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J Bacteriol, January 1998, p. 10-19, Vol. 180, No. 1
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Metabolic Roles of a Rhodobacter
sphaeroides Member of the 
32 Family
Russell K.
Karls,1
Jacqueline
Brooks,2
Peter
Rossmeissl,1
Janelle
Luedke,1 and
Timothy
J.
Donohue2,*
Department of
Bacteriology1 and
Graduate Program in
Cell and Molecular Biology,2 University of
Wisconsin
Madison, Madison, Wisconsin 53706
Received 8 August 1997/Accepted 28 October 1997
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ABSTRACT |
We report the role of a gene (rpoH) from the
facultative phototroph Rhodobacter sphaeroides that encodes
a protein (
37) similar to Escherichia coli
32 and other members of the heat shock family of
eubacterial sigma factors. R. sphaeroides 37
controls genes that function during environmental stress, since an
R. sphaeroides 
RpoH mutant is ~30-fold more sensitive
to the toxic oxyanion tellurite than wild-type cells. However, the
RpoH mutant lacks several phenotypes characteristic of E. coli cells lacking 32. For example, an
R. sphaeroides RpoH mutant is not generally defective in phage morphogenesis, since it plates the lytic virus RS1,
as well as its wild-type parent. In characterizing the response of
R. sphaeroides to heat, we found that its growth
temperature profile is different when cells generate energy by aerobic
respiration, anaerobic respiration, or photosynthesis. However, growth
of the RpoH mutant is comparable to that of a wild-type strain under each of these conditions. The RpoH mutant mounted a heat shock response when aerobically grown cells were shifted from 30 to 42°C,
but it exhibited altered induction kinetics of ~120-, 85-, 75-, and
65-kDa proteins. There was also reduced accumulation of several
presumed heat shock transcripts (rpoD PHS,
groESL1, etc.) when aerobically grown RpoH
cells were placed at 42°C. Under aerobic conditions, it appears that
another sigma factor enables the RpoH mutant to mount a heat shock
response, since either RNA polymerase preparations from an RpoH
mutant, reconstituted E37, or a holoenzyme containing a
38-kDa protein (38) each transcribed E. coli
E32-dependent promoters. The lower growth temperature
profile of photosynthetic cells is correlated with a difference in
heat-inducible gene expression, since neither wild-type cells or the
RpoH mutant mount a typical heat shock response after such cultures
were shifted from 30 to 37°C.
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INTRODUCTION |
The heat shock response is a
universal phenomenon that allows cells to survive a multitude of
environmental stresses (28). This response is characterized
by a rapid, transient increase in the rate of synthesis of a highly
conserved set of polypeptides known collectively as heat shock proteins
(HSPs) (14, 28). While increased expression of HSPs is often
seen after a stress, most of these gene products are present at
significant levels in steady-state cells to promote protein synthesis,
folding, and intracellular localization (28). Molecular
chaperone function for HSPs has been demonstrated in key cellular
processes such as DNA replication, cell division, or maintenance of
active protein conformations during conditions that cause cytoplasmic
stress (13, 14, 28, 43).
A molecular picture for what triggers the heat shock response is
beginning to emerge. In the enteric bacterium Escherichia coli, an alternative sigma factor (32) that
recognizes heat shock gene promoters (15) increases in abundance, stability, and activity under conditions that cause the
accumulation of misfolded cytoplasmic proteins (38). Use of
alternate sigma factors could be a common way to control the eubacterial heat shock response, since proteins related to E. coli 32 have been identified from a diverse group
of proteobacteria (4, 24, 27, 32, 41). These other
32 family members are believed to function in much the
same manner as E. coli 32, becoming more
abundant or active in response to thermal or metabolic stimuli that
produce cytoplasmic stress.
While control of the heat shock response by E. coli
32 is considered the eubacterial paradigm, other ways to
regulate procaryotic HSP synthesis exist. An additional alternate sigma
factor (E) recognizes the E. coli
32 gene (rpoH) and several other heat shock
promoters in response to signals that generate periplasmic stress
(11). In several other eubacteria, a cis-active
inverted repeat called CIRCE (for controlling inverted repeat of
chaperone expression) negatively regulates heat shock gene expression
(1, 19, 46). Indeed, recent experiments suggest that
individual Bradyrhizobium japonicum heat shock genes are
regulated by both members of the 32 family and CIRCE
elements (1, 27).
This work sought to identify the function of a member of the
32 family from the facultative phototroph
Rhodobacter sphaeroides. Previous experiments
implicated a 37-kDa R. sphaeroides protein (37) that reacted with antibody against E. coli 32 as a member of the RpoH family
(16). The observation that several E. coli heat
shock promoters were transcribed by R. sphaeroides RNA
polymerase samples that contained this 37-kDa protein supported the
provisional designation of 37 as a member of the
32 family (16). The recent finding that
R. sphaeroides E37 transcribes a promoter
(cycA P1) for an essential component of the R. sphaeroides photosynthetic apparatus like cytochrome
c2 suggested that 37 might have
functions outside its commonly accepted role in HSP synthesis
(21). Since little is known about the ability of
32 family members to recognize genes other than those
which encode HSPs, we were particularly interested in asking if
E37 or its target genes contribute to expression or
assembly of proteins that function in biological energy
generation by R. sphaeroides.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
E.
coli (Table 1) strains were grown at
37°C in Luria-Bertani medium (33). R. sphaeroides strains (Table 1) were routinely grown at 30°C in
Sistrom's succinate-based minimal medium A (37). For growth
of cells by anaerobic respiration in the dark, dimethyl sulfoxide
(DMSO) was used as a terminal electron acceptor in Sistrom's minimal
medium containing 20 mM glucose and 0.2% yeast extract (7).
Plating efficiency of phage RS1 was determined with aerobically grown
cells (6).
Tellurite sensitivity was tested by diluting aerobically grown cells
(~10
9 cells/ml) sufficiently to produce ~100 colonies
per plate (
23).
To score for growth phenotypes associated
with loss of
37, solid media were used routinely. Where
indicated, growth curves
with liquid cultures were performed to test
phenotypes associated
with the
rpoH allele.
Isolation of R. sphaeroides rpoH.
An E. coli RpoH mutant containing the R40 mutation (CAG12517
[44]) was used to isolate an R. sphaeroides
gene that recognizes heat shock promoters. While E. coli
RpoH strains are unable to grow above 20°C, the R40 mutation
allows growth of this strain up to 40°C (17). To identify
genes that restore phage growth to CAG12517, this tester strain was
infected with a lytic R. sphaeroides NCIB8253 
gt11
library (40) at a multiplicity of infection of 0.05.
Chromosomal mapping and Southern blot analysis.
Restricted
R. sphaeroides genomic DNA was separated with a CHEF-DR II
apparatus (Bio-Rad Laboratories, Richmond, Calif.) (39). For
Southern blot analysis (33), restricted R. sphaeroides DNA was transferred to membranes, hybridized with
nick-translated 32P-labeled rpoH probes
(Gibco-BRL, Gaithersburg, Md.), washed at moderate stringency (two
5-min washes at room temperature with a solution consisting of 1× SSPE
[1× SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA {pH 7.7}] and 0.1% sodium dodecyl sulfate [SDS] and two
15-min washes at 45°C with 0.1× SSPE-0.1% SDS), exposed to a
PhosphorImaging screen, and visualized with ImageQuant software
(Molecular Dynamics, Sunnyvale, Calif.).
DNA sequence analysis.
DNA sequencing used either
Taq DNA polymerase and deazanucleotide triphosphates
(Promega, Inc., Madison, Wis.) or automated sequencers at the
University of Wisconsin Madison Biotechnology Center. A list of
plasmids and primers used to sequence the ~1.5-kb segment of R. sphaeroides DNA in pPJR19 is available upon request. DNA sequences
containing rpoH were analyzed with software from the
University of Wisconsin Genetics Computer Group, Madison, Wis.
(5).
Construction of a RpoH mutant.
To produce an R. sphaeroides RpoH mutant, a StyI restriction fragment
internal to rpoH was replaced with a spectinomycin resistance (Spr) gene. Specifically, pPJR19 was digested
with StyI, and the ends were made blunt with T4 DNA
polymerase and ligated with a 2-kb SmaI restriction fragment
carrying an omega (
) cartridge encoding Spr
(31). The resultant plasmid (pPJR26) was digested with
ApaI to produce a restriction fragment containing the
interrupted (rpoH1::Spr) gene.
After the ends of this restriction fragment were made blunt, it was
purified and cloned into pSUP202, a mobilizable suicide vector
(36), which had been digested with PstI and
EcoRI and treated with T4 DNA polymerase. The resulting
plasmid (pPJR29) was used to place a
rpoH1::Spr allele in R. sphaeroides 2.4.1 (7). Screening Spr cells
(25 µg/ml) for those sensitive to tetracycline (1 µg/ml) identified
a strain (RpoH1) where the
rpoH1::Spr allele replaced a
wild-type gene by homologous recombination (8). One
Tcr strain (RpoH26), in which
rpoH1::Spr was integrated into
the chromosome along with the suicide plasmid, was used to aid genomic
mapping of rpoH (see Results).
Rates of protein synthesis.
Rates of protein synthesis were
determined before and after log-phase aerobically grown R. sphaeroides cultures (~109 cells/ml) were shifted
from 30 to 42°C. At indicated times, 1-ml samples were removed,
labeled with 50 µCi of [35S]Transmet (Amersham Corp.,
Arlington Heights, Ill.) for 1 min, and chased with a mixture of 1 M
cysteine and 1 M methionine for 2 min. At this time, cold
trichloroacetic acid was added to a final concentration of 5%,
proteins were harvested by centrifugation (13,000 × g,
10 min), the supernatant was aspirated, and the precipitate was washed
twice with ice-cold 80% acetone. After evaporation of residual liquid
under vacuum, 100 µl of solubilization buffer containing 10%
-mercaptoethanol was added (33). Samples of equal
radioactivity were separated by SDS-12.5% polyacrylamide gel
electrophoresis prior to analysis and quantitation on a PhosphorImager with ImageQuant software (Molecular Dynamics).
For monitoring the rates of protein synthesis under photosynthetic
conditions, cells were grown to mid-exponential phase (~5
× 10
8 CFU/ml) at 30°C in 15-ml screw-capped tubes at a
light intensity
of 10 W/m
2. Protein synthesis was monitored
as described above.
Primer extension assays.
RNA from cells grown aerobically
(45) was used in primer extension assays (2) with
these promoter-specific oligonucleotides: rpoD
PHS, 5'-CCTCGACCGCCTCCTCGATTTCCT-3'
(3a); groESL1,
5'-CACGGTCATGCAGCGTTTG-3' (19); rrnB,
5'-AAGACAAAACAAACCGAGACGCCA-3' (10). Products were separated on denaturing polyacrylamide gels (2) prior to estimation of their levels on a PhosphorImager with ImageQuant software.
In vitro transcription assays.
Conditions for preparation of
R. sphaeroides RNA polymerase, reconstitution of core enzyme
samples with potential sigma factors, and their use for in vitro
transcription assays with plasmid templates have been described
previously (21).
Nucleotide sequence accession number.
DNA sequences
containing rpoH were deposited with accession no. U82397.
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RESULTS |
Identification of R. sphaeroides rpoH.
E. coli
RpoH mutants are unable to support growth because they are
limited for the HSPs that are required for phage DNA replication
(13). To identify a gene product that supports infection, an E. coli RpoH strain was infected with an
R. sphaeroides genomic library in the lytic gt11 vector
(40). Phage DNA was purified (33), digested with
EcoRI to yield a 4.3-kb restriction fragment, and the DNA
was cloned into pGEM-7Zf(+) to yield pPJR18. To confirm that heat shock
gene expression was dependent on R. sphaeroides DNA, this
plasmid was shown to support 44°C growth and confer sensitivity
to an E. coli RpoH mutant (data not shown). In addition,
a plasmid (pPJR19) containing a smaller, ~1.5-kb, restriction
fragment (Table 1) activated an rpoD
PHS::lacZ fusion (data not shown). The
following experiments indicate that the responsible R. sphaeroides gene (rpoH) encodes a sigma factor related
to proteins in the eubacterial 32 family.
R. sphaeroides RpoH is a member of the
32 family.
The R. sphaeroides DNA
that activates E. coli heat shock gene expression encodes a
298-amino-acid protein (33.7 kDa) whose putative start codon
(coordinate 418 in accession number U82397) begins 10 bases downstream
of a potential ribosome binding site (AGAGG). The deduced protein has a
high degree of amino acid similarity to eubacterial proteins in the
32 family (Fig. 1); it
displays 68% identity to Caulobacter crescentus 32 (32, 41), 66% identity to
Agrobacterium tumefaciens 32 (26),
58% identity to B. japonicum 32
(27), and 40% identity to E. coli
32 (15, 18). The similarity of R. sphaeroides RpoH to 32 family members extends
throughout the protein, but the considerable conservation in regions
4.2 (
35 recognition) and 2.4 (10 recognition) explains why
E37 transcribes E. coli 32
promoters (16, 21; also see below). The amino acid
similarity is particularly striking in a unique and highly conserved
9-amino-acid insertion [Q(R/K)KLFFNLR], designated the RpoH box
(26) (Fig. 1), that has been implicated in DnaK-mediated
turnover of E. coli 32 (25). Other
features conserved between R. sphaeroides RpoH and
related proteins from nonenteric eubacteria include an insertion in
region 3.1 and a C-terminal extension (Fig. 1). It appears that the
known 32 family members can provisionally be separated
into two groups (Fig. 1), since proteins from the 
proteobacteria
(R. sphaeroides, C. crescentus, A. tumefaciens, Zymomonas mobilis, and B. japonicum) are more similar to each other and larger than their
counterparts from 
proteobacteria (E. coli,
Haemophilus influenzae, Pseudomonas aeruginosa,
and Citrobacter freundii). The second amino acid residue in
the RpoH box also seems to be a distinguishing feature; it is a lysine
in proteins from the proteobacteria and arginine in those
from the proteobacteria.
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FIG. 1.
Alignment of 32 family members (generated
by using the PILEUP program [5]). Identical amino
acids are indicated in boldface. The broken lines above the alignment
denote conserved regions of eubacterial sigma factors (20).
GenBank accession numbers for 32 family members are as
follows: C. crescentus, U39791; A. tumefaciens,
D50828; Z. mobilis, D50832; B. japonicum, U55047;
H. influenzae, U32713; P. aeruginosa, D50052;
C. freundii, X14960; and E. coli, U00039. RpoH
box and helix-turn-helix (H-T-H) sequences are underlined. Gaps
introduced to maximize alignment are indicated by dots. Asterisks
indicate the end of the protein. Abbreviations: Rsph, R. sphaeroides; Ccre, C. crescentus; Atum,
A. tumefaciens; Zmob, Z. mobilis; Bjap, B. japonicum; Hinf, H. influenzae; Paer, P. aeruginosa; Cfre, C. freundii; Ecoh, E. coli.
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Mapping rpoH.
When wild-type genomic DNA was probed,
rpoH mapped to an 1,100-kb AseI restriction
fragment (39; data not shown). To better position
rpoH, we took advantage of a strain (RpoH26) containing an
additional AseI restriction site from the suicide plasmid
pPJR29 (Table 1). When RpoH26 DNA is treated with AseI, the
~1,100-kb AseI restriction fragment was digested in two
~500-kb fragments (data not shown). This places rpoH
near the center of the ~1,100-kb AseI fragment or at
coordinate ~2250 ± 50 kb on chromosome I.
An R. sphaeroides RpoH strain is not temperature
sensitive.
To determine the role of R. sphaeroides
37, an rpoH insertion-replacement was
generated in a wild-type strain. Because we expected a RpoH mutant
to be temperature sensitive, like its E. coli counterpart (44), selection for the
rpoH1::Spr allele was initially
performed at temperatures between 10 and 30°C. To our surprise, cells
lacking RpoH (i.e., ones that are both Spr and Tc
sensitive) were obtained at all temperatures. To confirm the genotype
of these presumptive RpoH mutants, Southern blot analysis with
rpoH, suicide plasmid (pSup202), and spc-specific probes was used to show that the
rpoH::Spr allele had been
incorporated by an even number of crossover events (data not shown).
Respiratory phenotypes associated with the loss of R. sphaeroides 37.
Both wild-type and
RpoH cells grew at temperatures up to 42°C under either aerobic
conditions or when DMSO served as an anaerobic electron acceptor (Table
2). Loss of 37 did not
alter the ability of the lytic bacteriophage RS1 (6) to
infect cells, since this virus plated at wild-type efficiency on
RpoH cells at all temperatures tested (Table 2). Thus, we conclude
37 is not required for viability, phage replication, or
energy generation in the presence or absence of oxygen.
Because resistance to toxic compounds often requires HSPs (
28,
43), we compared aerobic sensitivity of wild-type and
RpoH
cells to the heavy metal oxyanion tellurite. Wild-type cells plate
at
100% efficiency at tellurite concentrations up to 300 µM, but
the
plating efficiency of the
RpoH mutant decreases by some 4
orders of
magnitude between 10 and 300 µM tellurite (Fig.
2).
Tellurite resistance in both strains
results in oxyanion reduction,
since all colonies exhibited the black
phenotype diagnostic of
metal deposition (
23). At tellurite
concentrations greater than
100 µM, plating efficiency of the
RpoH
mutant plateaus at ~10
3, suggesting that there is
37-independent pathway for heavy metal reduction.
Finally, the
RpoH
mutant appears incapable of growth at tellurite
concentrations
higher than 300 µM, since colonies were not observed
when as many
as 10
8 cells are plated.
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FIG. 2.
Plating efficiency of aerobically grown R. sphaeroides strains at different tellurite concentrations.
Viability of wild-type cells and the RpoH mutant is expressed as the
number of CFU per milliliter of original culture at each tellurite
concentration.
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Protein synthesis after aerobically grown cells are shifted to
42°C.
To monitor the ability of cells to mount a heat shock
response, protein synthesis was examined before and after aerobically grown cultures were placed at 42°C. In wild-type cells, a pattern of
heat-inducible proteins was observed that is characteristic of that
seen in other mesophilic eubacteria (14, 43). For example,
the synthesis rate of several low-molecular-weight proteins (presumed
Hsp10 family members) as well as those of ~65 (presumed Hsp60), 75 (presumed Hsp70), 85 (presumed Hsp90), and 120 kDa (presumed Clp family
member) increased rapidly and transiently after wild-type aerobically
grown cells were shifted from 30 to 42°C (Fig.
3). The response observed in wild-type
cells can provisionally identify three general classes of
heat-responsive proteins. The first class includes three proteins
(~120, 85, and 65 kDa) whose synthesis rate increased within 5 min
after a shift to 42°C and then returns to a preshift rate within 20 min (Fig. 4A, B, and D). The synthesis
rates of three other proteins (~75, 17, and 14 kDa) also increased
rapidly when wild-type cells were placed at 42°C, but they remained
elevated even 20 min after the temperature shift (Fig. 4C, I, and J).
The third class includes two proteins (~38 and 35 kDa) whose
synthesis rate decreased rapidly when wild-type cells were
shifted to 42°C (Fig. 4E and F).
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FIG. 3.
Protein synthesis after aerobically grown wild-type and
RpoH cells are shifted from 30 to 42°C. Strains, temperatures, and
sampling times (in minutes [indicated by prime symbols]) after the
shift are indicated over the gel. The migration of prestained molecular
mass standards (Gibco-BRL) was used to estimate the apparent molecular
masses of the indicated proteins.
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FIG. 4.
Relative synthesis rates of selected proteins before and
after aerobically grown cells are shifted to 42°C. The x
axes show the minutes after exposure to 42°C (1 indicates cells
sampled 1 min before the temperature shift). The y axes show
the relative pixel intensity of individual proteins in Fig. 5.
Wild-type () and RpoH ( )
cells are shown.
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When an aerobically grown culture of the
RpoH mutant was shifted to
42°C, the synthesis rates of the 38- and 35-kDa proteins
were
indistinguishable from those in wild-type cells (Fig.
3 and
4E and F).
However, other heat-inducible proteins in wild-type
cells behaved
differently in the
RpoH mutant. For example, there
was no detectable
increase in the synthesis rate of the heat-inducible
120- and 85-kDa
proteins when the
RpoH mutant was placed at 42°C
(Fig.
4A and B).
Within 5 min after the
RpoH mutant was incubated
at 42°C, there
was also smaller increases in the synthesis rates
of the heat-inducible
75- and 65-kDa proteins (Fig.
4C and D).
By 20 min after the
RpoH
mutant was placed at 42°C (Fig.
4C),
the synthesis rate of the 75-kDa
protein approximated that seen
in cells grown at 30°C. Despite these
differences, heat induction
of several potential HSPs in the
RpoH
mutant is a likely explanation
for the aerobic growth of cells lacking
37 at temperatures up to 42°C (Table
2).
The RpoH mutant contains elevated levels of presumed heat shock
transcripts after aerobically grown cells are shifted to 42°C.
Primer extension assays were performed to test if loss of
37 altered heat shock promoter function. For this
analysis, we chose to monitor several R. sphaeroides
promoters, cycA P1, rpoH PHS and
groESL1, which have significant similarity to
the E. coli 32 consensus sequence (Fig.
5D). One other, R. sphaeroides
rrnB (10, 30), has promoter elements related to the
E. coli 70 and 32 consensus
sequences (Fig. 5D).
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FIG. 5.
Transcript levels before and 30 min after aerobically
grown wild-type and RpoH cells were shifted to 42°C. (A) Primer
extension analysis (~8 µg of RNA per lane) of the rrnB,
rpoD PHS, and groESL1
transcripts at the indicated temperatures. (B) Primer extension
analysis (~8 µg of RNA per lane) of cycA P1-specific
transcripts at the indicated temperatures. (C) Transcript abundance
(pixel intensity) from panels A and B. Induction ratios denote the
increase at 42°C relative to the 30°C level. (D) Comparison of
potential R. sphaeroides promoters with the E. coli 32 consensus sequence; matches are denoted by
boldface type.
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Levels of
cycA P1,
rrnB,
rpoD
P
HS, and
groESL1-specific
transcripts increased ~2-, 3-, 8- and 17-fold, respectively, 30 min
after aerobically grown wild-type cells were placed at 42°C (Fig.
5A
to C). If the abundance of these primer extension products
is taken as
an estimate of promoter function, then loss of
37 alters
their heat inducibility because there were reproducibly
smaller
increases in the abundance of the
rrnB (~1.1-fold),
cycA P1 (~1.3-fold),
rpoD P
HS
(~2.7-fold), and
groESL1 (~10-fold)
transcripts
30 min after the
RpoH mutant was placed at 42°C (Fig.
5A to C).
The residual increase in
rpoD P
HS and
groESL1 transcript levels
after the
RpoH
mutant was shifted to 42°C suggests that some
other system is
increasing promoter function when cells that lack
37 are
placed at a higher temperature.
Heat shock promoters are recognized by multiple R. sphaeroides sigma factors.
To ask if recognition of heat
shock promoters by another RNA polymerase holoenzyme could explain how
aerobically grown RpoH cells mount a heat shock response, E. coli dnaK P1, htpG, and rpoD
PHS promoters were tested for function in vitro with
R. sphaeroides RNA polymerase preparations. Enzyme
preparations from wild-type or RpoH cells (21)
transcribed all of these E. coli heat shock promoters and
produced identically sized transcripts (Fig.
6) which are indistinguishable in size to
those generated by E. coli 32
(16). The lower transcript abundance with equivalent amounts of RNA polymerase from the RpoH mutant probably reflects a reduced level of the cognate holoenzyme in cells that lack 37.
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FIG. 6.
Transcription of E. coli heat shock
promoters, dnaK P1, htpG, and
rpoD PHS, by mixtures of R. sphaeroides RNA polymerase holoenzymes. Wild-type (w.t.) and
rpoH R. sphaeroides RNA polymerase (RNAP) were used
(+). The RNA1 transcript is a 70-dependent product from
the origin of DNA replication on all templates (21).
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To identify what
R. sphaeroides enzyme(s) recognized
these
E. coli 32 promoters, RNA polymerase
holoenzymes were reconstituted by adding
potential sigma factors from
the
RpoH mutant to a core preparation
(
21). This analysis
identified an ~38-kDa protein (
38) that allows
transcription of
E. coli dnaK P
1 when it is
added
to core RNA polymerase (Fig.
7).
This same 38-kDa protein directs
transcription of
R. sphaeroides cycA P1 when reconstituted with
core RNA
polymerase subunits (
21). Thus, both E
38 and
E
37 recognize heat shock promoters.
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FIG. 7.
R. sphaeroides E38
recognizes E. coli dnaK P1. RNA polymerase
holoenzymes were reconstituted by adding potential sigma factors (lanes
1 to 28) from the RpoH mutant to a core RNA polymerase preparation
from the RpoH mutant (21). The protein in lane 20 was the
38-kDa polypeptide (38) that allowed core RNA polymerase
to transcribe the cycA P1 heat shock promoter
(21).
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Loss of 37 alters the response of photosynthetic
cells to increased temperature.
Given the known function of HSPs
like GroESL in assembly of photosynthetic functions such as
ribulose-1,5-bisphosphate carboxylase (19), we asked if
photosynthetic cells were particularly sensitive to the loss of
37. We were surprised to find that wild-type R. sphaeroides has a lower temperature maximum under
photosynthetic conditions, since no growth was observed on solid media
at either 37 or 42°C (Table 2). However, loss of 37
does not make photosynthetic cells temperature sensitive, since the
RpoH strain grew normally at temperatures up to 30°C (Fig. 8 and Table 2). Photosynthetic RpoH
cells grown at 30°C with moderate light (22) contained
levels of light-harvesting bacteriochlorophyll-protein complexes that
are indistinguishable from a wild-type strain (Table 2). Thus, loss of
37 does not have a significant effect on photosynthetic
growth or assembly of pigment-protein complexes at 30°C.
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FIG. 8.
Photosynthetic growth of wild-type cells (top) and the
RpoH mutant (bottom) at 30°C and after a temperature shift from 30 to 37°C. Photosynthetic growth of wild-type cells ( ) and those
lacking 37 ( ) at 30°C and the response seen when
wild-type cells (+) or the RpoH ( ) mutant are shifted from 30 to
37°C are shown. The broken vertical line indicates when the
photosynthetic cultures of wild-type and RpoH cells were placed at
37°C.
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During these experiments, we noted a difference in the response of
wild-type and
RpoH cells to increased temperature under
photosynthetic conditions. When a wild-type photosynthetic culture
was
shifted from 30 to 37°C, there was typically an ~1.5- to 2.5-fold
increase in culture turbidity after the temperature increase (Fig.
8).
These turbidity increases cease long before those in a control
culture
maintained at 30°C. Microscopic examination of wild-type
photosynthetic cultures several hours after the shift to 37°C
revealed a significant number of paired cells or doublets in the
population (data not shown). One explanation for this behavior
is that
wild-type photosynthetic cultures only complete existing
rounds of DNA
replication or cell division after the temperature
is elevated.
Unfortunately, exposure to 37°C under photosynthetic
conditions is
bactericidal (data not shown), so we were unable
to observe
increases in CFU after photosynthetic cells were placed
at
37°C. In the
RpoH mutant, increases in culture turbidity cease
rapidly when photosynthetic cultures are shifted from 30 to 37°C
(Fig.
8). There are no microscopic indications for the formation
of
cell doublets after photosynthetic cultures of the
RpoH mutant
were
placed at 37°C (data not shown). Compared with wild-type
cells,
photosynthetic cells lacking
37 seem unable to complete
previously initiated cell division cycles
after exposure to 37°C.
To gain additional insight into potential reasons for this behavior, we
monitored bulk protein synthesis before and after
photosynthetic cells
were shifted from 30 to 37°C. At 30°C, there
are significant
differences between the protein synthesis pattern
of photosynthetic and
aerobically grown cultures (compare Fig.
3 to Fig.
9), but the protein synthesis patterns of
wild-type
and
RpoH cells are indistinguishable both before and after
photosynthetic
cells were incubated at 37°C (Fig.
9). Thus, the
different growth
response when photosynthetic cultures are shifted to
37°C (Fig.
8) cannot reflect a cessation of translation after the
temperature
increase. Of equal significance, no detectable heat shock
response
was observed when photosynthetic wild-type or
RpoH cells
were
shifted to 37°C (compare Fig.
3 and
9); only proteins of ~62
and
17 kDa exhibited a significant increase in their synthesis rate
when photosynthetic cultures of either strain were shifted to
37°C
(Fig.
9). Thus, the failure to mount a heat shock response
at 37°C
under photosynthetic conditions is a likely explanation
for why
wild-type and
RpoH cells are unable to grow at this temperature.
View larger version (63K):
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|
FIG. 9.
Protein synthesis before and after photosynthetic
wild-type and RpoH cells are shifted from 30 to 37°C. Temperatures
and sampling times (in minutes [indicated by prime symbols]) after
the shift are indicated over the gel. The migration of molecular mass
standards (Gibco-BRL) indicated to the left of the gel were used to
estimate the apparent sizes of the indicated proteins.
|
|
| |
DISCUSSION |
It was previously suggested that R. sphaeroides
E37 recognized E. coli heat shock promoters
because an RNA polymerase fraction containing a 37-kDa protein
transcribed these genes in vitro (16). The amino acid
similarity between R. sphaeroides RpoH and proteins in
the eubacterial heat shock sigma factor family, its ability to restore
bacteriophage sensitivity to an E. coli
32 null mutant, and the heat shock gene expression seen
when 37 is expressed in E. coli cells lacking
32 indicate that E37 recognizes promoters
related to the E. coli 32 consensus sequence.
The observation that RNA polymerase preparations from an R. sphaeroides RpoH mutant lack this 37-kDa protein further suggests that rpoH encodes 37
(21). Additional conclusions and questions generated by this characterization of R. sphaeroides RpoH are presented
below.
R. sphaeroides RpoH is essential only under limited
conditions.
Growth of cells lacking 37 at the same
temperatures as their wild-type counterparts was surprising when one
considers that E. coli RpoH mutants cannot grow at
temperatures above 20°C (44). The ability of R. sphaeroides RpoH cells to support infection by the lytic
RS1 virus at all temperatures tested also contrasts with the inability
of E. coli RpoH mutants to propagate phages such as (which served as the basis for our isolation of R. sphaeroides rpoH). Indeed, only two conditions were found
where the R. sphaeroides RpoH mutant had a phenotype.
One difference between wild-type and
RpoH cells was the increased
aerobic sensitivity of cells lacking
37 to the toxic
heavy metal oxyanion tellurite (
23). Increased
sensitivity
of the
R. sphaeroides RpoH mutant to tellurite could
reflect a limitation of HSPs or other members of a presumed
37 regulon. While an
R. sphaeroides RdxA
mutant has a tellurite-sensitive
phenotype reminiscent of the
RpoH
mutant (
29), it is not known
if this or proteins directly
involved in heavy metal reduction
are transcribed by
E
37. Alternatively, one promoter for the cytochrome
c2 gene (
cycA P1) is transcribed by
R. sphaeroides E
37 (
21). If
cytochrome
c2 transferred electrons to the
membrane-bound
reductase implicated in tellurite reduction
(
23), then increased
cycA P1 activity might be
required for heavy metal resistance.
This might also explain why
tellurite reduction was blocked in
cytochrome
c2
null mutants (
23).
Another phenotype associated with loss of
R. sphaeroides
37 was the rapid cessation in culture turbidity
increases when photosynthetic
cells were shifted from 30 to 37°C.
This behavior of the
RpoH
mutant does not reflect a total block in
macromolecular synthesis,
since translation continues after
photosynthetic cells are shifted
to 37°C. However, the failure of
both the
RpoH mutant and wild-type
cells to mount a heat shock
response at 37°C under photosynthetic
conditions suggests they are
limited for the HSPs needed to progress
through the cell cycle.
If a component of the photosynthetic apparatus
were temperature
sensitive, this behavior could reflect a lack
of energy for HSP
function after such cells are placed at 37°C.
R. sphaeroides mounts a heat shock response in the
presence and absence of 37.
From the universal
nature of the heat shock response, it is not surprising that synthesis
rates of a number of proteins and the transcript levels from several
potential heat shock genes increase after aerobic cells are placed at
42°C (42). Heat induction of several proteins was
transient, since the synthesis rate of several putative HSPs (Clp,
Hsp90, and Hsp60 homologs) decreased to a new steady state within 20 min after temperature up shift. Other potential HSPs (Hsp70 homologs
and several lower-molecular-weight polypeptides) continued to be
synthesized at an increased rate even after 20 min at 42°C. Such a
persistent induction of HSPs is not seen in well-studied systems like
E. coli (14, 43).
When the aerobic heat shock response is analyzed, induction of ~75-,
65-, and 17-kDa proteins seems partially dependent on
37, since the magnitude or timing of their synthesis is
altered
when the
RpoH mutant is shifted to 42°C. If the
heat-inducible
~65-kDa protein is a product of the
R. sphaeroides groESL1 operon
(
19), then
this reduction probably reflects altered promoter
function in cells
lacking
37, since we observed a diminished increase in
this transcript 30
min after the
RpoH mutant was placed at 42°C.
Heat induction
of another group of presumed HSPs (~120 and 85 kDa)
could be totally
dependent on
37, since their rate of
synthesis is not measurably increased in
the
RpoH mutant.
The temperature maxima of R. sphaeroides varies
with its mode of energy generation.
We were surprised by the
selective inability of R. sphaeroides to grow or mount a
heat shock response under photosynthetic conditions at temperatures
37°C. Photosynthetic temperature maxima of ~35°C have been
reported for many wild-type R. sphaeroides strains
(9), yet we have shown that strain 2.4.1 grows and mounts a
heat shock response at 42°C when it is generating energy by aerobic
or anaerobic respiration. This conditional difference in temperature
profile does not reflect an inability to synthesize photosynthetic
pigments, since cells using DMSO as an anaerobic electron acceptor at
42°C have colony pigmentation characteristic of the presence of
bacteriochlorophyll-protein complexes (data not shown).
R. sphaeroides has two sigma factors which
recognize heat shock promoters.
One likely reason why cells
lacking 37 mount a heat shock response is a 38-kDa
protein (38) that directs core RNA polymerase to
recognize 32 promoters like E. coli dnaK
P1 and R. sphaeroides cycA P1
(21). A similar duplication of RpoH function exists in
B. japonicum where a second related gene is evident in
genomic Southern blots probed with E. coli rpoH
(27). At the stringency conditions we employed, no
additional sequences related to R. sphaeroides rpoH were
observed in genomic Southern blots (data not shown). Until information
is available on 38, its sequence and functional
similarity to members of the 32 family remain open
questions.
A
B. japonicum rpoH mutation also does not
cause temperature sensitivity, presumably because this
proteobacterium contains
other related sigma factors (
27).
Thus, it will be interesting
to see if the existence of a second sigma
factor that transcribes
heat shock genes extends to the other
eubacteria from which proteins
in the
32 family have
been identified.
The presence of a CIRCE element in the
R. sphaeroides
groESL1 operon (
19) suggests that other
mechanisms can contribute
to increased heat shock gene expression. The
discovery that mutations
which increase activity of an
R. sphaeroides E homolog alters expression of
several genes (
30a,
34,
35),
including one recognized by
37 (
21), reveals additional potential
components of this bacterium's
response to environmental or metabolic
stress. Experiments are
in progress to define the roles and metabolic
signals for these
alternative
R. sphaeroides sigma
factors.
| |
ACKNOWLEDGMENTS |
These experiments were supported by grant NIH GM37509 to
T.J.D. J.B. was supported by NIH Biotechnology predoctoral
training grant GM08349 to UW-Madison. P.R. was supported by NIH grant
GM36278 and the Fred-Bascom Professorship from the University of
Wisconsin Foundation to Carol A. Gross.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department
of Bacteriology, University of WisconsinMadison, 1550 Linden Dr.,
Madison, WI 53706. Phone: (608) 262-4663. Fax: (608) 262-9865. E-mail: tdonohue{at}bact.wisc.edu.
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Abstract
Introduction
