JOURNAL OF BACTERIOLOGY, Aug. 2006, p. 5712–5721
0021-9193/06/$08.00⫹0 doi:10.1128/JB.00405-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 16
Activity of Rhodobacter sphaeroides RpoHII, a Second
Member of the Heat Shock Sigma Factor Family
Heather A. Green and Timothy J. Donohue*
Department of Bacteriology, University of Wisconsin—Madison, Madison, Wisconsin 53706
Received 22 March 2006/Accepted 31 May 2006
The ability to regulate gene expression through the use of
alternate sigma factors enables bacteria to respond quickly to
changes in the environment. These secondary sigma factors
guide RNA polymerase to promoter elements whose target
sequences differ from those of the primary sigma factors,
thereby directing the transcription of a specific set of genes
(44). One such rapid response to environmental perturbations,
known as the heat shock response, uses an alternate sigma
factor to increase the expression of proteins needed to maintain an intracellular milieu conducive for protein folding.
While these heat-inducible gene products have traditionally
been referred to as the heat shock proteins, increased heat
shock protein expression is also induced by other stress conditions (11, 33). We are interested in alternate sigma factors
that play a role in the heat shock or other stress responses of
the ␣-proteobacterium Rhodobacter sphaeroides.
In the ␥-proteobacterium Escherichia coli, 32 (encoded by
rpoH) is the alternate sigma factor responsible for recognizing
heat shock gene promoters (12). As a member of the 70 family
of eubacterial sigma factors, 32 recognizes unique promoter
sequences centered at positions ⫺10 and ⫺35 relative to the
transcriptional start site (44). All 32-like proteins are defined
by a conserved region known as the “RpoH box”; they also
contain conserved sequences in regions 2.4 and 4.2 that recognize the ⫺10 and ⫺35 elements, respectively, of heat shock
promoters (29). While E. coli, with its single rpoH gene, sets
the stage for much of what we know about bacterial heat shock
regulation, several ␣-proteobacteria are known to encode multiple RpoH homologs. Three rpoH-like genes in Bradyrhizobium japonicum have been reported (31, 32), while Sinorhizobium meliloti contains two rpoH genes (36, 37). Individual
RpoH family members from these bacteria can completely, or
partially, complement the temperature sensitivity of an E. coli
rpoH mutant (31, 36, 37), indicating that they are functionally
similar to 32. In both Rhizobium species, however, each of the
RpoH homologs appears to have different but overlapping
roles in the organism’s response to stress (31, 32, 36, 37).
Past work established that R. sphaeroides RpoHI (previously
called 37) was a member of the 32 family of alternate sigma
factors, since the rpoHI gene complemented the inability of an
E. coli 32 mutant to support phage growth (19). In addition, a
⬃37-kDa protein isolated from RNA polymerase preparations
transcribed several E. coli heat shock promoters in vitro when
reconstituted with core RNA polymerase. The R. sphaeroides
RpoHI-null mutant (⌬RpoHI), however, mounted a typical
heat shock response, implying the existence of a second system
by which this bacterium could activate heat shock promoters
(19, 24). Further evidence supporting this idea came from in
vitro transcription assays that demonstrated that a ⬃38-kDa
protein (previously called 38) purified from ⌬RpoHI cells
recognized both a known E. coli 32 promoter (19) and the
R. sphaeroides cycA P1 promoter, which contains sequence
elements related to the E. coli 32 promoter consensus (24).
The identity of this ⬃38-kDa protein, however, and its possible similarity to other alternate sigma factors were unknown at the time.
In this report, we illustrate that the rpoHII gene encodes a
second alternate sigma factor of the 32 family in R. sphaeroides. We also show that recombinant RpoHI and RpoHII
can each transcribe several heat shock promoters when reconstituted with core RNA polymerase from either E. coli or R.
sphaeroides. While both RpoHI and RpoHII recognize R. sphaeroides promoters that resemble the E. coli 32 consensus,
there are differences in the ability of each protein to recognize
individual promoters in vitro. We discuss the possibility that
each R. sphaeroides RpoH homolog may regulate different, yet
* Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin—Madison, Madison, WI 53706. Phone:
(608) 262-4663. Fax: (608) 262-9865. E-mail: tdonohue@bact.wisc.edu.
5712
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We have identified a second RpoH homolog, RpoHII, in the ␣-proteobacterium Rhodobacter sphaeroides.
Primary amino acid sequence comparisons demonstrate that R. sphaeroides RpoHII belongs to a phylogenetically distinct group with RpoH orthologs from ␣-proteobacteria that contain two rpoH genes. Like its previously identified paralog, RpoHI, RpoHII is able to complement the temperature-sensitive phenotype of an
Escherichia coli 32 (rpoH) mutant. In addition, we show that recombinant RpoHI and RpoHII each transcribe
two E. coli 32-dependent promoters (rpoD PHS and dnaK P1) when reconstituted with E. coli core RNA
polymerase. We observed differences, however, in the ability of each sigma factor to recognize six R. sphaeroides
promoters (cycA P1, groESL1, rpoD PHS, dnaK P1, hslO, and ecfE), all of which resemble the E. coli 32 promoter
consensus. While RpoHI reconstituted with R. sphaeroides core RNA polymerase transcribed all six promoters,
RpoHII produced detectable transcripts from only four promoters (cycA P1, groESL1, hslO, and ecfE). These
results, in combination with previous work demonstrating that an RpoHI mutant mounts a typical heat shock
response, suggest that while RpoHI and RpoHII have redundant roles in response to heat, they may also have
roles in response to other environmental stresses.
VOL. 188, 2006
ACTIVITY OF R. SPHAEROIDES RpoHII
overlapping, regulons in response to one or more environmental stress signals.
MATERIALS AND METHODS
in seven 1-ml fractions with Tris-EDTA buffer (10 mM Tris-HCl [pH 8.0] and 0.1
mM EDTA) supplemented with 0.75 M NaCl and 40% propylene glycol. Fractions containing core RNA polymerase subunits, as visualized by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), were combined and
dialyzed against 1 liter of TGED medium (10 mM Tris-HCl [pH 7.9], 10%
glycerol, 0.1 mM EDTA, and fresh 0.1 mM dithiothreitol) containing 47.5%
glycerol and 100 mM NaCl. E. coli core RNA polymerase was purchased from
Epicenter Technologies, Inc. (Madison, WI). All protein concentrations were
determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules,
CA). The proteins were stored at ⫺20°C prior to use.
In vitro transcription assays. Core RNA polymerase, whether from E. coli or
R. sphaeroides, was reconstituted with either His6-RpoHI or His6-RpoHII by
incubating the proteins for 1 h at 32°C in transcription buffer (40 mM Tris-HCl
[pH 7.9], 2 mM EDTA, 5 mM MgCl, 100 mM KCl, 1 mM dithiothreitol, 100
g/ml acetylated bovine serum albumin). The mixture was added to a 20-l
reaction mixture containing 20 nM supercoiled plasmid template in transcription
buffer and incubated for 25 min at 32°C. Transcription assays were initiated with
the addition of a mixture of ribonucleotide triphosphates at final concentrations
of 250 M GTP, CTP, and ATP; 25 M UTP; and 1 Ci [␣-32P]UTP (3,000
Ci/mmol). Reaction mixtures were incubated at 32°C for 25 min and terminated
with 6 l of 95% (wt/vol) formamide loading buffer (6). Samples were heated to
95°C and resolved on 6% polyacrylamide–7 M urea gels (19). The transcripts
were visualized and quantified using the Molecular Dynamics (Sunnyvale, CA)
PhosphorImaging system and ImageQuant software.
Identification of candidate RpoH-dependent promoters from R. sphaeroides.
The R. sphaeroides genome (GenBank accession numbers CP000143 to
CP000147) was scanned using the program PromScan (http://www.promscan
.uklinux.net) to search for candidate 32-like promoter sequences. This query
used DNA sequences upstream of four R. sphaeroides promoters whose in vivo
transcript levels increased upon an increase in temperature (cycA P1, groESL1,
rpoD, PHS, and rrnB) (19). We chose to analyze candidate promoters positioned
upstream of genes encoding known proteins in other bacteria.
RESULTS
Primary sequence similarity of R. sphaeroides RpoH homologs. The R. sphaeroides genome sequence predicts that this
␣-proteobacterium encodes two members of the heat shock
sigma factor family, RpoHI (19) and RpoHII (see below). R.
sphaeroides RpoHII shares 46% amino acid identity (64% similarity) with its paralog, R. sphaeroides RpoHI (19), and 36%
amino acid identity with E. coli 32 (23) (Fig. 1A). RpoHII
shares the greatest degree of amino acid identity with RpoH
proteins from ␣-proteobacteria known to contain two RpoH
factors. It displays 50% amino acid identity to Mesorhizobium
loti RpoH-like sigma factor C, 47% identity to Bartonella quintana RpoH2, 46% identity to a Brucella melitensis 32 factor,
and 42% identity to Sinorhizobium meliloti RpoH2 (36, 37).
Together, R. sphaeroides RpoHII and RpoHI are most similar,
displaying ⬃81% and ⬃84% amino acid identities, respectively, to the cognate RpoH proteins of two marine heterotrophs of the Roseobacter clade, Jannaschia helgolandensis (42)
and Silicibacter pomeroyi (27). These values are consistent with
a phylogenetic tree of 32 homologs (Fig. 2), which predicts
that six of these proteins form a distinct cluster with R. sphaeroides RpoHII. In contrast, R. sphaeroides RpoHI falls into a
larger cluster with proteins from ␣-proteobacterial species that
contain either one or more rpoH genes.
Figure 1B and C shows amino acid alignments of RpoH
proteins from ␣-proteobacteria known to contain multiple
rpoH genes and E. coli 32. All proteins in the heat shock
family of alternate sigma factors contain the RpoH box, a
conserved stretch of nine amino acids in region C (29) that has
been implicated in the regulation of E. coli 32 activity (3, 18,
28). The second amino acid residue in the RpoH box is characteristically a lysine (K) in proteins from the ␣-proteobacteria
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Bacterial strains and growth conditions. E. coli DH5␣ was used as a plasmid
host. Cells were grown in Luria-Bertani (LB) medium (38) supplemented with
ampicillin (100 g/ml) as necessary. For the complementation assays, we grew E.
coli CAG9333, an rpoH mutant capable of growth at 40°C (22), in LB medium
supplemented with ampicillin (25 g/ml) when required. All E. coli cells were
grown at 37°C unless otherwise indicated.
Comparison of amino acid sequences of RpoH-like proteins. The amino acid
alignments and the phylogenetic relationships of RpoHI and RpoHII were determined by comparing their amino acid sequences with those of other RpoH
homologs (DDBJ/EMBL/GenBank accession numbers are in parentheses):
Agrobacterium tumefaciens (accession number D50828), Alcaligenes xylosoxydans
(accession number AB009990), Bartonella quintana (accession numbers
BQ12120 [rpoH2] and BQ02820 [rpoH1]), Bradyrhizobium japonicum (accession
numbers U55047 [rpoH1], Y09502 [rpoH2], and Y09666 [rpoH3]), Brucella
melitensis (accession numbers AB3287 [gene BMEI0280] and AD3299 [gene
BMEI0378]), Buchnera aphidicola (accession number BAU35400), Caulobacter
crescentus (accession number U39791), Citrobacter freundii (accession number
X14960), Coxiella burnetii (accession number AF120928), Enterobacter cloacae
(accession number D50829), Escherichia coli K-12 (accession number U00096),
Haemophilus influenzae (accession number U32713), Hydrogenophilus thermoluteolus (accession number AB009991), Jannaschia sp. strain CCS1 (genome draft
in progress for gene 3800 and gene 400 [JGI]), Mesorhizobium loti (accession
numbers MLR3862 [RpoH-like sigma factor C] and MLR3741 [sigma factor]),
Methylovorus sp. strain SS1 (accession number AF177466), Myxococcus xanthus
(accession numbers X55500 [sigB], L12992 [sigC], and AF023661 [sigE]), Proteus
mirabilis (accession number D50830), Pseudomonas aeruginosa (accession number S77322), Pseudomonas putida (accession number AB025418), Ralstonia sp.
strain CH34 (accession number J05278), Rhodobacter capsulatus (accession number AF017436), Rhodobacter sphaeroides (accession numbers U82397 [rpoHI]
and CP000143 [rpoHII]), Rickettsia prowazekii strain Madrid E (accession number AJ235271), Serratia marcescens (accession number D50831), Silicibacter
pomeroyi (accession numbers SPO0406 [rpoH-1] and SPO1409 [rpoH-2]), Sinorhizobium meliloti (accession numbers AF128845 [rpoH1] and AF149031
[rpoH2]), Stigmatella aurantiaca (accession numbers U27311 [sigC] and Z14970
[sigB]), Vibrio cholerae (accession number U44432), Xanthomonas campestris pv.
campestris (accession number AF042156), and Zymomonas mobilis (accession
number D50832).
Amino acid alignments were generated using ClustalW and BoxShade 3.21.
The phylogenetic tree was created using PAUP* version 4.0beta (Sinauer Associates, Sunderland, Mass.).
Plasmid constructions. The rpoHI and rpoHII genes were amplified from an R.
sphaeroides 2.4.1 cosmid (pU18106) and genomic DNA, respectively, with 2.5
units of Pfu polymerase (Stratagene, La Jolla, CA) using primers specific for each
gene. The PCR products were cloned into pUC18 and pUC19 in each orientation
relative to the lac promoter. N-terminal hexahistidine-tagged RpoHI and RpoHII
were obtained by cloning either rpoHI or rpoHII into pET-15b at the NdeI and
NdeI-BamHI sites, respectively (Novagen, Madison, WI). The cloned portions
of the resulting plasmids, pHAG7 and pHAG16, were sequenced to ensure
that they encoded the desired His6-tagged versions of RpoHI and RpoHII,
respectively.
Plasmids used as in vitro transcription templates were derived from either
pRKK96 (34) or pRKK137 (24), which both carry the spot 42 transcription
terminator (1). Transcription templates containing R. sphaeroides cycA P1 as well
as the E. coli dnaK P1 and rpoD PHS promoters have been described previously
(19, 24). Candidate R. sphaeroides promoters (groESL1, rpoD PHS, dnaK, hslO,
and ecfE) were PCR amplified from 2.4.1 genomic DNA as described above and
sequenced with vector-specific primers to guarantee that the desired region was
cloned in the proper orientation. All potential promoter regions lie within 150 bp
of the predicted start of translation, except rpoD PHS, which is positioned ⬃400
bp upstream of the open reading frame start. Sequences of primers used in this
study are available upon request.
Proteins used for in vitro transcription assays. Conditions for expression and
purification of His6-RpoHI, His6-RpoHII, and R. sphaeroides core RNA polymerase were described previously (1), with the following exceptions. Core RNA
polymerase was obtained from an ⌬RpoHI null strain (19) by affinity chromatography (⬃40 g of cells) on a ⬃2-ml resin bed containing the 4RA2 monoclonal
antibody against the ␣ subunit of E. coli RNA polymerase (Richard Burgess,
University of Wisconsin—Madison). Proteins bound to the column were eluted
5713
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GREEN AND DONOHUE
J. BACTERIOL.
and an arginine (R) in those from the ␥-proteobacteria (19).
Not surprisingly, both RpoH paralogs from R. sphaeroides contain a lysine at this position. While the third amino acid residue
in the RpoH box from ␥-proteobacteria has invariably been a
lysine, this residue is either an arginine or a lysine in proteins
related to RpoHI from the ␣ subdivision (45). In contrast,
proteins that cluster phylogenetically with R. sphaeroides
RpoHII contain an uncharged amino acid, either alanine (A),
serine (S), or valine (V), in the analogous position. This alignment also illustrates that R. sphaeroides RpoHII and its most
closely related homologs have considerably less amino acid
conservation than the RpoHI orthologs (Fig. 1B) in regions 2.1
and 2.2, which comprise a domain implicated in the binding of
core RNA polymerase (7, 13).
Function of R. sphaeroides rpoH genes in an E. coli rpoH
mutant. Since R. sphaeroides RpoHII has significant amino acid
conservation in regions 2.4 (⫺10 promoter recognition) and
4.2 (⫺35 promoter recognition) (Fig. 1B and C), we predicted
that it should recognize heat shock promoters. To test this
hypothesis, we asked whether the R. sphaeroides rpoHII gene
could complement the temperature-sensitive phenotype of the
E. coli 32-null strain CAG9333. While this tester strain lacks
a functional copy of rpoH, a compensatory R40 mutation that
results in enhanced GroES and GroEL synthesis allows for
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FIG. 1. Alignments of proteobacterial RpoH proteins. (A) Alignment of R. sphaeroides RpoHI and RpoHII and E. coli 32. Identical/similar
amino acid residues are shaded. Numbers on the left indicate the amino acid position relative to the start of each protein. Regions that are
conserved among eubacterial sigma factors are denoted by the numbers below the sequences. Region C and the RpoH box are also shown. Gaps
introduced to maximize the alignment are indicated by dashes. (B) Partial alignment of RpoH homologs from eight ␣-proteobacteria containing
multiple rpoH genes and E. coli 32 (␥ subgroup) showing the amino acid sequence from region 2.1 to the end of region C. (C) Region 4.2 of the
proteins listed in B showing the predicted helix-turn-helix (H-T-H) DNA-binding motif. For simplicity, the “. . .” indicates an intervening sequence
between region C and region 4.2. Abbreviations: Bqui, Bartonella quintana; Bjap, Bradyrhizobium japonicum; BmelAB3287, Brucella melitensis
RpoH gene BMEI0280; BmelAD3299, B. melitensis RpoH gene BMEI0378; Ecoli, Escherichia coli; Jann3800, Jannaschia sp. strain CCS1-3800/
CDS; Jann0400, Jannaschia sp. strain CCS1-0400/CDS; MlotRpoHC, Mesorhizobium loti RpoH-like sigma factor C; MlotMLR3741, M. loti sigma
factor; RsphRpoH1, R. sphaeroides RpoHI; RsphRpoH2, R. sphaeroides RpoHII; Spom, Silicibacter pomeroyi; Smel, Sinorhizobium meliloti. See
Materials and Methods for accession numbers.
VOL. 188, 2006
ACTIVITY OF R. SPHAEROIDES RpoHII
5715
growth at up to 40°C (22). When we introduced a plasmid
containing rpoHII downstream of the lac promoter into
CAG9333 and tested for growth at 37°C (permissive temperature) and 44°C (restrictive temperature), we found that rpoHII
was sufficient to restore growth at 44°C. In contrast, CAG9333,
containing a control plasmid (pUC18), did not grow at 44°C
(Table 1). We had previously shown that R. sphaeroides rpoHI
TABLE 1. Complementation of an E. coli
⌬RpoH mutant (CAG 9333)a
Complementing
gene
37°C
44°C
rpoD PHS::lacZ
expressionb
rpoHI
rpoHII
Nonec
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫺
a
All phenotypes were scored on solid medium.
Scored by monitoring a LacZ phenotype (see the text); “⫹,” blue; “⫺,”
white.
c
Cells containing pUC18 control plasmid.
b
supported phage growth of an E. coli 32 mutant (19), so we
expected a copy of this gene to allow for growth at 44°C.
CAG9333 also carries a chromosomal rpoD PHS::lacZ fusion
that can be used to score for transcription from this known
heat shock promoter (22). We demonstrated in previous work
that RNA polymerase holoenzymes purified from either R.
sphaeroides wild-type or ⌬RpoHI cells were able to transcribe
the E. coli rpoD PHS promoter in vitro (19). As expected,
strains carrying either rpoHI or rpoHII formed blue colonies on
plates containing X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) and IPTG (isopropyl--D-thiogalactopyranoside)
(Table 1). These results indicate that the rpoHII gene product
as well as the rpoHI gene product can functionally replace 32,
presumably because they are capable of binding E. coli core
RNA polymerase and transcribing one or more heat shock
promoters.
Recombinant RpoHI and RpoHII can direct transcription
from E. coli heat shock promoters in vitro. To test the hypothesis that recombinant RpoHI and RpoHII can direct transcription from E. coli heat shock promoters in vitro, we reconsti-
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FIG. 2. Phylogenetic tree of 32 homologs from 31 different proteobacterial species analyzed by the neighbor-joining method using PAUP*
4.0beta (Sinauer Associates, Sunderland, Mass.). Sigma factor names other than RpoH are shown in parentheses. The subdivisions (␣, , ␥, and
␦) of the proteobacteria are shown to the right of the tree. As indicated by the numbers to the right of each branch point, 100 pseudoreplicates
were used in this bootstrap analysis. Only values greater than 70% are shown. See Materials and Methods for accession numbers.
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J. BACTERIOL.
TABLE 2. Comparison of promoters from R. sphaeroides and
E. coli recognized by RpoHI and RpoHII
Promoter
⫺35 regiona
Spacer
length (bp)
⫺10 regiona
R. sphaeroides
cycA P1
groESL1
rpoD PHS
dnaK
hslO
ecfE
GTATTGATT
CCGTTGACA
CCCTTGAAA
CGCTTGCAG
CCCTTGGTC
CCCTTGAGG
13
14
14
14
13
14
ACCTATAT
TCATATCT
ACCCATGT
CCCTATAT
GCCTATAT
GACCATCT
E. coli
rpoD PHS
dnaK P1
CCCTTGAAA
CCCTTGATG
14
13
GACGATAT
CCCCATTT
32 consensus
CNCTTGAAA
13/14
CCCCATNT
32
Those bases matching the E. coli consensus sequence (11) are shown in
boldface type. The N indicates no base preference.
tuted E. coli core RNA polymerase with histidine-tagged
versions of either RpoHI or RpoHII and assayed transcription
from two known E. coli heat shock promoters, rpoD PHS and
dnaK P1 (Table 2). In order to determine the amount of sigma
factor required to produce optimal transcription from each
promoter, we performed multiple-round transcription using a
fixed amount of E. coli core RNA polymerase (37.5 nM) and
increasing amounts of either RpoHI (5.75 nM to 115 nM) or
RpoHII (2 nM to 40 nM). As expected, transcript abundance
from rpoD PHS and dnaK P1 increased with increasing concentrations of either RpoHI or RpoHII (titration curves represent-
FIG. 3. In vitro transcription of E. coli rpoD PHS and dnaK P1 promoters by E. coli core RNA polymerase reconstituted with either purified
R. sphaeroides RpoHI (A) or RpoHII (B). Shown are products from multiple-round transcription assays performed in the presence of increasing
amounts of either RpoHI (A) (5.75, 11.5, 34.5, 57.5, and 115 nM) or RpoHII (B) (2, 4, 12, 20, and 40 nM) with a constant amount of core RNA
polymerase (37.5 nM). Lanes marked with an asterisk contain products from assays using a control template in the presence of either a 3:1 (A) or
a 1:1 (B) molar ratio of the indicated RpoH sigma factor to core RNA polymerase. Panels C and D represent the quantification and summary of
transcription results from A and B as well as additional assays. The relative transcript abundance (pixel intensity) from each promoter was
measured by pixel intensity on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and adjusted for background.
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a
ing an average of several assays are shown in Fig. 3C and D).
In the presence of RpoHI, we reproducibly obtained optimum
transcript levels from both promoters when the sigma factor
was in an approximately twofold molar excess over core RNA
polymerase (Fig. 3C). In comparison, adequate transcription
from reaction mixtures containing RpoHII required an approximately equimolar ratio of sigma to core RNA polymerase
(Fig. 3D) over the course of several different assays. The transcripts derived from each of these promoters (Fig. 3A and B)
are identical in length to those synthesized by E. coli E32 (20),
indicating that pure RpoHI and RpoHII each recognize the
established heat shock promoters.
The results of these in vitro transcription assays also revealed that different levels of transcript were produced from
rpoD PHS compared to dnaK P1 when the E. coli core was
reconstituted with either RpoHI or RpoHII. At the concentration of purified RpoHI required for optimal transcription, the
RNA product was twofold more abundant from the dnaK P1
promoter than from the rpoD PHS promoter (Fig. 3C). Similarly, greater than twofold more transcript resulted from the
dnaK P1 promoter than from the rpoD PHS promoter in the
presence of a 1:1 molar ratio of RpoHII to core (Fig. 3D).
Recombinant RpoHI and RpoHII can direct transcription
from R. sphaeroides heat shock promoters in vitro. To provide
further evidence that RpoHI and RpoHII are members of the
32 family, we tested their ability to transcribe the R. sphaeroides cycA P1 and groESL1 promoters (Table 2). Primer
extension assays indicate that the abundance of cycA P1 and
groESL1-specific transcripts increases when either R. sphaeroides wild-type or ⌬RpoHI cells mount a heat shock response
VOL. 188, 2006
ACTIVITY OF R. SPHAEROIDES RpoHII
5717
(19); hence, we predicted that these promoters would be recognized by RNA polymerase containing either RpoHI or
RpoHII in vitro. We also tested the more physiologically relevant holo-RNA polymerase in these reactions by reconstituting
the individual RpoH proteins with core enzyme isolated from
R. sphaeroides. To eliminate RpoHI activity from our core
RNA polymerase preparations, we purified this enzyme from an
RpoHI mutant (19). The in vivo levels of RpoHI and RpoHII
differ vastly. Protein gels of purified holo-RNA polymerase
(24), as well as Western blot analyses of RpoHI and RpoHII
levels in crude cell extracts (data not shown), indicate that
RpoHI is considerably more abundant than RpoHII under
aerobic growth conditions. This R. sphaeroides core RNA polymerase does not contain any detectable RpoHII activity with
these promoters by multiple-round transcription (data not
shown).
When we performed multiple-round transcription using a
fixed amount of R. sphaeroides core RNA polymerase (160
nM) and increasing amounts of RpoHI (20 nM to 800 nM), we
found that increasing the concentration of RpoHI stimulated
transcription from both cycA P1 and groESL1 (results from a
representative assay are shown in Fig. 4A). While maximal
levels of the groESL1 transcript appeared to require a sigmato-core ratio of only 0.5 in the presence of RpoHI, we chose to
focus on a ratio of sigma to core that was optimal for both
promoters tested. Thus, with this reconstituted RNA polymerase holoenzyme, transcript abundance from cycA P1 was ap-
proximately twofold greater than that from groESL1 when the
molar ratio of sigma to core was 1:1 (Fig. 4C). Similarly, when
we increased the molar concentration of pure RpoHII (5 nM to
160 nM) in the presence of a constant concentration of core
RNA polymerase (160 nM), the transcript abundance from
both promoters increased, reaching a maximum at 80 to 160
nM RpoHII (Fig. 4B). At all levels of RpoHII tested, however,
we detected only low levels of transcript produced from groESL1.
Consequently, RpoHII-dependent transcription from cycA P1 was
approximately 16-fold greater than that from groESL1 when the
sigma-to-core RNA polymerase molar ratio was approximately
1:1 (Fig. 4D).
If transcript abundance is taken as an estimate of promoter
function, cycA P1 is transcribed with greater efficiency than
groESL1 in vitro when core RNA polymerase is reconstituted
with either RpoHI or RpoHII. On the other hand, if one
analyzes the ⫺10 and ⫺35 regions of cycA P1 and groESL1, it
is the R. sphaeroides groESL1 promoter that is most similar in
sequence to the E. coli 32 consensus (Table 2), intimating that
sequence comparisons alone may not be good indicators of
promoter recognition by either RpoHI or RpoHII (see Discussion). The molar concentrations of purified RpoH proteins
essential to observe optimal transcription were similar to that
seen when we used E. coli core RNA polymerase in reactions
instead. However, as indicated by control experiments, each of
the E. coli heat shock promoters that we tested are stronger
than the R. sphaeroides promoters analyzed (data not shown).
Downloaded from http://jb.asm.org/ on August 15, 2015 by guest
FIG. 4. In vitro transcription of R. sphaeroides cycA P1 and groESL1 promoters after adding either RpoHI (A) or RpoHII (B) to R. sphaeroides
core RNA polymerase. Shown are products from multiple-round transcription assays performed in the presence of increasing amounts of either
RpoHI (A) (20, 40, 80, 160, 480, and 800 nM) or RpoHII (B) (5, 10, 20, 40, 80, and 160 nM) with a constant amount of core RNA polymerase
(160 nM). Panels C and D represent the quantification and summary of transcription results from A and B and depict typical titration curves for
these two assays. The products marked with an asterisk in B are of unknown origin, but control experiments indicate that they are derived from
an RpoHII-dependent promoter on the vector (pRKK96) used to generate these transcription templates (data not shown). The relative transcript
abundance (pixel intensity) from each promoter was measured by pixel intensity on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and
adjusted for background.
5718
GREEN AND DONOHUE
FIG. 5. In vitro transcription of R. sphaeroides cycA P1, groESL1,
rpoD PHS, dnaK P1, hslO, and ecfE promoters by either RpoHI (230
nM) or RpoHII (160 nM) reconstituted with either R. sphaeroides (160
nM) (A) or E. coli (37.5 nM) core RNA polymerase (B). Open arrows
denote transcription products of the expected length from each promoter. The product marked with an asterisk in the 4th, 6th, and 12th
lanes is of unknown origin, but control experiments indicate that it is
derived from an RpoHII-dependent promoter on the vector (pRKK96)
used to generate these transcription templates (data not shown).
transcript in assays where RpoHII is added to either R. sphaeroides (Fig. 5A, lane 8) or E. coli (Fig. 5B, lane 8) core RNA
polymerase suggests that this gene either lacks an RpoHIIdependent promoter or contains one whose activity is below
the detection level of this assay (see Discussion). Control experiments showed that transcript production from these six
promoters is dependent on either RpoHI or RpoHII, because
core RNA polymerase alone, from either E. coli or R. sphaeroides, does not produce a detectable product from any of the
six heat shock promoters (data not shown).
These in vitro transcription assays also revealed that an
additional transcript is generated by one of our transcription
templates (pRKK96) that is dependent on the presence of
RpoHII (see transcription products marked with asterisks in
Fig. 4B and 5A and B). The only difference between pRKK96
and pRKK137, the other template used in these assays, is the
presence of a ⬃2-kbp spectinomycin resistance cartridge
cloned upstream of the promoter. Thus, it would appear that
this RpoHII-dependent transcript is derived from either an
uncharacterized promoter within the spectinomycin resistance
cartridge or one created by the insertion of this element into
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Recognition of additional R. sphaeroides promoters by
RpoHI and RpoHII. To further investigate promoters recognized by RpoHI and RpoHII, we examined the ability of reconstituted RNA polymerase holoenzymes to transcribe other
R. sphaeroides candidate target genes. Having established the
sigma-to-core ratios that are required for efficient transcription
from the cycA P1 and groESL1 promoters, we chose four additional promoters to assay under these in vitro transcription
conditions: rpoD PHS, dnaK P1, hslO (10, 16), and ecfE (9).
Each of these promoters contains sequences related to the E.
coli 32 consensus promoter sequence (Table 2). We knew
from previous results that rpoD PHS-specific transcripts increase in both wild-type and ⌬RpoH1 cells upon a temperature
increase (19). The remaining three promoters (dnaK P1, hslO,
and ecfE) were selected after querying the R. sphaeroides genome with a matrix-driven program, PromScan (40), that utilized the ⫺10 and ⫺35 regions of known R. sphaeroides heat
shock promoters.
RpoHI reconstituted with R. sphaeroides core RNA polymerase stimulated transcription from the two control promoters, cycA P1 and groESL1, as well as from each of the four
other test promoters, rpoD PHS, dnaK P1, hslO, and ecfE (Fig.
5A). When we reconstituted RpoHII with R. sphaeroides core
enzyme, we detected transcription products from each of the
promoters except rpoD PHS and dnaK P1 (Fig. 5A, lanes 6 and
8). The transcript lengths from each set of promoters transcribed by the two different holoenzymes were identical except
for hslO (Fig. 5A, lanes 9 and 10), which reproducibly yielded
a transcript that was 1 to 2 nucleotides shorter in the presence
of RpoHII. One possible explanation for this feature of the
hslO promoter is that RpoHII-containing RNA polymerase
either initiates or terminates transcription at a different position than core enzyme reconstituted with RpoHI.
The lack of a detectable RpoHII-dependent transcript from
the dnaK promoter region (Fig. 5A, lane 8) suggests that the
dnaK operon may not be controlled by this sigma factor (see
Discussion). The absence of a detectable transcript from
rpoD PHS (Fig. 5A, lane 6) using holoenzyme containing
RpoHII, however, was surprising, since cells lacking RpoHI
still experience an increase in rpoD-specific mRNA from
this promoter after a heat shock (19). One feasible explanation for the failure to detect transcription from either
rpoD PHS or dnaK P1 is that a protein(s) present in our R.
sphaeroides core preparation reduces transcription in the
presence of RpoHII (see Discussion).
To test for the presence of such an activity in our R. sphaeroides core RNA polymerase, we repeated the above-described transcription reactions using a highly purified preparation of E. coli core RNA polymerase. As predicted by the
above-described results (Fig. 5A), E. coli core RNA polymerase reconstituted with RpoHI transcribed all six promoters
(Fig. 5B). However RNA polymerase holoenzyme formed by
adding E. coli core enzyme to RpoHII generated detectable
transcripts from only five R. sphaeroides promoters: cycA P1,
groESL1, rpoD PHS, hslO, and ecfE (Fig. 5B). The presence of
an rpoD PHS transcript from assays using RpoHII and E. coli
core RNA polymerase is consistent with the hypothesis that
our R. sphaeroides core RNA polymerase may contain a factor(s) capable of inhibiting transcription from this promoter
(see Discussion). The inability to detect a dnaK P1-specific
J. BACTERIOL.
VOL. 188, 2006
ACTIVITY OF R. SPHAEROIDES RpoHII
pRKK137. Simple inspection of the relevant sequences did
not identify a candidate promoter, so additional experiments
are under way to define this proposed RpoHII-dependent
promoter.
DISCUSSION
constitute the three most highly conserved RpoH box residues
among RpoH homologs (Fig. 1B). Alternatively, the third
residue in the RpoH box of proteins that form a distinct
cluster with RpoHII is either an alanine, serine, or valine,
deviating from the positively charged arginine or lysine otherwise found in this position. To our knowledge, no one has
studied the effects that altering this position has on 32’s, or
another RpoH factor’s, ability to bind core RNA polymerase. If the RpoH box indeed has a regulatory role, as many
lines of evidence suggest, the presence of an uncharged
residue at this position may influence the activity of proteins
sharing the clade with RpoHII.
Regions 2.1 and 2.2 are conserved among all members of the
70 superfamily. Not surprisingly, amino acid substitutions in
these regions alter core RNA polymerase binding (7, 13). In
the case of E. coli 32, amino acid substitutions in region 2.2
reduced its affinity for core RNA polymerase (17), while amino
acid substitutions in region 2.1 suggested that this region may
bind DnaK (15, 35). These observations concur with the view
that DnaK and core RNA polymerase compete for binding to
specific regions of 32 (45, 46). The proteins most closely
related to R. sphaeroides RpoHII, however, show less primary
amino acid conservation, as a group, in regions 2.1 and 2.2 than
their respective paralogs (Fig. 1B). The relative plasticity in the
primary amino acid sequences of regions 2.1 and 2.2 among the
RpoHII clade may modify their affinities for core RNA polymerase or, possibly, the binding of chaperones that could negatively regulate their activity (14, 41). Thus, the differences
in promoter activity that we have observed for RpoHI and
RpoHII may in part be due to their relative efficiencies in
binding core RNA polymerase, as reflected by the amino acid
sequence variations in regions 2.1 and 2.2 and the RpoH box.
While domains 2 and 4 of the 70-type sigma factors are
structurally conserved, it is the primary amino acid sequence
variation in regions 2.4 and 4.2 that accounts for differences in
promoter recognition among the diverse members of this superfamily (13). In light of the fact that RpoHI and RpoHII each
recognize several heat shock promoters, it is not surprising that
regions 2.4 and 4.2 of these two proteins show little sequence
variation from other 32 family members. In addition, both
RpoHI and RpoHII share several amino acid residues with
regions 2.4 and 4.2 of E. coli 32 that are believed to contact
the ⫺10 and ⫺35 elements of the groE promoter (21).
Promoter recognition by RpoHI and RpoHII. Of the six R.
sphaeroides promoters that we examined, all were transcribed
by either R. sphaeroides or E. coli core RNA polymerase reconstituted with RpoHI. When recombinant RpoHII was combined with R. sphaeroides core enzyme, however, we were unable to detect transcripts from two promoters, rpoD PHS and
dnaK P1. This was surprising, since rpoD PHS is one of three
promoters we chose to analyze based on the knowledge that
their transcript abundance increases in ⌬RpoHI cells after a
heat shock (19). On the other hand, when RpoHII was added
to E. coli core RNA polymerase, we detected a product from
rpoD PHS. The lack of a product from rpoD PHS in the presence
of RpoHII and the R. sphaeroides core enzyme may have been
due to a protein, or proteins, in the core RNA polymerase
preparation that reduces in vitro transcription from this particular promoter. Another feasible explanation is that promoter escape from R. sphaeroides rpoD PHS may be more
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Previous studies identified R. sphaeroides RpoHI as a member of the 32 family of alternate sigma factors (19). Several
lines of evidence, however, intimated the existence of a second sigma factor that recognized heat-inducible promoters
(19, 24). In this work, we provide evidence that R. sphaeroides RpoHII is this second sigma factor. Amino acid sequence alignments show that RpoHII is a member of the 32
family. Like its paralog, RpoHI, expression of RpoHII complements the growth defect of a temperature-sensitive E.
coli 32 mutant and initiates transcription from the E. coli
32-dependent promoter rpoD PHS both in vivo and in vitro.
Through the use of purified components, we demonstrate
that RpoHI and RpoHII recognize a number of known and
presumed heat shock promoters from R. sphaeroides. We also
identify differences in the abilities of RpoHI and RpoHII to recognize individual promoters in vitro.
RpoHII is a second sigma factor that recognizes R. sphaeroides heat shock promoters. Previously, two proteins copurifying with R. sphaeroides RNA polymerase were eluted from
SDS-PAGE gels, individually reconstituted with core enzyme,
and shown to transcribe the E. coli dnaK P1 and R. sphaeroides
cycA P1 promoters (19, 24). One of these proteins, 37, was
tentatively identified as RpoHI, but the identity of the other
protein, known as 38, was not known. Several lines of evidence link RpoHII to the 38 activity described by previous
studies. Both recombinant RpoHII (this work) and gel-purified
38 (19) recognized the heat-inducible promoters dnaK P1 and
cycA P1, and both proteins have similar apparent molecular
weights when analyzed by SDS-PAGE (data not shown). Accordingly, antibody raised to His-tagged RpoHII reacts with a
protein of similar molecular weight in R. sphaeroides RNA
polymerase preparations (data not shown). In addition to identifying RpoHII as a second member of the 32 family in R.
sphaeroides, our experiments provide the first in vitro analysis
of promoter recognition by a protein belonging to the clade
containing RpoHII.
Comparison of RpoH primary amino acid sequences. There
are now seven organisms, all members of the ␣-proteobacteria,
predicted to contain two rpoH genes. R. sphaeroides RpoHII
falls into a distinct phylogenetic clade of alternate sigma factors whose primary amino acid sequences differ somewhat
from their respective paralogs, most noticeably in conserved
regions 2.1 and 2.2 and the RpoH box. The RpoH box lies in
region C, a stretch of 23 amino acids that initially was implicated in the DnaK-mediated degradation of 32 (25, 28). Subsequent studies indicated that instead, region C directly interacts with core RNA polymerase and that the RpoH box may
give 32 a competitive advantage over other sigma factors in
binding to this enzyme (3, 18). For example, amino acid substitutions in the fifth and sixth residues (F136L and F137E) of
the E. coli 32 RpoH box (132QRKLFFNLR140) each decreased the binding of core RNA polymerase (3, 18). These
two phenylalanines, together with L135, the fourth residue,
5719
5720
GREEN AND DONOHUE
or RpoHII, dnaK P1 was a stronger promoter than rpoD PHS.
Among the E. coli 32-dependent promoters that have been
studied, those with higher activities tend to more closely mimic
the 32 consensus sequence (43). Indeed, the ⫺10 element of
E. coli dnaK P1 is a perfect match to the 32 consensus. In
addition, a recent study demonstrated that a tryptophan
(W108) in the boundary between regions 2.3 and 2.4 of 32
contacts a conserved ⫺13 C-G base pair (21). The presence
of a tryptophan residue at this position in both RpoHI and
RpoHII may explain why these sigma factors transcribe E. coli
dnaK P1, which contains this conserved base pair, with greater
efficiency than rpoD PHS.
The number of R. sphaeroides promoters tested, along with
the variation in their sequences, makes it difficult to propose a
consensus promoter for either RpoHI or RpoHII. The fact that
these two sigma factors do not recognize an identical repertoire of promoters in vitro makes constructing such a consensus for R. sphaeroides even more challenging. A future interest
is to determine whether R. sphaeroides has separate RpoHIand RpoHII-specific target genes that allow this ␣-proteobacterium to respond to different stress, or other environmental,
signals. For example, recent studies have shown that RpoHII is
part of the RpoE regulon (2). RpoE is required for R. sphaeroides to mount a transcriptional response to singlet oxygen
(2). Consequently, RpoHII may directly regulate the expression of genes whose products are required to mitigate the
damaging effects of this reactive oxygen species as well as play
a role with RpoHI in responding to stress caused by elevated
temperatures.
ACKNOWLEDGMENTS
This study was supported by grants GM37509 and GM75273 (National Institute of General Medical Sciences) and DE-FG0205ER15653 (Department of Energy) to T.J.D.
We are grateful to Matias Cafaro for constructing the phylogenetic
tree and to Yann Dufour for his computer expertise. We also thank
Rachelle Stenzel and Archna Bhasin for their discussions and thoughtful reading of the manuscript.
REFERENCES
1. Anthony, J., H. A. Green, and T. J. Donohue. 2003. Rhodobacter sphaeroides
RNA polymerase and its sigma factors. Methods Enzymol. 370:54–65.
2. Anthony, J. R., K. L. Warczak, and T. J. Donohue. 2005. A transcriptional
response to singlet oxygen, a toxic byproduct of photosynthesis. Proc. Natl.
Acad. Sci. USA 102:6502–6507.
3. Arsene, F., T. Tomoyasu, A. Mogk, C. Schirra, A. Schulze-Specking, and B.
Bukau. 1999. Role of region C in regulation of the heat shock gene-specific
sigma factor of Escherichia coli, 32. J. Bacteriol. 181:3552–3561.
4. Artsimovitch, I., V. Svetlov, L. Anthony, R. R. Burgess, and R. Landick. 2000.
RNA polymerases from Bacillus subtilis and Escherichia coli differ in recognition of regulatory signals in vitro. J. Bacteriol. 182:6027–6035.
5. Bardwell, J. C. A., and E. A. Craig. 1984. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous.
Proc. Natl. Acad. Sci. USA 81:848–852.
6. Barker, M. M., T. Gaal, C. A. Josaitis, and R. L. Gourse. 2001. Mechanism
of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on
transcription initiation in vivo and in vitro. J. Mol. Biol. 305:673–688.
7. Burgess, R. R., and L. Anthony. 2001. How sigma docks to RNA polymerase
and what sigma does. Curr. Opin. Microbiol. 4:126–131.
8. Cowing, D. W., J. C. A. Bardwell, E. A. Craig, C. Woolford, R. W. Hendrix,
and C. A. Gross. 1985. Consensus sequence for Escherichia coli heat shock
gene promoters. Proc. Natl. Acad. Sci. USA 82:2679–2683.
9. Dartigalongue, C., H. Loferer, and S. Raina. 2001. EcfE, a new essential
inner membrane protease: its role in the regulation of heat shock response
in Escherichia coli. EMBO J. 20:5908–5918.
10. Graf, P. C., and U. Jakob. 2002. Redox-regulated molecular chaperones.
Cell. Mol. Life Sci. 59:1624–1631.
11. Gross, C. A. 1996. Function and regulation of the heat shock proteins, p.
1382–1399. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin,
Downloaded from http://jb.asm.org/ on August 15, 2015 by guest
efficient when RpoHII is added to E. coli core RNA polymerase. For example, Artsimovitch and coworkers reconstituted E.
coli 32 with either Bacillus subtilis or E. coli core RNA polymerase and demonstrated that the two holoenzymes differed
vastly in their promoter selectivities (4). Their results suggested that while promoter recognition resides mainly in the
sigma subunit, promoter utilization depends primarily on core
RNA polymerase and that contacts made between the core
subunits and promoter DNA may either facilitate or inhibit
promoter escape (4). Among the R. sphaeroides promoters that
we examined, rpoD PHS is most similar to the E. coli 32
consensus in both the ⫺10 and ⫺35 regions, suggesting that
specific contacts made between E. coli core RNA polymerase
and this promoter may result in more efficient transcription
than those made with the R. sphaeroides core enzyme.
Why the R. sphaeroides dnaK P1 promoter was not transcribed by either the E. coli or R. sphaeroides core enzymes in
the presence of RpoHII is unknown at this time. The gene
product DnaK is an Hsp70 protein (5, 8), and we saw a diminished increase in the synthesis rate of a ⬃75-kDa protein in
⌬RpoHI cells after a heat shock relative to wild-type cells (19).
If this ⬃75-kDa protein is DnaK, a second mechanism, independent of RpoHII, may be responsible for the heat induction
of this promoter in cells lacking RpoHI. In contrast, the heatinduced synthesis of a 76-kDa protein in S. meliloti appears to
be totally dependent on RpoH1 (37). While it is conceivable
that dnaK P1 is simply not recognized by RpoHII, the ⫺10
region of dnaK P1 is almost identical to that of the strongest R.
sphaeroides promoter for RpoHII that we tested, cycA P1. The
⫺35 regions of these two promoters, however, share only the
TTG motif, and previous studies have shown that a point
mutation in the ⫺35 region of cycA P1 (from ⫺34TTGA⫺31 to
⫺34
TTGC⫺31) reduced activity by ⬃60% (24). Since dnaK P1
has a C in this position (TTGC), RpoHII may have a greater
requirement for an A in the TTGA motif of the ⫺35 element
than RpoHI.
If we use transcript abundance as an estimate of promoter
strength, the strongest R. sphaeroides promoter that we tested
with either RpoHI or RpoHII was cycA P1. Despite this, the
⫺10 and ⫺35 elements of cycA P1 have fewer matches to the
E. coli 32 consensus sequence than the other five promoters
examined (Table 2). Thus, the canonical idea of what makes a
strong heat shock promoter may not apply to R. sphaeroides
RpoHI and RpoHII. Less-than-optimal heat shock promoter
sequences for other ␣-proteobacteria have been noted (26, 30,
39). Based on the upstream regions of nine dnaKJ and groESL
operons from eight ␣-proteobacteria, Segal and Ron constructed a putative consensus sequence (CTTG[17 to 18 bp]C
YTAT-T--G) that differs from the E. coli 32 consensus at
several positions (39). Only the R. sphaeroides dnaK P1 and
hslO promoter regions, however, abide by this proposed consensus sequence. The existence of relatively weak promoters
that are regulated by two or more sigma factors may represent
a mechanism that evolved to more subtly regulate gene expression in the absence or presence of different stress conditions.
Based on transcript abundance alone, both of the E. coli
promoters that we examined, dnaK P1 and rpoD PHS, were
considerably stronger than any of the six R. sphaeroides promoters analyzed. Furthermore, whether a holoenzyme was
generated by mixing E. coli core RNA polymerase with RpoHI
J. BACTERIOL.
VOL. 188, 2006
12.
13.
14.
15.
16.
17.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
5721
of the 32 polypeptide is involved in DnaK-mediated negative control of the
heat shock response in Escherichia coli. Proc. Natl. Acad. Sci. USA 91:
10280–10284.
Nakahigashi, K., H. Yanagi, and T. Yura. 1995. Isolation and sequence
analysis of rpoH genes encoding 32 homologs from gram negative bacteria:
conserved mRNA and protein segments for heat shock regulation. Nucleic
Acids Res. 23:4383–4390.
Narberhaus, F., M. Kowarik, C. Beck, and H. Hennecke. 1998. Promoter
selectivity of the Bradyrhizobium japonicum RpoH transcription factors in
vivo and in vitro. J. Bacteriol. 180:2395–2401.
Narberhaus, F., P. Krummenacher, H. Fischer, and H. Hennecke. 1997.
Three disparately regulated genes for 32-like transcription factors in
Bradyrhizobium japonicum. Mol. Microbiol. 24:93–104.
Narberhaus, F., W. Weiglhofer, H. M. Fischer, and H. Hennecke. 1996. The
Bradyrhizobium japonicum rpoH1 gene encoding a 32-like protein is part of
a unique heat shock gene cluster together with groESL1 and three small heat
shock genes. J. Bacteriol. 178:5337–5346.
Neidhardt, F. C., and R. A. VanBogelen. 1987. Heat shock response, p. 1334–
1346. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M.
Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella
typhimurium: cellular and molecular biology, vol. 2. American Society of
Microbiology, Washington, D.C.
Newman, J. D., M. J. Falkowski, B. A. Schilke, L. C. Anthony, and T. J.
Donohue. 1999. The Rhodobacter sphaeroides ECF sigma factor, E, and the
target promoters cycA P3 and rpoE P1. J. Mol. Biol. 294:307–320.
Obrist, M., and F. Narberhaus. 2005. Identification of a turnover element in
region 2.1 of Escherichia coli 32 by a bacterial one-hybrid approach. J.
Bacteriol. 187:3807–3813.
Oke, V., B. G. Rushing, E. J. Fisher, M. Moghadam-Tabrizi, and S. R. Long.
2001. Identification of the heat-shock sigma factor RpoH and a second
RpoH-like protein in Sinorhizobium meliloti. Microbiology 147:2399–2408.
Ono, Y., H. Mitsui, T. Sato, and K. Minamisawa. 2001. Two RpoH homologs
responsible for the expression of heat shock protein genes in Sinorhizobium
meliloti. Mol. Gen. Genet. 264:902–912.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y.
Segal, G., and E. Z. Ron. 1995. The dnaKJ operon of Agrobacterium tumefaciens:
transcriptional analysis and evidence for a new heat shock promoter. J. Bacteriol. 177:5952–5958.
Studholme, D. J., M. Buck, and T. Nixon. 2000. Identification of potential
N-dependent promoters in bacterial genomes. Microbiology 146:3021–3023.
Tomoyasu, T., T. Ogura, T. Tatsuta, and B. Bukau. 1998. Levels of DnaK
and DnaJ provide tight control of heat shock gene expression and protein
repair in Escherichia coli. Mol. Microbiol. 30:567–581.
Wagner-Dobler, I., H. Rheims, A. Felske, R. Pukall, and B. J. Tindall. 2003.
Jannaschia helgolandensis gen. nov., sp. nov., a novel abundant member of
the marine Roseobacter clade from the North Sea. Int. J. Syst. Evol. Microbiol. 53:731–738.
Wang, Y., and P. L. deHaseth. 2003. Sigma 32-dependent promoter activity
in vivo: sequence determinants of the groE promoter. J. Bacteriol. 185:5800–
5806.
Wosten, M. M. 1998. Eubacterial sigma-factors. FEMS Microbiol. Rev. 22:
127–150.
Yura, T., M. Kanemori, and M. T. Morita. 2000. The heat shock response:
regulation and function, p. 3–18. In G. Storz and R. Hengge-Aronis (ed.),
Bacterial stress responses. ASM Press, Washington, D.C.
Zhao, K., M. Liu, and R. R. Burgess. 2005. The global transcriptional
response of Escherichia coli to induced 32 protein involves 32 regulon
activation followed by inactivation and degradation of 32 in vivo. J. Biol.
Chem. 280:17758–17768.
Downloaded from http://jb.asm.org/ on August 15, 2015 by guest
18.
K. B. Low, B. Magasanik, W. S. Reznikoff, M. Rily, M. Schaechter, and H. E.
Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular
biology, 2nd ed., vol. 1. American Society for Microbiology, Washington,
D.C.
Grossman, A. D., J. W. Erickson, and C. A. Gross. 1984. The htpR gene
product of E. coli is a sigma factor for heat-shock promoters. Cell 38:383–
390.
Gruber, T. M., and C. A. Gross. 2003. Multiple sigma subunits and the
partitioning of bacterial transcription space. Annu. Rev. Microbiol. 57:441–
466.
Guisbert, E., C. Herman, C. Z. Lu, and C. A. Gross. 2004. A chaperone
network controls the heat shock response in E. coli. Genes Dev. 18:2812–
2821.
Horikoshi, M., T. Yura, S. Tsuchimoto, Y. Fukumori, and M. Kanemori.
2004. Conserved region 2.1 of Escherichia coli heat shock transcription factor
32 is required for modulating both metabolic stability and transcriptional
activity. J. Bacteriol. 186:7474–7480.
Jakob, U., W. Muse, M. Eser, and J. C. A. Bardwell. 1999. Chaperone activity
with a redox switch. Cell 96:341–352.
Joo, D. M., N. Ng, and R. Calendar. 1997. A 32 mutant with a single amino
acid change in the highly conserved region 2.2 exhibits reduced core RNA
polymerase affinity. Proc. Natl. Acad. Sci. USA 94:4907–4912.
Joo, D. M., A. Nolte, R. Calendar, Y. N. Zhou, and D. J. Jin. 1998. Multiple
regions on the Escherichia coli heat shock transcription factor 32 determine
core RNA polymerase binding specificity. J. Bacteriol. 180:1095–1102.
Karls, R. K., J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue. 1998.
Metabolic roles of a Rhodobacter sphaeroides member of the 32 family. J.
Bacteriol. 180:10–19.
Karls, R. K., D. J. Jin, and T. J. Donohue. 1993. Transcription properties of
RNA polymerase holoenzymes isolated from the purple nonsulfur bacterium
Rhodobacter sphaeroides. J. Bacteriol. 175:7629–7638.
Kourennaia, O. V., L. Tsujikawa, and P. L. deHaseth. 2005. Mutational
analysis of Escherichia coli heat shock transcription factor sigma 32 reveals
similarities with sigma 70 in recognition of the ⫺35 promoter element and
differences in promoter DNA melting and ⫺10 recognition. J. Bacteriol.
187:6762–6769.
Kusukawa, N., and T. Yura. 1988. Heat shock protein GroE of Escherichia
coli: key protective roles against thermal stress. Genes Dev. 2:874–882.
Landick, R., V. Vaughn, E. T. Lau, R. A. VanBogelen, J. W. Erickson, and
F. C. Neidhardt. 1984. Nucleotide sequence of the heat shock regulatory
gene of E. coli suggests its protein product may be a transcription factor. Cell
38:175.
MacGregor, B. J., R. K. Karls, and T. J. Donohue. 1998. Transcription of the
Rhodobacter sphaeroides cycA P1 promoter by alternate RNA polymerase
holoenzymes. J. Bacteriol. 180:1–9.
McCarty, J. S., S. Rudiger, H.-J. Schonfeld, J. Schneider-Mergener, K.
Nakahigashi, T. Yura, and B. Bukau. 1996. Regulatory region C of the E. coli
heat shock transcription factor, 32, constitutes a DnaK binding site and is
conserved among eubacteria. J. Mol. Biol. 256:829–837.
Minder, A. C., F. Narberhaus, M. Babst, H. Hennecke, and H. M. Fischer.
1997. The dnaKJ operon belongs to the 32-dependent class of heat shock
genes in Bradyrhizobium japonicum. Mol. Gen. Genet. 254:195–206.
Moran, M. A., A. Buchan, J. M. Gonzalez, J. F. Heidelberg, W. B. Whitman,
R. P. Kiene, J. R. Henriksen, G. M. King, R. Belas, C. Fuqua, L. Brinkac, M.
Lewis, S. Johri, B. Weaver, G. Pai, J. A. Eisen, E. Rahe, W. M. Sheldon,
W. Ye, T. R. Miller, J. Carlton, D. A. Rasko, I. T. Paulsen, Q. Ren, S. C.
Daugherty, R. T. Deboy, R. J. Dodson, A. S. Durkin, R. Madupu, W. C.
Nelson, S. A. Sullivan, M. J. Rosovitz, D. H. Haft, J. Selengut, and N. Ward.
2004. Genome sequence of Silicibacter pomeroyi reveals adaptations to the
marine environment. Nature 432:910–913.
Nagai, H., H. Yuzawa, M. Kanemori, and T. Yura. 1994. A distinct segment
ACTIVITY OF R. SPHAEROIDES RpoHII