Supporting Information
Figure S1 Scatter plot of RpoHI versus FLAG-RpoHI dependent change in gene transcription levels.
(TIF)
Table S1 RpoHI and RpoHII target genes.
(XLS)
Sequence analyses
Predicted promoter sequences of RpoHI and RpoHII
target genes.
(XLS)
Table S2
DNA sequences were manipulated using custom Python scripts.
Operon structure predictions for R. sphaeroides 2.4.1 were obtained
from VIMSS MicrobesOnline (http://www.microbesonline.org/
operons/) [26]. The promoter sequences predicted to be
recognized by RpoHI and RpoHII were discovered using
PLOS Genetics | www.plosgenetics.org
Table S3 Bacterial strains and plasmids.
(XLS)
11
September 2012 | Volume 8 | Issue 9 | e1002929
Convergent Transcriptional Stress Responses
B-MK SI HAG TJD. Contributed reagents/materials/analysis tools: YSD
B-MK SI HAG TJD. Wrote the paper: YSD B-MK SI HAG TJD.
Author Contributions
Conceived and designed the experiments: YSD B-MK SI HAG TJD.
Performed the experiments: YSD B-MK SI HAG. Analyzed the data: YSD
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