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Journal of Bacteriology, December 1998, p. 6674-6680, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The yvyD Gene of Bacillus subtilis Is under
Dual Control of 
B and H
Kathrin
Drzewiecki,
Christine
Eymann,
Gerhard
Mittenhuber, and
Michael
Hecker*
Institut für Mikrobiologie und
Molekularbiologie, Ernst-Moritz-Arndt-Universität, D-17487
Greifswald, Germany
Received 28 July 1998/Accepted 6 October 1998
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ABSTRACT |
During a search by computer-aided inspection of two-dimensional
(2D) protein gels for 
B-dependent general stress
proteins exhibiting atypical induction profiles, a protein initially
called Hst23 was identified as a product of the yvyD gene
of Bacillus subtilis. In addition to the typical
B-dependent, stress- and starvation-inducible pattern,
yvyD is also induced in response to amino acid depletion.
By primer extension of RNA isolated from the wild-type strain and
appropriate mutants carrying mutations in the sigB and/or
spo0H gene, two promoters were mapped upstream of the
yvyD gene. The B-dependent promoter drives
expression of yvyD under stress conditions and after
glucose starvation, whereas a H-dependent promoter is
responsible for yvyD transcription following amino acid
limitation. Analysis of Northern blots revealed that yvyD
is transcribed monocistronically and confirmed the conclusions drawn
from the primer extension experiments. The analysis of the protein
synthesis pattern in amino acid-starved wild-type and relA
mutant cells showed that the YvyD protein is not synthesized in the
relA mutant background. It was concluded that the stringent response plays a role in the activation of H. The
yvyD gene product is homologous to a protein which might modify the activity of 54 in gram-negative bacteria. The
expression of a L-dependent (L is the
equivalent of 54 in B. subtilis)
levD-lacZ fusion is upregulated twofold in a yvyD mutant. This indicates that the yvyD gene
product, being a member of both the B and
H regulons, might negatively regulate the activity of
the L regulon. We conclude that (i) systematic,
computer-aided analysis of 2D protein gels is appropriate for the
identification of genes regulated by multiple transcription factors and
that (ii) YvyD might form a junction between the B and
H regulons on one side and the L regulon
on the other.
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INTRODUCTION |
The highly sensitive two-dimensional
(2D) protein gel electrophoresis technique combined with the
computer-aided evaluation of 2D gels is a very powerful tool for the
analysis of the global control of gene expression (1, 51,
59). The transcription of the majority of bacterial genes is
organized in regulons that are controlled by global regulators such as
repressors, activators, or alternative sigma factors. We used the 2D
gel electrophoresis methodology to describe the heat stress stimulon of
Bacillus subtilis. This heat stress stimulon could be
dissected into regulons by searching for proteins that follow the same
induction pattern and by analyzing mutants in global regulatory genes
(for a review, see reference 20). Increasing
attention will be paid to this proteomic approach (59),
bearing in mind that a lot of still-unknown regulons were discovered by
the sequencing of the B. subtilis genome and should be
analyzed in the near future (28).
The largest regulon in the heat stress stimulon is the
B-dependent general stress regulon of B. subtilis, which presumably contains more than 100 genes (4,
5, 20). The function of this regulon was totally unknown until
1994. By the identification of several B-dependent
general stress protein genes we and others have obtained evidence that
some of these proteins may provide an unspecific, multiple, and
prospective general stress resistance to a nongrowing, starving, or
stressed B. subtilis cell which is no longer able to
grow and divide (for a review, see reference
21).
B-dependent stress genes are strongly induced by heat,
salt, acid, or ethanol stress as well as by energy depletion (17, 20). The proteins and/or genes belonging to the B
regulon follow this typical expression pattern, which can be visualized
by a computer-aided evaluation of 2D gels (4). However, we
also found general stress proteins which were characterized by a
slightly modified induction pattern. In addition to the
characteristic B induction pattern the protein YvyD
(formerly Hst23), identified by N-terminal sequencing
(4), showed a strong induction by amino acid starvation
(4, 57). In this paper, we describe this atypical induction
profile of yvyD. We identified
besides the
B-dependent promotera second promoter which is
recognized by EH and which is responsible for the
induction of yvyD by amino acid starvation. H
is used for the transcription of many genes expressed during the
transition from exponential growth to the stationary phase (6, 24,
38, 39, 42, 50, 60-63). Such a dual control of a general stress
gene by B and H was already described by
Varón et al. (52) for the csb40 operon.
These results show that the 2D protein gel electrophoresis technique is
also a useful approach for defining a network of interacting regulons
or modulons. We suggest that yvyD (and presumably other genes or operons such as csb40) may form a junction in a
global regulatory network between the B regulon,
the H regulon, and most likely the stringent response also.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and culture conditions.
The
bacterial strains and plasmids used in this study are listed in Table
1. Escherichia coli DH5
was
routinely grown in Luria-Bertani medium and used as the host for
cloning experiments (44). B. subtilis
strains were cultivated with vigorous agitation at 37°C in a
synthetic medium described previously (48). For heat shock
and osmotic or ethanol stress experiments, exponentially growing cells
of B. subtilis were shifted from 37 to 48°C or were exposed to either 4% (wt/vol) NaCl or 4% (vol/vol) ethanol.
Deprivation of glucose, amino acids, or nitrogen was achieved by
cultivating bacteria in the synthetic medium with growth-limiting
amounts of glucose (0.05%, wt/vol), amino acids (62.4 µM lysine,
62.4 µM tryptophan), or (NH4)2SO4
(1 mM). To generate an artificial amino acid starvation (2,
19), DL-norvaline was added at an optical density
at 500 nm (OD500) of 0.5 to a final concentration of 0.05%
(wt/vol). B. subtilis BKD11 and BKD12 were cultivated in the synthetic medium with 0.2% (wt/vol) glucose (repressing conditions) or with 0.2% (wt/vol) fructose (inducing conditions) (30).
Construction of B. subtilis mutant
strains.
B. subtilis BKD2, BKD3, and BKD11 were
constructed by transformation of chromosomal DNA from various
B. subtilis strains into the wild-type strain IS58 or
the isogenic sigB mutant BEK38. B. subtilis
BKD1 and BKD12 were constructed by transformation of the wild-type IS58
or BKD11 with the nonreplicative plasmid pKD11. Correct integration was
proved by Southern blotting.
Primer extension and Northern (RNA) blot analysis.
Total RNA
of B. subtilis BGH1, BKD2, BKD3, IS58 (BR16), and IS56
(BR17) was isolated from exponentially growing or stressed cells by the
acid phenol method described by Majumdar et al. (29) with
some modifications (54). The 5' end of the yvyD
mRNA was identified by primer extension as described previously
(58). The oligonucleotide
5'-CTTCACATCAGCATCCACGC-3' labelled with
[
-32P]ATP at the 5' end was used as the
primer. Northern blot analysis was performed as described previously
(58) with a digoxigenin-labelled RNA probe which was
synthesized in vitro with T7 RNA polymerase and the linearized plasmid
pKD2 as a template.
Plasmid constructions.
The primers P1
(5'-TTGACCAAATTTTTGCGGAG-3') and P2
(5'-TCATCACACGCCTATTTTAG-3') were used for the construction
of the plasmid pKD1 (see Fig. 1). The resulting PCR product, after
amplification of chromosomal DNA of strain IS58, was cloned into
pBluescript II KS(+) linearized with EcoRV. pKD1 contains
the entire yvyD gene. The plasmid pKD2 harboring an internal
fragment of yvyD was constructed in a similar way. The
primers P3 (5'-AGTCTAAGGTTGAGGTTACG-3') and P4
(5'-GGTACACGACATTTGTAAGG-3') were used for the amplification of chromosomal DNA of strain IS58, and the resulting fragment was
cloned into pBluescript II KS(+) linearized with EcoRV. The plasmid pKD11 was constructed by cloning a 1,490-bp Kmr
cassette from plasmid pGD780 (16) within the
BglII site of the yvyD gene in the plasmid pKD1.
-Galactosidase assays.
The assay for -galactosidase
activity was performed as described previously (53).
2D polyacrylamide gel electrophoresis.
Labelling of cells,
2D polyacrylamide gel electrophoresis, and protein identification on 2D
gels were performed as described previously (4, 14).
Computer methods.
Alignments were performed with the
Genetics Computer Group package at default settings. Databases used
were those of the EMBL and GenBank. Figure 2 was generated by the
program BOXSHADE.
General methods.
Plasmid isolation, restriction enzyme
analysis, transformation of E. coli, ligation of DNA
fragments, and filling in of 3' termini with Klenow fragments of DNA
polymerase I were performed according to standard protocols
(44). Chromosomal DNA from B. subtilis was
isolated with a genomic DNA purification kit (Promega). Transformation
of B. subtilis was carried out according to the method
described by Hoch (22).
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RESULTS |
Identification of Hst23 as the product of the yvyD
gene.
YvyD (Hst23) has been described as a general stress protein
of B. subtilis (4). In 2D protein gels, YvyD
is characterized by the typical B-dependent stress and
starvation induction pattern; however, in contrast to most products of
the B-dependent general stress genes YvyD is also
induced by amino acid starvation (57).
The sequence encoding the N terminus of YvyD shows 100%
identity to
orf189, which is located between the
fli operon and the
secA gene in
B. subtilis (
7) (Fig.
1). A computer-aided search
for
similarities with other proteins showed an identity of 30
to 40%
between this open reading frame encoding a 189-amino-acid
polypeptide
and
54 modulating factors of various gram-negative
bacteria (
33) (Fig.
2).
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FIG. 1.
Schematic representation of the yvyD region
in the chromosome of B. subtilis. The yvyD
gene is located between the fli operon and the
secA gene. A rho-independent terminator is located upstream
and downstream of yvyD (7). The locations of the
primer pair P1-P2, used for construction of plasmid pKD1, and the
primer pair P3-P4, used for the construction of plasmid pKD2, are
indicated. The BglII site within yvyD was used
for the construction of the yvyD mutant by the insertion of
a kanamycin resistance cassette in plasmid pKD1, resulting in plasmid
pKD11.
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FIG. 2.
Multiple, partial alignment (approximately 100 amino
acids at the N terminus) of YvyD with proteins functioning as
54 modulating factors from gram-negative bacteria. Bs,
B. subtilis; Ac, Acinetobacter calcoaceticus
(orf2) (10); Ae, Alcaligenes eutrophus
(Ralstonia eutropha) (orf130) (55);
Av, Azotobacter vinelandii (second open reading frame)
(32); Bj, Bradyrhizobium japonicum
(orf203) (27); Ec, E. coli
(orfII) (25); Kp, K. pneumoniae
(orf95) (33); Pp, Pseudomonas putida
(orf102) (26); St, Salmonella
typhimurium (36); Tf, Thiobacillus
ferrooxidans (orf3) (3); C, consensus
sequence.
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During the
B. subtilis genome sequencing project,
orf189 was renamed
yvyD (
28). In this
paper, we refer to the gene encoding
Hst23 (Orf189) as
yvyD.
Mapping of the yvyD promoters and Northern blots.
The transcriptional regulation of yvyD was analyzed by
primer extension (Fig. 3) and Northern
blotting (Fig. 4). By the primer extension technique two transcriptional start sites, separated by 5 nucleotides, were found. The potential 
10 and 35 regions of
the upstream promoter are similar to those of known
B-dependent genes. The downstream promoter revealed high
similarities to promoters recognized by RNA polymerase containing
H. In exponentially growing cells of the wild-type
strain IS58, transcription is mainly initiated at the
H-dependent promoter (Fig. 3). In a sigH sigB
double mutant there is no transcription of yvyD at all,
supporting the hypothesis that only B and
H are involved in transcriptional regulation (Fig. 4).
Northern blot analyses showed that yvyD is transcribed
monocistronically. A 600-bp signal was detected as the main transcript
(Fig. 4). Both sigma factors contribute to the expression pattern. In a H mutant the transcription of yvyD depends
solely on EB, showing the typical induction profile for
B-dependent genes: yvyD is strongly induced
by heat, salt, or ethanol stress as well as by glucose starvation (Fig.
3 and 4). In a B mutant, however, no heat stress
induction occurred at the H-dependent promoter,
but an induction by glucose starvation (Fig. 3 and 4) and another by
amino acid starvation (data not shown) did. However, the transcription
at the H-dependent promoter appeared to show a
delayed induction in response to starvation. Surprisingly, we also
found a H-dependent induction by ethanol
treatment. Concerning the transcriptional regulation of
yvyD, we note that the induction by physical stress and
glucose starvation depends on B and that the induction
by amino acid starvation depends solely on H (Fig. 3).
When both sigma factors are activated (by ethanol stress and glucose
starvation) EB most probably is involved to a larger
extent in the transcription of yvyD than EH.
However, the possibility that there really is a competition between
both sigma factors for the transcription of yvyD needs further investigation.
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FIG. 3.
Determination of the transcriptional start sites of
yvyD. (A) Primer extension analysis of RNA. The
B. subtilis wild-type strain IS58, the sigB
mutant strain BGH1, and the sigH mutant strain BKD2 were
exposed at an OD500 of 0.5 to various stresses as described
in Materials and Methods. For RNA isolation, bacteria were harvested
before exposure (C, control) and 6 min after exposure to the different
stressors (H, heat [48°C]; S, salt [4% NaCl]; E, ethanol [4%
ethanol]). In cases of nutrient starvation bacteria were harvested at
transient phase (glucose depletion [GT0]) or 30 min after
entry into stationary phase (deprived of glucose [GS], amino acids
[AS], or nitrogen [NS]). A total of 5 µg of each RNA preparation
was used for each primer extension reaction. Lanes T, G, C, and A show
the sequencing ladder obtained with the same primer as that used for
primer extension. (B) DNA sequence of the yvyD promoter
region. Potential start sites are indicated by asterisks. The
B- and H-dependent promoters are printed
in bold. The consensus sequence for B-dependent
promoters is taken from the work of Hecker et al. (20), and
the consensus sequence for H-dependent promoters is
derived from the work of Haldenwang (17). The ribosomal
binding site is underlined, and the translational start codon is shown
in bold.
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FIG. 4.
Northern blot analysis of stress-inducible
yvyD transcription. Total RNA was isolated from
B. subtilis IS58, BGH1, BKD2, and BKD3 after exposure
to various stress conditions as described in the legend to Fig. 3. A
total of 5 µg of RNA was applied in each lane. The locations of RNA
molecular size markers and the size of the yvyD transcript
are marked. C, control; S, salt; E, ethanol; H, heat; GT0,
glucose depletion at transient phase; GS, glucose depletion at
stationary phase.
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YvyD induction by amino acid starvation in the relA
mutant.
The synthesis of YvyD was strongly stimulated by norvaline
treatment, which triggers a stringent response via leucine and isoleucine limitation (2, 19). In accordance with the
findings of Wendrich and Marahiel (57), this induction did
not occur in a relA mutant (Fig.
5). Figure 5 also demonstrates that the ribosomal protein RplJ is subjected to the characteristic stringent response, i.e., the continued synthesis in the relA mutant
versus downregulated synthesis in the wild type. Transcriptional
studies by Northern blotting provided additional evidence that
yvyD is strongly induced by amino acid depletion at the
H-dependent promoter only in the wild type and not in
the relA mutant (Fig. 6).
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FIG. 5.
Sections of 2D protein gels in the YvyD region. Strains
BR16 (wild type) and BR17 (relA) were grown to an
OD500 of 0.5. For panels B and C an artificial depletion of
amino acids was generated by the addition of DL-norvaline
to a final concentration of 0.05%. Labelling of cells with
[35S]methionine (5 µCi/ml) was performed after 15 min
of incubation with DL-norvaline for 3 min. (A) Control
(strain BR16, exponentially growing cells, labelling at an
OD500 of 0.5, and no DL-norvaline addition);
(B) strain BR16 after DL-norvaline treatment; (C) strain
BR17 after DL-norvaline treatment. The locations of the
proteins ClpP, SodM, GreA, AhpC, and RsbW and of the unidentified
proteins a, b, c, d, and e are included as references. Note in panel C
that the YvyD protein is no longer induced in the relA
mutant and that the protein RplJ is not subjected to the stringent
response.
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FIG. 6.
Northern blot analysis of stress induction of
yvyD transcription in a relA mutant. Total RNA
was isolated from B. subtilis BR16 and BR17 before (C,
control) and after depletion of amino acids (AT0, transient
phase; AS, 30 min after entry into stationary phase). A total of 2.5 µg of RNA was applied in each lane. The locations of RNA molecular
size markers and the size of the yvyD transcript are marked.
Note that induction of yvyD transcription does not occur in
the relA mutant strain.
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Analysis of L-dependent gene expression in a
yvyD mutant.
L in B. subtilis is the equivalent of 54 in gram-negative
bacteria (9). Because it had been shown that
54-dependent transcription was elevated by 25% in a
strain of Klebsiella pneumoniae carrying a mutation in the
gene homologous to yvyD (orf95) (33),
we examined the transcription of the L-dependent
lev operon in B. subtilis. The
lev operon is induced by fructose and repressed by glucose
in the medium (30).
The
-galactosidase activity originating from a
levD-lacZ
fusion was measured in the wild-type strain BKD11 and in the
yvyD mutant strain BKD12. In the presence of fructose the
expression
of
levD-lacZ was elevated twofold in the
yvyD mutant relative
to that in the wild type (Fig.
7). The relative amount of the
sigL transcript is not increased in the
yvyD
mutant relative to
that in the wild type as revealed by slot blot
analysis (data
not shown). These results indicate that YvyD might
influence the
activity of
L in
B. subtilis.
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FIG. 7.
-Galactosidase synthesis originating from a
levD-lacZ fusion in the wild type and the yvyD
mutant. Strains BKD11 (levD-lacZ) and BKD12 (levD-lacZ
yvyD) were grown in minimal medium containing either 0.2%
glucose (repressing conditions) (BKD11 [ ] and BKD12
[ ]) or 0.2% fructose (inducing conditions) (BKD11 [ ] and
BKD12 [ ]) as the carbon source. At the times indicated, samples
were removed and assayed for -galactosidase activity (corresponding
open symbols).
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| |
DISCUSSION |
By a careful and comprehensive computer-aided inspection and
matching of various 2D gels loaded with radioactively labelled proteins
from growing, starved, or stressed B. subtilis cells, it is possible to proceed from a 2D protein index to a more global analysis and description of the gene expression network (1). The proteomic research approach (59) relying mainly on the
highly sensitive 2D protein gel electrophoresis technique is a useful approach not only for the definition of stimulons and regulons (20, 51) but, as shown in this study, also for the discovery and preliminary analysis of genes controlled by more than one regulatory circuit.
B-dependent general stress genes show an expression
pattern induced by heat, salt, acid, or ethanol stress on the one hand and by glucose, oxygen, or phosphate starvation on the other. During a
search for general stress proteins exhibiting atypical induction
profiles we found YvyD, which shows the typical
B-dependent induction pattern. However, the strong
induction of YvyD caused by amino acid starvation did not fit with the
B-dependent induction profile. In this paper it is shown
that in addition to the B promoter, yvyD
contains a second, H-dependent promoter responsible for
this atypical induction profile. Our results indicate that a presumably
small subset of B-dependent genes are also controlled by
H, extending the inducing environmental stimuli to amino
acid or nitrogen starvation. The first member of this
B-H modulon was described by Varón
et al. (52), who found the csb40 operon to be
under this dual control. The second open reading frame of the
csb40 operon (orf2) showed a significant
resemblance to desiccation proteins occurring in dried leaves of
plants. The description of a new member of this stationary-phase or
general stress modulon seems to support the conclusion of Varón
et al. (52) that H may be more broadly
involved in stress response than previously suggested. Very recently,
Gaidenko and Price (13) found H to be
involved in the stress resistance of B. subtilis.
However, the number of genes whose expression is controlled by both
sigma factors seems to be rather low, because from about 30 to 40 general stress proteins whose expression profiles were analyzed by a
computer-aided evaluation, only Hst23 showed this atypical induction
pattern (4).
The H regulon and the expression of the spo0H
gene have been intensively investigated. H has been
described as a sigma factor responsible for the transcription of
several genes expressed at the transient phase at the start of
sporulation and competence development (for reviews, see references 15 and 23). Several genes under
H control, which are expressed early in the transition
period, also possess a A-dependent promoter; some genes
of the H regulon are not directly involved in the
sporulation pathway, e.g., citG, rpoD, and the
ureABC operon (6, 39, 50, 60). Early-sporulation
genes, spo0A, spo0F, kinA,
spoVG, and spoVS, are transcribed by
EH (11, 38, 42, 43, 63). The
spoIIA operon, encoding the prespore- and forespore-specific
sigma factor F, is transcribed exclusively by
EH (61, 62). The expression of
spo0H itself increases as the culture enters the late
logarithmic stage of growth (56). An acidification of the
internal pH negatively influences spo0H expression (8). The decreasing level of the transition state regulator AbrB, whose synthesis is repressed by phosphorylated Spo0A, seems to be
responsible for the transient derepression of spo0H
(11, 35, 46, 47, 56, 63).
We noticed some interesting details about the expression of
yvyD. Our transcriptional data appeared to show that in
response to glucose starvation the B-dependent promoter
is activated earlier than the H-dependent one.
Furthermore, the H-dependent induction of
yvyD by ethanol in the sigB mutant background is
a result which deserves future attention. The most promising result,
however, is that in a relA mutant yvyD is no
longer induced in response to amino acid starvation. The
relA-dependent induction of yvyD occurs at the
H-dependent promoter because B-dependent
genes are not induced after ppGpp accumulation (31). An
intriguing explanation for this result could be that H
requires the stringent response for its activity regardless of the
mechanism of this proposed activation. It is tempting to speculate that
ppGpp is somehow involved in the derepression of spo0H.
However, several reports indicate that spo0H expression is
regulated at different levels of gene expression, including even the
posttranslational level (12, 18, 41, 56). Recently, it has
been found that YvyD is accumulated in clpP and
clpX mutants (14). This observation can be
explained by the recent finding that H is a substrate
for Clp proteases (34). Further studies are necessary to
elucidate the still-putative relationship between SigH activity and the
stringent response.
The function of YvyD is still unknown. The protein shows 30 to 40%
identity to 54 modulating factors of gram-negative
bacteria; the yvyD gene is transcribed monocistronically. In
contrast to B. subtilis, the genes encoding the
54 modulating factors are organized in operons coding
for 54-like factors (3, 10, 25-27, 32, 33, 36,
55) and for proteins highly similar to components of the
phosphotransferase system (37) in gram-negative bacteria.
Reizer et al. (40) proposed that the phosphotransferase
system catalyzed protein phosphorylation functions in the regulation of
54-dependent gene expression and supposed a link between
carbon and nitrogen metabolic pathways. The 54
modulating proteins do not seem to act as repressors but may inhibit
the activity of the sigma factor via a still-unknown mechanism (33). In a yvyD mutant of B. subtilis, transcription of the L-dependent operon
levD seems to be elevated, indicating that YvyD may also
exert a negative effect on L activity. Despite the fact
that we do not yet know the physiological role of YvyD, the outstanding
location of yvyD in the gene regulation network linking
B and H on the one side with
L on the other might promise a very important function
of this protein in the genetic network of the transition phase which
needs further investigation.
| |
ACKNOWLEDGMENTS |
We thank M. Débarbouillé for the generous gift of
strain QB5081, D. Zeigler of the Bacillus Genetic Stock
Center for plasmid pGD780, T. Msadek for strain BH41, and E. Krüger for strain BEK38. We are grateful to C. Scharf for help
with computer work and to P. J. Piggot and two anonymous referees
for critical and helpful comments on the manuscript.
This work was supported by grants from the DFG, the BMBF, and Fonds der
Chemischen Industrie to M.H.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie und Molekularbiologie,
Ernst-Moritz-Arndt-Universität, F.L.-Jahnstr. 15, D-17487
Greifswald, Germany. Phone: 49-3843-864200. Fax: 49-3834-864202. E-mail: hecker{at}microbio7.biologie.uni-greifswald.de.
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Journal of Bacteriology, December 1998, p. 6674-6680, Vol. 180, No. 24
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Abstract
Introduction
