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Journal of Bacteriology, January 1999, p. 83-90, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Multiple Small Heat Shock Proteins in
Rhizobia
Martin
Münchbach,1
Andreas
Nocker,2 and
Franz
Narberhaus2,*
Protein Chemistry
Laboratory1 and
Mikrobiologisches
Institut,2 Eidgenössische Technische
Hochschule, CH-8092 Zürich, Switzerland
Received 22 July 1998/Accepted 28 October 1998
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ABSTRACT |
Seven genes coding for small heat shock proteins (sHsps) in
Bradyrhizobium japonicum have been identified. They are
organized in five operons that are coordinately regulated by ROSE, a
negatively cis-acting DNA element. The deduced sHsps can be
divided into two separate classes: class A, consisting of proteins that
show similarity to Escherichia coli IbpA and IbpB, and
class B, whose members display significant similarity to other sHsps
from prokaryotes and eukaryotes. Two-dimensional gel electrophoresis
and Edman sequencing revealed the presence of at least 12 sHsps in
B. japonicum, indicating a remarkable abundance of sHsps in
this organism. Three additional members of class A and two potentially
novel heat shock proteins were identified on the basis of their amino
termini. The presence of multiple sHsps was also demonstrated for a
variety of Rhizobium and Bradyrhizobium species
by immunoblot analysis and two-dimensional gel electrophoresis. An
extensive database survey revealed that, in contrast to the rhizobia,
other bacteria contain maximally two sHsps whereas many plants have
been reported to possess a sHsp superfamily.
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INTRODUCTION |
All organisms so far examined
respond to a sudden increase in growth temperature by inducing the
synthesis of a number of heat shock proteins (Hsps). Some of these
proteins are also important during normal growth. The regulation,
structure, and function of several Hsps have been studied in great
detail. The chaperone machineries GroES/GroEL and DnaK/DnaJ/GrpE, for
example, are involved in diverse processes such as protein folding and
protein degradation, assembly of protein complexes, and transport of
proteins across membranes. Their function appears to be highly
conserved between prokaryotes and eukaryotes (reviewed in references
7 and 40).
Comparatively little is known, however, about small Hsps (sHsps). In
contrast to the highly conserved DnaK and GroEL proteins, sHsps show
much less sequence similarity. This protein family is characterized by
the following criteria: (i) a molecular mass typically between 12 and
30 kDa; (ii) a conserved central domain, referred to as the
-crystallin domain; (iii) formation of large oligomeric complexes,
ranging from 150 to 800 kDa; (iv) ATP-independent chaperone activity
(9, 27, 51). The latter concept, however, has been
challenged by the observation that ATP enhances the molecular chaperone
activity of B-crystallin (36). According to the present model, sHsps bind to denatured proteins accumulated under stress conditions and maintain them in a folding-competent state (15, 32). Recently, the crystal structure of a sHsp from
Methanococcus jannaschii has been solved (29).
Twenty-four monomers form a hollow spherical complex with a total of 14 "windows" that might allow polypeptides to enter the complex.
Not surprisingly, most of the work on sHsps has been conducted with
eukaryotic members of this superfamily because they are related to the
-crystallin proteins of the vertebrate eye lens (26).
-Crystallins play a structural role in maintaining lens stability
and transparency but notably they are also expressed in nonlenticular
tissues, e.g., in heart, muscle, and kidney (3). A
remarkable abundance of sHsps was reported in heat-stressed plants. Up
to 30 different sHsps comprising six different classes are induced
after a temperature upshift, depending on the plant species. Each gene
family encodes proteins localized in a distinct cellular compartment
(51).
Most bacteria appear to have only a small number of heat shock
proteins. The completed genome sequences indicate that Mycoplasma genitalium completely lacks any gene coding for sHsps
(19). M. jannaschii encodes one and
Escherichia coli encodes two sHsps (8, 17). The
first hint that Rhizobiaceae may be an exception in that
they possess a larger set of sHsps was provided by a two-dimensional gel analysis by Michiels et al. (34). The authors compared
the induction of Hsps in a heat-tolerant and a heat-sensitive
Rhizobium strain and observed eight heat-inducible protein
spots in extracts from the temperature-sensitive strain. By contrast,
the tropical, heat-tolerant strain induced only two sHsps.
In the process of elucidating the complex regulatory network that
controls the heat shock response of Bradyrhizobium
japonicum, the nitrogen-fixing root-nodule symbiont of soybean, we
identified six genes encoding sHsps (37, 38). They are
organized in four operons that are located in an extended heat-shock
gene cluster. Each operon is preceded by a conserved DNA element of
approximately 100 bp that is positioned between the transcription start
and the start codon of the first gene. This element was designated ROSE
(for Repression Of heat Shock gene Expression), and several lines of
evidence suggest that it serves as a binding site for a putative
repressor protein under non-heat shock conditions (37).
Here we report on a bacterial sHsp superfamily comprising at least 12 members. The seven B. japonicum sHsps identified so far can
be grouped into two distinct classes. We monitored the induction of
sHsps under various stress conditions and examined their heat shock
induction by two-dimensional gel electrophoresis. Finally, we provide
evidence that the presence of a sHsp superfamily is not restricted to
B. japonicum but might be widespread in the Rhizobiaceae.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
B.
japonicum 110spc4 was grown aerobically at 28°C in
PSY medium (43) supplemented with 0.1% (wt/vol) arabinose
and 100 µg of spectinomycin per ml. YEM medium (12)
supplemented with 10 mM KNO3 was used for anaerobic
B. japonicum cultures. Bradyrhizobium sp.
(Parasponia) ANU289 and Bradyrhizobium sp.
(Lupinus) ATCC 10319 were propagated in PSY medium with
0.1% (wt/vol) arabinose. TY medium (5) was used to grow
Rhizobium leguminosarum bv. viciae 897, Rhizobium
etli (formerly R. leguminosarum bv. phaseoli) 8002, R. leguminosarum bv. trifolii ATCC 14480, Rhizobium sp. strain NGR234, and Sinorhizobium
meliloti 2011. E. coli cells were grown in
Luria-Bertani medium (35) supplemented with ampicillin (200 µg/ml) if required.
DNA manipulations and sequence analysis.
Recombinant DNA
techniques were performed according to standard protocols
(45). Chromosomal DNA was isolated as described previously
(23). Southern blot hybridizations using DIG
(digoxigenin-11-dUTP)-labeled DNA probes were performed according to
the manufacturer's instructions (Boehringer GmbH, Mannheim, Germany).
The 111-bp ROSE1 probe was produced by PCR using plasmid
pRJ5035, a pUC18 derivative containing a 1.7-kb HindIII
insert carrying the hspA gene region (38), and
the oligonucleotides Sig36 (5'-CGCCGCGACAAGCGGTCC-3') and Sig37 (5'-GTCCTCATAGCCAAATCCTCC-3'). Plasmid DNA was
sequenced by the chain termination method (46) with a Model
373 DNA sequencer (Applied Biosystems, Foster City, Calif.). The DNA
sequence was analyzed with the software package of the Genetics
Computer Group of the University of Wisconsin
Madison (UWGCG) (version
8.0) or the National Center for Biotechnology Information network
server. Multiple sequence alignments were generated with the PILEUP
program provided by the UWGCG software.
Western blot (immunoblot) analysis.
Crude cell extracts were
prepared, separated on sodium dodecyl sulfate-12% polyacrylamide
gels, and transferred to nitrocellulose membranes as described
previously (39). Bacteroid extracts were prepared as
described elsewhere (16). Anti-E. coli IbpA serum (2) was kindly provided by A. Easton (St. Louis, Mo.) and
was used in 1,500-fold dilution. Primary rabbit antibodies were
detected with the Chemiluminescence Western Blotting Kit (Boehringer GmbH).
Two-dimensional gel electrophoresis and Edman sequencing.
Two-dimensional gel electrophoresis, protein elution, concentration,
electrotransfer, and N-terminal sequencing were performed as described
elsewhere (42).
Transcript mapping.
RNA isolation and primer extension
analysis was performed as described elsewhere (4). The
oligonucleotides AN1 (5'-CTGAACATAGTCTGCCAGGTTGAACTGC-3') and AN18 (5'-CCGTTTCAACGAGGTCGAAAAGGC-3') were used to
determine the hspH transcription start site.
Nucleotide sequence accession numbers.
The nucleotide
sequences described here have been deposited in the EMBL, GenBank, and
DDBJ databases under the following accession numbers: U55047
(hspA, hspB, and hspC gene region),
AJ003064 (hspD, hspE, and hspF gene
region), and AJ010144 (hspH gene region).
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RESULTS AND DISCUSSION |
Two classes of sHsps in B. japonicum.
Six genes coding
for small Hsps (hspA, -B, -C,
-D, -E, and -F) have recently been
identified in a heat shock gene cluster of B. japonicum
(37, 38). They are organized in four operons together with
some additional heat shock genes. Heat-inducible transcription of each
operon is mediated by ROSE, a novel regulatory element that consists of
approximately 100 bp and precedes the first gene of each operon
(37). We identified a putative fifth ROSE-dependent operon
by using a ROSE1 fragment as a probe in Southern
hybridization experiments (data not shown). Two hybridizing fragments,
a 5.8-kb BamHI fragment and a 5.6-kb SalI
fragment, were subsequently cloned and found to contain the
hspH gene region (Fig. 1A). No
additional heat shock genes were present up- or downstream of
hspH. An amino acid sequence comparison of the deduced small
Hsps revealed that they fall into two distinct classes, as indicated in
Fig. 1A and shown more precisely in Fig.
2. Class A contains only bacterial
proteins, namely the B. japonicum proteins HspA, -B, -D, -E,
and -H, E. coli IbpA and IbpB, and Legionella pneumophila GspA. It is evident from the alignment that proteins belonging to this class are highly similar to each other throughout their entire length (between 34 and 73% positional amino acid sequence
identity). The similarity is not restricted to the -crystallin domain but extends into the flanking amino- and carboxy-terminal regions. Class B proteins are much more divergent in length, sequence, and phylogenetic origin. They include prokaryotic as well as eukaryotic members from a wide variety of organisms. The similarity between class
A proteins and class B proteins is rather low (around 20% amino acid
sequence identity). Although the degree of homology within class B is
significant (between 30 and 60% identical amino acids), only the
B. japonicum proteins HspC and HspF reach the latter,
highest score. The identity among the other members is generally
between 30 and 35%. Identical amino acids are almost exclusively
displayed in the -crystallin domain, and the flanking regions are
highly variable in length and sequence (with the exception of HspC and
HspF).
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FIG. 1.
Physical map of five ROSE-dependent heat shock operons
of B. japonicum and determination of the transcription start
site upstream of ROSE5. (A) Schematic representation of the
ROSE-dependent operons. The ROSE elements (1 through 5) are represented
by black boxes. Class A and class B small heat shock genes are
indicated. No significant open reading frames were identified
downstream of hspD and hspH. (B) Primer extension
analysis to determine the transcription start site upstream of
hspH. The extension product of primer AN18 is shown. The
same primer was used for the corresponding sequencing reaction
(TCGA).
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FIG. 2.
Amino acid sequence alignment of sHsp representatives
from class A and class B. Regions corresponding to the N-terminal
domain, -crystallin domain, and C-terminal extension are indicated
(9, 33). The consensus sequences (ConA and ConB) are defined
by capital letters when an amino acid is present in all eight aligned
sequences and by lowercase letters when an amino acid is present in at
least five of the eight sequences. Amino acids that conform with the
consensus are indicated by asterisks. Amino acids appearing in both
consensus sequences are connected by a line. The complete sequence of
the deduced proteins is shown and designated as follows: HspA to HspH,
B. japonicum HspA to HspH (references 37
and 38 and this work); IbpA and IbpB, E. coli IbpA and IbpB (2); GspA, L. pneumophila
GspA (1); SP21, S. aurantiaca SP21
(24); YocM, B. subtilis YocM (31);
Cace, C. acetobutylicum Hsp18 (47); Salb,
S. albus Hsp18 (48); Mjan, M. jannaschii Hsp16.5 (8); Gmax, Glycine max
Hsp17.5 (11).
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The
B. japonicum hspH gene is preceded by a typical
70-type promoter and a ROSE element with high sequence
similarity to all
previously identified ROSE elements (
37).
With the exception
of one nucleotide (a G instead of a C at the
ROSE
1-equivalent
position +32), all previously described
conserved ROSE nucleotides
were conserved in the ROSE
5
sequence. In particular, the nucleotides
in the promoter-distal half of
ROSE are highly conserved (data
not shown). Transcription of
hspH was heat inducible, and the
transcription start site
was located at the expected position
just upstream of
ROSE
5, as determined by primer extension (Fig.
1B). Thus,
all presently known sHsp genes of
B. japonicum are
coordinately regulated by ROSE, a negatively
cis-acting DNA
element
that precedes each class A gene and presumably serves as a
repressor
binding site under normal growth conditions (
37).
The two-dimensional
gel analysis revealed that the degree of induction
varied from
protein to protein (see below), suggesting that
posttranscriptional
or posttranslational mechanisms might contribute to
their regulation.
Heat-induced expression of class A genes in other
organisms (the
E. coli ibpAB operon and the
L. pneumophila gspA gene) is dependent
on a
32-type
promoter (
1,
2) whereas several class B genes are
under
negative control. Transcription of the
Streptomyces albus hsp18 gene is subject to repression by the OrfY protein at low
temperatures (
49). Transcriptional repression has also been
proposed to control the expression of
Clostridium acetobutylicum hsp18,
Leuconostoc oenos hsp18, and
Synechococcus
vulcanus hspA (
28,
44,
47). These genes are transcribed
from a typical
70-type housekeeping promoter, which
implies that additional mechanisms
must prevent their expression during
normal growth. However, the
exact control mechanisms have not been
elucidated
yet.
Induction of sHsps in B. japonicum.
An as-yet-undefined
set of B. japonicum sHsps was recognized by an antiserum
raised against the 15 N-terminal amino acids of E. coli IbpA
which is similar to the B. japonicum class A proteins (2, 39). Three cross-reacting bands were detected in
extracts from heat-shocked B. japonicum cells, and the
fastest migrating band was absent in a hspBC mutant
(B. japonicum 5069) indicating that the antiserum
specifically recognized the HspB protein (39). Immunoblots
of two-dimensional gels revealed that the serum indeed recognizes
several class A proteins (HspB, HspD, HspH, and spots 4, 8, and 10)
(data not shown; compare with Fig. 4). We monitored the kinetics of
sHsp induction in B. japonicum by using this antiserum. Extracts from cells harvested before and at different time points after
a heat shock from 28 to 43°C were analyzed (Fig.
3A). The first faint signal was observed
at 5 min after the heat shock. The accumulation of sHsps continued
until the level reached a maximum approximately 60 min after the
temperature upshift. This elevated level was maintained for at least
another hour. In a separate experiment, we determined how a shift to
various temperatures affected the induction of sHsps. The amount of
sHsps increased in proportion to the severity of the shift (Fig. 3B). A
shock from 28 to 37°C was sufficient to induce the complete set of
immunodetectable sHsps, but a shift to 40°C and in particular to
43°C was much more efficient. Next, we analyzed whether other stress
conditions could induce the synthesis of class A sHsps. Cultures grown
at 28°C did not induce sHsps during the onset of, or in, stationary phase (data not shown). Extracts from bacteroids that had been isolated
from soybean root nodules did not contain detectable amounts of sHsps
(data not shown). Neither continuous growth under anaerobic conditions
nor a shift of aerobically grown cultures to high-salt (0.3 M NaCl)
conditions or to a highly oxidizing environment (0.001%
H2O2) elicited a significant response (Fig. 3B). However, the addition of ethanol (5%) to a culture led to the
production of sHsps, albeit to a much lesser extent than a heat shock.
This result suggests that both a temperature shift and an ethanol shock
trigger a signal that is finally transduced to induce the synthesis of
sHsps in B. japonicum.
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FIG. 3.
Immunoblot analyses of B. japonicum extracts
by using anti-E. coli IbpA serum. (A) B. japonicum was grown to mid-exponential phase at 28°C. After a
reference sample (28°C) had been taken, the culture was shifted to
43°C and samples were collected at the time points indicated. (B)
Experiment similar to that shown in panel A, but the cultures were
shifted to the temperatures indicated (37, 40, or 43°C), or ethanol
(E) (5%), NaCl (N) (0.3M), or H2O2 (H)
(0.001%) were added. One extract (anaerobic) originated from a culture
grown under anaerobic conditions at 28°C. (C) Fractionation of
heat-shocked B. japonicum extracts. Normally grown cells
(28°C) and heat-shocked cells (48°C) were passed four times through
a French pressure cell at 110 MPa. The soluble (supernatant) and
insoluble (pellet) fractions of the heat-shocked cells were separated
by centrifugation at 12,000 × g for 30 min. The
apparent molecular mass (in kilodaltons) of a reference protein
(lysozyme) is indicated on the right.
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Many studies indicate that certain sHsps in animals, plants, and
bacteria are regulated by a variety of environmental and
developmental
cues. Developmental synthesis of sHsps in eukaryotes
is often
tissue-specific in contrast to the coordinate heat shock
induction of
sHsps in almost all tissues. Constitutive, but low
expression of Hsp27,
a mammalian sHsp, was observed in different
cell types. This protein
plays a role in regulating the dynamics
of actin filaments and probably
confers stability to actin fibers
(reviewed in reference
3). Expression of plant sHsps during
pollen
development and seed and fruit maturation has been reported.
Again,
only a subset of the sHsps reacts to the developmental
signals, and
their expression is temporally and spatially controlled
(
51). A number of bacterial sHsps can also be induced by
developmental
signals, although heat shock often is the major elicitor.
L. pneumophila GspA is expressed during intracellular
infection of macrophages
and mycobacterial Hsp16 might also be induced
in response to stresses
encountered during an infection process
(
1,
52). The
Bacillus subtilis CotM protein is
developmentally induced during sporulation
and
Stigmatella
aurantiaca SP21 is synthesized during sporulation
and fruiting
body formation (
24,
25). Induction of
C. acetobutylicum Hsp18 was demonstrated during a metabolic shift
from acid to solvent
production (
41,
47). By contrast, our
investigation indicates
that at least the immunodetectable
B. japonicum sHsps are classical
heat stress
proteins.
Small Hsps aggregate after heat shock in vivo.
Extracts of
heat-shocked B. japonicum cells were separated into a
soluble and insoluble fraction. The immunodetectable sHps were almost
exclusively found in the pellet fraction (Fig. 3C), indicating that
they form insoluble aggregates after heat shock. Whether these
aggregates consist only of sHsps (homo- or heterooligomers) or whether
substrate proteins are bound to the sHsps cannot be determined at present.
Identification of B. japonicum sHsps by two-dimensional
gel analysis.
The presence of at least seven genes coding for
sHsps in B. japonicum prompted us to investigate the
induction of such proteins by comparative two-dimensional gel
electrophoresis (Fig. 4). The positions
of DnaK and GroEL are indicated for comparison (Fig. 4A and B). Note
that B. japonicum contains five groESL operons and that the GroEL spot represents a composite of several GroEL proteins (16, 36a). At least 11 small proteins were
reproducibly upregulated after a heat shock and visible on
Coomassie-stained two-dimensional gels. GroES1, HspB, -C,
-D, -E, and -H were identified by N-terminal sequencing of the
collected protein spots from several gels (Fig. 4B). A comparison of
the sHsp pattern after heat shock in the wild type and the
hspBCdegP mutant 5069 confirmed the identity of the HspB and
HspC spots because they were in fact missing in the mutant (Fig. 4C and
D). HspA and HspF could not be identified. HspA may not be detectable
due to a cathodic drift in the first dimension (calculated isoelectric
point of 8.42). The amount of HspF is probably too low to be detectable
because HspE, the product of the first gene of the hspEForfG
operon, is also barely visible. The amino termini of proteins 4, 8, and
10 (MRTYDLTP, MRTYDFLP, and MRSYDFSPLWRSTXTG, respectively; compare
with Fig. 2) indicated that B. japonicum contains at least
three additional class A sHsps whose structural genes and regulatory
elements have yet to be identified. The amino-terminal sequence of two
proteins (ALYEHVFL and AGTVEQKL for spots 2 and 5 in Fig. 4,
respectively) did not show similarity to class A or class B proteins or
any other proteins in the databases which suggests that there might be
additional sHsp classes in B. japonicum. In summary, we
predict that B. japonicum contains a total of at least 12 sHsps.
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FIG. 4.
Two-dimensional gel electrophoresis of B. japonicum extracts. Crude extracts of cells grown at 28°C (A) or
of cells shifted from 28 to 43°C for 30 min (B) were separated and
stained with Coomassie blue. DnaK, a set of GroEL proteins, and sHsps
are circled. The positions numbered 1 to 10 in panel A correspond to
the proteins listed in Table 1. HspB, -C, -D, -E, and -H that were
identified by amino-terminal sequencing are labeled in panel B as B, C,
D, E, and H, respectively. Induction of sHsps in B. japonicum wild type (WT) (C) and the hspBCdegP mutant
B. japonicum 5069 (D) (34). Relevant sections of
two-dimensional Coomassie-blue stained gels are shown. The positions of
HspB (labeled B) and HspC (labeled C) which are present in the wild
type but missing in the mutant are indicated by an arrow. Other sHsps
in panels C and D are circled.
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A set of sHsps is present in other rhizobia.
In order to test
whether a superfamily of sHsps is present in other rhizobial species,
we screened a variety of Bradyrhizobium and
Rhizobium strains by immunoblot analysis using the
anti-E. coli IbpA serum. Heat induction of one or several
bands was observed in each case, indicating that all species tested
possess class A-type sHsps (data not shown). To monitor the
heat-induced proteins more accurately, we performed two-dimensional gel
electrophoresis of extracts from six rhizobial species. Between 3 and
10 potential sHsps were observed in each strain (Fig.
5; Table
1). In summary, we conclude that the
existence of a sHsp family is not restricted to B. japonicum
but occurs in many rhizobial species.
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FIG. 5.
Induction of sHsps in Bradyrhizobium and
Rhizobium strains. Crude extracts of cells grown at 28°C
or of cells shifted from 28 to 43°C for 30 min were separated by
two-dimensional gel electrophoresis and stained with Coomassie blue.
Only the section of the gel containing sHsps (in the range between
approximately 10 and 20 kDa) is shown. Potential sHsps are circled.
Spots marked by rectangles were not considered in Table 1. Based on
their apparent molecular masses they might represent GroES proteins
(cf. Fig. 4) or other proteins that do not belong to the sHsp family.
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The presence of multiple sHsps in a bacterium is a rather uncommon
feature. A literature and database survey that included
the 35 microbial genomes that are completed or currently being
sequenced,
revealed that bacteria other than rhizobia encode either
no or
maximally two sHsps (Table
1). For example, no
-crystallin-like
protein was found in the genomes of the pathogens
M. genitalium,
H. influenzae,
Helicobacter
pylori, and
Borrelia burgdorferi.
The available
sequence of
Rhodobacter capsulatus, an
-proteobacterium
and close relative of rhizobia, also did not reveal any sHsp.
One or
two sHsps are encoded in the genome of a number of eubacteria
and
archaebacteria and in yeast. Interestingly, if one of these
organisms
contains two sHsps, they always belong to the same
class.
The existence of a sHsp superfamily comprising defined classes is well
established in plants (Table
1). For example, the
sequences of 10 soybean (
Glycine max) sHsps are deposited in the
public
databases. They clearly fall into class B but have been
further
subdivided in different subfamilies. Six groups were classified:
two
classes (class I and II) localized to the cytosol, and one
class each
localized to the chloroplast, endoplasmic reticulum,
mitochondrium, and
membrane compartment (
51). The homology between
individual
members of these classes is restricted to only a few
amino acids in the
-crystallin domain. An phylogenetic analysis
suggested that the
abundance of plant sHsps arose from an ancient
gene duplication or
amplification more than 150 million years
ago that was followed by
sequence divergence (
51). A similar
gene multiplication
event with subsequent diversification might
have occurred in
B. japonicum, giving rise to the unusual broad
spectrum of bacterial
sHsps. The localization of six
B. japonicum genes
(
hspA to
hspE) encoding sHsps in a heat shock
gene cluster
probably supports this assumption. Five human sHsps have
been
described (
14). The ongoing genome sequencing projects
will
reveal whether sHsp superfamilies are common in
mammals.
It is unclear why the rhizobia analyzed in this work contain multiple
sHsps whereas most other organisms do not. The relative
abundance of
rhizobial sHsps after a heat shock certainly implies
an important
cellular function offering an advantage in their
natural environment.
Short periods of intense sunlight, for example,
might cause protein
damage. When chaperones become temporarily
overloaded with potential
substrates, sHsps might play an important
role as buffer for otherwise
aggregation-prone enzymes. In agreement
with a recent model (
15,
32), one can imagine that this reservoir
of folding-competent
proteins will later be refolded by the cellular
chaperone machineries
under conditions when their capacity becomes
available again. A
tropical
Rhizobium strain that is adapted to
high
temperatures apparently does not require multiple sHsps because
it
contains only two small heat-inducible proteins (
34). The
reason for the heat tolerance of this strain is unknown. Bacteria
which
thrive as mammalian pathogens live in an environment with
more or less
constant temperatures and may be able to cope without
a sophisticated
heat shock response. Their lifestyle is reflected
by a comparatively
small number of sHsp genes in their
genome.
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ACKNOWLEDGMENTS |
We are grateful to Hauke Hennecke and Peter James for generous
support, continuous interest in our work, and helpful comments on the
manuscript. Hans-Martin Fischer and Evelyne Bauer are acknowledged for
providing B. japonicum extracts. Rhizobium
strains were obtained from Michael Göttfert. We thank Alan Easton
for the generous gift of antisera and Wolfgang Weiglhofer for
performing the experiment whose results are shown in Fig. 3A.
This study was supported by grants from the Swiss National Foundation
for Scientific Research and the Swiss Federal Institute of Technology,
Zürich.
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FOOTNOTES |
*
Corresponding author. Mailing address:
Mikrobiologisches Institut, Eidgenössische Technische Hochschule,
Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland. Phone:
41-1-632-2586. Fax: 41-1-632-1148. E-mail:
fnarber{at}micro.biol.ethz.ch.
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Journal of Bacteriology, January 1999, p. 83-90, Vol. 181, No. 1
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Abstract
Introduction
