From the
Exogenously provided glycine betaine can efficiently protect Bacillus subtilis from the detrimental effects of high
osmolarity environments. Through functional complementation of an Escherichia coli mutant deficient in glycine betaine uptake
with a gene library from B. subtilis, we have identified a
multicomponent glycine betaine transport system, OpuA. Uptake of
radiolabeled glycine betaine in B. subtilis was found to be
osmotically stimulated and was strongly decreased in a mutant strain
lacking the OpuA transport system. DNA sequence analysis revealed that
the components of the OpuA system are encoded by an operon (opuA) comprising three structural genes: opuAA, opuAB, and opuAC. The products of these genes exhibit
features characteristic for binding protein-dependent transport systems
and in particular show homology to the glycine betaine uptake system
ProU from E. coli. Expression of the opuA operon is
under osmotic control. The transcriptional initiation sites of opuA were mapped by high resolution primer extension analysis, and two opuA mRNAs were detected that differed by 38 base pairs at
their 5` ends. Synthesis of the shorter transcript was strongly
increased in cells grown at high osmolarity, whereas the amount of the
longer transcript did not vary in response to medium osmolarity.
Physical and genetic mapping experiments allowed the positioning the opuA operon at 25° on the genetic map of B.
subtilis.
Monitoring and adapting to changes in environmental conditions
are critical processes that determine the survival of microorganisms
and their successful long term competition for a given habitat. In its
soil environment, Bacillus subtilis encounters often osmotic
challenges due to frequent variations in the availability of water.
Since the cell envelope is permeable to water, drying and wetting of
the soil alters the osmotic conditions and hence triggers the flux of
water across the cell membrane. Active and timely adaptation reactions
are thus required to avoid cell lysis under low osmolarity or
dehydration of the cytoplasm under high osmolarity growth conditions
(1, 2). Despite the importance of changes in the environmental
osmolarity for growth and survival of B. subtilis, the
specific physiological and genetic adaptation mechanisms to such
environmental challenges are in general rather poorly understood.
The exposure of B. subtilis to a hypersaline environment
triggers the induction of a set of general and salt-specific stress
proteins, indicating that increased salt concentration alerts the cell
to adverse growth conditions(3) . The expression of a number of
genes in this general stress regulon is determined by the alternative
transcription factor
A central part of the physiological
response of B. subtilis to high osmolarity stress is the
intracellular accumulation of inorganic and organic osmolytes that
serve to counterbalance intracellular versus extracellular
osmolarity and consequently help to maintain a turgor optimal for cell
growth. An increase in medium osmolarity stimulates turgor-sensitive
transport systems that mediate rapid accumulation of K
In B. subtilis the importance of exogenously provided
glycine betaine for the efficient adaptation to a high osmolarity
environment is firmly established, but the route of glycine betaine
uptake in this model system for Gram-positive bacteria is unknown. We
have begun to characterize the mechanisms of glycine betaine uptake in B. subtilis, and we report in this paper the identification
and analysis of a binding protein-dependent transport system (OpuA) for
this osmoprotectant.
The
ability of strain MKH13(pBKB1) to grow at high osmolarity in the
presence of 1 mM glycine betaine was shown to be dependent on
the presence of plasmid pBKB1 by retransformation into strain MKH13.
There was no growth of MKH13(pBKB1) in high osmolarity minimal medium
(MMA agar plates with 0.8 M NaCl) lacking this osmoprotectant.
Osmoprotection by glycine betaine requires the intracellular
accumulation of this compound(1, 2) . We therefore
measured the initial [1-
We
measured the initial uptake activity for radiolabeled
[1-
The deduced
amino acid sequences of the opuAA, opuAB, and opuAC genes exhibit features characteristic for binding
protein-dependent transport systems (37, 38) and, in
particular, show striking homology to the components of the glycine
betaine binding protein-dependent transport system ProU from E.
coli(18) . The opuAA gene encodes a hydrophilic
protein of 418 amino acid residues (M
The
last gene in the operon, opuAC, encodes a 293-amino acid
residue hydrophilic protein with a predicted M
Using a DNA
probe covering opuA (Fig. 8A), we performed
Southern hybridization experiments with chromosomal DNA prepared from
strains JH642 (opuA
The uptake of glycine betaine confers a high level of osmotic
tolerance in B. subtilis and thus is an important facet in the
stress response of this soil microorganism to high
osmolarity(9) . Glycine betaine is a preferred osmoprotectant in B. subtilis because the endogenous accumulation of proline is
strongly reduced under high osmolarity growth conditions when glycine
betaine is present in the culture medium(7) . The data presented
here show that glycine betaine transport in B. subtilis is
under osmotic control and involves at least two transport systems. We
have characterized one of these transporters in some detail and
identified it as a multicomponent, binding protein-dependent transport
system, OpuA.
Bacterial binding protein-dependent transport systems
are members of a superfamily of prokaryotic and eukaryotic
transporters, known as ATP-binding cassette (ABC) transporters or
traffic ATPases (37, 38). These transporters couple hydrolysis of ATP
to nutrient and ion uptake or to the translocation of drugs,
polysaccharides, peptides and proteins across biological membranes.
Because the substrate for the opuA-encoded ABC uptake system
is metabolically inert in B. subtilis and serves an
osmoprotective function(9) , OpuA can be classified as part of
the cellular defense machinery that permits this soil microorganism to
cope with high osmolarity environments. Binding protein-dependent
transport systems exhibit a very high affinity toward their substrate
and can mediate unidirectional solute accumulation against a steep
concentration gradient(37, 38) . Consequently,
transporters such as the OpuA system are especially well suited to
scavenge their substrate effectively from the environment even when it
is present at a very low concentration and still attain a high
intracellular level of the transported compound. Glycine betaine is
synthesized by plants (52) and is brought in a varying supply
into the habitat of B. subtilis through the degradation of
plant tissues, thus necessitating effective mechanisms for the active
acquisition of this important osmoprotectant.
Characteristic for the
binding protein-dependent transport systems of Gram-negative bacteria
is the presence of a soluble, ligand-binding, periplasmic protein that
serves to capture the substrate and deliver it to the membrane-bound
components. Binding of glycine betaine to the periplasmic ProX proteins (Fig. 9) from E. coli and S. typhimurium has
been demonstrated directly(17, 41, 53) . Since
Gram-positive bacteria have no periplasm, it has been proposed that
extracellular proteins anchored via lipid modifications in the
cytoplasmic membrane can serve the physiological function of
periplasmic proteins from Gram-negative
bacteria(42, 43) . The components of the OpuA transport
system show homology to those from the binding protein-dependent
glycine betaine uptake system ProU from E. coli (Fig. 4). The amino acid sequence of the ATPases (OpuAA and
ProV) and the integral inner membrane components (OpuAB and ProW) from
both systems show extensive identity, but the substrate-binding
proteins from the OpuA and ProU systems are not well conserved (Fig. 4). Such low level conservation of the ligand-binding
proteins has also been observed for several other pairs of ABC
transporters of Gram-negative and Gram-positive
microorganisms(54) . The OpuAC protein is essential for the
OpuA-mediated glycine betaine transport in B. subtilis (Fig. 1), and its processing is inhibited by globomycin (Fig. 5B), indicating that OpuAC is a lipid-modified and
extracellular substrate-binding protein. The overall organization and
subunit composition of the B. subtilis OpuA system is shown in Fig. 9and is compared with its counterpart, ProU, from the
Gram-negative bacterium E. coli.
Experiments with a opuAA-lacZ protein
fusion showed that the amount of OpuA is responsive to changes in the
osmotic strength of the environment. High osmolarity growth conditions
induce opuA expression, and two differently regulated
promoters direct the transcription of the opuA operon; one is
osmotically controlled (opuA P-1), whereas the second (opuA P-2) does not respond to the osmotic stimulus (Fig. 6). The putative -10 and -35 regions of the opuA P-1 and opuA P-2 promoters show homology (Fig. 6A) to the consensus sequence of
In addition to their unusual
-10 regions, both the osmotically regulated proU and opuA P-1 promoters deviate in the length of their spacer
regions between the -35 and -10 sequences from the
consensus 17-bp distance and contain suboptimal spacings of 16 and 18
bp, respectively (Fig. 6B). Expression of the
osmotically regulated proU operon from E. coli is
sensitive to changes in DNA topology(57, 58) . RNA
polymerase appears to make specific contacts with both the -10
and -35 regions, and the relative orientation of these sequences
is an important determinant for the efficiency of transcription
initiation(59) . Promoters with a 16- or 18-bp spacer sequence
might therefore respond sensitively to environmentally controlled
alterations in DNA topology, and both the E. coli proU and B. subtilis opuA P-1 promoters might thus be members of a
class of DNA twist-sensitive promoters(60) .
DNA sequences
located upstream and downstream of the osmoregulated E. coli proU promoter and the nucleoid-associated DNA binding protein H-NS and
HU contribute to the finely tuned genetic control of proU expression in response to changes in medium
osmolarity(25, 58, 61, 62, 63) .
Our identification of the osmoregulated opuA P-1 promoter from B. subtilis is an important first step in identifying the DNA
sequences required in cis to mediate osmotically controlled
transcription and in unravelling the signal transduction pathway that
allows B. subtilis to sense changes in the environmental
osmolarity and convert this information into a genetic response that
finally leads to increased opuA expression.
We thank R. Brückner, M. Itaya, J. Lucht, M.
Marahiel, W. Schumann, P. Stragier, and D. Zeigler from the BGSC for
generously providing plasmids and bacterial strains. We are
particularly grateful to M. Inukai (Sankyo Pharmaceutical Co.; Japan)
for the kind gift of the antibiotic globomycin. The expert technical
assistance of S. Kneip and the help of V. Koogle in preparing the
manuscript are greatly appreciated. We thank R. Thauer for continued
support.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, which serves to control a
regulatory network responsive to stationary phase signals and
growth-limiting conditions(4, 5, 6) . However, B. subtilis mutant strains lacking the
protein are not at a survival disadvantage compared with the wild
type when exposed to osmotic shock or extreme desiccation under
laboratory conditions(6) . Therefore, it is uncertain whether
members of the
-controlled general stress regulon
play a direct role in the adaptation of B. subtilis to high
osmolarity environments.
in the cell, which, in turn, restores turgor and permits cell
growth to resume(7, 8) . This initial reaction is
followed by a cellular response that replaces ionic osmolytes, which
are deleterious at high concentrations, with organic osmolytes, which
are more compatible with the normal physiological and structural
requirements of the bacterial cell(1, 2) . In B.
subtilis proline is the predominant organic osmolyte synthesized
in defined medium by cells exposed to a hypersaline
environment(7) . However, several hours are required to reach a
proline level that is sufficient for osmoprotection, leaving the cell
at a growth disadvantage in harsh high osmolarity
environments(9) . B. subtilis can more efficiently
respond to high osmolarity by accumulating glycine betaine(9) .
This potent osmoprotectant is widely found in nature and has been
adopted across the microbial, plant, and animal kingdoms as an
effective compatible solute(1, 2) . Glycine betaine can
be synthesized by B. subtilis from its precursor choline or
taken up directly from the
environment(7, 9, 10) . A strong increase in the
growth rate and the proliferation under environmental conditions that
are otherwise strongly inhibitory for B. subtilis can be
attained when glycine betaine can be directly accumulated from the
growth medium(7, 9, 10) . The presence of uptake
systems for glycine betaine has been reported for a variety of
Gram-negative and several Gram-positive
bacteria(11, 12, 13, 14, 15) ,
but the details of such transport systems have been studied at the
molecular level only in Escherichia coli and Salmonella
typhimurium. Here, two glycine betaine transport systems, ProP and
ProU, have been
characterized(1, 2, 16, 17, 18) .
Bacterial Strains and Plasmids
The B.
subtilis strain JH642 (trpC2, pheA1; BGSC(
)1A96), a derivative of the B. subtilis wild-type strain 168, was constructed by J. A. Hoch and was
obtained from M. Marahiel. Strain MO1099 (trpC2, pheA1, amyE::ery; BGSC 1A717) has been
described(19) . Strain TIBS57 (amyE3, aroI10;
BGSC 1A474) was used for the genetic mapping of the opuA operon. The B. subtilis strains BKB4
(
(opuA::neo)1]) and BKB7
(
(opuA::tet)2) are derivatives of JH642
and were constructed by transforming JH642 with EcoRI
restriction fragments isolated from plasmids pBKB11
(
(opuA::neo)1; Fig. 1), and
pBKB52 (
(opuA::tet)2; Fig. 1),
respectively, and selecting for kanamycin-resistant (BKB4) or
tetracycline-resistant (BKB7) colonies on LB agar plates. The E.
coli K-12 strain MKH13 (
(putPA) 101
(proP)2
(proU) 608)
is a derivative of strain MC4100 (20) and is entirely deficient
in glycine betaine uptake(21) . The E. coli B strain
BL21(
DE3) was employed for the selective expression of genes under
T7
10 control (22). The low copy number plasmid pHSG575 (23) was used for the construction of a B. subtilis gene library and for the construction of a number of subclones
carrying various segments of the opuA region (Fig. 1).
Strain DH5
(Life Technologies, Inc.) was used to propagate the B. subtilis gene library. The low copy number T7 expression
plasmids pPD100 and pPD101 (24) were used to express the genes
of the opuA operon under the control of the T7
10
promoter. Plasmid pJL29 is a pBR322-derived vector for the construction
of protein fusions to a truncated `lacZ gene(25) ; it
was used here for the isolation of the
(opuAA-lacZ)hyb1 hybrid gene. The E. coli-B. subtilis shuttle vector pRB373 has been
described(26) . DNA cartridges encoding genes conferring
resistance to kanamycine or tetracycline were isolated from plasmids
pAT21 and pBEST307, respectively(27, 28) .
Figure 1:
Physical and genetic organization of
the cloned opuA region. A map of a plasmid, pBKB1, which
carries the entire opuA operon, is shown. The exact positions
and transcriptional orientations of the opuAA, opuAB,
and opuAC genes were inferred from DNA sequence analysis. The
extent of the DNA segment present in the various deletion derivatives
and subclones of plasmid pBKB1 are indicated by the lines.
Plasmids pBKB11 and pBKB52 are deletion derivatives of pBKB1 and carry,
in addition, gene cartridges encoding kanamycin (kan) or
tetracycline (tet) resistance, respectively. The kan and tet gene cartridges are not drawn to scale.
Osmoprotection by glycine betaine was assayed by monitoring the growth
of the E. coli mutant strain MKH13 lacking both the ProP and
ProU glycine betaine transport systems and harboring the various
pBKB1-derived plasmids on glucose minimal plates containing 0.8 M NaCl and 1 mM glycine betaine. Growth of the strains was
scored after 3 days of incubation at 37
°C.
Growth Conditions, Media, and Chemicals
Bacteria
were grown aerobically at 37 °C in LB, MMA glucose (0.2%), or
Spizizen's minimal medium (SMM) supplemented with 0.5% glucose,
20 mg/liter L-tryptophan, 18 mg/liter L-phenylalanine, and a solution of trace elements (29, 30).
The osmolarity of the various media was increased by addition of NaCl
from a highly concentrated (5 M) stock solution. The
osmolarity of media was determined with a vapor pressure osmometer
(model 5500; Wescor Inc., Logan, UT). Expression of the opuA genes under T710 control was carried out in cells grown in
M9 minimal medium (29) supplemented with 0.2% casaminoacids. To
select derivatives of strain MKH13 synthesizing glycine betaine
transporters encoded by cloned B. subtilis DNA, we used MMA
minimal agar plates containing 0.2% glucose as the carbon source, 0.8 M NaCl and 1 mM glycine betaine. Plates for the
detection of extracellular
-amylase (AmyE) activity contained 1%
starch in LB agar plates. Production of AmyE by B. subtilis strains was detected by flooding the colonies grown on LB starch
plates with Gram's iodine stain (0.5% (w/v) iodine, 1% (w/v)
potassium iodide) for 1 min and scoring for zones of clearing around
the colony after decanting the stain(31) . The AroI phenotype of B. subtilis strains was tested by scoring the growth of
colonies on SMM minimal plates either lacking or containing 20 mg/liter L-tryptophan, 18 mg/liter L-phenylalanine, and 20
mg/liter L-tyrosine; AroI
strains can not
grow on minimal plates lacking these aromatic amino acids. The
antibiotics ampicillin, chloramphenicol, tetracycline, and kanamycin
were used in E. coli at a final concentration of 100, 30, 5,
and 15 µg/ml, respectively. Kanamycin, tetracycline, and
erythromycin were used in B. subtilis at a final concentration
of 5, 10, and 0.5 µg/ml, respectively. The cyclic peptide
antibiotic globomycin, a specific inhibitor of signal peptidase
II(32) , was kindly provided by M. Inukai from Sankyo
Pharmaceutical Co. (Japan). A stock solution (10 mg/ml) of globomycin
was prepared in dimethyl sulfoxide and added to cultures to a final
concentration of 120 µg/ml. Radiolabeled
[1-
C]glycine betaine (55 mCi/mmol) was purchased
from ARC (American Radiolabeled Chemicals Inc., St. Louis, MO),
radiolabeled [
S]methionine (523 mCi/mmol) was
obtained from ICN (Meckenheim, Germany), and
[
S]dATP (1000 Ci/mmol) was from Amersham Corp.
Methods Used with Nucleic Acids
Routine
manipulations of plasmid DNA, the isolation of chromosomal DNA from B. subtilis, and the detection of homologous sequences by
Southern hybridization were all carried out by standard
techniques(33) . Sequencing of double-stranded plasmid DNA and
of single-stranded DNA segments cloned in M13BM20 or M13BM21
(Boehringer Mannheim) was carried out using the Sequenase 2.0 kit (U.S.
Biochemical Corp.) and the conditions recommended by the supplier.
Sequencing reactions were primed with a number of synthetic
oligonucleotide primers spaced along the opuA region. The
entire 5.2-kb EcoRI insert present in plasmid pBKB1 (Fig. 1) was used to generate a probe by random nucleotide
labeling using DIG-dUTP (DIG DNA labeling and detection kit, Boehringer
Mannheim). Hybridization of the labeled DNA fragment to EcoRI-
and PstI-digested chromosomal DNA isolated from various B.
subtilis strains was performed according to the instructions of
the supplier, and the hybridization products were detected using
LumiPhos 530 (U.S. Biochemical Corp.). Total RNA was prepared from
cultures grown to mid-log phase (A = 0.5)
in either LB medium or LB medium of high osmolarity (0.5 M NaCl added) of the B. subtilis strain JH642 carrying the opuAA-lacZ fusion plasmid pBKB56 essentially as
described by Völker et al.(5) . The RNA was
further purified by passage through a Quiagen tip-100 column as
suggested by the supplier (Diagen, Düsseldorf, Germany). The total
amount of RNA isolated was spectrophotometrically (A
) determined; an A
of 1
corresponds to approximately 40 µg/ml RNA(33) . For the
primer extension reaction, a synthetic primer
(5`-GAACTGCCTTCTTTGTTTGTTTCCC-3`), complementary to the opuAA mRNA (position 283-307 bp; see Fig. 3) was hybridized
with 5 µg of RNA and extended with avian myeloblastosis virus
reverse transcriptase (U.S. Biochemical Corp.) in the presence of
radiolabeled [
S]dATP at a final concentration of
1 µCi/ml. The size of the reaction products was determined on a 4%
DNA sequencing gel under denaturing conditions and visualized by
autoradiography. A sequencing ladder produced by using the same primer
was run on the same sequencing gel to determine the precise 5` ends of
the opuA mRNAs. Transformation of competent B. subtilis cells with plasmids and linear DNA fragments was done according to
routine procedures(31) .
Figure 3:
Nucleotide sequence of the opuA operon. The determined DNA sequence of the opuA operon
and the deduced amino acid sequences of the OpuAA, OpuAB, and OpuAC
proteins are shown. The proposed ATG start codons for these genes are boxed, and putative ribosome-binding sites (rbs) are overlined. The transcription initiation sites for the opuA mRNAs (mRNA-1, mRNA-2) are indicated by arrows, and the
corresponding putative -10 and -35 regions are marked. An
inverted repeat downstream from the opuAC gene is indicated by
a pair of arrows. The positions of recognition sites for a
number of restriction enzymes are boxed.
Construction of Plasmids
A library of chromosomal
DNA segments from the B. subtilis wild-type strain JH642 was
prepared by cleaving chromosomal DNA with EcoRI and ligating
the resulting restriction fragments into the EcoRI site in the
polylinker of the lacZ -complementing plasmid pHSG575.
The DNA of the recombinant plasmids was transformed into strain
DH5
, and colonies were selected on LB plates containing
chloramphenicol, isopropyl-1-thio-
-D-galactopyranoside (1
mM), and x-gal (40 µg/ml). Approximately 90% of the
obtained transformants (40,000 colonies) carried plasmids with cloned B. subtilis DNA as judged from their LacZ
phenotype. All colonies were pooled and grown for 2 h in LB
medium with chloramphenicol; the plasmid DNA was then extracted and
used to transform the E. coli strain MKH13. Plasmids pBKB13,
pBKB14, pBKB17, pBKB18, pBKB38, and pBKB46 were constructed by deleting
defined restriction fragments from the opuA
plasmids pBKB1 and religating the plasmid backbones (Fig. 1). Plasmids pBKB15, pBKB35, and pBKB39 were isolated by
cloning appropriate restriction fragments isolated from plasmid pBKB1
into the vector pHSG575 (Fig. 1). Plasmid pBKB33 carries the
entire opuA operon on a 4-kb EcoRI-EcoRV
restriction fragment (Fig. 1) that has been cloned into the
polylinker sequence of the T7
10 expression vector pPD100, thus
positioning opuA under T7
10 control. The same
restriction fragment was inserted in the reverse orientation with
respect to the T7
10 promoter present in the vector pPD101,
yielding plasmid pBKB34. Plasmid pBKB44, which expresses the opuAA
opuAB
genes under
T7
10 control was constructed by deleting a 636-bp NsiI
fragment carrying most of the opuAC gene (Fig. 1) from
the opuA
plasmid pBKB33. To achieve
expression of the opuAA
gene under T7
10
control, a 1.7-kb EcoRI-HpaI restriction fragment (Fig. 1) was inserted into the vector pPD100, yielding plasmid
pBKB43. A plasmid expressing the opuAC
gene
under T7
10 control was constructed by isolating a 1.4-kb ApaLI-NotI fragment from pBKB1; the overhanging ends
of the restriction fragments were filled in with Klenow enzyme and then
ligated into the SmaI site of plasmid pPD100. Plasmids
positioning the opuAC gene under T7
10 control (pBKB58)
or aligning it in the reverse orientation with the T7
10 promoter
(pBKB57) were identified by restriction analysis. To construct an opuAA-lacZ gene fusion, a 1.3-kb EcoRI-SnaBI restriction fragment from pBKB1 (Fig. 1) was cloned into the EcoRI and SmaI
sites of the `lacZ fusion vector pJL29, yielding plasmid
pBKB54. In this plasmid, the reading frames of opuAA and `lacZ are properly aligned across the SnaBI and SmaI junction, thus generating a hybrid protein fusion,
(opuAA-lacZ)hyb1. The entire hybrid
gene was transferred from plasmid pBKB54 on a 4.4-kb EcoRI-DraI restriction fragment into the E.
coli-B. subtilis shuttle vector pRB373, which had been
cut with EcoRI and SmaI; this construction resulted
in plasmid pBKB56.
Transport Assays for Radiolabeled Glycine
Betaine
Uptake of glycine betaine in B. subtilis and E. coli was measured using [1-C]glycine
betaine (55 mCi/mmol) as a substrate. The cells were grown to
mid-exponential phase (A
=
0.15-0.5) in minimal medium with glucose as the carbon source and
used immediately for the transport assay. E. coli strains were
grown in MMA or MMA with 0.2 M NaCl, and B. subtilis strains were grown in SMM or SMM with 0.4 M NaCl. The
uptake assay contained [1-
C]glycine betaine at a
final substrate concentration of 10 µM (5.5 mCi/mmol) in a
total reaction volume of 2 ml. Samples (0.3 ml) were taken at various
times and filtered through 0.45 µm-pore-size filters (Schleicher
and Schuell GmbH, Dassel, Germany). The cells were washed with 20 ml of
isotonic minimal salts, and the radioactivity retained on the filters
was determined in a scintillation counter. Protein concentrations were
determined from total cell extracts using the Bio-Rad protein assay
with acetylated bovine serum albumin as the standard. The cell extracts
were prepared by passing the B. subtilis cells five times
through a French press cell at 103,000 kilopascals.
Preparation of Total Cell Extracts, SDS-Polyacrylamide
Gel Electrophoresis, and Immunological Detection of the
OpuAA`-
Cultures (20 ml in a
100-ml Erlenmeyer flask) of strain JH642 carrying the opuAA-lacZ fusion plasmid pBKB56 were grown overnight
at 37 °C in LB medium or LB medium with 0.5 M NaCl. The
optical density (A-Galactosidase Hybrid Protein
) of the cultures was
determined and adjusted to A
=5. From each
culture, 2-ml portions were withdrawn, the cells were collected by
centrifugation and resuspended in 150 µl of TE (10 mM Tris-HCl, 1 mM EDTA, pH = 8.0), and 15 µl of
lysozyme (10 mg/ml in H
O) was added. The cell suspension
was then incubated for 8 min at 37 °C in a water bath, 50 µl of
4-fold concentrated sample buffer (final concentration, 0.06 M Tris, pH 6.8, 5% SDS, 10% glycerol, 3% dithiothreitol, 0.001%
bromphenol blue) was added, and the cells were lysed by incubation for
5 min at 95 °C. To reduce the viscosity of the cell extract, 2
µl of benzon nuclease (Merck) was added and incubated for 10 min at
37 °C, followed by another short (5-min) incubation at 95 °C.
Aliquots of the cell extracts were then immediately loaded onto 7%
SDS-polyacrylamide gels(34) , and the proteins were visualized
by staining with Coomassie Brilliant Blue (33). For the immunological
detection of the OpuAA`-
-galactosidase hybrid protein, total
cellular extracts were electrophoretically separated on 7%
SDS-polyacrylamide gels and transferred onto a sheet of Immobilon
(Millipore, Eschwege, Germany). The bound proteins were probed with a
rabbit
-galactosidase antiserum, and the antigen-antibody
complexes were visualized with a second goat anti-rabbit immunoglobulin
G alkaline phosphatase-conjugated antibody (Sigma) using
5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium chloride
(Boehringer Mannheim) as substrates.
Expression of the opuA Gene Products under T7
Control
Plasmids carrying various genes from the opuA operon under the transcriptional control of the T710
promoter were transformed into strain BL21(
DE3) to selectively
visualize the opuA-encoded proteins. These plasmids are pBKB33 (opuAA
opuAB
opuAC
), pBKB44 (opuAA
opuAB
), pBKB43 (opuAA
),
and pBKB58 (opuAC
). Plasmids pBKB34 and
pBKB57 carrying the opuA
operon and opuAC
gene, respectively, improperly aligned
with respect to the T7 promoter were used as controls. Cultures (20 ml
in 100-ml Erlenmeyer flasks) of strain BL21(
DE3) carrying the
various plasmids were grown in M9 minimal medium with 0.2% casaminoacids to mid-log phase (A
= 0.5-0.7); the cells were washed with M9 minimal medium,
resuspended in 20 ml of M9 minimal medium supplemented with 0.2% methionine assay medium (Difco), and grown for 1 h at 37 °C.
T7
10-mediated gene expression was initiated by adding
isopropyl-1-thio-
-D-galactopyranoside to a final
substrate concentration of 1 mM, after 30 min rifampicin was
added (200 µg/ml) to inhibit the E. coli RNA polymerase.
After a 1-h incubation at 37 °C, 1-ml portions of the cultures were
withdrawn, the proteins were labeled for three min with
[
S]methionine (final concentration, 5.2 nCi/ml),
and the cells were collected by centrifugation and resuspended in 50
µl of SDS-sample buffer. Portions (30 µl) of the cell extracts
were loaded onto 12% SDS-polyacrylamide gels, the proteins were
electrophoretically separated, and radiolabeled peptides were
visualized by autoradiography. To inhibit signal peptidase II, the
antibiotic globomycin was added to the appropriate cultures 10 min
prior to the radiolabeling of the proteins.
Computer Work
Analysis of nucleotide sequences
from the opuA region and the alignment and analysis of protein
sequences was performed with the Lasergene program from DNA Star
(DNASTAR, Ltd., London) on an Apple Macintosh II computer. Multiple
sequence alignments were done at the National Center for Biotechnology
Information (NCBI) using the BLAST programs and the current versions of
the available data bases (November, 1994)(35) . The nucleotide
sequence of the opuA region (see Fig. 3) reported in
this paper has been submitted to the GenBank/EMBL Data
Bank with accession number U17292.
Cloning of the Structural Genes for a Glycine Betaine
Transport System
To clone genes from B. subtilis that
code for glycine betaine transporters, we capitalized on the growth
properties of the E. coli mutant strain MKH13. This strain is
defective for glycine betaine synthesis and also lacks the glycine
betaine transport systems, ProP and ProU(21) . Therefore, it is
severely impaired in its ability to cope with a high osmolarity
environment and, in contrast to its parental strain MC4100, cannot grow
in high osmolarity media containing the osmoprotectant glycine betaine.
We reasoned that it should be possible to functionally complement the
deficiency of strain MKH13 in glycine betaine uptake with the
appropriate B. subtilis genes. A gene bank of EcoRI
restriction fragments was prepared from chromosomal DNA of the B.
subtilis strain JH642 in the low copy number cloning vector
pHSG575 (Cm) and transformed into MKH13 by selecting for
Cm
colonies on LB agar plates. These transformants were
then replica-plated on high osmolarity minimal plates (MMA agar plates
with 0.8 M NaCl) containing 1 mM glycine betaine to
search for MKH13 derivatives that could grow under these selective
conditions. Such strains were readily obtained, and each of the 46
MKH13 derivatives analyzed carried the same pHSG575-derived plasmid
containing a 5.2-kb EcoRI restriction fragment. A restriction
map of one of these plasmids, pBKB1, is shown in Fig. 1.
C]glycine betaine uptake
in cultures of strain MKH13(pBKB1) grown in low osmolarity or high
osmolarity minimal media at a final substrate concentration of 10
µM. Glycine betaine transport activity was readily
detectable in cultures of MKH13(pBKB1), and we found that this
transport activity was under osmotic control (Fig. 2A).
Thus, plasmid pBKB1 encodes an osmotically controlled uptake system for
glycine betaine from B. subtilis. We designate this glycine
betaine transporter as OpuA (osmo protectant uptake) and refer to its
structural gene(s) as opuA.
Figure 2:
OpuA-mediated glycine betaine transport.
Uptake of radiolabeled [1-C]glycine betaine was
assayed in low and high osmolarity grown cultures at a final substrate
concentration of 10 µM. A, the E. coli mutant strain MKH13 (ProP
ProU
) harboring plasmid pBKB1 (opuA
) was grown in MMA (
) or MMA with
0.2 M NaCl (▴) to mid-log phase and assayed for glycine
betaine uptake. Strain MKH13 carrying the vector plasmid pHSG575 grown
in MMA with 0.2 M NaCl (♦) was used as a control. B, the B. subtilis strains JH642 (opuA
) (
,
) and BKB4 (opuA
) (
, ▪) were grown in SMM
(
,
) or SMM with 0.4 M NaCl (
, ▪) to
mid-log phase, and glycine betaine uptake was then
determined.
To identify the approximate
position of the opuA gene(s) within the cloned DNA segment
from the B. subtilis chromosome, we subcloned defined
restriction fragments from plasmid pBKB1 into the low copy number
vector pHSG575 and also constructed a number of deletion derivatives of
pBKB1 (Fig. 1). Each of these plasmids was introduced by
transformation into strain MKH13, and the ability of these
transformants to grow in high osmolarity minimal media in the presence
of 1 mM glycine betaine was tested. The results from these
complementation experiments are summarized in Fig. 1. It is
apparent that a large portion of the cloned 5.2-kb EcoRI
fragment is required to mediate glycine betaine uptake activity. Thus,
OpuA is most likely a multicomponent glycine betaine transport system.
Mutations in opuA Strongly Impair Glycine Betaine
Transport Activity in B. subtilis
OpuA mediates glycine betaine
uptake in E. coli. To investigate the role of the opuA-encoded transport system for glycine betaine transport in B. subtilis, we constructed two opuA mutations on
plasmid pBKB1 and then recombined them by homologous recombination into
the B. subtilis chromosome. For the construction of the
(opuA::neo)1 mutation, an HpaI
restriction fragment was deleted from plasmid pBKB1 and substituted by
a gene cartridge encoding a kanamycin determinant, yielding pBKB11 (Fig. 1). The
(opuA::tet)2 mutation was constructed in an analogous way by removing an NsiI DNA fragment from plasmid pBKB1 and inserting a
tetracycline resistance gene as the selective marker, resulting in
plasmid pBKB52 (Fig. 1). Both opuA mutations destroyed
the plasmid pBKB1-encoded glycine betaine uptake activity in the E.
coli strain MKH13 (Fig. 1). We isolated the
(opuA::neo)1 and the
(opuA::tet)2 constructs from plasmids
pBKB11 and pBKB52, respectively, as EcoRI restriction
fragments and then used this DNA to transform the B. subtilis strain JH642 to either kanamycin resistance
(
(opuA::neo)1) or tetracycline
resistance (
(opuA::tet)2). One
transformant from each experiment was purified, and the proper
integration of the
(opuA::neo)1 (strain
BKB4) or
(opuA::tet)2 (strain BKB7)
mutation into the B. subtilis genome via a double
recombinational cross-over event was proven by Southern hybridization
using a DNA probe derived from plasmid pBKB1 (data not shown).
C]glycine betaine of the opuA
B. subtilis strain JH642 and
its
opuA derivatives BKB4 and BKB7 in cells grown in low
osmolarity and high osmolarity media with low substrate concentration
(10 µM). An efficient and osmotically stimulated glycine
betaine transport activity was present in the wild-type strain JH642 (Fig. 2B). In contrast, both opuA mutations
strongly impaired glycine betaine uptake; this is documented in Fig. 2B for the
(opuA::neo)1 deletion present in strain BKB4. Thus, opuA encodes an
osmotically controlled glycine betaine transport system in B.
subtilis. We note that neither of the chromosomal opuA deletions present in the B. subtilis strains BKB4 and
BKB7 abolish glycine betaine uptake entirely (Fig. 2B).
Thus, besides OpuA, at least one additional glycine betaine transporter
must exist in B. subtilis, and the pattern of glycine betaine
uptake in the opuA mutants indicates that this transport
activity is also under osmotic control (Fig. 2B). In the
above described glycine betaine uptake experiments, the high osmolarity
media were prepared by adding NaCl to the growth media. In analogous
transport assays in which NaCl was replaced by an isoosmolar
concentration of KCl, glucose, or maltose, glycine betaine uptake
activity in strains JH642 (opuA
) and BKB4
(
(opuA::neo)1) was stimulated to an
extent similar to that shown in Fig. 2B (data not
shown). Thus, stimulation of glycine betaine transport in B.
subtilis growing in a high osmolarity environment is a true
osmotic effect since it can be triggered with either ionic or nonionic
osmolytes.
opuA Encodes a Binding Protein-dependent Transport
System
To characterize the nature of the opuA-encoded
glycine betaine transporter more closely, we determined the DNA
sequence of a 3.4-kb DNA segment from pBKB1 that covers the region
necessary for glycine betaine uptake activity (Figs. 1 and 3). Analysis
of the sequenced DNA segment revealed the presence of three open
reading frames that are oriented in the same direction and constitute
the opuA locus (Fig. 3). Downstream of opuA, a
region is present that harbors a DNA structure with dyad symmetry. This
inverted repeat is bracketed by runs of AT base pairs, indicating that
it possibly could function as a Rho-independent bidirectional
transcriptional terminator(36) . The opuA locus
consists of three structural genes (opuAA, opuAB, and opuAC), and their tight physical organization strongly
suggests that they are genetically organized in an operon. The
intergenic region between the opuAA stop codon (TAA) and the
ATG start codon of the opuAB gene is only one nucleotide in
length, and the ribosome-binding site for opuAB is thus
present in the preceding opuAA coding region (Fig. 3).
The genetic organization of the opuAB and the opuAC junction is even more tightly spaced; here, the ATG start codon of opuAC is part of the TGA stop codon for opuAB (Fig. 3). Each of the three genes is preceded at an
appropriate distance by a potential ribosome-binding site, which for
the opuAB and opuAC genes is entirely contained in
the coding region of the preceding gene (Fig. 3).
46,473), and
a comparison of the OpuAA protein with protein sequences present in the
data libraries revealed strong sequence identities to many prokaryotic
and eukaryotic proteins involved in ATP hydrolysis. Such a close
relatedness in the amino acid sequence is a hallmark of the energizing
components of binding protein-dependent transport
systems(37, 38) . The alignment of the OpuAA protein
with the corresponding polypeptide (ProV) from the ProU system from
both E. coli and S. typhimurium is shown in Fig. 4A. Approximately 58% of the amino acid residues
are identical among all three proteins, and only a single gap needs to
be introduced to achieve a good alignment of the protein sequences over
their entire length. Sequence conservation is particularly apparent in
the N-terminal half of the OpuAA and ProV proteins and is pronounced
around the Walker A and B ATP-binding motifs (Fig. 4A).
Figure 4:
Alignment of the sequences of the
components of the OpuA transport system with those of ProU. A,
the amino acid sequence of the opuA-encoded ATPase from B.
subtilis is compared with the homologous protein ProV from E.
coli (18) and S. typhimurium (64). The regions of the
three proteins corresponding to the Walker A and B ATP-binding motifs
are overlined. B, comparison of the OpuAB protein
from B. subtilis and the corresponding ProW protein from E. coli. C, alignment of the sequences of the
processed glycine betaine-binding protein ProX from E. coli and the OpuAC protein from B.
subtilis.
The opuAB reading frame codes for a quite hydrophobic
polypeptide (M 30,250) that is homologous to the
integral inner membrane protein ProW of the E. coli ProU
transport system. Analysis of the topology of the E. coli ProW
protein with phoA and lacZ fusions has revealed that
ProW has seven transmembrane segments with the carboxyl terminus in the
cytoplasm and the amino terminus in the periplasm (39).
(
)The ProW and OpuAB proteins show extensive sequence
homology (47% identity) over their entire length and can be aligned
without introducing a single gap into the amino acid sequence. Thus,
the topology of OpuAB and ProW appears to be similar. The OpuAB protein
(282 amino acids) is considerably smaller than ProW (354 amino acids).
Most of the reduced size of OpuAB can be ascribed to a deletion
removing 55 amino acids present in the N-terminal region of ProW
thought to be exposed in the periplasmic space(39) .
A small segment (amino acids 183-203) of OpuAB displays
limited homology to integral inner membrane components of other binding
protein-dependent transport systems from both Gram-negative and
Gram-positive bacteria(40) . It has been speculated that these
residues contribute to an interaction site for the ATPases of the
binding protein-dependent transporters(37, 38) .
of
32,218. The OpuAC protein is likely the substrate-binding protein
component for the OpuA glycine betaine transport system. Its homologue,
ProX, in the E. coli ProU system is a periplasmic protein (17,
41) that is initially synthesized with an N-terminal signal sequence
extension(18) . The first 20 amino acids of the opuAC-encoded protein exhibit the features of a secreted
protein and show the characteristic signatures of a lipoprotein signal
sequence. There is a positively charged N-terminal end, followed by a
highly hydrophobic stretch of amino acids and a string of amino acids
(Leu-Ala-Ala-Cys) that conforms to the consensus sequence recognized by
signal peptidase II(42, 43) . As a rule, the cysteine
residue constitutes the N terminus of the proteolytically processed
protein and is modified through the covalent attachment of lipids. This
lipid modification of the N terminus serves to anchor the extracellular
protein in the cytoplasmic membrane, and such lipoproteins have been
described as substrate-binding proteins for a number of binding
protein-dependent transport systems in Gram-positive
bacteria(42, 43) . The substrate binding proteins, ProX
and OpuAC, show the least sequence conservation (33% identity in a
46-amino acid segment) among the components of the ProU and OpuA
transport systems (Fig. 4C). Only a limited number of
residues in the central part of the OpuAC and ProX proteins can be
aligned, whereas the N-terminal and C-terminal ends of both proteins
are entirely different (Fig. 4C).
Identification of the opuA Gene Products
To
identify the proteins encoded by the opuA operon, we used the
T7 RNA polymerase and T710 promoter expression
system(22, 24) . We constructed a set of T7 expression
plasmids carrying either the entire opuA operon (opuAA, opuAB, opuAC; pBKB33), the first two
structural genes (opuAA, opuAB; pBKB44), or just the
first gene of the opuA locus (opuAA; pBKB43). These
plasmids were transformed into the E. coli strain
BL21(
DE3), which carries a chromosomal copy of the structural gene
for the T7 RNA polymerase under the control of the lacPO regulatory sequences(22) . T7
10 promoter-mediated
expression of the various opuA-encoded genes was initiated by
adding isopropyl-1-thio-
-D-galactopyranoside to the
cultures, and the translated proteins were then labeled with
[
S]methionine. We were able to express the opuA-encoded proteins under T7
10 control in E. coli, but many degradation products of the proteins were visible (Fig. 5A), indicating that the OpuA proteins from B.
subtilis were relatively unstable when produced in the
heterologous E. coli host. Such protein instability has also
been observed when components for the binding protein-dependent
iron-hydroxamate transport system from B. subtilis were
expressed in E. coli under T7
10 control(44) . A
comparison of the plasmid pBKB33-, pBKB44-, and pBKB43-encoded proteins
allowed us to visualize and identify the components of the OpuA system.
The opuA
plasmid pBKB33 directed the
synthesis of three proteins with a apparent molecular mass of 47,000
daltons (OpuAA), 24,000 daltons (OpuAB), and 30,500 daltons (OpuAC).
The 30,500-dalton protein was absent when the opuAA
opuAB
plasmid pBKB44 was used to mediate
gene expression, thus identifying this polypeptide as the product of
the opuAC gene. The same protein is synthesized in strain
BL21(
DE3) carrying just the opuAC gene under T7
10
control on plasmid pBKB58 (Fig. 5B, lane 5). A
47,000-dalton protein was produced in cells expressing only opuAA from plasmid pBKB43, (Fig. 5A, lane 3),
thus identifying this polypeptide as the OpuAA protein. Both the
47,000-dalton protein (OpuAA) and the 24,000-dalton protein were
synthesized when the opuAA
opuAB
genes (plasmid pBKB44) were expressed
under T7
10 control (Fig. 5A, lane 2).
Thus, the 24,000-dalton protein must be OpuAB. None of these opuA-encoded proteins were produced when an opuA
-containing restriction fragment was
cloned into the T7 expression vector in an orientation reversed with
respect to that present in plasmid pBKB33 (Fig. 5A;
compare lanes 1 and 4). The apparent molecular masses
of the OpuAA (47,000-dalton) and the OpuAC (30,500-dalton) proteins
compare favorably with the molecular masses deduced for OpuAA (46,473
daltons) and OpuAC (30,235 daltons for the proteolytically processed
but unmodified polypeptide) from the DNA sequence (Fig. 3) of
their structural genes. In contrast, the apparent molecular mass of the
OpuAB protein (24,000 daltons) as calculated from its electrophoretic
mobility on a 12% SDS-polyacrylamide gel, deviates considerably from
the molecular mass predicted for this protein from the opuAB DNA sequence (30,250 daltons). The OpuAB protein constitutes a
quite hydrophobic integral membrane protein, and its apparent
electrophoretic behavior is therefore not too surprising.
Figure 5:
Expression of the opuA-encoded
proteins under the control of the T710 promoter. A,
T7
10-mediated gene expression and radiolabeling of proteins was
performed in derivatives of strain BL21(
DE3) carrying plasmids
pBKB33 (opuAA
, opuAB
, opuAC
) (lane 1), pBKB44 (opuAA
, opuAB
) (lane 2), pBKB43 (opuAA
) (lane 3), and pBKB34 (opuA
, but improperly aligned with respect to
the T7
10 promoter) (lane 4). B, effect of the
antibiotic globomycin on the processing of the pro-OpuAC protein.
T7
10-mediated gene expression and radiolabeling of proteins was
performed in derivatives of strain BL21(
DE3) carrying plasmids
pBKB33 (opuAA
, opuAB
, opuAC
) (lanes 1 and 2), pBKB57 (opuAC
, but improperly aligned with respect
to the T7
10 promoter) (lanes 3 and 4), and
pBKB58 (opuAC
) (lanes 5 and 6). The samples of lanes 2, 4, and 6 were treated with globomycin. The proteins were
electrophoretically separated on a 12% SDS-polyacrylamide gel and
visualized by autoradiography. The positions of the OpuAA, OpuAB,
OpuAC, and pro-OpuAC proteins are indicated by arrows. The
molecular mass of marker proteins is indicated on the right.
OpuAC Probably Is a Lipoprotein
As outlined above,
the OpuAC protein is likely to carry lipid modifications at its N
terminus, anchoring it in the membrane. One characteristic feature of
such lipoproteins is the inhibition of the proteolytical processing of
their signal sequence by the cyclic peptide antibiotic
globomycin(32, 45) . To test whether OpuAC is indeed a
lipoprotein, we expressed the entire opuA operon and the opuAC gene alone under T710 control in the presence or
absence of globomycin. The presence of globomycin inhibited completely
the processing of the OpuAC protein and resulted in the accumulation of
its precursor molecule, pro-OpuAC (Fig. 5B). In
contrast, globomycin had no influence of the electrophoretic mobility
of the OpuAA and OpuAB proteins (Fig. 5B, lanes 1 and 2). Thus, the selective block imposed by globomycin
on pro-OpuAC processing strongly indicates that OpuAC is a lipoprotein
with an amino-terminal cysteine-lipid anchor for the mature protein.
Osmoregulation of opuA Expression
The
OpuA-mediated glycine betaine transport activity is osmotically
modulated (Fig. 2). To test whether this was due (at least in
part) to osmotic control of opuA transcription and to identify
the opuA promoter(s), we mapped the transcription initiation
sites by primer extension analysis. A 1.3-kb EcoRI-SnaBI restriction fragment carrying the opuA upstream region and most of the opuAA coding
sequence was used to construct a
(opuAA-lacZ)hyb1 protein fusion in the E. coli-B. subtilis shuttle vector pRB373. The
resulting plasmid, pBKB56, was transformed into the B. subtilis strain JH642 to increase the gene dosage for the opuA regulatory region. Total RNA was then prepared from log-phase
cultures grown either in LB medium or in LB medium with increased
osmolarity (LB + 0.5 M NaCl). An opuA-specific
primer was annealed to the RNA isolated from the low and high
osmolarity grown cells and extended with avian myeloblastosis virus
reverse transcriptase in the presence of
[
S]dATP. The reaction products were then
separated on a DNA sequencing gel and visualized by autoradiography.
Two opuA-specific mRNA species were detected that differed in
size (38 bp) at their 5` ends (Fig. 6C). Synthesis of
the shorter transcript (mRNA-1) is under osmotic control, and the
amount of this mRNA increases strongly in high osmolarity grown cells.
In contrast, production of the longer transcript (mRNA-2) was not
influenced by the osmolarity of the growth medium (Fig. 6C). Thus, expression of the opuA operon
is mediated by two promoters that respond differently to changes in
medium osmolarity. Inspection of the DNA sequence upstream of the
initiation sites of mRNA-1 and mRNA-2 revealed the presence of putative
-35 (consensus sequence: TTGACA) and -10 (consensus
sequence: TATAAT) sequences that could possibly constitute promoters
recognized by a form of RNA polymerase complexed with the main
vegetative
factor (
) of B. subtilis (Fig. 6A)(46) .
Figure 6:
Structure of the opuA regulatory
region and mapping of the opuA transcription initiation sites. A, DNA sequence of the opuA regulatory region.
Putative -10 and -35 sequences and the ribosome-binding
site (rbs) for the opuAA gene are overlined.
The exact positions of the transcription initiation sites for the opuA mRNA-2 and for the opuA mRNA-1 are indicated by arrows. B, comparison of the promoter regions of the
osmoregulated proU and proP from E. coli with those of the osmotically controlled opuA P1 promoter
from B. subtilis. C, mapping of the start sites for
the opuA mRNAs. Total RNA was prepared from cells of strain
JH642(pBKB56) grown in LB or LB with 0.5 M NaCl, hybridized to
a primer complementary to the opuA mRNA, and extended with
reverse transcriptase in the presence of radiolabeled
[S]dATP. DNA sequencing reactions primed with
the same synthetic oligonucleotide used for the primer extension
reaction were employed as a standard to size the opuA mRNAs.
We monitored the
influence of medium osmolarity on opuA expression with the aid
of the (opuAA-lacZ) hyb1 hybrid gene
present on plasmid pBKB56. This protein fusion encodes a hybrid protein
that carries at its amino terminus a large segment of the OpuAA protein
and at its carboxyl terminus an almost complete
-galactosidase. To
test whether synthesis of the OpuAA`-
Gal hybrid protein was under
osmotic control, we grew the B. subtilis strain JH642(pBKB56)
overnight in LB medium and LB medium with 0.5 M NaCl, prepared
total cell extracts, and separated the proteins electrophoretically on
a 7% SDS-polyacrylamide gel (Fig. 7A). Consistent with
the influence of medium osmolarity on opuA-directed
transcription, we detected a strong increase in the production of the
hybrid OpuAA`-
Gal protein in high osmolarity grown cells. This
hybrid protein cross-reacted with an antiserum directed against
-galactosidase (Fig. 7B). We observed that
increased synthesis of the large OpuAA`-
Gal hybrid protein
resulted in the formation of insoluble aggregates, which displayed no
-galactosidase activity. The protein from the E. coli ProU system, ProV, analogous in function to OpuAA from B.
subtilis, is a membrane-associated protein, and the clumping of a
ProV`-
Gal hybrid protein has also been reported(47) .
Figure 7:
Osmoregulated expression of a opuAA-lacZ protein fusion in B. subtilis. A, SDS-polyacrylamide gel electrophoresis of total protein
extracts from the B. subtilis strain JH642 containing plasmids
pRB373 (vector; lanes 1 and 2) or plasmid pBKB56
(((opuAA-lacZ) hyb1); lanes 3 and 4) grown in LB medium or LB medium with 0.5 M NaCl. The position of the OpuAA`-
Gal hybrid protein is
indicated by the arrow, and the molecular mass of marker
proteins (M) is indicated on the left. Only the upper
portion of the gel is shown; the proteins were stained with Coomassie
Brilliant Blue. B, proteins of the samples displayed in A (lanes 1-4) and the marker proteins (M)
containing
-galactosidase were electrophoretically separated by
SDS-polyacrylamide gel electrophoresis, transferred to a nylon
membrane, and reacted with an antiserum against
-galactosidase.
Physical and Genetic Mapping of the opuA Operon
A
comparison of the nucleotide sequence of the 3.4-kb opuA region (Fig. 3) to the DNA data base revealed two short DNA
sequences of 81 and 31 bp that matched (with the exception of a single
mismatch in each DNA segment) the DNA sequence from 2910-2991 bp
and 3339-3370 bp, respectively (Fig. 3). These matching
sequences are located upstream of the amyE gene at 25° on
the B. subtilis genetic map(48) . They represent
junction points of repeating units amplified in a mutant strain showing
hyperproduction of an extracellular -amylase (AmyE) and increased
resistance to the antibiotic tunicamycin (tmrB)(49, 50) . The identification of these
junction point sequences within the opuA region suggested that
the opuA operon is located in the vicinity of the amyE gene. We therefore carried out both physical and genetic mapping
experiments to test this assumption and to position the opuA operon on the B. subtilis genetic map.
amyE
), BKB4 (
(opuA::neo)1 amyE
),
and MO1099 (opuA
(amyE::ery)). As expected, the opuA probe hybridized to a single 5.2-kb EcoRI fragment in
chromosomal digests of the opuA
strains JH642
and MO1099 but recognized a smaller EcoRI restriction fragment
(4 kb) in the chromosomal digest of the
(opuA::neo)1 strain BKB4 (Fig. 8B). Thus, the DNA probe used detects specifically
the opuA region in the B. subtilis genome. Two
closely spaced PstI restriction sites are present in the amyE gene (Fig. 8A), both of which were removed
during the construction of the
(amyE::ery)
mutation (19) (Fig. 8A). The opuA DNA
probe detected an approximately 12-kb PstI restriction
fragment in a chromosomal digest of strain JH642 (and a correspondingly
smaller 10.8-kb restriction fragment from strain BKB4), but an
approximately 20-kb PstI fragment was found in the
(amyE::ery) strain MO1099 (Fig. 8B). The larger size of the hybridizing
chromosomal PstI restriction fragment from strain MO1099
results from the fusion of two adjacent PstI fragments (Fig. 8A). Taken together, these data show that opuA and amyE are physically located close to one another. The amyE gene has been positioned by DNA hybridization next to the
end of a NotI restriction fragment on the physical map of the B. subtilis chromosome(51) . Consistent with this
previous report, we found a NotI restriction site downstream
of the opuA operon (Fig. 1).
Figure 8:
Physical mapping of the opuA operon. A, restriction map and genetic organization of
the opuA, amyE, tmrB, and aroI regions around 25° on the B. subtilis genetic map.
The genetic and physical data for this diagram were compiled from the
literature (48-50). All EcoRI, NotI, and PstI sites are shown, but only the relevant HpaI
sites are indicated. The positions of the
(opuA::neo)1 deletion in strain BKB4
and the
(amyE::ery) mutation in strain MO1099
are marked. The locations and directions of transcription of the opuA operon and the amyE gene are indicated by arrows, and the position of the EcoRI restriction
fragment used as an opuA-specific probe in Southern
hybridization experiments are indicated. B, Southern blot of
chromosomal DNA of strains JH642 (lanes 1 and 4),
BKB4 (lanes 2 and 5), and MO1099 (lanes 3 and 6) cut with EcoRI (lanes 1, 2, and 3) or PstI (lanes 4, 5, and 6), respectively. The position of DNA standard
fragments is shown on the left.
The linkage between opuA and amyE was also apparent when we performed a
genetic mapping experiment using the DNA transformation technique.
Chromosomal DNA from strain BKB4
((opuA::neo)1 amyE
aroI
) was used to transform the B.
subtilis strain TIBS57 (opuA
amyE3
aroI10) to kanamycin resistance. This latter strain was used as
the recipient because it carries both an amyE mutation and an
alteration in the aroI, gene which is closely linked to the tmrB locus (Fig. 8A). Transformants of strain
TIBS57 were selected on LB agar plates containing kanamycin and were
then tested for both their AmyE phenotype on starch-containing agar
plates and their AroI phenotype on minimal plates lacking the aromatic
amino acids Tyr, Phe, and Trp. From 197 kanamycin-resistant
transformants characterized, 176 (89%) were found to be AmyE
and AroI
, attesting to the tight genetic
linkage between the opuA operon and the amyE gene.
Consistent with the expected greater genetic distance between opuA and aroI (Fig. 8A), only a minor portion
(21 of 197) of the kanamycin-resistant transformants of strain TIBS57
had acquired simultaneously both the amyE
and aroI
genes from strain BKB4. Taken together,
these physical and genetic mapping experiments allow us to
unambiguously position the opuA operon at 25° on the B. subtilis genetic map, and we conclude from the physical map
of opuA that this operon is transcribed in a clockwise fashion
on the B. subtilis chromosome.
Figure 9:
Model for the proposed organization of the
binding protein-dependent glycine betaine transport systems ProU and
OpuA. Glycine betaine uptake through the ProU system in the
Gram-negative bacterium E. coli is compared with that mediated
by the OpuA system of the Gram-positive microorganism B.
subtilis. The ProV and OpuAA proteins are ATP-binding subunits
that couple ATP hydrolysis to the transport process. The ProW and OpuAB
proteins are hydrophobic integral inner membrane (IM)
proteins. The substrate-binding proteins of the transport systems are
depicted within the periplasm (ProX) in E. coli and anchored
to the membrane by an amino-terminal lipoyl moiety in B. subtilis (OpuAC). Permeation of glycine betaine () across the E.
coli outer membrane (OM) occurs through the general
diffusion pores OmpC and OmpF (65).
The
(opuA::neo)1 mutation (Fig. 1)
present in strain BKB4 removes both the genes for the substrate-binding
protein (OpuAC) and the integral inner membrane component (OpuAB), thus
inevitably destroying the functioning of the OpuA system entirely. A
strongly reduced glycine betaine transport at low substrate
concentration (10 µM) reflects the loss of the
OpuA-mediated transport activity (Fig. 2B). However, the
presence of the
(opuA::neo)1 deletion
does not completely abolish glycine betaine uptake and hence uncovers
the existence of a second transport pathway for this osmoprotectant in B. subtilis. This second glycine betaine transport system is
under osmotic control (Fig. 2B), implying that it is
also involved in the defense against the deleterious effects of high
osmolarity.
-dependent promoters (46), and hence both promoters
are likely transcribed by an RNA polymerase complex containing the main
vegetative
factor (
). The alternative
transcription factor
is an important regulatory
element for a large network of stress proteins of B. subtilis whose synthesis increases after exposure of the bacterial cell to
salt(4, 5, 6) . We have tested glycine betaine
uptake in several sigB mutants and found no difference from
their sigB
parents, indicating that
does not play a central role in the regulation of
the glycine betaine uptake systems of B. subtilis.
(
)The distance of 17 bp between the -35 and
-10 boxes in the opuA P-2 promoter matches the ideal
distance between -35 and -10 regions of
-dependent promoters, whereas the osmoregulated opuA P-1 promoter has a suboptimal spacing of 18 bp (Fig. 6A). Although both opuA promoters can
direct the synthesis of substantial amounts of mRNA (Fig. 6C), they do not conform closely to the -35
and -10 consensus sequences of
-dependent
promoters (Fig. 6, A and B). In particular, the
osmoregulated opuA P-1 promoter is unusual since it contains
in its -10 region a string of three consecutive GC base pairs.
Interestingly, both the osmoregulated proU and proP promoters from E. coli also exhibit -10 regions
rich in GC base pairs (Fig. 6B), and each of these
promoters contains a TG motif characteristic for an extended -10
region that can partially compensate for inefficient -35
regions(55, 56) . A point mutation in the E. coli
proU -10 region altering one of the GC base pairs to an AT
base pair increases the basal level of proU expression at low
osmolarity but does not alter its osmotic regulation(47) . It is
thus likely that the unusual -10 region of the B. subtilis
opuA P-1 promoter makes an important contribution to the low basal
level of the opuA P-1 transcript in the absence of osmotic
stress (Fig. 6C).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.