J Biol Chem, Vol. 274, Issue 41, 29358-29365, October 8, 1999
The Escherichia coli ssuEADCB Gene Cluster Is
Required for the Utilization of Sulfur from Aliphatic Sulfonates and Is
Regulated by the Transcriptional Activator Cbl*
Jan R.
van der Ploeg
,
Roksana
Iwanicka-Nowicka§,
Tomasz
Bykowski§,
Monika M.
Hryniewicz§, and
Thomas
Leisinger¶
From the Institut für Mikrobiologie, Swiss
Federal Institute of Technology, ETH-Zentrum, CH-8092 Zürich,
Switzerland and § Institute of Biochemistry and Biophysics,
Polish Academy of Sciences, 02-106 Warsaw, Poland
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ABSTRACT |
The growth properties of an Escherichia
coli strain carrying a chromosomal deletion of the
ssuEADCB genes (formerly designated ycbPONME)
indicated that the products of this gene cluster are required for the
utilization of sulfur from aliphatic sulfonates. Sequence similarity
searches indicated that the proteins encoded by ssuA,
ssuB, and ssuC are likely to constitute an ABC
type transport system, whereas ssuD and ssuE
encode an FMNH2-dependent monooxygenase and an
NAD(P)H-dependent FMN reductase, respectively (Eichhorn, E., van der Ploeg, J. R., and Leisinger, T. (1999) J. Biol. Chem. 274, 26639-26646). Synthesis of 
-galactosidase from a
transcriptional chromosomal ssuE'-lacZ fusion
was repressed by sulfate or cystine and depended on the presence of a
functional cbl gene, which encodes a LysR-type
transcriptional regulator. Electrophoretic mobility shift assays with
the ssu promoter region and measurements of -galactosidase from plasmid-encoded ssuE'-'lacZ fusions
showed that full expression of the ssu operon required the
presence of a Cbl-binding site upstream of the 
35 region. CysB, the
LysR transcriptional regulator for the cys genes, was not
required for expression of a chromosomal ssuE'-lacZ fusion
although the ssu promoter region contained three
CysB-binding sites. Integration host factor could also occupy three
binding sites in the ssu promoter region but had no
influence on expression of a chromosomal ssuE'-lacZ fusion.
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INTRODUCTION |
When Escherichia coli is deprived of sulfate or
cysteine, a set of proteins is synthesized de novo or at
increased levels (1, 2). Some of these sulfate starvation-induced (Ssi)
proteins, the sulfate-binding protein (Sbp) and
O-acetylserine lyase (CysK), are components of the
assimilatory sulfate reduction pathway leading to cysteine (3). Two
other proteins whose synthesis is completely repressed when sulfate is
present in the growth medium have been identified as the TauD and TauA
proteins, which are encoded in the tauABCD gene cluster
required for the utilization of taurine (2-aminoethanesulfonate) as a
sulfur source (2). The proteins encoded by tauABC probably
function as an ABC transporter for taurine, whereas TauD is an
-ketoglutarate-dependent dioxygenase which catalyzes the
incorporation of oxygen into taurine and thereby leads to the
liberation of sulfite (4).
There is a clear difference between expression of the cys
genes, required for reduction of sulfate and biosynthesis of cysteine, and that of the tau genes. Whereas the tau genes
are completely repressed by sulfate (5), expression of the
cys genes is only partly repressed (3). The cys
genes are positively regulated by the LysR-type (6) transcriptional
activator CysB and the inducer N-acetylserine (7).
Expression of the tau genes requires CysB, but also Cbl,
another LysR-type transcriptional activator with 41% amino acid
sequence identity to CysB (8). cbl mutants are unable to
utilize taurine as well as a range of other aliphatic sulfonates as
sulfur source and many of the Ssi proteins were absent or present in
reduced amounts in a cbl mutant grown under sulfate-starvation conditions (5). Cbl therefore appears to be a
transcription activator for genes whose expression is induced by
sulfate starvation.
In this study we report the identification of the ssuEADCB
genes (for sulfonate-sulfur
utilization), whose products include two of the remaining
Ssi proteins, Ssi4 and Ssi6. It will be shown that the ssu
genes require cbl for expression and that they encode a set
of proteins that enables E. coli to utilize aliphatic
sulfonates other than taurine as a sulfur source.
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EXPERIMENTAL PROCEDURES |
Chemicals--
All chemicals used as sulfur sources were of the
highest quality available and were obtained from Fluka (Buchs,
Switzerland), except for isethionate and lanthionine, which were
purchased from Aldrich. Oligonucleotides were obtained from Microsynth
(Balgach, Switzerland).
Bacterial Strains, Plasmids, Phages, and Genetic
Procedures--
Strains of E. coli and plasmids used in
this study are listed in Table I. All
strains were grown at 37 °C either in LB medium or sulfur-free
minimal medium (2) supplemented with 0.2% glucose and a sulfur source
(described in the text) to a final concentration of 0.25 mM. When required, growth media contained ampicillin (100 µg/ml), tetracycline (15 µg/ml), chloramphenicol (20 µg/ml), or kanamycin (50 µg/ml). Amino acids were added at the following concentrations: leucine, isoleucine, and valine at 10 µg/ml and trypthophan at 4 µg/ml.
P1 transduction was performed as described (9).
Mapping and Cloning of Genes Encoding Ssi6 and
Ssi4--
Designed on the basis of part of the N-terminal sequence of
Ssi6 (LNMFWFLPTH; see Ref. 1), oligonucleotide 2D6N
(5'-TIAA(C/T)ATGTT(C/T)TGGTT(C/T)(C/T)TICCIACICA-3') was labeled
at its 3' end using digoxigenin-ddUTP and terminal transferase
(Roche Molecular Biochemicals). Total DNA was isolated from E. coli MC4100 using the hexadecyltrimethyl ammonium bromide method
(10) and digested with the restriction enzymes that had been used to
create the E. coli chromosomal restriction map (11) with the
exception of BglI. Restriction fragments were separated by
agarose gel electrophoresis, blotted to a Hybond N membrane (Amersham
Pharmacia Biotech) in 20× SSC, and hybridized overnight to 100 pmol of
digoxigenin-labeled oligonucleotide 2D6N at 45 °C in 10 ml of
hybridization buffer (5× SSC, 0.1% lauroylsarcosine, 0.02% SDS, 1%
blocking reagent). The membrane was washed twice in 2× SSC, 0.1% SDS
at room temperature and twice in 0.5× SSC, 0.1% SDS at 37 °C.
Detection of the hybridized probe was performed with disodium
3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl
phosphate (Roche Molecular Biochemicals) according to the manufacturer.
The sizes of the restriction fragments that hybridized to the probe
were estimated and fitted to the physical restriction map of E. coli according to Heurgué-Hamard et al. (12). For
localization of the gene encoding Ssi4, oligonucleotide 2D4N
(5'-ATGCGIGTIAT(T/C)ACI(T/C)TIGCIGG-3'), derived from the N-terminal
sequence of Ssi4 (MRVITLAG; see Ref. 1), was used in the same way as
described above.
DNA Manipulations and Construction of Plasmids--
For plasmid
isolation, restriction enzyme digestion, and transformation, published
procedures were used (10).
Plasmid pME4210, containing the complete ssuEADCB gene
cluster plus 326 bp1 upstream
of the ssuE start codon, was constructed as follows. Plasmid
pME4204 consists of a 0.33-kb EcoRI/BamHI
fragment obtained by PCR with primers JP12 and JP13 (see below). The
24-bp BamHI/MunI fragment from plasmid pME4204
was replaced by the 1.15-kb BamHI/MunI fragment
from pME4180 to yield plasmid pME4206. Subsequently the 1.4-kb
EcoRI/BamHI fragment from pME4206 and the 4.2-kb
EcoRI/BamHI fragment from plasmid pME4180 were
ligated to EcoRI-digested pBluescriptKS to yield pME4210.
To replace two mutations present in the ssuEADCB sequence,
the ClaI/SalI fragment was substituted by the
ClaI/SalI fragment from pME4281, which contains
an insert generated by PCR amplification from total DNA from E. coli strain EC1250 (14). Since pME4210 contained ClaI
and SalI sites in the vector backbone, the
SacI/HindIII fragment from pME4210 was cloned in
pUC19 to give plasmid pME4220. The ClaI/SalI
fragment from pME4281 was then used to replace the ClaI/SalI fragment in pME4220, thereby yielding
plasmid pME4221.
Sequencing and Sequence Analysis--
The dideoxy chain
termination method of Sanger et al. (14) was used to
sequence DNA fragments cloned into pBluescript KS or pGEM7 using an
Applied Biosystems 373 DNA sequencer. Nucleotide and protein sequences
were analyzed with GCG (Wisconsin Package Version 9.0, Genetics
Computer Group, Madison, WI) and compared with the latest releases of
the EMBL, Swiss-Prot, and TREMBL data bases.
The nucleotide sequence described is deposited in the EMBL data base
under accession number AJ237695.
Construction of a Combined ssuEADCB Deletion and Transcriptional
ssuE'-lacZ Fusion--
For the simultaneous construction of a deletion
in ssuEADCB and a chromosomally encoded transcriptional
ssuE'-lacZ fusion, the promoterless lacZYA genes
from pRS415 (15) were recovered as a BamHI/StuI
fragment and cloned in the BglII/StuI site of pME4210, yielding pME4230. The complete insert from pME4230 was subsequently cloned as NotI/SalI fragment in pKO3
(16). The resulting plasmid, pME4231, was transferred to strain EC1250
by electroporation. Colonies that appeared at 42 °C were suspended in LB medium and appropriate dilutions plated out on LB medium containing 5% sucrose. The resulting colonies were then replica-plated on LB plates containing chloramphenicol and sucrose.
Chloramphenicol-sensitive colonies were selected and tested for growth
on butanesulfonate and for expression of lacZ on minimal
plates containing tryptophan, glutathione as a sulfur source, and
5-bromo-4-chloro-3-indolyl--D-galactopyranoside. One such colony was purified and the strain was designated SE40.
Construction of ssuE'-'lacZProtein Fusions--
For the
construction of ssuE'-'lacZ fusions, PCR was
employed to amplify different fragments of the region upstream of
ssuE and to introduce BamHI and EcoRI
restriction sites in order to facilitate cloning in plasmid pRS552
(15). The forward primers, whose positions at different regions
upstream of the transcription start site are given in parentheses,
were as follows: JP12 (287), 5'-CTTCGTGAATTCGATAATGCC-3'; JP14 (144),
5'-CGAGGAATTCATTGATTCAAC-3'; JP15 (35),
5'-TTTGGAATTCTCTTGTCTCTCC-3'; JP18 (98),
5'-TATGCGTGAATTCTAAAGCC-3'; JP21 (211),
5'-ATACGAATTCCAATAAGTGA-3'; JP24 (80),
5'-CCCTTTCTTTAGTTTGAATTCAG-3'; JP25 (52),
5'-GATACACAGAATTCATATTTGG-3' (the nucleotides
changed to introduce restriction sites are underlined). The
reverse primer used for all fusions was JP13 (+64),
5'-TGATGGGATCCATACTCTCT-3'. The resulting
PCR products were digested with EcoRI and BamHI and ligated to plasmid pRS552 which had been cut with the same enzymes.
The inserts of all plasmids were sequenced to confirm that no errors
had been introduced during the PCR.
For the generation of plasmid pME4234, containing mutations in the
IHF-binding site IHF1, PCR was performed with the primers IHF2 (141)
(5'-GTAATTCAAAGCTTCAACATC-3') and T7
(5'-TAATACGACTCACTATAGGG-3') using pME4204 as a template. The product
was digested with BamHI and HindIII. In a
second PCR reaction, primers IHF1
(5'-ATGTTGAAGCTTTGAATTAC-3', complementary to
primer IHF2) and T3 (5'-ATTAACCCTCACTAAAGG-3') were used with template
pME4204, and the product was digested with EcoRI and
HindIII. Both fragments were combined and ligated to
EcoRI/BamHI-digested pBluescriptKS to yield
pME4234. The BamHI/EcoRI fragment from pME4234
was subsequently cloned in pRS552 to give pME4240. For construction of
pME4232, containing mutations in IHF2, primers IHF3 (181),
5'-ATTTAATCGATTCATGAATAT-3', and IHF4, 5'-CATGAATCGATTAAATTAAG-3' (partially complementary
to primer IHF3), were used in the same way as described above for
construction of pME4234 but by using ClaI instead of
HindIII for digestion of the PCR products. The
BamHI/EcoRI fragment from pME4232 was cloned in
pRS552 to give pME4233. Plasmid pME4209, containing mutations in both
IHF1 and IHF2, was constructed as described above for plasmid pME4232
but by using pME4234 as template DNA for PCR. Plasmid pME4291 was
obtained by cloning the EcoRI/BamHI fragment from
pME4209 in pRS552.
Primer Extension Analysis--
For primer extension analysis,
strain EC1250 was grown in minimal medium with sulfate or
butanesulfonate as sulfur source to an optical density at 600 nm of
approximately 0.8. Isolation of total RNA and primer extension analysis
was according to Babst et al. (17) using primer JP19
(5'-CGCGCATATTCCAGCAAGGAGCTGGA-3'; 71 to 45 bp downstream from the
translation start; Fig. 3A) and about 100 µg of RNA. The
products were loaded on a 6% polyacrylamide gel together with a
sequencing reaction done with the same primer and plasmid pME4181
(Table I) by using the fmol cycle sequencing kit (Promega, Madison, WI).
Enzyme Assays--
-Galactosidase activities were assayed in
cells taken from late log-phase cultures according to the method of
Miller (9) with o-nitrophenylgalactoside as a substrate.
DNA Binding Assays--
DNA fragments containing various
portions of the ssu promoter region were generated by PCR,
5'-labeled with [
-32P]ATP by using T4 polynucleotide
kinase (Promega), and purified by QIAquick spin columns (Qiagen). The
primers used for PCR are described above. Proteins used were highly
purified CysB from Salmonella typhimurium obtained from
N. M. Kredich, partially purified Cbl from E. coli (6),
and highly purified IHF from E. coli (18) obtained from A. Sirko.
Conditions for DNA-protein binding reactions and the electrophoretic
mobility shift assay were as described previously (5). Briefly,
incubation mixtures (20 µl) contained approximately 10 ng of labeled
DNA fragment and 2 µg of sonicated calf thymus DNA per ml to reduce
nonspecific binding, in a buffer consisting of 40 mM
Tris-HCl (pH 8.0), 10 mM MgCl2, 100 mM KCl, 1 mM dithiothreitol, and 100 µg/ml
bovine serum albumin. After incubation with given protein (5 min at
37 °C), samples were separated on a 5% acrylamide/bisacrylamide (82:1) gel in 0.05 M Tris borate/EDTA buffer (pH 8.3) for
1.5 h at 10 V/cm.
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RESULTS |
Cloning and Sequence of ssuEADBC--
The genes encoding the
proteins Ssi4 and Ssi6 were mapped to the chromosome of E. coli as described under "Experimental Procedures." For both
genes, the best fit to the restriction map of E. coli was
obtained at 21.4 min downstream of pepN (results not shown). The genes encoding Ssi4 and Ssi6 were located on plasmid pJP30, which
consists of a 22-kb chromosomal HindIII fragment cloned in
pACYC184 (19). A 5.5-kb SspI/EcoRI fragment from
pJP30 was cloned in pBluescript KS to yield plasmid pME4180 (Fig.
1). We sequenced 4499 bp from the
SspI site until the end of the pepN gene (20),
which corresponds with the genome sequence of E. coli from
996,943 to 992,445 (21). Analysis of the sequence indicated that
immediately downstream and opposite to the direction of transcription
of pepN five open reading frames were present (Fig. 1). The
corresponding genes have been designated ssuE,
ssuA, ssuD, ssuC, and ssuB.
These genes have previously been designated ycbP,
ycbO, ycbN, ycbM, and ycbE,
respectively (21).
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Fig. 1.
Organization of the E. coli
ssuEADCB gene cluster. Relevant restriction sites are
shown, and restriction fragments used for construction of plasmids are
indicated. The EcoRI sites marked with + were constructed by
using PCR. The location of the mutations in plasmids is indicated
with asterisks. Replacement of the chromosomal
ssuEADCB genes by lacZYA gave strain SE40.
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Sequence Analysis of ssuEADCB--
The sequence established by us
showed two differences with respect to the published sequence of the
E. coli genome (21). The T residues at bp 2626 and at bp
2927 were C residues in the sequence of the E. coli genome.
The amino acid sequence of SsuE showed similarity to the
NADH-dependent FMN reductase MsuE from Pseudomonas
aeruginosa (22) and to SsuE from Pseudomonas putida,
involved in utilization of methanesulfonate and aliphatic sulfonates as
sulfur sources, respectively. It has been demonstrated recently that
the purified SsuE protein from E. coli catalyzes the
reduction of flavins by NADH or NADPH (13).
SsuD was similar in sequence to FMNH2-dependent
monooxygenases involved in utilization of sulfonates as sulfur sources
(22, 23).2 In addition, SsuD
showed sequence similarity to a class of monooxygenases of diverse
substrate range which require FMNH2 for their activity. The
SsuD protein from E. coli has recently been purified and was found to act as an FMNH2-dependent oxygenase,
liberating sulfite from a range of aliphatic sulfonates (13).
The sequences of SsuB and SsuC were similar to ATP-binding proteins and
membrane components, respectively, of members of the ABC transporter
superfamily (not shown; see Ref. 24). Moreover, the sequences of the
proteins encoded by ssuA, ssuB, and
ssuC were significantly similar to those of the proteins
that constitute putative ABC transporters from E. coli
involved in utilization of taurine (2), from Bacillus
subtilis involved in utilization of aliphatic sulfonates (SsuA,
SsuB, and SsuC respectively; see Ref. 23), and from P. putida involved in utilization of aliphatic and aromatic
sulfonates (SsuA, SsuB and SsuC respectively).2
The ssuEADCB Gene Cluster Is Required for Utilization of Aliphatic
Sulfonates as Sulfur Source--
Since the synthesis of both Ssi4 and
Ssi6 is up-regulated when sulfate or cysteine are absent from the
growth medium (1), it was likely that the ssuEADCB gene
cluster is involved in utilization of sulfur sources other than
cysteine or sulfate. To study the function and expression of
ssuEADCB, the complete gene cluster was deleted and replaced
by the promoterless lacZYA genes (Fig. 1; see
"Experimental Procedures"). The resulting strain SE40 was unable to
utilize a broad range of aliphatic sulfonates as a sulfur source,
except for taurine. Sulfur sources that supported growth of the wild
type strain EC1250 but not of the mutant SE40 included ethanesulfonate,
propanesulfonate, butanesulfonate, pentanesulfonate, hexanesulfonate,
ethanedisulfonate, octanesulfonate, decanesulfonate, isethionate,
sulfoacetate, MOPS, HEPES, MES, and PIPES. Both the mutant and wild
type strains were unable to use methanesulfonate, cysteate,
dodecanesulfonate, or sulfosuccinate as sulfur source. We were
therefore unable to confirm that E. coli utilizes
methanesulfonate and cysteate as a source of sulfur (25, 26). The
mutant strain was not affected in utilization of sulfur from sulfate,
cysteine, cystine, lanthionine, methionine, glutathione, or taurine.
The ssuEADCB gene cluster is hence required for utilization
of aliphatic sulfonates as a sulfur source except for taurine, for
which the desulfurization system is encoded by the tauABCD
gene cluster (2).
Plasmid pME4180, originating from pJP30 and supposed to contain the
complete ssuEADCB operon plus 205 bp upstream of the
ssuE start, was unable to complement mutant SE40. We
presumed that the inability to complement the mutant might be caused by
the differences found between the ssuEADCB sequence
originating from plasmid pJP30 and that from the E. coli
genome. The mutation in ssuD at bp 2626 led to an amino acid
change from arginine to cysteine, whereas the mutation in
ssuC at bp 2927 was silent. The
ClaI/SalI restriction fragment on plasmid pME4220
(Fig. 1) that contained these two differences was therefore replaced by
a ClaI/SalI fragment that had been amplified by
PCR from genomic DNA of E. coli strain EC1250 (see
"Experimental Procedures"). The resulting plasmid, pME4221, could
complement mutant SE40. Thus, pJP30 and the plasmids derived from it
contained a mutation in SsuD that resulted in the inability to grow
with aliphatic sulfonates. Eichhorn et al. (13) have shown
that the SsuD protein with this mutation is inactive.
Determination of Transcription Initiation Site--
The
transcription start site of ssuEADCB was determined by
primer extension analysis, using RNA isolated from strain EC1250 grown
with butanesulfonate or sulfate as sulfur source as a template for
reverse transcriptase. A single band was visible in the reaction with
RNA from butanesulfonate-grown cells, which was absent when RNA from
sulfate-grown cells was used (Fig. 2).
The transcriptional start site was determined 50 bp upstream of the
translational start site of ssuE (Fig.
3A).
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Fig. 2.
Identification of the transcription start
site of ssuEADCB. RNA was isolated from cells
grown with sulfate (lane 1) or butanesulfonate (lane
2) as a sulfur source and reverse-transcribed with primer JP19. A
sequence ladder generated with the same primer and plasmid pME4181 is
also shown.
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Fig. 3.
A, nucleotide sequence of the
ssuEADCB promoter region. The transcription start site is
underlined and depicted with an arrow. The 35
and 10 regions of the ssu promoter are boxed.
Potential ribosome-binding sites are double underlined, and
the start of ssuE and ycbQ is indicated with the
amino acid sequence of their translation products. The amount of
promoter region used for construction of ssuE'-'lacZ fusions
is indicated together with the plasmid designation. The putative
IHF-binding sites IHF1, IHF2, and IHF3 are boxed, and
nucleotides that have been mutated are in bold type, with
the mutations introduced shown below the sequence. The
position of primer JP19 is indicated by an arrow above the sequence. B, -galactosidase
activities (in Miller units) in late exponential phase grown cells of
E. coli EC1250 carrying plasmid-encoded
ssuE'-'lacZ fusions. Each value represents the mean of at
least three different experiments.  , cystine;  ,
butanesulfonate;  , sulfate;  , butanesulfonate + sulfate;  ,
glutathione.
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Regulation of Expression of Translational ssuE'-'lacZ
Fusions--
To determine the minimal region required for sulfate
starvation-regulated expression of ssuEADCB, plasmids with
translational ssuE'-'lacZ fusions comprising
different fragments of the region upstream of the ssuE gene
were made. In all constructs, the fusion contained the first amino acid
from SsuE. -Galactosidase activity was measured in cells grown with
different sulfur sources (Fig. 3B). Cells grown with
glutathione or butanesulfonate as a sulfur source and containing
plasmids pME4205 and pME4208 exhibited high levels of
-galactosidase, whereas growth with cystine or sulfate prevented
expression. In cells containing plasmids pME4243, pME4196, pME4197, and
pME4245 -galactosidase was partially expressed upon growth with
sulfate or cystine. Cells containing plasmid pME4246 showed low levels
of -galactosidase activity that were not dependent on the sulfur
source, whereas cells containing plasmid pME4192 exhibited barely
detectable levels of -galactosidase activity. These results are
consistent with the results obtained from primer extension analysis.
The fusion lacking the 35 region was inactive, whereas the fusion
containing both 35 and 10 regions resulted in low but non-regulated
expression. Apparently, 62 bp preceding the transcription start point
were required and sufficient for high level expression of the
ssu genes, but wild type regulation of expression by sulfate
and cystine was dependent on at maximum 203 bp preceding the
transcription start point.
Expression of ssuEADCB Requires Cbl--
A chromosomally encoded
transcriptional ssuE'-lacZ fusion was constructed as
described under "Experimental Procedures." Expression of
-galactosidase from this fusion was measured in strain EC1250 grown
with different sulfur sources (Table II).
High levels were found in cells grown with lanthionine or taurine,
whereas djenkolate, glutathione, and methionine resulted in
intermediate levels of -galactosidase. Sulfate and cystine acted as
repressing sulfur sources. The ssu and also the
tau genes, which are regulated in a similar fashion (2), are
therefore only expressed in the absence of sulfate or cystine from the
growth medium. Sulfate and cystine are apparently preferred over
taurine or other sulfonates as sulfur source.
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Table II
Sulfur source dependent expression of -galactosidase from a
transcriptional ssuE'-lacZ fusion on the chromosome of E. coli SE40
-Galactosidase activity was determined in the late exponential phase
of growth and is given in Miller units. Each value represents the mean
of at least two independent experiments.
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Previously it has been shown that both LysR-type transcriptional
activators, CysB and Cbl, were necessary for expression of the
tauABCD genes (5). Synthesis of -galactosidase from the chromosomal ssuE'-lacZ fusion was also virtually
absent in a cbl mutant grown with the derepressing sulfur
sources glutathione or lanthionine (Table
III), which demonstrated that expression of ssu required the presence of Cbl. This is consistent with
our previous observations that a cbl mutant cannot utilize
sulfonates as sulfur source and that the TauD, Ssi4 (SsuE), and Ssi6
(SsuD) proteins are not synthesized in such a mutant (5).
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Table III
Expression of ssu requires the transcriptional activator Cbl but not
CysB
Cells were grown to the late exponential phase with the indicated
sulfur source, and -galactosidase activity was determined (in Miller
units). Each value represents the mean of two independent experiments.
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The synthesis of Cbl is dependent on the presence of CysB (8), and
expression of ssu therefore requires indirectly the presence
of CysB. To determine whether CysB was also directly involved in
expression of ssu, we measured expression of the
ssuE'-lacZ fusion in strains containing plasmid pMH176,
where cbl was expressed at low levels from the trc
promoter (10) by omitting IPTG as inducer. In both a
cbl and a cysB mutant harboring plasmid pMH176, -galactosidase activity could be measured (Table III). CysB is therefore not directly involved in expression of the ssu
genes but serves exclusively as activator of expression of the
cbl gene. This is surprising because we have previously
found such a direct involvement of CysB in the expression of the
tau gene cluster (5). CysB might even prevent full
expression of the ssu genes, since the levels of
-galactosidase in the cysB mutant containing pMH176 were
higher than in the cbl mutant containing the same plasmid
(Table III).
Binding of CysB and Cbl Proteins to ssu Promoter--
In order to
characterize further the mechanism of regulation of the ssu
operon, we have tested the ability of CysB and Cbl proteins to interact
with various portions of the ssu promoter region by an
electrophoretic mobility shift assay. The longest DNA fragment used in
this assay contained the sequence from position 287 to +64 relative
to the transcription start site (the same region was included in
plasmid pME4205 used for -galactosidase assays). As shown in Fig.
4A, both CysB and Cbl formed
complexes with this fragment in a protein
concentration-dependent manner; binding of Cbl resulted in
a single complex, and CysB was able to give several complexes. The
picture of these complexes formed by both proteins with the
ssu promoter was very similar to that obtained earlier for
the 266 to +51 tau promoter fragment where a single
binding site for Cbl and at least two binding sites for CysB were
identified (5). Therefore we believe that the complexes designated as
C1 (Fig. 4A) of either CysB and Cbl with the 351-bp ssu promoter fragment represent occupation of a single
binding site by each protein. Similarly as with the tau
promoter region (5), acetylserine and thiosulfate stimulated CysB
binding to the ssu promoter region that resulted in
increased amounts of the C1 complex (Fig. 4A, lanes 5-7).
Higher order complexes C2 and C3 were clearly seen after longer
exposure of the gel (Fig. 4A, lane 7a) and could represent
occupation of additional binding sites by the CysB protein.
Acetylserine had no effect on Cbl binding (not shown). Thiosulfate had
an opposite effect on Cbl binding than that observed with CysB, a
slight reduction of C1 complex formation with Cbl was observed with the
ssu promoter fragment (Fig. 4A, lane 11).
Essentially a similar picture of complexes formed by CysB and Cbl was
obtained with a 208-bp ssu promoter fragment that extended
from 144 to +64 relative to the transcription start site, suggesting
that all the binding sites for Cbl and CysB were contained within this
fragment (not shown). Two more 5'-shortened ssu promoter
fragments were also used as promoter probes, one starting from position
98 and the second from position 5 to +64 relative to the
transcription start. The former fragment was able to bind CysB and Cbl,
and the latter fragment bound only CysB (Fig. 4B). The above
results indicate that the Cbl-binding site in the ssu
control region lies within the 50-bp sequence preceding the 35 region
of the promoter. The ability of CysB to bind to a site lying within the
region from 35 to +64 is in accordance with the possible function of
this protein as a repressor of ssu expression.
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Fig. 4.
Binding of Cbl and CysB to the ssu regulatory region in a gel mobility shift assay. A,
the 351-bp DNA fragment used as a probe was obtained by PCR with
primers JP12 and JP13, 5'-labeled, and incubated with highly purified
CysB protein or partially purified Cbl protein at concentrations
indicated (in µg/ml). O-Acetylserine (OAS) was
added at 5 mM (lane 5) or 10 mM
(lane 6) and thiosulfate (TS) at 5 mM. C1 (primary complexes) and C2 and C3 (higher order
complexes seen better after longer exposure of the gel, exemplified by
lane 7a) are discussed in the text. B, binding of
CysB and Cbl (at concentrations indicated in µg/ml) to a 99-bp PCR
fragment amplified with primers JP13 and JP15 (lanes 1-6)
or to a 162-bp fragment obtained with primers JP13 and JP18
(lanes 7-12). C indicates complexed DNA.
FP in A and B represents free probe
DNA.
|
|
Integration Host Factor Binds to the ssu Promoter Region but Has No
Influence on Expression from a Chromosomal ssuE'-lacZ Fusion--
As
shown above, -galactosidase synthesis from the shorter
plasmid-encoded ssuE'-lacZ fusions (plasmids pME4243,
pME4196, pME4197, and pME4245) was not as strongly repressed by sulfate and cystine as that from the longer fusions (those present on pME4205
and pME4208; Fig. 3B). We noticed that the shorter fusions lacked one or two putative binding sites for IHF (Fig. 3B)
and speculated that these might be important for exerting repression by
sulfate and cystine. By using electrophoretic mobility shift assays,
the formation of several complexes of IHF with the ssu promoter region was demonstrated (Fig.
5). Binding of IHF to the 275-bp
ssu promoter fragment extending from position 211 to +64 resulted in formation of at least 3 complexes, designated C1, C2, and
C3 (Fig. 5A, lanes 8-10). The C1 complex could represent an
occupation of a single, high affinity binding site by IHF (possibly the
IHF1 site) as it was still present when a high excess of unspecific DNA
was included in the binding reaction (lanes 11 and
12). Binding of IHF to the 208-bp fragment devoid of the
IHF2 site (from 144 to +64) gave a more rapidly migrating specific
complex C1 and additional complexes that were lost in the presence of
an excess of unspecific DNA (Fig. 5A, lanes 2-6). The C1
complex may represent occupation of the IHF1 site by IHF, and its
relatively fast migration can be explained by the position of this site
close to the end of the fragment used as a probe; IHF bound to such
site causes less DNA bending than in the case of central location of
binding (27).
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Fig. 5.
Binding of IHF to the ssu regulatory region. A, purified IHF was incubated with
two 5'-labeled PCR fragments as follows: a 208-bp fragment obtained
with primers JP14 and JP13 (lanes 1-6) and a 275-bp
fragment obtained with primers JP21 and JP13 (lanes 7-12).
Incubation mixtures 5, 6, 11, and 12 contained 10 µg/ml sonicated
calf thymus DNA instead of 2 µg/ml used routinely in all other
binding reactions. C1, C2, and C3 complexes are discussed in the text.
B, IHF was incubated with three different 351-bp DNA
fragments amplified with primers JP12 and JP13 on template pME4204
(probe I, wild type ssu promoter), template pME4232 (probe
II, mutated IHF2 site), or template pME4234 (probe III, mutated IHF1
site). Primary complexes (C1) and higher order complexes (C2 and C3)
are discussed in the text. A and B,
concentrations of purified IHF are indicated in µg/ml; FP
shows free probe DNA.
|
|
We also compared binding of IHF to three ssu promoter
fragments of the same size (351 bp) derived from either pME4204
template (wild type, probe I), pME4232 template (probe II, IHF2 site
changed by site-directed mutagenesis; see "Experimental
Procedures"), or pME4234 (probe III, IHF1 site changed). As shown in
Fig. 5B, the C1 complex formed by IHF with wild type
ssu promoter (probe I, lanes 1-3) was also
present with the fragment containing a mutated IHF2 site (probe II,
lanes 4-6), but it was lost when the fragment with the
mutated IHF1 site was used (probe III, lane 7-9). The
fragment devoid of a functional IHF1 site was still able to form two
complexes with IHF, which suggests the presence of at least three
binding sites for this protein in the wild type ssu
regulatory region. The C2 and C3 complexes seen with the wild type
fragment may represent binding of two and three IHF molecules, respectively, to this region. The C3 complex was lost when the IHF2
site was mutated (lane 6); therefore, the C2 complex seen with this fragment (probe II) may represent occupation of IHF1 and an
additional site "IHF3" (Fig. 3). Binding of IHF downstream of the
35 region was confirmed using a fragment 35 to +54 relative to the
transcription start (not shown).
By using butanesulfonate as sulfur source for growth, -galactosidase
activity from the different plasmid encoded ssuE'-'lacZ fusions was measured in a wild type strain (MC1000) and in an isogenic
himA mutant (EC2643), which is unable to synthesize one of
the subunits of the heterodimeric IHF protein (Ref. 28; Fig. 6). Whereas activity was not changed in
the himA mutant containing plasmids pME4196 and pME4197, it
was significantly reduced in the mutant containing plasmid pME4205 or
pME4208. The latter two plasmids contain both IHF1 and IHF2, whereas
the former plasmids lack one or both binding sites. The presence of
binding sites IHF1 or IHF2 therefore appeared to have a negative effect
on expression of -galactosidase from plasmid encoded
ssu'-'lacZ fusions in a himA mutant but not in
the wild type strain. However, mutation of either binding site IHF1,
IHF2, or both IHF1 and IHF2 on plasmid pME4205 also resulted in
similarly decreased levels of -galactosidase in the himA
mutant EC2643, indicating that the binding sites themselves are not
involved.
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|
Fig. 6.
-Galactosidase activities (in
Miller units) in early exponential phase-grown cells of E. coli MC1000 (wild type) or EC2643 (himA)
grown with butanesulfonate and carrying plasmid-encoded
ssuE'-lacZ fusions. Each value is the mean of at
least three different experiments.
|
|
Sulfate-, cystine-, butanesulfonate-, or glutathione-grown cells of
EC1250 containing a plasmid-encoded ssuE'-'lacZ fusion and
mutations in IHF1 (on plasmid pME4240) or in IHF2 (on plasmid pME4233)
and both in IHF1 and IHF2 (on plasmid pME4291) had similar levels of
-galactosidase as cells containing the control plasmid pME4205. This
indicated that neither IHF1 nor IHF2 has influence on expression of the
ssu genes in a wild type background.
No difference in expression of -galactosidase from the chromosomal
ssuE'-lacZ fusion between wild type and a himA
mutant could be observed in the exponential or the stationary growth phase. We therefore conclude that the positive effect of IHF on expression is only seen with plasmid-encoded fusions, although it
cannot be excluded that IHF influences expression of the chromosomal ssuE'-lacZ fusion under conditions that have not
been tested.
| |
DISCUSSION |
It has been known for sometime that E. coli can utilize
sulfonates as sulfur source under aerobic conditions (2, 25, 26).
Sulfonates utilized include not only naturally occurring compounds,
like taurine or isethionate, but also industrially produced compounds
such as MOPS or HEPES. In this study we have identified the
ssuEADCB gene cluster, which encodes proteins for the
utilization of sulfur from a wide range of aliphatic sulfonates, and
whose expression is repressed by sulfate. Repression by sulfate of
organosulfur utilization was also observed in B. subtilis, P. aeruginosa, and Rhodococcus (22, 23, 29, 30).
Therefore, sulfate is preferred as sulfur source over organosulfur compounds.
The wide substrate range of the ssuEADCB system contrasts
with that of the tauABCD system, for which taurine appears
to be the only substrate (2, 4). Since tau mutants are still
able to use aliphatic sulfonates (2) and the ssu mutant
studied in this work was still able to use taurine, both systems
apparently have a complementary substrate range. The key enzyme for the
desulfonation of aliphatic sulfonates is the
FMNH2-dependent oxygenase encoded by
ssuD, which has been purified and characterized (13). The reduction of FMN is carried out by the NAD(P)H-dependent
FMN reductase, encoded by ssuE (13).
Expression of the ssu genes required the presence of Cbl,
which binds just upstream of the 35 region of ssu. Removal
of the Cbl-binding site resulted in low levels of -galactosidase
from plasmid-encoded ssuE'-'lacZ fusions. In addition,
expression of -galactosidase was absent from a chromosomal
ssuE'-lacZ fusion in a cbl mutant. These data
strongly suggest that the Cbl protein acts as a transcriptional
activator for ssu gene expression. It is not surprising that
this promoter needs an activator since the 10 sequence only matches 3 out of 6 nucleotides of the consensus sequence. Binding of Cbl in the
close vicinity of the 35 region may involve a direct contact of this
activator with RNA polymerase, a mechanism suggested for many other
LysR-type transcriptional regulators (6).
The role of CysB in the expression of ssu is more difficult
to understand. Contrary to its involvement in expression of the tauABCD operon (5), CysB does not seem to be directly
required for expression of ssuEADCB. There is, however, at
least one CysB-binding site located overlapping the 35 and 10
regions, and occupation of this site by CysB could prevent RNA
polymerase from binding to the ssu promoter. The possible
function of CysB as a repressor is confirmed by our findings that in
the presence of Cbl delivered from a plasmid, a cysB mutant
expressed a transcriptional ssuE'-lacZ fusion at higher
levels than a cbl mutant containing this fusion.
Although the direct involvement of CysB at the ssu promoter
is not completely clear, CysB is required for expression of
cbl (8) and therefore is an indirect activator of the
ssu operon. A cysB mutant was unable to
synthesize -galactosidase from a chromosomally encoded
ssuE'-lacZ fusion, unless the cbl gene was expressed from the trc promoter. Activation of transcription
of the genes involved in biosynthesis of cysteine from sulfate and of
the cbl gene requires the binding of CysB to activating
sites just upstream of the 35 region, the presence of acetylserine as
an inducer molecule, and the absence of sulfide or thiosulfate, which
function as anti-inducers (7). Cysteine has a negative effect on the
synthesis of acetylserine through feedback inhibition of serine
transacetylase by cysteine. Since expression of cbl is
regulated as part of the cys regulon, the presence of
cysteine in the growth medium leads to repression of cbl
(8). This explains why the ssu genes are not expressed in
cysteine-grown cells. In sulfate-grown cells however, cbl is
still synthesized, and repression of tau and ssu
gene expression by sulfate must be caused by a different mechanism that
involves the Cbl protein.
The structure of the C-terminal part of CysB (residues 88-324), which
comprises the binding site for the co-inducer acetylserine, has
recently been determined (31). The protein contains two / domains
that enclose a cavity. Unexpectedly, a sulfate ion could be modeled in
this cavity, although sulfate has no effect on activation of
transcription by CysB (32). It was speculated that this cavity serves
as the binding site for both acetylserine and for the anti-inducer
thiosulfate. The Cbl protein is very similar in sequence to CysB, and
many of the residues that were thought to interact with the inducer and
the anti-inducer are conserved. This could indicate that Cbl and CysB
recognize the same molecules as inducer and anti-inducer. But it is
more likely that the anti-inducer for Cbl is different from that for
CysB and that this difference causes repression by sulfate of
ssu and tau genes. Sulfate itself could act as
anti-inducer for Cbl. Alternatively, it is possible that sulfite, the
product of TauD- or SsuD-catalyzed desulfonation of taurine or
alkanesulfonates, acts as anti-inducer.
Even though expression of both the tau and ssu
genes is repressed by sulfate and regulated by Cbl, the operons are
probably regulated in a different manner. This is substantiated by
several observations. First, expression of the tau genes
requires the direct involvement of CysB, whereas expression of the
ssu genes was not dependent on the presence of CysB. Second,
binding of Cbl to the tau promoter occurred at 112 to 68
relative to the transcription start (5), which is different from the
Cbl-binding site at the ssu promoter just upstream of the
35 region. Third, the sequences of both promoter regions did not
reveal any similarity, except for their high AT content.
It has previously been observed that IHF mutants showed reduced growth
on limiting amounts of inorganic sulfur sources and on djenkolate (33,
34), which supported the assumption that IHF is required for full
expression of cysJIH in stationary phase but not during
exponential phase (34). The presence of several binding sites for IHF
in the intergenic region between the ssu operon and
ycbQ suggested that IHF could also be involved in regulation of ssu, but we were unable to demonstrate it. On the other
hand, it has to be considered that IHF may affect the expression of ycbQ, which encodes a hypothetical fimbrial-like protein.
IHF has previously found to be required for DNA inversion that controls phase variation of type 1 fimbriae in E. coli (35).
In conclusion, we have shown that the ssu genes are required
for utilization of sulfur from sulfonates. Our results have
demonstrated that there are at least three different proteins that
interact with the promoter region of the ssu genes: Cbl,
CysB, and IHF. The region from 62 to 0 relative to the transcription
start is needed for high level expression, which is probably brought
about by transcription activation by the Cbl protein. Full repression by cysteine and sulfate requires also the region from 203 to 62,
and although there are binding sites for IHF in this region, IHF does
not appear to be involved.
| |
ACKNOWLEDGEMENTS |
We thank Michael Kertesz for helpful
discussions. We also thank N. M. Kredich for a kind gift of CysB
protein and A. Sirko for IHF protein. We acknowledge J. Tommassen and
G. M. Church for gifts of plasmids.
| |
FOOTNOTES |
*
This work was supported in part by grants from the Federal
Institute of Technology, Zürich and from the Polish State
Committee for Scientific Research, Project Number 6PO4A05216).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ237695.
¶
To whom correspondence should be addressed: Institut für
Mikrobiologie, ETH-Zentrum, CH-8092 Zürich, Switzerland. Tel.: 41 1 632 33 24; Fax: 41 1 632 11 48; E-mail:
leisinger@micro.biol.ethz.ch.
2
P. Vermeij and M. A. Kertesz, unpublished observations.
| |
ABBREVIATIONS |
The abbreviations used are:
bp, base pair;
kb, kilobase pair;
PCR, polymerase chain reaction;
DTT, dithiothreitol;
IHF, integration host factor;
MES, 2-(N-morpholino)ethanesulfonic acid;
MOPS, 3-(N-morpholino)propanesulfonic acid;
PIPES, piperazine-1,4-bis(2-ethanesulfonic acid).
| |
REFERENCES |
| 1.
|
Quadroni, M.,
Staudenmann, W.,
Kertesz, M.,
and James, P.
(1996)
Eur. J. Biochem.
239,
773-781[Medline]
[Order article via Infotrieve]
|
| 2.
|
van der Ploeg, J. R.,
Weiss, M. A.,
Saller, E.,
Nashimoto, H.,
Saito, N.,
Kertesz, M. A.,
and Leisinger, T.
(1996)
J. Bacteriol.
178,
5438-5446[Abstract/Free Full Text]
|
| 3.
|
Kredich, N. M.
(1996)
in
Escherichia coli and Salmonella: Cellular and Molecular Biology
(Neidhardt, F. C.
, Curtiss, R., III
, Ingraham, J. L.
, Lin, E. C. C.
, Low, K. B.
, Magasanik, B.
, Reznikoff, W. S.
, Riley, M.
, Schaechter, M.
, and Umbarger, H. E., eds)
, pp. 514-527, American Society for Microbiology, Washington, D. C.
|
| 4.
|
Eichhorn, E.,
van der Ploeg, J. R.,
Kertesz, M. A.,
and Leisinger, T.
(1997)
J. Biol. Chem.
272,
23031-23036[Abstract/Free Full Text]
|
| 5.
|
van der Ploeg, J. R.,
Iwanicka-Nowicka, R.,
Kertesz, M. A.,
Leisinger, T.,
and Hryniewicz, M. M.
(1997)
J. Bacteriol.
179,
7671-7678[Abstract/Free Full Text]
|
| 6.
|
Schell, M. A.
(1993)
Annu. Rev. Microbiol.
47,
597-626[CrossRef][Medline]
[Order article via Infotrieve]
|
| 7.
|
Kredich, N. M.
(1992)
Mol. Microbiol.
6,
2747-2753[CrossRef][Medline]
[Order article via Infotrieve]
|
| 8.
|
Iwanicka-Nowicka, R.,
and Hryniewicz, M. M.
(1995)
Gene (Amst.)
166,
11-17[CrossRef][Medline]
[Order article via Infotrieve]
|
| 9.
|
Miller, J. H.
(ed)
(1992)
A Short Course in Bacterial Genetics
, pp. 268-274, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
|
| 10.
|
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. E., Seidman, J. G., Smith, J. A., and Struhl, K.
(eds)
(1987)
Current Protocols in Molecular Biology
, Wiley Interscience, New York
|
| 11.
|
Kohara, Y.,
Akiyama, K.,
and Isono, K.
(1987)
Cell
50,
495-508[CrossRef][Medline]
[Order article via Infotrieve]
|
| 12.
|
Heurgué-Hamard, V.,
Mora, L.,
and Buckingham, R. H.
(1995)
Nucleic Acids Res.
23,
2801-2802[Free Full Text]
|
| 13.
|
Eichhorn, E.,
van der Ploeg, J. R.,
and Leisinger, T.
(1999)
J. Biol. Chem.
274,
26639-26646[Abstract/Free Full Text]
|
| 14.
|
Sanger, F.,
Nicklen, S.,
and Coulson, A. R.
(1977)
Proc. Natl. Acad. Sci. U. S. A.
74,
5463-5467[Abstract/Free Full Text]
|
| 15.
|
Simons, R. W.,
Houman, F.,
and Kleckner, N.
(1987)
Gene (Amst.)
53,
85-96[CrossRef][Medline]
[Order article via Infotrieve]
|
| 16.
|
Link, A. J.,
Phillips, D.,
and Church, G. M.
(1997)
J. Bacteriol.
179,
6228-6237[Abstract/Free Full Text]
|
| 17.
|
Babst, M.,
Hennecke, H.,
and Fischer, H.-M.
(1996)
Mol. Microbiol.
19,
827-839[CrossRef][Medline]
[Order article via Infotrieve]
|
| 18.
|
Sirko, A.,
Zehelein, E.,
Freundlich, M.,
and Sawers, G.
(1993)
J. Bacteriol.
175,
5769-5777[Abstract/Free Full Text]
|
| 19.
|
Tommassen, J.,
van der Ley, P.,
van der Ende, A.,
Bergmans, H.,
and Lugtenberg, B.
(1982)
Mol. Gen. Genet.
185,
105-110[CrossRef]
|
| 20.
|
Foglino, M.,
Gharbi, S.,
and Lazdunski, A.
(1986)
Gene (Amst.)
49,
303-309[CrossRef][Medline]
[Order article via Infotrieve]
|
| 21.
|
Blattner, F. R.,
Plunkett, G., III,
Bloch, C. A.,
Perna, N. T.,
Burland, V.,
Riley, M.,
Collado-Vides, J.,
Glasner, J. D.,
Rode, C. K.,
Mayhew, G. F.,
Gregor, J.,
Davis, N. W.,
Kirkpatrick, H. A.,
Goeden, M. A.,
Rose, D. J.,
Mau, B.,
and Shao, Y.
(1997)
Science
277,
1453-1474[Abstract/Free Full Text]
|
| 22.
|
Kertesz, M. A.,
Schmidt-Larbig, K.,
and Wüest, T.
(1999)
J. Bacteriol.
181,
1464-1473[Abstract/Free Full Text]
|
| 23.
|
van der Ploeg, J. R.,
Cummings, N. J.,
Leisinger, T.,
and Connerton, I. F.
(1998)
Microbiology
144,
2555-2561[Abstract]
|
| 24.
|
Higgins, C. F.
(1992)
Annu. Rev. Cell Biol.
8,
67-113[CrossRef]
|
| 25.
|
Roberts, R. B., Abelson, P. H., Cowie, D. B., Bolton, E. T., and Britten, R. J.
(eds)
(1955)
Sulfur Metabolism
, pp. 318-405, Carnegie Institution, Washington D. C.
|
| 26.
|
Uria-Nickelsen, M. R.,
Leadbetter, E. R.,
and Godchaux, W., III
(1993)
J. Gen. Microbiol.
139,
203-208[Medline]
[Order article via Infotrieve]
|
| 27.
|
Engelhorn, M.,
and Geiselmann, J.
(1998)
Mol. Microbiol.
30,
431-441[CrossRef][Medline]
[Order article via Infotrieve]
|
| 28.
|
Bykowski, T.,
and Sirko, A.
(1998)
Biochimie (Paris)
80,
987-1001[Medline]
[Order article via Infotrieve]
|
| 29.
|
Hummerjohann, J.,
Küttel, E.,
Quadroni, M.,
Ragaller, J.,
Leisinger, T.,
and Kertesz, M. A.
(1998)
Microbiology
144,
1375-1386[Abstract]
|
| 30.
|
Li, M. Z.,
Squires, C. H.,
Monticello, D. J.,
and Childs, J. D.
(1996)
J. Bacteriol.
178,
6409-6418[Abstract/Free Full Text]
|
| 31.
|
Tyrrell, R.,
Verschueren, K. H.,
Dodson, E. J.,
Murshudov, G. N.,
Addy, C.,
and Wilkinson, A. J.
(1997)
Structure
15,
1017-1032[CrossRef]
|
| 32.
|
Hryniewicz, M. M.,
and Kredich, N. M.
(1991)
J. Bacteriol.
173,
5876-5886[Abstract/Free Full Text]
|
| 33.
|
Freundlich, M.,
Ramani, N.,
Mathew, E.,
Sirko, A.,
and Tsui, P.
(1992)
Mol. Microbiol.
6,
2557-2563[CrossRef][Medline]
[Order article via Infotrieve]
|
| 34.
|
Sirko, A.,
W gle ska, A.,
and Hulanicka, D.
(1998)
Mol. Gen. Genet.
258,
174-177[CrossRef][Medline]
[Order article via Infotrieve]
|
| 35.
|
Eisenstein, B. I.,
Sweet, D. S.,
Vaughn, V.,
and Friedman, D. I.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
6506-6510[Abstract/Free Full Text]
|
| 36.
|
Casadaban, M. J.
(1976)
J. Mol. Biol.
104,
541-555[CrossRef][Medline]
[Order article via Infotrieve]
|
| 37.
|
Jagura-Burdzy, G.,
and Hulanicka, D.
(1981)
J. Bacteriol.
147,
744-751[Abstract/Free Full Text]
|
| 38.
|
Silhavy, T. J., Berman, M. L., and Enquist, L. W.
(eds)
(1984)
Experiments with Gene Fusions
, p. xi, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
|
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ABSTRACT
INTRODUCTION
