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Journal of Bacteriology, October 1998, p. 5448-5453, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The 20S Proteasome of Streptomyces
coelicolor
István
Nagy,1,2
Tomohiro
Tamura,2
Jos
Vanderleyden,1
Wolfgang
Baumeister,2 and
René
De Mot1,*
F. A. Janssens Laboratory of Genetics,
Catholic University of Leuven, B-3001 Heverlee,
Belgium,1 and
Max-Planck-Institut
für Biochemie, D-82152 Martinsried, Germany2
Received 20 July 1998/Accepted 13 August 1998
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ABSTRACT |
20S proteasomes were purified from Streptomyces
coelicolor A3(2) and shown to be built from one 
-type
subunit (PrcA) and one 
-type subunit (PrcB). The enzyme
displayed chymotrypsin-like activity on synthetic substrates
and was sensitive to peptide aldehyde and peptide vinyl sulfone
inhibitors and to the Streptomyces metabolite lactacystin. Characterization of the structural
genes revealed an operon-like gene organization (prcBA)
similar to Rhodococcus and Mycobacterium
spp. and showed that the subunit is encoded with a 53-amino-acid
propeptide which is removed during proteasome assembly. The upstream
DNA region contains the conserved orf7 and an AAA ATPase
gene (arc).
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TEXT |
Bacterial intracellular proteases
have a variety of functions, including processing of precursor forms
(such as removal of signal peptides in exported proteins), degradation
of aberrant or damaged proteins (resulting from mutations or
environmental stress), and inactivation of proteins that play key roles
in regulatory processes. Such a diversity of tasks implicates the
involvement of a complex set of proteolytic enzymes, as exemplified by
the well-characterized Escherichia coli system
(12). Some of these proteases, like Lon, have homologues in
Archaea and Eucarya, whereas others seem to have
a restricted interdomain distribution. Proteasomes were thought to be
absent from Bacteria, until 20S proteasomes were discovered
in the actinomycete Rhodococcus (42).
In eukaryotic cells, the 20S proteasome constitutes the proteolytic
core of the 26S proteasome, which contains several accessory proteins
(including ATPases) that enable selective proteolysis of
ubiquitin-tagged substrates (9, 43), but such a 20S
proteasome-associated regulatory entity has not yet been identified in
Bacteria or Archaea. The 20S proteasome belongs
to the group of self-compartmentalizing proteases, in which the active
sites are confined to an interior compartment, created by self-assembly
of a number of subunits (4, 21). The barrel-like structure
of the 20S proteasome results from the stacking of four seven-membered
rings: the two outer rings are composed of -type subunits and the
two inner rings are built from -type subunits, in which the active
sites are generated by autocatalytic processing during proteasome
assembly. In eukaryotes such as yeast, different -type and -type
subunits (seven of each) generate the 20S complex (14). In
archaebacteria such as Thermoplasma acidophilum, the
composition with only one - and one -type subunit is of minimal
complexity (20), which has facilitated elucidation of the
novel catalytic mechanism involving the N-terminal threonine of the subunit generated upon autocatalytic maturation (37, 38). An
intermediate degree of complexity is provided by the 20S proteasome of
Rhodococcus erythropolis NI86/21, which is built from two
different - and -type subunits (42, 49). The
disclosure of proteasome-like genes by genomic sequencing of the
related nocardioform actinomycetes Mycobacterium leprae and
Mycobacterium tuberculosis (8, 22) and the
subsequent characterization of the 20S proteasome genes in
Mycobacterium smegmatis (18) revealed that the
1414 subunit composition, as found in
archaebacteria, also occurs in eubacteria. In this communication, we
report the biochemical and genetic characterization of the 20S
proteasome from a phylogenetically distant actinomycete, Streptomyces coelicolor strain A3(2).
Purification of 20S proteasomes from S. coelicolor.
S.
coelicolor A3(2) was grown at 30°C for 3 days in medium
containing casein (10 g/liter), yeast extract (5 g/liter), glucose (5 g/liter), glycine (5 g/liter), and 5 mM MgCl2. Cells
harvested from 3 liters of culture were washed with 50 mM HEPES buffer
(pH 8.0) and resuspended in 100 ml of this buffer containing lysozyme (1 mg/ml). The cell suspension was kept on ice for 2 h. All
further steps were carried out at 4°C, unless specified otherwise.
DNase I (200 U) was added to the lysate, which was cleared by
centrifugation at 61,700 × g for 1 h. Twenty
milliliters of supernatant (containing about 230 mg of protein) was
loaded on a Sepharose 6B column (3.2 by 86 cm; Pharmacia) and eluted
with 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM dithiothreitol
(DTT) and 20% (vol/vol) glycerol (buffer A) at a flow rate of 46 ml/h.
Fractions (5 ml) were collected and assayed for proteinase activity by
using the synthetic substrate succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC) (Bachem).
The fluorigenic synthetic peptide (10 nmol) was incubated for 15 to 60 min at 37°C in 50 mM Tris-HCl buffer (pH 8.0) with the enzyme samples
in a total reaction volume of 100 µl. The reaction was stopped by
adding 100 µl of 10% (wt/vol) sodium dodecyl sulfate (SDS), and the
fluorescence was measured to estimate the release of the
7-amido-4-methylcoumarin moiety. The active, high-molecular-mass fractions from three Sepharose 6B runs were pooled and loaded on a
DEAE-Sephacel column (2.2 by 10 cm; Pharmacia) equilibrated with buffer
A. Bound proteins were eluted with a 0 to 0.5 M NaCl linear gradient in
400 ml of buffer A. Fractions of 4 ml were collected. The fractions
with proteolytic activity eluting at approximately 300 mM NaCl were
pooled and dialyzed against 10 mM potassium phosphate buffer (pH 7.0)
containing 1 mM DTT and 20% glycerol. The dialyzed sample was applied
to a hydroxyapatite column (1.4 by 6 cm; Bio-Rad) equilibrated with 10 mM potassium phosphate buffer containing 20% (vol/vol) glycerol. A 10 to 300 mM potassium phosphate linear gradient (100 ml) was used for
elution, and 1.5-ml fractions were collected. Fractions (1.5 ml) with
proteolytic activity on Suc-LLVY-AMC and which eluted at approximately
85 mM potassium phosphate were pooled and dialyzed against 25 mM Tris-HCl (pH 7.5) containing 1 mM DTT and 20% glycerol (buffer B).
This sample was further purified on a Q Sepharose column (1.2 by
6 cm; Pharmacia). Fractions of 1 ml were collected during
linear gradient elution with 200 to 600 mM NaCl (50 ml). Fractions (1 ml) with proteolytic activity, eluted at about 470 mM NaCl, were again
pooled and dialyzed against buffer B. The final purification step
involved linear gradient elution (0 to 0.6 M NaCl in 40 ml) from a Mono
Q column. The fractions with proteasomes, eluted at approximately 480 mM NaCl, were dialyzed against buffer A and used for further
characterization. Table 1 presents an
overview of the purification procedure.
Characterization of the S. coelicolor 20S
proteasome.
Electron micrographs of negatively stained proteasomes
show the two characteristic views (end-on and side-on) of the
barrel-like 20S proteasome (Fig. 1).
SDS-polyacrylamide gel electrophoresis analysis showed that the
20S proteasome preparation was homogeneous, revealing two bands of
equal intensities with estimated relative molecular weights of 24,400 ( subunit) and 29,700 ( subunit) (Fig.
2). Purification of 20S proteasomes from
the archaea Methanosarcina thermophila (24) and
Pyrococcus furiosus (2), together with analyses
of complete archaeal genomes (Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and
Methanococcus jannaschii [overview at
http: //www.tigr.org/tigr_home/tdb/mdb/mdb.html]), indicate that most
archaeal 20S proteasomes have an 1414
subunit composition. However, we noted that the ongoing genomic
sequencing of Pyrococcus horikoshii has already disclosed
two distinct -type subunit genes. The increased level of subunit
complexity reported for the R. erythropolis NI86/21 20S
proteasome (42, 49) has not been observed for 20S
proteasomes from other eubacteria (actinomycetes), namely
Mycobacterium spp. (8, 18) and S. coelicolor (this work).
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FIG. 1.
Electron micrograph of 20S proteasomes from S. coelicolor, negatively stained with uranyl acetate (3),
showing ring-shaped end-on views representing projections along the
cylinder axis and rectangular side views corresponding to projections
perpendicular to the cylinder axis. Bar, 100 nm.
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FIG. 2.
Tricine-SDS-polyacrylamide gel electrophoresis analysis
of 2.28 µg of purified S. coelicolor 20S proteasomes
(right lane). The sizes of the marker proteins (left lane) are 97.4, 66.2, 45.0, 31.0, 21.5, and 14.4 kDa (from top to bottom). The proteins
were stained with Coomassie brilliant blue.
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The substrate specificity of the
Streptomyces 20S proteasome
was determined at 37°C with various synthetic substrates
(Bachem).
The highest activity was obtained with
Suc-LLVY-AMC (15.4 µmol
h
1 mg
1 = 100%). Substantially lower activities were measured with two
other chymotryptic substrates,
benzoyl- oxycarbonyl-Gly-Gly-Leu-7-amido-4-methylcoumarin (0.78
µmol
h
1 mg
1 = 5%) and
succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin (0.15
µmol
h
1 mg
1 = 1%), but no such activity was
detectable with succinyl-Leu-Tyr-7-amido-4-methylcoumarin.
Trypsin-like
activity was assayed with
benzoyloxy-carbonyl-Gly-Gly-Arg-7-amido-4-methylcoumarin,
tert-butyl-oxycarbonyl-Leu-Arg-Arg-7-amido-4-methylcoumarin, ben-zoyloxycarbonyl-Ala-Arg-Arg-7-amido-4-methylcoumarin,
and benzoyl-Val-Gly-Arg-7-amido-4-methylcoumarin, whereas benzoyloxycarbonyl-Leu-Leu-Glu-
-naphthylamide (Z-LLE-
NA)
was used as a peptidyl-glutamyl peptidase substrate; however,
neither
of these hydrolytic activities were demonstrated for the
20S proteasome
from
Streptomyces. This substrate spectrum is quite
similar
to that reported for the 20S proteasome from
Rhodococcus,
with high activity on chymotryptic substrates but no other
detectable
peptidase activities (
42). High chymotrypsin-like
activity is
also a feature of the proteasomes from
T. acidophilum (
1,
10)
and
P. furiosus
(
2). Remarkably, the
M. thermophila proteasome
displays about 40% higher activity on Z-LLE-
NA than on Suc-LLVY-AMC
(
23).
The effects of some known proteasome inhibitors on the activity of the
Streptomyces proteasome were determined (Fig.
3). Lactacystin
(Affiniti) was a more
potent inhibitor of the
Streptomyces 20S
proteasome than
acetyl-Leu-Leu-norleucinal (Ac-LLnL) (Sigma) and
benzoyloxycarbonyl-Leu-Leu-Leu-vinylsulfone (Z-LLL-VS). Very similar
inhibition patterns were reported for the chymotrypsin-like activities
of eukaryotic proteasomes preincubated with these inhibitors
(
6).
For the
Thermoplasma 20S proteasome,
lactacystin is a moderately
potent inhibitor (15% residual activity
with 100 µM lactacystin
[
41]). Unlike Ac-LLnL, both
lactacystin and Z-LLL-VS irreversibly
inactivate proteasomes by
covalent modification of the active-site
threonine (
6,
11,
25). Remarkably, lactacystin is produced
by a
Streptomyces strain (
29). It will be of interest
to investigate
what may be the physiological implications for
Streptomyces cells
that would produce both a major
intracellular protease and a potent
specific inhibitor of this enzyme.
This is reminiscent of the
regulation of activity of a trypsin-like
protease that is essential
for aerial mycelium formation by the
autogenous inhibitor leupeptin
(acetyl-leucine-leucine-arginal) and a
leupeptin-inactivating
enzyme in
Streptomyces exfoliatus
(
17).
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FIG. 3.
Inhibition of the S. coelicolor 20S
proteasome by Ac-LLnL, Z-LLL-VS, and lactacystin. Purified proteasome
(0.912 µg) was incubated with inhibitor in 100 µl of Tris-HCl (pH
8.0) at room temperature for 1 h. Subsequently, 20 nmol of
Suc-LLVY-AMC was added. After incubation at 37°C for 1 h, the
reaction was stopped (by addition of 100 µl of 10% SDS) and the
amount of 7-amido-4-methylcoumarin released was measured.
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The optimum temperature for Suc-LLVY-AMC hydrolysis by the
Streptomyces 20S proteasome was 55°C (data not shown),
which is
substantially lower than the temperature optima reported
for
T. acidophilum (85°C to 91°C [
1,
10]),
P. furiosus (95°C
[
2]),
and
M. thermophila (70°C to 75°C
[
23]). This difference probably
reflects the
thermophilic nature of these archaebacteria, as opposed
to
Streptomyces with an optimal growth temperature of about
30°C.
Cloning and characterization of the 20S proteasome structural genes
from S. coelicolor.
From S. coelicolor cells
grown in glycine-containing medium and treated with lysozyme (1 mg/ml)
at 30°C for 30 min, total DNA was isolated according to the method of
Nagy et al. (26). A digoxigenin-labeled 960-bp
SacI-EcoRI fragment from the R. erythropolis prcB2A2 operon
(42) was used as a probe for Southern hybridization (hybridization temperature, 60°C; washing was performed at 60°C with 7.5 mM sodium citrate containing 75 mM NaCl and 0.1% SDS) with
total S. coelicolor DNA restricted by several endonucleases. Two hybridizing fragments (5.1-kb KpnI and 8.0-kb
SphI fragments), covering a 10.6-kb region with the
proteasome genes and flanking DNA, were cloned in pUC18
(generating pFAJ2594 and pFAJ2609, respectively). These clones
were selected by colony hybridization with the Rhodococcus DNA probe from size-fractionated sublibraries. Subcloned fragments were subjected to automated sequence analysis (A.L.F. sequencer; Pharmacia), and subsequences were assembled with the
ASSEMGEL program of PCGENE (IntelliGenetics). Potential codon
regions were identified with the GCWIND program (40),
relying on codon usage bias in genomes with high G+C contents.
Two open reading frames (ORFs) (
prcB,
prcA)
with high homology to known eubacterial proteasome genes and
organized in a similar
way were identified (Fig.
4). The N-terminal sequence determined
for the smaller subunit of the purified 20S proteasome (TTIVAVTF)
was
identical to the deduced amino acid sequence of PrcB after
removal of a
53-amino-acid propeptide, characteristic of
-type
subunits (Fig.
5A). The calculated molecular mass of the
mature
PrcB protein (24,428 Da) is in excellent agreement with the
estimated
Mr of 24,400. The propeptides of
eubacterial
subunits are considerably
longer (up to 65 residues for
PrcB
1 of
R. erythropolis) than those
of
archaebacterial
subunits, which typically consist of fewer
than 10 amino acids. Zühl et al. (
48) demonstrated that the
propeptides of the
Rhodococcus proteasome
subunits play
a crucial
role: they support the initial folding of the
subunits
and promote
the maturation of the holoproteasomes, in which the
N-terminal
active-site residue, threonine, becomes available
following autocatalytic
release of the propeptides. Such a
chaperone-like function has
also been assigned to the propeptide of a
eukaryotic
-type proteasome
subunit (
7). A comparison of
the currently available eubacterial
propeptide sequences (Fig.
5A),
reveals that two residues (HG)
preceding the processing site (G-T
peptide bond) are strictly
conserved, as well as several residues in
the middle part of the
propeptide, whereas little conservation is
apparent in the remainder
of the sequences. It is likely that the
central box with the consensus
sequence
SSFX(D/E)(F/Y/L)LX
4PEXLP is important for the function
of
the eubacterial propeptide. Such a box is not obviously present
in
eukaryotic propeptides, although a number of them contain the
SF or AF
sequence. No N-terminal sequence was obtained for the
larger subunit,
indicating that the amino terminus was blocked,
a general
characteristic of
-type subunits (
42). The estimated
Mr of this subunit (29,700) is somewhat higher
than the calculated
molecular mass of 27,883 Da.
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FIG. 4.
Gene organization of the proteasome structural genes
(prcB, prcA) and the flanking regions from
M. tuberculosis (MT) (GenBank accession no. Z97559;
14,030 bp), R. erythropolis (RE) (GenBank accession no.
U26422 and this work; 13,000 bp), and S. coelicolor (SC)
(this work; 10,612 bp). The fragment shown for Rhodococcus
represents the second proteasome operon. The ORFs in the respective DNA
regions are numbered independently. For M. tuberculosis, the
ORF numbering of cosmid MTCY261 is used. The box labeled IS represents
one of the sixteen copies of IS6110 present in the genome of
M. tuberculosis H37Rv (8). Conserved ORFs and
homologous genes are shown as filled arrows, and the extent of sequence
conservation is indicated (numbers representing percentages of
identical amino acids are circled).
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FIG. 5.
Multiple sequence alignments of the proteasome
-subunit propeptides (A), the ORF7 sequences (B), and the N-terminal
coiled-coil regions of ARC homologues (C) from the following
actinomycetes: M. leprae (Ml), M. smegmatis (Ms),
M. tuberculosis (Mt), R. erythropolis (Re1 and
Re2, or Re), and S. coelicolor (Sc). Differential
shading (three levels in panels A and B; two levels in panel C)
reflects the degree of sequence conservation (identical or similar
amino acids) at a given position. The propeptide cleavage sites are
marked with an arrow, and the first 10 residues of the processed subunits are shown in panel A. In panel C, the hydrophobic amino acids
in positions 1 and 4 of the heptad repeats of the proposed coiled-coil
structures of ARC proteins are labeled  and  , respectively, and
proline residues flanking coiled-coil segments are marked (in black on
a shaded background).
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Organization of the proteasome genomic regions in
actinomycetes.
Although purification and biochemical
characterization of 20S proteasomes is currently limited to
Rhodococcus (42) and
Streptomyces (this work), genomic sequencing of
M. tuberculosis (8) and M. leprae, and subsequent genetic analysis with M. smegmatis (18), now enables a comparison of the
proteasome regions of representative actinomycetes to be
made. To extend the known DNA sequence of the upstream region of
prcB2A2 of R. erythropolis NI86/21, the flanking sequences of the arc
gene (46) were determined from the original 
FAJ2029 clone
(42).
Figure
4 reveals a patchy distribution of conserved genes and ORFs
(apparently restricted to actinomycetes) with several interjacent
ORFs
that are not conserved between the different actinomycete
species. The
20S proteasome structural genes of
S. coelicolor,
prcB and
prcA, are separated by 61 bp. However,
in
Rhodococcus (
42) and
Mycobacterium
(
8,
18) spp., the
prcB stop codon
overlaps the
prcA start codon, suggesting a translational coupling.
The
small conserved
orf7, which overlaps the respective
prcB genes
in
Rhodococcus and
Mycobacterium, ends 580 bp upstream of the
Streptomyces prcB gene. The
Streptomyces ORF7 (72 amino acids)
is somewhat larger than its counterparts from
Rhodococcus and
Mycobacterium (63 to 64 amino
acids) but is quite similar, particularly
in the C-terminal half,
which contains a high proportion of acidic
residues (Fig.
5B). The
calculated pI value for
Streptomyces ORF7
(3.7) and those of
its homologues (3.6 to 3.7) are very similar.
At present, it is not
known whether this conserved ORF7 may exert
a proteasome-related
function. A possible ORF (
orf8) is present
between
orf7 and
prcB and partially overlaps the latter,
but no
homologues were identified via database searches. Using a
proteasome-specific
probe, the proteasome genes have been mapped to the
AseI-I fragment
(cosmid I4) of the
S. coelicolor genome (
16,
32).
Other conserved ORFs in the upstream region of
S. coelicolor
are
orf6,
arc, and
orf1 (partially
sequenced). The
arc gene product
of
R. erythropolis, ARC (for AAA ATPase forming ring-shaped complexes),
was recently characterized (
46). The linkage with proteasome
genes and striking similarities in domain topology with those
AAA-type
ATPases forming part of the eukaryotic 26S proteasomes
(
5,
30) suggest a proteasome-related function for ARC, but
this
remains to be proven. A feature of the
Rhodococcus ARC
(and
of the putative mycobacterial homologues) which it shares
with
proteasomal ATPases (
33) is the presence of a
potential N-terminal
coiled-coil structure (
46). By using
the COILS server
(
http://www.isrec.isb-sib.ch/software/COILS_form.html),
such an N-terminal segment was also predicted for
Streptomyces ARC (Fig.
5C).
Unlike the gene organization in
Mycobacterium and
Rhodococcus, no ORFs are positioned between
arc
and another conserved (actinomycete-specific)
ORF,
orf6, in
Streptomyces (Fig.
4). It is notable that the deduced
ORF6
sequence has significant homology (about 40% identity) to
the putative
orf9 product (
27). A close homologue of ORF9
(partially
sequenced) appears to be present in
S. coelicolor
as well (Fig.
4). In
M. tuberculosis, but not in
M. leprae (
27), the equivalent
of
orf9
is located in another part of the genome (cosmid MTCY49
[GenBank
accession no.
Z73966]). The presence of a copy of
the promiscuous
insertion sequence IS
6110 downstream of the proteasome
genes
in
M. tuberculosis (Fig.
4) suggests that a genomic
rearrangement
may have taken place.
The
Streptomyces orf1 and its homologues in
Rhodococcus (
orf16) and
Mycobacterium
(
orf14) encode putative methyltransferases
with significant
homologies to putative gene products from several
archaebacteria (up to
37% identity for
P. horikoshii [GenBank
accession
no.
AB009528]). Collectively, these ORFs are distantly
related to
L-isoaspartyl protein carboxyl methyltransferases from
various organisms; these are involved in the repair of
L-isoaspartyl
residues present in damaged proteins
(
45). For the remaining,
nonconserved
Streptomyces ORFs, homology searches revealed similarity
to
ferredoxins for ORF3, to LacI-related repressors (including
S. coelicolor MalR [
44]) for ORF4, and
to transmembrane transporter
proteins for ORF5, but the extent of the
homology (<40% identity)
did not allow reliable predictions of their
possible functions.
Conclusions.
The presence of 20S proteasomes in
Rhodococcus, Mycobacterium, and
Streptomyces spp. suggests that this cytoplasmic protease is
widespread among actinomycetes (gram-positive bacteria with high
G+C contents). The presence of proteasome-like genes in other representative actinomycete species (Amycolatopsis
methanolica, Brevibacterium linens, Clavibacter
michiganense, and Corynebacterium glutamicum) was
confirmed by Southern hybridization with a rhodococcal proteasome
gene probe (28). Furthermore, a polyclonal antibody raised
against Rhodococcus proteasomes and cross-reacting with the
S. coelicolor -type subunit revealed the
presence of putative -type subunits in the SDS-insensitive,
Suc-LLVY-AMC-active fractions obtained by glycerol gradient
centrifugation of cell extracts from these species (28).
Recently, proteasome genes have also been identified in
Frankia sp. strain ACN 14a/ts-r (31).
Genomic sequencing data support the notion that 20S proteasome genes
are confined to actinomycetes. In many other bacteria,
including
gram-positive bacteria (such as
Bacillus spp.), a
structurally
different self-compartmentalizing protease, HslVU, is
present
(
19,
34). This protease is built from the
Clp-related ATPase
subunit HslU (
13,
39) and from the
proteolytic HslV subunit,
which shows some sequence homology
(
22) and similarity in catalytic
mechanism (
6,
47) to
-type proteasome subunits. In
E. coli,
a
synergistic action of HslVU with other ATP-dependent proteases
(such as
Lon) in controlling the turnover of
32 (sigma factor
directing transcription of heat shock genes) and
abnormal proteins has
been observed (
15). Lon homologues have
been identified in
mitochondria, archaebacteria, and eubacteria,
including the
fast-growing, nonpathogenic
M. smegmatis (
35).
To
our surprise, we were unable to identify a gene for a Lon homologue
in
the genome of the slow-growing
M. tuberculosis H37Rv
(
8).
It will be of interest to see whether this may also be
the case
for other pathogenic mycobacterial species, such as
M. leprae.
Recently, it was shown that specific inhibition of proteasomes with
Z-LLL-VS severely affected cell viability of the archaeon
T. acidophilum following exposure to heat shock (
36).
However,
an
M. smegmatis mutant lacking proteasomes showed
no obvious phenotypic
alterations (
18), indicating that an
efficient backup system
may exist in actinomycetes. The elucidation of
the in vivo function
of 20S proteasomes in actinomycetes should benefit
from the identification
of this protease and its structural genes in
Streptomyces, since
molecular genetic tools are much more
advanced for this genus
than for other actinomycetes.
Nucleotide sequence accession numbers.
The nucleotide
sequences of the S. coelicolor proteasome genes
(including flanking DNA) and the nucleotide sequence of the arc-containing DNA region upstream of the R. erythropolis prcB2A2 genes have been
deposited in the GenBank database under accession no. AF086832 and
AF088800, respectively.
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ACKNOWLEDGMENTS |
S. coelicolor A3(2) and Amycolatopsis
methanolica NCIB 11946 were provided by J. Dusart (University of
Liège, Liège, Belgium) and L. Dijkhuizen (University of
Groningen, Groningen, The Netherlands), respectively. Mapping of the
proteasome genes was carried out by H. Kieser and D. Hopwood (John
Innes Centre, Norwich, United Kingdom). We are indebted to M. Bogyo
(Harvard Medical School, Boston, Mass.) for providing the
proteasome inhibitor Z-LLL-VS. M.-N. Pouch (Max Planck
Institute of Biochemistry, Martinsried, Germany) kindly communicated
results prior to publication and provided the bacterial proteasome
antibodies. The GCWIND program was obtained from D. Shields (Trinity
College, Dublin, Ireland).
These studies were supported by a grant from the Fund for Scientific
Research
Flanders (Belgium) to R.D.M., a Senior Research Associate
with this fund. Support from the Human Frontier Science Program to W.B.
is gratefully acknowledged.
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FOOTNOTES |
*
Corresponding author. Mailing address: F. A. Janssens Laboratory of Genetics, Kardinaal Mercierlaan 92, B-3001
Heverlee, Belgium. Phone: 32 16 32 96 81. Fax: 32 16 32 19 66. E-mail:
rene.demot{at}agr.kuleuven.ac.be.
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Journal of Bacteriology, October 1998, p. 5448-5453, Vol. 180, No. 20
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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